the living cell -...

X

X

The Living Cell

Living cells are a complex network of interacting biopolymers, ions, and metabolites.

Complex cellular processes cannot be observed when the components are in their purified, isolated forms.

Thus, we are forced outside of the artificial confines of a test tube and into the dynamic living cell.

There is a burgeoning interest in developing methods where a reporter tag can be attached to a cellular component for aid in visualization and/or isolation.

David Goodshell, artist and biologist

Prescher, J. A.; Bertozzi, C. R. Nat. Chem. Biol. 2005, 1, 13-21.

X

X

Bioorthogonal Chemistry - Requirements

water as the solvent

Sletten, E. M.; Bertozzi, C. R. Angew. Chem. Int. Ed. 2009, 48, 6974-6998.

ambient temperature

fast reaction kinetics

physiological pH

reaction product must be stable

no cross-reactivity

cell-permeable reagents

non-toxic reagents

X

X

A Brief Consideration of Reaction Kinetics

most bioorthogonal reactions are bimolecular in nature with second-order rate constants

Lim, R. K. V.;Lin, Q. Chem. Commun. 2010, 46, 1589-1600.


A B A B

k2 = 2.3 M-1s-1, [A] = [B] = 1 µM, time = 1 hr

~ 0.8 % labeling of product

[product] is approximately k2 [A]0[B]0t k2 is typically anywhere from 10-3 to 103 M-1s-1

X

X

Biosynthesis of mucin-type O-linked glycoproteins

Hang, H. C.; Yu, C.; Kato, D.; Bertozzi, C. R. Proc. Natl. Acad. Sci. USA 2003, 100, 14846-14851.

Dube, D. H.; Prescher, J. A.; Quang, C. N.; Bertozzi, C. R. Proc. Natl. Acad. Sci. USA 2006, 103, 4819-4824.

OHO

HO

NH

OH

N-AcetylglucosamineGlcNAc

N-AcetylegalactosamineGalNAc

O

Me

O

HO

NH

O

Me

OH

OH OH OH

GalNAc1-kinase

O

HO

NH

O

Me

OPO3-2

OH OH

GalNAc-1-P

GlcNAcsalvagepathway

OHO

HO

NH

OH

O

Me

OP

OP

O

ON

NH

O

O

OHHO

O O OO

UDP-GlcNAc

X

X

Biosynthesis of mucin-type O-linked glycoproteins

Hang, H. C.; Yu, C.; Kato, D.; Bertozzi, C. R. Proc. Natl. Acad. Sci. USA 2003, 100, 14846-14851.

Dube, D. H.; Prescher, J. A.; Quang, C. N.; Bertozzi, C. R. Proc. Natl. Acad. Sci. USA 2006, 103, 4819-4824.

UDP-GlcNAcGalNAc-1-P

UDP-GalNAcpyrophosphorylase

UDP-Glc/GlcNAcC4-epimerase

O

HO

NH

OH

O

Me

OP

OP

O

ON

NH

O

O

OHHO

O O OO

UDP-GalNAc

OH

X

X

Bioorthogonal Chemistry

R''NH2

O

R'R

N

R'R

R''

OMe

O

PPh2

RN3 NHR

O

P(O)Ph2

RN3

NN N

R

RSR'

RSR'

condensation chemistry

Staudingerligation

[3+2] cycloadditions

olefin chemistry

X

X

Condensation Chemistry

Cornish, V. W.; Hahn, K. M.; Schultz, P. G. J. Am. Chem. Soc. 1996, 118, 8150-8151.

Zhang, Z.; Smith, B. A. C.; Wang, L.; Brock, A.; Cho, C.; Schultz, P. G. Biochemistry 2003, 42, 6735-6746.

! Unnatural amino acid mutagenesis was performed on T4 lysozyme

O

Me

NH2

CO2H

O

Me

O

CO2H

NH2

incorporated at two solvent

accessible sites - Ser44 and Ala82

5% efficiency 30% efficiency

determined by catalytic activity, SDS-PAGE, and autoradiography

X

X


Mahal, L. K.; Yarema, K. J.; Bertozzi, C. R. Science 1997, 276, 1125-1128.

! Cell surface remodeling

OHO

HO

HN

OH

HO

O

O

Me

ManLeva sialic acid analogue

O

CO2-

O

HO

HN

OMe

O

HO

OH

OH

O

NHNH2R

HydrazideO

CO2-

O

HO

HN

OMe

N

HO

OH

OH

HN

ONH

O

5

S

NHHN

O

4

biotin

Keppler, O. T.; Horstkorte, R.; Pawlita, M.; Schmidts, C.; Reutter, W. Glycobiology 2001, 11, 11R- 18R.

ManNAc. We selected three human celllines—Jurkat (T cell–derived), HL-60(neutrophil-derived), and HeLa (cervicalepithelial carcinoma)—to test the biosyn-thetic conversion of ManLev to the cor-responding cell surface–associated, unnat-ural sialic acid (Fig. 1A). Cells were treat-ed with ManLev, and the presence of ke-tone groups on the cell surface wasdetermined by the chemoselective ligationof a hydrazide-based probe, biotinamido-caproyl hydrazide (Fig. 1B). The cells wereanalyzed by flow cytometry after beingstained with FITC (fluorescein isothiocya-nate) avidin (15). The Jurkat, HL-60, andHeLa cells treated with ManLev showed alarge increase in fluorescence intensitycompared to cells treated with buffer orthe natural derivative ManNAc (Fig. 2).ManLev-treated cells that were stainedwith FITC-avidin alone, without prior bi-otinamidocaproyl hydrazide treatment,showed only a background fluorescence.These results indicate that ManLev-treat-ed cells express cell surface–associated ke-tone groups and can be chemoselectivelydecorated with hydrazide conjugates evenin the presence of serum.

We performed a series of experiments todemonstrate that the ketone groups are dis-played on the cell surface in the form ofmodified sialoglycoconjugates. Jurkat cellswere treated with tunicamycin, an inhibitorof N-linked protein glycosylation, before in-cubation with ManLev (16, 17). We antic-ipated a dramatic reduction in ketone ex-pression on the basis of the observation thatmost mature (and therefore sialylated) oligo-saccharides on Jurkat cells are found on N-linked rather than O-linked glycoproteins(18). Indeed, ketone expression resultingfrom ManLev treatment was inhibited bytunicamycin in a dose-dependent fashion(Fig. 3A), suggesting that the ketone groupsare presented on oligosaccharides and arenot nonspecifically associated with cell sur-face components. In contrast, ketone expres-sion in HL-60 and HeLa cells was unaffectedby tunicamycin, but was instead blocked bya-benzyl N-acetylgalactosamine, an inhibi-tor of O-linked glycosylation, consistentwith the high expression of mucin-like mol-ecules on myeloid- and epithelial-derivedcell lines.

Although we predicted that ManLevwould be converted into the correspond-ing sialoside, we addressed the possibilitythat ketone expression resulted from con-version of ManLev to N-levulinoyl glu-cosamine (GlcLev) by the enzyme thatinterconverts ManNAc and GlcNAc (12).In that GlcNAc is incorporated into mostglycoproteins, GlcLev might have manyavenues for cell surface expression. Flowcytometry revealed only a background lev-

el of fluorescence, suggesting that unnat-ural sialosides are the major biosyntheticproducts of ManLev.

