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Fluorogenic Probes for Live-Cell Imaging of Biomolecules

By

Wen Chyan

B.S. ChemistryMassachusetts Institute of Technology, 2013

Submitted to the Department of Chemistryin Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy in Biological Chemistry

at the

Massachusetts Institute of Technology

June 2018

0 2018 Massachusetts Institute of Technology. All rights reserved.

Signature redacted__Signature of Author:

Department of ChemistryApril 30, 2018

-Signature redactedCertified by:

Ronald T. RainesFirnich Professor of Chemistry

Signature redacted Thesis Supervisor

Accepted by:MASSACHUSETTS INSTITUTE Robert W. Field

OF TECHNOLOGY Haslam and Dewey Professor of ChemistryI I I Chairman, Departmental Committee on Graduate Students

JUN 20 2018

LIBRARIESARCHIVES

This doctoral thesis has been examined by a Committee of the Department of Chemistry as

follows:

Signature redactedBarbara Imperiali

Class of 1922 Professor of Chemistry and Professor of BiologyCommittee Chairman

-Signature redacted\ Ronald T. Raines

Pro ssor of Chemistryesis Supervisor

Signature redactedWtfatthew D. Shoulders

/hitehead Career Development Associate Professor

2

Fluorogenic Probes for Live-Cell Imaging of Biomolecules

by

Wen Chyan

Submitted to the Department of Chemistry on April 30, 2018in Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy in Biological Chemistry

ABSTRACT

Fluorogenic probes, small-molecule sensors that unmask brilliant fluorescence upon exposure tospecific stimuli, are essential tools for chemical biology. Probes that detect enzymatic activity canbe used to illuminate the complex dynamics of biological processes at a level of spatiotemporaldetail and sensitivity unmatched by other techniques. This dissertation describes the developmentof new fluorophore chemistries to expand our current fluorogenic probe toolkit and the subsequentapplication of these probes to study dynamic cell transport processes.

Chapter 1. Enzyme-Activated Fluorogenic Probes for Live-Cell and In Vivo Imaging. Chapter1 reviews recent advances in enzyme-activated fluorogenic probes for biological imaging,organized by enzyme classification. This review surveys recent masking strategies, differentmodes of enzymatic activation, and the breadth of current and future probe applications. Keychallenges, such as probe selectivity and spectroscopic requirements, are described in this chapteralong with therapeutic and diagnostic opportunities that can be accessed by surmounting thesechallenges.

Chapter 2. Electronic and Steric Optimization of Fluorogenic Probes for BiomolecularImaging. In many fluorogenic probes, the intrinsic fluorescence of a small-molecule fluorophoreis masked by ester masking groups until entry into a cell, where endogenous esterases catalyze thehydrolysis of esters, generating fluorescence. The susceptibility of masking groups to spontaneoushydrolysis is a major limitation of these probes. Previous attempts to address this problem haveincorporated auto-immolative linkers at the cost of atom economy and synthetic adversity. In thischapter, I report on a linker-free strategy that employs adventitious electronic and stericinteractions in easy-to-synthesize probes. I find that halogen-carbonyl n-sc* interactions and acylgroup size are optimized in 2',7'-dichlorofluorescein diisobutyrate. This probe is relatively stableto spontaneous hydrolysis but is a highly reactive substrate for esterases both in vitro and in cellulo,yielding a bright, photostable fluorogenic probe with utility in biomolecular imaging.

Chapter 3. Cellular Uptake of Large Monofunctionalized Dextrans. Dextrans are a versatileclass of polysaccharides with applications that span medicine, cell biology, food science, andconsumer goods. In Chapter 3, I apply the electronically stabilized probe described in Chapter 2to study the cellular uptake of a new type of large monofunctionalized dextran that exhibits unusualproperties: efficient cytosolic and nuclear uptake. This dextran permeates various human cell typeswithout the use of transfection agents, electroporation, or membrane perturbation. Cellular uptake

3

occurs primarily through active transport via receptor-mediated processes. Thesemonofunctionalized dextrans could serve as intracellular delivery platforms for drugs or othercargos.

Chapter 4. Paired Nitroreductase-Probe System to Quantify the Cytosolic Delivery ofBiomolecules. Cytosolic delivery of large biomolecules is a significant barrier to therapeuticapplications of CRISPR, RNAi, and biologics such as proteins with anticancer properties. InChapter 4, I describe a new paired enzyme-probe system to quantify cytosolic delivery ofbiomolecules-a valuable resource for elucidating mechanistic details and improving delivery oftherapeutics. I designed and optimized a nitroreductase fusion protein that embeds in the cytosolicface of outer mitochondrial membranes, providing several key improvements over unanchoredreporter enzymes. In parallel, I prepared and assessed a panel of nitroreductase-activated probesfor favorable spectroscopic and enzymatic activation properties. Together, the nitroreductasefusion protein and fluorogenic probes provide a rapid, generalizable tool that is well-poised toquantify cytosolic delivery of biomolecules.

Chapter 5. Future Directions. This chapter outlines several future directions for expanding thescope of fluorogenic probes and developing new biological applications. Additionally, Chapter 5is followed by an appendix describing a tunable rhodol fluorophore scaffold for improvedspectroscopic properties and versatility. Overall, the work described in this thesis illustrates thepower of enzyme-activated fluorogenic probes to provide fresh insight into dynamic biologicalprocesses, with direct implications for improved therapeutic delivery.

Thesis Supervisor: Ronald T. RainesTitle: Firmenich Professor of Chemistry

4

ACKNOWLEDGEMENTS

First and foremost, I am immensely grateful to Professor Ronald T. Raines for the opportunityto pursue fluorophore research in his lab. Professor Raines' contagious enthusiasm for new ideasfosters creativity and his many accomplishments in science, innovation, and entrepreneurship area true inspiration to me and many others. On a personal level, Ron's mentorship and support hasbeen pivotal for smooth navigation of key transitions in my career development and researchprogress.

I would also like to express my sincere gratitude to Dr. Luke D. Lavis for the opportunity tospend 6 weeks in his lab at HHMI as a visiting scientist. The practical skills and design thinking Ilearned from the Lavis lab jump-started my own research efforts. In particular, Todd Gruber andJonathan Grimm were exceedingly kind and generous with their time at HHMI.

Many thanks to thesis committee members Professors Barbara Imperiali and MatthewShoulders for their advice and feedback on my thesis.

Much of my research benefited greatly from close collaboration with fellow Raines labmembers, for whom I am truly grateful. Henry Kilgore, an essential collaborator and thoughtpartner, has never ceased to amaze me with his breadth of knowledge and seemingly infinite fountof ideas. Trish Hoang and Valerie Ressler, both biochemists of many talents, were an absolutepleasure to work with and significantly augmented the impact of our research projects. Manythanks to Brian Gold for his contributions towards conceptualizing probe electronic stabilization.I also would like to thank Aubrey, Brian Graham, Caglar, Emily, Ian, Jesus, Jim, John, Joelle,Kalie, Leland, Lindsey, Lucas, Matt, Robert, and Thom for making the Raines lab such a fantasticplace to work.

The impact my parents Oliver and Jin-Jian have had on my education and upbringing isimpossible to overstate, as are my mother's sacrifices to provide every possible opportunity formy brother and me. As the youngest in a family of chemists, it is humbling to complete my degreein the same department as my father, albeit some 30 years later. Although life has sent my brotherYieu and me to different corners of the country since our time together in college, I look backfondly on the fun times and shenanigans we have had over the years.

MIT also holds special significance as the place I first met my wife Joyce. Since starting ourjourney together almost a decade ago, we have navigated life's tumultuous waves hand in hand. Iam especially grateful for Joyce's love and support during my last year of undergraduate studiesand the second year of graduate school-two particularly challenging transitional periods.

In addition to my physical family, I thank God for the "chance" meeting with Ed Kao that ledto finding my spiritual family in ABSK YA and Antioch Baptist Church. It is rare to find believerswho speak and live the truth with such boldness, and I treasure the brief period of time I have hadto learn from Pastors Dave, Thomas, and Sang Peter, and also Ed, Peter, Tony, and the group.Most importantly, I thank Jesus Christ for His faithfulness and grace to one as unworthy as I.

But one thing I do: forgetting what lies behind and straining forward to what lies ahead, Ipress on toward the goal for the prize of the upward call of God in Christ Jesus."

(Philippians 3:13-14)

In loving memory of my grandparents

Chyan Cheng-Fu 101-14 (1925-2017) and Chyan Cheng-Chiao $1%R (1927-2015)

5

TABLE OF CONTENTS

ABSTRACT................................................................................................................................... 3

ACKNOW LEDGEM ENTS ..................................................................................................... 5

TABLE OF CONTENTS ....................................................................................................... 6

LIST OF FIGURES.................................................................................................................... 13

LIST OF SCHEM ES .................................................................................................................. 17

LIST OF TABLES...................................................................................................................... 19

LIST OF ABBREVIATIONS ................................................................................................ 20

CHAPTER 1................................................................................................................................ 25

1.1 Introduction............................................................................................................... 26

Fluorogenic Probes. ................................................................................................. 26

Probe Design for in Vivo and Live-Cell Imaging. .................................................. 27

Enzym es Targeted by Fluorogenic Probes. ............................................................. 30

1.2 Oxidoreductases (EC 1).......................................................................................... 32

Enzymes Acting on the CH-NH2 Group of Donors (EC 1.4). ................................ 32

Enzym es Acting on NADH or NADPH (EC 1.6).................................................... 34

Enzymes Acting on Other Nitrogenous Compounds as Donors (EC 1.7)............... 37

Enzym es Acting on Sulfur Groups as Donors (EC 1.8). ......................................... 37

Enzymes Acting on Paired Donors, with Incorporation or Reduction of Molecular

Oxygen (EC 1.14) .................................................................................................... 37

1.3 Transferases (EC 2) ................................................................................................ 39

Acyltransferases (EC 2.3) ....................................................................................... 40

Enzym es Transferring Alkyl or Aryl Groups (EC 2.5).......................................... 40

6

Enzymes Transferring Phosphorous-Containing Groups (EC 2.7).......................... 41

1.4 H ydrolases (EC 3) ................................................................................................... 41

Enzym es A cting on Ester Bonds (EC 3.1.1)............................................................ 41

Enzym es A cting on Phosphate Bonds (EC 3.1.3).................................................... 44

G lycosidases (EC 3.2.1) .......................................................................................... 44

Enzym es A cting on Peptide Bonds (EC 3.4) .......................................................... 45

Enzym es A cting on Cyclic Am ides (EC 3.5.2). ...................................................... 47

1.5 C onclusions................................................................................................................ 48

1.6 A cknow ledgem ents ................................................................................................ 49

CH A PTER 2 ................................................................................................................................ 50

2.1 Introduction............................................................................................................... 51

2.2 R esults and D iscussion.......................................................................................... 53

Tuning A cyl Probe Stability with H alogenation...................................................... 53

Optim ization of the A cyl M asking Group. ............................................................. 58

2.3 C onclusions................................................................................................................ 66

2.4 A cknow ledgem ents ................................................................................................ 66

2.5 Experim ental............................................................................................................. 67

G eneral Inform ation................................................................................................ 67

Instrum entation. .......................................................................................................... 67

Optical Spectroscopy ................................................................................................ 68

Spontaneous Probe Hydrolysis. ............................................................................... 68

PLE-Catalyzed Probe H ydrolysis. .......................................................................... 69

7

Cell Culture and Live Cell Imaging ........................................................................ 69

In Cellulo Probe Hydrolysis...................................................................................... 69

Photobleaching ............................................................................................................ 70

Computational Procedures ....................................................................................... 70

Synthesis of 2',7'-Dichlorofluorescein...................................................................... 70

Syntheiss of Diesters 2.1-2.5 and 2.11-2.17, and Esters 2.7-2.9. ......................... 71

Synthesis of 2',7'-Dichlorofluorescein Diacetoxymethyl Ether 2.18....................... 75

N M R Spectra .............................................................................................................. 76

IR Spectra.................................................................................................................... 91

Dihedral-Angle Scans .............................................................................................. 97

Cartesian Coordinates and Total Energies................................................................ 107

CH APTER 3.............................................................................................................................. 116

3.1 Introduction............................................................................................................. 117

3.2 Results and Discussion............................................................................................ 119

3.3 Conclusions.............................................................................................................. 136

3.4 Acknow ledgem ents ................................................................................................. 137

3.5 Experim ental........................................................................................................... 137

M aterials. .................................................................................................................. 137

General Procedures. .................................................................................................. 138

Instrum entation. ........................................................................................................ 138


UV-Visible and Fluorescence Spectroscopy............................................................ 139

8

Synthesis of A lkyne Probe 3.1.................................................................................. 140

Synthesis of Carboxyl Probe 3.2............................................................................... 141

Ellm an's A ssay . ........................................................................................................ 141

Synthesis of Conjugate 3.3. ...................................................................................... 141



Enzym atic U nm asking of Conjugate 3.3 .................................................................. 143

A ggregation A ssay .................................................................................................... 143

D extran Stability A ssay. ........................................................................................... 144

Linker Stability A ssay ............................................................................................... 144

Branching A ssay . ...................................................................................................... 145

Tim e-Course Im aging. .............................................................................................. 145

4 'U I ............................................ Ca.......................................145

Pearson's Colocalization Coefficient........................................................................ 146

Com petition A ssay .................................................................................................... 146

N M R Spectra ............................................................................................................ 147

CH A PTER 4.............................................................................................................................. 149

4.1 Introduction............................................................................................................. 150

4.2 R esults and D iscussion............................................................................................ 152

Design and Optimization of an Outer Mitochondrial Membrane-Anchored NTR

Fusion Protein........................................................................................................... 152

D esign and Testing of N itroreductase-A ctivated Probes.......................................... 159

9

4.3 Conclusions.............................................................................................................. 166

4.4 Acknow ledgem ents ................................................................................................. 166

4.5 Experim ental........................................................................................................... 166

General Experim ental. .............................................................................................. 166

General Optical Spectroscopy................................................................................... 168

UV -Visible and Fluorescence Spectroscopy. ........................................................... 168

Quantum Yield Determ ination.................................................................................. 168

Cloning of TOM 20-m Scarlet-N TR Fusion Protein................................................. 168

General Cell Culture. ................................................................................................ 169

General Transfection of Fusion Protein into HeLa Cells.......................................... 169

Cell Viability Assays. ............................................................................................... 169

Cell Viability A ssay Based Post-transfection........................................................... 169

M TS Cell Proliferation A ssay................................................................................... 170

General Confocal Live-cell Imaging. ....................................................................... 170

N TR Activation Kinetics A ssay................................................................................ 170

N TR A ctivity Assay in Live HeLa Cells .................................................................. 171

Synthesis of N itrobenzyl Coum arin 4.2.................................................................... 171

Synthesis of N aphthalim ides 4.3-4.5 and 4.9-4.10.................................................. 172

N M R Spectra ............................................................................................................ 175

CH A PTER 5.............................................................................................................................. 181

5.1 Extending Electronic Stabilization to Other Fluorophore Scaffolds................. 182

5.2 Biomolecule Imaging Using Electronically-Stabilized Probes............................ 184

10

5.3 Promoting Intracellular Delivery of Biomolecular Payloads via Dextran

Conjugation............................................................................................................. 185

5.4 Probing Mechanisms of Dextran Uptake and Endosomal Escape..................... 187

5.5. Quantifying Cytosolic Delivery with a Nitroreductase-Probe System............... 188

Applying the Nitroreductase-Probe System to Biomolecules................ 188

Optimizing Contrast Ratio, Background, and Responsiveness. ............................... 189

Quantifying Differences in Rates of Biomolecule Uptake and Internalization in

M atched C ell L ines................................................................................................... 190

APPENDIX A ............................................................................................................................ 192

A.1 Introduction............................................................................................................. 193

Xanthene Dyes and Rhodols..................................................................................... 193

D y e S ynth esis............................................................................................................ 196

A.3 Results and Discussion............................................................................................ 199

Preparation of Rhodols via Cross-Coupling Chemistry............................................ 199

Rhodol Spectroscopic Properties. ............................................................................. 200

A.4 Future Directions .................................................................................................... 205

Fluorogenic Azetidinyl Rhodols............................................................................... 206

Halogen-Halogen Interactions for Fluorescence Lifetime Extension and

Bathochromic Shifts in Azetidinyl Rhodols. ............................................................ 207

n--+ * Stabilization of Azetidine Rhodols and Fluorogenic Rhodol Probes. ........... 207

"Single-hit" Rhodol Fluorogenic Probes with Enhanced Enzyme-Response ................

K in etics. .................................................................................................................... 2 0 8

11

A .5 A cknow ledgem ents ................................................................................................. 208

A .6 Experim ental........................................................................................................... 209

General Inform ation.................................................................................................. 209

Instrum entation. ........................................................................................................ 209


pH Titrations............................................................................................................. 210

W ater-D ioxane Titrations......................................................................................... 210

Com putational Procedures........................................................................................ 211

Synthesis of Fluorescein Ditriflate A .0..................................................................... 211

Synthesis of Rhodol Triflates A .1-A .5..................................................................... 211

Synthesis of Rhodols A .6-A .7.................................................................................. 213

Synthesis of Rhodols A .8-A .10................................................................................ 214

Synthesis of Rhodol Esters A .11-A 12...................................................................... 216

Synthesis of Rhodol Ethers A.13-A .14.................................................................... 217

N M R Spectra........................................................................................................... . 218

REFERENCES......................................................................................... 231

12

LIST OF FIGURES

Figure

Figure

Figure

Figure

Figure

Figure

1.1.

1.2.

1.3.

1.4.

1.5.

2.1.

Figure 2.2.

Figure 2.3.

Figure 2.4.

Figure 2.5.

Figure 2.6.

Figure 2.7.

Figure 2.8.

Figure 2.9.

Schematic representation of enzyme-activated fluorogenic probes................... 27

Structures of probes activated by oxidoreductase enzymes (EC 1.4-EC 1.6)...... 33

Structures of probes activated by transferase enzymes (EC 2)......................... 39

Structures of probes activated by hydrolases in classes EC 3.1-EC 3.3. .......... 42

Structures of probes activated by hydrolases in classes EC 3.4 and EC 3.5......... 46

Graphs showing the time-course for the spontaneous hydrolysis of probes

2.1-2.5 as measured by the generation of fluorescence.................................... 55

Graph showing the strength of n--*w interactions in compounds 2.6-2.10

as calculated with second-order perturbation theory. ...................................... 57

3D Rendering of the 2D potential-energy surface for 2-chlorophenyl

acetate (2.8) in Figure 2.4C ................................................................................ 59

Calculated potential energy surfaces generated by scanning the Ca-Cb-O-Cc

dihedral angle of compounds 2.6-2.9................................................................ 60

Graphs showing the effect of acyl groups on the hydrolytic stability of

2',7'-dichlorofluorescein probes in vitro and in cellulo. ................................... 61

Graphs showing the time-course for the hydrolysis of probes 2.1, 2.3,

and 2.11-2.18 as measured by the generation of fluorescence......................... 62

Kinetic traces and Michaelis-Menten plots for the unmasking of fluorogenic

p ro b es.................................................................................................................... 6 4

In cellulo hydrolysis of fluorogenic probes 2.1, 2.3, and 2.11-2.18. ................ 65

Rate of fluorophore photobleaching in live HeLa cells under continuous

illum ination ...................................................................................................... . . 66

13

Figure 3.1.

Figure 3.2.

Figure 3.3.

Figure 3.4.

Figure 3.5.

Figure 3.6.

Figure 3.7.

Figure 3.8.

Figure 3.9.

Figure 3.10.

Figure 3.11.

Characterization of dextran conjugates............................................................... 120

Uptake of conjugate 3.3 by hum an cells............................................................. 121

Confocal microscopy images showing the time-course for the uptake

of conjugate 3.3 (10 pM ) by live HeLa cells...................................................... 122


of conjugate 3.3 (10 tM ) by live H 1299 cells.................................................... 123


of conjugate 3.3 (10 pM ) by live H 1299 cells.................................................... 124

Confocal microscopy images of live HeLa cells incubated with 1 00-kDa

TAMRA-dextran (A-D) or 70-kDa TAMRA-dextran (E-H) at 10 pM for

30 m in at 37 C . .................................................................................................. 12 6

Confocal microscopy images of live HeLa cells incubated for 30 min with

conjugate 3.4 (A-D) or conjugate 3.5 (E-H) at 10 pM for 30 min at 37 C...... 127

Graph showing the average fluorescence signal per cell measured in

live HeLa cells incubated with dextran conjugates............................................. 127

Graphs showing the acid stability of components of conjugate 3.3 upon

incubation in 1.0 M H Cl for 1 h.......................................................................... 128

C4 HPLC traces of probe 3.1 (black) and conjugate 3.3 (color) after

incubation for 1 h in 1.0 M HCl (A), DMEM containing FBS (B), or HeLa

cell ly sate (C ). ..................................................................................................... 12 9

Graphs showing the time-course of the hydrodynamic radius (Rh) Of

dextran Dl upon treatment with 1.0 M HCl at 25 'C (A) or 60 'C (B)....... 129

14

Figure 3.12.

Figure 3.13.

Figure 3.14.

Figure 3.15.

Figure 3.16.

Figure 3.17.

Figure 3.18.

Figure 4.1.

Figure 4.2.

Figure 4.3.

Figure 4.4.

Figure 4.5.

Figure 4.6.

Graphs showing the effects of concentration on the hydrodynamic

radius (Rh) of dextran D1 (A), dextran D2 (B), and dextran D3 (C). ................. 129

1H-NMR spectra of nigerose (top) and isomaltose (bottom) in D 20.................. 131

'H-NMR spectra of kojibiose (top) and maltose (bottom) in D 20. .................... 132

'H-NMR spectra of dextran D1 (top) and dextran D4 (bottom) in D20............. 133

'H-NM R spectrum of dextran D2 in D2 0........................................................... 134

Confocal microscopy images showing the effect of temperature on

the uptake of probe 3.1 (5 ptM; A and B) and conjugate

3.3. (5 jM ; C and D ) by HeLa cells. .................................................................. 135

Graph showing the effect of increasing concentrations of unlabeled

dextran on the uptake of conjugate 3.3 by live HeLa cells................................. 136

Schematic representation of (A) enzyme and (B) probe components of the

paired NTR-probe system for detecting cytosolic uptake of biomolecules. ...... 152

Validation and optimization of transfection of HeLa cells with NTR

fusion protein plasm id......................................................................................... 154

Images depicting NTR fusion protein expression levels in live HeLa cells....... 156

Images depicting NTR fusion protein expression levels over time in live

H eL a cells. .......................................................................................................... 15 7

Localization of NTR fusion protein to mitochondria as a function

of D N A concentration......................................................................................... 158

Graphs of normalized absorbance and emission profiles of 4.1-4.6 in

10 m M HEPES-NaOH buffer, pH 7.3................................................................ 161

15

Figure 4.7.

Figure 4.8.

Figure 4.9.

Figure 5.1.

Figure 5.2.

Figure

Figure

A.1.

A.2.

Toxicity profile of fluorogenic naphthalimide probes as determined

by M TS cell proliferation assay.......................................................................... 162

Kinetic traces and Michaelis-Menten plots for the unmasking of

fluorogenic probes 4.1 and 4.2............................................................................ 163

Graph of fluorescence fold increase upon exposure of probes 4.1-4.6

to n itroreductase.................................................................................................. 165

Extension of electronic and steric acyl masking group stabilization to

other fluorophore scaffolds and masking groups................................................ 183

Promoting cytosolic delivery of proteins via conjugation to

m onofunctionalized dextrans.............................................................................. 186

Titrations of rhodols A.6-A.10 in solutions of varying pH or solvent polarity.. 201

Graphs of spectroscopic properties for rhodol dyes. A.6-A.14,

fluorescein, and rhodam ine................................................................................. 204

16

LIST OF SCHEMES

Scheme 1.1.

Scheme 1.2.

Scheme 2.1.

Scheme 2.2.

Scheme 2.3.

Scheme 2.4.

Scheme 2.5.

Scheme 3.1.

Scheme 4.1.

Scheme 5.1.

Scheme 5.2.

Scheme A.1.

Scheme A.2.

Common fluorescence modulation methods in enzyme-activated fluorogenic

probes, with probe and enzyme examples for each method. ............................ 28

Representative examples of two predominant mechanisms of activation

in auto-immolative linkers: (A) elimination and (B) acyl transfer. .................. 29

Auto-immolative linkers (red) inserted between fluorophores and esterase

targets to enhance stability................................................................................ 52

Halogenated fluorescein diacetate probes.......................................................... 53

Halogenated m odel com pounds......................................................................... 56

Acylated 2',7'-dichlorofluorescein probes. ........................................................ 61

Optimized geometry of the butyryl ester moiety in probe 2.12........................ 63

(A) Synthetic route to conjugates 3.3, 3.4, and 3.5 from commercial dextrans

Dl and D3. (B) In cellulo enzymatic activation of conjugate 3.3. ..................... 119

Structure of nitroreductase-activated fluorogenic probes 4.1-4.6 and

parent fluorophores 4.7-4.12 released upon reduction of probe nitro groups.... 159

Application of bioconjugable electronically-stabilized acyl probes

to polysaccharides, nucleic acids, and lipids. ..................................................... 184

Structures of bioconjugable 4-nitronaphthalimide probe 4.5, control fluorophore

4.11, bioconjugable probe 5.6, control esterase-activated probe 5.7, improved

nitroreductase-activated probe 5.8, and control esterase-activated probe 5.9.... 189

Structures of the xanthene dye core structure and common xanthene

dye scaffolds. ................................................................................................... 194

Open-close equilibrium in xanthene dyes....................................................... 194

17

Scheme A.3.

Scheme

Scheme

Scheme

A.4.

A.5.

A.6.

Scheme A.7.

Scheme A.8.

Comparison of (A) "dual-hit" and (B) "single-hit" unmasking

mechanisms for fluorogenic probes based on xanthene dyes. ............................ 196

Location and effects of common substitutions on rhodol dyes........................... 196

Previous synthetic routes to xanthene dyes. ....................................................... 198

Synthesis of rhodols via stochastic Buchwald-Hartwig cross-coupling

of various nitrogen-containing moieties with fluorescein ditriflate.................... 200

Differences in tautomer equilibria in (A) amine-type rhodols and (B) amidic

rh o d o ls................................................................................................................. 2 02

Proposed rhodols for studying electronic effects and fluorogenic probe

ap p lication s. ........................................................................................................ 2 05

18

LIST OF TABLES

Table 1.1. Targeted enzymes, applications, and in cellulo or in vivo imaging with enzyme-

activated probes organized by enzyme commission number (EC) and subclass.. 31

Table 2.1. Spectroscopic attributes and pKa values of unmasked halogenated

fluorescein diacetate probes.............................................................................. 54

Table 2.2. Carbonyl stretching frequencies, donor-acceptor geometries and interaction

energies, and hydrolytic stabilities of compounds 2.1-2.10............................. 56

Table 2.3. Kinetic parameters were obtained by fitting Michaelis-Menten plots

generated from the initial rates of probe-unmasking in 10 mM HEPES-NaOH \

buffer, pH 7.3, containing PLE (Figure 2.6)..................................................... 65

Table 3.1. Pearson's correlation coefficients (r) for localization of a fluorogenic

dextran conjugate and either Hoechst 33342 nuclear stain or LysoTrackerTM

acidic vesicle stain . ............................................................................................. 126

Table 3.2. Dextran branching as determined by 1H-NMR spectroscopy............................. 130

Table 4.1. Spectroscopic properties of NTR-activated probes and parent fluorophores

4.1-4.12 measured in 10 mM HEPES-NaOH buffer, pH 7.3............. 162

Table 4.2. Kinetic parameters of nitroreductase activation and maximal fold

increase in fluorescence calculated from Table 4.1 for representative probes... 162

Table 5.1. Improved spectroscopic properties and activation rates of rhodamine

probe 5.6 compared to naphthalimide probe 4.5 and coumarin probe 5.6.......... 190

Table 5.2. Representative adherent matched cell lines amenable to screening

w ith the nitroreductase-probe system . ................................................................ 191

Table A.1. Key properties of fluorescein, rhodol, and rhodamine............................................. 195

19

LIST OF ABBREVIATIONS

ADEPT

AFU

ALKBH3

ALP

AM

ATCC

AU

BHQ2

BlaC

BODIPY

Btk

CHO

CMV

CRISPR

Cy5

CYP450

DABCYL

DIEA

DMAP

DMEM

DMSO

20

Antibody-directed enzyme-prodrug therapy

Arbitrary fluorescence units

a-Ketoglutarate-dependent dioxygenase alkB homolog 3

Alkaline phosphatase

Acetoxymethoxy

American Type Culture Collection

Arbitrary units

Black hole quencher 2

P-Lactamase from M tuberculosis

Boron-dipyrromethene

Bruton's tyrosine kinase

Chinese hamster ovary

Cytomegalovirus

Clustered regularly interspaced short palindromic repeats

Cyanine 5

Cytochrome P450

4-((4-(Dimethylamino)phenyl)azo)benzoic Acid

Diisopropylethylamine

4-Dimethylaminopyridine

Dulbecco's Modified Eagle's Medium

Dimethyl sulfoxide

DNA

DPBS

Sx Ji

EC

EDANS

ELISA

ER

ESIPT

ESI-QIT-MS

EtOAc

FADH2

FBS

FDA

FMN

FRET

FT-IR

GDEPT

GFP

GST

HEK

HEPES

hiPSC

Deoxyribonucleic acid

Dulbecco's phosphate-buffered saline

Dielectric constant or extinction coefficient

Brightness

Enzyme commission

5-((2-Aminoethyl)aminonaphthalene-1-sulfonic acid

Enzyme-linked immunosorbent assay

Endoplasmic reticulum

Excited state intramolecular proton transfer

Electrospray ionization quadrupole-ion trap mass spectrometry

Ethyl acetate

Flavin adenine dinucleotide (reduced form)

Fetal bovine serum

Food and Drug Administration

Flavin mononucleotide

F6rster resonance energy transfer

Fourier transform infrared

Gene-directed enzyme-prodrug therapy

Green fluorescent protein

Glutathione S-transferase

Human embryonic kidney

2 [4-(2-Hydroxyethyl)- 1 -piperazinyl]ethanesulfonic acid

Human induced pluripotent stem cells

21

HL Human promyelocytic leukemia

hNQO1 Human NAD(P)H:quinone oxidoreductase

HPLC High performance (pressure) liquid chromatography

HRMS High-resolution mass spectrometry

HTC Hepatoma tissue culture

IC5 0 Half maximal inhibitory concentration

IEFPCM Integral equation formalism polarizable continuum model

IR Infrared

IUBMB International Union of Biochemistry and Molecular Biology

kcat Enzyme turnover number

KM Michaelis-Menten constant

Iem Emission maximum wavelength

labs Absorbance maximum wavelength

Iex Excitation wavelength

LCMS Liquid chromatography mass spectrometry

mAb Monoclonal antibody

MAO Monoamine oxidase

MeOD Methanol-D4

MeOH Methanol

mSc Monomeric red fluorescent protein (mScarlet)

mScarlet Monomeric red fluorescent protein

NAD+ Nicotinamide adenine dinucleotide (oxidized form)

NADH Nicotinamide adenine dinucleotide (reduced form)

22

NADPH Nicotinamide adenine dinucleotide phosphate (reduced form)

NBO Natural bond orbital

NMR Nuclear magnetic resonance

nt Nucleotide

NTR Nitroreductase

OMM Outer mitochondrial membrane

PBS Phosphate-buffered saline

PDB Protein Data Bank

PET Photoinduced electron transfer

pKa Acid dissociation constant

PLE Pig (porcine) liver esterase

PrEC Prostate epithelial cells

PTP Protein tyrosine phosphatase

Pyr Pyridine

QSY21 2-[6-(1,3-Dihydro-2H-isoindol-2-yl)-9- {2-[(4-{[(2,5-dioxopyrrolidin-1-

yl)oxy]carbonyl}piperidin-1-yl)sulfonyl]phenyl}-3H-xanthen-3-

ylidene]-2,3-dihydro-1H-isoindolium

RFU Relative fluorescence unit

RI Ribonuclease inhibitor

RNA Ribonucleic acid

RNAi Ribonucleic acid interference

RNase 1 Human pancreatic ribonuclease

RNase A Bovine pancreatic ribonuclease

23

ROS Reactive oxygen species

RT Room temperature

Up Hammett para substituent constant

tI12 Half-life

TEM-1 P-Lactamase from E. coli

TFA Trifluoroacetic acid

THF Tetrahydrofuran

TICT Twisted internal charge transfer

TLC Thin-layer chromatography

TML Trimethyl lock

TOM Translocase of the outer membrane

TrxR Thioredoxin reductase

TXN Thioredoxin

UV Ultraviolet

'P Quantum yield

WT Wild type

XPhos 2-Dicyclohexylphosphino-2',4',6'-triisopropylbiphenyl

24

CHAPTER 1

Enzyme-Activated Fluorogenic Probes for Live-Cell and in VivoImaging

25

1.1 Introduction

Fluorogenic Probes. Fluorogenic probes are latent fluorophores that reveal signal in response to

specific chemical reactions, environmental changes, or interactions with analytes. 1-3 Fluorogenic

probes are generally prepared by chemically modulating the fluorescence of a parent fluorophore,

rendering it nonfluorescent until activation by specific triggering events. Because of their high

sensitivity and ability to monitor diverse events selectively, fluorogenic probes are essential

members of the sensor toolkit for chemistry and biology.4' 5 Furthermore, the ubiquitous role of

enzymes in complex biochemical pathways underscores the need for dynamic probes able to

maintain selectivity and sensitivity in biological environments and model systems.

Enzyme-activated fluorogenic probes (Figure 1.1), which utilize enzymatic activity to trigger

generation of fluorescence, address this need by providing a versatile platform for selectively

monitoring enzymes in vivo and in cellulo. Early enzyme-activated probes were based on xanthene

dye scaffolds and detected galactosidases, phosphatases, lipases, and esterases." Shortly

afterwards, rudimentary live cell imaging was demonstrated with cell-permeable probes,' 0 leading

to modem probe applications including cell viability assays," diagnostic tests,'2 and immunoassay

technologies such as ELISA.13 Recent innovations in probe chemistry and design continue to drive

development of new imaging techniques and applications utilizing enzyme-activated fluorogenic

probe. Chapter 2 and Appendix A outline several advances in probe chemistry, the applications of

which are detailed in Chapter 3 and Chapter 4.

26

Figure 1.1. Schematic representation ofenzyme-activated fluorogenic probes.

EnzymeActivation

Probe Design for in Vivo and live-Cell Imaging. The two overarching themes for fluorogenic

probe design and applications are the spectroscopic properties of the parent fluorophore and the

method employed to mask its fluorescence. Key properties of the parent fluorophore include

brightness (the product of quantum yield and extinction coefficient), wavelengths and shapes of

both excitation and emission peaks, pH effects on fluorescence, and resistance to photobleaching.

Recent trends in fluorophore scaffolds include reduction of phototoxicity and autofluorescence

background with far-red,' 4 near-IR,' 5 and two-photon probes' 6 and fine-tuning of spectroscopic

properties and brightness.17 18 Whereas parent fluorophore properties determine the post-activation

performance of a probe, the fluorescence masking strategy typically governs its target and enzyme

responsiveness. In the context of enzyme-activated probes, masking strategies are arguably of

greater importance than parent fluorophore identity because enzymes often interact directly or

indirectly with masking moieties. Accordingly, this chapter focuses on enzyme-activated masking

strategies and imaging applications, abstaining from extensive discussion of parent fluorophore

chemistry and spectroscopic properties previously covered elsewhere. 1720

Optimal fluorescence masking methods are chemically stable, respond selectively to the

desired event, and completely eliminate fluorescence and absorption at the excitation wavelength

via quenching, masking groups, or other chemical modifications. Three main methods to modulate

27

fluorescence in enzyme-activated fluorogenic probes are depicted in Scheme 1.1. Although

masking groups to block constitutive fluorescence (Scheme 1.1A) are most frequently used,

quenching strategies based on F6rster resonance energy transfer (FRET) or photoinduced electron

transfer (PET) effects (Scheme 1.1 C) can provide high modularity and, in some cases, ratiometric

imaging. Other common themes for enzyme activation include fluorophore precipitation and auto-

immolative linkers that can improve probe stability and performance. 2 ' Two most frequently

encountered auto-immolative motifs-elimination and acyl transfer-are used to release the

fluorophore payload rapidly and spontaneously (Scheme 1.2, Chapter 4).

Modulation methodExpe

EC 1-EC 3 o 0 OH ALP

1.1 P

EC 1 N02 NTR

I1.2

C FRET/PET Bu

EC 1-EC 3 0hNQO1

. HN 0

1.3 0

Scheme 1.1. Common fluorescence modulation methods in enzyme-activated fluorogenicprobes, with probe and enzyme examples for each method. Enzymes for each method aredenoted above each arrow. (a) Enzymatic cleavage of blocking groups. (b) Enzymatic conversionof blocking groups into other functional groups. (c) Enzymatic release of FRET or PET quenchers.

Because the intracellular space is a dense heterogeneous mixture of molecules, organelles, and

other cell structures, the complexity of biological systems is more faithfully represented by live

cell and in vivo models than by fixed-cell or isolated enzyme experiments. 2 2 ,'2 Additionally, the

rich trove of dynamic cell processes that can be studied in live cells and in vivo probes is not

28

accessible to fixed-cell imaging or similar disruptive methods. As a result, probe technologies

developed using live cell and in vivo models are more immediately translatable to therapeutic,

diagnostic, and clinical applications."," In fact, fluorogenic probes and masking groups are often

inspired by prodrug and inhibitor design strategies. 16 2 6 2 7

Operating in the crowded cellular environment imposes additional design constraints on

enzyme-activated probes for biological imaging applications. These constraints include optimizing

rates and specificities of enzymatic activation, directing probe uptake and localization, enhancing

probe stability, and minimizing toxicity. Probe stability, enzyme specificity, and rate of activation

are heavily influenced by the method of fluorescence modulation. In probes that employ a masking

or blocking strategy (Scheme 1.LA-B), a single covalently attached group serves as both the

enzyme-responsive moiety and fluorescence masking group. In contrast, probes utilizing

quenching techniques may require the addition of a separate enzyme-responsive group (Scheme

1.1C). Conversely, the modularity of masking and enzyme-responsive groups provides a

convenient method of adapting fluorophore scaffolds to target different enzymes.

A R1 0 ' R Elimination

~ A'R R

R1 -CO 2H R2 -OH Quinone methide

B H R N Acyl Transfer 1 0

NHR 2 NHR2

o R'-CO 2H 0 R2 -NH 2 TML Lactone

Scheme 1.2. Representative examples of two predominant mechanisms of activation in auto-immolative linkers: (A) elimination and (B) acyl transfer. Enzymatic activity first releases themasking group (R'), followed by rapid rearrangement or cyclization to release the fluorophore(R2).

