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.     

Pinensins: The First Antifungal Lantibiotics

Lantibiotics (lanthionine‐containing antibiotics) from Gram‐positive bacteria typically exhibit activity against Gram‐positive bacteria. The activity and structure of pinensin A ( 1 ) and B ( 2 ), lantibiotics isolated from a native Gram‐negative producer Chitinophaga pinensis are described. Surprisingly, the pinensins were found to be highly active against many filamentous fungi and yeasts but show only weak antibacterial activity. To the best of our knowledge, lantibiotic fungicides have not been described before. An in‐depth bioinformatic analysis of the biosynthetic gene cluster established the ribosomal origin of these compounds and identified candidate genes encoding all of the enzymes required for post‐translational modification. Additional encoded functions enabled us to build up a hypothesis for the biosynthesis, export, sensing, and import of this intriguing lantibiotic.     

Deuterium labeled peptides give insights into the directionality of class III lantibiotic synthetase LabKC

The biosynthesis of a considerable number of ribosomally synthesized peptide antibiotics involves the modification of Ser and Thr residues of a precursor peptide. This post-translational processing is performed by one or multiple modifying enzymes encoded in the biosynthetic gene cluster. We present a deuterium-label based enzyme assay, utilizing a series of peptide substrates with α-deuterated Ser, for the determination of the dehydration order during the biosynthesis of class III lantibiotic labyrinthopeptin A2. Remarkably, the data show that, in contrast to other modifying enzymes of class I and II lantibiotics, LabKC has a C- to N-terminal processing mode. This surprising finding, which we consider relevant for the biosyntheses of other class III lantibiotics, underlines significant differences of this class of modifying enzymes compared to other investigated systems.     

Mass spectrometry in the investigation of β-lysine-containing peptides

Mass spectrometry can be used for amino acid sequence determination in β-lysine-containing peptides and for the identification of α- and β-lysine residues in peptides, as well as for amide bond type determination in β-lysine peptides. The peptide bond in streptothricin D is shown to be formed through the participation of an ε-amino group of the L-β-lysine residue.     

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.     

HBTU Mediated Synthesis of α,β-Unsaturated γ-Lactams from E-α,β-Unsaturated γ-Amino Acids

The α,β-unsaturated γ-lactams have been found in many biologically active peptide natural products. Due to their biological activities, extensive efforts have been made in the literature to synthesize the α,β-unsaturated γ-lactams. Here, we are reporting the spontaneous transformation of E-α,β-unsaturated γ-amino acids into α,β-unsaturated γ-lactam through in-situ activation of free carboxylic acid using peptide coupling reagent HBTU and base DIPEA at room temperature. The transformation also involves the E→Z isomerization of α,β-unsaturated γ-amino acids. The reaction is also compatible with the peptides consisting of E-α,β-unsaturated amino acids at the C-terminus. The α,β-unsaturated γ-lactams were isolated in very good yields. Even though the reaction required very mild conditions, the products were isolated in the form of a racemic mixture. However, the products can be separated under a chiral environment. No α,β-unsaturated γ-lactams were observed if the reaction was performed in the presence of free amines. In addition, no racemization was observed during the peptide synthesis. The analysis of the reactions of various substrates revealed that amide NH and γ-CH are important for lactamization. No α,β-unsaturated γ-lactams or E→Z isomerization products were observed in the case of N-Me-(E)-α,β-unsaturated γ-amino acids, whereas in the case of E-α,β-unsaturated γ,γ-dimethyl amino acid α,β-unsaturated γ-lactam was isolated, however, with low yield.     

Reactions of β-heteroatom substituted α,β-unsaturated ketones with dimethylsulfonium methylide

β-Alkoxy- and β-alkylthio α,β-unsaturated ketones react with dimethylsulfonium methylide (DMSM) to give a furan, while β-anilino α,β-unsaturated ketones give the pyrrole. β,β-Bis(alkylthio) α,β-unsaturated ketones react with DMSM to afford the methylene inserted products, λ,λ-bis(alkylthio) β,λ-unsaturated ketones.     

