recent advances in copper catalyzed azide/alkyne cycloadditions: prototypical “click” reactions...
TRANSCRIPT
Recent Advances in Copper Catalyzed Azide/Alkyne Cycloadditions: Prototypical “Click” Reactions
Shane Mangold
Kiessling Group
February 14th 2008
2
Historical Perspective of Azide/Alkyne Cycloadditions
L. Pauling. Proc. Natl. Acad. Sci. USA 1933, 19, 860-867; Huisgen, R. Angew. Chem. Int. Ed. 1963, 2, 633-696 Sharpless, K.B. et al. Angew. Chem. Int. Ed 2002, 41, 2596-2599; Meldal,M.J. et al. J. Org. Chem. 2002, 67, 3057-3064
R'' R'N3 N NN
R'
R''
1
5
+80oC N NN
R'
R''
1
4
+
R N3 R N N N R N N NH2R N N N
1933- Dipolar nature of azide first recognized by Linus Pauling
1960- Mechanism of 1,3-dipolar cycloaddition of azidesand alkynes pioneered by Rolf Huisgen
2001- Copper catalyzed 1,3-Dipolar cycloaddition by Sharpless/Meldal
R'' R'N3N N
NR'
R''
1
4
+ Cu(I)
rt
3
Defining a “Click” Chemistry Reaction
“ A click reaction must be modular, wide in scope, high yielding, create only inoffensive by-products (that can be removed without chromatography), are stereospecific, simple to perform and that require benign or easily removed solvent. ”
- Barry Sharpless
Kolb, H.C.; Finn, M.G.; Sharpless, B.K. Angew. Chem. Int. Ed. 2001, 40, 2004-2021.
4
Reactions that meet the “Click” Criteria
Kolb, H.C.; Finn, M.G.; Sharpless, B.K. Angew. Chem. Int. Ed. 2001, 40, 2004-2021.
R
[O]
X
R
C=C Additions
HX
R Nuc
Catalyst
Nucleophilic Ring Opening
X = O, NR
R'
O
R''
N
Non-Aldol Carbonyl Chemistry
RO-NH2
[O]
RO
OR
Diels-Alder
OR
R
N
N
N
R
R'
Cu(I) catalyzed Huisgen1,3-dipolar cycloaddition
R-N3
Cu(I)
5
Copper Catalyzed Azide/Alkyne Cycloaddition (CuAAC)
• Thermodynamic and kinetically favorable (50 and 26 kcal/mol, respectively)
• Regiospecific
• Chemoselective
• 107 rate enhancement over non-catalyzed reaction
• Triazole stable to oxidation and acid hydrolysis
R'' R'N3
N NN
R'
R''
1
4
Cu(I)
+
Rostovtsev et al. Angew. Chem. Int Ed. 2002, 41, 2596-2599
6
CuAAC Catalytic Cycle
Himo, F. et al. J. Am. Chem. Soc, 2005, 127, 210-216.Ahlquist, M., Fokin, V.V. Organometallics 2007, 26, 4389-4391.
