Dehydroamino Acids Research Articles

Beneficial Soil Fungus Kills Predatory Nematodes with Dehydropeptides Translocating into the Animal Gut.

Mortierella alpina is a mold fungus that has gained attention for its positive correlation with soil health, plant growth, and applications as a crop biocontrol agent to suppress the threats of nematode pests. To date, the mechanisms underlying the protective traits of M. alpina against these plant parasites have remained elusive. Here we report that abundantly produced peptidic biosurfactants, malpinin A-D, exhibit robust inhibitory activity against nematodes. Nematode assays with malpinin congeners and chemically synthesized analogues revealed that the dehydro amino acid is critical for activity, whereas the N-terminal amino acid residues modulate the lipophilicity. Complementary imaging by fluorescence microscopy and Raman microspectroscopy, using externally fluorescence-labeled, semisynthetic malpinin or a biosynthetically alkyne-tagged probe generated by precursor-directed biosynthesis, visualized the translocation and enrichment of malpinin in the gut of the model nematode Caenorhabditis elegans. Our findings provide valuable insight into the use of M. alpina as a biocontrol agent, emphasizing the ecologically significant role of malpinins as a protective trait. In addition to solving a long-standing riddle, these findings have translational value for applications in agriculture.

N-Protection Dependent Phosphorylation of Dehydroamino Acids to Build Unusual Phosphono-Peptides.

An efficient Mn(III)-promoted phosphorylation of dehydroalanine (Dha) has been developed to give unusual α-amino acids bearing phosphonates/phosphine oxides and β-vinyl phosphonates/phosphinates depending on N-protection of amino acid. N,N-diprotected dehydroalanine reacted with H-phosphonates and H-phosphine oxides to give structurally diverse phosphorylated α-amino acids through conjugate addition of phosphorous radical generated by Mn(OAc)3.2H2O. Whereas, a highly Z-selective phosphorylation was observed in the case of mono N-Boc protected dehydroalanine via cross dehydrogenative coupling to give (Z)- β -vinyl phosphono amino acid. The method is successfully applied to short peptides to derive unusual phosphono-peptides under mild conditions.

Illuminating Substrate Preferences of Promiscuous F420H2-Dependent Dehydroamino Acid Reductases with 4-Track mRNA Display.

Stereoselective reduction of dehydroamino acids is a common biosynthetic strategy to introduce d-amino acids into peptidic natural products. The reduction, often observed during the biosynthesis of lanthipeptides, is performed by dedicated dehydroamino acid reductases (dhAARs). Enzymes from the three known dhAAR families utilize nicotinamide, flavin, or F420H2 coenzymes as hydride donors, and little is known about the catalysis performed by the latter family proteins. Here, we perform a bioinformatics-guided identification and large-scale in vitro characterization of five F420H2-dependent dhAARs. We construct an mRNA display-based pipeline for ultrahigh throughput substrate specificity profiling of the enzymes. The pipeline relies on a 4-track selection strategy to deliver large quantities of clean data, which were leveraged to build accurate substrate fitness models. Our results identify a remarkably promiscuous enzyme, referred to as MaeJC, that is capable of installing d-Ala residues into arbitrary substrates with minimal recognition requirements. We integrate MaeJC into a thiopeptide biosynthetic pathway to produce d-amino acids-containing thiopeptides, demonstrating the utility of MaeJC for the programmable installation of d-amino acids in ribosomal peptides.

Total Synthesis and Stereochemical Assignment of Enteropeptin A.

The total synthesis and structural elucidation of the antimicrobial sactipeptide enteropeptin A is reported. Enteropeptin A contains a thioaminoketal group with an unassigned stereochemical configuration that is embedded in a highly unusual thiomorpholine ring. In this synthesis, a linear peptide containing a dehydroamino acid and a pendant cysteine residue is subjected to Markovnikov hydrothiolation by a dithiophosphoric acid catalyst. This cyclization reaction forms the central thiomorpholine ring found in the enteropeptins. Both diastereomers at the unassigned thioaminoketal stereocenter of enteropeptin A were prepared, and their comparison to an authentic standard allowed for the unambiguous stereochemical assignment of the natural product to be of the D configuration. This inaugural total synthesis of enteropeptin A represents the first total synthesis of a sactipeptide reported to date. Moreover, the strategy disclosed herein serves as a general platform for the synthesis of stereochemically defined thiomorpholine-containing peptides, which may enable the discovery of new cyclic peptide antibiotics.