Über einen neuartigen synthetischen Zugang zu β,γ-ungesättigten α-Aminocarbonsäuren

A New Synthetic Route to β,α-Unsaturated α-Amino Acids A versatile new synthetic pathway for the preparation of βγ-unsaturated α-amino acids ( 1 ) is presented. Cu(I)-catalyzed addition of ethyl isocyanoacetate ( 2 ) to α-chloro carbonyl compounds ( 3 ) gives 5-chloroalkyl-2-oxazolin-4-carboxylates ( 4 ) in high yields. A reductive elimination on 4 by means of zinc yields the N-formyl derivatives of βγ-unsaturated α-amino carboxylates ( 5 ), which on acid hydrolysis lead to the free amino acids 1 . The five different βγ-dehydro-α-amono acids 1b-1f have been prepared by this method.     

12/14/14‐Helix Formation in 2:1 α/β‐Hybrid Peptides Containing Bicyclo[2.2.2]octane Ring Constraints

The highly constrained β‐amino acid ABOC induces different types of helices in β urea and 1:1 α/β amide oligomers. The latter can adopt 11/9‐ and 18/16‐helical folds depending on the chain length in solution. Short peptides alternating proteinogenic α‐amino acids and ABOC in a 2:1 α/β repeat pattern adopted an unprecedented and stable 12/14/14‐helix. The structure was established through extensive NMR, molecular dynamics, and IR studies. While the 1:1 α‐AA/ABOC helices diverged from the canonical α‐helix, the helix formed by the 9‐mer 2:1 α/β‐peptide allowed the projection of the α‐amino acid side chains in a spatial arrangement according to the α‐helix. Such a finding constitutes an important step toward the conception of functional tools that use the ABOC residue as a potent helix inducer for biological applications.     

Carbon-13 chemical shifts of α,β-unsaturated acids

Carbon-13 spectra of 59 α,β-unsaturated carboxylic acids have been measured. Large differential shieldings of ethylenic carbons in the cis and trans isomers of acrylic acid derivatives were found (altogether 15 isomeric Z- and E-pairs of acids were investigated). The origin of differential shieldings is complex and both molecular ground states as well as changes in excited states appear to be involved. All measured ¹³C chemical shifts can be described by additive parameters that provide a straightforward new technique by which structural assignments can be made for a wide variety of isomeric mono-, di- and tri- substituted α,β-unsaturated acids.     

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.     

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.     

An engineered lantibiotic synthetase that does not require a leader peptide on its substrate

Ribosomally synthesized and post-translationally modified peptides are a rapidly expanding class of natural products. They are typically biosynthesized by modification of a C-terminal segment of the precursor peptide (the core peptide). The precursor peptide also contains an N-terminal leader peptide that is required to guide the biosynthetic enzymes. For bioengineering purposes, the leader peptide is beneficial because it allows promiscuous activity of the biosynthetic enzymes with respect to modification of the core peptide sequence. However, the leader peptide also presents drawbacks as it needs to be present on the core peptide and then removed in a later step. We show that fusing the leader peptide for the lantibiotic lacticin 481 to its biosynthetic enzyme LctM allows the protein to act on core peptides without a leader peptide. We illustrate the use of this methodology for preparation of improved lacticin 481 analogues containing non-proteinogenic amino acids.     

Massenspektrometrische Untersuchung von Amiden. VIII—Vinyloge α-Spaltung und Nachbargruppenbeteiligung bei der Fragmentierung von Piperididen α,β-ungesättigter Carbonsäuren

The electron-impact-induced fragmentation of piperidides from β-substituted α, β-unsaturated carboxylic acids proceeds preferentially via 4-membered cyclic transition states. The development of a 6-membered ring, which is typical for α,β-unsaturated esters, occurs only to a minor extent. The mechanism of the reaction is best rationalised by the assumption of a vinylogous α-cleavage and a simultaneous neighbouring group participation of the amide function     