29

Enzymes Targeted by Fluorogenic Probes. Enzymes are conventionally named and classified

by the types of reactions they catalyze. Although naming conventions have evolved in the past few

decades, the International Union of Biochemistry and Molecular Biology (IUBMB) has

established a standardized enzyme classification system consisting of six main classes:

oxidoreductases (EC 1), transferases (EC 2), hydrolases (EC 3), lyases (EC 4), isomerases (EC 5),

and ligases (EC 6).28 The majority of enzyme-activated probes target enzymes in classes EC 1-EC

3. The sparsity of probes targeting EC 4-EC 6 can be partially attributed to the inefficient and

difficult fluorescence modulation mechanisms for chemical transformations catalyzed by these

enzymes. Probes targeting oxidoreductases in EC 1 employ the widest variety of fluorescence

modulation strategies, whereas probes targeting EC 2 and EC 3 enzymes primarily rely on

FRET/PET quenching or masking groups (Scheme 1.1). Although probes for the three main

enzyme classes EC 1-EC 3 have existed for several decades, recent advances in probe chemistry

and design have rejuvenated the field with methods to tune spectroscopic properties and enzyme

specificity. 17,26

In subsequent sections of this chapter, I describe advances in enzyme-activated probes for

biological imaging, organized by enzyme type. Each section contains descriptions of targeted

enzyme classes or subclasses, modes of enzyme activation, and applications to live cell and in vivo

imaging. A summary of enzymes, probes, applications, and model cell lines and organisms can be

found in Table 1.1.

30

EC Enzyme(s) A pplications Cell lines/Tissues Organisms Probe

1.4 Monoamine oxidase Parkinson's disease diagnosis, inhibitor screening Hep-G2, SH-SY5Y D. melanogaster, Mice 1.4,1.51.6 NAD(P)H:quinone Rapid cancer cell screening, tissue resection A549, HT29, H446, H596, OVCAR-3 - 1.3,1.6, 1.7

oxidoreductaseNitroreductases Antibiotic-resistant pathogen identification - E. faecium, S. aureus, K. pneumoniae, 1.8

A. baumanniiOrganelle-specific imaging in hypoxic tumor cells A549, HEK293, HeLa, HTC1 16, rat liver - 1.2, 1.9-1.14,

1.16Selective mitochondrial imaging and drug delivery A549, BT474, DU145, W138 - 1.15

1.7 Azoreductase Orthogonal reporter system A549, HEK293T, HeLa, NIH-3T3 - 1.171.8 Thioredoxin reductase Thioredoxin reductase-selective imaging in live cells Hep-G2 - 1.181.14 ALKBH3 Prostate cancer targeted therapy and inhibitor screening B16, HeLa Zebrafish 1.19

Tyrosinase Vitiligo and Parkinson's disease diagnosis PC3, U2OS - 1.202.3 y-Glutamyltranspeptidase Intraoperative fluorescence imaging SHIN3, SKOV3, whole mice colon - 1.21, 1.222.5 Glutathione Transferase Isoform-selective glutathione transferase imaging HL60 - 1.232.7 Bruton's tyrosine kinase Single-step selective kinase imaging Jurkat, Namalwa - 1.243.1 .1 Carboxylesterases Super-resolution study of enzymatic activity CHO, Drosophila S2, HEK293, HeLa, - 1.25

primary neurons, WBF344, brain sliceOrthogonal probe-enzyme pair, improved probe properties HeLa - 1.26, 1.27Theranostics hiPSC neurons, neuro-2A 1.28Pathogen profiling and detection M. tuberculosis 1.29Prodrug activation and multicolor imaging of ER esterases HeLa, HT1080, SK-N-SH 1.30Study of endocytic processes in cancer cells HeLa, HTB125, HTB126 1.31

3.1.3 Alkaline phosphatase Monitoring excreted phosphatases, near-IR imaging of ALP HeLa, Hep-G2, U-20S tissue, Saos-2 tissue D. melanogaster, Mice 1.32, 1.33Protein tyrosine Two-photon visualization of PTP HEK293, HeLa, Hep-G2 S. saprophyticus, E. faecalis, A. baumannii, 1.1, 1.34phosphatase S. aureus

3.2 p-Galactosidase Tracking cell senescence, quantification of cytosolic delivery C6, HeLa , SK-MEL-103 Mice 1.35, 1.36Glucocerebrosidase Study of lysosomal storage disorders Primary human fibroblasts 1.37P-Glucaronidase Deep tissue tumor imaging Mice 1.38

3.4 p-Alanyl aminopeptidase Pathogen detection P. aeruginosa, S. marcescens, B. cepacia 1.39Hepsin matriptase Prostate cancer imaging DU145, LNCaP, PC3, PrEC Mice 1.40Cathepsin B and S Deep tissue tumor imaging of lyososomal cathepsins HEK293, HeLa, KB, MCF-7, MDA-MB-231, Mice 1.42, 1.43

NIH-3T3, primary dendritic cells, U87Caspase-3 Visualization of pre-apoptotic enzymatic activity HeLa 1.44ER aminopeptidase Two-photon ER-targeted deep tissue redox imaging HeLa 1.45

3.5 p-Lactamases Detection and labelling of antibiotic-resistant pathogens - E. coli, M. tuberculosis 1.46, 1.47

Table 1.1. Targeted enzymes, applications, and in cellulo or incommission number (EC) and subclass.

vivo imaging with enzyme-activated probes organized by enzyme

1.2 Oxidoreductases (EC 1)

Oxidoreductases are a highly diverse class of redox enzymes that act on a wide variety of

substrates, often with cofactors NAD(P)H and flavin mononucleotide (FMN). These enzymes are

further classified as dehydrogenases or oxidases depending on the nature of the redox reaction and

the final electron acceptor. Oxidases transfer electrons to molecular oxygen as the acceptor,

whereas dehydrogenases remove hydrogens from a donor in a NADH- or FADH2-dependent

manner. Of the 22 different types of oxidoreductases, most fluorogenic probes target those classes

relevant to pathogen detection and cancer cell imaging, diagnosis, and treatment. Key foci of recent

work involve creating tools to investigate cancer biomarkers, tumor cell hypoxia, and

neurodegenerative disease.

Enzymes Acting on the CH-NH2 Group of Donors (EC 1.4). Oxidoreductases of subclass EC

1.4 include enzymes such as monoamine oxidase (MAO) that are associated with the outer

mitochondrial membrane and catalyze the oxidative deamination of amines to aldehydes.2 9

Although both MAO-A and MAO-B isoforms are mitochondria-bound and abundant in the brain,

they are differentially localized at a cellular and tissue level and have different substrate

preferences. Isoforms MAO-A and MAO-B are effective inhibitor targets for the treatment of

Parkinson's disease, as both MAO enzymes are important to maintain hormone and

neurotransmitter homeostasis.2 9 Two-photon probes 1.4 and 1.5 were recently developed to

selectively monitor MAO-A and MAO-B activity in deep tissue imaging.16,26 Probe 1.4 is based

on a resorufin scaffold with a linker connecting the fluorophore and propylamine enzyme-

32

-1

EC 1.6 - Quinone OxidoreductaseC1

H 2N -,O N0 0

1.4 N 0

0 0 0

H2N O N H HN O N

1.5 0 1.6 1.7 o

,OMe

N N

N

0 Ne2 O O

NOO N N021.8 1.9 1.10 1.11

0.7 0.1 0.2 n 'C02N 1 0N

/

F F

BB B B Br B / Br RO \ R = (CH2CH 20),CH,1.12 N02 R 1.13

0 2N "N3

ta- I I 0 0 oIN- 0

AN> NC -CN

1.14 O2N'C 1.15 1.16

Bu 0 N NH2N ON 0 + N_

'kTFA 0 N-N

N-N 0 NH2 NN AcOf

HN 0 HO0 -

1.17 1.18 1.19 1.20

Figure 1.2. Structures of probes activated by oxidoreductase enzymes (EC 1.4-EC 1.6). Enzyme-reactive moieties (red) and fluorophore scaffolds (blue) are highlighted.

reactive moiety. MAO-A specificity is conferred by the ortho-halogenated linker, which is inspired

by the MAO-A inhibitor clorgiline and also serves as an auto-immolative linker that undergoes

1,6-elimination to release a quinone methide. Probe 1.5 is based on the acedan scaffold and derives

its selectivity for MAO-B from a carbamate linker moiety, inspired by the MAO-B inhibitor

33

EC 1.4 - Monoamine Oxidase

pargyline and optimized by in silico molecular docking. Probes 1.4 and 1.5 demonstrate the utility

of drug- and inhibitor-inspired probe design and enable selective imaging of MAO-A and MAO-

B in live cells as well as in Drosophila and mice models of Parkinson's disease.

Enzymes Acting on NADH or NADPH (EC 1.6). The most frequently encountered enzymes in

EC 1.6 are quinone oxidoreductases and nitroreductases (NTR). Although nitroreductases have

also historically been assigned to EC 1.5 and 1.7 subclass depending on their mechanism of action,

most nitroreductases targeted by probes in Table 1 are of the EC 1.6 subclass. The characteristic

feature of quinone oxidoreductases and nitroreductases is their dependence on NAD(P)H cofactor

as an electron source.

Quinone oxidoreductases such as the human cancer tumor-linked NAD(P)H:quinone

oxidoreductase isozyme 1 (hNQO1), are upregulated in many tumors and constitute promising

therapeutic targets. 30 hNQO1 regulates the degradation of p53, p73a, and p3 3 tumor suppressors

in breast, lung, liver, stomach, and kidney tumors, among others. 30 Probes 1.3, 1.6, and 1.7 target

hNQO 1 via a quinone propionic acid motif, whose redox potential was carefully tuned to quench

the naphthalimide fluorophore by PET. 3 - Upon two-electron reduction by hNQO 1, the resultant

hydroquinone undergoes lactonization spurred by gem-dimethyl substituents, in a manner akin to

the trimethyl lock moiety (see EC 3.1.1, probe 1.31).27 In probe 1.3 and 1.6, the lactonization

directly restores fluorescence, whereas in probe 1.7, an additional auto-immolative rearrangement

step is required. Nonetheless, hNQOl activation of probe 1.7 was found to be two orders of

magnitude faster than 1.3 or 1.6, largely due to steric factors. Probe 1.7 also benefits from reduced

phototoxicity and background autofluorescence. As a result of the favorable toxicity profiles and

enzyme response of the naphthalimide probes, rapid identification of hNQO1-positive cells was

34

achieved with 1.7 in under 10 minutes with positive to negative ratios of over 500. More recently,

a cyanine-based probe with similar masking group strategy was applied to 3D tumor spheroids and

pre-clinical ovarian cancer mouse models.

Similar to quinone oxidoreductases, two-electron nitroreductases are homodimers that employ

NAD(P)H to reduce nitrogen-containing functional groups with the help of flavin mononucleotide

as cofactor. NTR activity is largely absent in most non-cancerous human tissue but highly

prevalent in bacteria, with E. coli and E. cloacae nitroreductases of particular interest.3 5 36

Interestingly, E. cloacae NTR was first isolated from bacteria found to metabolize TNT at a

munitions factory.3 5 Recent work with bacterial nitroreductases has focused on rapid identification

of pathogens, an application which benefits greatly from the low background and rapid response

of fluorogenic probes. Probe 1.8 consists of a Cy5.5 fluorophore linked to a NTR-responsive

nitroimidazole quencher.12 As a result of its cationic lipophilic nature, 1.8 readily penetrates both

Gram-positive and Gram-negative bacteria and undergoes NTR-responsive rapid regeneration of

fluorescence. Probe 1.8 was utilized to rapidly identify and distinguish between key antibacterial-

resistant pathogens E. faceium, S. aureus, K pneumonia, A. baumannii, and P. aeruginosa.

Additionally, bacterial nitroreductases have utility as orthogonal reporter enzymes because of

the relative absence of NTR activity in human cells. One such application is described in Chapter

4, in which exogenously introduced E. coli NTR serves as the reporter enzyme component of a

paired enzyme-probe system for detecting cytosolic delivery of biomolecules in human cells.

Inspired by recent reports of NTR activity in hypoxic tumor cells, probes 1.2 and 1.9-1.14

were created to study hypoxia-dependent nitroreductase activity in tumor cell lines, which is

largely attributed to cytochrome P450 reductase and similar reductases.3 14 3 Probes 1.2, 1.9, 1.10,

were highly selective for nitroreductase in the presence of other biologically relevant reducing

35

agents, and were successfully applied to imaging of human A549, HCT1 16, and HeLa cells.

Spatial differences in nitroreductase activity between different cellular compartments can be

studied using targeted probes such as 1.12, a cationic conjugated polymer that accumulates in the

nucleus, or 1.11, which contains a lysosome-targeting morpholine moiety. These probes are

selective and highly responsive to nitroreductases, enable targeted imaging, and span several

regions of the visible spectrum, enabling further studies of hypoxic tumor masses and other

hypoxia-related diseases such as stroke and cardiac ischemia.

In consideration of the putative prokaryotic origin of mitochondrion and nitroreductases, probe

1.15 was created to search for intramitochondrial NTR activity in normoxic tumor cells.2 4 The

cationic and lipophilic nature of probe 1.15 enabled selective mitochondrial accumulation in live

A549 cells and selective NTR response over other background reductases. Probe 1.15 revealed

intramitochondrial nitroreductase activity that was attenuated by bacterial NTR inhibitors. This

strategy was then expanded to prepare a prodrug version of Antimycin A for targeted release in

mitochondria, which showed enhanced biological activity in W138, BT474, and DU145 cancer

and non-cancer cells.

Unlike the nitroaromatic enzyme-reactive moieties in probes 1.8-1.15, probe 1.16 incorporates

an aryl azide masking group. Surprisingly, probe 1.16 was selective for CYP450 enzymes (EC

1.14) rather than cytochrome P450 reductases as expected.44 This orthogonal response of aryl

azide masking groups to CYP450 could be exploited for imaging and delivery applications. On

the other hand, effective reduction of probe 1.16 in several different cancer cell lines suggests

stability concerns for aryl azide reagents commonly used for proximity proteomics and

photoaffinity labeling.4 5

36

Enzymes Acting on Other Nitrogenous Compounds as Donors (EC 1.7). As an alternative to

nitroreductase-responsive probes, probe 1.17 is responsive to E coli azoreductase (EC 1.7.1.6),

which reduces the azo bond linking the rhodamine scaffold and the dimethylaniline auto-

immolative linker.46 Reduction of the azo bond reverses PET quenching but also enables

irreversible elimination to release the parent rhodamine green dye. The probe-azoreductase pair

can be used as an orthogonal reporter in any cell line amenable to transfection including HeLa,

A549, HEK293T, and NIH3T3 cells. The slight susceptibility of the azo bond to non-specific

bioreduction under hypoxic conditions is, however, a potential limitation to the scope of

application.

Enzymes Acting on Sulfur Groups as Donors (EC 1.8). Thioredoxin reductase (TrxR) is a

cornerstone of the thioredoxin pathway as the only known reductase of thioredoxin.4 7 Similar to

nitroreductases described above, TrxR is a homodimer requiring FMN and NADPH cofactors. To

overcome the limitations of tedious and time-intensive existing TrxR activity assays, probe 1.18

enables selective rapid imaging of TrxR in live cells.4 ' TxrR selectivity arises from the five-

membered cyclic disulfide attached to a naphthalimide fluorophore via a carbamate linker.

Reduction by TxrR generates a thiolate that cleaves the carbamate linker to form a stable cyclic

carbonothioate, releasing the naphthalimide fluorophore. Probe 1.18 resists nonspecific activation

by reducing agents and closely related enzymes, as demonstrated by in vitro assays and in cellulo

imaging in Hep-G2 cells with and without TxrR inhibitors.

Enzymes Acting on Paired Donors, with Incorporation or Reduction of Molecular Oxygen

(EC 1.14). Probe 1.19 enables direct measurement of a-ketoglutarate-dependent dioxygenase alkB

37

homolog 3 (ALKBH3) activity, also known as prostate cancer antigen- I.4 ALKBH3 preferentially

demethylates 1 -methyladenine in single stranded DNA or RNA, and elevated levels of ALKBH3

were correlated with increased invasiveness and cell survival in cancer cells.5 0 Probe 1.19

incorporates an electron-deficient 1-methyladenine quencher and adjacent pyrene fluorophores

into a single-strand of DNA. Enzymatic demethylation by ALKBH3 attenuates the 1-

methyladenine PET quenching, thus restoring pyrene fluorescence. Enzyme-optimized probe 1.19

possesses two pyrene nucleosides on the 3' side of 1 -methyladenine with symmetric poly-A tails

flanking both 3' and 5' ends for a total length of 10-12 nt. The combination of length and

positioning imbues 1.19 with kinetic parameters (KM and kcat) mirroring native substrates and

unusual selectivity for the ALKBH3 homolog out of nine other homologs. As evidenced by flow

cytometry and live cell imaging with prostate cancer cell line PC3, 1.19 enables direct

measurement of ALKBH3 activity in lieu of laborious immunohistochemistry or in vitro

enzymatic activity assays.

Another target of fluorogenic probes in subclass EC 1.14 is tyrosinase, an enzyme that converts

phenols into ortho-quinones and that limits the rate of melanin biosynthesis.5 ' Although abnormal

tyrosinase levels have been implicated in Parkinson's disease and vitiligo, detection and

quantification of these enzymes is often complicated by cross-reactivity of probes with reactive

oxygen species (ROS). To circumvent this limitation, near-IR probe 1.20 incorporates a new

tyrosinase-responsive masking group with an additional methylene inserted between the hemi-

cyanine fluorophore and aromatic ring, rendering 1.20 impervious to ROS oxidation.5 ' Elevated

tyrosinase levels in murine melanoma B 16 cells relative to those in HeLa cells were demonstrated

by live-cell imaging with cell-permeable 1.20 and corroborated by ELISA. Imaging with 1.20 in

38

zebrafish revealed previously unknown asymmetric distributions of tyrosinase between the yolk

sac and tail.

1.3 Transferases (EC 2)

Transferases facilitate the transfer of functional groups from donor to acceptor substrates, and

accommodate an extensive array of groups such as sulfuryl, phosphoryl, methyl, amino acyl, and

acetyl groups.2 8 Although transferases are essential to key biochemical pathways, fewer

fluorogenic probes targeting transferases have been reported than those targeting the more

frequently studied oxidoreductases (EC 1) or hydrolases (EC 3). This dichotomy is in part due to

the difficulty of designing selective masking groups that can clearly distinguish transferase activity

from that of hydrolases and other enzymes. In fact, early observations of transferase activity

attributed the activity to a combination of hydrolases and other enzymes instead of a single

transferase enzyme.52 Recent probes that have achieved selective activation by transferases rely

on transfer group mimetics (probe 1.21 and 1.22),"' 5 specific acceptors (probe 1.23),"- or

transferase recognition moieties (probe 1.24).56

HH2N HH02C12 Br G0,H 0

Is N2 0O NH

NH 2 H \/ NC N22N HN NH,HH2 NN H N F

.Si N 0C-aN2/Na-H0 2C 1 1 ,- N' F"/

00 H F

0 0 N1.22 - 1.23 1.24 H 0

Figure 1.3. Structures of probes activated by transferase enzymes (EC 2). Enzyme-reactivemoieties (red) and fluorophore scaffolds (blue) are highlighted.

39

Acyltransferases (EC 2.3). Probes 1.21 and 1.22 are activated by y-glutamyltransferase-catalyzed

transfer of a glutamate masking group, releasing indocyanine or silarhodamine fluorophores. y-

Glutamyltransferase is one of approximately 32 aminoacyltransferases that act on amines to

transfer peptide bonds. 28 y-Glutamyltransferase-activated probes are of particular interest for

oncologic operations because y-glutamyltransferase is highly expressed in a variety of cancers.5 7

Fluorogenic probes for such intraoperative applications require low background and rapid

activation in order to be effective. Probes 1.21 and 1.22 achieve these requirements-both share

the same rapid-response masking group and minimize autofluorescence via red or near-infrared

emission. Both probes were utilized successfully to image tumors in mouse intestines.

Enzymes Transferring Alkyl or Aryl Groups (EC 2.5). Glutathione S-transferase (GST) is an

important enzyme for the detoxification of xenobiotic substances through transfer of glutathione

for subsequent metabolic decomposition. The structural variety of encountered xenobiotics

exerts evolutionary pressure towards either a few enzymes with high substrate promiscuity or

many substrate-specific enzymes. Accordingly, multiple isoforms of human GST have evolved,

with at least eight subclasses present in varied cellular locations including the cytosol,

mitochondria, and microsomes.58 These defensive enzymes are also frequently commandeered by

tumor cells to acquire resistance to anticancer drugs. Probe 1.23 and similar probes enable selective

imaging of a and pi GST isoforms via a transfer-optimized arylsulfonyl group." GST catalyzes

nucleophilic aromatic substitution of glutathione in 1.23 to form a Meisenheimer complex, which

subsequently collapses to release the parent fluorophore. Selectivity for a and p isoforms is

achieved by tuning aryl substituents, and probe 1.23 was applied effectively for GST imaging in

HL60 cells.

40

Enzymes Transferring Phosphorous-Containing Groups (EC 2.7). Kinases are integral to

signaling pathways and exhibit extraordinary substrate variety. Identifying and spatiotemporal

tracking of individual kinase activity has required multistep protocols involving introduction of

non-native proteins.5 9 , 60 Probe 1.246 and analogous compounds 6' were developed as small-

molecule alternatives capable of single-step imaging of specific kinases. Probe 1.24 consists of

three components-a Bruton's tyrosine kinase (Btk) inhibitor-based recognition moiety, a

fluorophore-quencher pair, and a kinase-cleavable linker. A cysteine residue in the active site of

Btk (Cys481) covalently attaches to the probe, inducing elimination of the quencher. Real-time

imaging of Btk was demonstrated in live Namalwa cells using probe 1.24. Because kinase

specificity of probe 1.24 is derived from the inhibitor mimic, this strategy is generally applicable

to any kinase with accessible, selective small-molecule inhibitors.

1.4 Hydrolases (EC 3)

Hydrolases, which catalyze the hydrolytic cleavage of chemical bonds, are preeminent targets

of enzyme-activated fluorogenic probes. Early work with enzyme-activated probes was

predominately occupied by hydrolase-activated probes, which continue to be important tools for

intracellular drug delivery, 62 imaging of dynamic cell processes, 2' as well as diagnostics and

therapeutics. 63-65

Enzymes Acting on Ester Bonds (EC 3.1.1). Esterases are particularly amenable to repurposing

for drug delivery and diagnostic applications. Masking negatively charged carboxylic acids with

esters is a proven technique to enhance intracellular delivery of sensors, biomolecules,6 6 and

therapeutic agents. 62 Recent advances in esterase-activated probes include improvements to probe

41

performance and utility (1.25-1.27) and new applications in therapeutics, pathogen detection, and

cell physiology (1.28-1.31).

0 0 N 0 0 0 00_1 -yl -7 0~1~~~ 0

0 CI CI01 N, 0

/ 0 0201.25 1.26 1.27

00 00

1.28 ' 1.29 1.30

00

N O N ,,,,

1.31 40

0, ,OHP-OH HO 0 OH

I-HO_'P-0

0 - -O

0~H '0 B?60 N Q6

NH HO 0 0CI~c1N0

1.32 1.33 1.34

AcO NH OH R H

LO /=N (HONNFROH) ,N.O FA. l O_00 0 O-I SOOAc 0 HOH 0N___OH NH

OMe 0 H0 0

N RNase A 0 O H NHR2 H COOH

R, = DABCYL or BHQ2

Br R2 = EDANS or BODIPY H1.35 1.36 1.37 1.38

Figure 1.4. Structures of probes activated by hydrolases in classes EC 3.1-EC 3.3. Enzyme-reactive moieties (red) and fluorophore scaffolds (blue) are highlighted.

Probe 1.25 is a dual-input probe for super-resolution imaging of enzymatic activity. Many

super-resolution microscopy techniques require photoactivatable probes, which is achieved in 1.25

by replacing the traditional xanthene lactone with a diazo moiety. 67 Because the esterase-reactive

masking group is installed independently of the diazo moiety, this strategy can also be used to

42

target other classes of enzymes in live cell imaging. Probe 1.26 provides significantly enhanced

stability, brightness, and photostability due to generally applicable electronic and steric

optimization of the fluorophore and esterase-labile masking group (see Chapter 2).68 Probe 1.27,

along with introduction of exogenous pig liver esterase, forms a probe-enzyme pair that is

orthogonal to esterases in human cells.69 This enzyme-probe system unlocks an additional

dimension of selective delivery and demonstrates the feasibility of overlaying multiple enzymes

catalyzing similar reactions in a single biological system, while also preserving neatly discernable

enzymatic responses.

In recent applications, esterase-activated fluorogenic probes 1.28 and 1.29 were shown to

target bacterial enzymes relevant to botulism and tuberculosis. Probe 1.28 is a combination

therapeutic and diagnostic agent, or "theranostic", with a dual purpose masking group that is also

a potent inhibitor of botulinum neurotoxins. 25 A hydroxamate linker enables 1.28 to undergo

esterase-catalyzed activation of the prodrug and fluorogenic probe after freely diffusing across the

plasma membrane, with enhanced intracellular delivery and effectiveness in live neurons. Probe

1.29 is a far-red sensor with variable lipid tails for profiling bacterial esterase and lipase

activities.7 0 By testing samples with a battery of probes containing different lipid moieties, a

characteristic esterase fingerprint can be used to distinguish M tuberculosis from similar bacterial

strains. In contrast to bacterial probes 1.28 and 1.29, probes 1.30 and 1.31 function in human cell

lines to provide refined spatiotemporal control and detailed physiological information. Probe 30 is

one of a family of probes tuned to selectively report endoplasmic reticulum esterase activity, with

potential for application as a drug release trigger.7 1 Probe 1.31 contains a phosphatidylglycerol

moiety, allowing it to embed in the outer surface of cells and monitor endocytic events.2 '

Internalization of probe 1.31 allows intracellular esterases to unmask fluorescence, a feature which

43

was used to demonstrate fundamental differences in rates of endocytosis in matched cancer and

non-cancer cell lines (see Chapter 5).

Enzymes Acting on Phosphate Bonds (EC 3.1.3). Minimizing background and deep tissue

imaging are common themes of recent probes 1.32-1.35 targeting alkaline phosphatases (ALP) or

protein tyrosine phosphatases (PTP)-enzymes that play key roles in disease pathogenesis and cell

regulation. 72ALP probe 1.32 was designed to detect secreted phosphatases, triggering excited state

intramolecular proton transfer (ESIPT) fluorescence enhancement and irreversibly staining the

surrounding area via fluorophore precipitation. Probe 1.32 was able to distinguish cells with

different physiological profiles in heterogeneous tumor tissues.73 In contrast to the sedimentary

nature of 1.32, near-IR probe 1.33 was designed for dynamic imaging of ALP in live mice." Probe

1.34, which targets PTP, is a two-photon acyloxymethyl ketone probe conjugated to cell

penetrating peptides to facilitate organelle specific detection at tissue depths of up to 100 pM.7 4

Far-red PTP probe 1 provides reduced phototoxicity and background autofluorescence along with

enhanced cellular uptake compared to existing PTP probes. Probe 1 was used to image PTP activity

in HeLa cells and to identify S. aureus from a panel of similar human pathogens.14

Glycosidases (EC 3.2.1). Glycosidase-activated probes typically incorporate monosaccharides as

masking groups or cleavable linkers for FRET quenching. Probes 1.35 and 1.36 are activated by

P-galactosidase and are based on naphthalimide and xanthene dye scaffolds, respectively. 5 ' 76

Two-photon probe 1.35 is designed to identify cell senescence in live SK-MEL-103 cells and

xenograft tumor imaging experiments. 76 Bioconjugable probe 1.36, together with E. coli f-

galactosidase installed in the cytosol, forms a probe-enzyme system for quantification of

44

exogenous protein entry into the cytosol of HeLa cells.75 Similar probe-enzyme pairs with near-IR

emission profiles have been developed for general imaging applications in other cell lines." Probe

1.37 detects lysosomal glucocerebrosidase activity, deficiency of which is a hallmark of Gaucher's

disease,7 8 via cleavage of the Cl ether bond to release the parent fluorophore. The modularity of

the linkers, quenchers, and fluorophores used provides access to a variety of wavelengths for

imaging fibroblasts. Probe 1.38 is similarly modular but instead employs masking groups activated

by P-glucaronidase, a promising enzyme for prodrug-activation.7 The linker between masking

group and near-IR fluorophore contains a latent ortho-quinone methide electrophile that covalently

binds the activated probe to the P-glucaronidase enzyme, a trapping motif also seen in P-lactamase

probe 1.47 (EC 3.5.2). Trappable probe 1.38 was successfully used to visualize both subcutaneous

and deep tissue liver tumors in mice.

Enzymes Acting on Peptide Bonds (EC 3.4). Proteases, enzymes that cleave peptide bonds, act

with varying degrees of substrate discrimination. Targeting highly specific proteases with enzyme-

activated fluorogenic probes can be achieved by incorporating peptides or peptide mimics as

masking groups or linkers. Depending on the desired enzyme target, these protease-recognition

moieties can range from a single amino acid to more than 20 residues. Although activation

mechanisms in protease-activated probes have been fairly constant, recent advances have

significantly improved probe performance, selectivity, and breadth of applications.

Probe 1.39 detects P-alanyl aminopeptidase activity in Pseudomonas aeruginosa, a multi-drug

resistant pathogen commonly found in hospital-acquired infections.8 0 The P-alanyl masking group

45

H 2N N V

1.39

HAc-DEVD -N,--'.

0

0 CN

1.44

HNO

H2N,-k N

Y 1.45

HAc-KOLR N N N NH2

0Y

1.40

HNCy5

H HNNQSY21N N N NNH

1.41

Fluorescein , 0 NH2

H NHH 2N N N N

VHY 0,r N N --*' ' H 'DADBCYL0 H ~ 0 HO

1.42

NH2

0

HCbzHN kN

N(NH 0

1.43

Ph 0 O O O Ph F

O 0 oN ~ 0OH X0.

1.46 M 1.47Me

Figure 1.5. Structures of probes activated by hydrolases in classes EC 3.4 and EC 3.5. Enzyme-reactive moieties (red) and fluorophore scaffolds (blue) are highlighted.

of the probe was applied to resorufin, naphthalimide, and other fluorophore scaffolds to create

panels of probes with clinical utility for identifying P. aeruginosa in culture. 8 '

Probes 1.40-1.43 are designed for tumor visualization to improve diagnosis and intraoperative

inspection during surgery. Probe 1.40 contains a single KQLR peptide masking group that is

selectively cleaved by hepsin matriptase in prostate cancer.8 2 By leveraging the pH dependence

fluorescence of the masked probe relative to that of the parent hydroxymethyl rhodamine

fluorophore, high contrast ratios are achieved even with the use of only a single masking group.

The diagnostic utility of 1.40 was broadly established in live-cell and mouse-model imaging

46

EC 3.4 -- Proteases

experiments. Probes 1.41-1.43 target cathepsins, a group of proteases activated in the acidic

environment of lysosomes. Probe 1.41 is a non-peptide activity-based probe containing an

electrophilic moiety that first selectively labels cathepsin S and then eliminates a quencher

(QSY21), resulting in covalently labeling the enzyme with a fluorophore (Cy5). 83 Probe 1.41,

when deployed with nonspecific cathepsin-activated sister probes, has been instrumental for in

vivo imaging of syngeneic mammary tumors and profiling pathways of endolysosomal proteolysis

in live dendritic cells. Probes 1.42, 1.43, and others incorporate short peptides targeting cathepsin

B and have been used for selective imaging of cathepsin B in a wide variety of cell lines (Table

).65,84

Remaining protease probes 1.44-1.45 rely on amino acid or peptide masking groups to target

different proteases. Upon cleavage of the DEVD peptide in 1.44 by apoptotic protease caspase-3,

the released monomer forms hydrophobic excimers with long-wavelength fluorescence.85 Excimer

formation spurs precipitation of aggregates, enabling live cell imaging of location-specific caspase

activity without risk of fluorophore diffusion after cell apoptosis. Probe 1.45, which targets

endoplasmic reticulum (ER) aminopeptidase 1, is a two-photon fluorescent probe that undergoes

a bicyclic urea cyclization to release an ER-targeted naphthalimide.8 6 Selective enzymatic

hydrolysis of the appropriate amide bond by the desired enzyme target, rather than nonspecific

cleavage of thiourea and carbamate linkers in probes 1.42, 1.43, and 1.45, was confirmed by the

absence of undesirable background signal upon incubation with non-target proteases.

Enzymes Acting on Cyclic Amides (EC 3.5.2). Because of their clinical significance in antibiotic

resistance, j-lactamases are the most frequent targets for fluorogenic probes in this class. With

more than 890 P-lactamases identified to date, these cyclic amide hydrolases seriously challenge

47

the efficacy of current antibiotics portfolios." Advances in p-lactamase-activated probes focus

primarily on improvements to the mechanism of activation and enzymatic specificity, enabling

clinically-crucial identification of different antibiotic-resistant bacteria. Probe 1.46 selectively

targets BlaC, a P-lactamase overexpressed in Mycobacterium tuberculosis.63 Specificity is attained

by tuning substituents to complement the substrate recognition loop in BlaC. As a result, probe

1.46 is 8,900-fold more responsive to BlaC than other homologous P-lactamases such as TEM-1,

and can effectively identify M tuberculosis in human sputum and in live-cell imaging. Although

not as selective as probe 1.46, probe 1.47 conveniently generates a highly reactive Michael

acceptor upon P-lactamase activity, reminiscent of P-glucaronidase probe 1.38.4 The nascent

electrophile forms covalent bonds with nucleophilic residues of P-lactamase before probe diffusion

occurs, enabling spatiotemporal tracking of the enzyme. The utility of this approach was

demonstrated in P-lactamase-expressing E coli.

1.5 Conclusions

Enzyme-activated fluorogenic probes are highly sensitive tools for biological imaging

applications. In this thesis, I describe how advances in probe design and application expand the

toolkit for studying enzymatic activity and provide generalizable methods for imaging in live cell

and in vivo model systems.

Advances focus on two primary areas-enhanced specificity of enzyme activation and

improved probe chemistries. The obtainable spatiotemporal and physiological information for

imaging experiments is directly related to the degree of enzyme specificity. Expanding the palette

of single enzyme-specific probes (i.e., 1.23 and 1.41) and even isoform specific probes (i.e., 1.4

and 1.5) probes provides higher-resolution information to identify the location and function of an

48

individual enzyme amidst the intracellular tapestry. Alternatively, new orthogonal enzyme-probe

systems involving probes like 1.27 and 1.36 can be used to selectively interrogate of biological

processes while minimizing undesirable noise from endogenous enzymes. In Chapter 4, I report

the development and validation of one such system for detecting cytosolic delivery of

biomolecules. This enzyme-probe system consists of an exogenously introduced nitroreductase

that selectively activates a paired fluorogenic probe to monitor biomolecule delivery.

Improved probe chemistries can enhance the photophysical performance of parent fluorophore

scaffolds and improve probe enzymatic responses.2 ' Tuning probe masking groups and enzyme

recognition moieties, as demonstrated in probes 1.26 and 1.41, yield greater stability, reduced

background, and enhanced activation kinetics. Chapter 2 details the development of probe 1.26,

which was subsequently applied to live-cell imaging of biomolecules in Chapter 3. In Appendix

A, I outline design principles and potential applications for a new, tunable rhodol fluorophore

scaffold that can be used to build multifunctional probes with improved enzymatic activation

kinetics. Furthermore, incorporation of recently-developed tunable' 7 and multi-input88' 89

fluorophore scaffolds into new probes will facilitate design of sophisticated biological imaging

applications and provide new insight into fundamental cell physiology.

1.6 Acknowledgements

Related work in our laboratory was supported by grant ROI GM044783 to R.T.R. (NIH). W.C.

was supported by an NSF Graduate Research Fellowship.

49

CHAPTER 2

Electronic and Steric Optimization of Fluorogenic Probes forBiomolecular Imaging

Contribution: Chemical synthesis and characterization of fluorescent probes, determination offluorescent properties, hydrolysis assays, enzyme kinetics assays, probe response assays, confocalmicroscopy, photostability assays, composition of manuscript and figures. Computational studies,IR spectroscopy, chemical synthesis and characterization of model compounds were performed byH.R. Kilgore. Computational study design was conducted in part by B. Gold.

This chapter has been published, in part, under the same title. Reproduced with permission fromChyan, W; Kilgore, H.R.; Gold, B.; Raines, R.T. Electronic and Steric Optimization ofFluorogenic Probes for Biomolecular Imaging. J Org. Chem. 2017, 82, 4297-4304. C 2017American Chemical Society.

50

2.1 Introduction

Fluorogenic probes with specific responses to physiological events or environmental

conditions are invaluable for deciphering complex biological processes.' 19 Masked probes are a

class of fluorogenic probes in which a pendant functional group attenuates the fluorescence of a

fluorophore.' 20 Fluorescence is restored upon removal of the masking group by an enzyme-

catalyzed or uncatalyzed chemical reaction. In cell biological applications, masking groups are

frequently designed to serve as substrates for esterases,.9 ' 9 phosphatases, 9 2 ,9 azoreductases,9 4 or

cytochrome P450s.95 Caged fluorophores (which are also known as photoactivatable fluorophores)

are related but are activated instead by illumination at specific wavelengths.

Fluorogenic probes that are substrates for esterases are of special interest because they can be

activated by an endogenous intracellular enzyme (see Chapter 1.4).70, 97-'00 Conjugation of

fluorogenic esterase-activated probes to biomolecules can provide detailed spatiotemporal

information about biomolecular uptake and localization in live cells.21 ' 90 These biomolecule-probe

conjugates are, however, typically internalized by endocytic vesicles and can be exposed therein

to acidity as low as pH 4.5,101 making insensitivity to low pH essential to probe function.

Halogenation of xanthene dyes is a reliable strategy for altering spectroscopic properties and

tuning dye pKa to match those desired for biological applications. Oregon green, which is a

fluorescein derivative in common use, is fluorinated at the 2' and 7' positions.102 Although

fluorogenic probes based on fluorinated and chlorinated scaffolds are available from commercial

vendors, little is known about the effects of halogenation on probe stability. Prior work with

fluorinated derivatives demonstrated improved photostability, but accompanied by the accelerated

spontaneous loss of masking groups.1 03 In contrast, a report of an unusually stable chlorinated

probe3 suggested that halogens other than fluorine merit attention. With fluorinated Oregon green,

51

destabilization of the masked substrate was thought to stem from inductive effects resulting in

lowered pKa of the conjugate acid of the fluorescein leaving group.1 00 Accordingly, design

strategies for stable fluorogenic esterase probes have relied heavily on interjecting auto-

immolative linkers with a higher pKa between the low pKa fluorophore and the site of enzymatic

cleavage (Scheme 2. 1).103 Platforms for such auto-immolative linkers include the acetoxymethyl

(AM) ether,1 00 103 quinone methides,' and the trimethyl lock.2 7 90, 9' The beneficial stability

provided by auto-immolative linkers does, however, come at the expense of a longer synthetic

route to add atoms that are, ultimately, unnecessary.

acetoxymethyl ether oScheme 2.1. Auto-immolative linkers (red)

iiY H inserted between fluorophores and esteraseIZ1 0 N targets to enhance stability.

quinone methide precursor trimethyl lock

The AM ether linker has a small size and facile synthetic accessibility compared to other auto-

immolative linkers.1 00 Still, AM ether masking groups are installed on fluorescein by O-alkylation,

which often yields undesirable ether-ester mixed byproducts from O-alkylation of the 2-carboxyl

group.1 04 105 Addition of a 6-amido group for bioconjugation exacerbates the problem by shifting

the equilibrium away from the "closed" lactone form of a fluorescein derivative and towards the

"open" quinoid form. Accordingly, we sought a simple "linker-free" probe.