Enzymatic Formation of an Aminovinyl Cysteine Residue in Ribosomal Peptide Natural Products.

The carboxyl-terminal (C-terminal) S-[(Z)-2-aminovinyl]-cysteine (AviCys) analogs have been identified in four families of ribosomally synthesized and post-translationally modified peptides (RiPPs): lanthipeptides, linaridins, thioamitides, and lipolanthines. Within identified biosynthetic pathways, a highly reactive enethiol intermediate, formed through an oxidative decarboxylation catalyzed by a LanD-like flavoprotein, can undergo two types of cyclization: a Michael addition with a dehydroamino acid or a coupling reaction initiated by a radical species. The collaborative actions of LanD-like proteins with diverse enzymes involved in dehydration, dethiolation or cyclization lead to the construction of structurally distinct peptide natural products with analogous C-terminal macrocyclic moieties. This concept summarizes existing knowledge regarding biosynthetic pathways of AviCys analogs to emphasize the diversity of biosynthetic mechanisms that paves the way for future genome mining explorations into diverse peptide natural products.

Selective Aza-Michael Addition to Dehydrated Amino Acids in Natural Antimicrobial Peptides.

We report the efficient and site selective modification of non-canonical dehydroamino acids in ribosomally synthesized and post-transationally modified peptides (RiPPs) by β-amination. The singly modified thiopeptide Thiostrepton showed an up to 35-fold increase in water solubility, and minimum inhibitory concentration (MIC) assays showed that antimicrobial activity remained good, albeit lower than the unmodified peptide. Also the lanthipeptide nisin could be modified using this method.

Understanding selectivity of nucleophilic addition to β-formyl-α-haloenoates: A synthetic and theoretical investigation

The reactions of β-formyl-α-haloenoates with nitrogen-centered nucleophiles are described. DFT calculations have been used to better understand what factors influence and determine the chemo- and regioselectivity of the nucleophilic addition of amines to such systems. The easy synthetic approaches to the functionally substituted enoate derivatives including tertiary dehydroamino acid esters and furan-2(5H)-ones provides highlight of this work.

Characterization of a LanC-free pathway for the formation of an ll-MeLan residue and an alloAviMeCys residue in the newly identified class V lanthipeptide triantimycins.

The thioether-connected bis-amino acid lanthionine (Lan) residues are class-defining residues of lanthipeptides. Typically, the cyclization step of lanthionine formation, which relies on the addition of a cysteine to an unsaturated dehydroamino acid, is directed either by a standalone cyclase LanC (class I) or by a cyclase domain (class II-IV). However, the pathways of characterized class V members often lack a known cyclase (domain), raising a question on the mechanism by which their multi-macrocycle systems are formed. Herein, we report a new RiPP gene cluster in Streptomyces TN 58, where it encodes the biosynthesis of 3 distinct class V lanthipeptides-termed triantimycins (TAMs). TAM A1∼A3 share an N-terminal ll-MeLan residue, and only TAM A1 contains an additional internal ll-Lan residue. TAM A1 also has a C-terminal (2S, 3R)-S-((Z)-2-aminovinyl)-3-methyl-d-cysteine (alloAviMeCys) residue, which is distinct from the previously reported (2S, 3S)-AviMeCys residue in other RiPPs. Gene deletion, heterologous coexpression, and structural elucidation demonstrated that the cyclization for an ll-MeLan formation occurs spontaneously and is independent of any known lanthionine cyclase. This study provides a new paradigm for lanthionine formation and facilitates genome mining and engineering efforts on RiPPs containing (Me)Lan and (allo)Avi(Me)Cys residues.

Supramolecular bidentate rhodium(I) or iridium(I) phosphine and oxazoline amino acid bioconjugates as selective catalysts for enantioselective reactions.