Ambidextrous α,γ‐Hybrid Peptide Foldamers

Molecular chirality is ubiquitous in nature. The natural biopolymers, proteins and DNA, preferred a right‐handed helical bias due to the inherent stereochemistry of the monomer building blocks. Here, we are reporting a rare co‐existence of left‐ and right‐handed helical conformations and helix‐terminating property at the C‐terminus within a single molecule of α,γ‐hybrid peptide foldamers composed of achiral Aib (α‐aminoisobutyric acid) and 3,3‐dimethyl‐substituted γ‐amino acid (Adb; 4‐amino‐3,3‐dimethylbutanoic acid). At the molecular level, the left‐ and right‐handed helical screw sense of α,γ‐hybrid peptides are representing a macroscopic tendril perversion. The pronounced helix‐terminating behaviour of C‐terminal Adb residues was further explored to design helix–Schellman loop mimetics and to study their conformations in solution and single crystals. The stereochemical constraints of dialkyl substitutions on γ‐amino acids showed a marked impact on the folding behaviour of α,γ‐hybrid peptides.     

A concise stereoselective synthesis of orthogonally protected lanthionine and beta-methyllanthionine

Lantibiotics such as nisin are active against most Gram-positive bacteria and constitute an important class of antibacterial agents. These ribosomally synthesized peptides contain either one or both of the unusual amino acids meso-lanthionine (m-Lan) or beta-methyllanthionine (beta-MeLan). Nucleophilic ring opening of sulfamidates allows facile preparation of stereochemically pure derivatives of m-Lan and beta-MeLan with orthogonal protection for solid phase synthesis of lantibiotic analogues.     

Artificial β‐Double Helices from Achiral γ‐Peptides

Double helices are not common in polypeptides and proteins except in the peptide antibiotic gramicidin A and analogous l,d ‐peptides. In contrast to natural polypeptides, remarkable β‐double‐helical structures from achiral γ‐peptides built from α,β‐unsaturated γ‐amino acids have been observed. The crystal structures suggest that they adopted parallel β‐double helical structures and these structures are stabilized by the interstrand backbone amide H‐bonds. Furthermore, both NMR spectroscopy and fluorescence studies support the existence of double‐helical conformations in solution. Although a variety of folded architectures featuring distinct H‐bonds have been discovered from the β‐ and γ‐peptide foldamers, this is the first report to show that achiral γ‐peptides can spontaneously intertwine into β‐double helical structures.     

αvβ3‐ or α5β1‐Integrin‐Selective Peptidomimetics for Surface Coating

Engineering biomaterials with integrin‐binding activity is a very powerful approach to promote cell adhesion, modulate cell behavior, and induce specific biological responses at the surface level. The aim of this Review is to illustrate the evolution of surface‐coating molecules in this field: from peptides and proteins with relatively low integrin‐binding activity and receptor selectivity to highly active and selective peptidomimetic ligands. In particular, we will bring into focus the difficult challenge of achieving selectivity between the two closely related integrin subtypes αvβ3 and α5β1. The functionalization of surfaces with such peptidomimetics opens the way for a new generation of highly specific cell‐instructive surfaces to dissect the biological role of integrin subtypes and for application in tissue engineering and regenerative medicine.     

3-Amino-2H-Azirines. Synthons for α,α-Disubstituted α-Amino Acids in Heterocycle and Peptide Synthesis [New Analytical Methods (43)]

The recent upswing in peptide chemistry has been accompanied by an increasing interest in nonproteinogenic amino acids. These include the α,α-disubstituted glycines, the best known of which is Aib (2-aminoisobutyric acid, 2-methylalanine). These α-amino acids occur in natural oligopeptides such as the peptaibols, a class of membrane-active ionophores that has been isolated from fungal cultures. The twofold substitution at the α-C atom of the amino acids severely restricts the conformational freedom of the peptides and causes particular secondary structures to be favored; thus, α, α-disubstituted α-amino acids induce the formation of β turns or helices. 3-Amino-2H-azirines are ideal synthons for the construction of oligopeptides, cyclic peptides and depsipeptides (peptolides) containing such α,α-disubstituted α-amino acids. The presence of the ring strain in these molecules means that they can be used in peptide coupling without the need for additional activating reagents. Using 3-amino-2H-azirines a large array of heterocycles containing α, α-disubstituted α-amino acids as structural elements within their skeleton can be synthesized. The driving force in these reactions is the release of the strain on the three-membered ring, which usually takes place in a ring-expansion reaction. The mechanistic elucidation of these reactions, which can be quite complex, contains some surprises.     

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.