Bioconjugate Chemistry On Proteins Bioconjugate Chemistry = joining of biomolecules to other biomolecules, small molecules, and polymers by chemical or biological means Purpose

• Labelling • Conjugation / Crosslinking • Immobilization • Modification  introducing functional groups, Challenge

Bioconjunction = Chemistry on Proteins

 residue specific & biocompartible • water as solvent • pH 6-8 • different functional groups • temperatur • complex • low reactant concentrations • non toxic reagents Amino acids

• Aliphatic AS Aromatic AS

• hydrophobic core inside the protein • hardly accessible • no reactive groups • Polar AS

• hydrophilic • near surface  accessible • PTMs (glycosylated) • same nucleophility as water

# 5 Amino acids

• Ionizable Amino acis  Chemistry possible

Reactive groups  Amines Thiols Hydroxyl Carboxyl

# 6 Cysteine

Chemistry of Reactive Groups

• Amines • Thiols • Carboxylates • Hydroxyls

• Unspecific Reactions – Photoreactive Chemistry

# 7 Amines – Acylation or Alkylation

NHS Esters

• Hydrolysis (increases with pH) • in phosphate, carbonate-bicarbonate, HEPES or borate buffers (not TRIS) at pH 7.2 to 8.5 for 0.5 to 4 hours at room temperature or 4°C

Sulfo-NHS

 watersoluble, non cell-permeable

# 8 Amines – Acylation or Alkylation

• Isothiocyanate / Isocyanate • Epoxides

• Acyl- Azides, Sulfonylchloride • Imidoester

• Aldehydes, Ketones / Reductive Amination

# 9 Thiols –Alkylation/ Disulfide Exchange

• Haloacetals •Thiol-Disulfide Exchange

• Maleimides

z.B. Pyridyl Disulfides

• Aziridines / Epoxides

• Acrylonitril / Vinylsulfone-Derivatives

# 10 Carboxylate –Activation needed

• Carbodiimides EDC

• Carbonyldiimidazol

# 11 Hydroxyls – unspecific chemistry

• Epoxides  also for –SH, NH2…

• Activation Chemistry • Carbonyldiimidazol • N,N -Disuccinimidyl Carbonate or N -Hydroxysuccinimidyl Chloroformate

• Glyco/Carbonyl reactive chemistry: Periodate Cleavage + Amine or Hydrazide or Hydroxylamine

# 12 Non-specific –Photoreactive Reagents

• UUU = UV-light, Unspecific, U can decide when

• Diazirine

• Benzophenone

• Aryl Azide / Halogenated Aryl Azides

# 13 C/N Terminus

Native Chemical Ligation  similar to intein splicing

• No protection groups necessary • Chemoselective • < 300 AA

# 14 Application

• Antigen preparation for small molecules • e.g nucleosides, drugs, peptides, sugars

• Drug-antibody conjugates

• PEGylation of proteins

• Crosslinking for structural and interaction proteomics

# 15 • Antigen Preparation for small molecules

Referat Markus Mustermann 18.01.2016 # 16 Antibody Production

• Rabbits can generate only a vigorous immune response against various small molecule haptens conjugated to immunogenic carrier proteins

# 17 Antibody Production Hapten/Carrier Conjugation Techniques

 Carboxyl-to-Amine Conjugation using EDC

often Self-Conjugations / not necessary a disadvantage

 Sugars:

Periodat-Cleavage /Reductive Amination

# 18 Antibody Production Hapten/Carrier Conjugation Techniques

Maleimide Method higher yields with less polymerization, producing a conjugate preparation with superior immunoassay characteristics.

