BioconjugationBioconjugation TechniquesTechniques

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 Lysine, other reactive amines O O O R' R' O N N R' H O imine formation acylation C NaBH CN O S 3 isothiocyanate reduction

RHN R' RHN R' O

RHN NH-R' Thiourea Bioconjugation of Common Functional Groups

O (aspartic acid, glutamic acid, other reactive R O carboxylic acids) N O H O HO N SO Na N N N 3 N N C EDC O N CDI Sulfo-NHS (EDC) R O O O SO3Na O O R N H N R O N C N N N H O

R'-NHNH2 R'-OH R'-NH O 2 O R OR' O NHR' NHNHR' R ester R amide hydrazide 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 (Cysteine, other reactive thiols) O

N R' H N R' S N R' I S O O maleimide alkylation pyridyl disulfide O NH R' S R' N R' RS R S O RS O Thioether Disulfide Michael add. product

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

NH2 NH SH

O O

N O S O H N S NH2OH O H O N SH

O SATA O

COOH C SR O

S NH2 O H2N S C S NH2 N S EDC H NH2 N COOH H

O

O O O OH N H O

COOH C NH2 O

NH2 H2N

EDC O

NH2 N C H H O O N O

O O H Site-Selective incorporation of fluorophores to study protein conformation

FRET pair

Hohsaka, Nature Methods, 2006, 923 Nucleic Acid Biolabeling DNADNA DMTO B DMTO O B SynthesisSynthesis O

O O O CN P O O CN P O B I2 / H20 O O B (oxidation) O

O H O

99.5% H+ (deprotection) DMTO B O (coupling) HO 5' B O O O CN P N O Phosphoramidite Nucleic Acid Biolabeling 5’-amino-DNA 5’-thio-DNA

5'OH OH Sequence-labeled amino-DNA N N N N N N N N N N N N N N 3' N N 3’-amino DNA N N N N

MMT : monomethoxytrityl T : trityl Nucleic Acid Biolabeling

Fluorescein

Cy3

TAMRA Nucleic Acid Biolabeling –enzymatic incorporation

Glen Research Biotinylation

The Biotin- Streptavidin Interaction O Biotin (Vitamin-H):

HN NH • Can be conjugated to different biomolecules OH • Possesses one of the strongest S binding interactions with O avidin/streptavidin (K ~ 10-15 M) d Biotin

avidin / streptavidin

• Contains four identical binding sites for interaction with biotin Biotinylation O O

HN NH HN NH

O -O O N S S O O O Biotin NaO3S Biotin amino reactive

O

HN NH

O H N O N S H N O

O thiol reactive streptavidin

Biomolecule Organic fluorophore tagged with biotin tagged with biotin fluorescence quenched

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

O O HN NH -- H +2 (PF6 )2 N S O N HN NH N N Ru + AVIDIN N N H +2 N S N 4 O N 70000 60000 N N 50000 O Ru Luminescence HN NH H +2 N 40000 N N Intensity S O N N 30000 N N Ru N N 20000 N 10000 4 0 500 550 600 650 700 750 Wavelength Ruthenium Bipyridine as Chromophore

Emission from triplet MLCT N N N Ru 2+ N N N

MLCT

• Long lifetime ~ 1 μs • 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 O O n m X Y

O O O O N O O N O N O O O

O O O O O O O O Biomolecule NH

N O O O N N N O 2PF6 Ru N N Micelle N ~ 10,000 luminescent H N centers S O N H

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