Ideally, proof of the cell surface expres-sion of ketone-bearing sialic acids wouldinvolve abrogating the fluorescence signalby treatment with sialidase enzymes. How-ever, the commercially available sialidaseswere found to be inactive against N-levu-linoyl sialosides, in accordance with theirknown reduced activity against sialosideswith other unnatural N-acyl groups (19).We therefore evaluated the effects ofManLev treatment on the amount of nor-mal sialic acid on Jurkat cells, expecting areduction. Indeed, the amount of sialicacid released from ManLev-treated cells

by sialidase digestion, as quantified byhigh pH anion exchange chromatography(HPAEC), was approximately 10 timeslower than that released from ManNAc-treated cells (13, 20).

There are two possible explanations forthe observed reduction in normal sialic acidon ManLev-treated cells: (i) normal sialicacid is replaced with the unnatural sialicacid during incubation with ManLev, or (ii)the biosynthesis of all sialosides is sup-pressed during incubation with ManLev.Inhibition of sialoside biosynthesis wouldcause an increase in exposed terminal ga-lactose residues, the penultimate residue inthe majority of sialoglycoconjugates. Wetherefore examined the effect of ManLev

Fig. 1. Biosynthetic incorpora-

tion of ketone groups into cell

surface–associated sialic acid.

(A) N-Levulinoyl mannosamine

(ManLev, ML) is metabolically converted to the corresponding cell surface sialoside. (B) Cells

displaying ketone groups can be chemoselectively ligated to hydrazides under physiological condi-

tions through the formation of an acyl hydrazone. Cell surface ketones were conjugated to biotinami-

docaproyl hydrazide to provide a tag for subsequent detection with FITC-avidin. In principle, any

hydrazide-derivatized molecule can be used to selectively remodel the surface of ketone-expressing

cells.

Fig. 2. Ketone expression in Jurkat, HL-60, and HeLa cells. Cells treated with buffer alone or with

ManNAc showed only background fluorescence. Cells treated with ManLev showed up to a 30-fold

increase in fluorescence above background. The fluorescence increase was dependent on both bio-

tinamidocaproyl hydrazide (Bio) and FITC-avidin (Av) treatment. Results were similar in three replicate

experiments for each cell line.

SCIENCE z VOL. 276 z 16 MAY 1997 z www.sciencemag.org1126

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! Cell surface remodeling

cells were analyzed by flow cytometry after staining with FITC (fluorescein isothiocyanate) avidin

X

X



Addition of tunicamycin, a known inhibitor of N-linked protein glycosylation, inhibits ketone expression with ManLev treatment in Jurkat cells.

O

HO

HO

NH O

HO

O

HO

HO

N NH

O

O

O

NHAc

OHHO

HO

O

Me

Me

tunicamycin

Ketone expression was blocked on HL-60 and HeLa cells via alpha-benzyl N-acetylgalactosamine, an inhibitor of O-linked glycosylation.

OHO

HO

NH

OH

O

Me

alpha-benzyl N-acetylglucosamine

O

X

X



! Selective drug delivery?

O

Me O

H2NHN Biotin

N

Me

HN

O

Biotin

N

Me

HN

O

Biotin S

S

drug

avidin

N

Me

HN

O

Biotin

selective drugdelivery

S

S

drug

avidin

X

X



! Selective drug delivery?

ricin

inhibits protein synthesis

toxicity of the conjugate was dependent on the expression of ketones

cells with high ketone expression (~700,000 ketones per cell)were sensitive to lethal doses of ricin

LD50 between 1 to 10 nM

cells with low ketone expression (~50,000 ketones per cell) showed no toxicity

indicates the potential for cell surface engineering to support selective drug delivery

X

X


R''NH2

O

R'R

N

R'R

R''

OMe

O

PPh2

RN3 NHR

O

P(O)Ph2

RN3

NN N

R

RSR'

RSR'


Staudingerligation


olefin chemistry

X

X

Azide - A Powerful Chemical Reporter

N N N

Griffin, R. J. Prog. Med. Chem. 1994, 31, 121.

Hendricks, S. B.; Pauling, L. J. Am. Chem. Soc. 1925, 47, 2904.

Saxon, E.; Bertozzi, C. R. Science 2000, 287, 2007.

absent from biological systems

possesses orthogonal reactivity to most biological functional groups

the azide is small, so biological perturbation is minimal

first used as a chemical reporter in 2000 in the Staudinger Ligation

edly with the extensive biotinylation of azido-sugar-treated cells reacted with phosphine 5(Fig. 5A). We conclude that the chemoselectiveligation reaction proceeds as designed withoutcomplications arising from nonspecific amineacylation.

To satisfy the requirement of chemical or-thogonality, both participants in the reactionmay not engage functional groups endogenousto cells. Triarylphosphines are mild reducingagents, which raises the the possibility of disul-fide bond reduction as an undesirable side re-action. We addressed this issue by incubatingJurkat cells with triarylphosphine 4, an interme-diate in the synthesis of 5, and quantifying theappearance of free sulfhydryl groups on the cellsurface with iodoacetylbiotin and FITC-avidin(Fig. 5B). After 1 hour in the presence of 1 mM4, no detectable increase in free sulfhydrylgroups was observed relative to cells exposed toiodoacetylbiotin alone. In a positive control ex-periment, we incubated Jurkat cells with thetrialkylphosphine TCEP (1 mM, 1 hour), acommercial disulfide bond reducing agent. Amarked increase in free cell-surface sulfhydrylgroups was observed in this case (Fig. 5B). Weconclude that triarylphosphines such as 4 and 5are essentially unreactive toward disulfidebonds under these conditions, rendering liga-tion with azides the predominant pathway forreactivity.

In a side-by-side comparison with our pre-viously reported cell surface ketone reaction(2), the cell surface Staudinger process wassuperior in several respects. Using the samereagent concentrations, azidosugar metabolismfollowed by phosphine reaction produced two-fold higher fluorescence than ketosugar metab-olism followed by hydrazide reaction. This mayreflect either a faster reaction at the cell surfaceor more efficient metabolism of azidosugar 3 ascompared with ketosugar 2 (Fig. 1). The azidehas a major advantage over the ketone in that itsreactivity is unique in a cellular context owingto its abiotic nature. Ketones, by contrast,

abound inside cells in the form of metabolitessuch as pyruvic acid and oxaloacetate. Themodified Staudinger reaction is chemically or-thogonal to ketone ligations and should allowtandem modification of cell surfaces with thetwo chemistries.

The susceptibility of azides to reductionduring the metabolic process warrants someconsideration in light of the reducing potentialof the cell’s interior. Monothiols such as gluta-thione can reduce alkyl azides at alkaline pH,but the rates of such reactions under physiolog-ical conditions are insignificant on the timescale of our experiments (13). Correspondingly,metabolic studies of the azido drug AZT(azidothymidine) showed 90% recovery of theazide, either in its administered form or metab-

olized to the glucuronidated compound, withoutsignificant reduction (14).