CuLxR'
N N N
R2
CuLx
HR'
CuLx
23 kcal/mol
18 kcal/mol
HR'
H+
CuLxR'
N N N
R2
CuLxR'
N N N
R2
CuLx
N N N
R1
R2
N NN
R CuLx
H+
N NN
R H
R2
R2
[CuLx]
RDS
7
CuAAC Chemistry Applications
• Peptide/Protein Modification
• Therapeutics
• Combinatorial Synthesis
• Polymer Functionalization
• Materials/Surface Chemistry
8
CuAAC as a Route to Cyclic Tetrapeptide Analogues
• Cyclic peptides important antimicrobial agents
• More stable to enzymatic degradation and better cellular uptake than linear chain form
• Conformational restriction allows better understanding of receptor-ligand interactions
• Difficult to synthesize due to strain energy of cyclization in transition state
Rich, D.H. et al. Tetrahedron 1988, 44, 685-695
N
NH
N
HN
O
O
O
HO
O
cyclo-[Pro-Val-Pro-Tyr]
9
Synthesis of Tetrapeptide Analogue cyclo-[Pro-Val-(triazole)-Pro-Tyr]
• Cyclo-[LPro-LVal-LPro-LTyr] is a tyrosinase inhibitor isolated from L. helveticus
• Previous attempts at synthesis had failed due to epimerization upon cyclization
• Hypothesize ring contraction mechanism of CuAAC may help promote cyclization
Van Maarseveen, J.H. et al. Org. Lett. 2006, 8, 919-922
N
NH
N
HN
O
O
O
HO
O
cyclo-[Pro-Val-Pro-Tyr]
N
N
N
HN
O
O
O
N
N
HO
cyclo-[Pro-Val-(Triazole)-Pro-Tyr]
10
1,2,3-Triazoles as Peptide Bond Isosteres
• Triazole and peptide bond both possess large dipole (5D, 3.7D, respectively)
• N2 and N3 lone pairs serve as hydrogen bond acceptors
• C distance comparable
• Triazole mimics planarity of amide bond
Kolb, H.C., Sharpless, B.K. Drug. Disc. Today. 2003, 8, 1128-1136.
3.9 Å
5.1 Å
H2NNH
COOH
R1
O R2
H2N
N
NN
R1
COOH
R2
11Bock, V.D., et al. Org. Lett. 2006, 8, 919-922
Retrosynthesis
Pathway "A"
H2NN
O
NNN O
N
CO2H
OBn
N
N
N
HN
O
O
O
NN
BnO
Triazole Formation:Pathway "B"
Peptide BondFormation Pathway "A"
Pathway "B"
N3N
NH
N
O O
BnO
O
N3
O
N
CO2tBu
BocHNN
O
BnO
12Bock, et al. Org. Lett. 2006, 8, 919-922
Synthesis of Cyclic Tetrapeptide Analogue
BocHNN
O
OBn
(1)
N3
O
N
CO2tBu
H2NN
O
NNN O
N
CO2H
OBn
N
N
N
HN
O
O
O
NN
BnO
1) CuI, DIPEA5:1 MeCN:THF
2) TFA: CH2Cl274%
(2)
1 + 2
no product formation
1 + 2EDCI, HOBt, DIPEA
N3N
NH
N
O O
BnO
O
N
N
N
HN
O
O
O
NN
BnOCuBr, DBU
Toluene, 70%
Pathway A
Pathway B
DCM, 70%
13Bock, V.D. et al. Org. Biomol. Chem., 2007, 5, 971-975
Tyrosinase Inhibition
Compound Tyrosinase Activity IC50 / mM
Cyclo-[Pro-Tyr-Pro-Val] 1.5
Triazole analogue 2 0.6
Triazole analogue 3 0.5
Triazole analogue 4 1.6
N
NH
NO
O
HO
N
N
NO
O
NN
HON
NN
O NNN
N
NH
N
HN
O
O
O
HO
O
N
N
N
HN
O
O
O
NN
HO
cyclo-[Pro-Tyr-Pro-Val] 2 3 4
14
Outline
• Peptide/Protein Modification– Peptide Macrocyclization
• Therapeutics – Multivalent carbohydrate vaccines
• Inhibitors
• Chemoenzymatic Functionalization
• Materials Science/Polymers
15
Anticancer Vaccines Through Extended Cycloaddition Chemistry
• To exploit antitumor immune response, induce antibodies against carbohydrate antigens
• Protein Scaffold upon which
carbohydrates are attached is important for eliciting antibody production
• Drawback is that monovalent carbohydrate/antibody interactions are weak
Wan, Q., Chen, J., Chen, G., Danishefsky, S.J. J. Org. Chem. 2006, 71, 8244-8249
Glycopeptide N3 Peptide
HN
O
Glycopeptide NNN
Peptide
HN
O
m
m
+
CuAAC
GlycopeptideHN
SHO
PeptideN
O
OO
n
PeptideN
O
OO
NH
O S
ConjugationLow Yielding
n
Glycopeptide
16Wan, Q., Chen, J., Chen, G., Danishefsky, S.J. J. Org. Chem. 2006, 71, 8244-8249.
