Orthogonally protected lanthionines: synthesis and use for the solid-phase synthesis of an analogue of nisin ring C

[structure: see text] Lanthionine, a thioether analogue of cystine, is a key component of the lantibiotics, a family of modified peptides bearing multiple thioether bridges resulting from posttranslational modifications between side chains. It is also used as a conformational constraint in medicinally active peptides. We have explored two synthetic routes to give lanthionine, orthogonally protected with Alloc/allyl and Fmoc groups. One route utilized a carbamate-protected iodoalanine that yielded a mixture of diastereoisomers, and one utilized a trityl-protected iodoalanine, formed via a Mitsunobu reaction, that gave the single desired lanthionine, in complete regio- and diastereoselectivity. We then used this orthogonally protected lanthionine in the solid-phase synthesis of an analogue of a fragment of nisin containing its ring C. The chemoselective deprotection of the allyl and Alloc groups of the incorporated lanthionine unit was followed by regio- and stereoselective cyclization on resin to give the desired lanthionine-bridged peptide.     

Effect of bioengineering lacticin 3147 lanthionine bridges on specific activity and resistance to heat and proteases

Lacticin 3147 is a lantibiotic with seven lanthionine bridges across its two component peptides, Ltnα and Ltnβ. Although it has been proposed that the eponymous lanthionine and (β-methyl)lanthionine (Lan and meLan) bridges present in lantibiotics make an important contribution to protecting the peptides from thermal or proteolytic degradation, few studies have investigated this link. We have generated a bank of bioengineered derivatives of lacticin 3147, in which selected bridges were removed or converted between Lan and meLan, which were exposed to high temperature or proteolytic enzymes. Although switching Lan and meLan bridges has variable consequences, it was consistently observed that an intact N-terminal lanthionine bridge (Ring A) confers Ltnα with enhanced resistance to thermal and proteolytic degradation.     

The synthesis of active and stable diaminopimelate analogues of the lantibiotic peptide lactocin S

Lantibiotic peptides are potent antimicrobial compounds produced by Gram-positive bacteria. They can be used in food preservation, and some also show potential for clinical applications. Unfortunately, some of these peptides can be susceptible to inactivation by oxidation of the sulfur-containing amino acid lanthionine, limiting their use. Here we describe the synthesis and testing of diaminopimelate analogues of the lantibiotic lactocin S. These analogues were designed to improve the oxidative stability of the peptide by replacing the sulfur in lanthionine with a methylene unit. Lanthionine was systematically replaced with diaminopimelate during solid-phase peptide synthesis to produce several analogues. One analogue, A-DAP lactocin S, was found to retain full biological activity in addition to displaying increased stability. This is the first time a synthetic lanthionine ring analogue of a lantibiotic has retained natural activity levels. This methodology is potentially very promising for use in producing more stable, medically relevant lantibiotics.     

Fracturing rings to understand lantibiotics

Haloduracin is a bacterially produced antibiotic system of two alkali-stable peptides (Halalpha and Halbeta) that have extensive posttranslational modifications, including lanthionine rings. Now, Cooper et al. (2008) revise the structure of Halbeta and demonstrate that some of the lanthionine rings are not essential for bioactivity.     

Synthesis of orthogonally protected lanthionines

Synthetic approaches to the lantibiotics, a family of thioether-bridged antimicrobial peptides, require flexible synthetic routes to a variety of orthogonally protected derivatives of lanthionine 1. The most direct approaches to lanthionine involve the reaction of cysteine with an alanyl beta-cation equivalent. Several possibilities exist for the alanyl beta-cation equivalent, including direct activation of serine under Mitsunobu conditions: however, the low reactivity of sulfur nucleophiles in the Mitsunobu reaction has previously precluded its use in the synthesis of the lantibiotics. We report here a new approach to the synthesis of protected lanthionine, using a novel variant of the Mitsunobu reaction in which catalytic zinc tartrate is used to enhance the nucleophilicity of the thiol. In the course of these studies, we have also demonstrated that the synthesis of lanthionine from trityl-protected beta-iodoalanines is prone to rearrangement, via an aziridine, to give predominantly trityl-protected alpha-iodo-beta-alanines, and hence norlanthionines, as the major products.     

