NH NH N O NH O S N - Home - Macmillan Group...Jewett, J. C.; Bertozzi, C. R. Chem. Soc .Rev.2010,...
Transcript of 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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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
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Baathula, K.; Xu, Y.; Quian, X. Nature Protocols 2011, 6, 1990-1997.!http://papoian.chem.umd.edu!
! Reactions to an organic chemist
Click Reactions For Post-Translational Protein Modification
total control of reaction parameters
vs
environment controlled
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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
Click Reactions For Post-Translational Protein Modification
- simple (minimal components)- readily available reagents- use benign solvents (H2O)- simple product isolation(non-chromatographic methods)- irreversible
N
NNN3Protein
Cargo Protein Cargo
fast
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Jewett, J. C.; Bertozzi, C. R. Chem. Soc . Rev. 2010, 39, 1272-1279.!
! Two Approaches: Protein Engineering or Direct Functionalization
Click Reactions For Post-Translational Protein Modification
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
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! Why Do We Want Bioorthoganol Reactions?
Click Reactions For Post-Translational Protein Modification
Mechanistic Biology Pharmaceuticals
Biomaterials
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Click Reactions For Post-Translational Protein Modification
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
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Click Reactions For Post-Translational Protein Modification
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
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! Which residues do we target for Click and How?
Click Reactions For Post-Translational Protein Modification
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
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! Which residues do we target for Click and How?
Click Reactions For Post-Translational Protein Modification
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
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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
Click Reactions For Post-Translational Protein Modification
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
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Sletten, E. M.; Bertozzi, C. R. Agnew. Chem. Int. Ed. Engl. 2009, 48, 6974-6998. and references therein!
! Classical Methods for Cysteine Modification
Click Reactions For Post-Translational Protein Modification
Cys modification
RS
S
Reagent Product
SS
OI
NH
SO
NH
N
O
OS
N
O
O
SH S
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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
Click Reactions For Post-Translational Protein Modification
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
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Tyson, E. L.; Ament, M. S. Yoon, T. P. J. Org. Chem. 2013, 78, 2046-2050.!
! Cysteine: Thiol-ene
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
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Tyson, E. L.; Ament, M. S. Yoon, T. P. J. Org. Chem. 2013, 78, 2046-2050.!
! Cysteine:Thiol-ene
Click Reactions For Post-Translational Protein Modification
Yoon et al. 2013
SH PhRu(bpz)3(PF6)2 (0.25 mol %)
Blue LEDs, MeCN, 2h4:1
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
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Tyson, E. L.; Ament, M. S. Yoon, T. P. J. Org. Chem. 2013, 78, 2046-2050.!
! Cysteine: Thiol-ene
Click Reactions For Post-Translational Protein Modification
Yoon et al. 2013
SH RRu(bpz)3(PF6)2 (0.25 mol %)
Blue LEDs, MeCN, 2h4:1
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
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Tyson, E. L.; Ament, M. S. Yoon, T. P. J. Org. Chem. 2013, 78, 2046-2050.!
! Cysteine: Thiol-ene
Click Reactions For Post-Translational Protein Modification
Yoon et al. 2013
SH RRu(bpz)3(PF6)2 (0.25 mol %)
Blue LEDs, MeCN, 2h4:1
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'
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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
Click Reactions For Post-Translational Protein Modification
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)
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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)
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.!Tyson, E. L.; Ament, M. S. Yoon, T. P. J. Org. Chem. 2013, 78, 2046-2050.!
! Cysteine: Thiol-ene Bio-orthogonal variation
Click Reactions For Post-Translational Protein Modification
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
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!Tyson, E. L.; Ament, M. S. Yoon, T. P. J. Org. Chem. 2013, 78, 2046-2050.!
! Cysteine: Thiol-ene Bio-orthogonal variation
Click Reactions For Post-Translational Protein Modification
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
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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
Click Reactions For Post-Translational Protein Modification
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
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Vinogradova, E. V.; Zhang, C.; Spokoyny, A. M.; Pentelute, B. L. Buchwald, S. L. Nature 2015, 526, 687-691.!
! Cysteine: Metal-mediated Cross Coupling
Click Reactions For Post-Translational Protein Modification
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
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Vinogradova, E. V.; Zhang, C.; Spokoyny, A. M.; Pentelute, B. L. Buchwald, S. L. Nature 2015, 526, 687-691.!
