chemical biology

What is a bioorthogonal ligation reaction

a reaction that joins 2 components together without interfering with biological processes or molecules

5 features of a bioorthogonal ligation reaction

high yielding and rapid (even at low concentrations)

stable products and few (non-toxic) byproducts

temperatures and pHs that are compatible with biological systems

not affected by water, salts and biological functional groups

chemoselective as use functional groups not present in biology to control reaction

4 examples of bioorthogonal reactions

staudinger ligation

CuAAC

SpAAC

tetrazine ligation (IEDDA)

applications of bioorthogonal ligation reactions

‘smuggling’ useful functional groups onto areas of the cell which are difficult to study for later attachment of a label for studying.

staudinger ligation vs staudinger reduction

ligation is a modified version of the reduction (which converts azides to amines), where the aza-ylid is trapped by an internal electrophile.

rate of staudinger ligation

RDS is first step so rate depends on conc of both reagents - 2nd order.

put staudinger ligation mechanism on crib sheet

advantages of azides as bioorthogonal groups

not found in biological systems

don’t react with most groups in biological systems

small and non polar so azide analogues have similar structures to the unmodified molecule

fluorogenic labelling with staudinger ligation example

when P LP is available, it quenches the fluorophore, but when the reaction occurs and forms P=O group, the molecule becomes intensely fluorescent as the LP is no longer available

evaluation of staudinger ligation

slow rate as second order, depends on conc of azide and phosphine. Slow reaction overall

Phosphine reagents not very stable - oxidised by air and metabolic enzymes

thermal azide-alkyne cycloaddition vs copper catalysed

only copper reaction can occur at room temp and be selective for 1,4 triazole

rate comparison values of different bioorthogonal ligations on crib sheet

CuAAC mechanism

controversy over exact mechanicm, not true cycloaddition due to Cu involvement

click chemistry

reactions that are spring loaded for a single trajectory with a high thermodynamic driving force (usually >20kcalmol-1)

3 applications of CuAAC

drug discovery with library assembly

assembly in polymer and materials chemistry

imaging of cells with metabolic incorporation of sugar analogues onto cell surface gycans

disadvantages of CuAAC

Cu(I) is toxic and can generate ROS - not idea for imaging live cells.

BUT can use ligands to chelate Cu(I) to reduce amount needed and avoid contact with surroundings which would cause damage

reagents for CuAAC for biological applications

CuSO4, TCEP (reducing agent), TBTA (chelating ligand)

basis of SpAAC

energy of alkyne raises with ring strain to promote the reaction without a catalyst (with 163 degree bond angle)

is SpAAC regioselective

NO

put SpAAC mechanism on crib sheet!

further increasing reactivity of SpAAC reaction

EWGs in ring lower alkyne LUMO

more Sp2 centres in ring increases strain and rate

SpAAC evaluation

live cell compatible

cyclooctynes difficult to synthesise

rate is slower than CuAAC

side reactions of cyclooctyne, e.g. with thiols

molecular orbital diagrams and species for IEDDA reaction on crib sheet!

tetrazine ligation mechanism on crib sheet

increasing reactivity of tetrazine and alkene/alkyne

tetrazine - add EWGs onto ring to make diene more e- poor

alkene/alkyne - add EDGs to make more electron rich. strained dienophiles react faster as pre-distorted to a similar shape as the TS. E.g. trans-cyclooctene (TCO)

sterics also affects rate

are alkenes or alkynes more reactive for tetrazine ligation

alkenes as more s character than alkynes

put how to predict regiochemistry of tetrazine ligation products on crib sheet

issue with very reactive tetrazines

tend to be less stable and have issues with aq solubility

evaluation of tetrazine ligation

becoming increasingly popular for fast cellular imaging

size of the groups involved is not ideal

reactants can be hard to synthesise and unstable (e.g. cyclooctenes)

how to use multiple tags selectively

exploit reactivity of groups, e.g. TCO will react first with tetrazines then cyclooctynes

what is a bioorthogonal dissociation reaction

reactions that break chemical bonds in a biocompatible way

how to biorthogonal dissociation reactions tend to work

reagent or trigger generates an unstable intermediate which collapses to form the product with bond cleavage

staudinger reduction mechanism on crib sheet

staudinger reaction as a bioorthogonal dissociation reaction application

azide is inactive drug, amine is active

applications of bioorthogonal dissociation reactions

activating prodrugs

cleaving antibody-drug conjugates to release drug at desired site of action

what does staudinger reaction use

TCEP as a reducing agent

bioorthogonal dissociation by tetrazine ligation mechanism on crib sheet!

PTM definition

covalent addition of functional groups or other proteins or the removal of groups or parts of a protein

PTM basics

around 400 known PTMs, which add diversity and rapidly fine tune protein structure and function

Phosphorylation

introduces phosphate group (on crib sheet) which has negative charge. Can be recognised by other proteins

Acetylation

neutralises positive charge of amines. Can disrupt salt bridges. Catalysed by acyltransferase and removed by de-acylase

acetylation of histones reduces electrostatic interactions of DNA, making it more loosely packed

Glycosylation in covid

N-glycosylation sites on spike protein

O-GlcNAcylation basics

addition of N-acteyl glucosamine to O on Ser/Thr

addition catalysed by O-GlcNAc transferase (OGT) and removal catalysed by O-GlcNAcase (OGA)

mechanisms of OGT and OGA on crib sheet

designing OGA inhibitors

mimic structure of oxazoline intermediate - with thiazole group which can’t be hydrolysed, leads to less removal of O-GlcNAc groups.