The delivery of azides to cell surfacesthrough other carbohydrate biosynthetic path-ways could significantly expand applicationsof cell surface engineering. Azides and phos-phines are abiotic structures both inside andoutside cells, which raises the exciting possi-bility that their ligation could proceed in theintracellular environment. Given existingpowerful methods for incorporating unnaturalbuilding blocks into other biopolymers, oneneed not be restricted to cell surface oligo-saccharides as hosts for these chemical han-dles (15, 16). Azido–amino acids, for exam-ple, could be introduced into proteins andlater targeted with phosphine probes. The

Fig. 4. (A) Analysis of cell sur-face reaction by flow cytom-etry. Jurkat cells (1.25 3 105

cells per milliliter) were cul-tured in the presence or ab-sence (control) of acetylated 3(20 mM for 3 days). The cellswere washed twice with 1 mlof buffer (0.1% fetal bovineserum in PBS, pH 7.4) and di-luted to a volume of 240 ml.Samples were added to 60 mlof a solution of 5 (5 mM inPBS, pH 7.4) and incubated atroom temperature for 1 hour.The cells were washed and re-suspended in 100 ml of buffer, then added to 100 ml of FITC-avidin stainingsolution (1: 250 dilution in PBS). After a 10-min incubation in the dark at4°C, the cells were washed with 1 ml of buffer and the FITC-avidin stainingwas repeated. The cells were washed twice with buffer, then diluted to avolume of 300 ml for flow cytometry analysis. Similar results were

obtained in two replicate experiments. (B) Progress of reaction over time.Assays were performed as in (A) with 40 mM acetylated 3 and varying theduration of the reaction with compound 5. (C) pH profile of reaction. Assayswere performed as in (A) with 40 mM acetylated 3 and varying the pH of thebuffer used during incubation with compound 5.

Fig. 5. Specificity of the modified Staudingerreaction. (A) Cell surface biotinylation does notproceed by classical Staudinger azide reductionfollowed by nonspecific acylation. Jurkat cellswere cultured in the presence of acetylated 3as described in Fig. 4. Cell surface azides wereeither reduced intentionally with a trisulfon-ated triphenylphosphine or left unreduced.Phosphine oxide 6, the product of the classicalStaudinger reaction, was prepared indepen-dently and incubated with the cells (1 mM for1 hour). Analysis by flow cytometry was per-formed as in Fig. 4. (B) Triarylphosphines do notreduce disulfide bonds at the cell surface. Jurkatcells were incubated with a 1 mM solution oftriarylphosphine 4 or TCEP for 1 hour at roomtemperature. The cells were centrifuged (2 min,3000 g), washed with PBS, and diluted to avolume of 240 ml. Samples were combined with60 ml of a solution of iodoacetylbiotin (5 mM inPBS). After incubation in the dark at roomtemperature for 1.5 hours, the cells werewashed with buffer, stained with FITC-avidin,and analyzed by flow cytometry. In both (A)and (B), error bars represent the standard de-viation of two replicate experiments.

R E P O R T S

www.sciencemag.org SCIENCE V OL 287 17 MARCH 2000 2009

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The Staudinger Ligation

Jurkat cells were incubated with azidoacetylmannosamine at a concentration 20 mMfor three days.


Cell viability was tested with incubation for up to six days.

Cells were washed and reacted with phosphine for 1 hour at a concentration of 1 mM.

Stained with FITC-avidin and analyzed by flow cytometry.












R E P O R T S


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R E P O R T S


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X

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The Staudinger Ligation

Biological Controls -


Could a Staudinger reduction be taking place and the phosphine oxide localizesin or outside the cell?

Could the phosphine be reducing disulfide bonds and not be completely bioorthogonal?

P

O

OHHO

O

OHO

TCEP

X

X

Activation of a Fluorgenic Dye via the Staudinger Ligation

Lemieux, G. A.; de Graffenried, C. L.; Bertozzi, C. R. J. Am. Chem. Soc. 2003, 125, 4708-4709.

The lone pair of electrons on phosphorus quench the excited fluorophore

N O O

P

CO2Me

coumarin dye

not fluorescent

O

N3NH

Me

CH3CN/H2O

N O O

P

O

Ph

O

NH

O

NH

Me

strongly fluorescent

CO2H

NH2

N3

L - azidohomoalanine

Phosphine dye was reacted with recombinant murine dihydrofolate reductase bearing azidohomoalanine residues.

Unnatural amino acids incorporated during overexpression in a methionine auxotrophic E. coli strain.

Fluorescence was observed over background.

No washing or Western blotting necessary.

X

X


Hangauer, M. J.; Bertozzi, C. R. Angew. Chem. Int. Ed. 2008, 47, 2394.

A small problem

N O O

P

CO2Me

not fluorescent

N O O

P

O

Ph

strongly fluorescent

[O]

MeO2C

Oxidation of the probe by air would provide background fluorescence.

X

X


Hangauer, M. J.; Bertozzi, C. R. Angew. Chem. Int. Ed. 2008, 47, 2394.

Fluorescence resonance energy transfer (FRET) based probe

O

O

N

O

N

O

O

OHO

PPh2

N

Me

N

N

NO2

2

FRET

no fluorescence

O

NHR

N

O

N

O

O

OHO

PPh2

O

fluorescence

+

N

Me

N

N

NO2

HO

RN3, H2O

quencher

X

X

"Traceless" Staudinger Ligation

Soellner, M. B.; Nilsson, B. L.; Raines, R. T. J. Am. Chem. Soc. 2006, 128, 8820.

Peptide coupling by the traceless Staudinger ligation

N3 Peptide 2

O

SPeptide 1 PPh2

O

SPeptide 1 PPh2

NPeptide 2

N2

O

Peptide 1 NPeptide 2

PPh2

SH

H2OO

Peptide 1 NH

Peptide 2

Ph2P SH

O

Nilsson, B. L.; Kiessling, L. L.; Raines, R. T. Org. Lett. 2000, 2, 1939.

X

X

"Traceless" Staudinger Ligation

Dawson, P. E.; Muir, T. W.; Clarklewis, I.; Kent, S. B. H. Science 1994, 266, 776.

Traceless Staudinger ligation is reminiscent of native chemical ligation

O

SRPeptide 1

O

Peptide 2H2N

HS

thioesterificationO

SPeptide 1

H2NPeptide 2

O

S-N acyl shift O

NH

Peptide 1

SH

Peptide 2

O

X

X

Staudinger Ligation

Prescher, J. A.; Dube, D. H.; Bertozzi, C. R. Nature 2004, 430, 873.

The Staudinger ligation has been used to probe biomolecules within living animals.

OMe

O

PPh2

RN3 NHR

O

P(O)Ph2

Staudingerligation

Second-order kinetics where the rate-determining step is purported to be attack of the phosphine on the azide.

k = 10-3 M-1s-1 (very slow)

Lin, F. L.; Hoyt, H. M.; Van Halbeek, H.; Bergman, R. G.; Bertozzi, C. R. J. Am. Chem. Soc. 2005, 127, 2686.

High concentrations of the phosphine are often necessitated (> 250 uM)

Attempts to increase phosphine nucleophilicity has increased the suspectibility of phosphine oxidation.

X

X


R''NH2

O

R'R

N

R'R

R''

OMe

O

PPh2

RN3 NHR

O

P(O)Ph2

RN3

NN N

R

RSR'

RSR'


Staudingerligation


olefin chemistry

X

X

[3 + 2] Cycloadditions

Tornoe, C. W.; Christensen, C.; Meldal, M. J. Org. Chem. 2002, 67, 3057.

RN3

Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. Angew. Chem. Int. Ed. 2002, 41, 2596.

