Vijaya R. Pattabiraman

Rethinking amide bond synthesis

One of the most important reactions in organic chemistry—amide bond formation—is often overlooked as a contemporary challenge because of the widespread occurrence of amides in modern pharmaceuticals and biologically active compounds. But existing methods are reaching their inherent limits, and concerns about their waste and expense are becoming sharper. Novel chemical approaches to amide formation are therefore being developed. Here we review and summarize a new generation of amide-forming reactions that may contribute to solving these problems. We also consider their potential application to current synthetic challenges, including the development of catalytic amide formation, the synthesis of therapeutic peptides and the preparation of modified peptides and proteins.

Chemical Protein Synthesis by Chemoselective α‐Ketoacid–Hydroxylamine (KAHA) Ligations with 5‐Oxaproline

Total chemical synthesis is an important method for preparing proteins of biological interest and makes possible the incorporation of unnatural amino acids and site-specific modifications. Currently, chemical protein synthesis is served almost exclusively by the remarkable native chemical ligation (NCL), which allows unprotected peptide segments to be chemoselectively coupled under aqueous conditions. The requirements of the NCL, an N-terminal cysteine or cysteine-surrogate and C-terminal thioesters, have encouraged a search for alternative amide-forming ligation reactions. Although several mechanistically unique amide formations have emerged from these studies, none has yet proven to be readily applicable for protein synthesis by the combination of unprotected segments. As part of our efforts to develop a general peptideforming ligation we reported that peptide a-ketoacids and Nterminal hydroxylamines undergo chemoselective, reagentless couplings to give backbone amide bonds (KAHA ligation). We have demonstrated that the KAHA ligation is suitable for the synthesis of medium-sized peptides by ligations of unprotected segments, but we have struggled to perform the reactions in aqueous media typically employed for peptide solubilization and handling. Mechanistic studies of the ligation of a-ketoacids and O-unsubstituted hydroxylamines revealed a complicated pathway that is likely disturbed by water. In contrast, the ligations of O-Bz hydroxylamines give clean and rapid amide-forming ligations in water but are not suitable for a-peptide-derived substrates owing to facile elimination. These observations led us to search for O-substituted hydroxylamines that are stable to the conditions for ligation and solid-phase peptide synthesis while at the same time sufficiently reactive in ligations performed in aqueous solvent. We are now pleased to report that 5-oxaproline is a stable, easily prepared and incorporated N-hydroxyamino acid that gives clean ligations in the presence of water, thereby leading to a homoserine residue at the ligation site (Scheme 1). We demonstrate utility of KAHA ligation with 5-oxaproline by the total chemical synthesis of two small proteins from Mycobacterium, the prokaryotic-ubiquitin-like protein (Pup, 63 residues) and one of its target proteins, probable cold shock protein A (cspA, 66 residues), by the ligation of unprotected peptide segments prepared by Fmoc solidphase peptide synthesis (SPPS). Vasella et al. have reported an asymmetric synthesis of (S)-5-oxaproline. Using a modified version of this procedure, we prepared compound 3 by cycloaddition of ethylene and the nitrone formed from l-gulose hydroxylamine 1 and ethyl glyoxalate (2) at 30 bar (Scheme 2). The cycloadduct 3 was obtained as a 7:3 mixture of diastereomers, which gave a single stereoisomer after two recrystallizations from hexanes. The relative and absolute configuration of 3 was Scheme 1. The a-ketoacid–5-oxaproline ligation. R = aminoacid sidechain, DMSO= dimethylsulfoxide.

Synthesis of the Lantibiotic Lactocin S Using Peptide Cyclizations on Solid Phase

Lactocin S is a lantibiotic peptide with potent antibacterial activity against a range of gram-positive bacteria. Because of challenges in obtaining sufficient quantities of this compound from natural sources, the stereochemistry of the lanthionine residues in lactocin S had not been confirmed. This report describes the chemical synthesis of lactocin S on chlorotrityl polystyrene resin in 10% overall yield using intramolecular cyclization to form the lanthionine rings and employing fragment coupling for the two N-terminal residues. This represents the first report of solid-supported synthesis of a naturally occurring lantibiotic. Comparison to lactocin S isolated from Lactobacillus sakei L45 using a combination of HPLC, MS/MS sequencing, bacterial testing, and chiral GC-MS analysis confirmed the initially proposed structure and the stereochemistry of the DL-lanthionine residues.

Solid-Supported Synthesis and Biological Evaluation of the Lantibiotic Peptide Bis(desmethyl) Lacticin 3147 A2

