Bioconjugate Chemistry

On Proteins

Bioconjugate Chemistry

=

joining of biomolecules to other biomolecules, small

molecules, and polymers by chemical or biological means

• Labelling

• Conjugation / Crosslinking

• Immobilization

• Modification introducing functional groups,

Purpose

Bioconjunction = Chemistry on Proteins

residue specific & biocompartible

• different functional groups• complex

Challenge

• water as solvent• pH 6-8• temperatur• low reactant

concentrations• non toxic reagents

# 5

Amino acids

• Aliphatic AS Aromatic AS

• Polar AS

• hydrophilic

• near surface accessible

• PTMs (glycosylated)

• same nucleophility as water

• hydrophobic core inside the protein

• hardly accessible• no reactive groups

# 6

Amino acids

• Ionizable Amino acis Chemistry possible

Reactive groups AminesThiolsHydroxylCarboxyl

# 7

Cysteine

Chemistry of Reactive Groups

• Amines• Thiols• Carboxylates• Hydroxyls

• Unspecific Reactions – Photoreactive Chemistry

# 8

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

# 9

Amines – Acylation or Alkylation

• Isothiocyanate / Isocyanate

• Acyl- Azides, Sulfonylchloride

• Aldehydes, Ketones / Reductive Amination

• Epoxides

• Imidoester

# 10

Thiols –Alkylation/ Disulfide Exchange

• Haloacetals

• Maleimides

• Aziridines / Epoxides

• Acrylonitril / Vinylsulfone-Derivatives

•Thiol-Disulfide Exchange

z.B. Pyridyl Disulfides

# 11

Carboxylate –Activation needed

• Carbodiimides EDC

• Carbonyldiimidazol

# 12

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

# 13

Non-specific –Photoreactive Reagents

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

• Diazirine

• Benzophenone

• Aryl Azide / Halogenated Aryl Azides

# 14

C/N Terminus

Native Chemical Ligation similar to intein splicing

• No protection groups necessary• Chemoselective• < 300 AA

# 15

Application

• Antigen preparation for small molecules

• e.g nucleosides, drugs, peptides, sugars

• Drug-antibody conjugates

• PEGylation of proteins

• Crosslinking for structural and interaction proteomics

# 1618.01.2016Referat Markus Mustermann

• Antigen Preparation for small molecules

# 17

Antibody Production

• Rabbits can generate only a vigorous immune response against

various small molecule haptens conjugated to immunogenic carrier

proteins

# 18

Antibody ProductionHapten/Carrier Conjugation Techniques

Carboxyl-to-Amine Conjugation using EDC

often Self-Conjugations / not necessary

a disadvantage

Sugars:

Periodat-Cleavage /Reductive Amination

# 19

Antibody ProductionHapten/Carrier Conjugation Techniques

Maleimide Method

higher yields with less polymerization,

producing a conjugate preparation with superior immunoassay characteristics.

# 20

Antibody ProductionHapten/Carrier Conjugation Techniques

Cationized BSA

Creating a more immunogenic Carrier Protein

# 2118.01.2016Referat Markus Mustermann

• Antibody-drug conjugates

Antibody drug conjugates

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

# 2218.01.2016Referat Markus Mustermann

Antibody drug conjugates

Trastuzumab emtansine (T-DM1)

• For targeted cancer therapy (breast cancer)

• single trastuzumab molecule with several molecules of DM1

# 2318.01.2016

Antibody drug conjugates

Trastuzumab emtansine (T-DM1)

# 2418.01.2016

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

• targeted cancer therapy

(breast cancer)

• single trastuzumab

molecule with several

molecules of DM1

# 2518.01.2016Referat Markus Mustermann

• PEGylation of protein-based drugs

PEGylation

# 2618.01.2016

Advantages.

• Prolonged half-life

• Higher stability

• Water solubility

• Lower immunogenicity / antigenicity

PEGylation

# 2718.01.2016Referat Markus Mustermann

PEGLOTICASE

• Treatement against gout

• Porcine-like uricase

• metabolises uric acid to

allotoin

Decrease of immunogenicity

Long-term treatment possible

# 2818.01.2016Referat Markus Mustermann

• Crosslinking for structural and

interaction proteomics

# 29

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)

# 30

Chemical X-Linking

Design:

# 31

Chemical X-Linking

• biggest problem: Mono-links

• optimization of cross-linking

# 32

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

# 3318.01.2016Referat Markus Mustermann

• Bioorthogonal Chemistry

# 34

• What about site-specific Labelling?

Introduction of genetically encoded AS – Bioorthogonal

Reactions

Bioorthogonal Reactions

# 35

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 systemY = complementary group, reacts in a bio-compartible way with X

# 36

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

# 37

Bioorthogonal Chemistry

Cu-catalyzed Azid-Alkyne Cycloaddition „Click“

• Huisgen• Sharpless, Medal Nobelpriz 2001

# 38

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.

# 39

Biorthogonal Chemistry

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

C.R.Bertozzi, J.Am.Chem.Soc. 2004, 126,15046–15047

# 4003.02.14

Biorthogonal Chemistry

Tetrazine Ligation– Inverse Diels Alder/retro Diels-Alder

.M.Fox, J.Am.Chem.Soc. 2008, 130,13518–13519.

# 41

Biorthogonal Chemistry

– Photo-induced Tetrazole-Alken Cycloaddition

# 42

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.

# 43

Biorthogonal Chemistry

– Strain-promoted Alkyne-Nitrone Cycloaddition

• problem:Incorporation of nitrones into biomolecules