The secretion of peptides and proteins is essential for survival and ecological adaptation of bacteria. Dual-functional ATP-binding cassette transporters export antimicrobial or quorum signaling peptides in Gram-positive bacteria. Their substrates contain a leader sequence that is excised by an N-terminal peptidase C39 domain at a double Gly motif. We characterized the protease domain (LahT150) of a transporter from a lanthipeptide biosynthetic operon in Lachnospiraceae and demonstrate that this protease can remove the leader peptide from a diverse set of peptides. The 2.0 Å resolution crystal structure of the protease domain in complex with a covalently bound leader peptide demonstrates the basis for substrate recognition across the entire class of such transporters. The structural data also provide a model for understanding the role of leader peptide recognition in the translocation cycle, and the function of degenerate, non-functional C39-like domains (CLD) in substrate recruitment in toxin exporters in Gram-negative bacteria.

A method for site- and stereoselective peptide modification using a cyclic sulfamidate scaffold containing peptides is described. A peptide synthesis strategy allowing the rapid generation of mixed α/β-peptides incorporating a sulfamidate residue, derived from 2-methylisoserine, has been generalized. The unique electrophilic nature of this scaffold for nucleophilic substitution at a quaternary center with total inversion of its configuration, which was demonstrated computationally, allows for site-selective conjugation with various nucleophiles, such as anomeric thiocarbohydrates and pyridines. This strategy provides rapid access to complex thioglyco-α/β-conjugates and charged α/β-peptides.

Starting from commercially available ( S)-isoserine and effectively accessible ( S)-α-methylserine, enantiopure cyclic sulfamidates have been prepared as chiral building blocks for the synthesis of various S- and O-glycosylated amino acid derivatives, including unnatural variants of the Tn antigen, through highly chemo-, regio-, and stereoselective nucleophilic ring-opening reactions with carbohydrate C1- S- and C1- O-nucleophiles.

The reactivity of cyclic tertiary sulfamidates derived from α-methylisoserine strongly depends on the substitution at the C and N termini. These substrates are one of the very few examples able to undergo nucleophilic ring opening at a quaternary carbon with complete inversion of the configuration, as demonstrated both experimentally and computationally. When the sulfonamide is unprotected, the characteristic ring-opening reaction is completely silenced, which explains that the majority of the ring-opening reactions reported in the literature invoke N-alkyl or N-carbonyl-protected sulfamidates. Accumulation of negative charge at the NSO moiety in the transition state, especially when the sulfonamide NH is deprotonated, drastically raises the activation barrier for the nucleophilic attack. On the other hand, ester groups at the carboxylic position favor ring opening, whereas amides allow competition between the substitution and elimination pathways. Using pyridine as a nucleophilic probe, we have demonstrated both experimentally and computationally that a proper selection of the substitution scheme can enhance the synthetic scope of α-methylisoserine-derived sulfamidates, switching off and on the nucleophilic ring-opening in a controlled manner. This is particularly convenient for hybrid α/β-peptide synthesis, as demonstrated recently by our group.

The highly diastereoselective 1,4-conjugate additions of several nitrogen nucleophiles to chiral bicyclic dehydroalanines have been assessed effectively at room temperature in good to excellent yields without needing any catalyst or additional base. This methodology is general, simple, oxygen and moisture tolerant, high-yielding, totally chemo-and stereoselective. Significantly, most of the reaction adducts were obtained in nearly quantitative yield without column chromatography purification. This procedure offers an efficient and practical approach for the synthesis of Nβ-substituted α,βdiamino acids, such as 1-isohistidine, τ-histidinoalanine, β-benzylaminoalanine, β-(piperidin-1-yl)alanine, β-(azepan-1-yl)alanine and fluorescent and ciprofloxacincontaining amino acid derivatives.