In this chapter, we combine electronic and steric effects to create linker-free fluorogenic probes

with high hydrolytic stability, enzymatic reactivity, and photostability. We begin by characterizing

the effects of ortho-halogenation to identify an optimal substitution pattern. Then, we identify an

52

ideal acyl masking group after searching for a high rate of enzyme-catalyzed unmasking along

with a low rate of spontaneous hydrolysis. The ensuing probe is small and readily accessible, and

has superior photostability and enzymatic unmasking kinetics in vitro and in cellulo relative to

auto-immolative probes.

2.2 Results and Discussion

Tuning Acyl Probe Stability with Halogenation. Previous reports have hinted at a role for

halogenation in probe stability.' '00 We pursued this strategy, synthesizing halogenated fluorescein

diacetate probes 2.1-2.5 to characterize ortho-halogen effects (Scheme 2 .2 ).10 -109

X2 X2

0 , , ,0

Scheme 2.2. Halogenated fluoresceindiacetate probes.

2.1: XI = H, X2 = H 2.4: XI= Br, X2 = Br2.2: X1= F, X2 =H 2.5: X1= 1, X2 = 1

2.3: X' = C1, X2 = H

We began by assessing the spectrophotometric properties of the unmasked probes. The product

of the extinction coefficient and the quantum yield (E x P) accounts for both the amount of light

absorbed by a fluorophore and its quantum efficiency. This product is directly proportional to the

brightness of the dye. By this measure, the hydrolysis of chlorinated probe 2.3 provides the

brightest fluorophore.

53

Table 2.1. Spectroscopic attributes and pKa values of unmasked halogenatedfluorescein diacetate probes.Probe Aabs (nm) E (M- 1cm- 1) D E x c (M- 1 cm- 1) pKa2.1 490 9.3 x 104 0.92110 8.4 x 104 6.41102.2 492 8.6 x 104 0.92 7.9 x 104 4.71022.3 503 1.01 x 105 0.88 8.9 x 104 4.62.4 525 1.12 x 105 0.24111 2.7 x 104 3.81122.5 521 8.25 x 104 0.02111 1.6 x 103 3.8112

Next, we assessed spontaneous hydrolysis by incubating each compound in either a simple

buffer or a mammalian cell culture medium. The observed rates of spontaneous hydrolysis for

probes 2.1-2.5 varied with ortho substituents in the order F > H > Cl > Br > I (Figure 2.1).

Moreover, probes 2.1-2.5 exhibited increased stability relative to fluorinated probe 2.2, despite

their low pKa values (Table 2.1), suggesting that inductive electron-withdrawal is not the dominant

contributor to probe stability with larger halogen substituents.

We hypothesized that the resistance of probe 2.3 to hydrolysis was due to stabilization by a

donor-acceptor interaction. Specifically, donation of a lone-pair of electrons (n) from an ortho-

halo group into the antibonding orbital of the adjacent carbonyl group (,r*)-an n->wC*

interaction" 3-- could decrease the electrophilicity of the carbonyl group by raising the energy of

its 7r* orbital." 4 In addition, an intimate interaction with a halo group would shield one face of the

acyl group from nucleophilic attack by water.

54

.0

2.2

2.1. 2.3

0 , , , .4, 2.520 40

Time (h)60

2.22.1

2.32.4

2.5

20 40Time (h)

A

N

0

0.

60

To characterize the interaction between the halo and acyl groups in 2.1-2.5 at higher resolution,

we synthesized o-halophenyl acetates 2.6-2.10 (Scheme 2.3). Infrared carbonyl stretching

frequencies can report on electronic effects on carbonyl groups," 5 116 including n--*

interactions." 7 We found that the introduction of ortho halogens induced modest hypsochromic

shifts in the carbonyl stretching frequencies of 2.6-2.10 with magnitudes in the order: Cl > Br > F

> I > H, whereas the hypsochromic shifts observed in 2.1-2.5 followed the order: Br > F ~ Cl ~ I

> H (Table 2.2). The observed hypsochromic shifts in 2.1-2.5 follow a pattern similar to that of

known rate constants for the hydrolysis of o-halophenyl acetates." 8 Still, hypsochromic shifts of

2.1-2.5 and 2.6-2.10 did not follow a pattern based simply on electron-withdrawal. Accordingly,

we turned to quantum mechanical calculations to evaluate the origin of the anomalous

hypsochromic shifts and their implication in the observed hydrolysis trends.

55

Figure 2.1. Graphs showing the time-coursefor the spontaneous hydrolysis of probes2.1-2.5 as measured by the generation offluorescence. (A) Hydrolysis in 10 mMHEPES-NaOH buffer, pH 7.3.(B) Hydrolysis in OptiMEM cell culturemedium supplemented with FBS (10% v/v).

0

1.0-

0.5 -

B

N

0

0

U

U.u0

-

X' 0Scheme 2.3. Halogenated model compounds.2.6: XI = H 2.9 : X'= Br2.7: XI = F 2.10: X'= 12.8: XI= CI

Table 2.2. Carbonyl stretching frequencies, donor-acceptor geometries and interactionenergies, and hydrolytic stabilities of compounds 2.1-2.10.

Probe VC=o dx c (A)' ex...c=o En. AEx,c=o t/2(h)'(cm-1)a (kcal/mol)b (kcal/mol)(2.1 1766 3.06 80.9 - 0.20 4.7

2.2 1774 2.97 77.0 0.22 0.85 1.82.3 1774 3.25 84.7 0.54 0.98 112.4 1776 3 .5 0 d 3.55e 9 1 .0d, 92.1e 0 .2 5d, 0.33e 0 .4 6d, 0.54e 332.5 1774 3 .6 2 d 3.65e 9 1.0 d, 92.1e 0 .4 0d, 0.44e 0 .8 1d, 1.01e >2,000

2.6 1766 3.41 85.1 - 0.11 ND

2.7 1770 3.27 85.9 0.22 0.19 ND

2.8 1774 3.00 85.8 0.51 1.10 ND

2.9 1772 3.13 87.6 0.52 1.17 ND

2.10 1767 3.59 91.1 0.51 1.03 NDaMeasured with FT-IR spectroscopy. bCalculated for each X... C=O interaction in the optimizedgeometry. cExperimental half-life for spontaneous hydrolysis in OptiMEM containing FBS (10%v/v). dData for 4',5'-halo substituents. eData for 2',7'-halo substituents. ND, not determined.

We used second-order perturbation theory calculations provided by Natural Bond Orbital

(NBO) analysis"' to assess possible n-7r* interactions in compounds 2.1-2.5. The stabilizing

effects of n-+ir* interactions were pivotal for each compound but could not provide the sole

explanation for the observed trends. The zenith in n-r* interaction energies (En-,*) occurs when

X = Cl (Figure 2.22), suggesting that increasing the size of the halogen plays dichotomous roles.

Every favorable n--ir* interaction is counteracted, at least partially, by unfavorable Pauli repulsion

between the lone pair and 7r bonding orbital-a factor that is of increasing importance for larger

halogen atoms. The steric exchange energy (AEx,c=o), which is the energetic penalty associated

with the overlap of the lone pair and z bonding orbital, is substantial only in compounds bearing

56

larger halo substituents: Cl (2.3 and 2.8), Br (2.4 and 2.9), and I (2.5 and 2.10). Hence, we

proceeded to assess in greater detail how n)(r Pauli repulsion contributes to the reactivity of the

acyl masking groups in compounds 2.1-2.5.

0.6-

Figure 2.2. Graph showing the strength ofE 0.- 2.8 n--+r* interactions in compounds 2.6-2.10 as

calculated with second-order perturbationT06 theory. Data are listed in Table 2.2. Insert:

Ld NBO orbital rendering of n-,rr* interactions in2-chlorophenyl acetate (2.8).

0 .0 12.6 2.7 2.8 2.9 2.10

Compound

Potential energy surfaces can provide valuable insight into the interplay between n--> *

interactions and n)(w Pauli repulsion." 4 120 Favorable interactions dominate when the dx...c

distance and Ox -.c=o angle between the halo and carbonyl groups provide sufficient orbital overlap

for n-+7r* donation, generating a trough in the potential energy surface. This surface is manicured

further by unfavorable steric interactions [e.g., n)(w Pauli repulsion] when the value of d is too

small for a particular value of 0 (Figure 2.3).

To provide additional information, we calculated potential energy surfaces for compounds 2.6-

2.9 by scanning the Ca-Cb-O-Cc dihedral angle (Figure 2.4). The dominant feature in these

surfaces is a trough in which n--+* interactions are favorable and steric repulsion is minimal. The

most productive angle formed between an attacking nucleophile and carbonyl group for the

formation of a tetrahedral intermediate is the Btirgi-Dunitz trajectory.1 2 ' Due to the covalent nature

of n-7* interactions, energies are minimized near the Burgi-Dunitz trajectory. Moving along the

Btrgi-Dunitz trajectory (~107*), the unfavorable n)(w interaction dominates until d ~ 3.6 and 3.8

57

A for X = Cl and Br, respectively (Figures 2.4C and 2.4D), which recapitulates the length of the

C-X bond. When X = F (Figure 2.4B), the small van der Waals radii and weak overlap of 2p

orbitals restrict favorable conformations to relatively small values of d and 0. In the absence of

halo substituents, the surface has a singular trough (Figure 2.4A), unaltered by significant changes

in steric repulsion. Thus, the observed trend in carbonyl stretching frequencies (Table 2.2) is a

balance between n-+rc* interactions, n)(w Pauli repulsion, and through-bond inductive and

resonance effects. These findings, in conjunction with spectroscopic attributes (Table 2.1), anoint

2',7'-dichlorofluorescein-based probes as having an optimal combination of stability and

brightness.

Optimization of the Acyl Masking Group. Encouraged by the attributes endowed by

2',7'-dichlorination, we suspected that tuning the sterics of the acyl mask group could enhance

probe stability. In particular, an ideal acyl masking group could provide steric hindrance to

spontaneous hydrolysis without slowing the rate of enzymatic cleavage. 122 ' 123 Accordingly, we

synthesized a small library of fluorogenic probes with various acyl masking groups (Scheme 2.4).

Then, we assessed their susceptibility to spontaneous hydrolysis and enzymatic unmasking in

vitro.

As expected, the combination of steric and n-+7r* stabilization in probes 2.11-2.17 reduced the

rate of spontaneous hydrolysis significantly (Figure 2.5A). Nevertheless, the bulkier acyl groups

in probes 2.13-2.17 also diminished the rate of enzymatic hydrolysis in vitro (Figure 2.5B). The

isobutyryl masking groups in probe 2.12 provided the best combination of increased stability

(- 10-fold greater than that of fluorescein diacetate) and rapid enzymatic unmasking.

58

10

a6 -A

4 I I

Figure 2.3. 3D Rendering of the 2D potential-energy surface for 2-chlorophenyl acetate (2.8) inFigure 2.4C. Lowest energy conformations (blue) correspond to regions with favorable norTr*interactions and the ester in a syn conformation. Highest energy conformations (yellow) arecaused by unfavorable n)(Tr Pauli repulsion and rotational barriers. Although the anti conformationis not preferred, when 2-chlorophenyl acetate adopts a conformation with an n-+rr* interaction ofthe same strength as the syn conformation, the AE between the syn and anti conformations isonly 2.66 kcal/mol. Reducing the distance d and angle eleads to the highest energy conformation(red highlight) with a maximum difference of 11.77 kcal/mol at d = 2.92 A and e = 93.5*, as thecarbonyl carbon enters the plane of the phenyl ring.

59

120- 2.6 (X =H) 8

110- 6100-90- 480-0070-dC 260- C

2.8 3.0 3.2 3.4 3.6 4.0

B130 2.7 (X =F)

S10- 1'(*)20-

80-7060

3.0 3.2 3.4 3.6 3.8

C 130. 2.8 (X =CI)

120.

110-e(0)100-

90 -

80- jP-

3.0 3.2 3.4 3.6 3.8 4.0 4.2

130-

120-

110-

100-

90-

80-

3.2 3.4 3.6 3.8 4.0 4.2d (A)

10

8AE

6 (kcal/mol)4

2

0

10

AE(kcal/mol)

4

2

0

Figure 2.4. Calculated potential energysurfaces generated by scanning the Ca-Cb-O-Cr dihedral angle of compounds 2.6-2.9.Minimal energies (blue) follow a trough thatcorrelates with favorable n-+Tr* interactionsfor a given X ... Cc distance (d) and X- Cc=Oangle (0).

12

10

8AE

6 (kcal/mol)4

2

0

60

A

e(0)

AE(kcal/mol)

e(0)

R 0 0 O R

0 C1 C1 00

0

2.3: R = ',<' 2.13: R =

2.11: R =

2.12: R =

2.14: R =

2.15: R =

2.16: R =

2.17: R =

Scheme 2.4. Acylateddichlorofluorescein probes.

0 c /\ c

02.18

21 2 2 21 2. 24 . .1 217 .182.1 2.3 2.11 2.12 2.13 2.14 2.15 2.16 2.17 2.18

B 100-

RFU

50

- I2.1 2.3 2.11 2.12 2.13 2.14 2.15 2.16 2.17 2.18

300-

200-

RFU -

100- II

Figure 2.5. Graphs showing the effect of acylgroups on the hydrolytic stability of2',7'-dichlorofluorescein probes in vitro and incellulo. (A) Spontaneous hydrolysis after a24-h incubation in OptiMEM containing FBS(10% v/v). Raw data are shown in Figure 2.6B.Inset: structure of compound 2.18.(B) Hydrolysis by pig liver esterase in 1 h. Rawdata are shown in Figure 2.6C. (C) Hydrolysisby intracellular esterases in live HeLa cells.Data were quantified from images in Figure2.7. RFU: relative fluorescence units.

61

2',7'-

A 100-

RFU

50-

/

/

/

/

/

0

C

U- ' r2. 82.1 2.3 2.11 2.12 2.13 2.14 2.15 2.16 2.17 2.18

11 0 M-

a

OF:

A100-

RFU

50-

0-

B100-

RFU

50-

2.12

5 10Time (h)

15 20

0.5Time (h)

- 2.1- 2.3- 2.11- 2.12- 2.13- 2.14- 2.15- 2.16

- 2.17- 2.18

Figure 2.6. Graphs showing the time-course for the hydrolysis of probes 2.1,2.3, and 2.11-2.18 as measured by thegeneration of fluorescence. Probeconcentrations were 5 pM.(A) Spontaneous hydrolysis in 10 mMHEPES-NaOH buffer, pH 7.3.(B) Spontaneous hydrolysis in OptiMEMcontaining FBS (10% v/v). (C) Enzyme-catalyzed hydrolysis in 10 mM HEPES-NaOH buffer, pH 7.3, containing PLE(50 ng/mL). RFU: relative fluorescenceunits.

1.0

The resistance of probe 2.12 to spontaneous hydrolysis likely arises from a combination of

electronic and steric effects. In its optimized geometry, one face of its ester carbonyl group is

shielded from solvent water by an n-c* interaction with the ortho-chloro group (Scheme 2.5).

The other face is shielded by a methyl group. These effects might be less detrimental to enzyme-

catalyzed hydrolysis.

62

5 10 15 20Time (h)

I

C 100-

RFU

50-

00.0

0

2.12

Scheme 2.5. Optimized geometry of thebutyryl ester moiety in probe 2.12.

We evaluated the steady-state kinetic parameters for esterase-catalyzed hydrolysis of the

probes (Figure 2.7). Although the probes undergo two step hydrolysis, full fluorescence is

generated only after release of the second ester group. Accordingly, hydrolysis data can be reliably

fit to the Michaelis-Menten equation to obtain apparent kinetic parameters. Chlorinated probes

tended to interact more strongly with the enzymic active site than did unmodified or fluorinated

probes (Table 2.3). The additional steric bulk in butyryl probe 2.12 had only a modest effect on

the rate of enzymatic hydrolysis compared to acetyl probe 2.3. Optimized acyl probe 2.12, with

apparent kcat/KM = 1.8 x 106 M-'s' and KM = 4.6 pM, outperformed probes with auto-immolative

linkers (Table 2.3).

Human esterases often exhibit higher substrate specificity than does pig liver esterase.

Accordingly, we sought to corroborate in vitro kinetic data with in cellulo data to ensure probe-

applicability in human cells. Confocal images of live HeLa cells incubated with probes 2.11-2.17

confirmed the trends observed in vitro (Figures 2.6C and 2.8). Additionally, probe 2.12 showed

enhanced rates of enzyme-catalyzed unmasking compared to the analogous AM ether probe (2.18).

Finally, we monitored the fluorescence of the 2',7'-dichlorofluorescein scaffold in live cells under

constant illumination and found that probes based on this scaffold have superior photostability

(Figure 2.9).

63

2Substrate (pM)

4

10 20 30 40

Substrate (pM)

20Substrate (pM)

30

Figure 2.7. Kinetic traces and Michaelis-Menten plots for the unmasking of fluorogenic probes.Assays were performed in 10 mM HEPES-NaOH buffer, pH 7.3, containing PLE (9 ng/mL).Substrate was added at t = -60 s, and enzyme was added at t = 0. (A, B) Probe 2.3; kct/KM = 2.8x 106 M- 1s- 1 and KM = 1.8 pM. (C, D) Probe 2.12; kct/KM = 1.8 x 106 M- 1s-1 and KM = 4.6 pM.(E, F) 2.18; ktI/KM = 8.4 x 105 M- 1s- 1 and KM = 2.2 pM.

64

A 60 -

40-

LL

20-

B 0.15-

0.10-

0.05 -

a0

n-4

0 50

C

100

1.5-

Time (s)150

0.000

1.0-

0.5-

D 0.008-

0.006-

(I,

C

0.0

0.004-

0r0 50

E

100Time (s)

0.002-

0.000-150

15 -

10-

0

U-

F 0.08 -

(0

C

0.06-

0.04-

0.02 -

0 50 100Time (s)

1500.00

0 1'0

n

5-

Figure 2.8. In cellulo hydrolysis of fluorogenic probes 2.1, 2.3, and 2.11-2.18. HeLa cells wereincubated with a probe (5 pM) for 15 min, counterstained with Hoechst 33392, and imaged byconfocal microscopy. Quantitation shows that cells incubated with 2.3 or 2.12 had comparablelevels of fluorescence signal generation (Figure 2.5). Scale bars: 25 pm.

Table 2.3. Kinetic parameters were obtained by fitting Michaelis-Menten plots generated fromthe initial rates of probe-unmasking in 10 mM HEPES-NaOH buffer, pH 7.3, containing PLE(Figure 2.6).

Number of Auto-immolative kcat/KM KMFluorophore (Probe) Masking Groups Linker (M- 1s- 1) (pM)

fluorescein (2.1)103 2 1.4 x 106 112',7'-difluorofluorescein (2.2)103 2 2.9 x 101 5.32',7'-dichlorofluorescein (2.3) 2 2.8 x 106 1.8fluorescein1 03 2 AM ether 6.8 x 105 3.22',7'-difluorofluorescein1 03 2 AM ether 3.8 x 105 4.92',7'-dichlorofluorescein (2.18) 2 AM ether 8.4 x 105 2.22',7'-dichlorofluorescein (2.12) 2 1.8 x 106 4.6morpholino-urea-rhodamine 0 1 trimethyl lock 8.2 x 105 0.10

65

Figure 2.9. Rate of fluorophore photobleaching inlive HeLa cells under continuous illumination. Cells

2.1 were incubated with 2.1, 2.2, or 2.3 (4 pM) for 15o min, washed twice, and images were acquired at 2-

2.2 min intervals with continuous illumination between2.3 measurements. 2',7'-Dichlorofluorescein (which is

unmasked 2.3) had significantly greater resistanceo -to photobleaching than did either fluoresceinU- 0.0 I I (unmasked 2.1) or difluorofluorescein (unmasked

0 20 Time (min) 40 60 2.2).

2.3 Conclusions

We have described a new strategy for stabilizing esterase-activated fluorogenic probes. An

n-+7r* interaction between an ortho-halogen and pendant acyl group in 2',7'-fluorescein diacetate

deters spontaneous hydrolysis. n)(w Pauli repulsion from larger halo groups limits the benefit that

can be gained from this n---+w* interaction, and an optimum is achieved with chloro-substitution.

Spontaneous hydrolysis is deterred further with little effect on esterase-catalyzed cleavage when

the esters derive from isobutyric acid rather than acetic acid. Thus, 2',7'-dichlorofluorescein

diisobutyrate is a simple linker-free probe derived by optimizing electronic and steric effects.


We are grateful to Dr. C. L. Jenkins for her critical review of the manuscript. This work was

supported by grant ROl GM044783 to R.T.R. (NIH). W.C. was supported by an NSF Graduate

Research Fellowship. B.G. was supported by an Arnold 0. Beckman Postdoctoral Fellowship.

This work used data acquired at the National Magnetic Resonance Facility at Madison, which is

supported by Grant P41 GM103399 (NIH). The work also made use of a Thermo Q Exactive T M

Plus mass spectrometer (NIH grant S 10 OD020022), Phoenix Cluster at the UW-Madison HPC

center (NSF grant CHE-0840494), and Micro FT-IR spectrometer (NSF grant DMR-1 121288).

66

2.5 Experimental

General Information. Phenyl acetate (2.6), 2-iodophenyl acetate (2.10), and all other commercial

chemicals were from Sigma-Aldrich (St. Louis, MO), Fischer Scientific (Hampton, NH), or Alfa

Aesar (Haverhill, MA) and were used without further purification. Porcine liver esterase (PLE)

was from Sigma-Aldrich.

Chemical reactions were monitored by thin-layer chromatography (TLC) using EMD 250-rIm

silica gel 60-F 254 plates and visualization with UV illumination or KMnO4-staining. Flash

chromatography was performed with a Biotage Isolera automated purification system using pre-

packed SNAP KP silica gel columns.

All procedures were performed in air at ambient temperature (-22 'C) and pressure (1.0 atm)

unless specified otherwise. The phrase "concentrated under reduced pressure" refers to the removal

of solvents and other volatile materials using a rotary evaporator at water aspirator pressure (<20

torr) while maintaining a water-bath temperature below 40 'C. Residual solvent was removed from

samples at high vacuum (<0.1 torr), which refers to the vacuum achieved by mechanical belt-drive

oil pump.

All fluorogenic probes and fluorescent molecules were dissolved in spectroscopic grade

DMSO and stored as frozen stock solutions. For all applications, DMSO stock solutions were

diluted such that the DMSO concentration did not exceed 1% v/v.

Instrumentation. Absorbance data were acquired with an Agilent Cary 60 UV-vis spectrometer.

Hydrolysis kinetics were measured with a Tecan Infinite M1000 plate reader. All other

fluorescence data were acquired with a PTI QuantaMaster spectrofluorometer. 'H and "C NMR

67

spectra were acquired on Bruker Spectrometers at the National Magnetic Resonance Facility at

Madison (NMRFAM) operating at 500 MHz for 1H and 125 MHz for 13C. Mass spectrometry was

performed with a Q ExactiveTM Plus electrospray ionization quadrupole-ion trap (ESI-QIT-MS)

mass spectrometer at the Mass Spectrometry Facility in the Department of Chemistry at the

University of Wisconsin-Madison. IR spectra were acquired with a Micro FT-IR spectrometer at

the Materials Science Center of the University of Wisconsin-Madison. Microscopy images were

acquired with a Nikon Eclipse Ti inverted confocal microscope at the Biochemistry Optical Core

of the University of Wisconsin-Madison.

Optical Spectroscopy. UV-visible and fluorescence spectra were recorded by using 1-cm path

length, 4-mL quartz cuvettes or 1-cm path length, 1-mL quartz microcuvettes. Analyte solutions

were stirred with a magnetic stir bar. Quantum yields were determined by referencing probe

solutions to fluorescein (Xex = 495 nm; P = 0.95) in 0.1 M NaOH(aq).

FT-IR spectra were recorded on compounds solvated with a minimum quantity of

dichloromethane or dimethyl sulfoxide, and sandwiched between two sodium chloride windows.

FT-IR spectra were collected with 128 scans at 1200-3500 cm-1 and a resolution of 2 cm-1. A

background spectrum was taken of the solvent alone every 20 min. Plates were washed with

acetone (3 x) after recording the spectrum of each compound.

Spontaneous Probe Hydrolysis. Probe stocks were diluted to a final concentration of 5 piM in

300 ptL of either 10 mM HEPES-NaOH buffer, pH 7.3, or OptiMEM containing FBS (10% v/v).

Fluorescence was measured with a plate reader (Costar 96 well clear bottom, bottom measurement

68

mode) at 30-min intervals for 72 h. Hydrolysis data were fitted to single-phase decay curves with

GraphPad Prism software.

PLE-Catalyzed Probe Hydrolysis. PLE (168 kDa, >15 units/mg solid) was suspended in 10 mM

HEPES-NaOH buffer, pH 7.3, and diluted to appropriate concentrations before use in protein

LoBind tubes from Eppendorf. Initial rate measurements for each probe were acquired and the

resulting data was fit to the Michaelis-Menten equation in GraphPad Prism software to obtain

apparent kinetic parameters for the enzyme-catalyzed unmasking of probes.

Cell Culture and Live Cell Imaging. HeLa cells were from American Type Culture Collection

(Manassas, VA) and were maintained according to recommended procedures. Gibco brand

Dulbecco's Modified Eagle Medium (DMEM), fetal bovine serum (FBS), trypsin (0.25% w/v),

OptiMEM, and Dulbecco's PBS (DPBS) were from Thermo Fisher Scientific (Waltham, MA).

HeLa cells were grown in DMEM supplemented with FBS (10% v/v), penicillin (100 units/mL),

and streptomycin (100 pg/mL). For all imaging experiments, 8-well microscopy slides from Ibidi

(Madison, WI) were seeded with HeLa cells (105 cells/mL) 24 h before use. All imaging

experiments were performed in live cells without fixation. ImageJ software from the National

Institutes of Health (Bethesda, MD) was used for all image processing, signal quantification, and

colocalization measurements.124

In Cellulo Probe Hydrolysis. HeLa cells in 8-well microscopy slides were incubated with 5 pM

probe for 15 min, and counterstained with Hoechst 33342 (2 pg/mL) for 10 min at 37 'C. Cells

69

were washed twice and imaged with a confocal microscope. Background-subtracted cell

fluorescence signal was quantified in each image.

Photobleaching. HeLa cells in 8-well microscopy slides were incubated with 5 pM of 2.1, 2.2, or

2.3 for 15 min, and counterstained with Hoechst 33342 (2 ptg/mL) for 10 min at 37 'C. The cells

were washed twice and imaged with a confocal microscope using a 488-nm filter set. Images were

acquired every 2 min with continuous, constant-intensity illumination between acquisitions.

Normalized fluorescence signal was quantified with ImageJ software and plotted in Figure 2.9.

Computational Procedures. Geometry optimization calculations were performed with Gaussian

09, revision D.01 with the functional M06-2X and 6-311+g(2d,p) basis set. 2 5 Frequency

calculations were performed to ensure that the optimized structure was at a true minimum. All

calculations were performed in water with the integral formalism polarizable continuum model

(IEFPCM implicit solvent model as implemented in Gaussian 09). Potential-energy surfaces were

generated by varying the Ca-Cb-O-Cc dihedral using a relaxed scan. All calculations performed

on systems containing iodine were done by using the Stuttgart relativistic electron core potential

for treating iodine, whereas the 6-311+g(2d,p) basis set was used for all other atoms. NBO

calculations were performed with NBO 6.0.11 The pairwise steric exchange energy (AEx,c=o)

between the lone pair of a halo group and the wr orbital of a carbonyl group was calculated using

natural steric analysis as implemented in NBO 6.0. All energies include zero-point corrections.

Synthesis of 2',7'-Dichlorofluorescein. 4-Chlororesorcinol (14.4 g, 99.6 mmol) and phthalic

anhydride (7.3 g, 49.3 mmol) were dissolved in MeSO3H (50 mL), and the resulting solution was

70

heated at 90 'C for 24 h. After cooling to room temperature, the reaction mixture was added slowly

to 1 L of stirred ice water. The resulting suspension was filtered and triturated with cold water to

afford 2',7'-dichlorofluorescein as a yellow solid (18.1 g, 90.7% yield). 'H NMR (500 MHz,

(CD3) 2SO, 6): 11.08 (s, 2H), 8.01 (d, J= 7.7 Hz, 1H), 7.82 (t, J= 7.7 Hz, 1H), 7.75 (t, J= 7.6 Hz,

1H), 7.34 (d, J= 7.7 Hz, 1H), 6.91 (s, 2H), 6.66 (s, 2H). 13C NMR (125 MHz, (CD 3 ) 2 SO, 6): 168.3,

155.1, 151.5, 150.1, 135.9, 130.5, 128.2, 125.9, 125.1, 124.0, 116.3, 110.5, 103.7, 81.5. HRMS

(ESI-QIT) m/z: [M + H]+ Calcd for C2 HIC1205 400.9979; Found 400.9982.

Synthesis of Diesters 2.1-2.5 and 2.11-2.17, and Esters 2.7-2.9. To a suspension of phenol or

fluorescein derivative (0.15 mmol, 1 equiv) in DCM (2.0 mL) were added

4-dimethylaminopyridine (15 pmol, 0.1 equiv) and pyridine (0.33 mmol, 2.2 equiv). Acyl chloride

(0.33 mmol, 2.2 equiv) was added dropwise, and the resulting solution was stirred for 1 h or until

completion of the reaction. After dilution with water and extraction with DCM, the combined

organic extracts were washed with saturated aqueous NH 4Cl and brine, dried with MgSO4(s), and

concentrated under reduced pressure. Purification by column chromatography on silica gel (0-

40% v/v EtOAc in hexanes) afforded the title compounds as white solids or clear oils.

Characterization data for fluorescein diacetate (2.1) and 2',7'-difluorofluorescein diacetate (2.2)

were in accord with those reported previously.' 0 3

2', 7'-Dichlorofluorescein Diacetate (2.3). Off-white solid (68.6 mg, 94.2% yield). 'H NMR

(500 MHz, CDCl 3, 6): 8.08 (d, J= 7.6 Hz, 1H), 7.73 (dd, J= 26.5, 1.0 Hz, 2H), 7.22 (d, J= 7.6

Hz, 1H), 7.16 (s, 2H), 6.87 (s, 2H), 2.38 (s, 6H). "C NMR (125 MHz, CDCl 3, 6): 168.7, 168.1,

151.9, 149.8, 148.6, 136.0, 130.8, 129.1, 125.8, 125.8, 124.2, 122.8, 117.8, 112.9, 80.6, 20.8.

HRMS (ESI-QIT) m/z: [M + H]+ Calcd for C24Hl5C1207 485.0190; Found 485.0196.

71

Eosin Y diacetate (2.4). White solid (40.6 mg, 37% yield). 'H NMR (500 MHz, CDCl 3, 6): 8.13

(dd, J= 7.5, 1.0 Hz, 1H), 7.82 (t, J= 7.2 Hz, 1H), 7.77 (dt, J= 7.5, 3.8 Hz, 1H), 7.28 (d, J= 7.3

Hz, 1H), 7.07 (s, 2H), 2.48 (s, 6H). 13C NMR (125 MHz, CDCl 3, 6): 171.0, 169.5, 151.1, 150.6,

138.8, 133.7, 132.6, 128.7, 127.9, 126.7, 121.9, 115.4, 111.1, 82.8, 23.3. HRMS (ESI-QIT) m/z:

[M + H] Caled for C24HI3 Br4O 7 728.7389; Found 728.7390.

Erythrosin B Diacetate (2.5). White solid (44.2 mg, 32% yield). 'H NMR (500 MHz, CDC1 3,

6): 8.09 (d, J= 7.7 Hz, 1H), 7.78 (m, 1H), 7.72 (t, J= 7.4 Hz, 1H), 7.22 (m, 3H), 2.46 (s, 4H). 13 C

NMR (125 MHz, CDC1 3, 6): 168.4, 166.9, 154.0, 152.0, 137.4, 137.3, 136.1, 130.9, 125.9, 125.2,

124.1, 119.5, 84.4, 83.1, 21.4. HRMS (ESI-QIT) m/z: [M + H]+ Caled for C2 4HI3 40 7, 920.6835;

Found 920.6837.

2-Fluorophenyl Acetate (2.7). Clear oil (9 mg, 39% yield). 'H NMR (500 MHz, CDCl 3, 6):

7.22- 7.07 (m, 1H), 2.35 (s, 1H). 13C NMR (125 MHz, CDC1 3, 6): 168.5 (s), 154.2 (d, JC-F = 249

Hz), 138.2 (d, JC-F = 13.0 Hz), 127.2 (d, JC-F = 7.2 Hz), 124.6 (d, JC-F = 3.9 Hz), 123.8 (d, JC-F

0.8 Hz), 116.8 (d, JC-F = 18.6 Hz), 20.6 (s). HRMS (ESI-QIT) m/z: [M + H]+ Calcd for C8H8FO2,

155.0503; Found, 155.0502.

2-Chlorophenyl Acetate (2.8). Clear oil (7.9 mg, 31% yield). 'H NMR (500 MHz, CDC 3, 6):

7.46 (dd, J= 8.0, 1.6 Hz, 1 H), 7.33-7.27 (m, 1H), 7.20 (td, J= 7.7, 1.6 Hz, 1H), 7.15 (dd, J= 8.0,

1.6 Hz, 1H), 2.37 (s, 3H). 13C NMR (125 MHz, CDC 3, 6): 168.5, 146.9, 130.3, 127.7, 127.0,

126.8, 123.7, 20.6. HRMS (ESI-QIT) m/z: [M + H]* Calcd for C8H8ClO2 171.0207; Found

171.0206

2-Bromophenyl Acetate (2.9). Clear oil (9.4 mg, 29% yield). 'H NMR (500 MHz, CDCl 3, 6):

7.46 (dd, J= 8.0, 1.6 Hz, 1H), 7.33-7.27 (m, 1H), 7.20 (td, J= 7.7, 1.6 Hz, 1H), 7.15 (dd, J= 8.0,

1.6 Hz, 1H), 2.37 (s, 3H). 13 C NMR (125 MHz, CDCl 3, 6): 168.7, 148.4, 133.5, 128.7, 127.6,

72

123.9, 116.4, 21.0. HRMS (ESI-QIT) m/z: [M + NH4]+ Calcd for C8Hi NBrO 2 231.9968; Found

231.9965.

2', 7'-Dichlorofluorescein Dipropionate (2.11). White solid (66.2 mg, 86% yield). 'H NMR (500

MHz, CDC 3, 6): 8.08 (d, J= 7.6 Hz, 1H), 7.73 (dtd, J= 31.0, 7.5, 0.9 Hz, 2H), 7.22 (d, J= 7.6

Hz, 1H), 7.16 (s, 2H), 6.87 (s, 2H), 2.67 (q, J= 7.5 Hz, 4H), 1.31 (t, J= 7.6 Hz, 6H). 13C NMR

(125 MHz, CDCl 3, a): 171.6, 168.7, 152.0, 149.9, 148.7, 135.9, 130.8, 129.0, 125.8, 125.8, 124.1,

122.8, 117.7, 112.9, 80.6, 27.6, 9.1. HRMS (ESI-QIT) m/z: [M + H]* Caled for C2 6 H, 9Cl207

513.0502; Found 513.0503.

2', 7'-Dichlorofluorescein Diisobutyrate (2.12). White solid (60.1 mg, 74% yield). 'H NMR (500

MHz, CDCl 3, 6): 8.08 (d, J= 7.6 Hz, 1H), 7.74 (tdd, J= 15.0, 11.0, 4.1 Hz, 2H), 7.22 (d, J= 7.6

Hz, 1H), 7.14 (s, 2H), 6.87 (s, 2H), 2.96-2.79 (m, 2H), 1.36 (dd, J= 7.0, 4.0 Hz, 12H). 13C NMR

(125 MHz, CDCl 3, a): 174.1, 168.6, 152.0, 149.8, 148.6, 135.8, 130.7, 128.9, 125.7, 125.7, 124.0,

122.7, 117.5, 112.7, 80.5, 34.2, 18.9, 18.9. HRMS (ESI-QIT) m/z: [M + H]+ Calcd for C2 8H 2 3 C1207

541.0815; Found 541.0815.

2', 7'-Dichlorofluorescein Dipivalate (2.13). White solid (73.5 mg, 86% yield). 'H NMR (500

MHz, CDCl 3, a): 8.08 (d, J= 7.5 Hz, 1H), 7.72 (dtd, J= 29.1, 7.4, 1.0 Hz, 2H), 7.20 (d, J= 7.6

Hz, 1H), 7.13 (s, 2H), 6.87 (s, 2H), 1.40 (s, 18H). 13C NMR (125 MHz, CDCl 3, a): 175.6, 168.6,

152.0, 149.8, 148.8, 135.8, 130.6, 128.9, 125.7, 125.7, 124.0, 122.8, 117.4, 112.7, 80.6, 39.4, 27.1.

HRMS (ESI-QIT) m/z: [M + H]+ Calcd for C3 0H2 7 C1207 569.1128; Found 569.1128.

2', 7'-Dichlorofluorescein Di-tert-butylacetate (2.14). White solid (76.2 mg, 85% yield). 'H

NMR (500 MHz, CDCl 3, a): 8.08 (d, J= 7.6 Hz, 1H), 7.73 (dtd, J= 23.7, 7.4, 1.0 Hz, 2H), 7.20

(d, J= 7.5 Hz, 1H), 7.14 (s, 2H), 6.88 (s, 2H), 2.53 (s, 4H), 1.16 (s, 16H). 13C NMR (125 MHz,

CDCl 3, a): 169.3, 168.6, 152.0, 149.7, 148.5, 135.8, 130.6, 129.0, 125.7, 125.6, 124.0, 122.7,

73

117.5, 112.7, 80.5, 47.3, 31.1, 29.6. HRMS (ESI-QIT) m/z: [M + H]' Calcd for C32H31C12 07

597.1441; Found 597.1438.

2', 7'-Dichlorofluorescein Dicyclobutyrate (2.15). White solid (78 mg, 92% yield). 'H NMR

(500 MHz, CDCl 3, 6): 8.08 (d, J= 7.6 Hz, 1H), 7.79-7.68 (m, 2H), 7.22 (d, J= 7.6 Hz, 1H), 7.15

(s, 2H), 6.87 (s, 2H), 3.52-3.41 (m, 2H), 2.55-2.45 (m, 4H), 2.43-2.32 (m, 4H), 2.15-1.97 (m,

4H). 13C NMR (125 MHz, CDC 3,3): 172.5, 168.7, 152.1,149.9,148.7,135.9,130.8, 129.0,125.8,

125.8, 124.2, 122.9, 117.6, 112.9, 80.7, 38.0, 25.5, 25.4, 18.6. HRMS (ESI-QIT) m/z: [M + H]

Caled for C30H 23C12 07 565.0815; Found 565.0817.

