1. 2 what is click chemistry? reactions with the following characteristics: modular, wide in scope...
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Click Chemistry in Biological Systems
Trevor Mischki
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What is Click Chemistry?
• Reactions with the following characteristics:• Modular, wide in scope• Afford high yields w/o purification• Stereospecific• Generate inoffensive byproducts and operate in a
benign solvent
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Click Reactions
• Nucleophilic substitution
• Cycloadditions
• “Non-aldol” carbonyl
R
O: Nu R
OH
Nu
R' N
NN
R R'R N3
R NCO : NuNH
Nu
O
R
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Click Reactions
• Nucleophilic substitution
• Cycloadditions
• “Non-aldol” carbonyl
R
O: Nu R
OH
Nu
R' N
NN
R R'R N3
R NCO : NuNH
Nu
O
R
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[3+2] Dipolar CycloadditionR1 N
N+
R1 N-
N+
NN-
1,3-DipoleR1
N-
+N
N
R2
R3
NN
NR1
R2
R3
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Thermal
H+N
Cl-
HNN3
N+
NH2
Neat, 80oC, 6 days
N+
NH2
N+
NH2
HNNH
N N NHNH
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HNNH
N N
1,5 1,4
6
•Thermal: 1:1 mixture
Kolb. J. Am. Chem. Soc. 2004, 126, 12809
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Cu(I) Catalyzed
OPh
Ph N3
CuSO4 5H2O 1mol%Sodium Ascorbate 5mol%H2O : tBuOH 4:1, RT, 8h
NN
N
OPh
Ph
91%
O
O
N
NNPh
92%
N
NN
NN
N NHHN
NH2
NH
88%
N
N NHO
O
O Ph
88%
OH
H
H H
N
NN OH
OH
94%
Sharpless. Angew. Chem. Int. Ed. 2002, 41(14), 2596
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“this is a very robust catalytic process, which is so insensitive to the usual reaction parameters as to strain credulity”
-V.V. Rostovtsev, L.G. Green, V.V. Fokin, K.B. Sharpless. Angew. Chem. Int. Ed. 2002, 41(14), 2596-2599
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Catalytic Cycle
R1 CuLn-1
NN
N R2
R1 CuLn-1
[LnCu]+
R1 H
NN
N
R1
R2
CuLn-2
N-
R1
N+
R2N
NN
N
CuLn-2C
R2
R1
N N+
N-R2
Direct
Stepwise
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Autocatalytic?
• Rate acceleration during formation of dendrimers
• Binding is tetradentate• Prevents oxidation and disproportionation• Improves catalytic activity
N
NNN
NN N
NN
NBn
Bn
Bn
Cu NNN N
NN
Bn
Bn
N
NN
N
Bn
Forkin. Org. Lett. 2004, 6, 2853
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Optimized Cycloaddition Conditions
• CuSO4 – 1mM
• Ligand (tris(triazoyl) amine) – 2mM• Reducing agent (tris(carboethyoxy)phosphine) – 2mM
HN
O
HN
O
N33
O
OO
HO2C
OH
O
60
N NNProtein
Dye
conditions
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Applications
• Library synthesis• in situ inhibitor formation• Bioconjugation
– Activity based protein profiling (ABPP)– Cell Surface Modification– Non-canonical amino acids
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Protein Synthesis
DNA
RNA
Protein•Active•Modified•“Stored”
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Proteomics
• Genomics– The study of an organism’s genome and use of it’s
genes
• Proteomics– The identification and functional assignment of all
proteins in the proteome
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Methods for Analyzing the Proteome
