Transcript Recent Advances in Copper Catalyzed 1,3

Recent Advances in Copper Catalyzed
Azide/Alkyne Cycloadditions:
Prototypical “Click” Reactions
Shane Mangold
Kiessling Group
February 14th 2008
Historical Perspective of
Azide/Alkyne Cycloadditions
1933- Dipolar nature of azide first recognized by Linus Pauling
R N3
R N N N
R N N NH2
R N N N
1960- Mechanism of 1,3-dipolar cycloaddition of azides
and alkynes pioneered by Rolf Huisgen
+ N3 R'
R''
N 1
N R'
N
80oC
5 R''
+
N 1
N R'
N
R'' 4
2001- Copper catalyzed 1,3-Dipolar cycloaddition by
Sharpless/Meldal
R''
+
N3 R'
Cu(I)
rt
N 1
N R'
N
R'' 4
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
2
Defining a “Click” Chemistry
Reaction
“ A click reaction must be modular,
wide in scope, high yielding,
create only inoffensive byproducts (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.
3
Reactions that meet the “Click” Criteria
X = O, NR
OR
HX
X
OR
R
R
R
C=C Additions
[O]
Diels-Alder
Nuc
Nucleophilic Ring
Opening
Catalyst
R
[O]
RO
O
R'
N
RO-NH2
R''
Non-Aldol Carbonyl
Chemistry
4
Kolb, H.C.; Finn, M.G.; Sharpless, B.K. Angew. Chem. Int. Ed. 2001, 40, 2004-2021.
Copper Catalyzed Azide/Alkyne
Cycloaddition (CuAAC)
• Thermodynamic and kinetically
favorable (50 and 26 kcal/mol,
respectively)
• Regiospecific
R''
+
N3 R'
Cu(I)
• Chemoselective
• 107 rate enhancement over noncatalyzed reaction
N 1
N R'
N
R'' 4
• Triazole stable to oxidation and acid
hydrolysis
Rostovtsev et al. Angew. Chem. Int Ed. 2002, 41, 2596-2599
5
CuAAC Catalytic Cycle
N
N
N
R2
[CuLx]
H
R
R'
H
CuLx
R'
H+
H+
N
N
R
H
N
R2
R'
23 kcal/mol
CuLx
R2
CuLx
N N N
R2
R2
N N N
R1
N N N
CuLx
RDS
18 kcal/mol
Himo, F. et al. J. Am. Chem. Soc, 2005, 127, 210-216.
Ahlquist, M., Fokin, V.V. Organometallics 2007, 26, 4389-4391.
R'
R2
CuLx
N
N
R'
N
CuLx
CuLx
6
CuAAC Chemistry Applications
• Peptide/Protein Modification
• Therapeutics
• Combinatorial Synthesis
• Polymer Functionalization
• Materials/Surface Chemistry
7
CuAAC as a Route to Cyclic
Tetrapeptide Analogues
• Cyclic peptides important antimicrobial
agents
O
• More stable to enzymatic degradation
and better cellular uptake than linear
chain form
N
O
HN
HO
NH
O
• Conformational restriction allows better
understanding of receptor-ligand
interactions
N
O
cyclo-[Pro-Val-Pro-Tyr]
• Difficult to synthesize due to strain
energy of cyclization in transition state
Rich, D.H. et al. Tetrahedron 1988, 44, 685-695
8
Synthesis of Tetrapeptide Analogue cyclo[Pro-Val-(triazole)-Pro-Tyr]
O
N
• Cyclo-[LPro-LVal-LPro-LTyr] is
a tyrosinase inhibitor isolated
from L. helveticus
O
HN
HO
NH
O
N
O
• Previous attempts at
synthesis had failed due to
epimerization upon
cyclization
cyclo-[Pro-Val-Pro-Tyr]
O
N
• Hypothesize ring contraction
mechanism of CuAAC may
help promote cyclization
N
HN
HO
N
O
N
N
O
cyclo-[Pro-Val-(Triazole)-Pro-Tyr]
Van Maarseveen, J.H. et al. Org. Lett. 2006, 8, 919-922
9
1,2,3-Triazoles as Peptide Bond Isosteres
3.9 Å
• Triazole and peptide
bond both possess large
dipole (5D, 3.7D,
respectively)
O
H2N
N
H
• Triazole mimics planarity
of amide bond
COOH
R1
• N2 and N3 lone pairs
serve as hydrogen bond
acceptors
• C distance comparable
R2
5.1 Å
H2N
R1
N
N
N
R2
COOH
Kolb, H.C., Sharpless, B.K. Drug. Disc. Today. 2003, 8, 1128-1136.
