nh nh n o nh o s n - home - macmillan group...jewett, j. c.; bertozzi, c. r. chem. soc .rev.2010,...

Click Reactions For Post-Translational Protein Modification

Steven BloomGroup Meeting

12/2/2015

NH

O

O

NH

O

OHN

NH

O

OH

NH

O

S

NH

O

NHNH

O

NH

Baathula, K.; Xu, Y.; Quian, X. Nature Protocols 2011, 6, 1990-1997.!http://papoian.chem.umd.edu!

! Reactions to an organic chemist


total control of reaction parameters

vs

environment controlled

Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Agnew Chem. Int. Ed. 2001, 2004-2021!Jewett, J. C.; Bertozzi, C. R. Chem. Soc . Rev. 2010, 39, 1272-1279.!

! Definition: "Click" Chemistry

Coined by K. Barry Sharpless in 1998

- Reactions that generate substances rapidly and reliably by joining small molecules together


- simple (minimal components)- readily available reagents- use benign solvents (H2O)- simple product isolation(non-chromatographic methods)- irreversible

N

NNN3Protein

Cargo Protein Cargo

fast

Jewett, J. C.; Bertozzi, C. R. Chem. Soc . Rev. 2010, 39, 1272-1279.!

! Two Approaches: Protein Engineering or Direct Functionalization


N3

H

O

FFF

F

engineered direct

RO

NH2

COOCH3

PPh2 R S PPh2

O

NN N

N

Functionalized Protein Conjugates

and many more...

Limited Toolbox

Reactions for selective residue manipulation

R NHNH2

O

! Why Do We Want Bioorthoganol Reactions?


Mechanistic Biology Pharmaceuticals

Biomaterials


H2N COOH

H

H2N COOH

CH3

H2N COOHH2N COOH H2N COOH H2N COOHH2N COOH

Glycine Alanine Valine Leucine Isoleucine Methionine Tryptophan

H2N COOH COOH

Phenylalanine Proline

H3C CH3 H3CCH3

CH3

CH3 SCH3

NH

NH

H2N COOH

OH

Serine Threonine Cysteine Tyrosine Asparagine GlutamineH2N COOH

CH3HO

H2N COOH

SH

H2N COOH

OH

H2N COOH

NH2

O

H2N COOH

NH2O

Non-polar

Polar


H2N COOH H2N COOH H2N COOHH2N COOH H2N COOH

Aspartic acid Glutamic acid Lysine Arginine Histidine

O

O

O ONH3

NH

NH2H2N

Acidic Basic

HNNH

! Which residues do we target for Click and How?


SH

Cysteine

NH3

Lysine

OH

Tyrosine

- uncommon residue- nucleophilic- good ligand for metals

- most abundant residue- strong nucleophile (as free amine)-vast literature for reactions of amines

- activated for EAS reactions

COOH

Aspartic or Glutamic acid Histidine Tryptophan

- prevalenceof coupling reactions in biology

- good ligand for metals- prone to acylation

- electrophilic addition to C3

NHNNH

Hackeng, T. M.; Griffin, J. H.; Dawson, P. E. P.N.A.S. 1999, 96, 10068-10073.!Malins, L. R.; Payne, R. J. Curr. Opin. Chem. Biol. 2014, 22, 70-78.!

! Cysteine- Native Chemical Ligation


peptide 1

O

SR + peptide 2

OH2N

HS

thioesterificationpeptide 1

O

S

H2NO

peptide 2

S-N acyl shift

peptide 1

O

NH

SH

O

peptide 2

C-terminus N-terminus

Sletten, E. M.; Bertozzi, C. R. Agnew. Chem. Int. Ed. Engl. 2009, 48, 6974-6998. and references therein!