• 2-D Gel Electrophoresis / staining + MS
Charge (pI)
MW
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Methods for Analyzing the Proteome
• 2-D Gel Electrophoresis / staining + MS• LC-MS/MS based
– Isotope coded affinity tagging (ICAT)
• Limitation: primarily measures protein abundance
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Activity Based Protein Profiling (ABPP)
•Proteins analyzed by function
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(-)-FR182877
O
HH
Me
O
O
MeH
HO
OH
Me
H H
H
Me
•Isolated 1998 from Streptomyces•Found to inhibit tumor cell growth
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(-)-FR182877
O
HH
Me
O
O
MeH
HO
OH
Me
H H
H
Me
•Isolated 1998 from Streptomyces•Found to inhibit tumor cell growth
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(-)-FR182877
O
HH
Me
O
O
MeH
HO
OH
Me
H H
H
Me
•Isolated 1998 from Streptomyces•Found to inhibit tumor cell growth
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Retrosynthesis of (-)-FR182877
OH
HORMe
OR
Br Me
H CO2R H
Me
H
OR
O
HH
Me
O
O
MeH
HO
OH
Me
H H
H
Me
O
Me
Me
OR
RO
MeRO
Br
CO2R
Evans. J. Am Chem. Soc. 2003, 125, 13531Sorensen J. Am Chem. Soc. 2003, 125, 5393
N
O
Me
OR
Me
Br
Br
OMe
RO
Me
OR
OR
(HO)2B
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Synthesis of Fragments: Evans
OH
6 Steps
48% Yield
NO
O
Bn
O
Me
OH
Me N
O
Me
OTBS
Me
Br
BrOTBS
5 Steps
57% Yield
OMe
OH OH
3 Steps
53% Yield
5 Steps
67% Yield
NO
O
Bn
O
Me
OH
OTBDPS
TBS O
Me
OTBS
OTBDPS
(HO)2B
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Macrocycle Formation: EvansN
O
Me
OTBS
Me
Br
Br
OMe
TBS O
Me
OTBS
OTBDPS
(HO)2B
O
Me
Me
OTBS
TBSO
MeTBSO
Br
CO2Et
Pd(PPh3)4, Tl2CO3
N
O
Me
OTBS
MeOMe
Me
TBDPSO
OTBS
OTBS
1) DIBAL, -78oC
2) EtO2CCN2, SnCl2
84% Yield
70% 2 Steps
1) TBAF, AcOH, DMF2) I2, PPh3, CH2Cl23) CsCO3, THF (0.005M)
71% 3 Steps
Br
O
Me
OTBS
MeEtO
Me
TBDPSO
OTBS
OTBS
Br
O
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Transannular Diels-Alder: Evans
O
Me
Me
OTBS
TBSO
MeTBSO
Br
CO2Et1) Ph2Se2O3, SO3-Pyr
TEA, THF, 23oC
2) hexanes, 50oC
63% yield
O
HH
Me
O
O
MeH
HO
OH
Me
H H
H
Me
O
HH
Me
TBSO
CO2Et
MeH
TBSO
OTBS
Me
H H
H
Br
1)HF-MeCN
2)Me3B3O3, Pd(dppf)Cl
DMF-H2O 100oC
O
HH
Me
HO
CO2Et
MeH
HO
OH
Me
H H
H
Me1) TMSOK, THF,2) Mukaiyama Reag,NaHCO3, CH2Cl2
63% 2 steps
62% 2 steps
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Activity Based Protein Profiling (ABPP)
Cravatt. J. Am Chem. Soc. 2004, 126, 1363
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Reporter Tags
O
N+
N
CO2-
O
OS
HN NH
H H
O
Biotin
•Target purification / isolation
•Avidin chromatography
Rhodamine
•Target detection
•Fluorescent probe
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Synthesis of Rhodamine Tag
rhodamine tag
O
O
N+
N
-O2CO
N
O
O H2N NHBoc
1) DMF, TEA
96% yield
NH
NH3+Rh
O
2) HClCl-
alkyne-NHS, TEA, MeOH
75% yield
HN
O
NH
O
O
N+
N
CO2-
4 4
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Synthesis of Biotin / Rhodamine Tag
NH
O
HN
O
NH
NH
OS
HN NH
H H
O
O
O N+N
HO
O
NHBoc
NHFmoc1)EDC, NHS, DMF2)Biotin-NH2, MeOH
62% 2 steps S
HN NH
H H
O
HN
O
HN
O
NHFmoc
NHBoc1) Morpholine, DMF2)NHS-Rh, Et3N, DMF
48% 2 steps
Bt
HN
O
HN
O
HN
NHBoc
O
Rh
Bt
HN
O
HN
O
HN
NH3+
O
RhHCl / Dioxane