10
Retrosynthesis
O
Triazole Formation:
Pathway "B"
N
HN
BnO
N
O
Peptide Bond
Formation Pathway "A"
N
N
N
O
Pathway "B"
Pathway "A"
BnO
OBn
N
H2N
N N
N
O
CO2H
O
N
N
N3
O
O
N
H
N
O
BnO
O
N3
CO2tBu
N
N
BocHN
O
Bock, V.D., et al. Org. Lett. 2006, 8, 919-922
11
Synthesis of Cyclic Tetrapeptide Analogue
OBn
O
N3
N
BocHN
CO2tBu
N
O
Pathway A
(1)
(2)
O
OBn
1
+
N
1) CuI, DIPEA
5:1 MeCN:THF
2
2) TFA: CH2Cl2
74%
N N
N
N
H2N
O
CO2H
BnO
N
HN
N
O
O
N
N
N
O
no product formation
Pathway B
BnO
O
EDCI, HOBt, DIPEA
1
+
2
DCM, 70%
N
O
N
N3
O
N
H
N
O
CuBr, DBU
Toluene, 70%
BnO
HN
N
O
N
O
Bock, et al. Org. Lett. 2006, 8, 919-922
12
N
N
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
O
O
N
HO
N
O
HN
NH
O
O
HO
N
HN
N
O
N
O
cyclo-[Pro-Tyr-Pro-Val]
Bock, V.D. et al. Org. Biomol. Chem., 2007, 5, 971-975
N
O
N
N
HO
N
N
O
N
NH
N
HO
N
N
N
N
N
O
2
N
O
3
O
4
13
N
N
Outline
• Peptide/Protein Modification
– Peptide Macrocyclization
• Therapeutics
– Multivalent carbohydrate vaccines
• Inhibitors
• Chemoenzymatic Functionalization
• Materials Science/Polymers
14
Anticancer Vaccines Through Extended
Cycloaddition Chemistry
•
To exploit antitumor immune
response, induce antibodies
against carbohydrate antigens
O
Glycopeptide
Glycopeptide
H
NN
H
N N
+
SH
3
O
m
•
Protein Scaffold upon which
carbohydrates are attached is
important for eliciting antibody
production
n
CuAAC
Conjugation
Low Yielding
Glycopeptide
Glycopeptide
•
Peptide
Peptide
OO
O
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
NH N N
N
O
S
H
N
OO
N
Peptide
O
m
Peptide
O
n
15
CuAAC of Multivalent Carbohydrate
Peptide Conjugate
OH OH
O
HO
AcNH
NHAc
H
N
O
N
N
N
O
O
HO
O
O
OH OH
AcNH O
NHAc
H
N
O
HN
Ala-Lys-Arg-Tyr-Lys-Phe-Ala-Lys-Ser-Ala
NH2
O
HN
NH
N3
O
O
Cu nanoparticle, PBS buffer, 65%
N
H
O
H
N
N
H
O
O
H
N
H
N
N
H
O
O
O
N
H
N
H
O
OH
HN
O
H
N
O
OH OH
O
OH
O
HN
O
OH OH
OH
H
N
AcNH O
NHAc
H
N
O
Wan, Q., Chen, J., Chen, G., Danishefsky, S.J. J. Org. Chem. 2006, 71, 8244-8249.
O
N
N
N
HO
AcNH O
NHAc
H
N
N
N
N
O
16
O
NH2
Template-Assembled Oligosaccharide
Epitope Mimics
• 2G12 antibody targets
oligomannose cluster (Man9) present on HIV-1 gp120
• Recognizes terminal Man12Man residues
• Man-4 had comparable
affinity to the antibody as
that of Man-9 moeity
OH
O
HO
HO
HO
O
HO
HO
HO
O
O
HO
HO
O
HO
HO
HO
HO
HO
HO
O
O
O
O
OH
OH
O
O
HO
O
AcNH
OH
O
HO
O
O
O
O
O
OH
17
Wang, J., Li, H., Zou, G., Wang, L-X. Org. Biomol. Chem., 2007, 5, 1529-1540.