! Classical Methods for Cysteine Modification


Cys modification

RS

S

Reagent Product

SS

OI

NH

SO

NH

N

O

OS

N

O

O

SH S

Hoyle, C. E.; Bowman, C. N. Agnew Chem. Int. Ed. 2010, 49, 1540-1573. and references therein!Tyson, E. L.; Ament, M. S. Yoon, T. P. J. Org. Chem. 2013, 78, 2046-2050.!

! Cysteine: Thiol-ene


initiatorR1 SH R1 S

R2

R1 SR2

R1 SH

R1 S

R1 SR2

H

common initiatorsthoether

AIBNR3Sn-Hdirect UV irradiation

Yoon et al. 2013

Ph SH-e-

Eox = 0.45 VPh SH

pKa =2.4

-H+Ph S

photocatalyst

Tyson, E. L.; Ament, M. S. Yoon, T. P. J. Org. Chem. 2013, 78, 2046-2050.!


Click reactions for Post-translational Protein Modification

Yoon et al. 2013

Ph SH-e-

Eox = 0.45 VPh SH

pKa =2.4

-H+Ph S

Ph SH PhRu(bpz)3(PF6)2 (0.25 mol %)

Blue LEDs, MeCN, 2h4:1

Ph SPh

H 98%

Ru(bpz)3(PF6)2

RuII

N

N

N

N

N

N

N

N

NN

NN

E1/2II*/I = + 1.35 V


! Cysteine:Thiol-ene


Yoon et al. 2013

SH PhRu(bpz)3(PF6)2 (0.25 mol %)


RS

Ph

H 98%R

SH

98% (2h)MeO

OSH

96% (1.5h)

SH

98% (1h)

SH

Me

Me SHMe

98% (8h) 86% (20h)

MeO

O

NHBocSH

97% (1.5h)

Thiol Scope




Yoon et al. 2013

SH RRu(bpz)3(PF6)2 (0.25 mol %)


SR

H 98%

n-Hex

99% (1h) 98% (1h) 98% (6h)

86% (2h) 88% (3h) 80% (3h)

Alkene Scope

Ph Ph

82% (2h)

AcO

HO BocHN Cl

93% (26h)

PhCO2Et




Yoon et al. 2013

SH RRu(bpz)3(PF6)2 (0.25 mol %)


SR

H 98%

Proposed Mechanism

Ph Ph

h! 450 nm

[Ru]II [Ru]I

O2O2

[Ru]II*RSH

RSH

-H+

RS

R'

RSR'

RSHRSH

R'

Tyson, E. L.; Niemeyer, Z. L.; Yoon, T. P. J. Org. Chem. 2014, 79, 1427-1436.!Tyson, E. L.; Ament, M. S. Yoon, T. P. J. Org. Chem. 2013, 78, 2046-2050.!

! Cysteine: Thiol-ene Bio-orthogonal variation


Yoon et al. 2013

PhRu(bpz)3(PF6)2 (0.25 mol %)Blue LEDs, MeCN [0.2M], 2h

4:1

SPh

H 97% (1.5h)

-

MeO

ONHBoc

SHNHBoc

O

MeO

- amino acid is limiting- work in dilute, aquesous media- exhibit high rate of reactivity

Bio-orthogonal variation

Yoon et al. 2014

Ru(bpz)3(PF6)2 (0.25 mol %)Blue LEDs, MeCN [0.1M], 0.5h

1:2.5

SH

99% (0.5h)

MeO

ONHBoc

SHNHBoc

O

MeO

NH2

H3C

RR

(0.5 equiv)

.!Tyson, E. L.; Ament, M. S. Yoon, T. P. J. Org. Chem. 2013, 78, 2046-2050.!



Yoon et al. 2014

Ru(bpz)3(PF6)2 (0.25 mol %)Blue LEDs, H2O [0.1M], 0.5h

HOOC

NH2

NH2

H3C

(0.5 equiv)

O

HN

O

NH

SH

COOH HOOC

NH2

O

HN

O

NH

S

COOH

R

RH

N3

60%

OO

OO

OH

75%

O OAcAcO

OAc

AcO

71%

HN NH

O

S

HH

O

O

77%

HO

OHN

O

O

SMe 76%

HO

OHN

O

O

HO 92%

exclusively

!Tyson, E. L.; Ament, M. S. Yoon, T. P. J. Org. Chem. 2013, 78, 2046-2050.!