CuAAC of Multivalent Carbohydrate Peptide Conjugate
OOH
HO
O
NHAc
OH
AcNH HN
O
N3
NH
OHN
NH
HN
NH
O
O
O
OHN
NH
HN
O
O
NH
OH
O
O
NH
NH2
HNHN O
HNO
HNO
Ala-Lys-Arg-Tyr-Lys-Phe-Ala-Lys-Ser-Ala
O
O
O
Cu nanoparticle, PBS buffer, 65%
OOH
HO
O
NHAc
OH
AcNH HN
O
NN
N
NNN
OOH
OHO
NHAc
OH
AcNH HN
O
NNN
OOH
HOO
NHAc
OH
AcNH HN
O
HN
NH2
OH
O
O
17
Template-Assembled Oligosaccharide Epitope Mimics
• 2G12 antibody targets oligomannose cluster (Man-9) present on HIV-1 gp120
• Recognizes terminal Man1-2Man residues
• Man-4 had comparable affinity to the antibody as that of Man-9 moeity
Wang, J., Li, H., Zou, G., Wang, L-X. Org. Biomol. Chem., 2007, 5, 1529-1540.
OHO
HO
HO OH
OHO
HO
HO O
OHO
HO
HO O
OHO
HO
HO OH
OHO
HO
HO O
OHO
HO
HO OH
OHO
HO
HO O
OHO
HO O
O
OHO
OHO
O
HOAcNH
O
HONHAc
HN
OH OH
OOO
Man-9
18
Template-Assembled Oligosaccharide Epitope Mimics
• Cyclic decapeptide shown to be better immunogen than linear form
• T-helper peptide previously shown to increase immunogenicity of conjugate
• Synthesize template consisting of decapeptide conjugated with T-helper peptide epitopes for IgG antibody production.
Wang, J., Li, H., Zou, G., Wang, L-X. Org. Biomol. Chem., 2007, 5, 1529-1540.
PK
K
KG
P
K
KKG
T - H e l p e rT - H e l p e r Mannose
1-2
1-3
1-2
1-2
1-2
1-3
1-2
1-2
1-3
1-2
1-2
1-3
19Wang, J., Li, H., Zou, G., Wang, L-X. Org. Biomol. Chem., 2007, 5, 1529-1540.
Synthesis of Man4
OOBz
HOO
O N3
OOPh
TMSOTf, DCM, 77%
2) 80% AcOHNaOMe/MeOH
OBzO
BzO
BzO OH
OBzO
BzO
BzO O
OAll
OBzO
BzO
BzO OAc
OBzO
BzO
BzO O
OBzO
BzO O
BzO
OAll
OBzO
BzO
BzO OAc
OBzO
BzO
BzO O
OBzO
BzO O
BzO
O
NH
CCl3
OBzOBzO
OAc
BzOO
NH
CCl3
TMSOTf, DCM, 82%
PdCl2, MeOH1)
2) CCl3CN, DBU
OHO
HO
HO OH
OHO
HO
HO O
OHO
HO
HO O
OHO
OH
O
HO
OO N3
Man4
1)
76% (2 steps)
20Wang, J., Li, H., Zou, G., Wang, L-X. Org. Biomol. Chem., 2007, 5, 1529-1540.