Cl-NHS-Alkyne,TEA, MeOH
75% yield
4
4
CO2-
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Synthesis of tagged FR182877
HOOH
H
H
H
H
H
HO
O
O
H
H
(-)-FR182877
2,4,6 collidine, CH2Cl2
77% yield
ClN3
O
9 HOO
H
H
H
H
H
HO
O
O
H
HN3
O
9
tag
CuSO4, sodium ascorbate,tBuOH/H2O
HOO
H
H
H
H
H
HO
O
O
H
HN
O
9
NNtag
~50% yield
•Prepared: (-)-FR182877 Rhodamine tag (+)-FR182877 Rhodamine tag (-)-FR182877 Rhodamine-Biotin tag
Cravatt. Ang. Chem. Int. Ed. 2003, 42, 5480
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Mouse Tissue Proteome
(-)-FR182877 – Rh tag
•0.1M electrophile•2mg/mL protein
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Mouse Tissue Proteome
(-)-FR182877 – Rh tag
Heat denatured
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Mouse Tissue Proteome
(-)-FR182877 – Rh tag
(+)-FR182877 – Rh tag
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Mouse Tissue Proteome
(-)-FR182877 – Rh tag
(-)-FR182877
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Mouse Tissue Proteome
(-)-FR182877 – Rh tag
(+)-FR182877
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Identification of Target
• Isolated using biotin-rhodamine tagged (-)-FR182877 via avidin chromatography and analyzed by MS
• Target protein is Carboxyl Esterase-1
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IC50 Determination
• Pre-incubate proteome with (+/-)-FR182877 then treat with Rhodamine tagged label
• IC50 = 34nM
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Activity of Carboxylesterase-1
• Broad spectrum serine hydrolase• Drug and xenobiotic metabolism
N O
OMe
O Ph
O
carboxyl esterase-1
H2O
EtOH
N O
OH
O Ph
O
N O
OEt
O Ph
O
carboxyl esterase-1
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Summary
• (+)-FR182877 is inactive• (-)-FR182877 target is carboxyl esterase-1
• Potent: IC50 34nM
• Selective: 1M gives 20X difference
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Limitations
• Ideal: measure activity in native environment• Reporter tag limits scope
– Bioavailability– Biological activities– Subcellular compartmentalization– Electrostatic interactions
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Click Chemistry ABPP
• General Concept– Dose electrophile tethered azide– Collect sample, homogenize, perform cycloaddition– Isolate and characterize target
in vivo in vitro
Cravatt. Chem & Biol. 2004, 11, 535
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“Click Chemistry”-ABPP
• Rh-tagged phenylsulfonate labels• Phenylsulfonate is a general label for cysteine proteases
SO
O
OR1
R1 = N3 RG N3
HN
O
NH
O
O
N+
N
CO2-
4 4R2
R1 = RG
R2 = Dye
R2 = N3 Dye N3
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“Click Chemistry”-ABPP
RG-N3 / Dye-Ξ
RG-Ξ / Dye-N3
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“Click Chemistry”-ABPP
RG-N3 / Dye-Ξ
RG-Ξ / Dye-N3
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“Click Chemistry”-ABPP
RG-N3 / Dye-Ξ
RG-Ξ / Dye-N3
45
Results
• PS-≡ / Rh-N3 reduced background labeling– Improved signal : noise– Allowed detection of low abundant proteins
• Successfully measured enzyme activity in vivo
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in vivo Bioconjugation