H
N
NHAc
Man-9
HO
HO
HO
HO
HO
HO
O
HO
O HO
OH HO
HO
O
Template-Assembled Oligosaccharide
Epitope Mimics
• Cyclic decapeptide shown to
be better immunogen than
linear form
1-2
1-2
1-2
1-2
1-2
1-2
1-2
1-3
• T-helper peptide previously
shown to increase
immunogenicity of conjugate
1-2
1-3
1-3
1-3
K
G
K
K
Wang, J., Li, H., Zou, G., Wang, L-X. Org. Biomol. Chem., 2007, 5, 1529-1540.
K
K
P
G
K
r
e
lp
e
H
reT
lp
e
-H
T
• Synthesize template
consisting of decapeptide
conjugated with T-helper
peptide epitopes for IgG
antibody production.
P
Mannose
18
Synthesis of Man4
BzO
BzO
BzO
OH
O
BzO
BzO
BzO
O
BzO
BzO
BzO
O
O
BzO
BzO
BzO
CCl3
NH
O
OAc
O
BzO
BzO
BzO
OAc
O
TMSOTf, DCM, 82%
BzO
BzO
BzO
OAll
OAc
O
BzO
BzO
BzO
O
1) PdCl2, MeOH
O
2) CCl3CN, DBU
76% (2 steps)
O
OAll
BzO
BzO
BzO
BzO
BzO
BzO
O
O
O
O
O
CCl3
NH
Ph
O
O
HO
HO
HO
O
OBz
O
O
HO
HO
HO
O
N3
1) TMSOTf, DCM, 77%
2) 80% AcOH
NaOMe/MeOH
HO
HO
HO
HO
HO
HO
OH
O
O
O
O
O
O
O
N3
Man4
O
OH
Wang, J., Li, H., Zou, G., Wang, L-X. Org. Biomol. Chem., 2007, 5, 1529-1540.
19
Template Synthesis of Man-4 Cluster
Dde
Dde
Dde
K
K
K
K
P
H2N
Dde
G
P
G
2% Hydrazine, DMF
NHBoc
H2N
K
K
K
P
G
K
NH2
K
84%
K
BocHN
NH2
77%
K
G
K
NHBoc
BocHN
R
R
O
O
NH
G
P
NH
HN
O
NH
K
K
K
K
K
K
BocHN
O
P
Man-4
N N
R
N N N
N N
N
N
O
CuSO4, Sodium Ascorbate
tBuOH:H2O (1:1); 90%
G
NHBoc
Wang, J., Li, H., Zou, G., Wang, L-X. Org. Biomol. Chem., 2007, 5, 1529-1540.
O
NH
G
P
Propynoic Acid, DCC
P
NH
R
N N
N
HN
O
NH
K
K
K
K
K
K
BocHN
O
P
G
NHBoc
20
Synthetic Vaccine Conjugate
R
R
N N
R
R
N N N
N N
N
N
O
O
NH
NH
G
O
R
N N
N
HN
O
NH
K
K
P
K
O
O
O
N3
O
NH
NH
G
O
O
O
NH
G
P
NH
NH
P
K
K
N N
N
N3
K
K
P
K
K
HN
G
R = Man-4
O
R2
HN
R2
T-helper
G
HN
N
H
CuSO4, Sodium Ascorbate
tBuOH:H2O (1:1), 70%
O
O
O
N3
O
NH
K
K
T-helper
P
K
O
HN
O
O
K
HN
R
O
K
K
O
N N
R
R
N N N
N N
N
N
N N
N
HN
O
O
R
R
O
NH2
H2N
N N
R
N N N
N N
N
N
O
0.5 M NaHCO3, ACN:MeOH, 90%
G
K
O
N
K
K
P
R
R2 =
O
O
N N
N
O
Fully synthetic
Vaccine
Kd = 2.64 M
Template with
Single Mannose
Kd > 20 M
Man9
Kd = 2 mM
Man4
Kd = 1.9 mM
T-helper
Wang, J., Li, H., Zou, G., Wang, L-X. Org. Biomol. Chem., 2007, 5, 1529-1540
21
Outline
• Protein Molecular Architecture
– Peptide Macrocyclization
• Multivalent Architecture
– Vaccine Conjugates
• Inhibitors
– Combinatorial Chemistry
• Chemoenzymatic Functionalization
• Materials Science/Polymers
22
Inhibitors of HIV-Protease by CuAAC
• HIV-Protease cleaves
proteins to yield active HIV
virus
O
Ph
O
O
O
N
H
N
O
S
OH
NH2
Amprenavir
• Amprenavir is HIV-protease
inhibitor used clinically since
1997.
