A Comparative Study of Bioorthogonal Reactions with

Azides

CHEM 258

Jodi Wyman

Agard, N. J.; Baskin, J. M.; Prescher J. A.; Lo A.; Bertozzi C. R. ACS Chem. Biol. 2006,10, 644.

2

Overview

Biomolecule tagging

Bioorthogonal chemical reporters

Scope of labeling reactions

Protein and live cell labeling

Reaction guide

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Green Fluorescent Protein (GFP)

Comprised of 238 amino acids Isolated from jellyfish Aequorea victoria Tripeptide Ser65-Tyr66-Gly67 (center of β–barrel) is the

chromophore Can be modified to fluoresce a variety of colors

Tsien R.; Annu. Rev. Biochem. 1998, 67, 509.

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GFP Applications and Limitations

Application: Monitoring proteins in living cells

Limitations: Structure perturbation (very large) Cannot be used for glycans, lipids, nucleic acids

or other small metabolites

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Bioorthogonal Chemical Reporters

Tags without direct genetic encoding Small molecule reporter Non-native, non-perturbing handles that can work in

living cells Can label biomolecules as long as biosynthetic

pathway will tolerate modified precursors

Prescher, J. A.; Bertozzi, C. R. Nat. Chem. Biol. 2005, 1, 13.

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Choosing a Bioorthogonal Chemical Reporter

Tolerated by cell machinery

Robust (avoid unwanted side reactions)

Rapid and selective labeling

Non-toxic (for use with live cells)

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Bioorthogonal Chemical Reporter:

Azide Advantages: Small Stable in physiological conditions Have metabolic precursors compatible with

existing cellular machinery Not found in many natural species Reacts only with soft nucleophiles (highly

selective)

RN

N+

N-

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Staudinger Ligation

Azide reacts with RPPh2 under mild conditions

Internal electrophilic trap forms amide linkage

Phosphines unreactive towards biological functional groups

O

O

R

N3R'

-N2

O

O

R

P +

N

O

R'

P h P h

R

-OCH3

O

NHR'

R

H2O

P P h 2 P + N- R'

P hP h

P P h 2

O

Saxon, E.; Bertozzi, C. R. Science, 2000, 287, 2007.

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Cu(I)-Catalyzed Azide-Alkyne Cycloaddition

Azide (1,3-dipole) can undergo reactions with activated alkynes

Forms triazole adducts Performed at physiological conditions Fast but has high cellular toxicity

R N3 + R'

Cu(I),ligand

N

N

N

R'

R

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Strain-promoted [3+2] Cycloaddition

Catalyst free [3+2] Can be performed on surface of living cells Increase reaction rate with addition of EWG

on cyclooctyne

R N3 +

R' R'

N

N

N

R

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Improve Strain [3+2] Cycloaddition Kinetics

Strategies: Remove phenyl ring EWG next to alkyne

O

NHRO

O

O NHR

F

NHR

O

1 2 3

O NH

O

3

S

NH

HNO

H

H

R=

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Synthesis of [3+2] Cycloaddition Probes

BrB r

AgClO4 , 10eqmethyl glycolate

60%

Br

O

C O2Me(i) NaOMe, DMSO, 0 oC(ii) H2 O

85%

O

O O H

(a) Pentafluorophenyltrifluoro-acetate, pyr

(b)RNH2, pyr, 27%

O

O NHR

67

8

2

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Synthesis of [3+2] Cycloaddition Probes

O

X

(i) LDA, THF, -78 o C(ii) Methyl 4-bromo-methylbenzoate-78 o C to 25 oC

10a: 43%, 10b: 80%9a: X=F9b: X=H

O

X

C O2Me

(i) KHMDS, THF, -78 oC(ii) Tf2 NPh, -78 o C to 25 oC

11a: 73%, 11b: 68%

OTf

X

CO 2Me

(i) LDA, THF, 0 o C(ii) LiOH, dioxane/H2O50 oC

12: 58%, 13: 22%

X

C O2H

10a: X=F10b: X=H

11a: X=F11b: X=H

12: X=F13: X=H

F

(a ) Pentafluorophenyltrifluoro-ace tate, DIEA

(b) RNH2, DIEA, 40%

NHR

O

3

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Kinetic Evaluation of Strained [3+2] Cycloaddition

NN+

N-

benzyl azide

Compound Reaction rate (M-1s-1)

12 4.3x10-3

1 2.4x10-3

8 1.3x10-3

13 1.2x10-3

F

CO2Me

12

O

OHO

1

O

O OH

8

H

CO2Me

13

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Biotinylated ProbesO

NHRO

O

O NHR

F

NHR

O

O

NHR

O

O

P Ph2

NHR

O

1 2 3

4 5

O NH

O

3

S

NH

HNO

H

H

R=

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Protein Labeling

Dehydrofolate reductase (DHFR) Replaced methionine residues with

azidohomoalanine

Noted: certain detergents used to solubilize the protein hindered click chemistry and Staudinger ligation

S

NH2

O

OH

methionine

N3

NH2

O

OH

azidohomoalanine

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Protein Labeling Results

Top: Time analysis of 100μM of reagent Bottom: Concentration dependence

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Protein Labeling in Presence of Cell Lysate

Top: Labeled proteins detected by Western blot Bottom: Total protein content determined by

Ponceau S

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Live Cell Labeling

Grew Jurkat cells with Ac4ManNAz which (expressed as SiaNAz on the cell surface)

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Live Cell Labeling

Varki, A.; Cummings, R. D.; Esko, J. D.; Freeze, H. H.; Stanley, P.; Bertozzi, C. R.; Hart, G. W.; Etzler, M. E. Essentials of Glycobiology, Second Ed., 2009, p. 686

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Live Cell Results

Mean fluorescence intensity (MFI)

Determined by flow cytometry

Click chemistry resulted in significant cell death

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Toxicity in Live Cells

Toxicity - Az

0.0

20.0

40.0

60.0

80.0

100.0

Click Cyclooctyne Phosphine

% Alive

0 uM

50 uM

100 uM

200 uM

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Specific Applications

Optimal reaction based on application Staudinger ligation: Surfaces of cells and live

organisms Click chemistry: Proteomic samples Strain-promoted [3+2]: Surfaces of cells

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Summary

Bioorthogonal chemical reporters are powerful tools for tagging specific biomolecules

Azide is an easy and versatile reporter A variety of chemical reactions to tag azide

can be performed depending on desired sensitivity