2', 7'-Dichlorofluorescein Dihexanoate (2.16). White solid (56.5 mg, 63% yield) 'H NMR (500

MHz, CDCl 3, 6): 8.08 (d, J= 7.5 Hz, 1H), 7.78-7.68 (m, 2H), 7.22 (d, J= 7.6 Hz, 1H), 7.15 (s,

2H), 6.87 (s, 2H), 2.63 (t, J= 7.5 Hz, 4H), 1.78 (dd, J= 14.9, 7.4 Hz, 4H), 1.46-1.34 (m, 8H), 0.93

(t, J= 7.0 Hz, 6H). 13C NMR (125 MHz, CDC1 3, 6): 171.0, 168.7, 152.0, 149.9, 148.7, 135.9,

130.8, 129.1, 125.8, 125.8, 124.2, 122.8, 117.6, 112.90, 80.6, 34.1, 31.3, 24.6, 22.4, 14.0. HRMS

(ESI-QIT) m/z: [M + H]' Caled for C32H31C1207 597.1441; Found 597.1441.

2', 7'-Dichlorofluorescein Di-2-propylvalerate (2.17). White solid (68.5 mg, 70% yield). 'H

NMR (500 MHz, CDCl 3, 6): 8.09 (d, J= 7.5 Hz, 1 H), 7.74 (tdd, J= 14.9, 10.8, 4.2 Hz, 2H), 7.21

(d, J= 7.6 Hz, 1H), 7.11 (s, 2H), 6.88 (s, 2H), 2.73-2.64 (m, 2H), 1.84-1.76 (m, 4H), 1.63-1.56

(m, 5H), 1.51-1.43 (m, 8H), 0.97 (td, J= 7.3, 2.2 Hz, 12H). 13C NMR (125 MHz, CDCl 3, 3): 173.6,

168.8, 152.1, 149.8, 148.6, 135.9, 130.8, 129.1, 125.8, 125.7, 124.1, 122.8, 117.6, 112.9, 80.6,

45.4, 34.6, 34.5, 20.8, 20.8, 14.1 HRMS (ESI-QIT) m/z: [M + H]' Calcd for C36H39C1207

653.2067; Found 653.2067.

74

Synthesis of 2',7'-Dichlorofluorescein Diacetoxymethyl Ether (2.18). Ag20 (145 mg,

0.63 mmol), 2',7'-dichlorofluorescein (0.25 mmol), and powdered activated 4-A molecular sieves

(208 mg) were added to an oven-dried round-bottom flask. Anhydrous CH3CN (4.0 mL) was

added, and the resulting suspension was stirred under N2(g) for 5 min. To this mixture was added

bromomethyl acetate (0.1 mL, 1.0 mmol) dropwise, and the resulting mixture was stirred under

N2(g) for 48 h. The reaction mixture was then diluted with DCM and filtered through a pad of

Celite*. Purification by column chromatography on silica gel (0-40% v/v EtOAc in hexanes with

constant 40% v/v DCM as cosolvent) afforded the title compound as a white solid (53.2 mg, 39%

yield). 'H NMR (500 MHz, CDCl 3, 6): 8.08 (d, J= 7.4 Hz, 1H), 7.72 (td, J= 7.4, 1.1 Hz, 2H),

7.21-7.14 (in, 1H), 7.09 (s, 2H), 6.79 (s, 2H), 5.86 (dd, J= 8.4, 6.5 Hz, 4H), 2.19 (s, 6H). 3C

NMR (125 MHz, CDCl 3, 6): 169.6, 168.8, 154.1, 151.9, 150.4, 135.7, 130.6, 129.4, 126.3, 125.7,

123.9, 119.7, 113.9, 104.1, 85.4, 81.2, 21.0. HRMS (ESI-QIT) m/z: [M + H]' Calcd for

C26H19C12 09 545.0401; Found 545.0400.

75

NMR Spectra

'H NMR ((CD 3 ) 2 SO) and 13C NMR ((CD 3) 2SO) Spectra of 2',7'-Dichlorofluorescein

HO 0 OH

CI Cl f

A

Ill

16 15 14 13 12 11 10 9 8 7 6PPM

5 4 3 2 1 0 -1 -2 -3

li C!L 40% a en L

HO 0 OH

I IC C

0

200 190 180 170 160 150 140 130 120 110 100 90PPM

80 70 60 50 40 30 20 10 0

76

iii

r..

I

'H NMR (CDC1 3) and 13C NMR (CDC1 3) Spectra of Compound 2.3N I~.. N N C 40C

. 0 0

0

Cl Cl

0

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -05PPM

o 0 0

210 200 '190 '180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 -10PPM

77

'H NMR (CDC1 3) and 13C NMR (CDC1 3) Spectra of Compound 2.4

Br Br

0 0

0

Br Br0

1 if ( i

10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5PPM

O\ |

Br Br

0 0 0

Br Br0

11.111M__200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10

PPM

78

I

'H NMR (CDC1 3) and 13C NMR (CDCL 3) Spectra of Compound 2.5

0 0

0

SIf JI

IA 1k .188~ ~.~0.4 E~

10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5PPM

4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0

"? a! qC "M7 "L1 Cr. mI A 9 r e-4 LA t- Mi

0 0N

~LJliuJ210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10

PPM

79

I

A

'H NMR (CDC1 3) and 13C NMR (CDC1 3) Spectra of Compound 2.7In MN N CD MWwoN Nr

I. ~ ..<rz <r <r <r.0'U

iii I I'I

0

9.5 9.0 8.5 8.0 7.5 7.0

Li "ItI LA

6.5 6.0 5.5 5.0 4.5 4.0PPM

3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 -1.0

'0d

I..-. p.

190 180 170 160 150 140 130 120 110 100 90PPM

80 70 60 50 40 30 20 10 0 -10

80

0 T

F

'H NMR (CDC13) and 13 C NMR (CDC1 3) Spectra of Compound 2.8

0

1 1~Ii

I iii ., Ioooo~ 0

fi

10.0 9.5 9.0 8.5

0

CX

8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5PPM

jrO 0 0I.- r<. kor4JM 14C 6

81

200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10PPM

'H NMR (CDC1 3) and 13C NMR (CDC1 3) Spectra of Compound 2.9M

Br

1 i

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0PPM

In C4

1.5 1.0 0.5 0.0 -0.5 -1.0

0r

B r

I i i

I 2 0 I I I 210 200 190 180 170

82

. I I I I I I I . I 'I I I I I I I I I160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10

PPM

C! a!

'H NMR (CDCL 3) and 13C NMR (CDC1 3) Spectra of Compound 2.11

0 0 0

Cl CI/\0

I /If

Ltia'in NV ~

8 q C 'i'

10.5 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5PPM

0 0

0

0

210 200 190 180 170

2.0 1.5 1.0 0.5 0.0

160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10PPM

83

C. 4 0%

8zjLr;

a

'H NMR (CDC1 3) and 13C NMR (CDC13) Spectra of Compound 2.12

0 0 0

CI CI

1/

IMy VI

I.

iii-9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5

PPM3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 -1.0

0n 00 0

X T0 0C Cl

I I|

200 190 180 170 160

V0 O

I n I

84

I

V0 ENIN EN

150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10PPM

I I . , - 9i I" . -8 -a

r0 8 NO

'H NMR (CDCL 3) and 13 C NMR (CDC1 3) Spectra of Compound 2.13

00

CI CI

/ / If I

-LLI -A4.Y

-4 %O

9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0PPM

1.5 1.0 0.5 0.0 -0.5

I q -,,

Cl C

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10PPM

85

'H NMR (CDC1 3) and 13C NMR (CDC1 3) Spectra of Compound 2.14q - M

-* 0 0 0 J~

C4 I I - '-Cl Cl0

if / 1

I M II

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -1.0PPM

O O 0 N LA O0r..LFr

wl V\? M f4 F-

Cl CI0

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10PPM

86

'H NMR (CDC1 3) and 1 3 C NMR (CDC13) Spectra of Compound 2.15r:", DLn eq" $ $P~jLO r,

CI Ci

I/

I U 11S

IIi

L 14,ii9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5

PPM1.0 0.5 0.0 -0.5

N;~ LM M

0 0 0

A I1 i: N \+, //

Cl Cl0

rlL wArIr. . r n LV : V_ _ _ _ _ _ __q r n n (n f ? 9i e n n r

10.5

LV I%LAM 00

190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0PPM

87

I I I I . . -T- T- , . . . I

I

I

'H NMR (CDC1 3) and 13C NMR (CDCL 3) Spectra of Compound 2.16

Cl C0

M" EO M 5 OMOnr4%0E~ %0 OOU v V-.. % 0C4E C4 r-4 E -D 6E C

I

I I I0 EN

If'

yr

IE E E

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5PPM

o ~-

0 0

Cl Cl

21 20 i 180 170 1 I 150210 200 190 180 170 160 150

r,4 c LA LA A EN rz E4mn q M rj N N

2.0 1.5 1.0 0.5 0.0 -0.5 -1.0

ff? l*e~ V I

1 r~.~iniiIinI iht

0 -10

88

, , ,1 , I -I-140 130 120 110 100 90 80 70 60 50 40 30 20 10

PPM

'

'

'H NMR (CDCL 3) and 13C NMR (CDCL 3) Spectra of Compound 2.17

I ICl C

0

1/ 1.

III

r 4 r4. 4 -6d 6 6

I ' lg I

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5PPM

.-. O O.

0 0 0

0

2 .5 1 . - . -0 .-1 .2.0 1.5 1.0 0.5 0.0 -0.5 -1.0

mm Ln 00G. *i

Cl

89

4~4~~4 ~

|

i i U I i I * |I -I I I I |

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10PPM

I

'H NMR (CDCL 3) and 13C NMR (CDC1 3) Spectra of Compound 2.18

0

Cl CI0

10.5 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5 -1.0PPM

Y In; 9 4 ' - I I I " 9 M

10 0 0 00

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10PPM

90

IR Spectra

IR Spectrum of Compound 2.1, 1200-3500 cm-1

100

000- Io

1500 2000 2500 3000 3500

Wavenumber (cm1 )

91

IR Spectrum of Compound 2.2, 1200-3500 cm-

100-

50-

01 0 1 O

F F0

0- I -1500 2000 2500 3000 3500

Wavenumber (cm-1)

IR Spectrum of Compound 2.3, 1200-3 500 cm-

100

50 -

0 0 0T

0-1I

1500 2000 2500 3000 3500

Wavenumber (cm-1)

92


100-

50 -

Br Br

Br Br

0

1500 2000 2500 3000 3500

Wavenumber (cm-1)

IR Spectrum of Compound 2.5, 1200-3500 cm-'

100-

50 -

0

1500 2000 2500 3000 3500

Wavenumber (cm-1 )

93

IR Spectrum of Compound 2.6, 1200-3500 cm'

100 -

IR 50-

0

1500 2000 2500

Wavenumber (cm-1)

IR Spectrum of Compound 2.7, 1200-3500 cm-'

100 -

50-

0

2500Wavenumber (cm-1)

94

0

3000 3500

1500 2000

0c3000 3500

I

I


100-

0-

50-

1500 2000 2500 3000 3500Wavenumber (cm-1)


100-

50 -

500

1500 2000 2500 3000 3500Wavenumber (cm-1)

95

IR Spectrum of Compound 2.10, 1200-3500 cm-

100-

F50-

0

1500 2000 2500 3000 3500Wavenumber (cm-1)

96

Dihedral-Angle Scans

Dihedral-angle scan of compound 2.6, Figure 2.4A.

d O E - Eo E (Hartrees)

3.293 130.348 2.4920 -460.06493.337 130.830 2.4881 -460.06493.377 130.173 2.4584 -460.06503.417 129.850 2.4848 -460.06493.455 130.060 2.5527 -460.06483.493 130.137 2.6428 -460.06473.532 130.265 2.7396 -460.06453.568 130.370 2.8440 -460.06443.603 130.253 2.9622 -460.06423.635 130.017 3.0943 -460.06403.666 129.816 3.2527 -460.06373.696 129.216 3.4341 -460.06343.723 128.880 3.6416 -460.06313.748 128.499 3.8806 -460.06273.771 127.927 4.1439 -460.06233.792 127.143 4.4226 -460.06193.809 125.971 4.7052 -460.06143.825 125.197 5.0035 -460.06093.839 124.485 5.3275 -460.06043.852 123.784 5.6767 -460.05993.863 122.953 6.0458 -460.05933.87 121.752 6.4231 -460.05873.875 120.497 6.8006 -460.05813.879 119.232 7.1848 -460.05743.881 117.981 7.5794 -460.05683.881 116.693 7.9885 -460.05623.88 115.410 8.4077 -460.05553.877 114.053 8.8259 -460.05483.87 112.514 9.2279 -460.05423.819 123.599 1.9130 -460.06593.813 122.209 1.7307 -460.06613.805 120.718 1.5436 -460.06643.795 119.118 1.3529 -460.06673.782 117.551 1.1656 -460.06703.767 116.121 0.9912 -460.06733.75 114.454 0.8314 -460.06763.73 112.551 0.6727 -460.06783.707 111.063 0.5387 -460.06803.68 109.653 0.4268 -460.06823.652 108.180 0.3363 -460.06843.622 106.613 0.2657 -460.0685

97

3.589 105.113 0.2116 -460.06863.553 103.647 0.1690 -460.06863.517 101.629 0.1174 -460.06873.478 99.978 0.0686 -460.06883.439 98.109 0.0305 -460.06893.399 96.117 0.0070 -460.06893.357 93.917 0.0152 -460.06893.312 92.105 0.0544 -460.06883.266 90.547 0.0819 -460.06883.218 88.434 0.0685 -460.06883.171 86.798 0.0326 -460.06883.128 84.731 0.0000 -460.06893.087 82.750 0.0229 -460.06893.046 79.949 0.0543 -460.06883.005 77.935 0.1032 -460.06872.966 75.911 0.1548 -460.06872.929 73.637 0.2035 -460.06862.895 71.811 0.2586 -460.06852.868 69.945 0.3303 -460.06842.84 68.064 0.4225 -460.06822.817 66.280 0.5369 -460.06802.799 64.556 0.6733 -460.06782.788 62.816 0.8295 -460.06762.78 60.988 0.9980 -460.06732.774 59.174 1.1641 -460.06702.771 57.680 1.3434 -460.06682.769 56.172 1.5227 -460.06652.769 54.759 1.6961 -460.06622.771 53.476 1.8625 -460.06592.774 52.242 2.0176 -460.06572.777 51.166 2.1554 -460.06552.785 49.963 2.2665 -460.06532.788 49.299 2.3247 -460.06522.79 49.023 2.3529 -460.06512.792 49.047 2.3550 -460.06512.789 49.248 2.3307 -460.06522.786 49.826 2.2777 -460.06532.778 51.002 2.1743 -460.06542.774 52.067 2.0394 -460.06562.771 53.297 1.8866 -460.06592.769 54.562 1.7215 -460.06622.769 55.954 1.5491 -460.06642.77 57.456 1.3704 -460.06672.773 58.963 1.1906 -460.06702.78 60.430 1.0184 -460.06732.789 62.143 0.8577 -460.06752.797 64.281 0.6957 -460.0678

98

2.814 66.013 0.5559 -460.06802.837 67.792 0.4381 -460.06822.863 69.661 0.3428 -460.06842.891 71.556 0.2681 -460.06852.924 73.381 0.2108 -460.06862.961 75.259 0.1664 -460.06862.999 77.626 0.1110 -460.06873.039 79.664 0.0607 -460.06883.08 81.892 0.0229 -460.06893.121 83.990 0.0090 -460.06893.165 86.488 0.0262 -460.06893.211 88.200 0.0635 -460.06883.259 89.757 0.0857 -460.0688

Dihedral-angle scan of compound 2.7, Figure 2.4B.

d CO E - Eo E (Hartrees)

2.995 78.425 0.0000 -559.30752.959 76.755 0.0445 -559.30752.927 74.900 0.1249 -559.30732.894 73.589 0.2674 -559.30712.866 72.627 0.4653 -559.30682.845 71.814 0.7330 -559.30642.831 71.091 1.0780 -559.30582.820 70.375 1.5001 -559.30512.812 69.622 1.9902 -559.30442.807 68.908 2.5356 -559.30352.806 68.173 3.1305 -559.3025-2.808 67.450 3.7696 -559.30152.811 66.723 4.4450 -559.30042.817 65.970 5.1453 -559.29932.825 65.266 5.8560 -559.29822.834 64.595 6.5632 -559.29712.846 63.869 7.2604 -559.29602.866 62.801 7.9396 -559.29492.890 61.714 8.5917 -559.29382.915 60.616 9.2107 -559.29282.958 58.839 9.7684 -559.29202.891 61.639 8.6453 -559.29382.869 62.689 7.9957 -559.29482.848 63.790 7.3185 -559.29592.835 64.543 6.6224 -559.29702.826 65.210 5.9160 -559.2981

99

2.818 65.909 5.2051 -559.29922.812 66.666 4.5033 -559.30042.808 67.391 3.8253 -559.30142.806 68.120 3.1828 -559.30252.807 68.845 2.5839 -559.30342.812 69.556 2.0342 -559.30432.819 70.305 1.5390 -559.30512.830 71.034 1.1107 -559.30582.844 71.761 0.7591 -559.30632.864 72.564 0.4850 -559.30682.891 73.494 0.2817 -559.30712.924 74.790 0.1349 -559.30732.958 76.248 0.0422 -559.30752.993 77.857 0.0068 -559.30753.030 79.805 0.0253 -559.30753.072 81.729 0.1204 -559.30733.124 83.991 0.2346 -559.30723.178 85.620 0.3457 -559.30703.234 87.422 0.4503 -559.30683.290 89.351 0.5357 -559.30673.344 91.866 0.5708 -559.30663.395 94.266 0.6215 -559.30653.444 96.603 0.6790 -559.30643.491 99.311 0.7526 -559.30633.536 101.569 0.8200 -559.30623.579 103.337 0.8913 -559.30613.623 105.029 0.9588 -559.30603.664 106.594 1.0138 -559.30593.702 108.314 1.0636 -559.30583.736 109.766 1.1231 -559.30573.768 111.289 1.1962 -559.30563.796 112.796 1.2891 -559.30553.821 114.220 1.4095 -559.30533.843 115.698 1.5578 -559.30503.861 117.527 1.7251 -559.30483.877 119.268 1.9008 -559.30453.891 121.122 2.0823 -559.30423.902 122.747 2.2699 -559.30393.912 124.299 2.4566 -559.30363.919 125.738 2.6404 -559.30333.924 127.064 2.8175 -559.30303.927 128.531 2.9788 -559.30283.927 129.798 3.0931 -559.30263.927 130.549 3.1386 -559.30253.927 130.568 3.1404 -559.30253.927 130.009 3.0945 -559.30263.927 128.682 2.9905 -559.3028

100

127.178 2.83213.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.2.

920912903892878862844823798770739705668627583540495449399348294238183128076033996

12512412212111911711511411211110910810610510310199.97.94.92.90.88.85.84.81.79.78.

.860

.424

.886

.249

.738

.694

.833

.332

.917

.423

.894

.435

.695

.144

.485

.728534217497052109169761133943944449

2.65562.47222.28582.09801.91731.73961.57151.42091.29811.20311.12871.06811.01820.96370.89750.82570.75840.69490.62580.57510.52320.45850.35500.24380.13040.03090.0000

3.925

Dihedral-angle scan of compound 2.8, Figure 2.4C.


367416464514563614668722773817856895

119120121122122122123123124123123123

144167443631688968288720085935960904

665868377207779385779720125527593963516767288644

-919-919-919-919-919-919-919-919-919-919-919-919

663866386637663666356633663166286626662566226619

101

-559.-559.-559.-559.-559.-559.-559.-559.-559.-559.-559.-559.-559.-559.-559.-559.-559.-559.-559.-559.-559.-559.-559.-559.-559.-559.-559.-559.

3.3.3.3.3.3.3.3.3.3.3.3.

3030303330363039304230453048305030533055305630573058305930603061306230633064306530663067306830703071307330753075

3.934 123.717 4.0791 -919.66163.973 123.417 4.3151 -919.66124.010 123.215 4.5499 -919.66084.044 123.225 4.7918 -919.66044.073 122.787 5.0584 -919.66004.098 121.862 5.3430 -919.65954.120 120.826 5.6255 -919.65914.141 119.999 5.9191 -919.65864.160 119.327 6.2304 -919.65814.177 118.588 6.5598 -919.65764.191 117.857 6.9046 -919.65714.204 117.189 7.2599 -919.65654.215 116.471 7.6218 -919.65594.224 115.675 7.9922 -919.65534.230 114.681 8.3775 -919.65474.232 113.440 8.7738 -919.65414.234 112.213 9.1717 -919.65344.234 111.058 9.5640 -919.65284.161 132.608 3.2021 -919.66304.157 130.721 3.0164 -919.66334.150 129.233 2.8154 -919.66364.141 127.520 2.6045 -919.66394.129 125.831 2.3827 -919.66434.114 124.208 2.1631 -919.66464.097 122.255 1.9513 -919.66494.078 120.455 1.7497 -919.66534.054 118.727 1.5618 -919.66564.029 116.940 1.3892 -919.66584.000 115.088 1.2355 -919.66613.969 113.348 1.1088 -919.66633.935 111.661 1.0166 -919.66643.898 109.521 0.9494 -919.66653.858 107.337 0.8929 -919.66663.813 105.476 0.8364 -919.66673.761 103.965 0.7686 -919.66683.706 102.277 0.6655 -919.66703.654 100.052 0.5440 -919.66723.602 97.855 0.4396 -919.66743.548 95.084 0.3377 -919.66753.494 92.897 0.2563 -919.66763.435 91.176 0.1790 -919.66783.380 89.612 0.1015 -919.66793.328 88.246 0.0373 -919.66803.281 86.519 0.0000 -919.66813.239 84.905 0.0061 -919.66803.203 83.587 0.0743 -919.66793.176 81.953 0.2021 -919.6677

102

3.1503.1243.1023.0823.0683.0583.0513.0453.0413.0393.0383.0383.0383.0393.0393.0313.0012.9152.9132.9212.9242.9282.9322.9362.9392.9432.9492.9562.9642.9742.9852.9983.0373.0993.1283.1613.1963.2353.2763.3203.367

81.06280.19879.20478.26277.43976.51675.67574.97274.43273.96773.59173.32273.15973.05473.26174.33277.35193.465102.031105.196106.599107.685108.544109.181109.584109.901110.246110.672111.131111.571111.984112.404112.655110.913111.754112.696113.861115.177116.557117.940119.159

103

0.42050.72231.09871.55062.07642.67073.32534.03344.79135.58806.40957.24248.07578.90939.734710.527911.260611.777011.693811.381310.947310.43979.88189.29248.69328.10277.52956.97786.45205.96085.51345.11784.47813.95633.58843.28273.03972.85942.74022.67842.6657 -919.6638

-919.6674-919.6669-919.6663-919.6656-919.6647-919.6638-919.6628-919.6616-919.6604-919.6592-919.6578-919.6565-919.6552-919.6539-919.6525-919.6513-919.6501-919.6493-919.6494-919.6499-919.6506-919.6514-919.6523-919.6532-919.6542-919.6551-919.6561-919.6569-919.6578-919.6586-919.6593-919.6599-919.6609-919.6618-919.6623-919.6628-919.6632-919.6635-919.6637-919.6638

Dihedral-angle scan of compound 2.9, Figure 2.4D.


3.495 118.020 2.5894 -3033.64743.543 118.900 2.5864 -3033.64743.585 119.643 2.6535 -3033.64733.628 120.591 2.7396 -3033.64713.682 121.864 2.8968 -3033.64693.739 122.076 2.9999 -3033.64673.786 121.607 3.1141 -3033.64653.837 122.273 3.3338 -3033.64623.889 121.652 3.4903 -3033.64593.930 121.659 3.6803 -3033.64563.971 121.515 3.8989 -3033.64534.014 121.574 4.1340 -3033.64494.055 121.759 4.3786 -3033.64454.093 122.090 4.6201 -3033.64414.126 121.790 4.8578 -3033.64384.154 120.708 5.0948 -3033.64344.181 120.284 5.3414 -3033.64304.205 119.733 5.6111 -3033.64264.227 119.294 5.9032 -3033.64214.246 118.891 6.2205 -3033.64164.263 118.291 6.5595 -3033.64114.276 117.219 6.9049 -3033.64054.289 116.452 7.2636 -3033.63994.300 115.806 7.6442 -3033.63934.310 115.033 8.0413 -3033.63874.318 114.075 8.4485 -3033.63804.324 113.034 8.8612 -3033.63744.330 112.096 9.2782 -3033.63674.253 135.793 3.6146 -3033.64574.253 133.656 3.4489 -3033.64604.249 131.729 3.2372 -3033.64634.243 129.890 3.0079 -3033.64674.233 128.165 2.7755 -3033.64714.221 126.218 2.5412 -3033.64754.207 124.351 2.3058 -3033.64784.189 122.479 2.0757 -3033.64824.168 120.808 1.8580 -3033.64854.145 119.173 1.6553 -3033.64894.118 117.535 1.4704 -3033.64924.088 115.881 1.3086 -3033.64944.056 114.029 1.1808 -3033.64964.020 112.183 1.0862 -3033.64983.976 110.460 1.0077 -3033.6499

104

3.9313.8833.8343.7843.7323.6673.6093.5543.5053.4583.4133.3663.3173.2803.2503.2253.2033.1853.1683.1543.1423.1333.1273.1243.1233.1243.1263.1273.1283.1253.1143.0933.0273.0233.0293.0353.0383.0403.0413.0433.0463.0503.0553.0623.0703.0803.093

108.516106.212104.489102.44499.74197.90195.80194.16492.33890.24988.23386.60085.52684.67083.89383.20782.39581.50980.58379.64178.71677.72876.81475.98975.25074.57274.06573.72473.61574.07975.41378.08692.161100.144102.921104.731105.901106.721107.255107.655108.020108.318108.648108.964109.312109.717110.128

0.90600.79630.71250. 61820.53830.41250.26830.16630.07850.01640.00000.02560.06870.16040.33540.59990.95381.39221.90962.49763.14573.85154. 60725.40696.24327.09687. 96678.84699.724710.584411.397612.134112.555812.540912.243811.811511.286110.704510.09149.46568.83958.22177.62217.04846.50415.99235.5232

-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.

65016502650465056507650965116512651465156515651565146513651065066500649364856475646564546442642964166402638863746360634663336322631563156320632763356344635463646374638463946403641164206427

105

3.1083.1293.1843.2113.2393.2693.3023.3403.3873.4353.495

110.110.108.109.110.111.112.113.114.116.118.

574043545364173137206412844428062

10467185211477014021111489617463646659535893

-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.-3033.

64346440644864556461646564696471647364746474

106

Cartesian Coordinates and Total Energies

Compound 2.1

Sum of electronic and zero-point Energies: -1450.6636 Hartrees

Atom x y z

2.326653001.175975001.245471002.507015003.661159003.548215002.250206002.577142000.000069000.00295500

-1.24369900-1.17147500-2.32064300-3.54348100-3.65914100-2.50650100-4.68419900-0.00148500-0.00260700-0.00184900-0.00422800-0.00341900-0.00089900-0.00462100-0.00516600-0.00372100-0.00585000-0.00175100-0.00016500-0.00218300-2.57881700-2.242047004.690972005.41767400

-5.418135006.625597006.312913007.23569500

-1.89867400-1.141312000.214002000.810474000.08226600

-1.27468100-2.954035001.869193001.01574200

-1.825124000.21137300

-1.14376800-1.90355300-1.28208000

0.074740000.80526100

-2.029872002.369221003.359742002.682981004.708060004.029686001.911260005.032334005.469298004.310811006.070551002.713594001.370972003.205902001.86387100

-2.95876200-2.01956800-2.43485900-2.43557700-3.19782800-4.06702500-2.56772500

-0-0-0

0-0-0-0

00

-0-0-0-0-0-0

0-0-0

0-1

0-2-2-1

0-3-1

1120

-0-0

00

-0-0-0

5669990035992900071364000008740019931500480932007918340022300700209945004652270007112800359641005663690048027700198919000011540074085200466090004929900081465900164565001582880057509700183184009344980020401600488732008193320063307100913263002230950079113100741161003320430033107900107803006865280075488500

107

7.19489400-6.62396000-7.23376900-6.30948800-7.194585005.09291800

-5.100628004.63798800

-4.63684500

-3-3-2-4-3-2-2

00

509095002010390057188600070169005119380020156900192970005431280053373300

0-0-0-0

011

-0-0

762277001102050075858800687947007591370046188800461037001405400014015100

Compound 2.2


Atom x y z

2.325829001.173904001.244196002.502990003.633937003.555224002.251016002.60135600

-0.00002700-0.00003900-1.24422500-1.17395200-2.32591000-3.55526100-3.63396300-2.50303600-4.698045000.000031000.000058000.000056000.000131000.000121000.000020000.000162000.000162000.000135000.00021500

-0.00000500-0.00008400

-2.02815700-1.26359000

0.102390000.71382500

-0.03800100-1.41168800-3.09220300

1.779207000.91172000

-1.951661000.10241100

-1.26354500-2.02813500-1.41164300-0.037907000.71389600

-2.139724002.258151003.259151002.557843004.606557003.903864001.778114004.917228005.376000004.175157005.953075002.625857001.27863900

-0.48252600-0.29093500-0.05085900-0.01661500-0.20972700-0.43971900-0.66596500

0.159348000.22174900

-0.35785000-0.05094300-0.29095300-0.48252400-0.43978300-0.20988900-0.01677300-0.68555700-0.47110800

0.48128400-1.82531800

0.13901400-2.18162200-2.57836500-1.21458000

0.90167100-3.23070300-1.53066000

1.816261001.63979600

108

HCHHH00HH

0HH0CCCHHHCHHH00FF

-0.00001700-2.60132900-2.251042004.697974005.58931200

-5.589342006.792777007.274736007.480762006.48240400

-6.79271700-7.27458500-6.48225700-7.480815005.37480700

-5.374868004.84389100

-4.84393100

3.1.

-3.-2.-2.-2.-3.-2.-3.-4.-3.-2.-4.-3.-1.-1.

0.0.

125130007792890009218900139794002843210028421100044280005023770016566800017582000444000050292800017893001653970084809900847814005390750053913600

2.0.

-0.-0.

0.0.

-0.-0.

0.-0.-0.-0.-0.

0.1.1.

-0.-0.

Compound 2.3


Atom x y z

2.322565001.171729001.242660002.500200003.645062003.550735002.249108002.58236400

-0.00000700-0.00016800-1.24276700-1.17197900-2.32290100-3.55099000-3.64518700-2.50024800-4.684430000.000060000.000129000.000059000.00019900

-2.02597200-1.27077200

0.084321000.68507200

-0.05134800-1.41547700-3.08221900

1.743421000.89146900

-1.954060000.08445700

-1.27062800-2.02568500-1.41505600-0.05095400

0.68533500-2.152807002.243837003.233809002.556392004.58199100

-0.-0.-0.

0.-0.-0.-0.

0.0.

-0.-0.-0.-0.-0.-0.

0.-0.-0.

0.-1.

0.

545487003389840005283200015485001873370046688900766916002327790023063100440588000528080033901500545612004669670018723500015599007292290044788700511895007965690018335100

109

906155001591560066588700685559003465570034658200107498009223840072405300489469001073060092253400488731007242060043975500439720001955620019577400

CCCCCCHHC0CCCCCC0CCCC

CHCHHHC00HH0CCCHHHCHHH00C'C'

6.7.7.

-6-7

440611002855460027745300

.70198600

.28485000-6.44007100-7.277860005.13335100

-5.13297100-5.206048005.20598200

3.903270001.78488400454

.90563100

.34328600

.18439700

0.000128000.000007000.000198000.000256000.000129000.000251000.000110000.000024000.00013600

-2.58228800-2.24954600

4.684052005.45808400

-5.458009006.70216300

-3-3-2

.49986900

.19069500.50584800

-4.05315100-3.49911700-2.25127500-2.25200400

0.701515000.70104000

-2.13979100-2.55714700-1.16456800

0.95312500-3.18545000-1.47021300

1.838290001.650100002.932397000.23299600

-0.76713900-0.729054000.345829000.34567500

-0.10227700-0.71609300-0.71794900

0.76436100-0.10237600-0.71944800-0.71479100

0.764281001.470463001.47035900

-0.10654300-0.10691000

Compound 2.4


Atom x y z

2.333643001.171232001.248180002.498467003.647150003.570694002.56848900

-0.000013000.00012500

-1.24813200-1.17105200-2.33338000

-1.36552300-0.587436000.798329001.408690000.64566300

-0.747753002.489384001.64198400

-1.265914000.79821000

-0.58754600-1.36575900

0.327145000.380409000.342285000.235731000.182595000.239976000.193744000.467465000.463566000.342268000.380425000.32719100

110

5.943785002.588102001.242582003.076732001.74367300

-3.08191900-2.15342600-2.49783800-2.49799900-3.19030000-4.05184500-2.50461000

CCCCCCHC0CCC

CCC0CCCCCHCHHHC00H0CCCHHHCHHH00BrBrBrBr

-3.57049600-3.64708700-2.49847900-4.71428800-0.00008500-0.00019700-0.00004100-0.00026800-0.00010300

0.00003800-0.00021300-0.00034400-0.00005900-0.00025700-0.00021200-0.00005400-0.00026200-2.568645004.714606005.21795600

-5.218286006.488289007.232638006.322897006.83599800

-6.48881100-6.32379000-7.23304100-6.836479004.66693000

-4.667611005.33918300

-5.339208002.22085400

-2.22041600

-0.748117000.645285001.40843500

-1.500976002.839752003.995187002.889186005.258582004.149153001.986495005.320036006.152492004.227467006.281980003.611899002.251434004.294449002.48911900

-1.50043900-1.89497100-1.89459900-2.65447000-1.98752800-3.47852800-3.02495900-2.65377000-3.47762500-1.98645200-3.02446300-1.62444300-1.62362200

1.464837001.46430000

-3.24094500-3.24116300

Compound 2.5


Atom x y z

2.330831001.173128001.242829002.48369800

-1.20026100-0.43845700

0.945314001.56788200

-0.0.0.0.

12202800036401001094190000710800

111

0.240024000.182564000.235669000.25155100

-0.458251000.29975400

-1.84359900-0.27964000-2.43630500-2.44354000-1.66705600

0.33222600-3.51681300-2.16429800

1.726340001.793156002.710561000.193659000.25142400

-0.96752300-0.96740600-0.78038600-0.34110900-0.08575900-1.74033400-0.78021800-0.08526600-0.34130300-1.74010100-1.99311900-1.993076000.005732000.005740000.358407000.35839700

CCCC

3.632133003.555566002.541173000.00000200

-0.00001100-1.24283200-1.17314400-2.33085300-3.55558300-3.63213900-2.48369600-4.691316000.000007000.00001700

-0.000001000.000019000.00000200

-0.000010000.000012000.00002800

-0.000004000.000014000.000026000.000007000.00001800

-2.541160004.691293005.39575200

-5.395709006.618089006.327808007.242552007.16015800

-6.61801700-7.24252100-6.32769600-7.160057005.02919800

-5.02912000-5.49876100-2.22460100

2.224567005.49876400

0.82443600-0.564389002.647502001.75651900

-1.119340000.94532500

-0.43844600-1.20023800-0.56435500

0.824469001.56790400

-1.303558003.104258004.096436003.413006005.443351004.759117002.639889005.763606006.206473005.037249006.800968003.456030002.108946003.948019002.64752400

-1.30360500-1.70311800-1.70319700-2.45514400-3.31839600-1.81017000-2.77083700-2.45526300-1.81030100-3.31847900-2.77102000-1.44786500-1.448021001.78455800

-3.29513800-3.29516200

1.78450200

-0.15637400-0.222574000.062603000.381192000.114454000.109422000.03639700

-0.12204500-0.22258500-0.15636500

0.00711900-0.44590700-0.30233700

0.65510300-1.65159700

0.32306200-1.99826300-2.41037000-1.02575100

1.09090400-3.04466000-1.33396400

1.983813001.800313003.075703000.06262300

-0.445883000.662134000.662108000.25832800

-0.34119000-0.361251001.143948000.25828900

-0.36126000-0.34126300

1.143903001.770801001.77078100

-0.30859900-0.19604600-0.19598900-0.30864900

112

Compound 2.6


Atom x y z

849350001794250083915200186599008387700017904000892944006987890030033900698100001404910016214000281457008053730020942700208864007249080029967700

0.00037400-1.20571600-1.21230100-0.000474001.211756001.206029000.00071000

-2.14574400-2.139287002.14638700

-0.00094200-0.00005200-0.00038500

0.001269000.88222600

-0.878886000.001500002.13840000

0.0.

-0.-0.-0.

0.0.0.

-0.0.

-0.0.

-0.1.1.1.2.

-0.

268692000797540029451300469403002959150007838200559025002210200045146400218578008941170000511200426872004638230070790400709396000417770045393000

Compound 2.7


Atom x y z

9264960038964000034991002278410077641000116413009794620002224700591905005066050002903600106363000352160073125000

-0-1-1-0

010

-2-2

21

-0-0

0

0543950033288900501487003889430088143200066612000776780020206700485538000734630094241200539561001811910021021200

0.230227000.11789800

-0.15707400-0.32328700-0.210106000.066069000.444635000.24463000

-0.250372000.14625100

-0.38365500-0.65754400

0.277465001.36809300

113

CCCCCCHHHH0C0CHHHH

-2.-2.-0.-0.-0.-2.-3.-2.-0.-2.

1.2.3.1.1.1.2.

-0.

CCCCCCHHHHF0C0

2.2.1.0.0.2.3.3.0.2.

-0.-1.-2.-1.

-3.41840800-3.57027000-3.53337900-4.14335100

-0.348463000.37280400

-1.34803500-0.17927700

-0.26565100-1.07064200-0.68467200

0.52543500

Compound 2.8


Atom x y z

969784004221340006869000271222008194200016926000023040000455990061564100583275000599130098971100686233003720560053934300471816000959660019668900

-0.29655200-1.54856000-1.66904600-0.536086000.716291000.84070200

-0.19804900-2.43338700-2.63093500

1.82129000-0.65387300-0.56898100-0.47896300-0.58758800

0.35382300-1.39922500-0.693132002.12595500

0.23919800-0.02158500-0.31762000-0.35637200-0.095926000.202849000.469906000.00436900

-0.524808000.40091100

-0.714561000.282045001.43754700

-0.28629300-0.81421700-1.00625000

0.51679500-0.15931600

Compound 2.9


Atom x y z

-3.06256100-2.58549100-1.24101200-0.37982400-0.85678400-2.19945200-4.10890900

0110

-0-0

0

51019200780930009651960087814600392502005805910036008500

0-0-0-0-0

00

23964100065676003672580036617800060243002435940047428200

114

CHHH

CCCCCCHHHH0C0CHHHC'

2.2.1.0.0.2.4.3.0.2.