Lantibiotics are a class of bacteriocins (antimicrobial peptides from bacteria) that undergo extensive post-translational processing. Their biosynthesis involves enzymatic dehydration of serine and/or threonine residues with subsequent intramolecular Michael addition of cysteine thiols to form lanthionine or b-methyllanthionine rings. Lantibiotics are produced by Gram-positive bacteria either as single peptide antibiotics (e.g., nisin A) or as two peptide systems. Many lantibiotics bind lipid II, the precursor of peptidoglycan, thereby hindering bacterial cell wall formation, and in some cases, creating pores in the membrane at nanomolar concentrations. They are generally nontoxic to mammals, and some are very active against Gram-positive bacteria that are resistant to methicillin (e.g., methicillin-resistant Staphylococcus aureus (MRSA)) and vancomycin (e.g., vancomycinresistant enterococcus (VRE)). They are already used in food preservation and have considerable potential in human medicine. The two-component lantibiotic, lacticin 3147, consists of A1 (1) and A2 (2) peptides that exhibit synergistic antimicrobial activity in nanomolar concentration (Figure 1). The mechanism involves initial binding of A1 (1) to lipid II. This complex is then recognized by lacticin A2 (2) to give a three-component assembly that promotes the formation of pores in the cell membrane. Studies on structure–activity relationships of these lantibiotics are being pursued to uncover the principles for designing new antibiotics. In this respect, the development of chemical methods for the synthesis of lantibiotics and their analogues has interested a number of research groups. 8] A solution-phase synthesis of nisin A represents the only total chemical construction of a lantibiotic. Recently, we reported a solid-supported synthesis of an inactive analogue of lacticin 3147 A2 wherein all of the lanthionine bridges were replaced by larger carbocyclic rings. We now describe a solid-phase synthesis of bis(desmethyl) lacticin 3147A2 (Lan-A2, 3), an analogue of A2 (2) that has the two b-methyllanthionine bridges replaced by lanthionines. Biological evaluation of 3 shows that it unexpectedly retains potent synergistic activity with A1, but loses its inherent independent antimicrobial activity, which indicates two independent mechanisms for the natural A2 peptide. The synthetic approach to 3 utilizes solid-supported (9Hfluoren-9-ylmethoxy)carbonyl (Fmoc) peptide synthesis with an orthogonally protected lanthionine precursor, which is coupled to the growing chain and eventually deprotected at the distal sites for intramolecular ring formation (Figure 2). The N-terminal residues (1–5) are synthesized in solution and coupled as a unit onto the peptide. The lanthionine protection was inspired by elegant studies by Tabor and co-workers for the preparation of the monocyclic ring of nisin. To obtain multigram quantities of orthogonally protected lanthionine 11] in a minimum number of steps, a combination of the phase-transfer conditions to make lanthionines reported by Zhu and Schmidt was used with the orthogonal protection scheme of Bregant and Tabor. Reaction of Aloc/ allyl-protected b-bromo-d-alanine (4) (Aloc = allyloxycarbonyl) as the electrophile with Fmoc/tBu-protected l-cysteine (5) as the nucleophile in the presence of (Bu)4NBr in EtOAc and NaHCO3 (0.5m, pH 8.5), gave orthogonally protected lanthionine, with the desired isomer predominating in a 9:1 ratio based on the C NMR spectrum (Scheme 1). Figure 1. Lacticin 3147 components A1 (1) and A2 (2). The bis(desmethyl) lanthionine analogue (R = H) of lacticin A2 is Lan-A2 (3). It is proposed that the a-carbon atoms of residues 26, 22, and 16 in 2, and alanine residues 9 and 12 have the d configuration.

Traceless Preparation of C‐Terminal α‐Ketoacids for Chemical Protein Synthesis by α‐Ketoacid–Hydroxylamine Ligation: Synthesis of SUMO2/3

A novel protecting group for enantiopure α-ketoacids delivers C-terminal peptide α-ketoacids directly upon resin cleavage and allows the inclusion of all canonical amino acids, including cysteine and methionine. By using this approach, SUMO2 and SUMO3 proteins were prepared by KAHA ligation with 5-oxaproline. The synthetic proteins containing homoserine residues were recognized by and conjugated to RanGAP1 by SUMOylation enzymes.

Formation and Rearrangement of Homoserine Depsipeptides and Depsiproteins in the α‐Ketoacid–Hydroxylamine Ligation with 5‐Oxaproline

The primary products of the chemical ligation of α-ketoacids and 5-oxaproline peptides are esters, rather than the previously reported amides. The depsipeptide product rapidly rearranges to the amide in basic buffers. The formation of esters sheds light on possible mechanisms for the type II KAHA ligations and opens an avenue for the chemical synthesis of depsiproteins.

Synthesis and biological activity of oxa-lacticin A2, a lantibiotic analogue with sulfur replaced by oxygen.

An oxidatively stable analogue 3 of lacticin 3147 A2 (2), wherein the sulfur atoms are replaced with oxygens, was synthesized using solution phase peptide synthesis and sequential on-resin cyclizations. Biological evaluation suggests that oxa-lacticin A2 (3) retains independent antimicrobial activity against Gram-positive bacteria but lacks the synergistic activity with natural lacticin A1 that is characteristic of the native lacticin A2 peptide.

Chemical Synthesis of the Highly Hydrophobic Antiviral Membrane-Associated Protein IFITM3 and Modified Variants

Abstract Interferon‐induced transmembrane protein 3 (IFITM3) is an antiviral transmembrane protein that is thought to serve as the primary factor for inhibiting the replication of a large number of viruses, including West Nile virus, Dengue virus, Ebola virus, and Zika virus. Production of this 14.5 kDa, 133‐residue transmembrane protein, especially with essential posttranslational modifications, by recombinant expression is challenging. In this report, we document the chemical synthesis of IFTIM3 in multi‐milligram quantities (>15 mg) and the preparation of phosphorylated and fluorescent variants. The synthesis was accomplished by using KAHA ligations, which operate under acidic aqueous/organic mixtures that excel at solubilizing even the exceptionally hydrophobic C‐terminal region of IFITM3. The synthetic material is readily incorporated into model vesicles and forms the basis for using synthetic, homogenous IFITM3 and its derivatives for further studying its structure and biological mode of action.

Synthesis of Bifunctional Potassium Acyltrifluoroborates

New bifunctional potassium acyltrifluoroborate (KAT) substrates have been synthesized in gram scale using optimized reaction conditions. Chemoselective transformation of functional groups in the presence of an acyltrifluoroborate has been demonstrated, and orthogonal reactions of bifunctional KAT reagents are reported. This allows for the incorporation of KAT moieties into peptides and dyes.