R'

NN N

R

R'

PhON N N

Ph

CuSO4.5H2O 1 mol%

sodium ascorbate 5%

H2O:tBuOH, RT

NN N

PhO

Ph

91%

In separate efforts, Meldal and Sharpless reported the copper-catalyzed azide-alkyne 1,3-dipolar cycloaddition (CuAAC).

First discovered by Arthur Michael in 1893.

In the 1950s, Rolf Huisgen proposed that the reaction proceeds through a 1,3-dipolar cycloaddition.

High temperatures and pressure required made this reaction largely impractical.

X

X

[3 + 2] Cycloadditions

Himo, F.; Lovell, T.; Hilgraf, R.; Rostovtsev, V. V.; Noodleman, L.; Sharpless, K. B.; Fokin, V. V. J. Am. Chem. Soc. 2005, 127, 210-216.

[LnCu]+

R CuLn-1

R CuLn-2

NR'N

N

NN

N

CuLn-2

R

R'

NNN

R'

R CuLn-1

CatalyticCycle

HR

N N N

R'

NNN

R'

R

alkyne

azide

1,4-disubstituted1,2,3-triazoles

copper acetylide

copper(III)metallacycle

X

X

Copper(I) Toxicity

Speers, A. E.; Adam, B. F.; Cravatt, B. F. J. Am. Chem. Soc. 2003, 125, 4686.

PhON N N

Ph

CuSO4.5H2O 1 mol%

sodium ascorbate 5%

H2O:tBuOH, RT

NN N

PhO

Ph

91%

Finn, Sharpless, and coworkers reported the first biomolecule coupling application of [3+2] cycloaddition through the attachment of dyes to the cowpea mosaic virus.

CuAAC is not widely employed, due to copper(I)'s toxicity.

Link, A. J.; Tirrell, D. A. J. Am. Chem. Soc. 2003, 125, 11164.

Chan, T. R.; Hilgraf, R.; Sharpless, K. B.; Fokin, V. V. Org. Lett. 2004, 6, 2853.

E. Coli stops dividing after exposure to 100 uM CuBr for 16 hours.

Mammalian cells and zebrafish embryos can survive low concentrations of copper (I) (< 500 uM) but considerable cell death is observed above 1 mM.

Wang, Q.; Chan, T. R.; Hilgraf, R.; Fokin, V. V.; Sharpless, K. B.; Finn, M. G. J. Am. Chem. Soc. 2003, 125, 3192-3193.

X

X

[3+2] Cycloaddition - Ring Strain

Alder, K.; Stein, G. Justus Liebigs Ann. Chem. 1931, 485, 211.

Strain-promoted azide cycloadditions were first investigated by Alder and Stein in the 1930s.

Wittig, G.; Krebs, A. Chem. Ber. 1961, 94, 3260.

N NN

In the 1960s, Krebs and Wittig commented that phenylazide and cyclooctyne

NN

N

"proceeded like an explosion"

massive bond angle deformation of the acetylene to 163o

~ 18 kcal/mol of ring strain

X

X

In Vivo Imaging of Zebrafish Embryos

Laughlin, S. T.; Baskin, J. M.; Amacher, S. L.; Bertozzi, C. R. Science 2008, 320, 664.

F

FO

O

HN

DIFO

copper-freeclick chemistryN3

imaging of dynamic biological processes usingthese chemistries could be complicated by slowreaction kinetics or reagent toxicity. The copper-free click reaction of azides with difluorinatedcyclooctyne (DIFO) reagents (10) overcomes theselimitations, suggesting its potential application toin vivo imaging.

We chose zebrafish as a model organism be-cause of their well-defined developmental pro-gram (11), emerging disease models (12), andamenability to optical imaging. The metabolicsubstrate peracetylated N-azidoacetylgalactosamine(Ac4GalNAz) was selected on the basis of itsknown incorporation into mucin-type O-linkedglycoproteins inmammalian cells andmice via theN-acetylgalactosamine (GalNAc) salvage pathway(13, 14) (fig. S1). We envisioned an imaging ex-periment (Fig. 1A) in which zebrafish embryosare incubated with Ac4GalNAz and their glycansare visualized by reaction with DIFO-fluorophoreconjugates (fig. S2).

Before performing imaging experiments, weconfirmed that the zebrafish glycan biosyntheticenzymes are permissive of the unnatural sugar.The zebrafish cell line ZF4 (15) was incubatedwith various doses of Ac4GalNAz, reacted witha DIFO–Alexa Fluor 488 conjugate (DIFO-488,fig. S2), and analyzed by flow cytometry (Fig. 1B).Robust dose-dependent metabolic labeling was ob-served, similar to that of mammalian cells (13, 14).We further characterized the azide-labeled celllysates by treatment with a DIFO–Flag peptideconjugate (10). The observed high-molecular-weight species were consistent with labeledglycoproteins (fig. S3). We then purified the Flag-containing species (16) and identified severalglycoproteins (b-hexosaminidase, b-integrin 1b,lysosome-associated membrane protein, nicastrin,scavenger receptor B, and Thy1) with known(17–19) or predicted (20) sites of mucin-type O-linked glycosylation (fig. S4). We concluded thatAc4GalNAz was metabolically incorporated intoglycoproteins in zebrafish-derived cells.

We next evaluated Ac4GalNAz labeling invivo. Zebrafish embryoswere incubated inmediacontaining either Ac4GalNAz or, as a control,peracetylated GalNAc (Ac4GalNAc) from 3 to120 hours post-fertilization (hpf). Whole-animallysates were then reacted with a phosphine-Flagprobe (21) (fig. S2) and analyzed (Fig. 1C).The labeled glycoproteins were refractory to di-gestion with peptide N-glycosidase F or chon-droitinase ABC (fig. S5), which suggests thatGalNAz is primarily incorporated into mucin-type O-linked glycoproteins.

To image azide-labeled glycans in vivo,we incubated zebrafish embryos with eitherAc4GalNAz or Ac4GalNAc from 3 to 72 hpfand then reacted the embryos with a DIFO–Alexa Fluor 647 conjugate (DIFO-647, fig. S2).Robust fluorescence was observed with virtuallyno background (Fig. 2A). Even after a 1-min re-action with DIFO-647, the Ac4GalNAz-treatedembryos displayed substantial fluorescence thatincreased in a time-dependent manner (Fig. 2B).We observed no toxicity or developmental abnor-malities resulting from treatment withAc4GalNAz

or any DIFO reagents (fig. S6 and supporting on-line material text).

We then assessed global patterns of glycosyl-ation by incubating embryos with Ac4GalNAzstarting at 3 hpf, followed by reaction withDIFO-647 at 12-hour intervals over a 5-day pe-riod. We observed azide-labeled glycans as earlyas 24 hpf (Fig. 2C and fig. S6). Starting at 60 hpfand continuing until at least 72 hpf, we observeda burst in fluorescence intensity in the jaw region,pectoral fins, and olfactory organs (Fig. 2, D andE). Thus, we focused on 60 to 72 hpf for more

1Department of Chemistry, University of California, Berkeley,CA 94720, USA. 2Department of Molecular and Cell Biology,University of California, Berkeley, CA 94720, USA. 3HowardHughes Medical Institute, University of California, Berkeley,CA 94720, USA. 4The Molecular Foundry, Materials SciencesDivision, Lawrence Berkeley National Laboratory, Berkeley, CA94720, USA.