! Cysteine: Metal-mediated Cross Coupling
Click Reactions For Post-Translational Protein Modification
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
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! Which residues do we target for Click and How?
Click Reactions For Post-Translational Protein Modification
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
-
Jewett, J. C.; Bertozzi, C. R. Chem. Soc . Rev. 2010, 39, 1272-1279.!
! Classical Lysine Modification
Click Reactions For Post-Translational Protein 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
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Jewett, J. C.; Bertozzi, C. R. Chem. Soc . Rev. 2010, 39, 1272-1279.!
! Classical Lysine Modification
Click Reactions For Post-Translational Protein Modification
NH
O
HN
NH2
S
physiological conditions NH
O
HN
HN
Cl
O OS
O O
NH
O
HN
NH2
N
physiological conditions NH
O
HN
HN
CO
O
NH
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McFarland, J. M.; Francis, M. B. J. Am. Chem. Soc. 2005, 127, 13490-13491.!
! Lysine Modification by Transfer Hydrogenation
Click Reactions For Post-Translational Protein Modification
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)
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McFarland, J. M.; Francis, M. B. J. Am. Chem. Soc. 2005, 127, 13490-13491.!
! Lysine Modification by Transfer Hydrogenation
Click Reactions For Post-Translational Protein Modification
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%
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McFarland, J. M.; Francis, M. B. J. Am. Chem. Soc. 2005, 127, 13490-13491.!
! Lysine Modification by Transfer Hydrogenation
Click Reactions For Post-Translational Protein Modification
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
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! Which residues do we target for Click and How?
Click Reactions For Post-Translational Protein Modification
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
-
Jewett, J. C.; Bertozzi, C. R. Chem. Soc . Rev. 2010, 39, 1272-1279.!
! Tyrosine Classical Modification
Click Reactions For Post-Translational Protein 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
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Jewett, J. C.; Bertozzi, C. R. Chem. Soc . Rev. 2010, 39, 1272-1279.!
! Tyrosine Classical Modification
Click Reactions For Post-Translational Protein 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
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Ban, H.; Gavrilyuk, J.; Barbas, C. F. III. J. Am. Chem. Soc. 2010, 132, 1523-1525.!
! Tyrosine Modification by Aqueous-ene reaction
Click Reactions For Post-Translational Protein Modification
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
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Ban, H.; Gavrilyuk, J.; Barbas, C. F. III. J. Am. Chem. Soc. 2010, 132, 1523-1525.!
! Tyrosine Modification by Aqueous-ene reaction
Click Reactions For Post-Translational Protein Modification
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)
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Ban, H.; Gavrilyuk, J.; Barbas, C. F. III. J. Am. Chem. Soc. 2010, 132, 1523-1525.!
! Tyrosine Modification by Aqueous-ene reaction
Click Reactions For Post-Translational Protein Modification
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)
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! Which residues do we target for Click and How?
Click Reactions For Post-Translational Protein Modification
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
-
Antos, J. M.; Francis, M. B. J. Am. Chem. Soc. 2004, 126, 10256-10257.!
! Tryptophan Modification
Click Reactions For Post-Translational Protein 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
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Antos, J. M.; Francis, M. B. J. Am. Chem. Soc. 2004, 126, 10256-10257.!
! Tryptophan Modification
Click Reactions For Post-Translational Protein Modification
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)
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Antos, J. M.; McFarland, J. M.; Lavarone, A. T.; Francis, M. B. J. Am. Chem. Soc. 2009, 131, 6301-6308.!
! Tryptophan Modification
Click Reactions For Post-Translational Protein Modification
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)
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Jewett, J. C.; Bertozzi, C. R. Chem. Soc . Rev. 2010, 39, 1272-1279.!
! Modification of other Residues
Click Reactions For Post-Translational Protein Modification
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
-
Jewett, J. C.; Bertozzi, C. R. Chem. Soc . Rev. 2010, 39, 1272-1279.!
! Modification of other Residues
Click Reactions For Post-Translational Protein Modification
NH
O
HN
NH
O
HN
0-5oC, 5 min
Glu sidechain
R NH2 R N C N R
COOHHNO
R
-
! Concluding Remarks
Click Reactions For Post-Translational Protein Modification
- 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
Click Reactions For Post-Translational Protein Modification
HN
NH2
SH
HO
O
OH
OH