Engineering dehydro amino acids and thioethers into peptides using lacticin 481 synthetase

Lantibiotics are peptide antimicrobials containing the thioether-bridged amino acids lanthionine (Lan) and methyllanthionine (MeLan) and often the dehydrated residues dehydroalanine (Dha) and dehydrobutyrine (Dhb). While biologically advantageous, the incorporation of these residues into peptides is synthetically daunting, and their production in vivo is limited to peptides containing proteinogenic amino acids. The lacticin 481 synthetase LctM offers versatile control over the installation of dehydro amino acids and thioether rings into peptides. In vitro processing of semisynthetic substrates unrelated to the prelacticin 481 peptide demonstrated the broad substrate tolerance of LctM. Furthermore, a chemoenzymatic strategy was employed to generate novel thioether linkages by cyclization of peptidic substrates containing the nonproteinogenic cysteine analogs homocysteine and beta-homocysteine. These findings are promising with respect to the utility of LctM toward preparation of conformationally constrained peptide therapeutics.     

Cacaoidin,First Member of the New Lanthidin RiPP Family

Lantibiotics are ribosomally synthesized and post-translationally modified peptides (RiPPs) characterized by the presence of lanthionine or methyllanthionine rings and their antimicrobial activity. Cacaoidin, a novel glycosylated lantibiotic, was isolated from a Streptomyces cacaoi strain and fully characterized by NMR, mass spectrometry, chemical derivatization approaches and genome analysis. The new molecule combines outstanding structural features, such as a high number of d -amino acids, an uncommon glycosylated tyrosine residue and an unprecedented N,N-dimethyl lanthionine. This latter feature places cacaoidin within a new RiPP family located between lanthipeptides and linaridins, here termed lanthidins. Cacaoidin displayed potent antibacterial activity against Gram-positive pathogens including Clostridium difficile. The biosynthetic gene cluster showed low homology with those of other known lanthipeptides or linaridins, suggesting a new RiPP biosynthetic pathway.     

Cacaoidin,First Member of the New Lanthidin RiPP Family

Lantibiotics are ribosomally synthesized and post‐translationally modified peptides (RiPPs) characterized by the presence of lanthionine or methyllanthionine rings and their antimicrobial activity. Cacaoidin, a novel glycosylated lantibiotic, was isolated from a Streptomyces cacaoi strain and fully characterized by NMR, mass spectrometry, chemical derivatization approaches and genome analysis. The new molecule combines outstanding structural features, such as a high number of d ‐amino acids, an uncommon glycosylated tyrosine residue and an unprecedented N,N‐dimethyl lanthionine. This latter feature places cacaoidin within a new RiPP family located between lanthipeptides and linaridins, here termed lanthidins. Cacaoidin displayed potent antibacterial activity against Gram‐positive pathogens including Clostridium difficile. The biosynthetic gene cluster showed low homology with those of other known lanthipeptides or linaridins, suggesting a new RiPP biosynthetic pathway.     

In vitro selection of functional lantipeptides

In this report we present a method to identify functional artificial lantipeptides. In vitro translation coupled with an enzyme-free protocol for posttranslational modification allows preparation of more than 10(11) different lanthionine containing peptides. This diversity can be searched for functional molecules using mRNA-lantipeptide display. We validated this approach by isolating binders toward Sortase A, a transamidase which is required for virulence of Staphylococcus aureus. The interaction of selected lantipeptides with Sortase A is highly dependent on the presence of a (2S,6R)-lanthionine in the peptide and an active conformation of the protein.     

Biomimetic synthesis of lantibiotics

The lantibiotics are a class of highly posttranslationally modified small peptide antibiotics containing numerous lanthionine and dehydroamino acid residues. We have prepared peptides containing multiple dehydroamino acids and cysteine residues in order to probe the biomimetic synthesis of the lantibiotics from their precursor peptides. A novel synthetic methodology was developed to allow the synthesis of multiple dehydroamino acid containing peptides. Cyclisations were rapid, quantitative and regiospecific. Remarkably the peptide sequences alone appear to contain sufficient information to direct a series of stereo- and regiospecific ring closures. Thus both the two linear peptides for the B and E-rings closed stereoselectively. In the case of the A-ring precursor peptide which contained two dehydroamino acids, cyclisation was again totally regioselective, although not totally stereoselective.     