-1.-1.-1.-3.-3.-3.-4.-0.

CCCCCCH

-3.25695400-0.84265700-2.562064000.942683001.861405001.551096003.241992003.477302003.284909003.953850000.32670400

2.630176002.94294200

-1.572990001.066277001.159204001.139950001.278694000.370525002.118915001.41648000

-1.87175900

-0.07004200-0.61005300

0.47845500-0.72726700

0.278780001.43600300

-0.28166200-0.83945200-0.97517900

0.52712500-0.06802900

115

HHH0C0CHHHBr

CHAPTER 3

Cytosolic Uptake of Large Monofunctionalized Dextrans

Contribution: Chemical synthesis and characterization of dextran conjugates, determination offluorescent properties, hydrolysis assays, cell culture, confocal microscopy, and experimentaldesign. Composition of manuscript and figures was conducted jointly. Dextran branching assays,dynamic light scattering assays, and stability assays were performed by H.R. Kilgore.

This chapter has been submitted for publication, in part, under the same title.

116

3.1 Introduction

Dextrans are glucose polymers with widespread applications in the modem clinic, laboratory,

and home. Dextrans are isolated from Lactobacillales, an order of gram-positive, low guanine-

cytosine content, nonsporulating bacteria.1 26 The constituent glucose units are linked through

a(1,6) glycosidic bonds, with occasional x(1,4) and a(1,3) glyosidic linkages being introduced by

biosynthetic promiscuity. 2 7

Many applications of dextrans leverage their non-immunogenic nature, large size, and

viscogenic properties.128 129 For example, dextrans are on the World Health Organization's "Model

List of Essential Medicines" due to their antithrombotic and volume-expanding properties, among

other beneficial effects. 3 0' 13' Recently, dextran nanoparticles have been used as the basis for a

small-molecule drug delivery platform.1 32 Immediately after uptake via endocytosis, these

nanoparticles degrade, allowing embedded small molecules to escape and diffuse into the

cytosol. 3 3 In the laboratory, fluorophore-conjugated dextrans serve as a tracking agent for macro-

and micropinocytosis, facilitating imaging of endocytosed particles or organisms and probing the

details of autophagy. 3 4-13 6 At home, dextrans are employed as thickening agents in cuisines and

as a base for cosmetics.'3 18

To provide a conjugation handle for fluorophores or other moieties, dextrans are functionalized

by either chemoselective reactions at the reducing-end'3 9 or nonselective reactions such as

periodate oxidation. 40 -142 Commercial fluorophore-conjugated dextrans are typically produced

using nonspecific functionalization followed by fluorophore conjugation that peppers the dextran

with up to 130 molar equivalents of dye.' 43 Because typical dyes are hydrophobic and interact with

lipids,'4 4 functionalizing dextrans with excess dye risks undesirable changes to structural and

117

surface properties. In addition, the nonselective processes that are used to polyfunctionalize

dextrans can impart structural damage and leave residual reactive moieties. 4 5

To overcome the limitations of current fluorophore-dextran conjugates, we sought to create a

fluorogenic dextran with minimal perturbation to the dextran by selectively conjugating a

fluorogenic probe to the reducing end. We chose to use a pH-independent, electronically stabilized

fluorogenic probe1 46 that is suitable for the next generation of agents to track endocytosis and

autophagy. The probe has ester moieties that mask a fluorescent signal until entry into cells, upon

which intracellular esterases cleave the masking groups and restore fluorescence. Conjugation of

the probe to dextrans enables precise spatiotemporal monitoring of cellular uptake. More

importantly, this fluorogenic dextran exhibits high contrast ratios and real-time imaging

capabilities. These advantages stem from the fluorogenic nature of the probe, which ensures little-

to-no background, even without washing of the cells. In contrast, commercially available

fluorescent dextrans are constitutively fluorescent and are not amenable to real-time imaging.

Finally, we compare the cellular uptake of a monofunctionalized dextran with that of

polyfunctionalized dextrans. The results revealed unanticipated differences between these two

types of dextrans, which suggest potential applications of monofunctionalized dextrans as a

cytosolic delivery platform.

118


Our fluorogenic probe (3.1) was conjugated to commercial 100-kDa and 70-kDa dextrans via

thiol-ene and N-hydroxysuccinimide-amine chemistry (Scheme 3.1A) to produce fluorogenic

dextrans 3.3, 3.4, and 3.5. 14 '8 To our knowledge, conjugate 3.3 is the first monofunctionalized

fluorogenic dextran. Conjugate 3.3 was prepared in good yield with no residual unconjugated small

molecule fluorophore contaminants (Figure 3.1). Upon incubation with pig liver esterases or upon

exposure to cytosolic esterases, the isobutyryl masking groups in conjugate 3.3 were cleaved to

effect total reclamation of fluorescence (Scheme 3.1 B, Figure 3.1).

(ran

1D2: X = SHorD3: X = NH 2

0 0 0B I

C1 0 -- C1 0Q

I CIO

0 -

NH0

/-S

0 0 0SI, I N0

-SH 0 -1

NH 0

'a, 3.3 (from 3.1 + Dl)

-0 0 0 Y--0 0 0 Y 1e C1 0C1 C100 00 I ",1 0-1

0W.. 0 or 0 0

NH 0NH -n

. (-n 3an

3.4 (from 3.1 + 112) 3.6 (from 3.2 + 113)

CellularEsterases

2 00

3.36ran

H

,N

0

Scheme 3.1. (A) Synthetic route to conjugates 3.3, 3.4, and 3.5 from commercial dextrans D1and D3. (B) In cellulo enzymatic activation of conjugate 3.3.

119

A

0 0 0

C'I v CI 00 0_

R ~3.1: R = NHCH2CH=CH 23.2: R = OH

A B C1.0- 1.0 3.0-

(D NN

EM2.0-0 0 probe 3.1

0.5- -0.5 - - conjugate 3.31 0-dextran D1

CC + PLE1.

00

. 0.0- -000.0w400 450 500 550 600 3.3 3.1 0 20 40

Wavelength (nm) Retention Time (min)

Figure 3.1. Characterization of dextran conjugates. (A) Spectra showing the response ofconjugate 3.3 (10 pM) to treatment with pig liver esterase (9 nM) for 30 min. (B) Eluted TLC plateshowing that purified conjugate 3.3 has no detectable contamination from residual small-moleculeprobe 3.1. (C) C4 HPLC traces showing that conjugate 3.3 has no detectable contamination fromsmall-molecule probe 3.1.

Next, we assessed the cellular uptake of conjugate 3.3 in HeLa cells by confocal microscopy.

To our surprise, we observed the fluorescent signal for conjugate 3.3 to be dispersed evenly

throughout the cytosol and nucleus (Figures 3.2A and 3.3-3.5), instead of the punctate staining

that is typical of commercially available fluorophore-dextran conjugates.' 49 Although mixed

cytosolic and vesicular uptake of dextrans was reported in a few studies using smaller

polyfunctionalized dextrans, 15-153 conjugate 3.3 seemed to far surpass these in the efficacy of its

cytosolic internalization, with no observable vesicular fluorescence.

120

A 150- HeLa Cells

100-*o o

S50--o-

0 10 20 3OTime (min)

B 1so- H1299 Cells

-U 100-

Tme (mDn

~0

- 1- 5

0-

0* 10 20 30

Time (min)

C 150s H460 Cells

1002

,0

2~ 50-0

0

0 10 20 30Time (min)

Figure 3.2. Uptake of conjugate 3.3 by human cells. Time-courses for the uptake of conjugate3.3 (10 pM, green signal) were obtained by summing the background-subtracted signal withinHeLa cells (A), H1299 cells (B), and H460 cells (C), counterstained with Hoechst 33342 stain(blue signal) for 15 min prior to imaging. Confocal microscopy was used to image the cellscontinuously from 0 to 30 min. Scale bars: 25 pm.

121

Figure 3.3. Confocal microscopy images showing the time-course for the uptake of conjugate 3.3(10 pM) by live HeLa cells. Image-acquisition began immediately after the on-stage addition ofconjugate 3.3 in 300 pL of OptiMEM. Cells were stained with Hoechst 33342. Scale bars: 25 pm.

122

Figure 3.4. Confocal microscopy images showing the time-course for the uptake of conjugate 3.3(10 pM) by live H1299 cells. Image-acquisition began immediately after the on-stage addition ofconjugate 3.3 in 300 pL of OptiMEM. Cells were stained with Hoechst 33342. Scale bars: 25 pm.

123

I

- -

Figure 3.5. Confocal microscopy images showing the time-course for the uptake of conjugate 3.3(10 pM) by live H 1299 cells. Image-acquisition began immediately after the on-stage addition ofconjugate 3.3 in 300 pL of OptiMEM. Cells were stained with Hoechst 33342. Scale bars: 25 pm.

We sought to validate our initial observations. The cytosolic dispersion of conjugate 3.3 was

replicated consistently across different dextran batches and HeLa cell passages. Further, the same

transport localization observed in HeLa cells (cervix adenocarcinoma) was observed in H 1299 and

H460 cell lines (non-small cell lung carcinoma), suggesting that probe entry into the cell was not

an artifact of cell type (Figures 3.2B and 3.2C). Indeed, conjugate 3.3 dispersed generally

throughout the cytosol and nucleus with only small deviations in rate of uptake between these three

cell types (Figure 3.2). Imaging analysis indicates that the same linear rate function is observed in

all cell types, suggesting the mechanism of cell entry is conserved within this set.

Having established the consistent cytosolic entry of conjugate 3.3 into mammalian cells, we

next compared conjugate 3.3 with commercially available fluorophore-dextran conjugates.

124

Although conjugate 3.3 displayed a dispersed signal within cells (Figure 3.2), commercial

polyfunctionalized tetramethylrhodamine-dextran conjugates of various sizes (TAMRA-dextran)

showed punctate staining (Figure 3.6).

To ensure that the hydrophobic fluorophore-masking group and linker components of

conjugate 3.3 did not alter the cell-penetrating properties of the dextran significantly, we also

prepared polyfunctional conjugates 3.4 and 3.5 (Scheme 3.1). Although 3.4 and 3.5 were labelled

with the same probe moiety via thiol-ene or NHS-ester chemistry, respectively, both failed to

reproduce the diffuse staining achieved by conjugate 3.3. Upon incubation of probes 3.4 and 3.5

with HeLa cells over 30 min, polyfunctionalized conjugates 3.4 and 3.5 behaved similarly to

TAMRA-dextrans, yielding highly punctate staining patterns indicative of being trapped within

endocytic vesicles (Figures 3.6 and 3.7). Accordingly, we concluded that the fluorogenic probe

does not perturb dextran transport. Similarly, having either a thioether or an amide in the linker

had no effect on cytosolic penetration properties, as conjugates 3.4 and 3.5 showed similar cellular

distributions. Further, conjugate 3.3 exhibited increased fluorescence signal relative to conjugates

3.4 and 3.5, indicating significantly higher uptake in consideration of the higher degree of dye

labeling in 3.4 and 3.5 (Figure 3.8). To confirm the dextran localization patterns quantitatively,

Pearson's correlation coefficients between each dextran and LysoTrackerTM (which is a stain for

acidified vesicles), or Hoechst 33342 (which is a stain for nuclei) were calculated from cell images

(n > 20, Table 3.1). The correlation coefficients confirmed that conjugate 3.3 was indeed

distributed throughout the cytosol whereas conjugates 3.4 and 3.5 correlated strongly to only the

LysoTrackerTM vesicle stain.

125

Table 3.1. Pearson's correlation coefficients (r) for localization of a fluorogenic dextran conjugateand either Hoechst 33342 nuclear stain or LysoTracker TM acidic vesicle stain.

Dextran Conjugate r(Hoechst 33342) r(LysoTracker T M)

3.3 0.19 0.04 0.02 0.053.4 -0.63 0.03 0.43 0.023.5 -0.42 0.02 0.46 0.05TAMRA-dextran -0.55 0.04 0.31 0.03

0

C

6

r-..

Figure 3.6. Confocal microscopy images of live HeLa cells incubated with 100-kDa TAMRA-dextran (A-D) or 70-kDa TAMRA-dextran (E-H) at 10 pM for 30 min at 37 *C. Images correspondto fluorescence of TAMRA-dextran (A,E), Hoechst 33342 (B,F), LysoTracker T M (C,G), andoverlay (D,H). Scale bars: 25 pm.

126

0

C),

0

Figure 3.7. Confocal microscopy images of live HeLa cells incubated for 30 min withconjugate 3.4 (A-D) or conjugate 3.5 (E-H) at 10 pM for 30 min at 37 *C. Images correspond tofluorescence of conjugates (A,E), Hoechst 33342 (B,F), LysoTrackerT M (C,G), and overlay (D,H).Scale bars: 25 pm.

500-

400-

3.3 3.4Conjugate

Figure 3.8. Graph showing the averagefluorescence signal per cell measured in liveHeLa cells incubated with dextran conjugates.Cells were incubated with conjugates 3.3, 3.4,or 3.5 (10 pM), washed, and imaged byconfocal microscopy. *, p < 0.05; ***, p <0.001.

3.5

127

D 300-2-

200-

100

0-

Next, we assessed the chemical stability of the linkers and dextran in conjugate 3.3. If any of

the linker components were to decompose, then the ensuing fluorescent fragments could diffuse

across a lipid bilayer into the cytosol. We were concerned, for example, about acid-catalyzed

hydrolysis following endocytosis. As endocytic vesicles mature, their pH drops to 4.6.4 155 We

were also concerned about enzyme-catalyzed hydrolysis, because endosomes contain

glycohydrolases.15 6 To test stability, we subjected conjugate 3.3 to conditions at least as harsh as

those encountered on the route to the cytosol. After treatment, we assessed its integrity by high-

performance liquid chromatography (HPLC) and dynamic light scattering (DLS). In HPLC

experiments monitoring fluorophore absorbance at 254 nm, intact conjugate 3.3 elutes rapidly

because its large size results in minimal interactions with column packing material. Any

fragmentation of conjugate 3.3 would be visible as a secondary peak with significantly longer

retention time, with free fluorogenic dye 3.1 (tR = 32.3 min) as reference. We found that conjugate

3.3 was stable in acid (even at 60 'C), growth medium, and cell lysates (Figures 3.9-3.11). These

data suggest that the cytosolic fluorescence (Figure 3.2) arises from intact conjugate 3.3.

A B CB 0 40-

N I3.3 3.1 .30 - 30

oO.5- 20- 010- 3 0-E

S0.0

0 20 40 60 0 10 20 30 40 0 10 20 30 40Retention Time (min) 2Rh (nm) 2Rh(m

Figure 3.9. Graphs showing the acid stability of components of conjugate 3.3 upon incubation in1.0 M HCI for 1 h. (A) C4 HPLC trace of conjugate 3.3 and, for reference, probe 3.1. Sizedistribution as measured by DLS before (B) and after (C) incubation in acid.

128

A1.0-

NI

El

C45C 0.0-

BB1.0-

0.5-

0.0'

0 20 40Retention Time (min)

PuC

1.0-

0.5-

0.0 -60 0 20 40

Retention Time (min)60 0 20 40

Retention Time (min)

Figure 3.10. C4 HPLC traces of probe 3.1h in 1.0 M HCI (A), DMEM containing FBS

(black) and conjugate 3.3 (color) after incubation for 1(B), or HeLa cell lysate (C).

B 15-

I

10-E

e~I

20 40 6Time (min)

I ~6*O 0 * 600. -. ** U p6 - 600

OSO ~6 *g* *

5-

v 6 20

Time (min)40

Figure 3.11. Graphs showing the time-course of thetreatment with 1.0 M HCI at 25 *C (A) or 60 *C (B).

hydrodynamic radius (Rh) of dextran D1 upon

B 15 _

10-

5-

2 4 6[Dextran] (mg/mL)

8

C 40

30-

20-

10-

0 2 4 6 8[Dextran] (mg/mL)

IL ~ I0 2 4 6

[Dextran] (mg/mL)

Figure 3.12. Graphs showing the effects of concentrationdextran D1 (A), dextran D2 (B), and dextran D3 (C).

on the hydrodynamic radius (Rh) of

129

60

A 15-

10-E

0-60

A 15-

C

C% 5

0

I A " ''

T

We sought to examine if there were any inherent structural differences in conjugate 3.3 relative

to other dextrans that might cause cell penetration. Physical characterization of dextrans typically

entail determining their average hydrodynamic radius (which is correlated with molecular mass)

and branching ratios.'12 7 157-159 We were especially interested in deviations amongst the parent

dextrans in our conjugates (D1-D3) as well as an unconjugated 100-kDa dextran (D4). We first

determined the hydrodynamic radii, which agreed closely with standard parameters for dextrans. 59

Then, we verified that aggregation was not playing a significant role, as changes in concentration

did not alter the hydrodynamic radius significantly (Figure 3.12).

The branching in a dextran can be calculated from 1H NMR peak areas that correspond to

a(1,6), a(1,4), and a(1,3) glycosidic linkages. We measured the branching ratios of all dextrans

using glucose disaccharides with a(1,6), u(1,4) and a(1,3) linkages as standards. The frequency of

a(1,4) branches between dextrans D1 and D3 varied by 3%, whereas dextran D4 was branched

half as frequently (Table 3.2, Figures 3.13-3.16). The dextrans showed twofold differences in the

frequency of rare a(1,3) branches, and no dextrans appeared to have detectable a(1,2) branches.

Protein-carbohydrate interactions can be highly specific,' 60 and additional studies are needed to

identify causal relationships between structural variations and the unusual transport properties of

dextran D1.

Table 3.2. Dextran branching as determined by 'H-NMR spectroscopy

Dextran a(1,6):a(1,4) a(1,6):a(1,3)D1 (100-kDa from Fina Biosciences) 30:1 230:1D2 (70-kDa from Thermo Fisher Scientific) 29:1 101:1D4 (100-kDa from Sigma-Aldrich) 46:1 166:1

130

OHHO 0

HO2 OHIn' UED ED LfE0ei4 ('4 "I ri HO

HO .OHH

0

6.3 6.0 5.7 5.4 5.1 4.8 4.5 4.2 3.9 3.6 3.3 3.0 2.7 2.4 2.1 1.8 1.5 1.2 0.9 0.6 0.3 0.PPM

OH

o -OH

0000H H

HO' "OH OHH

I.I.I.I.I... .................. ... . ... ... .. .. .6.3 6.0 5.7 5.4 5.1 4.8 4.5 4.2 3.9 3.6 3.3 3.0 2.7 2.4 2.1 1.8 1.5 1.2 0.9 0.6 0.3 0.PPM

Figure 3.13. 1H-NMR spectra of nigerose (top) and isomaltose (bottom) in D20.

131

OH

HOt. OH

'OH

HO O O

HO "OHOH

7.0 6.7 6.4 6.1 5.8 5.5 5.2 4.9 4.6 4.3 4.0 3.7 3.4 3.1 2.8 2.5 2.2 1.9 1.6 1.3 1.0 0.'PPM

HQ OHOH

HO 000 0

HO ' - --'OH

HO OH

IL6.3 6.0 5.7 5.4 5.1 4.8 4.5 4.2 3.9 3.6 3.3 3.0 2.7 2.4 2.1 1.8 1.5 1.2 0.9 0.6 0.3 0.

PPM

Figure 3.14. 'H-NMR spectra of kojibiose (top) and maltose (bottom) in D20.

132

100 KDa Fina Biosciences Dextran

MM'1,rCJOOOV0 *N

N ~ r N ~ C" " RRCLA LA Lm . inL1L AL

000

6.4 6.1 5.8 5.5 5.2 4.9 4.6 4.3 4.0 3.7 3.4 3.1PPM

2.8 2.5 2.2 1.9 1.6 1.3 1.0 0.7 0.4 0.1

100 KDa Sigma Aldrich Dextran

.5 6.3 6.1 5.9 5.7 5.5 5.3 5.1 4.9 4.7 4.5 4.3 4.1 3.9 3.7 3.5 3.3 3.1 2.9 2.7 2.5 2.3 2.1 1.9 1.7 1.5 1.3 1.1 0.9 0.7 0.5 0.3 0.1f1 (ppm)

Figure 3.15. 'H-NMR spectra of dextran D1 (top) and dextran D4 (bottom) in D 20.

133

kd 0L6 A Ui 6 00

70 KDa Thermofisher Dextran

hkIL0602Z1.1.fid ag;

d, f.6 6.3 6.1 5.9 5.7 5.5 5.3 5.1 4.9 4.7 4.5 4.3 4.1 3.9 3.7 3.5 3.3 3.1 2.9 2.7 2.5 2.3 2.1 1.9 1.7 1.5 1.3 1.1 0.9 0.7 0.5 0.3 0.1

i1 (ppm)

Figure 3.16. 'H-NMR spectrum of dextran D2 in D 20.

To better understand the transport process, we conducted mechanistic studies of the cellular

entry of conjugate 3.3. Lowering environmental temperature to 4 *C is known to inhibit active

transport (e.g., endocytosis), which is energy-dependent.1 61-163 At 4 *C, we observed no cellular

uptake of conjugate 3.3, indicating that its entry relies on active transport rather than passive

diffusion (Figures 3C and 3D). These data are in agreement with previous work showing dextran

uptake through a mixture of clathrin-mediated endocytosis and macropinocytosis,15 0 both energy-

dependent processes inhibited by low temperatures. In contrast, small-molecule probe 3.1 diffused

across the membrane and stained the cytosol similarly at 4 and 37 *C (Figures 3.17A and 3.17B).

134

Figure 3.17. Confocal microscopy images showing the effect of temperature on the uptake ofprobe 3.1 (5 pM; A and B) and conjugate 3.3. (5 pM; C and D) by HeLa cells. Scale bars: 25 pm.

Endocytic processes can largely be classified into three components-pinocytosis,

phagocytosis, and receptor-mediated endocytosis.' 33 Unlike the specific cargo-receptor

interactions required for receptor-mediated endocytosis, pinocytosis entails the ingestion of solutes

in a non-specific manner. Competition assays titrating labeled fluorescent dextran with unlabeled

dextran provide a means to study the extent of reliance on receptor-mediated transport.' 64 HeLa

cells were incubated with a constant concentration of conjugate 3.3 while varying the unlabeled

dextran, then imaged using confocal microscopy. Because the added unlabeled dextran does not

contain a fluorophore, any loss of signal is directly proportional to lowered uptake of conjugate

3.3. Quantification of the resulting cell images shows an exponential decay of the signal from

conjugate 3.3 versus increasing concentrations of unlabeled dextran (Figure 3.18). The exponential

(rather than linear) nature of the curve is diagnostic of a receptor-mediated process.' 6 5 An

asymptotic basal level of uptake was observed, which can be attributed to the rate of nonspecific

uptake via pinocytosis. Thus, the uptake of conjugate 3.3 occurs via a combination of receptor-

mediated endocytosis and pinocytosis.

135

U- 400-

C - " Figure 3.18. Graph showing the effect of.~300-

increasing concentrations of unlabeleddextran on the uptake of conjugate 3.3 by live

0 200- HeLa cells. Uptake was quantified by confocal-> microscopy after a 30-min incubation with

conjugate 3.3 (5 pM) and unlabeled dextran4 c 100-(0-75 pM).

D*3 0 _ _ _ __ _ _ __ _ _

20 40 60 80[Unlabelled Dextran] (pM)

3.3 Conclusions

We prepared a monofunctionalized fluorogenic dextran, conjugate 3.3, with improved imaging

capabilities compared to previous polyfunctionalized dextran-fluorophore conjugates. Our initial

intent was to use conjugate 3.3 as a fluorogenic tracker for endocytosis. Conjugate 3.3 is

impervious to pH fluctuations, easy to synthesize, and most importantly avoids the complications

associated with non-specific polyfunctionalization. Remarkably, conjugate 3.3 engages in highly

productive cellular transport into the cytosol. Although this renders conjugate 3.3 a poor tracker

of endocytosis, the rapid and disperse uptake observed with conjugate 3.3 suggests

monofunctionalized dextrans as a vehicle for cytosolic delivery. Structural characterization

following degradation experiments show that the cellular environment and its enzymatic

machinery do not degrade 3.3. Variations in the branching ratios or surface differences of dextrans

investigated could be responsible for various transport properties. Encouraged by the results of the

cellular uptake studies, we are currently investigating the mechanism of uptake while exploring

alternative cargos that could use monofunctionalized dextrans as a delivery system.

136


This work was supported by grant RO1 GM044783 (NIH). W.C. was supported by an NSF

Graduate Research Fellowship. NMRFAM was supported by Grant P41 GM103399 (NIH). The

Mass Spectrometry Facility at the University of Wisconsin-Madison was supported by Grant S 10

OD020022 (NIH). The Soft Materials Laboratory at the University of Wisconsin-Madison was

supported by Grants SlO RR013790 (NIH) and BIR-9512577 (NSF).

3.5 Experimental

Materials. Monothiodextran (100-kDa, Dl) was purchased in three batches (JZ134P62,

JZ13JP91, and MOS0099) from Fina Biosolutions (Rockville, MD). Polyaminodextran (70-kDa,

D3) and TAMRA-dextrans (70-kDa and 1 00-kDa) were from Thermo Fisher Scientific (Waltham,

MA). Unfunctionalized dextran (1 00-kDa, D4) was from Sigma-Aldrich (St. Louis, MO). Pig liver

esterase (PLE) was from Sigma-Aldrich. All other materials were from Sigma-Aldrich, Fischer

Scientific (Hampton, NH), or Alfa Aesar (Haverhill MA), and were used without further

purification.

HeLa, H460, and H1299 cell lines were from American Type Culture Collection (Manassas,

VA) and were maintained according to recommended procedures. Dulbecco's Modified Eagle's

Medium (DMEM), RPMI 1640 medium, fetal bovine serum (FBS), trypsin (0.25% w/v),

OptiMEM, and Dulbecco's phosphate-buffered saline (PBS) were from Thermo Fisher Scientific.


and streptomycin (100 pg/mL). H460 and H1299 cells were grown in RPMI 1640 medium

supplemented with FBS (10% v/v), penicillin (100 units/mL), and streptomycin (100 ptg/mL). For

137

all imaging experiments, 8-well microscopy slides from Ibidi (Madison, WI) were seeded with 105

cells/mL 24 h before use. All imaging experiments were performed in live cells without fixation.

ImageJ was used for all image-processing, signal-quantification, and colocalization

measurements.1 2 4 HeLa cell lysates were prepared by treating HeLa cells with M-PER mammalian

protein extraction reagent from Thermo Fisher Scientific (1 mL per 107 cells) with and without the

addition of protease inhibitor (Pierce Protease Inhibitor tablets). Lysates elicited fluorescence from

probe 3.1 immediately, indicative of enzymatic activity (data not shown).

General Procedures. Chemical reactions were monitored by thin-layer chromatography (TLC)

with EMD 250-jam silica gel 60-F254 plates visualized by UV illumination or KMnO4 stain. Flash

chromatography was performed on a Biotage Isolera automated purification system using pre-


The phrase "concentrated under reduced pressure" refers to the removal of solvents and other

volatile materials using a rotary evaporator at water aspirator pressure (<20 torr) while maintaining

the water-bath temperature of 40 'C. Residual solvent was removed from samples by the vacuum

(<0.1 torr) achieved by a mechanical belt-drive oil pump.

All procedures were performed at ambient temperature (-22 'C) and pressure (1.0 atm) unless

noted otherwise.

Instrumentation. 'H and 13 C NMR spectra were acquired on Bruker spectrometers at the National

Magnetic Resonance Facility at Madison (NMRFAM) operating at 500 MHz for 'H and 125 MHz

for 13 C. Electrospray ionization (ESI) mass spectrometry was performed with a Thermo Scientific

Q Exactive Plus instrument at the Mass Spectrometry Facility in the Department of Chemistry at

138

the University of Wisconsin-Madison. Dynamic light scattering data were acquired with a

Malvern Zetasizer Nano ZSP instrument at the Soft Materials Laboratory of the University of

Wisconsin-Madison. Microscopy images were acquired with a Nikon A1R-Si+ confocal

microscope (60x objective, GaAsP PMT detector, 405 nm/488 nm excitation laser), at the

University of Wisconsin-Madison Biochemistry Optical Core.

Fluorescence data were acquired with a PTI QuantaMaster spectrofluorometer. Absorbance

measurements were made with an Agilent Cary 60 UV-Vis spectrophotometer. Thiol-ene

conjugation reactions were performed with a Spectronics Spectrolinker XL-1500 UV crosslinker.

Dextran purity was verified with a Shimadzu LC-20 HPLC equipped with a Vydac C4 peptide

214TP510 column.

Optical Spectroscopy. All fluorogenic probes and fluorescent molecules were dissolved in

spectroscopic grade DMSO and stored as frozen stock solutions. For all measurements, DMSO

stock solutions were diluted such that the DMSO concentration did not exceed 1% v/v.

UV-Visible and Fluorescence Spectroscopy. Spectroscopy was performed using 1-cm path

length, 4-mL quartz cuvettes or 1-cm path length, 1 -mL quartz microcuvettes. Fluorescence

spectroscopy was performed on solutions that were stirred with a magnetic stir bar.

139

1. 'NH2HATU, DIEADMF

2. 0

O O O DMA py0 0

C 0 C1 DCM 0 C - 0 C0 O

HO2C O NH O

Synthesis of Alkyne Probe 3.1. 3',6'-Diacetyl-2',7'-dichloro-6-carboxyfluorescein' 66

(200 mg, 0.33 mmol), HATU (150 mg, 0.39 mmol), and diisopropylethylamine (144 p.L,

0.83 mmol) were dissolved in DMF (2 mL). Allylamine (50 ptL, 0.33 mmol) was added to the

resulting solution, which was then stirred for 2 h. After concentration under reduced pressure, the

residue was dissolved in EtOAc and washed with 1.0 M HCl and brine (3 x), dried with MgSO4(s),

and concentrated under reduced pressure. The residue was suspended in 10 mL of DCM. 4-

Dimethylaminopyridine (4.0 mg, 33 tmol) and pyridine (106.4 !.L, 1.32 mmol) were added to this

suspension. Isobutyryl chloride (139 pL, 1.32 mmol) was added dropwise, and the resulting

solution was stirred for 2 h. The mixture was diluted with water (50 mL) and DCM (50 mL). The

organic phase was washed with saturated aqueous NH4 Cl and brine, dried with MgSO4(s), and

concentrated under reduced pressure. Purification by column chromatography (0-60% v/v EtOAc

in hexanes) on silica gel afforded the title compound as a white solid (146 mg, 62% yield). 'H

NMR (500 MHz, CDCl 3, 6): 8.14 (d, J= 1.0 Hz, 2H), 7.51 (s, 1H), 7.13 (s, 2H), 6.84 (d, J= 3.5

Hz, 2H), 6.44 (t, J= 5.6 Hz, 1H), 5.89 (ddt, J= 16.1, 10.2, 6.0 Hz, 1H), 5.25 (dd, J= 17.1, 1.3 Hz,

IH), 5.18 (dd, J= 10.2, 1.2 Hz, 1H), 4.04 (tt, J= 5.9, 1.3 Hz, 2H), 2.89 (dt, J= 14.0, 7.0 Hz, 2H),

1.36 (dd, J= 7.0, 4.6 Hz, 13H). 13 C NMR (125 MHz, CDCl 3, 6): 174.34, 167.80, 165.37, 152.60,

149.71, 148.93, 141.79, 133.36, 130.21, 129.09, 127.92, 126.28, 123.08, 122.32, 117.93, 116.81,

113.00, 80.72, 43.04, 34.27, 18.96. HRMS-ESI (m/z) calcd for C32H28Cl2NO8, 624.1187; found,

624.1178.

140

0

HO 0 OH O 0 01 DMAP, py I I I

C _ 1 CI DCM O CI 0 Cl0

HO 2C o HO 2C

Synthesis of Carboxyl Probe 3.2. 2',7'-Dichloro-6-carboxyfluoresceinI (200 mg, 0.45 mmol) was

suspended in DCM. 4-Dimethylaminopyridine (5.5 mg, 45 tmol) and pyridine (144 pL, 1.8 mmol)

were added to the suspension. Isobutyryl chloride (189 pL, 1.8 mmol) was added dropwise, and

the resulting solution was stirred for 1 h. After dilution and extraction with DCM (3 x), the

combined organic extracts were washed with 1.0 M HCl and brine, dried with MgSO4(s), and

concentrated under reduced pressure. Purification by column chromatography on silica gel (30-

80% v/v EtOAc in DCM) afforded the title compound as a white solid (224 mg, 86% yield). 'H

NMR (500 MHz, CDCl 3, 6): 8.40 (dd, J= 8.0, 1.1 Hz, 2H), 8.17 (d, J= 8.0 Hz, 2H), 7.90 (s, 2H),

7.17 (s, 4H), 6.85 (s, 4H), 2.89 (dt, J= 14.0, 7.0 Hz, 4H), 1.36 (dd, J= 7.0, 2.3 Hz, 21H). 13 C NMR

(125 MHz, CDCl 3, 6): 183.29, 174.00, 173.99, 169.22, 167.47, 152.15, 149.66, 148.82, 136.28,

132.36, 129.61, 128.71, 125.93, 125.76, 122.90, 116.61, 112.92, 80.86, 34.13, 18.82. HRMS-ESI

(m/z): [M + H]' calcd for C2 9H2 3C 2 09, 585.0714; found, 585.0712.

Ellman's Assay. Ellman's assay167 was used to assess the concentration of free thiols in dextrans.

The assays was performed in 0.10 M sodium phosphate buffer, pH 8.0, containing EDTA (1.0

mM) using e= 14,150 M1cm' at 412 nm for reduced 5,5'-dithio-bis-(2-nitrobenzoic acid).

Synthesis of Conjugate 3.3. Fluorogenic probe 3.1 was conjugated to dextran D1, which has 0.82

(JZ134p62), 0.79 (JZ13JP91), or 0.31 (MOS0099) free thiols per dextran according to Ellman's

141

assay, by a thiol-ene reaction.168 169 The linker connecting the dextran and thiol in D1 is located

at the reducing end of the dextran and contains amide and ether moieties. All concentrations of

conjugate 3.3 were measured as total dextran concentration (labeled and unlabeled), and all three

batches showed identical same staining patterns and qualitative results. Quantitative fluorescence

measurements were acquired with consistent batches of conjugate 3.3.

Briefly, dextran DI (10 mg, 100 nmol) was dissolved in a solution of 200 pLL of 0.20 M sodium

acetate buffer, pH 4.0, 50 pL of acetonitrile, and 100 tL of DMSO. Glutathione (1.5 equiv), lithium

phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) initiator168 169 (2.5 equiv), and probe 3.1

(5 equiv) were added. The reaction mixture was irradiated with a 365-nm light source for 15 min.

The resulting solution was diluted with 800 pL of PBS, transferred into a 10-kDa MWCO Slide-

A-Lyzer MINI dialysis device from Thermo Fisher Scientific, and dialyzed against 1.0 L of PBS.

After 2 h, the dialysis buffer was replaced, and dialysis was continued for an additional 8 h. TLC

of conjugate 3.3 (60% v/v EtoAc in hexanes) showed a single baseline spot with no residual

unconjugated probe 3.1. The average hydrodynamic radius (Rh = 6.3 nm) of the dextran was

unchanged after conjugation. The absorbance spectrum of the dextran in aqueous 0.1 M NaOH

(which hydrolyzes the isobutyryl esters) was indicative of an average of 0.29 flourogenic moieties

per dextran molecule. The purity of conjugate 3.3 from unconjugated probe 3.1 was verified

further by comparing HPLC traces of probe 3.1, dextran D1, and conjugate 3.3, obtained using a

linear gradient of B (10-95% v/v) over 45 min at a flow rate of 5 mL/min (A: H20 containing

0.1% v/v TFA; B: acetonitrile containing 0.1% v/v TFA). Eluates were monitored at 254 nm.

Synthesis of Conjugate 3.4. Thiol groups were installed on 70-kDa polyaminodextran. Briefly,

the dextran (10 mg, 14 nmol) was reacted with succinimidyl 3 -(2-pyridyldithio)propionate (SDPD)

142

from Thermo Fisher Scientific according to the manufacturer's instructions. The resulting

polythiodextran (D3.3) was dissolved in sodium acetate buffer and conjugated with probe 3.1 as

described above for the synthesis of conjugate 3.3. After irradiation, conjugate 3.4 was dialyzed

overnight against 1.0 L of PBS. Conjugate 3.4 had an average of 11 fluorogenic moieties per

dextran molecule.

Synthesis of Conjugate 3.5. The NHS ester of probe 3.2 was generated by stirring probe 3.2 (10

ptmol) with N-chlorosuccinimide (10 tmol) in DCM for 1 h, followed by removal of solvent under

reduced pressure. The resulting NHS ester was used without further purification. 70-kDa

polyaminodextran (10 mg, 14 nmol) was dissolved in 1.0 mL of PBS. To this solution was added

100 piL of a 30 mM solution of the NHS ester in DMSO. The resulting solution was stirred gently

for 1 h, then dialyzed overnight against 1.0 L of PBS. Conjugate 3.4 had an average of 9

fluorogenic moieties per dextran molecule.

Enzymatic Unmasking of Conjugate 3.3. PLE (168 kDa, >15 units/mg solid) was suspended in

10 mM HEPES-NaOH buffer at pH 7.3, and the resulting solution was diluted to appropriate

concentrations before use in protein LoBind tubes from Eppendorf. Conjugate 3.3 (1 pM) in

10 mM HEPES-NaOH buffer, pH 7.3, was allowed to equilibrate with stirring in a cuvette for

5 min, after which PLE was added to final enzyme concentration of 9 nM. After stirring for 30 min,

the absorption and emission spectra (ex = 470 nm) were recorded.

Aggregation Assay. Solutions of dextrans were prepared in PBS at 1.0 mg/mL, 0.5 mg/mL, 0.25

mg/mL, 0.0125 mg/mL, and 0.00625 mg/mL, filtered through a 40-pim filter, and equilibrated for

143

30 min at 25 'C. The average hydrodynamic radius was measured by dynamic light scattering at

25 'C. Data were analyzed by the method of cumulants.17 0

Dextran Stability Assay. Solutions of dextrans were prepared in PBS at 1.0 mg/mL, filtered

through a 40-pm filter, and equilibrated for 30 min at 25 'C. Solutions were then acidified to

pH 0.4 by the addition of 1.0 M HCl, shaken thoroughly, and incubated for 15, 30, 45, or 60 min.

The solutions were then neutralized by the addition of 1.0 M NaOH, and the average hydrodynamic

radius was measured by dynamic light scattering at 25 'C.

