Beware non-benign alkyne

Alkynes will probably feature a lot in this blog due to their bioorthoganol nature – but beware, as a recent paper demonstrates, everything can change in the local environment of a protein.

Whilst investigating de-ubiquitinase enzymes (DUBs), the substrate ubiquitin (Ub) was C-terminally capped with propargyl amine, with idea of forming triazole linked conjugates.  On screening the propargyl intermediate in a fluorescent polarisation assay, it was discovered that the propargyl substrate inhibited the enzyme irreversibly with a 1:1 stoichiometry and covalent modification by MS and SDS-PAGE analysis.  The reaction could be abolished by preincubation of the DUB with the cysteine reactive N-ethyl maleimide, but not by the presence of excess propargyl amine or the thiol DTT.  The acid lability of the product suggested a vinyl thiolether, which was confirmed by X-ray crystallography.

The scope of the reaction was examine by varying the C-terminus and propargyl capping unit, briefly it was proposed that the terminal CH bond was important, steric hindrance at the 2-position hampered the reaction and C-terminal residues were important in the binding affinity. The authors proposed 3 mechanisms for this reaction (Figure 1);

(1) direct nucleophilic attack on the quaternary propargyl carbon(pathway b);

(2) formation of an allene followed by nucleophilic or radical attack (pathway a); and

(3) formation of a thiol radical in the enzyme active site followed by radical reaction with the alkyne or allene (pathway c or d).

Figure 1: Proposed mechanisms for the conjugation of a DUB with its propargylated substrate.

The radical mechanism was ruled out by performing the reaction in the presence of NO (a radical trap) generating reagents (Figure 2).  The formation of an allene in the active site could not be ruled out, but it was noted that the formation requires a strong base and the reaction is performed under acidic conditions. 

Figure 2: NO generating reagents used in the paper.

Having characterised the reaction, the paper goes on to describe activity based profiling using a fluorescent Ub conjugate and the purification of DUBs from cells using a resin bound propargyl Ub substrate.  Purification was possible as the reaction selectively forms a covalent bond with its target, enabling stringent washing and finally elution under acidic conditions.  The paper also demonstrates that the reaction is possible with a different class of proteases.  Modifying peptide aldehyde based cysteine protease inhibitors by direct conversion of the aldehyde to alkyne using Bestmann−Ohira reagent and increasing the peptide chain length to increase binding affinity, resulted in labelling of capsase-1.

There probably isn’t too much to fear when we lob alkynes into cells, as the reaction requires specific molecular recognition, before a catalytically active thiol will react with an alkynes.  The paper does describe a novel conjugation strategy that could be employed for other strategies, with a bit of carful design.