Click chemistry reagents and site specific conjugation

Designing a conjugation

1. Design the attachment point before the chemistry. An enzymatically installed tag, an unnatural amino acid or an engineered residue gives a single defined site. Random coupling to abundant residues gives a distribution that has to be characterised every batch.
2. Choose copper free chemistry for living systems. Copper catalysis is efficient and toxic to cells. Strain promoted variants proceed without it, more slowly, and are the route for live cell and in vivo work.
3. Start from characterised material. A conjugation on a peptide of unknown purity or net content produces an uninterpretable mixture. Establish purity, net peptide content and identity first.
4. Plan the purification of the conjugate. The product has to be separated from unreacted starting materials and from over-coupled species, and the yield is frequently modest. Budget the separation and the analytics.
5. Characterise the conjugate, not the components. Mass, degree of labelling and its distribution, aggregation state and retained activity. A conjugate described only by its intended structure has not been characterised.
6. Order named ligands as reagents. A well known signalling ligand ordered as a characterised protein comes with activity data. Ordering the sequence and making it yourself adds a folding project to the experiment.

Selectivity is the whole point

Bioorthogonal reactions exist so that a coupling can happen in a complex mixture without touching anything else. That selectivity is what allows labelling in cells and in animals rather than only in a tube.

Where the reaction is not selective, the conjugate is a mixture and every subsequent measurement is an average over it.

Homogeneity is easier to defend

A conjugate with one attachment site behaves consistently and can be characterised once. A distribution of species behaves as an average that shifts between batches.

Where a conjugate will be used more than a few times, the effort of designing a defined site repays itself quickly.

Research reagents, and how to compare two catalogue entries

Two entries for the same reagent differ on things a price cannot show: grade and the impurity specification, the lot documentation and whether a certificate of analysis is supplied, the storage and shipping condition, the pack size against your usage and the shelf life after opening, and whether the supplier will state lot-to-lot consistency. For anything entering a reported result, the certificate and the lot record are what make the result defensible later, which is why the cheapest line is not always the cheapest purchase.

Ellman's reagent and the thiols it counts

Ellman's reagent reacts with free thiols to give a coloured product measured at 412 nanometres, which is how free cysteine content is quantified in a protein or a buffer, and it is the standard check before and after a conjugation. Disulphides are invisible to it unless the sample is reduced first, which is exactly how bound and free thiols are told apart.

Cumate and an inducible expression switch

Cumate is the inducer of a bacterial operator system adapted for mammalian expression, chosen where the more common antibiotic based systems are unsuitable because of resistance genes or leakiness. The practical questions are the inducer's cost at culture scale, its removal kinetics when expression has to be switched off, and whether the system's basal activity is low enough for a toxic product.

An amino acid supplier, and what an order has to state

An amino acid supplier is chosen by grade and form rather than by price: free base or hydrochloride, L or D, unprotected for media and protected for synthesis, at a purity suited to cell culture, peptide chemistry or analysis. Endotoxin and trace metal limits matter for culture. Labelled amino acids for tracing are a separate line with their own isotopic enrichment specification.

oligonucleotide conjugation and the chemistry chosen

oligonucleotide conjugation joins a nucleic acid to a protein, a dye or a lipid, and the chemistry follows the handle put on the oligonucleotide during synthesis: an amine, a thiol, an azide or an alkyne. Purification of the conjugate from unreacted material is the hard step, so the method is planned with the separation in mind rather than only the reaction.

A c3 spacer and what a spacer is for

A c3 spacer is a three carbon insert placed in an oligonucleotide to block extension, to position a modification away from the duplex or to break a sequence without changing its length. Because it blocks polymerase extension it is used as a three prime terminator in probes and primers, and its position rather than its chemistry is what the design has to specify.

An ammonium acetate solution and where it is preferred

An ammonium acetate solution is the volatile buffer for mass spectrometry, since it evaporates in the source where phosphate and Tris suppress ionisation, and it is also used for nucleic acid precipitation. It buffers weakly and over a narrow range, so it is chosen for volatility rather than for buffering strength, and its pH shifts as ammonia is lost.

A phosphate buffer solution and its limits

A phosphate buffer solution is the default physiological buffer and is the wrong choice in several common steps: it precipitates with calcium and other divalent cations, inhibits several enzymes, suppresses ionisation in mass spectrometry and buffers poorly above pH 8. Its pH also shifts on freezing, which is why frozen aliquots change between thaws.

Common questions

Why use copper free chemistry?

Because copper catalysis is toxic to cells and can damage proteins. Strain promoted reactions are slower and proceed without it, which is what makes live cell and in vivo labelling possible.

Random or site specific coupling?

Site specific wherever the conjugate has to be reproducible or characterised, because it gives one species. Random coupling is faster, cheaper and gives a distribution that must be measured each time.

What should a conjugate certificate state?

Mass confirmation, degree of labelling and its distribution, purity after conjugation, aggregate content and retained activity. Purity of the starting material alone is not enough.

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