Yoon et al. 2014

Ru(bpz)3(PF6)2 (0.25 mol %)Blue LEDs, H2O [0.1M], 0.5h

HOOC

NH2

NH2

H3C

(0.5 equiv)

O

HN

O

NH

SH

COOH HOOC

NH2

O

HN

O

NH

S

COOH

R

RH

h! 450 nm

[Ru]II [Ru]I

O2O2

[Ru]II*RSH

RS

R'

RSR'

RSHRSH

R'

ArNH2

ArNH3

ArNH4

Proposed MechanismEox = 0.72V

Eox = 0.5V

Kung. K. K.; Ko, H. M. Cui, J. F.; Chong, H. C.; Leung, Y.C.; Wong, M. K. Chem. Commun. 2014, 50, 11899-11902.!Vinogradova, E. V.; Zhang, C.; Spokoyny, A. M.; Pentelute, B. L. Buchwald, S. L. Nature 2015, 526, 687-691.!

! Cysteine: Metal-mediated Cross Coupling


NH

O

HN

SH

N NAu

N

R

MeO2S SO2Me

H2O

Wong et al. 2014

NAu

N

R

ONH

PCy2PriO

OiPrPd Ar

OHTf

MeCN:H2O, 5:95pH 7.5, 5 min Buchwald et. al 2015

NPd

ONH

Cy2P

OiPr

OiPr

NH

O

HN

S

R

N

NH

O

HN

SAr

S

NH2

H

O

HO

M = Fe, Cu, Mo, Pd, Pt....etc S

NH

H

O

HOM

Ar

S

S

Vinogradova, E. V.; Zhang, C.; Spokoyny, A. M.; Pentelute, B. L. Buchwald, S. L. Nature 2015, 526, 687-691.!



PCy2PriO

OiPrPd Ar

OTf

C(O)NH2NH2

HS0.1 M Tris, pH 7.5

CH3CN:H2O, 5:95, [10 µ!]

C(O)NH2NH2

SAr

ArPd cat. O-allylation of tyrosine (Francis et. al 2005)

electrophilicity of metal center tuneschemoselectivity

PdLn4

OEt2N

O

N

N

Fluorescent tags Bioconjugation handles

O O

quantitative

CHOTMS

Drug MoleculesMeN

O

N

N

NH

F

MeOVandetanib

NH2-RSNFYLGCAGLAHDKAT-C(O)NH2

Vinogradova, E. V.; Zhang, C.; Spokoyny, A. M.; Pentelute, B. L. Buchwald, S. L. Nature 2015, 526, 687-691.!



PCy2PriO

OiPrPd Ar

OHTf

20 mM Tris, 150 mM NaCl

CH3CN:H2O, 5:95, 30 minpH 7.5

antibody mimic

0.5 mM Tris

CH3CN:H2O, 1:1, 10 minpH 7.5

Peptide stapling

20 mM Tris, 150 mM NaCl

CH3CN:H2O, 5:95, 30 minpH 7.5

Trastuzumab

Pd cat. with vandetanib

Trastuzumab-vandetanib conjugate

SDrug

DAR = 4.4

SH SAr

PCy2PriO

OiPrPd ArCl

NH2-I-K-F-T-E-C-G-L-L-C-Y-Q-K-R-C(O)NH2

SH SH

NH2-I-K-F-T-E-C-G-L-L-C-Y-Q-K-R-C(O)NH2

S SPhPh

O

SH



SH

Cysteine

NH3

Lysine

OH

Tyrosine




COOH





NHNNH


! Classical Lysine Modification


NH

O

HN

NH2

N

O

O

O

O

physiological conditions NH

O

HN

HN O

Me

NH

O

OHMeO

O

CH3O

MeNMe

Cl

MeO O

O

N MeO

Me

O

S N

O

O

O

HN

DAR = 3.5

Kadcyla


! Classical Lysine Modification


NH

O

HN

NH2

S


O

HN

HN

Cl

O OS

O O

NH

O

HN

NH2

N


O

HN

HN

CO

O

NH

McFarland, J. M.; Francis, M. B. J. Am. Chem. Soc. 2005, 127, 13490-13491.!