O
R1
N N
N
R2
N
H
R4
R3
• Develop Amprenavir
analogue using CuAAC for
combinatorial screening
R2
O
R1
X
N3
H2N
R4
R3
23
Folkin, V, V. et al. J. Med. Chem. 2006, 49, 7697-7710
Synthesis of HIV Protease Inhibitor
BocHN
Ph
N
1) BnMgCl, THF
OMe
O
NHBoc
NHBoc
Ph
2) NaBH4, MeOH, -20oC
79% (2 steps)
1) MsCl, Et3N, DCM
Ph
OH
Ph
2) NaN3, DMF
Ph
N3
60% (2 steps)
dr: 90:10 anti:syn
Ph
NHBoc
H
O
NHBoc
NHBoc
BnMgCl/CuBrDMS, THF
Ph
60%
Ph
OH
1) MsCl, Et3N, DCM
2) NaN3, DMF
54% (2 steps)
Ph
Ph
N3
dr: 80:20 syn:anti
O
HN
1) TFA/DCM
2) cyclopentyl chloroformate
TEA, Toluene,
75% (2 steps)
O
O
Ph
Folkin, V.V. et al. J. Med. Chem. 2006, 49, 7697-7710
HN
Ph
N3
O
Ph
Ph
N3
24
Synthesis of HIV Protease Inhibitor
O
O
1) R
HN
O
Ph
Ph
N3
(36 Alkynes)
2) CuSO4, Cu(s)
t-BuOH/H2O (1:1) 50oC
> 90% conversion
HN
O
O
Ph
Ph
N
N
N
HN
Ph
O
Ph
N
N
N
R
Cl
N
N
O
O
HN
1) R
O
Ph
Ph
N3
HN
(36 Alkynes)
2) CuSO4, Cu(s)
t-BuOH/H2O (1:1) 50oC
> 90% conversion
Folkin, V.V. et al. J. Med. Chem. 2006, 49, 7697-7710
O
Ph
Ph
N
N
N
R
Ki = 23 nM
Ki of Amprenavir = 19 nM
25
Inhibitor Optimization
O
O
HN
H
O
H
O
Ph
1) n-BuLi (2 eq), THF, -78oC
Ph
2) (CH2O)n
N
Cl
N
N
N
N
Ki = 23 nM
Folkin, V.V. et al. J. Med. Chem. 2006, 49, 7697-7710
HN
Ph
O
Ph
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
N
N
N
Cl
OH
N
N
Ki = 8 nM
26
Outline
• Protein Molecular Architecture
– Peptide Macrocyclization
• Multivalent Architecture
– Vaccine Conjugates
• Inhibitors
– Combinatorial
• Chemoenzymatic Functionalization
– Metabolic Engineering
– Antibiotic Derivatization
• Polymers/Materials Science
27
Glycoproteomic Probes for
Imaging of Fucosylated Glycans in vivo
O
N
O
• Develop probe that is fluorescently active
when undergoing reaction, whereas
unreacted reagent remains traceless
non-fluorescent
OR2
• Fluorescent signal of naphthalimides
modulated by electron donating
properties of triazole
N3
OH
HO
OH
R2 = glycoprotein
O
• Incorporate azidofucose analog into
glycoproteins using biosynthetic pathway
O
N
N
N N
O
fucose OR2
strongly fluorescent
28
Wong, C.H. et al. Proc. Natl. Acad. Sci. 2006, 103, 12371-12376
Metabolic Oligosaccharide Engineering
glycoconjugate
O
N
N N
O
N3
O
O
N3
glycoconjugate
N3
L-fucose
FucTs
N3
O 1-P
N3
glycoconjugate
substrate
O GDP
Golgi
N3
O GDP
29
Wong, C-H., et al. Proc. Natl. Acad. Sci. 2006, 103, 12371-12376.