Linker Stability Assay. The stability of the linker and dextran components in conjugate 3.3 were

assessed by incubating conjugate 3.3 (10 pM) for 1 h in 1.0 mL of 1.0 M HCl, DMEM

supplemented with fetal bovine serum (FBS, 10% v/v), penicillin (100 units/mL), and

streptomycin (100 tg/mL), or HeLa cell lysate. The integrity of conjugate 3.3 was assessed by

HPLC using a Vydac C4 peptide 214TP5 10 column as compared against probe 3.1 eluted under

the same conditions, which were an isocratic wash for 10 min followed by a linear gradient of B

(10-95% v/v) over 45 min at 5 mL/min (A: H20 containing 0.1% v/v TFA; B: acetonitrile

containing 0.1% v/v TFA). Eluates were monitored at 254 nm, a wavelength that unconjugated

dextran D1 does not absorb significantly (Figure Si C). Note that the isobutyryl masking groups in

conjugate 3.3 are cleaved by some of the incubation conditions, unveiling the parent

dichlorofluorescein fluorophore conjugated to the dextran (see Scheme 1B). The difference in

polarity between unmasked and masked fluorophore does not significantly alter conjugate-

retention time due to its large size excluding it from interaction with the resin. Although the

presence or absence of the isobutyryl groups should materially affect retention times for dye-

144

containing degradation fragments, no such degradation was observed in any of the tested

incubation conditions.

Branching Assay. Solutions of dextrans D1-D4, kojibiose (a(1,2)), nigerose (a(1,3)), maltose

(a(1,4)), and isomaltose (a(1,6)) were prepared in D2 0. 'H-NMR spectra of each solution were the

recorded with 2048 scans. Disaccharide 'H-NMR spectra were used to establish the 'H chemical

shift of the proton attached to the anomeric carbon: a(1,3), 5.20-5.27 ppm; a(1,4), 5.30 ppm;

a(1,6), 4.85 ppm; and a(1,2), 5.33 ppm. These chemical shifts were in close agreement with values

reported previously.' 7 1 Peaks in the 1H-NMR spectra of dextrans D1-D4 that corresponded to these

shifts were integrated, and the branching of the dextrans was calculated from the values of these

integrals.

Time-Course Imaging. HeLa, H1299, or H460 cells in 8-well microscopy slides were incubated

with Hoechst 3342 (2 pg/mL) for 10 min and washed. Dextran-conjugated probe (10 pM) was

added to the well on stage, and images were acquired every 30 s. At each 30-s time point, the 408

nm and 488 nm excitation channels were acquired sequentially, with exposure time and excitation

intensities selected to prevent saturation in images taken at the final time point. No wash steps

were performed before or during imaging. The background-subtracted fluorescence signal in the

nucleus and total cell was quantified across all time points for individual cells with the program

ImageJ.

4 0C Internalization Imaging. HeLa cells in 8-well microscopy slides were incubated at 4 'C

with OptiMEM containing either conjugate 3.3 (10 pM) or small molecule 3.1 (5 pM) for 20 min.

145

The cells were counterstained with Hoechst 33342 (2 tg/mL) for 10 min at 4 'C, then washed

thoroughly with OptiMEM at 4 'C to ensure complete removal of residual conjugate 3.3 or probe

3.1 from the medium. Cells were then visualized with confocal microscopy at room temperature.

Pearson's Colocalization Coefficient. Pearson's colocalization coefficient was calculated for

dextrans and LysoTrackerTM or Hoescht 33342 by processing confocal images using an ImageJ

plugin.12 Regions of interest corresponding to individual cells were processed (n > 20), and the

means and standard deviations for each experiment are listed in Table S 1.

Competition Assay. HeLa cells in 8-well microscopy slides were incubated with conjugate 3.3 (5

pM) and dextran D1 (0-75 pM) for 30 min. Cells were counterstained with Hoescht 33342 (2

tg/mL) for 10 min, washed, and visualized with confocal microscopy at room temperature. Overall

uptake was calculated as the sum of cell area signal and compared. The resulting data was fit to a

single exponential decay function using GraphPad Prism software with R2 = 0.992, asymptote

89.6 10.6 RFU (95% CI, 47.1-114.9).

146

NMR Spectra

'H NMR (CDC13) and 13 C NMR (CDC1 3) spectra of compound 3.1Ln gj; w r r 00 8

o 0 0

-- I ICI Cl

00

H -

r f f

I .1

f

10.5 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5PPM

t g i 4 O !f 4 CI A A 4

C0 C nI l qi

/ \

0I CI0

H -

f . . ..

220 210 200 190 180 170 160 150 140 130 120 110 100PPM

90 80 70 60 50 40 30 20 10 0 -0

147

'H NMR (CDC1 3) and 13C NMR (CDC1 3) spectra of compound 3.2

-4 C Wr,~ r-. LA

w w 6 C 0606 -z r-: %6

UI I

-- y0 0 0

0CI ClHO 2C /

1hI'd,8 'I r

10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0PPM

2.5 2.0 1.5 1.0 0.5

Go F, V

0 0 0

0CI ClHO 2C / 0

210 200 190 180 170 160 150

wOD qM a C

- ~

140 130 120 110 100 90 80 70 60 50 40 30 20PPM

148

fp TT

r4 C4 ENA (E r-4

I-0.0 -0.5

10 0 -10

. . . . . . . . . . . . . . . . .

Ln r4

L'I

CHAPTER 4

Paired Nitroreductase-Probe System to Quantify the CytosolicDelivery of Biomolecules

Contribution: Chemical synthesis and characterization, determination of fluorescent properties,toxicity assays, enzyme kinetics assays, probe response assays, confocal microscopy, imageprocessing. Plasmid design, preparation, and transfection were performed by V.T. Ressler and T.T.Hoang. Experimental design was assisted in part by T.D. Gruber and L.D. Lavis.

149

4.1 Introduction

The biological activity of drugs is highly dependent on their transport properties, such as their

ability to cross cell membranes en route to desired targets. Orally delivered drugs must overcome

additional transport challenges compared to intravenously delivered drugs, including the highly

degrading gastrointestinal environment and traversing additional layers of epithelial cells. To

streamline drug development, several predictive tools have been developed for estimating

biological availability, 7 2 173 among which Lipinski's rule of five and its variants are used most

widely. 7 4 Although there are notable exceptions to Lipinksi's rules that have found clinical

success, Lipinski's guidelines have provided a useful framework for predicting drug-like character

and reducing development costs, particularly for small-molecule drugs.'

In contrast to synthetic small-molecule drugs, biologics-therapeutic agents based on large

biomolecules such as proteins, peptides, or nucleic acids-violate most, if not all of Lipinski's

rules. Not only do biologics have difficulty entering the bloodstream via oral routes, there are

currently no FDA-approved biologics with intracellular targets due to the difficulty of cytosolic

delivery.1 76 In fact, endocytosis-the most common route for cellular uptake of biomolecules-is

also a barrier to cytosolic delivery since biologics trapped in endocytic vesicles are slated for

degradation if unable to escape.'17 7 19 In spite of these challenges, there is clear clinical imperative

to understand and enhance this delivery process considering the vast therapeutic potential that can

be unlocked by generalizable cytosolic delivery strategies. For example, the number of druggable

intracellular targets of monoclonal antibodies (mAb) is estimated to be 3-4 times greater than the

number of surface targets. 8 0 More importantly, the potential for entirely new classes of protein

and nucleic acid therapeutic agents far exceed those of new mAb drugs. Cytosolic delivery remains

150

one of the most significant barriers to promising therapies such as CRISPR genome editing,' 8

RNAi gene silencing,18 2 and protein anticancer agents.' 8 3

Developing tools to quantify delivery is the first step towards formulating design rules for

cytosolic delivery of large biomolecules, akin to Lipinski's rules for small-molecule drugs.

Previous techniques have explored installing exogenous HaloTag,' 814 biotin ligase,'8 5 and P-

galactosidase 5 enzymes that generate a cytosolic signal by attaching or activating a reporter bound

to a protein of interest after cytosolic entry. Unfortunately, each of these methods suffers from

some combination of high background signal, tedious purifications, potentially transport-altering

protein modifications, low signal-to-background ratio, and lack of target generalizability. Ideal

systems for quantification of cytosolic delivery must possess three key traits-ability to resolve

faint, diffuse cytosolic signal amidst a background of vesicular signal, orthogonality to endogenous

enzymes, and minimal perturbation of the protein payload.

In this chapter, I report on a paired enzyme-probe system consisting of an exogenous

nitroreductase (NTR) enzyme and fluorogenic probes to quantify cytosolic delivery of

biomolecules (Figure 4.1). Selective cytosolic measurement is achieved by incorporating NTR into

a fusion protein anchored to the outer mitochondrial membrane (OMM). This anchoring ensures

that vesicular pools of probe-labeled biomolecules remain nonfluorescent and fluorescence signal

is measured only from cytosolic pools of biomolecules. I prepared and validated a panel of small-

molecule fluorogenic probes that provide high signal-to-noise contrast ratios, fast activation by

nitroreductase, and minimal perturbation to the proteins of interest. Together, the NTR fusion

protein and fluorogenic probes constitute an optimized toolkit well-poised for development and

validation of cytosolic delivery strategies.

151

A

OMM Expression ActivatorAnchor Reporter Enzyme

B

Masked BiomoleculeFluorophore

C

f~w

I~Tflhmflfl mm

Figure 4.1. Schematicrepresentation of (A) enzymeand (B) probe components ofthe paired NTR-probe systemfor detecting cytosolic uptake ofbiomolecules. (C) Diagram ofNTR-probe system In live cellstransfected with NTR fusionprotein anchored. to the outermitochondrial membrane(OMM). Cytosolic pools ofprobe-labeled biomoleculeremain masked andnonfluorescent in theendosome. Only cytosolicpools of the biomolecule areunmasked by OMM-embeddedNTR to generate fluorescence.


Design and Optimization of an Outer Mitochondrial Membrane-Anchored NTR Fusion

Protein. The enzyme-probe detection system consists of a fluorogenic probe and a cytosol-

accessible activating enzyme that can unmask probe fluorescence (Figure 4.1 A-B). The activating

enzyme component should fulfill three crucial requirements-complete cytosolic sequestration,

orthogonality to endogenous enzymes, and rapid rates of enzymatic activation. The first two

152

requirements ensure that only biomolecules in the cytosol are detected, whereas the final

requirement ensures complete detection over the desired experiment timescale.

Nitroreductase, a common bacterial enzyme that catalyzes reduction of nitroaromatic groups,

is particularly well-suited to serve as the reporter enzyme due to its orthogonality to endogenous

human reductases.18 6 Nitroreductases are classified by their mechanism of activity, with oxygen-

insensitive type I nitroreductases employing two-electron reduction of nitroaromatic groups to

amines. 186, 187 In contrast, type II nitroreductases are oxygen sensitive and undergo a one-electron

futile redox cycle to produce hydroxylamines via nitro anion radical intermediates.1 86-18 8 Type I

and type II nitroreductase activity are largely absent from the cytosol of human cells, with the

exception of type II nitroreductase activity observed in select cancer cell lines under hypoxic

conditions. 31,38,43 In fact, the only known type I nitroreductase in normoxic human cells are located

within the mitochondrial matrix, which is satisfyingly congruent with the putative bacterial origin

of mitochondria.2 4 Because of their orthogonality, bacterial nitroreductases have been particularly

useful for gene-directed enzyme-prodrug therapy (GDEPT)' 89 and antibody-directed enzyme-

prodrug therapy (ADEPT) applications. 9 0

In light of the extensive mechanistic studies and applications of E coli nitroreductases, we

selected type I nitroreductase NsfB from E coli for use in our enzyme-probe system. Specifically,

an NTR variant with a F 124W mutation was chosen for its ability to reduce small molecules rapidly

and selectively, with a seven-fold rate increase over wild-type NTR in vitro and in cultured cells.' 9 '

To ensure cytosolic exposure, we chose to anchor NTR to the outer surface of the mitochondria

using the TOM20 protein. TOM20 is a component of the mitochondrial TOM complex that

contains a short N-terminal transmembrane segment that embeds in the cytosolic face of the

153

A 2.5- B 3.0-

2.0-

C 2.0-X 1.5-

1.0-S11.0-

0.5-

0.0 0.0-0.0 0.5 1.0 2.5 5.0 10.0 0 12 24 36 48 60 72

[DNA] (pg/mL) Time (h)

C 250- D 150 + NTR

200 -

100-150 --

CC.2100- 0

50-X X

Wi Lii

0 0-WT 0.0 0.5 1.0 2.5 5.0 10.0 0 12 24 36 48 60 72

[DNA] (gg/mL) Time (h)

E 1.0-

0.8-

0.6-00 0.4-

0E2 0.2

0.0 5.0 10.0[DNA] (pg/mL)

Figure 4.2. Validation and optimization of transfection of HeLa cells with NTR fusion proteinplasmid. (A) Graph of cell count as a function of transfection DNA concentration. (B) Graph ofHeLa cell growth over time after transfection with 2.5 pg/mL DNA. (C) Graph of fusion proteinexpression as a function of transfection DNA concentration. Expression was quantified byconfocal microscopy of mScarlet fluorescence in live HeLa cells, normalized to cell count (Figure4.3). (D) Graph of fusion protein expression over time in HeLa cells transfected with 2.5 pg/mLDNA. Expression was quantified by confocal microscopy of mScarlet fluorescence in live HeLacells, normalized to cell count (Figure 4.4). (E) Graph depicting the degree of association betweenthe fusion protein and outer mitochondrial membrane (OMM) as a function of transfection DNAconcentration. OMM association was quantified by measuring Pearson's correlation coefficientbetween the NTR fusion protein and MitoTrackerTM Green FM mitochondrial stain in images oflive HeLa cells acquired by confocal microscopy (Figure 4.5).

154

oMM. 9 2 193 For the NTR-probe system, mitochondria are optimal intracellular tethering points

because they are discrete organelles that have no direct interactions with the cellular transport

system, in contrast to other membrane-bound organelles such as the endoplasmic reticulum or

nucleus. Moreover, anchoring to the mitochondrial membrane deters NTR association with

endocytic vesicles or cellular compartments and prevents premature activation of the fluorogenic

probes by adventitious NTR.

Accordingly, we incorporated NTR into a fusion protein consisting of TOM20, mScarlet

fluorescent protein, 9 4 and the F124W NsfB NTR (Figure 4.2F). The NTR, mScarlet, and TOM20

components were linked with flexible GSGSG repeats connecting the termini of each protein. To

prevent potential loss of activity, the NTR N-terminus was verified to be well-separated from the

enzymic active site. Gibson assembly was used to insert the construct into a pNeo mammalian

expression vector under the control of a strong CMV promoter.

The NTR fusion protein was introduced into HeLa cells through transient transfection, with no

marked toxicity to cells within experimental scope and timeframe (Figure 4.2A-B). Expression of

the NTR fusion protein scaled proportionally with DNA concentration (Figure 4.2C, Figure 4.3),

providing an optimal expression window of 40-60 hours post-transfection (Figure 4.2D, Figure

4.4). Most importantly, the NTR fusion protein exhibited selective localization to mitochondria at

or above DNA concentrations of 2.5 pg/mL, a crucial feature for the success of the overall

nitroreductase-probe system (Figure 4.2E, Figure 4.5). Previous work with a mitochondria-

targeted tagging system suggested errant construct localization could be of concern, 9 5 so it was

gratifying to see that the NTR fusion protein localized selectively to the OMM. Optimal

transfection conditions for NTR fusion protein expression in HeLa cells consist of incubation with

2.5 ptg/mL DNA for 48 h.

155

NTR Fusion

Figure 4.3. Images depictingNTR fusion proteinexpression levels in live HeLacells. Cells were transfectedwith various concentrations ofDNA for 48 h, counterstainedwith Hoechst 33342, andvisualized by confocalmicroscopy.

156

-j

-E

0DL

6D

-J

E0)

E0")

V-

Hoechst Overlay

Hoechst Overlay

Figure 4.4. Images depictingNTR fusion proteinexpression levels over time inlive HeLa cells. Cells weretransfected with 2.5 pg/mLDNA, counterstained withHoechst 33342, andvisualized by confocalmicroscopy.

co)U-)

NN-

157

0

N

':j.N

-CCo

NTR Fusion

MitoTracker

Figure 4.5. Localization of NTR fusion protein to mitochondria as a function of DNA concentration.HeLa cells were transfected with NTR fusion protein for 48 h, counterstained with MitoTrackerTMGreen and Hoechst 33342, then imaged by confocal microscopy. Pearson's correlation coefficient(r) was used to quantify mitochondrial association (Figure 4.2E).

158

NTR Fusion Hoechst Overlav

0 0 NO2 NTR 0 0 NH2

4.1 4.7NO 2

O NTR OH

4.2 HN=Z_ 4.8

R RO N 0 0 N 0

NTR

NO 2 NH2

4.3 R = CH 2CH 2 CH, 4.9 R=CH2CH2CH,4.4 R = Bz 4.10 R = Bz4.5 R = CH 2 CECH 4.11 R = CH2 CECH

0 2N NO2

N o o o N HO 0 0

F F NTR F

/ CO2H- 0 2 N-NH

4.6 4.12

Scheme 4.1. Structure of nitroreductase-activated fluorogenic probes 4.1-4.6 and parentfluorophores 4.7-4.12 released upon reduction of probe nitro groups.

Design and Testing of Nitroreductase-Activated Probes. Having optimized the NTR fusion

protein to serve as the reporter enzyme, we next sought to prepare and evaluate NTR-activated

fluorogenic probes for use as the second component of our enzyme-probe system. Compared with

constitutively fluorescent labels, fluorogenic probes offer significant improvements in detection-

sensitivity by masking vesicular signal from proteins trapped in endocytic compartments that

would otherwise impede the visualization of cytosolic proteins. Masking endocytic protein signal

is particularly important when attempting to quantify therapeutic agents with low partition ratios

between the cytosol and vesicles, because in such cases vesicular signal intensity could easily

register 106-108 times brighter than diffuse cytosolic signal intensity if left unmasked.'96 '9'

Currently, most confocal microscopy and live-cell fluorescence techniques provide dynamic range

159

on the order of 1 o4, which is insufficient to quantify cytosolic signal reliably in the presence of

unmasked vesicular signal.

Accordingly, we prepared and tested a panel of NTR-activated fluorogenic probes 4.1-4.6

inspired by recent advances in probe research (see Chapter 1.2, Scheme 4.1, Figure 4.6). Key probe

properties include rapid kinetics of NTR activation, overall brightness after activation, fold

fluorescence increase after activation, and selectivity of probe response. We first assessed the

spectroscopic properties for each of the six probes (Table 4.1) and found promising in vitro

responses to NTR (Figure 4.6).

Probe brightness and fold change in brightness are determined in large part by the properties

of the parent fluorophore. Probes 4.1-4.6 are presented in order of increasing emission wavelength

and are based on three core fluorophore scaffolds-coumarin (4.1-4.2), naphthalimide (4.3-4.5),

and xanthene dyes (4.6) as summarized in Table 4.1 and Table 4.2. The coumarin scaffold has

moderate brightness but operates in shorter spectral wavelengths, which can lead to greater

phototoxicity and autofluorescence background. The 4-substituted naphthalimide scaffold is

readily synthesized but significantly dimmer and less photostable. Naphthalimides do benefit from

unusually large Stokes shifts, which is the difference between excitation and emission

wavelengths. Coincidentally, naphthalimide probes 4.3-4.5 are structurally related to cancer drug

amonafide, which is a strong DNA intercalator and selective topoisomerase II inhibitor approved

for treatment of secondary acute myeloid leukemia. 98 Although naphthalimide probes will not be

able to reach the nucleus once conjugated to biomolecules of interest, we evaluated the toxicity of

naphthalimides 4.3-4.5 and 4.9-4.10 to establish working concentrations for small-molecule in

cellulo assays (Figure 4.7).

160

BA 1.0-LLo

0.5 -N -

E0

0.0-

1.0-

0.5-

0.0-500 600

I I T 1 1 i300 400 500 600

I~--

300 400 500Wavelegnth (nm)

,600

D 1.0

0.5 -

0.0

F 1.0 -

0.5-

0.0.300 400 500

Wavelegnth (nm)

Figure 4.6. Graphs of normalized absorbance and emission profiles of 4.1-4.6 in 10 mM HEPES-NaOH buffer, pH 7.3. Absorbance (black) and fluorescence (colored) spectra were recordedbefore (dashed lines) and after (solid lines) incubation with 1 pg/mL NTR and 100 pM NADPH.Coumarin (blue), naphthalimide (green), and xanthene (red) fluorogenic probes (A) 4.1, (B) 4.2,(C) 4.3, (D) 4.4, (E) 4.5, (F) 4.6 were assayed for NTR response.

161

300 400

C1.0-

06

Cn

E0z

0.0*

1.0 -

0.5 -

E

F6

a)

~0U)

0.0600

' --- -r

' i men -'

300 400 500 600

300 400 500 600

Table 4.1. Spectroscopic properties of NTR-activated probes and parent fluorophores 4.1-4.12 measured in 10 mM HEPES-NaOH buffer, pH 7.3.

Amax (nm) Aex (nm) Aem (nm) Emax (M- 1cm-1) Eex (M- 1cm- 1) P Eex x (

4.1 298 350 - 1.4 x 104 2.9 x 102 <0.01 <1024.2 310 390 - 1.2 x 104 1.1 x 103 <0.01 <1024.3 354 450 - 1.1 x 104 1.5 x 102 <0.01 <1024.4 354 450 - 8.5 x 103 1.1 x 103 <0.01 <1024.5 354 450 - 1.0 x 104 8.0 x 102 <0.01 <1024.6 317 488 - 1.7 x 104 1.1 x 103 <0.02 <1024.7 341 350 437 1.7 x 104 1.7 x 104 0.56 8.9 x 1034.8 329 390 449 1.3 x 104 4.6 x 103 0.80 3.7 x 1034.9 429 450 533 2.0 x 104 1.2 x 104 0.03 3.9 x 1024.10 431 450 532 1.6 x 104 1.1 x 104 0.04 3.7 x 1024.11 430 450 534 2.2 x 104 1.3 x 104 0.03 4.2 x 1024.12 488 488 511 7.2 x 104 8.7 x 104 0.98 8.5 x 104

Table 4.2. Kinetic parameters of nitroreductase activation andmaximal fold increase in fluorescence calculated from Table 4.1 forrepresentative probes.Probe KM kcat/KM (M- 1s-1) Fold AFI4.1 1.0 pM 1.8 x 103 1,7004.2 2.3 pM 2.3 x 104 2,2004.541 45.6 pM 3.3 x 104 3504.6191 1.6 mM 8.1 x 104 4,200

--- 4.3-0- 4.4- 4.5

I

-7 -6log[Probe]

-8 -7 -6log[Probe]

-5 -4

Figure 4.7. Toxicity profile of fluorogenicnaphthalimide probes as determined by MTScell proliferation assay. HeLa cells weretreated with probes 4.3-4.5 or 4.9-4.10 for 48h in 5% CO2 at 37 *C. (A) Cell viability curvesfor cells treated with 4-nitronaphthalimideprobes 4.3-4.5. (B) Cell viability curves forcells treated with 4-aminonaphthalimideprobes 4.9-4.10.

-5 -4

162

CU

N

0z

120-

100 -

80 -

60 -

40 -

20 -

0-

-20 - -8

B 120-

100-

- 80-CO 60-

40 -N

co 20-

0 0-z

-20 -

I-

I

T --

74-4.9--m- 4.10

A 300 -

200 -

U-

100 -

0- ---- 10

0 1 0Time (s)

B 2.5-

2.0-

1.5-

> 10-

0.5-

0.0'-150 C 5 10

Substrate (pM)

c 150-

100-

U-

50-

0

D 1.0

0.8 -

( 0.6 -C

0.4-

0.2 -

A % -0 50 100 150 0 5 10 15 20

Time (s) Substrate (pM)

Figure 4.8. Kinetic traces and Michaelis-Menten plots for the unmasking of fluorogenic probes4.1 and 4.2. Assays were performed in 10 mM HEPES-NaOH buffer, pH 7.3, containingnitroreductase (1 pg/mL) and NADPH (100 pM). Substrate was added at t = -60 s and enzymewas added at t = 0. (A, B) Probe 4.1; kct/KM = 2.3 x 104 M- 1s-1 and KM = 2.3 pM. (C, D) Probe4.2; kwt/KM = 1.8 x 103 M- 1s-1 and KM = 1.0 pM.

Probe 4.6 is built on the xanthene dye fluorescein, which benefits from high brightness and

photostability but also requires two masking groups to attenuate fluorescence completely.

Conversely, xanthene dyes also require two enzymatic events to restore fluorescence, which in

some cases can result in significantly slower unmasking kinetics. One key benefit to the xanthene

scaffold is the complete attenuation of both absorbance and fluorescence upon installation of two

masking groups, due to disruption of the conjugated xanthene core. As a result, probe 4.6 enjoys

the largest theoretical fold fluorescence increase between masked and unmasked forms compared

to other dye scaffolds (Table 4.2).

163

15 20--Ib

"I .....................

Whereas overall brightness and fold response are largely determined by the parent fluorophore,

the masking strategy primarily affects the kinetics of unmasking and fold-change in fluorescence

after activation. Two distinct strategies are used--direct installation of nitro groups onto parent

fluorophores and insertion of auto-immolative linkers between nitro groups and the fluorophore.

In probes 4.1 and 4.3-4.5, the electron deficient nitro groups directly quench fluorophores until

NTR activity restores fluorescence by reducing the nitro groups to electron rich amines (Scheme

4.1). Probes 4.2 and 4.6 incorporate quinone methide or substituted imidazole auto-immolative

linkers, which generate fluorescence upon reduction of the nitro group (Scheme 4.1). Because the

rate of linker immolation greatly outpaces the rate of NTR activation, the linkers in 4.2 and 4.6

were not expected to affect overall rates of activation significantly.

In vitro Michaelis-Menten kinetic parameters for representative probes 4.1, 4.2, 4.5, and 4.6

were compared to evaluate rates of NTR activation (Table 4.1). Activation rates of coumarin

probes 4.1 and 4.2 (Figure 4.8) were slower than those of naphthalimide or xanthene probes,

although coumarin probes exhibited the highest affinities for NTR enzyme. Interestingly, even

though activation of xanthene probe 4.6 requires removal of two masking groups, activation of 4.6

occurs rapidly. This observation can be explained by the fact that the nitroimidazole masking

groups in 4.6 have been optimized for reduction by E coli F124W NTR,' 9' an improvement that

outweighs the kinetic penalty of multiple masking groups.

In cellular imaging environments, the rate and specificity of probe activation are of equal

importance. Nonspecific activation of internalized probe would be disastrous because of signal

oversaturation from prematurely unmasked vesicular pools of protein (Figure 4.1). To gauge the

in cellulo performance of the probes, the fold increase in fluorescence and specificity of probes

4.1-4.5 and 5.8, an analog of probe 4.6 with an additional carbamate immolative linker between

164

the nitroimidazole masking group and parent fluorophore, were measured (Figure 4.9). Probes 4.2

and 5.8 stand out in these two parameters, with 79- and 134-fold increases in fluorescence after

activation. The relatively lower fluorescence change observed in probe 4.1 relative to probe 4.2 is

likely caused by its slower rates of NTR activation. Probes 4.3-4.5 have the lowest theoretical fold

change in fluorescence, but more importantly, appear to be partially unmasked by endogenous

enzymes in the absence of the NTR fusion protein. In consideration of the four key parameters-

brightness, fold change in florescence, activation rate, and specificity-probes 4.2 and 4.6 promise

the best combination of performance and selectivity. Direct application of probes 4.2 and 4.6 to

biomolecule payloads of interest can be accomplished by appending a bioconjugation handle at

the coumarin 4 position (4.2) or fluorescein 6 position (4.6) and quantifying cytosolic

internalization of the biomolecule-probe conjugate.

150_ Figure 4.9. Graph of fluorescence foldM NTR increase upon exposure of probes 4.1-4.5 to

100 -W nitroreductase. Probes 4.1-4.5 (2 pM) wereO100)- incubated with HeLa cells transfected with 2.5

pg/mL of NTR plasmid or carrier DNA plasmidfor 48 h. Fold increase in fluorescence for

< 50- probe 5.8 (2 pM), a close analog of probe 4.63> was measured in cell lysate because of the

_ __ transport properties of unmasked 4.6 and 5.8.4 1 4 4 4 M All measurements were normalized to the4.5 5.8 fluorescence signal of each probe in OptiMEM

without HeLa cells.

165

4.3 Conclusions

In this chapter, I described the design and optimization of an NTR fusion protein and panel of

fluorogenic probes for quantification of cytosolic delivery, a major barrier to biologic- and

macromolecule-based therapeutic agents with intracellular targets. The NTR fusion protein

selectively anchors to the cytosolic face of the outer mitochondrial membrane while preserving

nitroreductase activity. Probes 4.2 and 4.6 were found to display excellent brightness, NTR

response, and selectivity for NTR over endogenous enzymes. Accordingly, this optimized NTR-

probe system is well-poised to serve as a tool for studying and improving cytosolic biomolecule

delivery. Application of the NTR-probe system can be tested and further optimized by using

conjugable versions of probes 4.2 and 4.6 to label variants of human RNase 1, a protein with potent

anticancer activity.' 9 9


We are grateful to V.T. Ressler and T.T. Hoang for NTR fusion protein design and transfection

optimization, and L.D. Lavis and T.D. Gruber for helpful discussion about the F124W NTR

system. This work was supported by grant R01 GM044783 to R.T.R. (NIH). W.C. was supported

by an NSF Graduate Research Fellowship.

4.5 Experimental

General Experimental. Compounds 4.1, 4.7-4.8, and 4.11 were from Sigma-Aldrich (St. Louis,

MO). Compounds 4.6 and 4.12 were prepared as described previously.' 9 ' All other chemicals

were from Sigma-Aldrich, Thermo Fisher Scientific (Waltham, MA), or Invitrogen (Carlsbad,

166

CA) unless indicated otherwise, and were used without further purification. Aqueous solutions

were made with water that was produced with an Atrium Pro water purification system from

Sartorius (Bohemia, NY) and had resistivity 18 MQ-cm-1.

Chemical reactions were monitored by thin-layer chromatography (TLC) with EMD 250 Im

silica gel 60-F254 plates visualized by UV illumination or KMnO4 stain. Flash chromatography

was performed on a Biotage Isolera automated purification system using pre-packed SNAP KP

silica gel columns.

The phrase "concentrated under reduced pressure" refers to the removal of solvents and other

volatile materials using a rotary evaporator at water aspirator pressure (<20 torr) while maintaining

a water-bath temperature of 40 'C. Residual solvent was removed from samples at high vacuum

(<0.1 torr). The term "high vacuum" refers to vacuum achieved by mechanical belt-drive oil pump.

Phosphate-buffered saline (PBS) contained Na2HPO 4 (10 mM), KH2PO 4 (1.8 mM), NaCl (137

mM), and KCl (2.7 mM) at pH 7.3. All procedures were performed at room temperature (~22 'C)

and atmospheric pressure (1 atm) unless noted otherwise.

'H and 13 C NMR spectra were acquired on Bruker Spectrometers operating at 400 MHz for 'H

and 100 MHz for 13C. Mass spectrometry was performed on a Thermo-Fisher Q Exactive Hybrid

Quadrupole-Orbitrap Mass Spectrometer. In cellulo probe unmasking assays were performed on a

Tecan Infinite M1000 plate reader. All other in vitro fluorescence measurements were acquired

with a PTI QuantaMaster spectrofluorometer. Absorbance measurements were performed using

an Agilent Cary 60 UV-vis spectrophotometer. Confocal microscope images were acquired with

an RPI spinning disk inverted confocal microscope at the W.M. Keck Microscopy Facility at the

Whitehead Institute (Cambridge, MA).

167

General Optical Spectroscopy. All fluorogenic probes and fluorescent small molecules were

dissolved in spectroscopic grade DMSO and stored as frozen stock solutions. For all applications,

DMSO stock solutions were diluted such that the DMSO concentration did not exceed 1% v/v.

UV-Visible and Fluorescence Spectroscopy. Spectroscopy was performed using either 1-cm

path length, 4-mL quartz cuvettes or 1-cm path length, 1-mL quartz microcuvettes, with all

measurements taken at room temperature. Fluorescence spectroscopy samples were stirred with a

magnetic stir bar and acquired with excitation and emission slit widths set to 4 nm.

Quantum Yield Determination. Quantum yields for all compounds were measured in 10 mM

HEPES-NaOH buffer, pH 7.3 and compared to appropriate standards (quinine sulfate in 0.1 M

H2SO 4, fluorescein in 0.1 M NaOH).

Cloning of TOM20-mScarlet-NTR Fusion Protein. A DNA fragment encoding E coli F 124W

NTR with a linker peptide, GSGSGS, on the N-terminus was synthesized by IDT (Skokie, IL).

The gene was inserted into pNeo3 vector using Gibson Assembly (Gibson et al., 2009). The vector

contained a previously cloned gene to generate a TOM20-mScarlet fusion protein with a GSGSGS

linker peptide connecting the C-terminus of the TOM20 to the N-terminus of the mScarlet. The

insertion of the F124W NTR gene block yielded a gene encoding for a TOM20-mScarlet-NTR

fusion protein with a GSGSGS linker peptide between the C-terminus of the mScarlet and the N-

terminus of the F124W NTR. The sequence of the TOM20-mScarlet-NTR construct was

confirmed by DNA sequencing at Quintara Biosciences (San Francisco, CA).

168

General Cell Culture. HeLa (ATCC CCL-2) cells were obtained from American Type Culture

Collection (Manassas, VA) and maintained according to recommended procedures. Gibco-brand

Dulbecco's Modified Eagle's Medium (DMEM), fetal bovine serum (FBS), trypsin (0.25% w/v),

OptiMEM, and Dulbecco's PBS (DPBS) were from Thermo Fisher Scientific (Waltham, MA).


and streptomycin (100 ptg/mL). HeLa cell extract was prepared by treating 10' cells with CellLytic

M reagent from Sigma-Aldrich. Total protein content in cell extract was found to be 1.28 mg/mL

by Bradford assay.

General Transfection of Fusion Protein into HeLa Cells. HeLa cells were seeded in complete

medium in 8 well slides or 24 well plates at a density of 50 cells/tL. After 24 h, cells were

transfected with TOM20-mScarlet-NTR plasmid using Lipofectamine TM 3000 following the

manufacturer's protocol. Transfection efficiency was measured after 48 h by confocal microscopy.

Cell Viability Assays. HeLa cells were seeded in complete medium in 24-well plates at a density

of 50 cells/ptL. After 24 h, cells were transfected with varying amounts of TOM20-mScarlet-NTR

plasmid (0.0-10.0 pLg/mL) using Lipofectamine TM 3000. Cells were trypsinized 48 h after

transfection, then diluted in complete medium before being measured with a Countess FL

Automated Cell Counter (Thermo Fisher Scientific). Total cell count was recorded in biological

triplicate for each plasmid concentration.

Cell Viability Assay Based Post-transfection. HeLa cells were seeded in complete medium in

24 well plates at a density of 50 cells/ptL. After 24 h, cells were transfected with 2.5 ptg/mL

169

TOM20-mScarlet-NTR plasmid using Lipofectamine TM 3000. At 12 h intervals, cells were

trypsinized, then diluted in complete medium before being counted with a Countess FL Automated

Cell Counter (Thermo Fisher Scientific). Total cell count was recorded in biological triplicate for

each time point.

MTS Cell Proliferation Assay. HeLa cells were seeded in a 96-well plate at a density of 100

cells/ptL. After 24 h, cells were treated with varying concentrations of naphthalimide probes for 48

h. After washing with 200 ptL PBS, each well was treated with CellTiter 96@ AQueous tetrazolium

reagent from Promega according to the manufacturer's protocols. After incubation for 30 min at

37 0C, absorbance of each well was measured at 490 nm using a plate reader. Data were graphed

with GraphPad Prism software and fitted to sigmoidal dose-response curves. Measured IC50 values

were as follows: 2.1 piM (4.3), 4.8 pM (4.4), 1.4 jtM (4.5), >10 tM (4.9), >10 pM (4.10).

General Confocal Live-cell Imaging. For all imaging experiments unless noted otherwise, 8-well

microscopy slides from Ibidi (Madison, WI) were seeded with HeLa cells (50 cells/gL) and

incubated for 12 h to allow adhesion. Cells were then transfected with DNA for the desired time

interval, after which they were washed with 100 pL DPBS three times and counterstained with

Hoechst 33342 and MitoTrackerTM Green FM as needed. All imaging experiments were performed

in live cells without fixation. ImageJ200 was used for all image processing, signal quantification,

and colocalization measurements. 2 4

NTR Activation Kinetics Assay. NTR from E coli (24 kDa) was suspended in PBS and diluted

to appropriate concentrations before use in protein LoBind tubes from Eppendorf. Initial rate

170

measurements for probes 4.1 and 4.2 were acquired and the resulting data were fittedto the

Michaelis-Menten equation with GraphPad Prism software to obtain kinetic parameters for NTR-

catalyzed unmasking of probes.

NTR Activity Assay in Live HeLa Cells. HeLa cells were seeded in 96-well plates at a density

of 50 cells/pL and transfected with NTR or transfected with carrier DNA (WT). Probes 4.1-4.5 (2

gM) were added to wells and incubated for 1 h at 37 'C. Fluorescence was measured with a plate

reader (Xex/,{em 350/450 nm, 390/450 nm, 425/530 nm, and 488/520 nm). Fluorescence signal from

wells containing NTR and WT cells were normalized to fluorescence signal in medium without

cells (Figure 4.9). Measurements were carried out in triplicate. Because this assay depends on the

free diffusion of activated probe out of the cytosol into well medium, probe 4.6 was not compatible

with this quantification method. Instead, probe 4.6 (1 gM) and 100 pM NADPH were dissolved in

OptiMEM with and without WT HeLa cell lysate (total protein content 100 pg/mL). NTR from E.

coli (1 pg/mL) was added, and the resulting mixtures were stirred for 1 h at room temperature,

after which fluorescence measurements at iex/Xem 488/520 nm were acquired with a fluorimeter.

Measurements were normalized to probe 4.6 (1 pM) fluorescence intensity in 10 mM HEPES-

NaOH buffer, pH 7.3.

Synthesis of Nitrobenzyl Coumarin (4.2). To a solution of 7-hydroxycoumarin (100 mg, 0.62

mmol) in anhydrous acetonitrile (2 mL) was added a solution of 4-nitrobenzyl bromide (133 mg,

0.62 mmol), K2CO 3 (170 mg, 1.23 mmol), and activated powdered 4-A sieves (40 mg) in 6 mL

anhydrous acetonitrile. The resulting solution was stirred in the dark at room temperature for 24

h, then concentrated under reduced pressure. The residue was suspended in water (40 mL),

171

extracted with ethyl acetate (3 x 50 mL), and the organic portions were pooled and dried with

MgSO4(s). Purification by column chromatography on silica gel (0-20% v/v EtOAc in hexanes)

yielded the title compound as a white solid (115 mg, 62.5% yield). 'H NMR (400 MHz, CDCl 3,

6): 8.28 (d, J= 8.7 Hz, 2H), 7.65 (dd, J= 11.0, 9.2 Hz, 3H), 7.43 (d, J= 8.6 Hz, 1H), 6.94 (dd, J

= 8.6, 2.4 Hz, 1H), 6.88 (d, J= 2.3 Hz, 1H), 6.29 (d, J= 9.5 Hz, 1H), 5.25 (s, 2H). 13 C NMR (100

MHz, CDCl 3, 5): 161.07, 160.91, 155.80, 143.22, 143.09, 129.05, 127.73, 124.01, 113.77,113.02,

101.92, 69.06. HRMS (ESI-QIT) m/z: [M + H]' Calcd for C1 6HNNO5 298.0715; found 298.0716.