! Lysine Modification by Transfer Hydrogenation


NH

O

HN

NH2

NH

O

HN

HNN

N

IrH2O

SO42-

K2HPO4 buffer, pH 7.4

CHOPh

Ph

COOHNH2

NH2-MYKFARLAND-CO2H

NH2

N

N

IrH2O

SO42-

(20 µM)

HCO2Na (25 mM)K2HPO4 buffer (50 mM)

pH 7.4 [10 µM]

cat.

HCO2Na

COOHNH

NH2-MYKFARLAND-CO2H

NH

CHOPh

Ph

(37%)

Alk

(100 equiv)




NH

O

HN

NH2

NH

O

HN

HNN

N

IrH2O

SO42-

K2HPO4 buffer, pH 7.4 (50 mM)

CHOPh

Ph

(20 µM)

HCO2Na (25 mM)

Cytochrome c !-Chymotrypsinogen A(19 Lys) (14 Lys)

Myoglobin(19 Lys)

Ribonuclease(10 Lys)

+ 1 Lys 19% + 2 Lys 27%+ 3 Lys 22%+ 4 Lys 12%

18h

+ 1 Lys 47% + 2 Lys 27%+ 3 Lys 02%+ 4 Lys 00%

+ 1 Lys 15% + 2 Lys 00%+ 3 Lys 00%+ 4 Lys 00%

+ 1 Lys 41% + 2 Lys 00%+ 3 Lys 00%+ 4 Lys 00%




NH

O

HN

NH2

NH

O

HN

HNN

N

IrH2O

SO42-

K2HPO4 buffer, pH 7.4 (50 mM)

CHOPh

Ph

(20 µM)

HCO2Na (25 mM)

18h

Proposed Mechanism

N

N

IrH2O

SO42-N

N

IrH

SO42-

HCO2-CO2

RN Ph

RHN Ph

H



SH

Cysteine

NH3

Lysine

OH

Tyrosine




COOH





NHNNH


! Tyrosine Classical Modification


NH

O

HN

NH

O

HN

OH OH

aqueous buffer, rt

N2

R NN

R

bright organge

one of the earliest reported Modifications-1905


! Tyrosine Classical Modification


NH

O

HN

NH

O

HN

OH OH

Oxidative coupling

NH

O

HN

NH

O

HN

OH OH

Mannich reactionRCHO,PhNH2

OH

NH

O

HN

NH

O

HN

OH OR

PdLn

HN

R

R

Ni(OAc)2

OH

allyl-Pd reagents

Ban, H.; Gavrilyuk, J.; Barbas, C. F. III. J. Am. Chem. Soc. 2010, 132, 1523-1525.!

! Tyrosine Modification by Aqueous-ene reaction


NH

Me

O

HN

Me NH

Me

O

HN

Me

OH OHNN

N

O

O N

NHN

O

O

Ph

MeCN/H2O (1:1), 5 min

Competition experiments

65%quantitative with 3.3 equiv PTAD

sodium phosphate buffer, pH 7

(1.1 equiv)

exclusive Tyrosine Modification39%

NH2

HO

HS

OH

NH

NH2

NHN

COOH NH2

HO

HS

OH

NH

NH2

NHN

COOH

N

NNHO

OPh




NH

Me

O

HN

Me NH

Me

O

HN

Me

OH OHNN

N

O

O N

NHN

O

O

Ph

MeCN/H2O (1:1), 5 min

65%quantitative with 3.3 equiv PTAD

sodium phosphate buffer, pH 7

(1.1 equiv)