Template Synthesis of Man-4 Cluster
NNN
R NNN
R
NNN
R
NNN
R
PK
K
KG
P
K
KKG
NHNH
BocHNNHBoc
NH
HNO
O OO
PK
K
KG
P
K
KKG
NHNH
BocHNNHBoc
NH
HNO
O OO
PK
K
KG
P
K
KKG
NH2H2N
BocHNNHBoc
H2N
NH2
PK
K
KG
P
K
KKG
DdeDde
BocHNNHBoc
Dde
Dde
2% Hydrazine, DMF
84%
Propynoic Acid, DCC
77%
Man-4
CuSO4, Sodium AscorbatetBuOH:H2O (1:1); 90%
21Wang, J., Li, H., Zou, G., Wang, L-X. Org. Biomol. Chem., 2007, 5, 1529-1540
Synthetic Vaccine Conjugate
OO
N3
O
ON
O
O
0.5 M NaHCO3, ACN:MeOH, 90%
NNN
R NNN
R
NNN
R
NNN
R
PK
K
KG
P
K
KKG
NHNH
H2NNH2
NH
HNO
O OO O
NH
T-helper
CuSO4, Sodium AscorbatetBuOH:H2O (1:1), 70%
HN
HN
R2
R2
T-helper
T-helperR2 = O
ON
O NN
O
R = Man-4
O
O
O
O
OO
N3N3
NNN
R NNN
R
NNN
R
NNN
R
PK
K
KG
P
K
KKG
NHNH
HNHN
NH
HNO
O OO
NNN
R NNN
R
NNN
R
NNN
R
PK
K
KG
P
K
KKG
NHNH NH
HNO
O OO
Kd = 2.64 MFully synthetic Vaccine
Man4
Kd = 2 mM
Template withSingle Mannose Kd > 20 M
Man9
Kd = 1.9 mM
22
Outline
• Protein Molecular Architecture– Peptide Macrocyclization
• Multivalent Architecture – Vaccine Conjugates
• Inhibitors– Combinatorial Chemistry
• Chemoenzymatic Functionalization
• Materials Science/Polymers
23
Inhibitors of HIV-Protease by CuAAC
• HIV-Protease cleaves proteins to yield active HIV virus
• Amprenavir is HIV-protease inhibitor used clinically since 1997.
• Develop Amprenavir analogue using CuAAC for combinatorial screening
Folkin, V, V. et al. J. Med. Chem. 2006, 49, 7697-7710
O NH
O
NS
OH
PhO O
NH2
O
Amprenavir
R1 NH
N
O R2NN
R4
R1 X H2N R4
ON3
R2
R3
R3
24Folkin, V.V. et al. J. Med. Chem. 2006, 49, 7697-7710
Synthesis of HIV Protease Inhibitor
Ph
BocHN
O
NOMe
1) BnMgCl, THF 1) MsCl, Et3N, DCM Ph
NHBoc
Ph
N3
Ph
NHBoc
O
H BnMgCl/CuBrDMS, THF 1) MsCl, Et3N, DCM Ph
NHBoc
Ph
N3
Ph
NHBoc
Ph
OH
Ph
NHBoc
Ph
OH
2) NaBH4, MeOH, -20oC 2) NaN3, DMF
2) NaN3, DMF
Ph
HN
Ph
N3
1) TFA/DCM
2) cyclopentyl chloroformateTEA, Toluene,75% (2 steps)
O
O
Ph
HN
Ph
N3
O
O
dr: 90:10 anti:syn
dr: 80:20 syn:anti
79% (2 steps)
60%
60% (2 steps)
54% (2 steps)
25
Synthesis of HIV Protease Inhibitor
Ki of Amprenavir = 19 nM
Folkin, V.V. et al. J. Med. Chem. 2006, 49, 7697-7710
R
CuSO4, Cu(s)t-BuOH/H2O (1:1) 50oC
(36 Alkynes)
> 90% conversion
1)
2)
R
CuSO4, Cu(s)t-BuOH/H2O (1:1) 50oC
(36 Alkynes)
> 90% conversion
1)
2)
Ph
HN
Ph
N3
O
O
Ph
HN
Ph
N3
O
O
Ph
HN
Ph
N
O
O
Ph
HN
Ph
O
O
NN
NNN
R
R
Ki = 23 nM
Ph
HN
Ph
NNN
O
O
N N
Cl
26
Inhibitor Optimization
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
Folkin, V.V. et al. J. Med. Chem. 2006, 49, 7697-7710
1) n-BuLi (2 eq), THF, -78oC
2) (CH2O)n
Ph
HN
Ph
NNN
N N
ClOH
Ki = 8 nM
O
O
HO
H Ph
HN
Ph
NNN
N N
Cl
Ki = 23 nM
O
O
27
Outline
• Protein Molecular Architecture– Peptide Macrocyclization
• Multivalent Architecture – Vaccine Conjugates
• Inhibitors– Combinatorial