Intracellular Fucosylation
Fluorescent
probe
WGA-Dye
(Golgi Marker)
Wong, C-H., et al. Proc. Natl. Acad. Sci. 2006, 103, 12371-12376
Overlay
30
Chemoselective Functionalization
of Antibiotics by Glycorandomization
• Glycorandomization: Chemoenzymatic glycodiversity
of natural product based scaffolds
N3
N3
O
HO
HO
Add activating group
and enzyme
OH
OH
OH
OR
non-natural substrate
activated sugar
R'
N3
HO
HO
Add antibiotic
and enzyme
O
HO
HO
O
OH
CuAAC
O
R'
antibiotic
N
N
N
HO
HO
randomized library of
Antibiotic Derivatives
O
OH
O
glycosylated antibiotic
Thorson, J.S. et al. Org. Lett. 2005, 7, 1513-1515
antibiotic
31
Glycorandomization of Vancomycin
•
OH
Vancomycin: glycosylated
natural product isolated from the
bacteria Amycolatopsis
orientalis
NH2
HO
HO
O
HO
•
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
Hubbard, B.K., Walsh, C.T. Angew. Chem. Int. Ed. 2003, 42, 730-765
O
O
HO
O
O
O
O
HO2C
HO
Cl
OH
Cl
NH
•
O
O
H
N
N
H
O
OH
OH
N
H
O
H
N
NH
O O
H
N
NH2
vancomycin
32
Glycorandomization of Vancomycin
N3
N3
O
HO
HO
O
O P O
O
HO
HO
Thiamine Pyrophosphate
OH
O
OH
Nucleotidyltransferase
O
NH
O
O
O P O P O
O
O
N
vancomycin aglycon
O
GtfE
O
OH
N3
HO
HO
O
N
OH
O
O
HO
O
Cl
H
N
N
H
O
OH
HO
H
N
N
H
O
NH
HO2C
OH
(24 Alkynes)
NH
CuI, MeOH/H2O
70oC, 12h
O
O
N
R
Cl
O
O
NH2
O
R
R = COOH
N
HO
HO
O
OH
O
H
N
Twice as potent as
Vancomycin
OH
Thorson, J.S. et al. Org. Lett. 2005, 7, 1513-1515
33
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
34
DNA Microarrays Using CuAAC
Create ssDNA or RNA library
•
DNA microarrays (DNA chips)
useful for large scale parallel
analysis of gene expression
•
Chemistry used for
immobilization is limited by
cross-reactivity on surface
•
hybridize to surface
Add complementary
DNA strand with dye
Efficiency and Bioorthogonality
of CuAAC could overcome
existing limitations of
immobilization
Reinhoudt, D.A. et al. ChemBioChem, 2007, 8, 1997-2002
35
Transfer Printing of DNA Using
Dendritic Architectures
H3N
NH3
H3N
NN
O
Si
O
Si
H3 N
NH3
NN
NH3
O
Si
O
Si
Add alkyne modified ssDNA
O
Si
O
Si
H3N
PDMS
O
Si
NH3
O
Si
PDMS
Add Azide Coated Glass
N3
N3
N3
N3
N3
1) Add Cu(I)
DNA
N
N
N
DNA
N
N
N
DNA
N
N
N
2) Remove PDMS Stamp
3) Wash away unbound
dendrimer
H3N
NH3
NN
O
Si
O
Si
H3N
NH3
O
Si
PDMS
Reinhoudt, D.A. et al. ChemBioChem, 2007, 8, 1997-2002
36
O
Si
Synthesis of Alkyne Modified
DNA Monomer
TMS
O
I
O
NH
N
TBDMSO
TMS
NH
O
O
TBDMSO
PdCl2(PPh3)3
CuI, DIPEA, 92%
OTBDMS
N
1) TBAF
O
2) DMTrCl, pyridine
DMAP, 55% (2 steps)
O
OTBDMS
O
O
NH
N
DMTrO
O
N
Cl
P
NH
O
CN
N
DMTrO
O
O
O
THF, DIPEA, 70%
OH
Reinhoudt, D.A. et al. ChemBioChem, 2007, 8, 1997-2002
O
P
N
ssDNA
O
N
37
Surface Patterning of ssDNA
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
Oxime Functionalized Template
QuickTime™ and a
TIFF (Uncompressed) decompressor
are needed to see this picture.
CuAAC Functionalized Template
38
Reinhoudt, D.A. et al. J. Am. Chem. Soc. 2007, 129, 11593-11599
Future Directions: Target Guided
Synthesis (TGS)
•
Target Guided synthesis uses
enzyme for assembling its own
inhibitors in situ
N3
N3
N3
•
Kinetically controlled approach
by irreversible formation of
product
Add enzyme
N3
•
•
Chemoselectivity of
azide/alkyne reaction eliminates
byproducts that may alter
enzyme
In situ generated inhibitors
separated by LCMS and resynthesized for Ki determination
Krasinski, A. et al. J. Am. Chem. Soc. 2005, 127, 6686-6692
Enzyme
N N
N
Inhibited Enzyme
39
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
Mocharla, V.P. et al. Angew. Chem. Int. Ed. 2005, 44, 116-120
Whiting, M. et al. Angew. Chem. Int. Ed. 2006, 45, 1435-1439
40
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
41
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
42