*These authors contributed equally to this work.†To whom correspondence should be addressed. E-mail:[email protected]

Fig. 1. Ac4GalNAz is metabolicallyincorporated into zebrafish glycans.(A) Schematic depicting the use ofmetabolic labeling with Ac4GalNAzand copper-free click chemistry usingDIFO probes for the noninvasiveimaging of glycans during zebrafishdevelopment. (B) Flow cytometry anal-ysis of ZF4 cells metabolically labeledwith Ac4GalNAz. ZF4 cells were incu-bated with Ac4GalNAz (0 to 100 mM,3 days) and subsequently reacted withDIFO-488 (10 mM, 1 hour). Error barsrepresent the standard deviation fromthree replicate samples. (C) Immuno-blot analysis of lysates from zebrafishembryos at 120 hpf incubated withAc4GalNAc (Ac) or Ac4GalNAz (Az),probed with horseradish peroxidase–conjugated antibody to Flag (toppanel) or antibody to b-tubulin(bottom panel).

Fig. 2. In vivo imaging ofglycans during zebrafishdevelopment. (A and B)Zebrafish embryos weremetabolically labeled withAc4GalNAz (Az) or Ac4GalNAc(Ac) starting at 3 hpf. (A)Embryos were reacted at72 hpf with DIFO-647 for1 hour. The right panel in-dicates an exposure timethat is 20 times longer thanthat in the other two panels.(B) Embryos were reacted at72 hpf with DIFO-647 for1 to 60 min. Asterisks de-note autofluorescence. (C)Zebrafish embryos incu-bated with Ac4GalNAz orAc4GalNAc (fig. S6) startingat 3 hpf were reacted withDIFO-647 at 24 hpf andsubsequently at 12-hourintervals, viewed laterallyand ventrally (alternatingpanels). (D and E) Zebrafishfrom (C) imaged at highermagnification at 60 hpf (D)or 72 hpf (E), viewed laterally (left panels) and ventrally (right panels). Solid arrowhead, olfactory organ; openarrowhead, pectoral fin. Dotted line indicates the pharyngeal epidermis in the jaw region. Scale bars in (A) and(C), 500 mm; in (B), (D), and (E), 200 mm.

www.science ma g .org SCIE N C E V O L 320 2 M AY 2008 66 5

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on

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7,

20

12

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w.s

cie

nce

ma

g.o

rgD

ow

nlo

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ed

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m

X

X



First confirmed that zebrafish glycan biosynthetic enzymes are tolerant of the unnatural sugar.

Zebrafish cell line ZF4 was incubated with various doses of Ac4GalNAzm reacted with DIFO-488 and analyzed by flow cytometry.

Azide-labeled cell lysates were further characterized by treatment with a DIFO-Flag peptide conjugate.

Observed high-molecular weight species were consistent with labeled glycoproteins.

Flag-containing species (glycoproteins like b-hexosaminidase, b-integrin, nicastrin) were knownor predicted sites of mucin-type O-linked glycosylation.

X

X



From 60 hours post-fertilization (hpf) to 72 hpf, a burst in fluorescence intensity in the jaw region,pectoral fins, and olfactory organs.

These three areas are reexamined and reacted with DIFO-647 between 60 and 61 hpf and DIFO-488 between 61 and 62 hpf.

mouth region pectoral fin jaw region olfactory region

X

X



Three-dye bioimaging - DIFO-647 between 60 and 61 hpf, DIFO-488 between 62 and 62 hpf, andthen DIFO-555 between 72 and 73 hpf.

jaw region olfactory region

kinociliacells

X

X

DIFO Synthesis

Baskin, J. M.; Prescher, J. A.; Laughlin, J. T.; Agard, N. J.; Chang, P. V.; Miller, I. A.; Lo, A.; Codelli, J. A.; Bertozzi, C. R. Proc. Natl. Acad. Sci. USA 2007, 104, 16793.

OH

HO

1) NaH2)

Br

52%

OH

O

O

O

OSiEt3

O

Selectfluor

95%1.0:2.2

PCC

91%

1) LHMDS, - 78o C

2) TESCl

91%

O

O

F

O

O

F

+

cat. LHMDS, 99%, 1:1

X

X

DIFO Synthesis

Baskin, J. M.; Prescher, J. A.; Laughlin, J. T.; Agard, N. J.; Chang, P. V.; Miller, I. A.; Lo, A.; Codelli, J. A.; Bertozzi, C. R. Proc. Natl. Acad. Sci. USA 2007, 104, 16793.

O

O

F1) KHMDS, - 78 oC

2) TESCl

97%4:1 desired:undesired

OTES

O

F

Selectfluor

74%

O

O

F

F

RuCl3NaIO493%

O

O

HO2C

F

F

OTf

O

HO2C

F

F

LDA, 0 oC

11%

1) KHMDS, - 78 oC

2) Tf2NPh

47%O

HO2C

F

F

X

X

Cyclooctyne Analogues

Sletten, E. M.; Bertozzi, C. R. Org. Lett. 2008, 10, 3097.

NF

F

N

MeO

MeO

O

HOOC

HO

Ning, X. H.; Guo, J.; Wolfert, M. A.; Boons, G. J. Angew. Chem. Int. Ed. 2008, 47, 2253.

Sletten, E. M.; Bertozzi, C. R. Acc. Chem. Res. 2009, 44, 666-676.

DIFO, Bertozzi Lab

k = 0.076 M-1s-1

DIMAC, Bertozzi Lab

more water soluble

k = 0.003 M-1s-1

DIBO, Boons Lab

5 steps

nontoxic, k = 0.057 M-1s-1

O

HOOC

O

ON

OH

BARAC, Bertozzi Lab

k = 0.96 M-1s-1

O

OBuHO

Boons and Popik Labs

photocaged

k = 0.076 M-1s-1

X

X


R''NH2

O

R'R

N

R'R

R''

OMe

O

PPh2

RN3 NHR

O

P(O)Ph2

RN3

NN N

R

RSR'

RSR'


Staudingerligation


olefin chemistry

X

X

Inverse-Electron Demand Diels-Alder

Blackman, M. L.; Royzen, M.; Fox, J. M. J. Am. Chem. Soc. 2008, 130, 13518-13519.

N

N N

N

R

R

N

NN

NR

R

N

N

H

H

R

R

-N2

N

HN

O

O

O O

Trx SH

N

HN

O

O

O O

STrx

thioredoxin

k2 = 2000 M-1s-1

N NH

R R

O

NH

O

N

S

O

OTrx

N

N N

N

N

N

available 5-amino-2-cyanopyridine (7) and 2-cyanopyridine (6)

(Scheme 3b). The amino group of tetrazine 8 provides a handle

for functionalization via acyl transfer (e.g., 9).15

To illustrate compatibility of the tetrazine ligation with proteins,

we functionalized thioredoxin (Trx) with trans-cyclooctene deriva-

tive 10. Trx is a 11.7 kDa protein that contains a single disulfide.