• Chemoenzymatic Functionalization– Metabolic Engineering– Antibiotic Derivatization
• Polymers/Materials Science
28
Glycoproteomic Probes for Imaging of Fucosylated Glycans in vivo
• Develop probe that is fluorescently active when undergoing reaction, whereas unreacted reagent remains traceless
• Fluorescent signal of naphthalimides modulated by electron donating properties of triazole
• Incorporate azidofucose analog into glycoproteins using biosynthetic pathway
Wong, C.H. et al. Proc. Natl. Acad. Sci. 2006, 103, 12371-12376
NO O
O
OH
OR2
OHHO
N3
R2 = glycoprotein
NO O
N
N N fucose OR2
strongly fluorescent
non-fluorescent
29
Metabolic Oligosaccharide Engineering
Wong, C-H., et al. Proc. Natl. Acad. Sci. 2006, 103, 12371-12376.
L-fucose
glycoconjugatesubstrateFucTs
ON3
ON3
ON3
1-P
ON3
GDP
ON3
GDP
glycoconjugate ON
glycoconjugateON3
NN
Golgi
30
Intracellular Fucosylation
Fluorescentprobe
WGA-Dye(Golgi Marker)
Overlay
Wong, C-H., et al. Proc. Natl. Acad. Sci. 2006, 103, 12371-12376
31
Chemoselective Functionalization of Antibiotics by Glycorandomization
• Glycorandomization: Chemoenzymatic glycodiversity of natural product based scaffolds
Thorson, J.S. et al. Org. Lett. 2005, 7, 1513-1515
OHO
HO
OHOH
N3
OHO
HO
OROH
N3
activated sugar
OHO
HO
OOH
N3
Add antibioticand enzyme
OHO
HO
OOH
NCuAAC
NN
R'
randomized library ofAntibiotic Derivatives
R'
Add activating groupand enzyme
non-natural substrate
glycosylated antibiotic
antibiotic
antibiotic
32
Glycorandomization of Vancomycin
• Vancomycin: glycosylated natural product isolated from the bacteria Amycolatopsis orientalis
• Last defense against infections caused by methicillin-resistant Gram-positive bacteria such as Stapholococcus aureas
• Chemical and chemoenzymatic alterations to vancomycin impact both molecular target and organism specificity vancomycin
Hubbard, B.K., Walsh, C.T. Angew. Chem. Int. Ed. 2003, 42, 730-765
O
NH
O
HN
OHN
O
NH
O
NH2
O O
HN
OH
NH
HO
NH
HO2C
O
O
OCl
Cl
HO
OH
OH
O
HO
HO
O
O
HO
OH
NH2
33
Glycorandomization of Vancomycin
Thorson, J.S. et al. Org. Lett. 2005, 7, 1513-1515
Twice as potent as Vancomycin
O
NH
O
HN
OHN
O
NH
OHO
HO
OH O
N3
NH2
O O
HN
OH
NH
HO
NH
HO2C
O
O
OCl
Cl
HO
OH
OH
R
(24 Alkynes)
CuI, MeOH/H2O70oC, 12h
OHO
HO
OH O
N
N
N
RR = COOH
OHOHO
OH
N3
O P
O
O
OHOHO
OH
N3
O
Thiamine Pyrophosphate
Nucleotidyltransferase
vancomycin aglycon
GtfE
NH
O
ON
O
OH
OP
O
O
P O
O
OO
34
Outline
• Protein Molecular Architecture– Peptide Macrocyclization
• Multivalent Architecture – Vaccine Conjugates
• Inhibitors– Combinatorial
• Chemoenzymatic Functionalization– Metabolic Engineering– Antibiotic Derivatization
• Polymers/Materials Science– Surface Patterning with Dendritic Scaffolds
35
DNA Microarrays Using CuAAC
• DNA microarrays (DNA chips) useful for large scale parallel analysis of gene expression
