Selective Tryptophan Modification with Rhodium Carbenoids in Aqueous Solution

Abstract

A new transition metal-based reaction has been developed for the selective modification of tryptophan residues on protein substrates. After activation of vinyl-substituted diazo compounds by Rh2(OAc)4, the resulting metallocarbenoid intermediates were found to modify indoles in aqueous media despite competing reactions with water. Both N- and 2-substituted indole products were observed in the reaction. Following initial small-molecule studies, the reaction was performed on two protein substrates. Both myoglobin and subtilisin Carlsberg were modified readily in aqueous solution, and the tryptophan selectivity of the reactions was confirmed through MS analyses of trypsin digest fragments. It was also demonstrated that myoblobin concentrations as low as 10 muM still led to appreciable levels of modification. Reconstitution experiments confirmed that myoglobin retained its ability to bind heme following modification.

Selective chemical protein modification

“…A further example of intriguing TM catalysis was first reported by Antos and Francis 136 , utilising rhodium-generated carbenoids formed from diazo reagents for modification of tryptophan residues. While this reaction initially required quite harsh acidic conditions (pH 3), it was subsequently found that this was primarily to denature early protein substrates and hence to expose the reactive tryptophan residue; conjugations at pH 6 are now possible 137 .…”

Designing Enzyme-Like Catalysts: A Rhodium(II) Metallopeptide Case Study

“…There was established reactivity of these complexes toward tryptophan side chains [6], and we developed robust, general, and reliable methods for the synthesis and purification of rhodium(II) metallopeptides [7][8][9]. The metalation of unprotected peptides did not affect folding, and could even be used to enforce helical structure [7].…”

Chemoselektive Ligations‐ und Modifikationsstrategien für Peptide und Proteine

“…[255] 185 kann eine elektrophile aromatische Substitution an einem Tyr in einem Protein 186 eingehen und zu einer kovalenten Bindung in 187 führen. Diese Konjugation, die auch Trp-Aminosäuren in Proteinen funktionalisieren kann, [256,257] wurde auf die Verankerung von Peptidbindungsmarkern, insbesondere von FLAG-Epitopen [258] und LBTs, [246] an das Protein Chymotrypsinogen verwendet. Im Allgemeinen wurden Umsätze bis 84 % bei Peptiden mit mehreren Gly-Aminosäuren am Anilinmarker erzielt, was auf die notwendige Voraussetzung einer flexiblen Region für die Tyr-Funktionalisierung hinweist.…”