Upon reduction, the solvent exposed cysteine can be selectively

functionalized by maleimides.16 Thus, Trx (15 µM) was reducedwith tri(3-hydroxypropyl)phosphine (THP, 1 mM) and combined

with 10 (15 µM) in acetate buffer (pH 6). ESI mass spectral analysis(Figure 1b) indicated that most of the Trx had been consumed and

that the conjugate 11 had formed. Subsequent combination of 11

with 1b (30 µM) indicated that the formation of 12 was completewithin 5 min. A control experiment with the cis-cyclooctene

analogue of 10 gave the analogue of 11, but reaction with 1b did

not give the analogue of 12 even after 24 h.

HPLC was also used to monitor the bioconjugation reactions

that gave 11 and 12 and to demonstrate that the tetrazine ligation

to form 12 was fast and high yielding (see Supporting Information).

In summary, a new method for bioconjugation based on inverse-

electron demand Diels-Alder chemistry has been described. Thereaction proceeds with very fast rates and tolerates a broad range

of biological functionality.

Acknowledgment. This work was supported by NIH Grant

GM068640-01. We thank Colin Thorpe for donating Trx and Danny

Ramadan for HPLC assistance. We thank Ryan Mehl for insight

into the aqueous stability of 1b. We thank Colin Thorpe and John

Koh for insightful discussions.

Supporting Information Available: Experiments in which HPLC

was used to monitor bioconjugation reactions are described. Full

experimental details and 1H,13C NMR spectra are provided. This

material is available free of charge via the Internet at http://pubs.acs.org.

References

(1) (a) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed.2001, 40, 2004. (b) Kohn, M.; Breinbauer, R.Angew. Chem., Int. Ed. 2004,43, 3106. (c) Chen, I.; Ting, A. Y. Curr. Opin. Biotechnol. 2005, 16, 35.(d) Antos, J. M.; Francis, M. B. Curr. Opin. Chem. Biol. 2006, 10, 253.(e) Hahn, M. E.; Muir, T. W. Trends Biochem. Sci. 2005, 30, 26. (f)Soellner, M. B.; Nilsson, B. L.; Raines, R. T. J. Am. Chem. Soc. 2006,128, 8820. (g) Lin, F. L.; Hoyt, H. M.; van Halbeek, H.; Bergman, R. G.;Bertozzi, C. R. J. Am. Chem. Soc. 2005, 127, 2686. (h) Kolakowski, R. V.;Shangguan, N.; Sauers, R. R.; Williams, L. J. J. Am. Chem. Soc. 2006,128, 5695.

(2) Recent examples: Duckworth, B. P.; Xu, J. H.; Taton, T. A.; Guo, A.;Distefano, M. D. Bioconjugate Chem. 2006, 17, 967. (b) Gauchet, C.;Labadie, G. R.; Poulter, C. D. J. Am. Chem. Soc. 2006, 128, 9274. (c)Esser-Kahn, A. P.; Francis, M. B. Angew. Chem., Int. Ed. 2008, 47, 3751.

(3) (a) Wittig, G.; Krebs, A. Chem. Ber. 1961, 94, 3260.(4) (a) Agard, N. J.; Prescher, J. A.; Bertozzi, C. R. J. Am. Chem. Soc. 2004,

126, 15046. (b) Baskin, J. M.; Prescher, J. A.; Laughlin, S. T.; Agard,N. J.; Chang, P. V.; Miller, I. A.; Lo, A.; Codelli, J. A.; Bertozzi, C. R.Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 16793. (c) Codelli, J. A.; Baskin,J. M.; Agard, N. J.; Bertozzi, C. R. J. Am. Chem. Soc. 2008, 130, 11486.(d) Sletten, E. M.; Bertozzi, C. R. Org. Lett. 2008, 10, 3097. (e) Ning,X. H.; Guo, J.; Wolfert, M. A.; Boons, G.-J. Angew. Chem., Int. Ed. 2008,47, 2253.

(5) Song, W.; Wang, Y.; Qu, J.; Madden, M. M.; Lin, Q. Angew. Chem., Int.Ed. 2008, 47, 2832. (b) Song, W.; Wang, Y.; Qu, J.; Lin, Q. J. Am. Chem.Soc. 2008, 130, 9654.

(6) (a) Dantas de Araujo, A.; Palomo, J. M.; Cramer, J.; Seitz, O.; Alexandrov,K.; Waldmann, H. Angew. Chem., Int. Ed. 2006, 45, 296. (b) Latham-Timmons, H. A.; Wolter, A.; Roach, J. S.Nucleosides, Nucleotides NucleicAcids 2003, 22, 1495. (c) Yousaf, M. N.; Houseman, B. T.; Mrksich, M.Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 5992. (d) Yousaf, M. N.; Mrksich,M. J. Am. Chem. Soc. 1999, 121, 4286. (e) Seelig, B.; Jaschke, A.Tetrahedron Lett. 1997, 38, 7729.

(7) Boger, D. L. Chem. ReV. 1986, 86, 781.(8) Thalhammer, F.; Wallfahrer, U.; Sauer, J.Tetrahedron Lett. 1990, 31, 6851.(9) Hunter, D.; Nielson, D. G.; Tweakley, T., Jr. J. Chem. Soc., Perkin Trans.

1 1985, 2709, and references therein.(10) For Diels-Alder reactions of styrene and 1b in water: Wijnen, J. W.;

Zavarise, S.; Engberts, J. B. F. N.; Charton, M. J. Org. Chem. 1996, 61,2001.

(11) No reaction was observed by 1H NMR after 8 h when 1a (2 � 10-2 M)was combined with BuNH2 (2 � 10-2 M) or EtSH (2 � 10-2 M) inCD3OD. When a CD3OD solution of 1b (2 � 10-2 M) was combined withBuNH2 (2 � 10-2 M), 50% of 1b had reacted after 4 h. In a similarexperiment with EtSH (2 � 10-2 M), 50% of 1b had reacted after 10 min.When 1b (2 � 10-2 M) was allowed to stir in pure water, 20%decomposition of 1b was noted after 2 h.

(12) The product from trans-cyclooctene and 1a aromatizes upon workup. See:Padwa, A.; Rodriguez, A.; Tohidi, M.; Fukunaga, T. J. Am. Chem. Soc.1983, 105, 933.

(13) Royzen, M.; Yap, G. P. A.; Fox, J. M. J. Am. Chem. Soc. 2008, 130, 3760.(14) Soloducho, J.; Doskocz, J.; Cabaj, J.; Roszak, S. Tetrahedron 2003, 59,

4761.(15) Acyl transfer to 8 was most effective when aromatic acylating agents were

employed. When aliphatic acylating agents were used, undesired reactivity(presumably via ketene formation) competes with the rate of acylation. Itis likely that the rate of acyl transfer to 8 is slow because it is a veryelectron-deficient aniline. Sugars and peptides can be appended to aromaticacylating agents via terephthaloyl linkers, e.g.: (a) Angelastro, M. R.; Baugh,L. E.; Bey, P.; Burkhart, J. P.; Chen, T.-M.; Durham, S. L.; Hare, C. M.;Huber, E. W.; Janusz, M. J.; Koehl, J. R.; Marquart, A. L.; Mehdi, S.;Peet, N. P. J. Med. Chem. 1994, 37, 4538. (b) Kanie, O.; Grotenbreg, G.;Wong, C.-H. Angew. Chem., Int. Ed. 2000, 39, 4545. (c) Czifrak, K.;Hadady, Z.; Docsa, T.; Gergely, P.; Schmidt, J.; Wessjohann, L.; Somsak,L. Carbohydr. Res. 2006, 341, 947.