• Chemistry used for immobilization is limited by cross-reactivity on surface
• Efficiency and Bioorthogonality of CuAAC could overcome existing limitations of immobilization
Reinhoudt, D.A. et al. ChemBioChem, 2007, 8, 1997-2002
Create ssDNA or RNA library
hybridize to surface
Add complementaryDNA strand with dye
36
Transfer Printing of DNA Using Dendritic Architectures
Reinhoudt, D.A. et al. ChemBioChem, 2007, 8, 1997-2002
N N
NH3
NH3
H3N
H3N
PDMS
Si
N N
NH3
NH3
H3N
H3N
N
NN
DNA
N
NN
DNA
N
NN
DNA
OSi
OSi
O
Si
O
N3 N3 N3 N3 N3
Add alkyne modified ssDNA
PDMS
Si
OSi
OSi
O
Si
O
Add Azide Coated Glass
N N
NH3
NH3
H3N
H3N
PDMS
Si
OSi
OSi
O
Si
O
1) Add Cu(I)
3) Wash away unbounddendrimer
2) Remove PDMS Stamp
37
Synthesis of Alkyne Modified DNA Monomer
Reinhoudt, D.A. et al. ChemBioChem, 2007, 8, 1997-2002
NH
O
ON
O
OTBDMS
TBDMSO
I
TMS NH
O
ON
O
OTBDMS
TBDMSO
TMS
PdCl2(PPh3)3
CuI, DIPEA, 92%
1) TBAF
2) DMTrCl, pyridineDMAP, 55% (2 steps)
NH
O
ON
O
OH
DMTrO
NP
O
Cl
CN
THF, DIPEA, 70%
NH
O
ON
O
O
DMTrO
PO
NN
ssDNA
38Reinhoudt, D.A. et al. J. Am. Chem. Soc. 2007, 129, 11593-11599
Surface Patterning of ssDNA
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
Oxime Functionalized Template CuAAC Functionalized Template
39
Future Directions: Target Guided Synthesis (TGS)
• Target Guided synthesis uses enzyme for assembling its own inhibitors in situ
• Kinetically controlled approach by irreversible formation of product
• Chemoselectivity of azide/alkyne reaction eliminates byproducts that may alter enzyme
• In situ generated inhibitors separated by LCMS and re-synthesized for Ki determination
Krasinski, A. et al. J. Am. Chem. Soc. 2005, 127, 6686-6692
N3
N3N3
Enzyme
Inhibited Enzyme
N3
N NN
Add enzyme
40
Future Directions
• Target Guided Synthesis has created the most potent inhibitors of HIV Protease, Acetylcholine esterase, and Carbonic Anhydrase known.
• May lead to a revolution in drug discovery
Manetsch, R. et al. J. Am. Chem. Soc. 2004, 126, 12809-12818
Whiting, M. et al. Angew. Chem. Int. Ed. 2006, 45, 1435-1439
Mocharla, V.P. et al. Angew. Chem. Int. Ed. 2005, 44, 116-120
41
Conclusions
• Stepwise, non-concerted mechanism accounts for 1,4 regiospecificity
• Chemoselectivity of azide/alkyne cycloaddition allows for bioorthogonal conjugation and combinatorial screening
• Electronic properties of triazole serve as peptide bond mimics and modulate fluorescence of dyes
• High thermodynamic stability of triazole offers superior control for surface functionalization
42
Acknowledgements
• Laura Kiessling• Hans Reich • Kathleen Myhre• Kiessling Lab Members
Practice Talk Attendees• Chris Shaffer• Christie McGinnis• Emily Dykhuizen• Raja Annamalai• Chris Brown• Katie Garber• Margaret Wong• Aim Tongpenyai• Becca Splain