what is metabolic tagging

modified group metabolised and incorporated into cell structures which allows a bioorthogonal ligation reaction to introduce a tag. Small structural changes to the group are tolerated by the metabolic pathway

benefits of metabolic tagging

allows concepts that may be difficult to study to be explored

How to get a modified sugar to be taken up into cells for metabolic tagging

Use Ac groups as protecting groups for OHs to allow the sugar to pass through the hydrophobic cell membrane. Once in the cell the sugar is processed by esterases and converted to have a UDP leaving group. Once incorporated into desired structure, can then introduce group for bioorthogonal ligation

biotin extraction

biotin binds with very high affinity to streptavadin. Using streptavadin coated beads allows other proteins to be washed away and the protein to be isolated. Or streptavadin-enzyme conjugates allow identification

synthesis of GlcNAc probe on crib sheet

groups typically incorporated for metabolic tagging

azide or alkyne

N-myristolisation basics

attachment of myristol to N terminus of proteins. Affects localisation/folding/stability

N-myristolisation catalysis

catalysed by NMT with SCoA as a leaving group (nucleophilic attack by N terminus)

studying N-myristolisation with metabolic tagging

incorporate alkyne at end of chain (small enough difference to be tolerated)

Once inside cell, conversion to SCoA form. Then incorporated onto proteins and can be labelled and visualised with bio-orthogonal ligation reaction

basis of amino acid residue labelling

exploit nucleophilicity of some protein residues:

• Lys (most abundant)

• Tyr

• Met

• Cys (most nucleophilic but least abundant)

Lys modification reagents

activated carbonyls as electrophiles to form amide (put examples on crib sheet). Commerically available or easy to prepare and react fast

Evaluation of Lys modification

some reactivity with other residues (cys/Ser) - depends on microenvironment. Lys is common so hard to select for desired Lys

example of role of microenvironment in Lys modification

nearby His/Asp residues promote deprotonation of Lys which makes it more reactive so able to react with less reactive carbonyls (examples on crib sheet!). Selective as reaction is slower so prefers deprotonated Lys

Cys modification

can be selective due to high nucleophilicity and low abundance of Cys - less reactive electrophiles (common to use conjugate addition or alkylation (examples on crib sheet!))

issue with Cys modification

some Cys residues tied up in disulfide bonds - can introduce one cys with mutagenesis

mechanism for labelling disulfide bonds on crib sheet

labelling ‘hyper-reactive’ cysteines

depending on microenvironment, cys may be likely to be deprotonated, making it more nucleophilic so can react with most electrophiles and can be used to deveop covalent inhibitors.

Combining with bio-orthogonal ligation can allow identification of the most reactive Cys residue

how to identify sites of modification in a protein

intact protein mass spec

applications of protein labelling

• therapeutics (antibody-drug conjugates)

• fluorescently label proteins to study behaviour

• identify ‘hyper-reactive’ residues which could be useful to covalently inhibit

• attach to surfaces with biotin

what are activity based probes

chemical probes which selectively label active enzymes in a complex biological sample often by targeting active site nucleophiles

why are activity based probes useful

for enzymes that are tightly regulated, their overall levels doesn’t reflect their activity so need to quantify active proteases

role of catalytic triad in serine proteases

increases the nucleophilicity of ser

put serine protease mechanism on crib sheet!

fluorophosphonate inhibitors of serine proteases

electrophilic warhead which leads to covalent inhibition, can be combined with a fluorophore for visualisation of the active protein

put mechanism/structure on crib sheet!

application of serine protease inhibitors in drug discovery

if the enzyme is effectively inhibited, labelling with a fluorophore will be blocked

bacterial serine hydrolase mechanism - targeting His and Ser - put on crib sheet!

cysteine protease mechanism on crib sheet

S1 and P1 nomenclature for enzyme inhibition on crib sheet

how to increase selectivity for Cys proteases

manipulate the P1/P2/P3 positions

P2 position for cys proteases

leucine fits well into S2 position and mimics substrate - increases selectivity

useful warhead for cys proteases

epoxides, michael acceptors good for selective cys inhibition (e.g. acylamide)

basis of fluoresence assay to measure protease substrate specificity

attaching fluorophore to other side of peptide bond - when bond is cleaved, the fluorophore will be released and fluoresence will be generated

general structure of activity based probes

electrophilic warhead, linker or specificity element to improve binding, then tag (fluorophore or biotin)

how does biotin work

can be used to enrich and purify labelled proteins due to high affinity of biotin for streptavadin - using streptavadin coated beads means that other proteins are washed away (pull down), then the biotin bound proteins can be digested into fragments and analysed with LC-MS

how to determine which specific proteins are inhibited by an inhibitor

compare fluorescently labelled proteins before and after addition of the inhibitor. If the protease is inhibited, it won’t be fluorescently labelled

benefit of bioorthogonal tags over large probes

large probes have issues with cell permeability and may alter the binding behaviour of the protein, whereas smaller probes set up for a bio-orthogonal ligation can be more cell permeable and more likely to bind to the active site

different options for visualisation of fluorescent probes

can visualise in living cells or be used in SDS-PAGE

mechanism of inverting and retaining glycosidases on crib sheet

epoxide inhibitors of glycosidases basis

lack of oxygen in ring system means that covalent intermediate with enzyme is harder to hydrolyse as the O LP in the ring helpes to stabilise the positive charge on the intermediate by resonance

put structure of inhibtors on crib sheet

how is the site of modification on the protein determined

tryptic digest - figure out details!

consider watching back the SDS-page details from screencasts? or ask in workshop

how do quenched fluorescent probes work?