Ergothioneine in a peptide: Substitution of histidine with 2-thiohistidine in bioactive peptides

Ergothioneine (EGT) is the betaine of 2-thiohistidine (2-thioHis) and may be the last undiscovered vitamin. EGT cannot be incorporated into a peptide because the α-nitrogen is trimethylated, although this would be advantageous as an EGT-like moiety in a peptide would impart unique antioxidant and metal chelation properties. The amino acid 2-thioHis is an analogue of EGT and can be incorporated into a peptide, although there is only one reported occurrence of this in the literature. A likely reason is the harsh conditions reported for protection of the thione, with similarly harsh conditions used in order to achieve deprotection after synthesis. Here, we report a novel strategy for the incorporation of 2-thioHis into peptides in which we decided to leave the thione unprotected. This decision was based upon the reported low reactivity of EGT with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), a very electrophilic disulfide. This strategy was successful, and we report here the synthesis of 2-thioHis analogues of carnosine (βAH), GHK-tripeptide, and HGPLGPL. Each of these peptides contain a histidine (His) residue and possesses biological activity. Our results show that substitution of His with 2-thioHis imparts strong antioxidant, radical scavenging, and copper binding properties to the peptide. Notably, we found that the 2-thioHis analogue of GHK-tripeptide was able to completely quench the hydroxyl and ABTS radicals in our assays, and its antioxidant capacity was significantly greater than would be expected based on the antioxidant capacity of free 2-thioHis. Our work makes possible greater future use of 2-thioHis in peptides.