NH

Me

O

HN

Me

OHN

NHN

O

O

Ph

pH 2-12 for 24h120oC for 1h

NH

Me

O

HN

Me

OHN

NHN

O

O

Ph

new C-N bond stable to pH and temperature condtions(89 % recovered)




NH

O

HN

NH

O

HN

OH OHNN

N

O

O N

NHN

O

O

Ph

DMF/H2O (5:95), [30 µM]sodium phosphate buffer, pH 7.4

(3.3 equiv)

O

Rhodamine

O

Rhodamine

Chymotrypsinogen A Myoglobin Bovine Serum Albumin(81%) (8 %) (96 %)(3 Tyr) (2 Tyr) (21 Tyr)



SH

Cysteine

NH3

Lysine

OH

Tyrosine




COOH





NHNNH

Antos, J. M.; Francis, M. B. J. Am. Chem. Soc. 2004, 126, 10256-10257.!

! Tryptophan Modification


HN HN

N2

PhO

OOMe

3

0.5 mM Rh2(OAc)475 mM HONH2 HCl

ethylene glycol/ H2O (20:80)17h, 10µM

(4 equiv)RO2C

Ph

N

CO2R

Ph

(1.4:1)51%

H3C H3C

Possible Reaction pathways

HN

CH3

N-H insertioncyclopropaneintermediate N

CH3

RO2C

Ph

CH3

HN

RO2C

Ph

H3C

Antos, J. M.; Francis, M. B. J. Am. Chem. Soc. 2004, 126, 10256-10257.!



N2

PhO

OOMe

3

Rh2(OAc)4 (1.0 equiv)75 mM HONH2 HCl

ethylene glycol/ H2O (20:80)7h, pH 3.5

(100 equiv)

Myoglobin (100 µM)

Tryptophan Modification products

N2

PhO

OOMe

3

Rh2(OAc)4 (1.0 equiv)75 mM HONH2 HCl

ethylene glycol/ H2O (20:80)7h, pH 1.5

(100 equiv)

subtilisin Carlsberg (100 µM)

Tryptophan Modification product

(one Trp residue)

(2 Trp)

Antos, J. M.; McFarland, J. M.; Lavarone, A. T.; Francis, M. B. J. Am. Chem. Soc. 2009, 131, 6301-6308.!



N2

PhO

OOMe

3

100 µM Rh2(OAc)475 mM tBuNHOH

ethylene glycol/ H2O (20:80)7h, pH 6-7

(100 equiv)

Melittin (100 µM)

Tryptophan Modification products

tBuNHOH promotes carbenoid formation at higher pH 3-9

Rh

O

O

RhO

OO

O

OO O

ONH

HN

H

O-boundFavored

Rh

O

O

RhO

OO

O

OO N

NOH

OH

N-boundDisfavored

pH 6

(1 Trp)


! Modification of other Residues


NH

O

HN

NH

O

HN

HN

NH

NH2

H

O

NO2

O

H

pH 12-140-5oC, 15 min

HN

N

N

NO2

Arg sidechain

NH

O

HN

NH

O

HN

O

O

O

pH 4.0

His sidechain

NNH N

N

EtMeMe

O


! Modification of other Residues


NH

O

HN

NH

O

HN

0-5oC, 5 min

Glu sidechain

R NH2 R N C N R

COOHHNO

R

! Concluding Remarks


- Direct site-selective modification of proteins has evolved over the last 20 years

- Direct modification can be a powerful tool with widespread pharmaceutical and industrial applications

- Research to expand the number of available reactions and surface reidues which can be modified is needed

! Questions


HN

NH2

SH

HO

O

OH

OH

nh nh n o nh o s n - home - macmillan group...jewett, j. c.; bertozzi, c. r. chem. soc .rev.2010,...

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