Bioconjugation Techniques

Hanadi Sleiman Department of Chemistry, McGill University

References: Bioconjugate Techniques, Greg T. Hermanson, Academic Press, 1996 Principles of Fluorescence Spectroscopy, Joseph Lakowicz, Third Edition, Springer, 2006 Current Protocols in Nucleic Acid Chemistry, Wiley, e-Book @ McGill Molecular Probes (Invitrogen) http://probes.invitrogen.com/ Glen Research http://www.glenres.com/

Attachment of Fluorescent Labels to Biological Molecules

Biological Assays: Detection and monitoring of a protein or nucleic acid finding the function of genes/proteins,monitoring the progress of a disease, etc.

Cell Biology: mechanistic studies of biological transformations in cells

Genomics, Molecular Biology, Proteomics, Medical Diagnostics

Bioconjugation of Common Functional Groups

R-NH2
O R'
O O N

Lysine, other reactive amines

O

R' N C S
isothiocyanate

R'

acylation
O RHN R'

imine formation

NaBH 3CN
reduction

RHN
O RHN NH-R' Thiourea

R'

Bioconjugation of Common Functional Groups

O R
N H N C N

O
O N N

(aspartic acid, glutamic acid, other reactive carboxylic acids)

O HO N

SO3Na

EDC

CDI
R

Sulfo-NHS
(EDC)

O
O
H N

O
N N

O O N O

SO3Na

O
N C

N H

O R
OR'

R'-OH

R'-NHNH2

R'-NH2 O
NHNHR'

O R
R

NHR'

ester

hydrazide

amide

Protocol (eg, for EDC coupling): 1. Dissolve the protein in buffer (no amines or carboxylate buffers) 2. Dissolve the molecule to be coupled in same buffer at 10-fold molar excess to protein 3. Add the molecule solution to the protein solution 4. Add EDC (concentrated solution in water, freshly prepared) to the protein and molecule; EDC should be 10-fold molar excess 5. React 2 hours at RT 6. Purify the conjugate by gel filtration or dialysis

Bioconjugation of Common Functional Groups

RSH
O N O R'

(Cysteine, other reactive thiols)

H N R'

R'

S S

I O

maleimide
O N R'

alkylation

pyridyl disulfide

RS O

NH

R'

S R S Disulfide

R'

RS

Thioether

Michael add. product

Cysteine/cystine buried inside protein, reduction may cause disruption of protein structure.

Creation of new groups

NH2
O N O O O S

NH

SH

O
H N O O S
NH2OH H N O

SH

SATA

COOH

C O

SR

H2N

NH2

O C N H S S NH2

EDC

NH2
O O O

N H

COOH

O N H

OH O

COOH
H2N EDC NH2

C O

NH2

O NH2
O N O O O O H

N H

Site-Selective incorporation of fluorophores to study protein conformation

FRET pair

Hohsaka, Nature Methods, 2006, 923

Nucleic Acid Biolabeling

DMTO O B

DMTO O

DNA Synthesis

O CN O P O

O CN
B O

O P O O H B

I2 / H20

(oxidation)
O

H+

99.5%
DMTO O
HO 5' O

(deprotection)

(coupling)
B

CN

O P N

Phosphoramidite

Nucleic Acid Biolabeling 5-amino-DNA 5-thio-DNA

5' OH OH N N N N N N N N N N N N N N 3' N N N N N N
MMT : monomethoxytrityl T : trityl

Sequence-labeled amino-DNA

3-amino DNA

Nucleic Acid Biolabeling

Fluorescein

Cy3

TAMRA

Nucleic Acid Biolabeling enzymatic incorporation

Glen Research

Biotinylation The Biotin- Streptavidin Interaction

O

Biotin (Vitamin-H): Can be conjugated to different biomolecules Possesses one of the strongest binding interactions with avidin/streptavidin (Kd ~ 10-15 M) avidin / streptavidin Contains four identical binding sites for interaction with biotin
HN NH

OH S O

Biotin

Biotinylation
O HN NH

O HN O NH

O S O

O N O O S

Biotin

NaO3S

amino reactive
O HN NH

O O N O N H

H N S O

thiol reactive

streptavidin

Biomolecule tagged with biotin

Organic fluorophore tagged with biotin

fluorescence quenched

Mohamed Slim, H. F. Sleiman, Bioconjugate Chemistry, 2004, 15, 949

O HN NH S O H N

(PF6--)2
+2

HN S

NH O

H N N N N Ru N N

+2

4
70000 60000

+ AVIDIN

N N N Ru N

N N

Luminescence Intensity

50000
HN

O NH S O N N H N N N Ru N N
+2

40000 30000 20000 10000 0 500

550

600

650

700

750

Wavelength

Ruthenium Bipyridine as Chromophore

Emission from triplet MLCT

N

N N Ru N N
2+

MLCT

Long lifetime ~ 1 s Large Stokes Shift Resistance to photobleaching Electrochemiluminescence (generates light catalytically)
Chem. Rev. 2004, 104, 3003-3036

Bingzhi Chen, Kim Metera, Rachel Nassif, N. Sankaran

Ph O O m
O X N O O Y

O N O O N O O O OO O O
O O

O
O O O

2PF6

N N

N Ru N

N N

NH O

Biomolecule
N

O O

H N S N H O

Micelle ~ 10,000 luminescent centers

B. Chen, K. Metera, H. F. Sleiman, Macromolecules, 2005, 38, 1084-1090 B. Chen, H. F. Sleiman, Macromolecules, 2004, 37, 5866 37,

Fluorescence probe for DNA mismatches

Oregon Green

Barton, et al, JACS 2006

Is labeling of biomolecules always necessary?

cationic, conjugated polymer

yellow red yellow

Leclerc, M.; Ho, H. A.; Boissinot, M. WO 02/081735 A2, 2002. Gaylord, B. S.; Heeger A. J.; Bazan G. C. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 10954.

Click Chemistry Labeling of virus at all 60 sites

Cowpea mosaic virus

Cu(I)

Cu(I)

Finn, Sharpless, et al, J. AM. CHEM. SOC. 2003, 125, 3192

Staudinger Reaction: in vivo biological labeling

Bertozzi, et al, Nature, 2004, 430, 873

Broad comments: The biolabeling market is lucrative; kits available for most jobs Beware of black box attitude; need to understand the chemistry Think outside the Molecular Probes box New fluorescent labels: metal centers, quantum dots, conjugated polymers, etc. New trends: in vivo labeling with bio-orthogonal chemistry