# 19 Antibody Production Hapten/Carrier Conjugation Techniques

Cationized BSA Creating a more immunogenic Carrier Protein

# 20 • Antibody-drug conjugates

Referat Markus Mustermann 18.01.2016 # 21 Antibody drug conjugates

combining the specificity of antibodies with the potency of small molecules to create targeted drugs

Referat Markus Mustermann 18.01.2016 # 22 Antibody drug conjugates

Trastuzumab emtansine (T-DM1)

• For targeted cancer therapy (breast cancer) • single trastuzumab molecule with several molecules of DM1

18.01.2016 # 23 Antibody drug conjugates

Trastuzumab emtansine (T-DM1)

• targeted cancer therapy (breast cancer) • single trastuzumab molecule with several molecules of DM1

deliver toxins directly into tumor cell with the help of an antibody

18.01.2016 # 24 • PEGylation of protein-based drugs

Referat Markus Mustermann 18.01.2016 # 25 PEGylation

Advantages. • Prolonged half-life • Higher stability • Water solubility • Lower immunogenicity / antigenicity

18.01.2016 # 26 PEGylation

PEGLOTICASE • Treatement against gout • Porcine-like uricase • metabolises uric acid to allotoin

 Decrease of immunogenicity  Long-term treatment possible

Referat Markus Mustermann 18.01.2016 # 27 • Crosslinking for structural and interaction proteomics

Referat Markus Mustermann 18.01.2016 # 28 Chemical Crosslinking = To join (adjacent chains of a polymer or protein) by creating covalent bonds using a crosslinking reagent

 In combination with Mass-Spectrometry (Orbitrap)

# 29 Chemical X-Linking

Design:

# 30 Chemical X-Linking

• biggest problem: Mono-links • optimization of cross-linking

# 31 Chemical X-Linking / Label Transfer

SBED • Heterotrifunctional, photoreactive, thiol-cleavable label transfer reagents enable the tagging of a prey protein

• Biotin Label is transferred to interacting protein • Affinity enrichment possible

# 32 • Bioorthogonal Chemistry

Referat Markus Mustermann 18.01.2016 # 33 • What about site-specific Labelling? Introduction of genetically encoded AS – Bioorthogonal Reactions

 Bioorthogonal Reactions

# 34 Bioorthogonal Chemistry

 real time studies in living systems without cellular toxicity 1. cellular substrate is modified with a bioorthogonal functional group (chemical reporter) and introduced to the cell 2. probe containing the complementary functional group is introduced to react and label the substrate

X = bioorthogonal group, not present in the biological system Y = complementary group, reacts in a bio-compartible way with X

# 35 Bioorthogonal Chemistry

• Bioorthogonal Chemistry Strategies

– Cu-catalyzed Azid-Alkyne Cycloaddition „Click“ – Strain-promoted „Click“-Reaction – Staudinger Ligation – Tetrazine Ligation – Photo-induced Tetrazole-Alken Cycloaddition – Norbonene System – Strain-promoted Alkyne-Nitrone Cycloaddition

# 36 Bioorthogonal Chemistry

Cu-catalyzed Azid-Alkyne Cycloaddition „Click“

• Huisgen • Sharpless, Medal Nobelpriz 2001

# 37 Biorthogonal Chemistry

Staudinger Ligation • based on classic Staudinger reaction = Reduction of azides to amines • Bertozzi 2000: – trapping of product prior to hydrolysis  coupled product

– slow kinetics – PPh3 can be oxidized before

Science 2000, 787, 2002. # 38 Biorthogonal Chemistry

Strain-promoted „Click“-Reaction / (Cu-free)

C.R.Bertozzi, J.Am.Chem.Soc. 2004, 126,15046–15047 # 39 Biorthogonal Chemistry

Tetrazine Ligation – Inverse Diels Alder/retro Diels-Alder

.M.Fox, J.Am.Chem.Soc. 2008, 130,13518–13519. 03.02.14 # 40 Biorthogonal Chemistry

– Photo-induced Tetrazole-Alken Cycloaddition

# 41 Biorthogonal Chemistry

– Norbonene Click –Incorporation of norbonen via pyrrolysine system  Markus –balance between strain-promoted reactivity and stability.

Carell, Organic Letters 2009, 11, 2405–8, Angew Chem Int Ed Engl. 2012, 51, 4466-9. # 42 Biorthogonal Chemistry

– Strain-promoted Alkyne-Nitrone Cycloaddition

• problem: Incorporation of nitrones into biomolecules

# 43