Synthesis of Naphthalimides 4.3-4.5 and 4.9-4.10. 4-Nitro-1,8-naphthalic anhydride (100 mg,

0.41 mmol, equiv, 4.3-4.5) or 4-amino-1,8-naphthalic anhydride (87 mg, 0.41 mmol, 1 equiv 4.9-

4.10) was suspended in ethanol (2 mL), and the appropriate amine (0.41 mmol, 1 equiv) was added

to the suspension. The reaction vessel was heated to 70 'C for 4 h, then allowed to cool to room

temperature. The reaction mixture was concentrated under reduced pressure to yield title

compounds as light brown or orange solids. For products with residual amine or anhydride starting

materials, purification by column chromatography on silica gel (1-10% v/v methanol in

dichloromethane) yielded pure title compounds.

Propyl 4-nitronaphthalimide (4.3). Light tan powder (96 mg, 82.4% yield). 'H NMR (400 MHz,

CDCl 3, 5): 8.84 (dd, J= 8.7, 0.7 Hz, 1H), 8.75 (dd, J= 7.3, 0.7 Hz, 1H), 8.70 (d, J= 8.0 Hz, 1H),

8.41 (d, J= 8.0 Hz, 1H), 8.00 (dd, J= 8.6, 7.4 Hz, 1H), 4.16 (dd, J= 8.4, 6.8 Hz, 2H), 1.84-1.71

(m, 2H), 1.03 (t, J= 7.4 Hz, 3H). 13 C NMR (100 MHz, CDCl 3, 6): 163.33, 162.51, 149.55, 132.44,

132.44, 129.94, 129.94, 129.79, 129.79, 129.28, 129.28, 129.10, 127.05, 127.05, 123.91, 123.91,

123.68, 123.68, 123.06, 123.06, 42.34, 42.34, 21.32, 21.32, 11.49, 11.49. HRMS (ESI-QIT) m/z:

[M + H]' Calcd for Ci 5H 12N204 285.0875; found 285.0872.

172

Benzyl 4-nitronaphthalimide (4.4). Light brown powder (120 mg, 88.2% yield). 'H NMR (400

MHz, CDC 3, (): 8.85 (dd, J= 8.7, 0.9 Hz, lH), 8.76 (dd, J= 7.3, 0.8 Hz, 1H), 8.71 (d, J= 8.0 Hz,

1H), 8.41 (d, J= 8.0 Hz, 1H), 7.99 (dd, J= 8.7, 7.4 Hz, lH), 7.55 (d, J= 7.2 Hz, 2H), 7.32 (dd, J

= 15.4, 7.9 Hz, 3H), 5.39 (s, 2H). 13C NMR (100 MHz, CDCl 3, 6): 163.33, 162.51, 149.63, 136.55,

132.66, 130.02, 129.94, 129.45, 129.06, 128.56, 127.80, 127.07, 126.93, 123.89, 123.67, 122.96,

43.92. HRMS (ESI-QIT) m/z: [M + H]+ Caled for C1 9H12N2 04 333.0875; found 333.0876.

Propargyl 4-nitronaphthalimide (4.5). Light tan powder (86 mg, 75.0% yield). 'H NMR (400

MHz, (CD 3 ) 2SO, 6): 8.72 (d, J= 8.7 Hz, 1H), 8.69-8.61 (m, 2H), 8.56 (d, J= 8.0 Hz, 1H), 8.11

(dd, J= 8.6, 7.4 Hz, 1H), 4.80 (d, J= 2.4 Hz, 2H), 3.22 (t, J= 2.4 Hz, 1H). 13C NMR (100 MHz,

(CD 3) 2SO, 6): 162.25, 161.49, 149.43, 132.06, 130.18, 130.03, 129.23, 128.35, 126.22, 124.30,

122.82, 122.39, 78.88, 73.45, 29.43. HRMS (ESI-QIT) m/z: [M + H]* Calcd for Ci5 H8 N2 04

281.0484; found 281.0492.

Propyl 4-aminonaphthalimide (4.9). Orange powder (63 mg, 60.5% yield). 'H NMR (400 MHz,

(CD 3) 2 SO, 6): 8.61 (d, J= 8.4 Hz, 1H), 8.42 (d, J= 7.2 Hz, 1H), 8.19 (d, J= 8.4 Hz, 1H), 7.65 (t,

J= 7.8 Hz, 1H), 7.45 (s, 2H), 6.84 (d, J= 8.4 Hz, 1H), 4.02-3.91 (m, 2H), 1.70-1.56 (m, 2H), 0.90

(t, J= 7.4 Hz, 3H). 13C NMR (100 MHz, (CD3) 2 SO, 6): 163.80, 162.94, 152.70, 133.96, 131.00,

129.69, 129.29, 123.98, 121.79, 119.37, 108.16, 107.54, 40.73, 21.00, 11.42. HRMS (ESI-QIT)

m/z: [M + H]+ Calcd for C15 H1 4N202 255.1134; found 255.1130.

Benzyl 4-aminonaphthalimide (4.10). Dark orange powder (113 mg, 91.0% yield). 'H NMR

(400 MHz, CDC 3, 6): 8.63 (d, J= 8.3 Hz, 3H), 8.44 (d, J= 6.8 Hz, 3H), 8.21 (d, J= 8.4 Hz, 3H),

7.66 (t, J= 7.8 Hz, 3H), 7.50 (s, 2H), 7.34-7.25 (m, 12H), 7.21 (t, J= 6.9 Hz, 3H), 6.85 (d, J= 8.4

Hz, 3H), 5.21 (s, 6H). 13C NMR (100 MHz, CDC 3, 6): 164.31, 163.37, 153.35, 153.27, 138.45,

173

134.71, 131.75, 130.28, 130.03, 128.76, 127.94, 127.33, 124.53, 122.12, 119.81, 108.67, 108.61,

107.74, 42.90. HRMS (ESI-QIT) m/z: [M + H]' Calcd for C9H 4N202 303.1134; found 303.1161.

174

NMR Spectra

'H NMR (CDC1 3) and 13C NMR (CDC1 3 ) Spectra of 4.2.

CNM # L&f r -00%D W Q Cq r

I if [

Y4 r ?N

10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5T --I -' ' I .I I I I I ...I .I I .

6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5PPM

v I~

0

NO 2

O0,- O t /

2 2 I1' 180 170 1 0 I I I 1 14210 200 190 180 170 160 150 140 130 120 110 100

PPM

9 I ' I 0 " I 0 I 3 I 2 10 I 1 I90 80 70 60 50 40 30 20 10 0 -10

175

000

I6 NO2

0 0 /,a

Lnr1iLn

C. ON

'H NMR (CDCL 3) and 13C NMR (CDC1 3) Spectra of 4.3.

m w ui wr- <r- z

LT I

0 N O

0 2

I

10.0 9.5 9.0 8.5 8.0- I -.- I .I " ..

7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0PPM

1.5 1.0 0.5 0.0 -0.5

a; ; r9W M-

2 - I I I - I 1 5200 190 180 170 160 150I - I . I I '' I " I " I . I . I . I . I . I 1

140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10PPM

176

D v 6n m m

I

mo -

0 N 0

NO2

OD f %D Ln

\1 - -ji- -

C4

'H NMR (CDC1 3) and 13C NMR (CDC1 3) Spectra of 4.4.

06 6 6 ww ww 6 0 ~ N?8 z LnI OIrn -

lif f

9.0 8.6

("U 'O O(1 L

ON% r4 a N aNm ?

ON 0

N N

N02

8.2 7.8 7.6 7.4 7.2 7.0 6.8 6.4 6.0 5.6PPM

~*a' 8Nm0

I-,--

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60PPM

50 40 30 20 10 0 -10

177

'V

O N O

NO2

9.8 9.4 5.2 4.8I | | |

I

rN

M

T r""F I

IV 1 -7

'H NMR ((CD 3 ) 2 SO) and "C NMR ((CD 3 ) 2SO) Spectra of 4.5.

0 N O

NO 2

i1i I

.b+VI V '~1~'

r

I

-- l -IV

C-410CD

10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0PPM

3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5

%o w- mN m~ (nqN m

m mm N (NN

0 N O

NO2

L I

210 200 190 180 170 160 150 140 130 120 110 100PPM

908 70 6 50 40 3 20 10 0 -10

178

gj

L..L. .'

a a rn

m (n m

Ln mN V:r'l -

. .1. 1 .. I L L I..

'H NMR ((CD3)2SO) and 13C NMR ((CD 3 ) 2SO) Spectra of 4.9.QwN Lm

wO wO wO a66c 'zrZ ,OO, 0%

0 N 0

NH2

II"ALN I W

I If (

rI4 - rq-.

10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5PPM

4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5

(En00qin

0 NO

NH2

S~WWMJ~lJWN UL210 200 190 180 170 160 150 140 130 120 110 100

PPM90 80 70 60 50 40 30 20 10 0 -10

179

0% r"Q N N

, I . . . . . . . . . . . . . . . . . . . . . . . . . .

0 II IN I II I i U i 15 Im E -I I I i i I . m g Imi

I ~ ~ ~ ~ ~~~~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Ln MOD aD

CC

Cs Go N %0 V." 0 M? - Ln

CN CN M 0 g ".:C-4 C-4 N C4

CC?

4

1H NMR ((CD 3 ) 2 SO) and 13C NMR ((CD 3 ) 2SO) Spectra of 4.10.

I ffI I -F

mitt__n 0C! + C?~r

qCD~ V 0-

8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5PPM

4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 -0.5

Ode NI .n Wn W mDn~ PI N qN 6'1 G IN Z IN-~00

CO N 0

NH2

ii

200 190 180 170 160 150 140 130 120 110 100PPM

90 80 70 60 50 40 30 20 10 0

180

o N O

NH2

10.0 9.5 9.0

CHAPTER 5

Future Directions

181

5.1 Extending Electronic Stabilization to Other Fluorophore Scaffolds.

The stabilizing effects of ortho-halogenation in diacyl fluorescein fluorogenic probes

described in Chapter 2 are not unique to the fluorescein scaffold. Indeed, this stabilizing effect can

be extended to other fluorophore scaffolds and masking groups. Three fluorophores scaffolds are

of particular interest, given their frequent application as acyl-masked fluorogenic probes-

coumarin, resorufin, and Tokyo green (Figure 5. lA).7 0' 100, 103 Both electronic and steric

stabilization can be applied to acylated versions of these fluorophores. Preliminary results with

acetyl chlorocoumarins suggest that similar stability benefits to those in dichlorofluoresceins are

achievable (Figure 5. 1B). Additionally, chlorination of both scaffolds lowers fluorophore pKa, a

desirable effect that renders the unmasked parent fluorophore less sensitive to fluctuations in pH

(Figure 5.1 C). The addition of steric stabilization by acyl group screening would further augment

chlorocoumarin probe stability.

The ortho-halogenation motif can also be used to stabilize acyl groups slightly removed

from the fluorophore scaffold. We found that the acetoxymethoxy (AM) ether masking group,' 03

which interjects a methoxy linker between the fluorophore and acyl group, is significantly

stabilized by ortho-chlorination in dichlorofluorescein AM, but less so in chlorocoumarin AM

(Figure 5.1D). Although the acyl groups in AM ethers are separated by a linker, calculated

optimized structures suggest there is sufficient flexibility to adopt a configuration with significant

ortho-halogen stabilization. Additionally, the acyl group in AM ethers provides opportunity for

steric stabilization. 69 Further investigations into ortho-halogen stabilization of the AM ether and

other masking groups could greatly expand the scope of the improvements to probe stability

described in Chapter 2.

182

- U

HO 0 0

C 2H

X =H

x =CI

10pH

--HO 0 0

X=HX=CI

8pH

10

B1.0-

(DN -

0

-

0.0 -

D --1 1.0 -

'D

N

2

=0.5-

0

(ULL -

0.0-

1.0-

'a

a:0.5-C -0

0.0-

[.

10Time (h)

N) 0 0y

0 10Time (h)

Figure 5.1. Extension of electronic and steric acyl masking group stabilization to other fluorophorescaffolds and masking groups. (A) General structures of ortho-halogenated coumarin, Tokyogreen, and resorufin masked with isobutyryl groups. (B) Hydrolysis curves of 5 pM acetylcoumarins in 10 mM HEPES-NaOH buffer, pH 7.3 depicting the stabilizing effects of ortho-chlorination. (C) Normalized fluorescence of chlorinated and unchlorinated coumarin as a functionof pH. (D) Hydrolysis curves of 5 pM fluorescein and coumarin probes masked withacetoxymethoxy (AM) ethers in DMEM containing 10% (v/v) FBS. Fluorescein AM probes benefitmore significantly from electronic stabilization by ortho-chlorines than do coumarin AM probes.

183

AX =H

-U- XCIcc>

S5.1o1 0 0

0 C1 C1

5.2

C1

5.3

-0- X = HU X=CI

0

10

Time (h)

1.0-

0

0

LI. 0.5-

(D1.0-

0

CO

LL 0~0

4 6

20

X = H-U- X = CI

20

- -..------

,

, . .

. .

10 0 0X)O

.............

6

,

5.2 Biomolecule Imaging Using Electronically-Stabilized Probes.

__0 10 0 1_0 . C1 0

1 0

0

3.2

a'

OC1 C1 0o ' 0 '// 0

o - HN \ N ON -N N 0N

5.4 HO

0a 0 C

ac 'C- ~ 1 0000

0 HN

5.5

00 -'s ^

0-

Scheme 5.1. Application of bioconjugable electronically-stabilized acyl probes topolysaccharides, nucleic acids, and lipids. Structures of a bioconjugable 2,7-dichlorofluoresceindiisobutyrate probe covalently attached to a polysaccharide (3.2), ribonucleic acid (5.4), and lipid(5.5).

Valuable spatiotemporal information can be gathered by appending a conjugable

fluorogenic probe to a biomolecule of interest and tracking its transport via live-cell imaging. For

optimal performance, design of the probe-biomolecule conjugate should be matched with

experimental conditions, probe properties, and biomolecule characteristics. Some factors to

consider include experiment timescale, biological environment, and cell or organism type. In the

case of the stabilized acyl probes described in Chapter 2, these probes perform best when

conjugated to glycans, lipids, or nucleic acids, but not proteins with free amino groups. Although

stabilized acyl masking groups resist adventitious intermolecular hydrolysis by nucleophiles

present in FBS or cell media, the masking groups are not impervious to hydrolysis by amines

prepositioned for intramolecular attack. Accordingly, the judicious matching of probe properties

184

and the biomolecule of interest minimizes probe background. In addition to conjugate 3.2 used in

the dextran-uptake studies in Chapter 3, electronically stabilized acyl probes can be incorporated

into lipids for studying endocytosis (probe 5.5) or nucleic acids for transport studies (probe 5.4).

The modularity of the pendant 6-carboxy group of the dye accommodates most common

bioconjugation chemistries. In these applications, acyl dichlorofluorescein probes are significantly

easier to prepare than are other probes and offer improved spectroscopic properties.

5.3 Promoting Intracellular Delivery of Biomolecular Payloads via Dextran Conjugation.

In Chapter 3, we described the rapid and diffuse uptake of monofunctionalized dextrans

into the cytosol of three different cell lines. The most immediate application of these dextrans is

to harness their unusual transport properties to deliver proteins and nucleic acids, given the

increasing importance of biologic drugs2 0 1 and recent advances in genome editing technologies

such as CRISPR/Cas9.1 8 1 Dextrans are particularly well suited for this task given their non-

immunogenic nature, lack of inherent toxicity, and current widespread use in clinical

applications-traits that are likely to accelerate regulatory approval of delivery platforms based on

this strategy.

Initially, one could prepare several different protein-dextran conjugates with the same

linker chemistries used in the dextran-probe conjugates in Chapter 3 (Scheme 5.1, Figure 5.2).

Site-selective protein modification can be accomplished by a thermodynamically controlled diazo

group transfer followed by dextran conjugation (Kilgore and Raines, unpublished results). The

three proteins selected for conjugation-GFP, cytochrome c, and RNase 1-have varying

sequences and structures which would test the generalizability of the dextran strategy. GFP

provides an easily-monitored control due to its inherent fluorescence. Cytochrome c is a

mitochondrial protein involved in the electron transport chain and plays important roles in

185

apoptosis.20 2 Ribonucleases are of particular interest to the Raines group and others given their

potential application as a potent biologic therapeutic to treat a wide spectrum of cancers. 9 9 203

Depending on the modification site and orientation, conjugated dextran could attenuate enzymatic

activity but could also assist evasion from endogenous ribonuclease inhibitor (RI). 204-206 These two

competing effects, along with enhancement of conjugate uptake due to the dextran moiety, could

significantly improve the toxicity profile of a ribonuclease.

In cases where cytosolic release of the free protein is required, monofunctionalized

dextrans can be bioreversibly conjugated to proteins via disulfide linkers. Upon entry into a cell,

reduction of the disulfide bond would release the free protein. This strategy is most straightforward

with proteins that possess few solvent-accessible free cysteine residues or have a judiciously

installed cysteine, as is the case with P19C RNase 1, which contains only a single free cysteine.

Successful delivery of proteins by conjugation to dextrans could be followed by extension of the

strategy to nucleic acids.

A B C

Figure 5.2. Promoting cytosolic delivery of proteins via conjugation to monofunctionalizeddextrans. Proteins of interest include (A) GFP (PDB 1GFL), (B) Cytochrome c (PDB 3NBS), and(C) RNase 1 (PDB 1Z7X).

186

5.4 Probing Mechanisms of Dextran Uptake and Endosomal Escape

Exploiting the unusual transport properties of monofunctionalized dextrans to their full

potential requires a better understanding of the mechanism by which they enter the cytosol. In

Chapter 3, we found that dextran conjugates rely on active, receptor-mediated processes for their

cellular uptake. Our current model of dextran uptake consists of two distinct transport events that

appear to occur in rapid succession--endocytic uptake via receptor-mediated endocytosis and

macropinocytosis, followed by endosomal escape.

Previous studies of dextran uptake using a panel of inhibitors suggested that

polyfunctionalized dextrans are dependent on clathrin-mediated endocytosis and a basal level of

nonspecific uptake by macropinocytosis.15 0 In addition to repeating inhibitor studies for

monofunctionalized dextrans, high-throughput RNA interference (RNAi) studies can provide

detailed profiles of specific receptors important for monofunctionalized dextran uptake.

Alternatively, dextran-conjugated photoaffinity labels can be used to covalently crosslink dextrans

to close interaction partners, followed by identification via by tandem mass spectrometry. 207

Deciphering dextran endosomal escape, the second step in the cytosolic uptake process,

presents a greater challenge because our current understanding of endosomal escape is based

largely on peptide, protein, and small-molecule strategies.208 Dextrans are neutral and lack the

functional group diversity of their protein and nucleic acid counterparts, such that many previous

mechanistic explanations might not be directly applicable. 208 Despite their structural differences,

it is conceivable that dextrans perturb endosomal membrane stability via two proposed

mechanisms reminiscent of other delivery mechanisms-delocalized destabilization of the

endosomal membrane to increase porosity or penetration of the endosomal membrane by the

dextran, with subsequent extraction of conjugated cargo from the endosome. Liposomal leakage

187

assays can provide insight into the membrane-destabilizing effects of these conjugates.

Additionally, various combinations of polarity-sensitive fluorophores or fluorogenic probes can

be used to label the end of the dextran conjugate differentially and thereby investigate embedding

or penetration of vesicular membranes. A more comprehensive mechanistic understanding would

enable further optimization of the dextran platform for cytosolic delivery.

5.5. Quantifying Cytosolic Delivery with a Nitroreductase-Probe Reporter System

Applying the Nitroreductase-Probe System to Biomolecules. The nitroreductase-probe system

described in Chapter 4 is well poised for quantifying cytosolic uptake of biomolecules.

Bioconjugable 4-nitronaphthalimide probe 4.5 or alkynyl coumarin 5.6 are readily prepared and

can be appended to any protein of interest, with RNase 1 as the initial test protein (Figure 5.2C).

Probe 4.5 or 5.6 can be conjugated to RNase 1 via site-specific diazo transfer followed by

cycloaddition, and constitutively fluorescent RNase 1 conjugate can be prepared from 4-

aminonaphthalimide probe 4.11 or alkylnyl coumarin 5.7 as a complimentary control to measure

total endocytic uptake. After confirmation of expected conjugate localization patterns in HeLa

cells by confocal microscopy, cytosolic internalization can be quantified by flow cytometry to

obtain the fraction of cytosolic RNase 1 and timeframe of uptake.

188

O N 0 0 N 0 O2N

< O 0 HO 0 0

NO2 NH

2

4.5 4.11 5.6 5.7

0 2 N NO2 OAc AcO

N N H H ~N H HN .O N NO N N 0 N

0 - -0 0 ,-0

0 0 0 0

~J&H 0"WKO~ 1N NH -- 0 N -/ NH --

O 5.8 0 5.9

Scheme 5.2. Structures of bioconjugable 4-nitronaphthalimide probe 4.5, control fluorophore4.11, bioconjugable probe 5.6, control esterase-activated probe 5.7, improved nitroreductase-activated probe 5.8, and control esterase-activated probe 5.9.

Optimizing Contrast Ratio, Background, and Responsiveness. 4-Nitronaphthalimides exhibit

large Stokes shifts and moderate rates of enzymatic activation, but otherwise have inferior

spectroscopic properties to those of xanthene dyes. In particular, xanthene dyes are significantly

brighter (c x 0), more photostable, and less cytotoxic.68 As described in Chapter 4, the optimized

masking group in probe 4.6 and its bioconjugable variant 5.8 provides nitroreductase activation

rates greater than those of 4-nitronaphthalimides, even though probe 4.6 requires two enzymatic

events to unmasking versus single-step activation in 4.5 (Table 5.1).'9' Unfortunately, probe 5.8

and 5.9 are more difficult to access than bioconjugable naphthalimide (4.5) and coumarin (5.6)

probes. Thiol-conjugable probe 5.8 (Scheme 5.2) can be prepared via Buchwald-Hartwig cross-

coupling and provides significantly improved brightness and nitroreductase-responsiveness over

naphthalimide probes. Furthermore, contrast ratio and background signal can be improved by

replacing constitutively fluorescent 4.11 and 5.7 with fluorogenic probe 5.9, which is masked by

esterase-activated trimethyl lock moieties (Scheme 5.2).

189

Table 5.1. Improved spectroscopic properties and activation rates of rhodamine probe 5.6compared to naphthalimide probe 4.5 and coumarin probe 5.6. Aex, Aem, and E x c are measuredfor activated probe, with coumarin probe 5.6 approximated by values for 4.2.

Probe Aex (nm) Aem (nm) E x j (M- 1cm- 1) kcat/KM (M- 1s-1) KM4.5 429 532 6.3 x 102 3.3 x 10441 45.6 pM 41

5.6 390 450 3.7 x 103 2.3 x 104 2.3 pM5.8 496 517 6.8 x 104 8.1 x 104 191 1.6 mM'9'

Quantifying Differences in Rates of Biomolecule Uptake and Internalization in Matched Cell

Lines. Previous work using a fluorogenic lipid probe revealed that the cancerous breast cell line

HTB-126 exhibited elevated constitutive endocytosis compared to the non-cancerous matched

breast cell line HTB-125.2 Fundamental transport physiology differences in cancerous versus non-

cancerous cells can be leveraged for tumor-selective drug delivery and reduction of undesirable

toxicity in non-cancerous cells. Accordingly, profiling rates of transport processes in matched cell

lines can provide a valuable library of information for designing and targeting biologic drugs.

Using the optimized nitroreductase-probe system described in Chapter 4, differences in the overall

rate of endocytosis as well as the rate of endosomal escape of proteins of interest can be measured

across different cell lines (Table 5.2). The selected matched cell lines in Table 5.2 are adherent

tumor and normal cell lines harvested from the same individual, and should be screened for

baseline endogenous reductase activity before use with the nitroreductase-probe system. Tumor

cell lines identified to have the largest differences in rates of uptake and internalization correspond

to cancer and tissue types that are prime candidates for follow-up in vivo tests. Because cytosolic

uptake is the predominant barrier to intracellular targeting of biologics, the optimized

nitroreductase-probe system could be an invaluable tool for predicting and improving cell transport

properties of new chemotherapeutic agents.

190

Table 5.2. Representative adherent matched cell lines amenable to screening with thenitroreductase-probe system.Cancer type Tissue source Tumor cell line Normal cell lineAdenocarcinoma Lung NCI-H1I395 NCI-BL1 395Basal cell carcinoma Skin TE 354.T TE 353.SkCarcinoma Mammary gland Hs 605.T Hs 605.SkMelanoma Skin Hs 895.T Hs 895.SkOsteosarcoma Bone Hs 888.T Hs 888.LuSmall cell carcinoma Lung NCI-H2195 NCI-BL2195

191

APPENDIX A

Tunable Rhodol Scaffolds for Single-hit, MultifunctionalFluorogenic Probes

192

A.1 Introduction

Xanthene Dyes and Rhodols. The first xanthene dye was synthesized in 1871 by Adolf von

Baeyer who prepared a brilliant green dye-now known as fluorescein-via condensation of

resorcinol with phthalic anhydride. 20 9 Over the next 150 years, fluorescein and other xanthene dyes

have maintained their position as some of the brightest and most versatile scaffolds for

fluorescence applications in chemistry and biology.

Xanthene dyes are attractive fluorophores due to their unusual brightness and the ability to

easily modulate both absorbance and fluorescence of the dye, an important feature for fluorogenic

probes.' 9 The core xanthene structure consists of three conjugated rings with heteroatoms or

functional groups incorporated at various ring positions (Scheme A. 1). Most xanthene dyes have

a benzylic acid substituent at the 10 position that introduces tautomerization between quinoid and

lactone forms, often referred to as the open and closed forms (Scheme A.2A). The closed lactone

form disrupts the conjugated xanthene system, completely abolishing both absorbance and

emission transitions. The quinoid and quinonimine forms have strong absorbance and emission

transitions resulting in high extinction coefficients (c) and quantum yields (<P), the two contributors

to overall fluorophore brightness (e x (P).'9 Few other fluorophore scaffolds exhibit such stark

differences between a "bright" state and a "dark" state as seen in xanthene dye tautomers. More

importantly, the open-close tautomer equilibrium can be tuned by pH, solvent polarity, and

substitution patterns. 90 91, 210, 211 Understanding and exploiting rules governing these modifications

has yielded a diverse library of fluorophores (Scheme A. 1) and fluorogenic probes for applications

including pH sensors,212 , super-resolution microscopy,2 1 3 and disease diagnosis in

ophthalmology. 214 Because fluorogenic probe performance depends on comprehensive masking of

parent dye fluorescence, the open-close equilibrium in xanthene dyes provides the highest contrast

193

ratios out of any fluorogenic dye scaffold, with masked/unmasked contrast ratios exceeding

10,000 . 215

Xanthene Dye General Structure Common Xanthene Dye Scaffolds

X1 X1

R1 W R 2

X2

X2

y2 y1

y2

R = N- or 0-linked functional groupsW = 0, NR, Te, C(CH 3) 2, Si(CH3 )2X = alkyl, halogen, sulfonateY = carboxylic acid, alkyl, halogen, sulfonate

Ho 5- 0 HO 0 NH

' 1 '

7 8 CO 2H CO 2H

64

Fluorescein Rhodol

H2N O NH H2N \ i NH

CO 2H CO2H

Rhodamine Silarhodamine

Scheme A.1. Structures of the xanthenecommon xanthene dye scaffolds.

dye core structure (with the xanthene ring in bold) and

A HO 0 0 HO 0 OH

I |

CO 2H 0

Quinoid Lactone

B H2N 0 0 H 2N 0 OH HN 0 OHSI I

CO 2H / C2H

Quinoid Lactone Quinonimine

Scheme A.2. Open-close equilibrium in xanthene dyes. The quinoid and quinonimine forms arecharacterized by high fluorescence and strong absorbance at Aex. Conversely, the lactone haslittle to no fluorescence or absorbance at Aex. (A) Equilibrium in symmetric xanthene dyes. (B)Equilibrium in asymmetric xanthene dyes.

Two of the most commonly used xanthene dyes for fluorogenic probes are the fluorescein and

rhodamine scaffolds (Scheme A.1), symmetrical dyes with excellent spectroscopic properties

suitable for common laser lines and filters in imaging and fluorescence applications.' Rhodols are

194

asymmetric hybrids of the fluorescein and rhodamine scaffolds with both nitrogen and oxygen ring

substituents (Scheme A. 1). As can be predicted from their hybrid nature, rhodols properties such

as pKa, ;ex, uem are intermediate to those of fluorescein and rhodamine (Table A. 1). Nonetheless,

rhodols do possess several unique advantages that are attractive for fluorogenic probe applications.

Unlike fluorescein, which requires two modifications at the 3' and 6' positions to elicit completely

the non-fluorescent closed form, rhodols favor the closed form upon a single modification.216

Fluorescein and rhodamine probes modified with two masking groups recover a small portion of

fluorescence after removal of the first masking group, but require removal of both masking groups

("dual-hit") to fully restore fluorescence (Scheme A.3). In contrast, the kinetics of unmasking

rhodol fluorogenic probes are conceivably faster because only one step is required to restore

fluorescence completely ("single-hit", Scheme A.3). Rhodamines can be converted into single-hit

probes at the cost of additional synthetic steps and a reduction in contrast ratio and brightness.

Furthermore, rhodols possess three orthogonal sites for modification or functionalization, versus

two on fluorescein or rhodamine (Scheme A.4). In this case, the symmetric nature of fluoresceins

and rhodamines is a barrier to differential modification of the 3' and 6' positions, a challenge easily

overcome by the asymmetric rhodol dye. Finally, rhodols can accommodate either N- or O-linked

masking groups which are normally restricted to only rhodamines or fluoresceins respectively.

Table A.1. Key properties of fluorescein, rhodol, and rhodamine.Dye Aex(nm) Aem (nm) e (M- 1cm-1) 0 e x 0 (M- 1cm-1) pKaFluorescein" 490 514 9.3 x104 0.95 8.8 x104 6.3210Rhodo 2 17 493 512 7.0 x104 0.98 6.9 x104 5.5218Rhodamine' 9 496 517 7.4 x104 0.92 6.8 x104 4.3219

195

A RO 0 OR RO 0 0 HO 0

\ R R C02

- 0

B R'O 0 NHR2 HO O NR2

0 R

/\ R1COH

R'O 0 NR 2

x2 x2/\ 0

R 0

X = halogen, sulfonateR = various functional groups

Scheme A.3.Comparison of (A)"dual-hit" and (B)"single-hit"unmaskingmechanisms forfluorogenic probesbased on xanthenedyes.

Modification Effects

N Installation of bioconjugation handle(s)

R - Masking fluorescenceR Tuning (ex,2em,c- Charge modification- Alteration of cell permeability and retention

E Modification of charge and solubility

X : Stabilization of masking groupsImprovement of photostability

- Tuning ex, Lem, P- Tuning pKa

Scheme A.4. Location and effects of common substitutions on rhodol dyes.

Dye Synthesis. Despite the advantages of xanthene dye scaffolds, the chemical reactions to

prepare these dyes remained largely unchanged until the last three decades. Application of

methanesulfonic acid as both Lewis acid catalyst and solvent was a major advance that enabled

gram-scale preparation of fluorescein and derivatives at lower reaction temperatures and with

improved yield and purity (Scheme A.5A).n2 -m Even with improvements, the condensation

reaction can accommodate only a narrow scope of substituents at the 3' and 6' position. Attempts

to install primary amines and other nitrogen functional groups at the 3' and 6' positions introduce

competing side reactions that drastically reduce yield and increase difficulty of purification. This

196

problem is further compounded when asymmetrical xanthene dyes with different 3' and 6'

substituents are desired.

Preparation of asymmetrical rhodol dyes has historically relied on three synthetic routes, all of

which are disadvantageous in yield, scope, or difficulty (Scheme A.5B-D). Hydrolysis of

rhodamines at high temperatures over four days provides the desired rhodol in moderate yield but

requires starting materials that are themselves difficult to prepare and are restricted in scope of

nitrogen substituents (Scheme A.5B). 1 The other two routes involve preparation of a

benzophenone intermediate, effectively separating the one-pot condensation reaction in Scheme

A.5A into two distinct steps. In Scheme A.5C, fluorescein is hydrolyzed to yield a benzophenone,

which is then condensed with aminophenol to yield the rhodol. This approach starts with readily

accessible fluorescein, but the second condensation step is prone to polymerization and formation

of other byproducts unless a tertiary aminophenol is used. Alternatively, hydrolyzing substituted

rhodamines as the starting material for amine-substituted benzophenones improves yields, but is

subject to the same amine substitution restrictions (Scheme A.5D). All three of these methods

suffer from some combination of low yields, long reaction times, costly starting materials, and

restrictions on the scope of nitrogen-containing functional groups that can be incorporated.

Recently, Buchwald-Hartwig cross-coupling chemistry for aryl amines has been applied to

fluorophore synthesis (Scheme A.5E).4' 215, 224, 225 Buchwald-Hartwig cross-coupling provides a

convenient method to convert readily accessible fluorescein into rhodamines with unparalleled N-

substituent diversity. Although this method has been applied systematically to prepare and

characterize libraries of carborhodamines ,215 silarhodamines,225 and rhodamines,22 4 no such

investigation of the rhodol scaffold has been reported. In this work, we seek to establish a

generalizable synthetic route to substituted rhodols, understand the open-close equilibrium

197

dynamics and spectroscopic properties of fluorogenic rhodol probes, and apply our understanding

to develop new and improved single-hit fluorogenic probes.

A 0

C0

HO R+ 2 MeSOH

90 *C, 24 h19-98%

R=OH, NH2, NR2

B R2N 0 NR2 .

CO -

HOC N

C HO 0 0

CO 2H

D R2 N 0 NR2'

CO 2

E X Y X

I I0

X = OTf, CI, Br, IY = 0, C(CH3)2, Si(CH) 2

KOH(a)

100 C, 4 d30%

R 0 R

CO2 H

R2N 0 0

CO2H

HO2C

HO 0 0 OHKOH(a)

100 *C, 5h86% OH

HO 0 0 OHNaOH(e)

100 *C, 1h93% NR 2

R2N OH

TFA95 *C, 3 h

18%

HO OH

TFA90 *C, 12 h

77%

R2N 0 0

CO2H

R2N 0 0

CO 2H

H2NR RHN Y NR

Pd, Ligand

Base, Solvent CO H25-95% 2

Y = 0, C(CH )2, Si(CH) 2

Scheme A.5. Previous synthetic routes to xanthene dyes. (A) Synthesis of symmetric xanthenedyes by condensation.2 2 1 , 222 (B) Preparation of rhodol by hydrolysis of rhodamine . 2 18 (C)Preparation of rhodol by hydrolysis of fluorescein and condensation with aminophenol. 2 16 (D)Preparation of rhodol by hydrolysis of rhodamines and condensation with resorcinol.2 3 (E)Preparation of symmetric xanthene dyes by cross-coupling. 2 15 224, 225

198

A.3 Results and Discussion

Preparation of Rhodols via Cross-Coupling Chemistry. The inherent asymmetry of the rhodol

scaffold is the primary barrier to synthesis with cross-coupling chemistries. When preparing

rhodols from symmetrical starting materials, only modification of either the 3' or 6' position is

permissible. Introduction of this asymmetry can be accomplished by protecting group chemistry

or during cross-coupling. Protection with alkyl halides has been reported,2" but suffered from

nonspecific alkylation patterns in our hands and in other reports.226 227 In the presence of base, the

pendant carboxylate has greater nucleophilic character than does either of the 3' or 6' phenolates.

The relative reactivity of the carboxylate and phenolates also depends on the type of electrophile

used, with alkyl halides especially prone to reacting with the pendant carboxylate. Addition of a

6-carboxy substituent for bioconjugation or charge modification (Scheme A.4) further confounds

the desired alkylation with a total of four competing nucleophiles. As a result, attempts to mono-

protect fluorescein or 6-carboxyfluorescein produces an undesirable mixture of mono-, di-, and

tri-alkylated products. 226,227

Instead, we elected to synthesize rhodols by stochastic coupling of the nitrogen-containing

functional group to fluorescein ditriflate, which was previously demonstrated with core

unsubstituted rhodol (Scheme A.6). 224 Formation of the desired mono-coupled rhodol triflates

(A.1-A.5) was favored by stoichiometric control and ring deactivation effects of monotriflates

relative to the ditriflate starting material. Using this approach, rhodols A.6-A.10 were prepared in

moderate yields after hydrolysis of the residual triflate in A.1-A.5. Rhodols were modified further

with alkyl or acyl masking groups to create fluorogenic rhodols A.11-A.14.

199

HO 0 0 TfO 0 OTf HR', Pd2dba , TfO 0 RI

Tf20, Pyr XPhos, CsCO 3

CO H CH2C 2 0 Dioxane, 100 *C 0

2 93% 38-62%

A.1: RI = NHBocA.2: RI = NCH3BocA.3: R1 = N(CO)N(CH,) 2A.4: RI = N(CO)OCH,A.5: RI = N(CO)CH3

HO 0 R 1 R2 0 R1

aorb cordCO 2 H 0

0A.6: R1 = NH2 A.11: R 1 = N(CO)N(CH )2 R2 = (CO)CH,A.7: RI = NHCH, A.12: R 1 = N(CO)OCH3 R2 = (CO)CH3A.8: RI = N(CO)N(CH,)2 A.13: R' = N(CO)N(CH,)2 R2 = CH2O(CO)CH3A.9: R1 = N(CO)OCH, A.14: R 1 = N(CO)OCH3 R2 = CH2O(CO)CH3A.10: R1 = N(CO)CH,

Scheme A.6. Synthesis of rhodols via stochastic Buchwald-Hartwig cross-coupling of variousnitrogen-containing moieties with fluorescein ditriflate. (A) KOH, MeOH/THF, 1 h. (B) TFA,CH 2CI 2 , 5 h followed by KOH, MeOH/THF 1 h. (C) Bromomethyl acetate, MeCN, 4-A sieves, 48h. (D) Acetyl chloride, DMAP, pyridine, CH 2CI 2, 1 h.

Rhodol Spectroscopic Properties. Because the asymmetry in rhodols removes the degeneracy

between 3' and 6' substituents seen in fluorescein or rhodamine, the open-close equilibrium and

protonation states of rhodols have an added degree of complexity (Scheme A.2). This asymmetry

presents an opportunity to tune the rhodol equilibrium in ways not accessible with symmetric

xanthene eyes to enhance the brightness, contrast ratio, and unmasking kinetics of fluorogenic

probes based on the rhodol platform.