Solid supported chemical syntheses of both components of the lantibiotic lacticin 3147

Lantibiotics are antimicrobial peptides produced by bacteria. Some are employed for food preservation, whereas others have therapeutic potential due to their activity against organisms resistant to current antibiotics. They are ribosomally synthesized and posttranslationally modified by dehydration of serine and threonine residues followed by attack of thiols of cysteines to form monosulfide lanthionine and methyllanthionine rings, respectively. Chemical synthesis of peptide analogues is a powerful method to verify stereochemistry and access structure-activity relationships. However, solid supported synthesis of lantibiotics has been difficult due to problems in generating lanthionines and methyllanthionines with orthogonal protection and good stereochemical control. We report the solid-phase syntheses of both peptides of a two-component lantibiotic, lacticin 3147. Both successive and interlocking ring systems were synthesized on-resin, thereby providing a general methodology for this family of natural products.     

Versatile and stereoselective syntheses of orthogonally protected beta-methylcysteine and beta-methyllanthionine

[reaction: see text] Lantibiotics are a class of lanthionine (and/or beta-methyllanthionine)-containing peptides with antibioitic activity against Gram-positive bacteria. As part of our research effort directed toward the synthesis and mechanistic study of the lantibiotic peptide mersacidin (1), we report stereoselective syntheses of orthogonally protected beta-methylcysteine (beta-MeCys) and beta-methyllanthionine (beta-MeLan), two key nonnatural amino acid components of the mersacidin architecture.     

Bacterial lantibiotics: strategies to improve therapeutic potential

Lantibiotics are ribosomally-synthesised antimicrobial peptides produced by Gram-positive bacteria that are characterised by the presence of lanthionine and/or methyllanthionine residues. Other unusual post-translationally modified amino acids, most frequently dehydroalanine and dehydrobutyrine, can also be present. While it has been frequently suggested that these peptides have the potential to be utilised in a wide range of medical applications, to date no actual therapeutic applications have been convincingly described. More recently, however, they have been the focus of much attention as a consequence of improved biotechnological capabilities, an improved understanding of lantibiotic biosynthesis and mode of action, and their high specific activity against multi-drug resistant bacteria. This review concerns the fundamental analyses that have revealed the importance of individual amino acids in these peptides and has permitted the implementation of rational mutagenesis strategies ('intelligenetics') to alter individual residues with a view to ultimately widening the active pH range, improve stability, and enhance binding to cell wall targets with the ultimate aim of optimising their antimicrobial activity. It is hoped that as a consequence of this improved knowledge the most suitable application of individual lantibiotics will become apparent. It should also prove possible, in the near future, to generate tailor-made lantibiotics and utilise biosynthetic enzymes to incorporate modified amino acids into non-lantibiotic peptides. In the shorter term, the extensive characterisation of lantibiotics will be instrumental in reassuring drug industry regulators of their safety and facilitate the widespread application of these novel antimicrobial agents in medicine.     

Total synthesis of peptide antibiotic nisin

Total synthesis of a lanthionine peptide nisin was successfully achieved for the first time by application of new methods for preparations of dehydroalanine and lanthionine moieties, resulting in a confirmation of the proposed structure.     

Characterization of alkali induced formation of lanthionine, trisulfides, and tetrasulfides from peptide disulfides using negative ion mass spectrometry

Peptide disulfides are unstable under alkaline conditions, resulting in the formation of products containing lanthionine and polysulfide linkages. Electrospray ionization mass spectrometry has been used to characterize major species obtained when cyclic and acyclic peptide disulfides are exposed to alkaline media. Studies on a model cyclic peptide disulfide () and an acyclic peptide, oxidized glutathione, bis (^γGlu - Cys - Gly - COOH), are described. Disulfide cleavage reactions are initiated by the abstraction of C^αH or C^βH protons of Cys residues, with subsequent elimination of H₂S or H₂S₂. The buildup of reactive thiol species which act on intermediates containing dehydroalanine residues, rationalizes the formation of lanthionine and polysulfide products. In the case of the cyclic peptide disulfide, the formation of cyclic products is facilitated by the intramolecular nature of the Michael addition reaction of thiols to the dehydroalanine residue. Mass spectral evidence for the intermediate species is presented by using alkylation of thiol groups as a trapping method. Mass spectral fragmentation in the negative ion mode of the peptides derived from trisulfides and tetrasulfides results in elimination of S₂.     