• Cu(I) catalyzed [3+2] dipolar cycloaddition valuable tool for addition of tag in vitro
• Cu(I) is toxic
• in vivo ligation requires biocompatible reagents
47
Cell Surface Glycoconjugation
• Unnatural sugar tolerated by sialic acid biosynthetic pathway
• Conjugate to azide via Staudinger reaction
Bertozzi. Science. 2000, 287, 2007
48
Staudinger Reaction
P
NN+
-N
P
N
N2
R
PR
R
N N+
N-R'
R
PR
R
NR' R
P+R
R
N-R'
R
P+R
R
N N N-R'
R
P
RR N
N N
R'
-N2
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Aza-ylide Traps
R
PR
R
NR' R
P+R
R
N-R'
+H2O
-R3POH2N R'
+
-R3PO
R''
O
H R'' N
H
R'
R''
O
NH
+ R''' R'' N
NH
R'
R'''-R3PO
Biological applications require aqueous solvent
50
Intramolecular Staudinger
O OMe
NHO
Tag
PPh2
-N N+
NR
MeO O
NHO
Tag
P+Ph
Ph
N- R
PN
MeO
O
R
Ph
Ph
NHO
Tag
H2O
O NH
R
P
PhPh
O
NHO
Tag
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“Traceless” Staudinger
PPh2
O
O
R2
-N N+
NR
P+
O
R2
O
PhPh
-NR
P+
O- PhPh
N
R
R2
OH2OPPh2
OH OHN
RR2
O
P+
O
PhPh
NR
-O R2
52
Phosphine Synthesis
O OMe
O OH
NH2
1) NaNO2, HCl/H2O2) KI, H2O
57% Yield
O OMe
O OH
I
O OMe
O OH
PPh2
Pd(OAc)2(1%)Ph2PHEt3N, MeOH
OH
I
Pd(OAc)2(1%)Ph2PHKOAc, DMA
OH
PPh2
69% Yield
35% Yield
•Common intermediates allow for diversity
53
Synthesis of Non-Natural Sugar
• Peracylation aids bioavailability
O
NH2 HClHO
HOHO
OH
1) NaOMe/MeOH2) (ClCH2CO)2O
O
HNHO
HOHO
OH
O
Cl NaN3, DMFreflux
O
HNHO
HOHO
OH
O
N3
59% 2 Steps
Ac2O, DMAP,Pyridine
95% Yield O
HNAcO
AcOAcO
OAc
O
N3
54
Modulation of Cell Surfaces in Living Animals
O
HNAcO
AcOAcO
OAc
O
N3
1) Inject Ac4ManNaz for 7 Days
O OMe
NHO
Flag
PPh2
1eq
Bertozzi. Nature. 2004, 430, 873
55
Results
• No adverse affects associated with Ac4ManNAz or Phos-Flag
• Ac4ManNAz stable to serum esterases
• Labeled glycoproteins only observed in groups dosed with Ac4ManNAz
• Majority of azides ligated in vivo
56
Conclusions
Identified carboxylesterase-1 as target of
(-)-FR182877
Cu(I) catalyzed [2+3] dipolar cycloaddition allows for in vivo labeling of proteins
Staudinger ligation in a living animal!!
57
Acknowledgements
• Dan Wayner
• Greg Lopinski
• John Pezacki
• Bojana Rakic
• Jenny Clarke
• JL Brochu
• Corinne Stocco
58
[3+2] Cycloaddition of Biomolecule
CuSO4
(mM)
Ligand(mM)
TCEP(mM)
Loading%/60
- - - 3
- 2.0 2.0 <2
1.0 2.0 - <2
1.0 - 2.0 17
1.0 2.0 2.0 100
2.0 2.0 5.0 80
HN
O
HN
O
N33
O
OO
HO2C
OH
O
60
N NNProtein
Dye
conditions
Sharpless. J. Am. Chem. Soc. 2003, 125, 3192
59
[3+2] Cycloaddition of Biomolecule
CuSO4
(mM)
Ligand(mM)
TCEP(mM)
Loading%/60
- - - 3
- 2.0 2.0 <2
1.0 2.0 - <2
1.0 - 2.0 17
1.0 2.0 2.0 100
2.0 2.0 5.0 80
HN
O
HN
O
N33
O
OO
HO2C
OH
O
60
N NNProtein
Dye
conditions
Sharpless. J. Am. Chem. Soc. 2003, 125, 3192
60
(-)-FR182877
• Tethered to reporter tag via cycloaddition• Specifically labels one protein ~70kDa• Only natural enantiomer is active• Target identified as Carboxylesterase-1
– Potent: 34nM– Selective: >20X over other enzymes
61
“Click” – Activity Based Protein Profiling
• Orientation: Electrophile–≡ / Tag–N3
– Copper alkylidyne causes non-specific labelling
• Low abundance enzymes observed• Proteins labeled in vivo
62
Cell Surface Glycoconjugation
• No toxic effects from dosing unnatural sugar• Specific labeling of azido-sugar• Dose dependant fluorescence