(16) Kallis, G.-B.; Holmgren, A. J. Biol. Chem. 1980, 255, 10261.

JA8053805

Figure 1. (a) Rapid reactivity to form 12 was monitored by ESI-MS andHPLC. (b,c) Crude ESI-MS data for 11 and 12 in experiments that beganwith 15 µM Trx.

Scheme 3. Synthesis of trans-Cyclooctene and TetrazineDerivatives

J. AM. CHEM. SOC. 9 VOL. 130, NO. 41, 2008 13519

C O M M U N I C A T I O N SX

X


Blackman, M. L.; Royzen, M.; Fox, J. M. J. Am. Chem. Soc. 2008, 130, 13518-13519.

N

N N

N

R

R

N

NN

NR

R

N

N

H

H

R

R

-N2

k2 = 2000 M-1s-1

Trx = 11700 Da

Michael product = 12014 Da

Diels-Alder product = 12222 Da

9647 dx.doi.org/10.1021/ja201844c |J. Am. Chem. Soc. 2011, 133, 9646–9649

Journal of the American Chemical Society COMMUNICATION

dienophiles for the tetrazine!trans-cyclooctene ligation havebeen derived from cyclooct-4-enol,7 a derivative of which wasshown to adopt the crown conformation in a crystal structure.7

We recognized that that the eight-membered ring of bicyclo-[6.1.0]non-4-ene derivative 3 (Figure 3b), a trans-cyclo-octene annealed to cyclopropane with a cis ring fusion, wouldbe forced to adopt a strained conformation similar to that of 1b(Figure 2).16 Computation was used to predict whether theadded strain in 3 would manifest in faster reactions withtetrazines.

Transition state calculations in the gas phase for the inverseelectron demand Diels!Alder reaction between s-tetrazine andtrans-cyclooctene derivatives were studied by us at the M06L/6(311)þG(d,p) level.17,18 The reaction between trans-cyclooctenein the crown conformation (1a) and s-tetrazine proceeded with abarrier of ΔG‡ = 8.92 kcal/mol (Figure 3a). By comparison, thereaction of s-tetrazine and cis-fused bicyclo[6.1.0]non-4-ene 3proceeded with a significantly lower barrier of ΔG‡ = 6.95 kcal/mol (Figure 3b). These barriers are consistent with those thathave been calculated for other Diels!Alder reactions thatproceed with fast rate constants.19 These calculations predictthat the reaction of s-tetrazine with 3 would be 29 times fasterthan the reaction with 1a.20

We also computed the barrier for the reaction between s-tetrazineand trans-fused bicyclo[6.1.0]non-4-ene 4. Because 4 bears a

trans-ring fusion, the eight-membered ring adopts a crownconformation (similar to 1a) in its minimized conformation(Figure 3c). The barrier for the reaction between 4 ands-tetrazine, ΔG‡ = 8.24 kcal/mol, is similar to that for 1aand significantly higher than that for 3. Compounds 3 and 4are diastereomers and the cyclopropyl moiety is distant fromthe tetrazine in each transition state. We therefore concludethat the low barrier computed for the reaction of 3 withs-tetrazine is attributable to the higher strain of the eight-membered ring.

Based on these calculations, we sought to prepare compound7 (Scheme 1). Thus, a Rh-catalyzed reaction of ethyl diazoacetatein neat21c 1,5-cyclooctadiene gave 5 in 54% yield (along with 44%of the separable syn-isomer).21 DIBAL reduction of 5 gave theknown alcohol 6.21a,22 The flow-chemistry method developed inour laboratories was used to photoisomerize 6 to trans-isomer 7in 74% yield.7

During the completion of our studies, van Delft et al. elegantlydemonstrated that cyclooctyne-based bioconjugations can beaccelerated through fusion of a cyclopropane ring.21a This groupreported the synthesis of 6 and readily converted it to thecorresponding cyclooctyne derivative for bioorthogonal labelingand cell imaging using 3 þ 2 cycloaddition strategies.21a Therates of these conjugations were as high as 1.66 M!1 s!1 fornitrone cycloadditions.

Compound 7 combines with 2a to give product 8 in >95%yield by 1H NMR analysis (Scheme 2). As expected,6,14,23 theinitially formed 4,5-dihydropyrazine derivative 8 slowly iso-merizes in the presence of water to the 1,4-dihydropyrazinederivative 8b via the aminal intermediates 8a.24

The rate of the reaction between 7 and 2a was studied. As thereaction was too rapid for reliable rate determination by UV!viskinetics, we determined the relative rate by 1H NMR through acompetition experiment with trans-cyclooctene at 25 !C. NMRanalysis was conducted immediately upon mixing, and productmixtures were analyzed for the formation of conjugated 4,5-dihydropyrazine products 8 and 9 (Figure 4). Thus, competitionof 7 (10 equiv) and 1 (10 equiv) with 2a (6.5 mM) in CD3ODgave a 19:1 ratio of 8:9. As the rate of the reaction between 1 and2a had been previously measured to be k2 = 1140 M!1 s!1 ((40) inMeOH, these NMR experiments show the rate of reactionbetween 7 and 2a to be k2 = 22 000 M!1 s!1 (( 2000). Asinverse-demand Diels!Alder reactions of tetrazines show sig-nificant accelerations due to the hydrophobic effect,6,25 it ispossible that rates may be even faster in aqueous solvents.26

Figure 3. M06L/6-311þG(d,p)-optimized transition structures for theDiels!Alder reaction of s-tetrazine with the crown conformer of trans-cyclooctene (a), the cis-ring fused bicyclo[6.1.0]non-4-ene 3 (b), andthe trans-ring fused bicyclo[6.1.0]non-4-ene 4 (c). The barrier (8.24kcal/mol) for the reaction of 4 with s-tetrazine is 1.29 kcal/mol higherthan the analogous reaction of 3.

Scheme 1. Synthesis of a Highly Reactive Dienophile

X

X


Taylor, M. T.; Blackman, M. L.; Dmitrenko, O.; Fox, J. M. J. Am. Chem. Soc. 2011, 133, 9646-9649.

N

N N

N

k2 = 22,000 M-1s-1

H

HHO

N

N

N

HN

H

H

OH

N

N

CD3OD

trans-cyclooctene ring is designed via computation (M06L/6-311+G(d,p)-optimized transition structure)

X

X


Taylor, M. T.; Blackman, M. L.; Dmitrenko, O.; Fox, J. M. J. Am. Chem. Soc. 2011, 133, 9646-9649.

N

N N

N

k2 = 22,000 M-1s-1

H

HHO

N

N

N

HN

H

H

OH

N

N

CD3OD

trans-cyclooctene ring is designed via computation (M06L/6-311+G(d,p)-optimized transition structure)

9647 dx.doi.org/10.1021/ja201844c |J. Am. Chem. Soc. 2011, 133, 9646–9649

Journal of the American Chemical Society COMMUNICATION

dienophiles for the tetrazine!trans-cyclooctene ligation havebeen derived from cyclooct-4-enol,7 a derivative of which wasshown to adopt the crown conformation in a crystal structure.7

We recognized that that the eight-membered ring of bicyclo-[6.1.0]non-4-ene derivative 3 (Figure 3b), a trans-cyclo-octene annealed to cyclopropane with a cis ring fusion, wouldbe forced to adopt a strained conformation similar to that of 1b(Figure 2).16 Computation was used to predict whether theadded strain in 3 would manifest in faster reactions withtetrazines.