Lantibiotics—Ribosomally Synthesized Biologically Active Polypeptides containing Sulfide Bridges and α,β-Didehydroamino Acids

Lantibiotics are polycyclic peptide antibiotics containing intrachain sulfide bridges, formed from the thioether groups of the amino acids lanthionine and β-methyllanthionine. They also contain α,β-unsaturated amino acids such as didehydroalanine and didehydroaminobutyric acid. A knowledge of the lantibiotic biosynthetic steps and the enzymes involved makes possible a gene technological construction of analogous highly modified polypeptides. To the family of lantibiotics belong nisin, an important food preservative, epidermin, a highly specific therapeutic agent against acne, a series of enzyme inhibitors, as well as immunologically interesting active peptides. Lantibiotics are produced by ribosomal synthesis, starting from inactive precursor proteins (prelantibiotics). The latter are post-translationally converted into the active peptide antibiotics through enzymic modifications. The modifying enzymes effect dehydrations at the serine and threonine residues and stereospecific additions of the cysteine thiol groups to the resulting α,β-unsaturated double bonds, which lead to the formation of several sulfide bridges. Upon subsequent proteolytic cleavage of the leader peptide, the biologically active lantibiotic is formed. Conformational analyses of the lantibiotics, as well as of their prepeptides, enables one to obtain information about the mechanism and steps of the biosynthesis. Antibodies against synthetic prepeptide sequences, and modern instrumental methods for the analysis of peptides, allow structural elucidation of the biosynthetic intermediates.     

Synthesis of protected α-alkyl lanthionine derivatives

Protected α-alkyl lanthionine derivatives were synthesized in five steps starting from a known phenyloxazoline precursor. This approach involved the synthesis of a family of substituted cyclic sulfamidates and their regioselective opening by nucleophilic attack with a protected cysteine. This efficient multistep strategy affords various α-alkylated lanthionine derivatives in high yields.     

Chemical synthesis and biological activity of analogues of the lantibiotic epilancin 15X

Lantibiotics are a large family of antibacterial peptide natural products containing multiple post-translational modifications, including the thioether structures lanthionine and methyllanthionine. Efforts to probe structure-activity relationships and engineer improved pharmacological properties have driven the development of new methods to produce non-natural analogues of these compounds. In this study, solid-supported chemical synthesis was used to produce analogues of the potent lantibiotic epilancin 15X, in order to assess the importance of several N-terminal post-translational modifications for biological activity. Surprisingly, substitution of these moieties, including the unusual N-terminal D-lactyl moiety, resulted in relatively small changes in the antimicrobial activity and pore-forming ability of the peptides.     

Functional Genome Mining Reveals a Class V Lanthipeptide Containing a d-Amino Acid Introduced by an F420H2-Dependent Reductase

Lantibiotics are a type of ribosomally synthesized and post-translationally modified peptides (termed lanthipeptides) with often potent antimicrobial activity. Herein, we report the discovery of a new lantibiotic, lexapeptide, using the library expression analysis system (LEXAS) approach. Lexapeptide has rare structural modifications, including N-terminal (N,N)-dimethyl phenylalanine, C-terminal (2-aminovinyl)-3-methyl-cysteine, and d -Ala. The characteristic lanthionine moiety in lexapeptide is formed by three proteins (LxmK, LxmX, and LxmY), which are distinct from enzymes known to be involved in lanthipeptide biosynthesis. Furthermore, a novel F₄₂₀H₂-dependent reductase (LxmJ) from the lexapeptide biosynthetic gene cluster (BGC) is identified to catalyze the reduction of dehydroalanine to install d -Ala. Our findings suggest that lexapeptide is the founding member of a new class of lanthipeptides that we designate as class V. We also identified further class V lanthipeptide BGCs in actinomycetes and cyanobacteria genomes, implying that other class V lantibiotics await discovery.     

Facile Synthesis of Biologically Active Peptides Using Phase Transfer Reagents as C-Terminal Protecting Group

Phase-transfer reagents (basic, neutral, and acidic) can temporarily protect carboxyl groups by salt formation of C-terminal free amino acids or peptides during peptide synthesis. The salts of amino acids or peptides behave as RNH₂ rather than RNH₃⁺. At least there is a sufficient concentration of the free amine to act as a nucleophile under the reaction conditions. Many biologically active small peptides have been synthesized by this procedure. No racemization was detected. Unusual amino acids such as β-alanine, and ε-aminohexanoic acid can be incorporated into peptides in high yields.