We first sought to characterize the open-close equilibrium in the context of pH and solvent

polarity. Because only the open quinoid or quinonimine forms have absorbance at A > 250 nm,

measuring absorbance of visible-wavelength light provides direct insight into the open-close

equilibrium. Titration of rhodols A.6-A.10 in buffers spanning pH 4-10.5 (Figure A.1A, A.1B)

revealed two types of equilibrium behaviors. In amidic rhodols A.8-A.10, absorbance signal

200

- -6

4 6 8 10

-H

-

.

6 pH 8

-- A .7-- A .8+ A.9

-4- A.10

- - I1

Figure A.1. Titrations of rhodols A.6-A.10 insolutions of varying pH or solvent polarity. (A)Titration of amine-type rhodols A.6 and A.7 inbuffers ranging from pH 4 to pH 10.5. (B)Titration of amidic rhodols A.8-A.10 in buffersranging from pH 4 to pH 10.5. (C) Titrations ofrhodols A.6-A.10 in water-dioxane mixturesof varying polarity.10

20 40 60Dielectric constant (c)

80

decreases with pH until the closed, nonabsorbent lactone form predominates. The sigmoidal curves

in Figure A. 1 B suggest that a single protonation event in amidic rhodols governs interconversion

between a single open state and a closed state. This restriction can be explained by resonance

donation of the nitrogen lone pair into the adjacent carbonyl, which strongly disfavors the

quinonimine form (Scheme A.7B). Thus, the open-close equilibrium in amidic rhodols is largely

governed by protonation of the xanthene phenolate, with little contribution from the xanthene

201

A 1.0-

C.00.5-

~0

B1.0 -

C

. 0.5 -0CI)

C

CD

2u.0

U.u

1.0

0.5

0.0

nitrogen within physiologically relevant pH ranges. Thus, the tautomer equilibrium for amidic

rhodols is collapsed from three to two tautomers (Scheme A.7).

A H2N 0 0 H2N 0 OH HN 0 OH

CCO 2 H 0 CO 2H

Quinoid Lactone QuinonimineH H

B N N OH IN 0 OH

CO2H O CO 2H

Quinoid Lactone Quinonimine

Scheme A.7. Differences in tautomer equilibria in (A) amine-type rhodols and (B) amidic rhodols.Structures of tautomers in representative rhodols A.6 and A.10. Amine-type rhodols exist as allthree tautomers, whereas amidic rhodols are restricted to only the lactone and quinoid forms.

In contrast, titration of amine rhodols A.6 and A.7 shows a plateau at an intermediate

absorbance (Figure A.lA). In amine-type rhodols, the absence of adjacent carbonyl groups

encourages donation of amine electron density into the xanthene ring scaffold, favoring the open

quinonimine tautomer. Thus, the open-close equilibrium is governed by the pKa values of both the

ring anilinium and phenol groups, which are significantly reduced in xanthene dyes. For example,

the pKa of phenols in fluorescein is 6.3,"' compared to free phenol pKa of 10.0.228 Similarly, the

pKa of the anilinium ion in rhodols is expected to be several orders of magnitude lower than the

pKa of free anilinium ions in water (pKa = 4.6).228 Accordingly, amine-type rhodols with

unsubstituted phenols are unable to achieve full ring closure at physiological pH ranges.

Additionally, amine-type rhodols have higher pKa values than do amidic rhodols, another

consequence of stronger electron donation into the xanthene ring system. From a design

perspective, these results suggest that pH-sensor applications of rhodols should minimize

202

quinonimine formation by attenuating nitrogen electron-donation to ensure bimodal probe

responses and enhance contrast ratios.

Solvent polarity is another key factor affecting the open-close equilibrium in xanthene

dyes.21 5 The absorbance of rhodols A.6-A.10 was measured in mixtures of water and dioxane,

which are miscible but have starkly different polarities as can be approximated by their dielectric

constants (e). Gratifyingly, trends in ring closure mirror those seen in pH titrations-amidic

rhodols A.8-A.10 close as solvent polarity decreases, whereas amine-type rhodols A.6-A.7

maintain the open quinone and quinonimine forms in moderately nonpolar solvent mixtures.

Additionally, secondary amine rhodol A.7 favors open forms more strongly than primary amine

rhodol A.6, further confirming that the open-close equilibria is primarily governed by the electron-

donating character of the xanthene ring substituents in rhodol dyes.

Given the role of rhodol ring substituents in open-close equilibria, we next sought to assess

the feasibility of tailored rhodols as fluorogenic probes. We hypothesized that the electron-

donating character of the substituents would also be important for probe brightness and contrast

ratios, just as it was for pH and solvent polarity effects. Since the Hammett substituent constant

(up) is commonly used as a proxy for electron-donation character in fluorophores,9 0 2, 224 we

applied a similar measure to rhodols A.5-A.14 by calculating the average up value for the 3' and

6' substituents in the closed lactone form. The average up for rhodols A.5-A.14, fluorescein, and

rhodamine were plotted against extinction coefficients (c), quantum yields (0), and brightness (c

x P) in Figure A.2. Both the quantum yield and extinction coefficient varied inversely with up for

probes with average op values less than -0.4. Rhodols with average op values greater than -0.4

displayed consistently reduced extinction coefficients and quantum yields. These two trends are

further emphasized when brightness is plotted as a function of up, since brightness is the product

203

of extinction coefficient and quantum yield (Figure A.2B). As hypothesized, rhodol brightness is

highly dependent on the electron-donation character of the 3' and 6' substituents, with a linear

relationship observed between brightness and average up values above the up > -0.4 threshold.

The sharp transition from nonfluorescent to linearly increasing fluorescence at up -0.4 is

highly useful for single-step fluorogenic rhodol probes (Figure A.2B). Based on this curve, ideal

fluorogenic rhodols should have masking groups with average up values between -0.4 and -0.2.

Upon exposure to triggering events, the masking groups would be cleaved or converted into

strongly donating substituents with as low as possible op values. This difference in up values

ensures the largest contrast ratio by suppressing the masked state and enhancing the unmasked

fluorophore signal.

66

-0.8 -0.6 -0.4 -0.2 0.0

-0.8 -0.6 -0.4 -0.2 0.0

P

- 10

-0.5

-0.0

Figure A.2. Graphs of spectroscopicproperties for rhodol dyes. A.6-A.14,fluorescein, and rhodamine. (A) Graph ofextinction coefficients (V, R2 = 0.917) andquantum yields (0, R2 = 0.950) versusaverage Hammett substituent constant (op).Extinction coefficients were calculated at Amax> 250 nm. (B) Graph of probe brightness (E x

0, R2 = 0.960) versus average Hammettsubstituent constant (op). Average Hammettsubstituent constants are calculated from themean op values for 3' and 6' substituents ofeach dye in lactone form.

204

A 10.0

0

5.0

C)

0.0

B 10.0

0

S5.0

CO.

0.0

-

-

-

-

-

A.4 Future Directions

Rhodols for Open-Close Equillibration and Other Spectroscopic Properties

A

R1= HA.6-A.10

R 10 0 R2

0

- 0 R2=

Probe Stability and Lifetime

B HO 0 N

I X 7 F

0

X = C, Br, I

C HO 0 ' N ,

0 0 OEt

X = C, Br, I

NH2

A.6

N]

A.11, A.13

HN N

A.7

X

Nd

X = F, Cl, Br, I

0 -

A.12, A.14

H HN yNN N Y

0 0A.8, A.11, A.9, A.1

A.13 A.14

F F

Nd

OMe

NdN

Nd

I H

02, A.10

N CC 2 Me

"Single-hit" Probes for Enzyme Kinetics

N 0 R

I Enzyme

0

"Single-hit" Rhodol

R 0 R

O N Enzyme0

"Dual-hit" Fluorescein

NoR L= 2

Nitroreductase

0

Esterz

N 0 0

CO2 H

HO 0 0

CO 2H

OH

HO O

ise B3-galactosidase]

Scheme A.8. Proposed rhodols for studying electronic effects and fluorogenic probe applications.(A) Substituted rhodols for study of open-close equilibria and other spectroscopic properties. (B)Halogen-halogen interactions in the excited state of 3,3-difluoroazetidinyl rhodols could enhancefluorescence lifetimes. (C) n-r*n stabilization effects in ground-state 3-substituted azetidinylrhodols and model systems. (D) Enzyme-activated fluorogenic probes for kinetics and imagingstudies.

205

Fluorogenic Azetidinyl Rhodols. The sharp cutoff between fluorescent and nonfluorescent

rhodols is particularly impactful in the context of azetidine-modified dyes. Functionalization of

fluorophores with azetidines yielded significant and universal dye improvements in extinction

coefficients (e) and quantum yields (0P) by reducing twisted internal charge transfer (TICT) in

strained four-membered ring systems. 2 Unfortunately, symmetrical rhodamines that have

azetidine substituents at both 3' and 6' positions cannot be used as fluorogenic probes because the

azetidines are strong electron donors that prohibit the formation of the closed lactone form. This

inaccessibility of the masked, closed form prevents application of azetidinyl rhodamine scaffolds

to fluorogenic probes. In contrast, only one side of a rhodol is substituted with azetidine, leaving

the phenol free for modification with a masking group. Thus, the azetidine modification can be

applied to fluorogenic probes built on the rhodol scaffold, but not on other dye scaffolds.

Furthermore, compatible masking groups for azetidine rhodols can be predicted by calculating

average Hammett parameter constants op and comparing to the trends in Figure A.2. These

predictions can then be verified by preparing azetidinyl rhodols through cross-coupling and

masking group installation (Scheme A.6).

Recently, fine tunability of azetidinyl dyes was demonstrated with various 3-substited

azetidines. Although the 3 position in the azetidine ring is not conjugated with the dye scaffold,

through-space electronic effects enabled direct modulation of rhodamine emission wavelengths in

increments of 1-5 nm over a range of 50 nm." To study spectroscopic tuning of azetidinyl rhodol

fluorogenic probes, a library of rhodols with and without masking groups can be synthesized, some

of which have already been prepared (Scheme A.8A, Figure A. 1-2). Optimized azetidinyl rhodols

will have brilliant fluorescence when R2 = H and completely masked absorbance and fluorescence

when R2 is a masking group. These optimized azetidinyl rhodol probes could be applied to

206

biomolecule imaging upon facile addition of a bioconjugation handle at the 6 position of the rhodol

dye.

Halogen-Halogen Interactions for Fluorescence Lifetime Extension and Bathochromic

Shifts in Azetidinyl Rhodols. Previous studies of hetereocyclic descarboxyrhodamines with

varying ring sizes reported that in the ground state, the 3' and 6' azetidine rings and the xanthene

ring were coplanar.2 2 9 In contrast, our calculated structure of excited-state azetidinyl rhodol shows

perpendicular orientation of the azetidine ring and the xanthene core ring. This geometry presents

the opportunity to selectively stabilize the rhodol excited state via halogen-halogen interactions

between 3,3-dihaloazetidines and 2',7'-halogenated rhodol scaffolds. Two main effects are

predicted from this excited state stabilization-bathochromic shifts of excitation and emission

spectra and extension of the fluorescence lifetime. Long-wavelength fluorophores are desirable for

their decreased cell autofluorescence background and decreased phototoxicity in biological

imaging. Few levers are available to alter xanthene dye lifetimes, such that a tunable electronic

interaction to extend xanthene dye lifetimes (typically a few nanoseconds) would be invaluable. A

variety of 3,3-dihalogenated azetidines can be coupled with 2',7'-halogenated fluorescein

ditriflates to prepare appropriately halogenated rhodols for further study (Scheme A.8B).

n--* Stabilization of Azetidine Rhodols and Fluorogenic Rhodol Probes. Previously, we

reported stabilizing effects of n-+rc* interactions between isobutyryl masking groups and ortho-

chloro groups at the 2' and 7' positions of fluorescein. In azetidinyl rhodols, similar n->r*

stabilization can occur, with donation from the nonbonding orbital of the ortho halogen into the

Z* orbital of the 3-carboxylated azetidine (Scheme A.8C). Although the n-> c* interaction in

these rhodols stabilizes the fluorophore scaffold rather than the masking group, inherent probe

207

stability and fluorescence lifetime could be improved by this effect. The optimized structure of a

truncated model compound suggests that this interaction occurs even in the ground state (Scheme

A.8C, Eng = 0.71 kcal/mol), which would significantly alter both parent rhodol and fluorogenic

probe spectroscopic properties.

"Single-hit" Rhodol Fluorogenic Probes with Enhanced Enzyme-Response Kinetics. Three

representative enzymes were chosen to test the kinetics of enzyme-activated rhodol fluorogenic

probes-nitroreductase, esterase, and P-galactosidase (Scheme A.8D). These three enzymes were

selected for their variety of function, well-characterized rates of activation for dual-hit probes, and

commercial availability. A significant improvement in response rates is expected over dual-hit

fluorogenic probes, which not only have two equivalents of enzyme substrate per probe requiring

sequential activation. Three "single-hit" rhodol probes can be prepared as described in Scheme

A.6 and compared to the corresponding "dual-hit" fluorescein versions.

A.5 Acknowledgements

We are grateful to Henry R. Kilgore for his help with the computational portions of this work

and contributions to experimental design. This work was supported by grant RO1 GM044783 to

R.T.R. (NIH). W.C. was supported by an NSF Graduate Research Fellowship. This work used data

acquired at the National Magnetic Resonance Facility at Madison, which is supported by Grant

P41 GM 103399 (NIH). The work also made use of a Thermo Q ExactiveTM Plus mass spectrometer

(NIH grant S10 OD020022).

208

A.6 Experimental

General Information. All commercial chemicals were from Sigma-Aldrich (St. Louis, MO),

Fischer Scientific (Hampton, NH), or Alfa Aesar (Haverhill, MA) and were used without further

purification. Porcine liver esterase (PLE) was from Sigma-Aldrich.

Chemical reactions were monitored by thin-layer chromatography (TLC) using EMD 250-jam

silica gel 60-F2 54 plates and visualization with UV illumination or KMnO4-staining. Flash

chromatography was performed with a Biotage Isolera automated purification system using pre-


All procedures were performed in air at ambient temperature (~22 'C) and pressure (1.0 atm)

unless specified otherwise. The phrase "concentrated under reduced pressure" refers to the removal

of solvents and other volatile materials using a rotary evaporator at water aspirator pressure (<20

torr) while maintaining a water-bath temperature below 40 'C. Residual solvent was removed from

samples at high vacuum (<0.1 torr), which refers to the vacuum achieved by mechanical belt-drive

oil pump.

All fluorogenic probes and fluorescent molecules were dissolved in spectroscopic grade

DMSO and stored as frozen stock solutions. For all applications, DMSO stock solutions were

diluted such that the DMSO concentration did not exceed 1% v/v.

Instrumentation. Absorbance data were acquired with an Agilent Cary 60 UV-vis spectrometer.

Hydrolysis kinetics were measured with a Tecan Infinite M1000 plate reader. All other

fluorescence data were acquired with a PTI QuantaMaster spectrofluorometer. 'H and "C NMR

spectra were acquired on Bruker Spectrometers at the National Magnetic Resonance Facility at

209

Madison (NMRFAM) operating at 500 MHz for 'H and 125 MHz for 13 C. Mass spectrometry was

performed with a Q ExactiveTM Plus electrospray ionization quadrupole-ion trap (ESI-QIT-MS)

mass spectrometer at the Mass Spectrometry Facility in the Department of Chemistry at the

University of Wisconsin-Madison.

Optical Spectroscopy. UV-visible and fluorescence spectra were recorded by using 1-cm path

length, 4-mL quartz cuvettes or 1-cm path length, 1-mL quartz microcuvettes. Analyte solutions

were stirred with a magnetic stir bar. Quantum yields were determined by referencing probe

solutions to fluorescein ( =ex 495 nm; < = 0.95) in 0.1 M NaOH(aq).

pH Titrations. pH titrations for rhodols A.6-A.10 were carried out in appropriate 10 mM buffer

solutions for each pH range as follows: citrate (pH 4.0-6.2), phosphate (pH 5.8-8.0), tris (pH 7.8-

9.0), carbonate (pH 9.2-10.5). The absorbance of a 10 pM solution of each rhodol was measured

at Xmax, normalized, then plotted and fitted to a sigmoidal dose-response curve using GraphPad

Prism to obtain pKa values.

Water-Dioxane Titrations. The absorbance of rhodols A.6-A.10 in a variety of water-dioxane

mixtures was measured. To prepare water-dioxane mixtures, varying amounts of spectroscopic-

grade dioxane were mixed with water and the resulting solution vigorously stirred for 1 h. For each

data point, stock solutions of rhodol were added to the water-dioxane mixture and allowed to

equilibrate under stirring for 30 min. Measurements were carried out in triplicate and the resulting

plots for each rhodol were fit to sigmoidal dose response curves in GraphPad Prism.

210

Computational Procedures. Geometry optimization calculations were performed with Gaussian

09, revision D.01 with the functional M06-2X and 6-311+g(2d,p) basis set.' 25 Frequency

calculations were performed to ensure that the optimized structure was at a true minimum. All

calculations were performed in water with the integral formalism polarizable continuum model

(IEFPCM implicit solvent model as implemented in Gaussian 09). NBO calculations were

performed with NBO 6.0.11 All energies include zero-point corrections.

Synthesis of Fluorescein Ditriflate (A.0). Fluorescein (1 g, 1 equiv) was dissolved in 10 mL

dichloromethane and cooled to 0 'C. Pyridine (1.94 mL, 8 equiv) was added, followed by

trifluoromethanesulfonic anhydride (2.03 mL, 4 equiv), after which the ice bath was removed. The

reaction mixture was stirred at room temperature for 1 h, then diluted with water and extracted

with dichloromethane twice. The combined organics were washed with 1 M HCl (3 x) and brine,

dried with MgSO4(s), and concentrated under reduced pressure. Purification by column

chromatography (0-30% v/v EtoAc in hexanes) yielded the title compound as white crystals (1.568

g, 87.3% yield). 'H NMR (CDCl 3, 400 MHz, 6): 8.10 (d, J= 7.4 Hz, 1H), 7.75 (dt, J= 18.2, 7.3

Hz, 2H), 7.33 (d, J= 2.2 Hz, 2H), 7.22 (d, J= 7.4 Hz, 1H), 7.03 (dt, J= 27.3, 5.5 Hz, 4H). 13 C

NMR (CDCl 3, 100 MHz, 6): 168.45, 152.12, 151.31, 150.20, 135.81, 130.68, 129.98, 125.70,

125.59, 123.75, 123.42, 120.23, 119.29, 118.64 (q, 'JC-F= 321 Hz) 117.67, 117.04, 113.85, 110.68,

80.06. HRMS (ESI-QIT) m/z: [M+H]' Calcd for C22HioF609S2 596.9743, found 596.9728.

Synthesis of Rhodol Triflates A.1-A.5. An oven-dried roundbottom flask was charged with

Pd2dba3 (17 tmol, 0.05 equiv), XPhos (50 pmol, 0.15 equiv), Cs2CO3 (0.47 mmol, 1.4 equiv),

fluorescein ditriflate A.0 (0.34 mmol, 1 equiv), and the appropriate amine, amide, carbamate, or

211

urea (0.34 mol, 1 equiv). The vial was then sealed and purged three times with N2(g). Anhydrous

dioxane (2 mL) was thoroughly degassed and were added to the vial, followed by purging with

N2(g) three times again. The sealed flask was heated at 85 'C for 3 h. Reaction progress was

monitored by LCMS, and the reaction mixture was quenched at 3 h. The reaction mixture was

diluted with dichloromethane, filtered, and a small amount of Celite* was added to the filtrate, and

the suspension concentrated under reduced pressure. Purification by column chromatography (20-

100% v/v EtOAc in hexanes) yielded the title compounds as white foams.

Rhodol (Boc) triflate (A.1). White foam (110 mg, 58.3% yield). 1H NMR (500 MHz, CDCl 3,

3): 8.07-8.03 (m, 1H), 7.68 (dtd, J= 22.9, 7.4, 1.0 Hz, 2H), 7.61 (s, 1H), 7.24 (d, J= 2.4 Hz, 1H),

7.16 (d, J= 7.5 Hz, 1H), 6.96 (dd, J= 8.8, 2.4 Hz, 1H), 6.92 (td, J= 6.3, 3.2 Hz, 2H), 6.72 (d, J=

8.6 Hz, 1H), 6.68 (s, 1H), 1.53 (s, 9H). 13 C NMR (125 MHz, CDCl 3, 6): 169.20, 152.85, 152.34,

152.16, 151.46, 150.14, 141.10, 135.56, 130.33, 130.12, 128.65, 126.18, 125.49, 123.99, 122.62,

120.07, 119.79, 117.52, 116.85, 114.96, 114.88, 112.63, 110.70, 106.09, 81.56, 79.86, 28.39.

HRMS (ESI-QIT) m/z. [M+H]' Calcd for C2 6H2 oF3NO8 S 564.0936; found 564.0937.

Methyl rhodol triflate (A.2). White foam (83 mg, 42.9% yield). 'H NMR (500 MHz, CDCl 3,

6): 8.09 (d, J= 7.5 Hz, 1H), 7.77-7.68 (m, 2H), 7.30 (dd, J= 9.2, 2.2 Hz, 2H), 7.22 (d, J= 7.5 Hz,

1H), 7.07 (dd, J= 8.6, 2.1 Hz, 1H), 7.01 (dd, J= 8.8, 2.4 Hz, 1H), 6.96 (d, J= 8.8 Hz, 1H), 6.80

(d, J= 8.6 Hz, 1H), 3.33 (s, 3H), 1.52 (s, 9H). 13C NMR (125 MHz, CDCl 3, 6): 171.66, 156.85,

155.25, 154.69, 153.34, 152.76, 148.86, 138.19, 133.00, 132.76, 130.56, 128.70, 128.11, 126.62,

123.89, 122.64, 122.32, 119.53, 117.44, 115.65, 113.26, 84.01, 83.87, 39.62, 30.99. HRMS (ESI-

QIT) m/z. [M+H]* Calcd for C2 7H2 2F3NO8 S 578.1091; found 578.1089.

Dimethyl urea rhodol triflate (A.3). White foam (96 mg, 53.6% yield). 'H NMR (500 MHz,

CDCl 3, 6): 8.09 (d, J= 7.6 Hz, 1H), 7.79-7.66 (m, 2H), 7.60 (d, J= 2.2 Hz, 1H), 7.28 (d, J= 2.4

212

Hz, 1H), 7.21 (d, J= 7.5 Hz, 1H), 7.00 (dd, J= 9.4, 2.0 Hz, 2H), 6.94 (d, J= 8.8 Hz, 1H), 6.76 (d,

J= 8.6 Hz, 1H), 6.55 (s, 1H), 3.11 (s, 6H). 13C NMR (125 MHz, CDCl 3, 6): 169.29, 155.38, 152.89,

152.19, 151.37, 150.15, 141.64, 135.60, 130.33, 130.12, 128.54, 126.16, 125.47, 124.07, 122.63,

120.08, 119.77, 117.52, 116.83, 116.11, 114.97, 112.71, 110.75, 107.41, 81.65, 36.75. HRMS

(ESI-QIT) m/z: [M+H]+ Caled for C24 H1 7F3N2 0 7 S 535.0787; found 535.0775.

Methyl carbamate rhodol triflate (A.4). White foam (111 mg, 63.5% yield). 'H NMR (500

MHz, CDCl 3, 6): 8.06 (d, J= 7.3 Hz, 1H), 7.69 (dtd, J= 23.4, 7.4, 1.1 Hz, 2H), 7.59 (s, 1H), 7.26

(s, 1H), 7.18 (d, J= 7.5 Hz, 1H), 6.97 (dd, J= 8.8, 2.4 Hz, 2H), 6.92 (d, J= 8.8 Hz, 1H), 6.79 (s,

1H), 6.75 (d, J= 8.6 Hz, 1H), 3.82 (s, 3H). 13C NMR (125 MHz, CDCl 3, 3): 169.19, 153.71,

152.78, 152.11, 151.48, 150.18, 140.52, 135.62, 130.39, 130.15, 128.84, 126.18, 125.54, 124.00,

122.63, 120.08, 119.74, 117.52, 114.99, 113.20, 110.75, 106.32, 81.47, 52.86. HRMS (ESI-QIT)

m/z: [M+H]* Calcd for C2 3H14 F3N20 8 S 522.0471; found 522.0460.

Amide rhodol triflate (A.5). White foam (98.5 mg, 58.2% yield). 'H NMR (500 MHz, CDCl3,

6): 8.09-8.03 (m, 1H), 7.80 (d, J= 1.8 Hz, 1H), 7.69 (dd, J= 23.1, 1.2 Hz, 2H), 7.32 (s, 1H), 7.17

(d, J= 7.5 Hz, 1H), 7.03 (dd, J= 8.6, 2.1 Hz, 1H), 6.98 (dd, J= 8.8, 2.4 Hz, 1H), 6.92 (d, J= 8.8

Hz, 1H), 6.76 (d, J= 8.6 Hz, 1H), 2.22 (s, 3H). 13C NMR (125 MHz, CDC1 3, 6): 169.18, 168.58,

152.82, 152.08, 151.31, 150.19, 140.32, 135.64, 130.41, 130.13, 128.67, 126.09, 125.54, 124.01,

122.63, 120.08, 119.67, 117.52, 117.00, 115.88, 114.97, 113.99, 110.77, 107.75, 81.39, 24.91.

HRMS (ESI-QIT) m/z: [M+H]* Calcd for C2 3 H, 4 F3NO 7S 506.0521; found 506.0511.

Synthesis of Rhodols A.6-A.7. Rhodol (boc) triflate A.1 or A.2 (1 equiv) was dissolved in 5

mL of a 1:4 TFA/dichloromethane solution and stirred for 5 h at room temperature. The reaction

mixture was then concentrated under reduced pressure and redissolved in 5 mL of a 1:1

213

THF/MeOH solution. Aqueous 2 M KOH was added (4 equiv) and the mixture was stirred at room

temperature for 1 hour, after which complete hydrolysis was observed by LCMS. The mixture was

neutralized with 1 M HCl and evaporated to dryness to obtain the rhodol product as an orange or

red powder.

Rhodol (A.6). Red solid (63.7 mg, 98.5% yield). 'H NMR (500 MHz, (CD 3 ) 2 SO, 6): 10.37 (s,

1H), 8.01 (d, J= 7.4 Hz, 1H), 7.86 (s, lH), 7.70 (dt, J= 22.9, 7.2 Hz, 2H), 7.24 (d, J= 7.5 Hz,

1H), 7.11 (dd, J= 8.7, 1.7 Hz, 1H), 6.72 (d, J= 8.7 Hz, 1H), 6.62 (d, J= 8.9 Hz, 1H), 6.56 (d, J=

1.3 Hz, 1H), 6.48 (dd, J= 8.9, 2.1 Hz, IH). 13C NMR (125 MHz, (CD 3 ) 2SO, 6): 169.08, 168.61,

153.55, 151.329 141.88, 133.98, 129.82, 129.61, 128.50, 126.02, 125.25, 124.29, 124.28, 118.19,

116.11, 115.11, 114.05, 110.57, 105.72, 102.75. HRMS (ESI-QIT) m/z: [M+H]' Calcd for

C20H 13NO4 332.0917; found 332.0913.

Methyl rhodol (A.7). Dark orange solid (43 mg, 86.6% yield). 1H NMR (500 MHz, MeOD, 6):

8.38 (dd, J= 7.7, 1.2 Hz, 1H), 7.86 (dtd, J= 24.0, 7.5, 1.4 Hz, 2H), 7.48 - 7.42 (m, 1H), 7.24 (d,

J= 9.0 Hz, 1H), 7.19 (dd, J= 7.6, 6.1 Hz, 2H), 7.01 (dd, J= 9.0, 2.3 Hz, 1H), 6.97 (dd, J= 8.0,

1.9 Hz, 2H), 3.14 (s, 3H). 13C NMR (125 MHz, MeOD, 6): 169.89, 169.33, 164.30, 164.02, 163.28,

162.59, 159.40, 136.79, 135.29, 133.99, 133.75, 133.39, 132.96, 132.48, 121.25, 119.60, 118.51,

117.73, 104.55, 96.89, 31.74. HRMS (ESI-QIT) m/z: [M+H]+ Calcd for C2 1HI 5NO4 346.1074,

found 346.1071.

Synthesis of Rhodols A.8-A.10. Rhodol triflate A.3-A.5 (1 equiv) was dissolved in 5 mL of 1:1

THF/MeOH solution. Aqueous 2 M KOH was added (4 equiv) and the mixture was stirred at room

temperature for 1 h, after which complete hydrolysis was observed by LCMS. The mixture was

214

neutralized with 1 M HCl and evaporated to dryness to obtain the rhodol product as an orange or

red powder.

Dimethyl urea rhodol (A.8). Orange solid (71 mg, 98.6% yield). 'H NMR (500 MHz,

(CD 3)2 SO, 6): 10.14 (s, lH), 8.59 (s, lH), 8.00 (d, J= 7.6 Hz, 1H), 7.79 (td, J= 7.5, 1.1 Hz, 1H),

7.72 (td, J= 7.6, 0.8 Hz, lH), 7.67 (d, J= 2.1 Hz, lH), 7.27 (d, J= 7.7 Hz, 1H), 7.15 (dd, J= 8.7,

2.1 Hz, lH), 6.70 (t, J= 1.3 Hz, 1H), 6.60 (d, J= 8.7 Hz, 1H), 6.55 (d, J= 1.3 Hz, 2H), 2.93 (s,

6H). "C NMR (125 MHz, (CD3 )2 SO, a): 171.89, 162.64, 158.46, 155.66, 155.02, 153.99, 146.26,

138.77, 133.24, 132.20, 130.89, 129.21, 127.79, 127.17, 118.57, 115.75, 114.55, 112.62, 109.01,

105.41, 39.38. HRMS (ESI-QIT) m/z. [M+H]+ Calcd for C23H1 7N205 403.1289; found 403.1286

Methyl carbamate rhodol (A.9). Red solid (81 mg, 99% yield). 'H NMR (500 MHz, (CD3) 2SO,

6): 8.59 (s, 1H), 8.00 (d, J= 7.6 Hz, 1H), 7.79 (td, J= 7.5, 1.1 Hz, 1H), 7.72 (td, J= 7.6, 0.8 Hz,

1H), 7.67 (d, J= 2.1 Hz, 1H), 7.27 (d, J= 7.7 Hz, 1H), 7.15 (dd, J= 8.7, 2.1 Hz, 1H), 6.70 (t, J=

1.3 Hz, 1H), 6.60 (d, J= 8.7 Hz, 1H), 6.55 (d, J= 1.3 Hz, 2H), 2.93 (s, 6H). 13C NMR (125 MHz,

(CD 3 ) 2 SO, a): 170.20, 160.90, 155.30, 153.98, 153.28, 152.60, 142.80, 137.06, 131.56, 130.51,

129.92, 127.51, 126.11, 125.44, 115.65, 114.10, 114.05, 110.94, 106.40, 103.69, 84.03, 53.24.

HRMS (ESI-QIT) m/z: [M+H]* Caled for C22 H1 4NO 390.0972; found 390.0965.

Amide rhodol (A.10). Dark red solid (62 mg, 85.2 % yield). 'H NMR (500 MHz, (CD 3 ) 2 SO,

6): 10.43 (s, 1H), 8.04-7.95 (m, 1H), 7.87 (d, J= 1.8 Hz, 1H), 7.69 - 7.58 (m, 2H), 7.20 (dd, J=

6.4, 1.7 Hz, 1H), 7.11 (dd, J= 8.7, 2.0 Hz, 1H), 6.75 (d, J= 8.7 Hz, 1H), 6.66 (d, J= 9.0 Hz, 1H),

6.51 - 6.36 (m, 2H), 2.02 (s, 3H). 13 C NMR (125 MHz, (CD 3 ) 2 SO, a): 172.21, 172.20, 171.44,

157.54, 154.62, 145.31, 136.44, 136.37, 135.88, 135.81, 133.13, 132.63, 131.69, 130.19, 130.03,

215

129.36, 129.06, 118.28, 117.73, 108.58, 106.09, 27.23. HRMS (ESI-QIT) m/z. [M+H] Calcd for

C22 H14NO5 374.1023; found 374.1017.

Synthesis of Rhodol Esters A.11-A12. To a suspension of rhodol A.8 or A.9 (37 pmol, 1 equiv)

in dichloromethane (2 mL) were added 4-dimethylaminopyridine (3.7 imol, 0.1 equiv) and

pyridine (81 pmol, 2.2 equiv). Acetyl chloride (81 tmol, 2.2 equiv) was added dropwise, and the

resulting solution was stirred for 1 h or until completion of the reaction. After dilution with water

and extraction with dichloromethane, the combined organic extracts were washed with saturated

aqueous NH4Cl and brine, dried with MgSO4(s), and concentrated under reduced pressure.

Purification by column chromatography on silica gel (0-40% v/v EtOAc in hexanes) afforded the

title compounds as white solids.

Dimethyl urea rhodol acetate (A.11). White foam (9.6 mg, 5 8.4% yield). I H NMR (500 MHz,

CDCl 3, a): 8.03 (d, J= 7.6 Hz, 1H), 7.71-7.60 (m, 2H), 7.58 (d, J= 2.2 Hz, 1H), 7.17 (d, J= 7.6

Hz, 1H), 7.06 (d, J= 2.1 Hz, 1H), 6.93 (dd, J= 8.6, 2.2 Hz, 1H), 6.84 - 6.76 (m, 2H), 6.69 (d, J=

8.6 Hz, 1H), 6.46 (s, 1H), 3.05 (s, 6H), 2.32 (s, 3H). 13 C NMR (125 MHz, CDCl 3, 6): 169.62,

169.16, 155.18, 153.26, 152.03, 152.01, 151.74, 144.85, 141.61, 135.31, 129.99, 129.11, 128.51,

126.41, 125.22, 124.23, 117.50, 116.73, 115.42, 112.79, 110.56, 107.17, 82.52, 36.64, 21.28.

HRMS (ESI-QIT) m/z: [M+H]* Calcd for C25H20N206 445.1387; found 445.1394.

Methyl carbamate rhodol acetate (A.12). White foam (11 mg, 68.9% yield). 'H NMR (500

MHz, CDCl 3, 6): 8.04 (d, J= 7.5 Hz, 1H), 7.66 (dd, J= 24.9, 1.0 Hz, 2H), 7.56 (s, 1H), 7.18 (d, J

7.5 Hz, 1H), 7.09 (d, J= 1.7 Hz, 1H), 6.93 (dd, J= 8.6, 2.1 Hz, 1H), 6.84-6.77 (m, 2H), 6.73 (d,

J= 8.6 Hz, 2H), 3.81 (s, 3H), 2.33 (s, 3H). 13 C NMR (125 MHz, CDCl 3, a): 203.54, 186.67, 169.45,

169.12, 153.70, 153.14, 152.07, 151.93, 151.85, 140.22, 135.34, 130.07, 129.14, 128.87, 126.43,

216

125.31, 124.16, 117.66, 116.70, 114.47, 113.60, 110.57, 106.32, 82.24, 52.78, 21.29. HRMS (ESI-

QIT) m/z: [M+H]+ Caled for C24H17NO7 432.1078; found 432.1076.

Synthesis of Rhodol Ethers A.13-A.14. Ag20 (21.4 mg, 2.5 equiv), rhodol A.8 or A.9 (37

pimol, 1 equiv), and powdered activated 4-A molecular sieves (50 mg) were added to an oven-

dried round-bottom flask. Anhydrous CH3CN (1 mL) was added, and the resulting suspension was

stirred under N2(g) for 5 min. To this mixture was added bromomethyl acetate (14.5 piL, 4 equiv)

dropwise, and the resulting mixture was stirred under N2(g) for 48 h. The reaction mixture was

then diluted with dichloromethane and filtered through a pad of Celite*. Purification by column

chromatography on silica gel (0-40% v/v EtOAc in hexanes with constant 40% v/v

dichloromethane as cosolvent) afforded the title compound as a white solid.

Dimethyl urea rhodol acetoxymethoxy ether (A.13) (12 mg, 68.3% yield). HRMS (ESI-QIT)

m/z: [M+H]+ Calcd for C26H22N207 474.1427, found 474.1424.

Methyl carbamate rhodol acetoxymethoxy ether (A.14) (9.7 mg, 57.2% yield). HRMS (ESI-

QIT) m/z: [M+H]+ Calcd for C25HIgNO8 462.1189; found 462.1194.

217

NMR Spectra

'H NMR (CDCL 3) and 13C NMR (CDC1 3) Spectra of Compound A.0

TfO .. OTf

J I Jilt-O 0 OV

I tI~J-U&J

10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0PPM

4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0

TfO 0 OTf

I I

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210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0PPM

218

II

'H NMR (CDC13) and 13 C NMR (CDC13) Spectra of Compound A.1

TfO 0 NHBoc

- N

0If Il/

l C CiLWC qCN~ nO-r..

10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0PPM

1.5 1.0 0.5 0.0 -0.5

TfO 0 NHBoc

-.- 0

210 200 190 180 170 160 150 140 130 120 110 100PPM

90 80 70 60 50 40I ' I 10 i I1

30 20 10 0 --10

219

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., :

. . I I I . I I I . I . . I I I . . . I . . . . . . . . . . . . . .

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'H NMR (CDC1 3) and 13C NMR (CDC1 3) Spectra of Compound A.2

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1.5 1.0 0.5 0.0 -0.5

160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10PPM

220

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06

'H NMR (CDCL3) and 13C NMR (CDC13) Spectra of Compound A.3

f If' Ii I f

HTfO 0 N N

00

8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0PPM

a% LAu Cr I L O% A4 ,a C%0 LOmLnLA v mM -N "4(

H|TfO 0 N N

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140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10PPM

221

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'H NMR (CDC 3) and 13C NMR (CDC13) Spectra of Compound A.4

a 11, 113 ~oIr:r6? 0_ _ __-_N_ _-r r.rr, Dt

H ITfO O N N,

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H ITfO 0 N N

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0

210 200 190 180 170

II

i i I

m~mmmiinsmm -

222

f If I IJ

160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10PPM

'H NMR (CDC13) and 1 3 C NMR (CDC13) Spectra of Compound A.5

OqLj .- NN t2 I a' 2 -gg ;* er ,N000 O08N

HTfO OU N

0

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223

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'H NMR ((CD3)2SO) and 13C NMR ((CD 3 )2 SO) Spectra of Compound A.6

0

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224

10.5

p

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'H NMR (MeOD) and "C NMR (MeOD) Spectra of Compound A.7

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1H NMR ((CD 3)2SO) and 1 3 C NMR ((CD 3)2SO) Spectra of Compound A.8

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200 190 180 170 160 150 140 130 120 110 100 90 80 70 60PPM

50 40 30 20 10

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'H NMR ((CD 3 ) 2SO) and 13 C NMR ((CD 3 ) 2SO) Spectra of Compound A.10

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228

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'H NMR (CDC1 3) and 13C NMR (CDC1 3) Spectra of Compound A.11

1 1 iIf I f

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200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0PPM

229

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'H NMR (CDCL 3) and 13C NMR (CDCL 3) Spectra of Compound A.12

06~ r-6 r

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210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 -10PPM

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