Attaching one molecule to another without damaging either: why click chemistry reagents are chosen for bioorthogonal reactions that proceed in water at low concentration and why copper free variants exist for living systems, what a cyclic peptide, a lyophilized peptide, synthetic peptide synthesis and peptide production generally have to deliver before conjugation is attempted, where peptide modifications and an avi tag give a defined attachment point rather than a random one, what an amino acid supplier and specialised building blocks contribute to any of it, why heregulin and other named ligands are ordered as characterised reagents rather than as sequences, and what the conjugate itself has to be characterised on

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.

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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