Transition state calculations in the gas phase for the inverseelectron demand Diels!Alder reaction between s-tetrazine andtrans-cyclooctene derivatives were studied by us at the M06L/6(311)þG(d,p) level.17,18 The reaction between trans-cyclooctenein the crown conformation (1a) and s-tetrazine proceeded with abarrier of ΔG‡ = 8.92 kcal/mol (Figure 3a). By comparison, thereaction of s-tetrazine and cis-fused bicyclo[6.1.0]non-4-ene 3proceeded with a significantly lower barrier of ΔG‡ = 6.95 kcal/mol (Figure 3b). These barriers are consistent with those thathave been calculated for other Diels!Alder reactions thatproceed with fast rate constants.19 These calculations predictthat the reaction of s-tetrazine with 3 would be 29 times fasterthan the reaction with 1a.20

We also computed the barrier for the reaction between s-tetrazineand trans-fused bicyclo[6.1.0]non-4-ene 4. Because 4 bears a

trans-ring fusion, the eight-membered ring adopts a crownconformation (similar to 1a) in its minimized conformation(Figure 3c). The barrier for the reaction between 4 ands-tetrazine, ΔG‡ = 8.24 kcal/mol, is similar to that for 1aand significantly higher than that for 3. Compounds 3 and 4are diastereomers and the cyclopropyl moiety is distant fromthe tetrazine in each transition state. We therefore concludethat the low barrier computed for the reaction of 3 withs-tetrazine is attributable to the higher strain of the eight-membered ring.

Based on these calculations, we sought to prepare compound7 (Scheme 1). Thus, a Rh-catalyzed reaction of ethyl diazoacetatein neat21c 1,5-cyclooctadiene gave 5 in 54% yield (along with 44%of the separable syn-isomer).21 DIBAL reduction of 5 gave theknown alcohol 6.21a,22 The flow-chemistry method developed inour laboratories was used to photoisomerize 6 to trans-isomer 7in 74% yield.7

During the completion of our studies, van Delft et al. elegantlydemonstrated that cyclooctyne-based bioconjugations can beaccelerated through fusion of a cyclopropane ring.21a This groupreported the synthesis of 6 and readily converted it to thecorresponding cyclooctyne derivative for bioorthogonal labelingand cell imaging using 3 þ 2 cycloaddition strategies.21a Therates of these conjugations were as high as 1.66 M!1 s!1 fornitrone cycloadditions.

Compound 7 combines with 2a to give product 8 in >95%yield by 1H NMR analysis (Scheme 2). As expected,6,14,23 theinitially formed 4,5-dihydropyrazine derivative 8 slowly iso-merizes in the presence of water to the 1,4-dihydropyrazinederivative 8b via the aminal intermediates 8a.24

The rate of the reaction between 7 and 2a was studied. As thereaction was too rapid for reliable rate determination by UV!viskinetics, we determined the relative rate by 1H NMR through acompetition experiment with trans-cyclooctene at 25 !C. NMRanalysis was conducted immediately upon mixing, and productmixtures were analyzed for the formation of conjugated 4,5-dihydropyrazine products 8 and 9 (Figure 4). Thus, competitionof 7 (10 equiv) and 1 (10 equiv) with 2a (6.5 mM) in CD3ODgave a 19:1 ratio of 8:9. As the rate of the reaction between 1 and2a had been previously measured to be k2 = 1140 M!1 s!1 ((40) inMeOH, these NMR experiments show the rate of reactionbetween 7 and 2a to be k2 = 22 000 M!1 s!1 (( 2000). Asinverse-demand Diels!Alder reactions of tetrazines show sig-nificant accelerations due to the hydrophobic effect,6,25 it ispossible that rates may be even faster in aqueous solvents.26

Figure 3. M06L/6-311þG(d,p)-optimized transition structures for theDiels!Alder reaction of s-tetrazine with the crown conformer of trans-cyclooctene (a), the cis-ring fused bicyclo[6.1.0]non-4-ene 3 (b), andthe trans-ring fused bicyclo[6.1.0]non-4-ene 4 (c). The barrier (8.24kcal/mol) for the reaction of 4 with s-tetrazine is 1.29 kcal/mol higherthan the analogous reaction of 3.

Scheme 1. Synthesis of a Highly Reactive Dienophile

X

X


Lang, K.; Davis, L.; Torres-Kolbus, J.; Chou, C.; Deiters, A.; Chin, J. W. Nature Chem. 2012, Advanced Online Publication.

Genetically encoded norbornene directs site-specific cellular protein labelling

+N

N N

N

R

r.t.

aqueousO

O

O

ON

NH

R

Orthogonal synthetase/tRNA pair used to install a norbornene-containing amino acid

O

HN

NH2

O

OH

N

HMe

Pyrrolysine "The 22nd Amino Acid"

Genetically coded amino acid used by somemethanogenic archaea.

Pyrrolysyl-tRNA synthetase/tRNACUA isorthogonal to endogenous tRNAs andaminoacyl-tRNA synthetases in E. coli

and eukaryotic cells.

X

X




Orthogonal synthetase/tRNA pair used to install a norbornene-containing amino acid

+N

N N

N

R

r.t.

aqueousO

O

O

ON

NH

R

N N

N

NN

N

N

HN

NHBocO

HO

H2O:MeOH (95:5)

21 oC HO

N

NH

N

N

O

NH

NHBoc

k = 9 M-1s-1

X

X




+N

N N

N

R

r.t.

aqueousO

O

O

ON

NH

R

The fluorescence of certain probes increased by a 5-10 fold increase after the cycloaddition

OHN NH2

O

HN

N

N

N N

N

N

tetramethylrhomdamine(TAMRA)

"turn-on" fluorophorogenic probes -tetrazine can quench fluorophore viaenergy transfer

eGFP TAMRA Merged + DIC

2 (1 mM)

3 (1 mM)

eGFP TAMRA Merged + DIC

X

X



Cycloaddition tested with mutated epidermal growth factor receptor (EGFR-GFP)

O

HN OH2N

COOH

O

HN OH2N

COOH

Me

MeMe

fluorescent

10-3 to 10-1 M-1s-1

X

X

Photochemical 1,3-Dipolar Cycloaddition

Song, W.; Wang, Y.; Lin, Q. J. Am. Chem. Soc. 2008, 130, 9654.

Song, W.; Wang, Y.; Qu, J.; Madden, M. M.; Lin, Q. Angew. Chem. Int. Ed. 2008, 47, 2832.

Wang, Y.; Hu, W. J.; Song, W.; Lim, R. K. V.; Lin, Q. Org. Lett. 2008, 10, 3752.

N N

N

N

310 nm10 minutes

N2

N N

R

NN

R

NN

R

+

X

X

Future Outlook

Groups 15 elements have been particularly lucrative.

Perhaps larger elements in this group, like bismuth or antimony, may be of use.

Pericyclic reactions have been very promising as well, due to concertedmechanisms that leave little room for interruption from other components.

Applications with other sources of energy, such as light or ultrasound, may occur.

We may see the extension of chemical reporters to other small-molecule metabolites.


the living cell -...

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