This publication describes monodentate phosphine and oxazoline ligands attached to an amino acid ester and the application of their supramolecularly assembled rhodium(I) or iridium(I) complexes in asymmetric catalysis. The major feature of these complexes is the transmission of chirality from distant hydrogen bonded amino acids to the prochiral catalytic metal center ("backdoor induction"). The in situ generated homoleptic and heteroleptic rhodium(I) or iridium(I) precatalysts were studied by NMR, UV-VIS and CD spectroscopy as well as X-ray single crystal diffraction. In asymmetric hydrogenation of methyl α-acetamidocinnamate, rhodium(I) and iridium(I) complexes afforded complete conversions with enantioselectivities up to 85%, while iridium complexes proved to be more sensitive to the variation of reaction conditions, including catalyst loading, metal to ligand ratio and temperature. The hydrogenation of four other dehydroamino acid substrates resulted in similar conversion and selectivity as obtained with methyl α-acetamidocinnamate. The influence of the phosphine/oxazoline heteroleptic mixtures in catalysis was studied using both rhodium(I) and iridium(I) complexes. Finally, a homoleptic phosphine rhodium(I) complex was successfully applied in asymmetric hydroformylation of styrene and 1-octene with complete conversions and selectivity up to 40% ee for the branched styrene product.

Dehydroamino acid residues in bioactive natural products.

Covering: 2000 to up to 2023α,β-Dehydroamino acids (dhAAs) are unsaturated nonproteinogenic amino acids found in a wide array of naturally occurring peptidyl metabolites, predominantly those from bacteria. Other organisms, such as fungi, higher plants and marine invertebrates, have also been found to produce dhAA-containing peptides. The α,β-unsaturation in dhAAs has profound effects on the properties of these molecules. They display significant synthetic flexibility, readily undergoing reactions such as Michael additions, transition-metal-catalysed cross-couplings, and cycloadditions. These residues in peptides/proteins also exhibit great potential in bioorthogonal applications using click chemistry. Peptides containing contiguous dhAA residues have been extensively investigated in the field of foldamers, self-assembling supermolecules that mimic biomacromolecules such as proteins to fold into well-defined conformations. dhAA residues in these peptidyl materials tend to form a 2.05-helix. As a result, stretches of dhAA residues arrange in an extended conformation. In particular, peptidyl foldamers containing β-enamino acid units display interesting conformational, electronic, and supramolecular aggregation properties that can be modulated by light-dependent E-Z isomerization. Among approximately 40 dhAAs found in the natural product inventory, dehydroalanine (Dha) and dehydrobutyrine (Dhb) are the most abundant. Dha is the simplest dehydro-α-amino acid, or α-dhAA, without any geometrical isomers, while its re-arranged isomer, 3-aminoacrylic acid (Aaa or ΔβAla), is the simplest dehydro-β-amino acid, or β-enamino acid, and displays E/Z isomerism. Dhb is the simplest α-dhAA that exhibits E/Z isomerism. The Z-isomer of Dhb (Z-Dhb) is sterically favourable and is present in the majority of naturally occurring peptides containing Dhb residues. Dha and Z-Dhb motifs are commonly found in ribosomally synthesized and post-translationally modified peptides (RiPPs). In the last decade, the formation of Dha and Dhb motifs in RiPPs has been extensively investigated, which will be briefly discussed in this review. The formation of other dhAA residues in natural products (NPs) is, however, less understood. In this review, we will discuss recent advances in the biosynthesis of peptidyl NPs containing unusual dhAA residues and cryptic dhAA residues. The proposed biosynthetic pathways of these natural products will also be discussed.

Investigation into the mechanism of action of the antimicrobial peptide epilancin 15X.

Addressing the current antibiotic-resistance challenge would be aided by the identification of compounds with novel mechanisms of action. Epilancin 15X, a lantibiotic produced by Staphylococcus epidermidis 15 × 154, displays antimicrobial activity in the submicromolar range against a subset of pathogenic Gram-positive bacteria. S. epidermidis is a common member of the human skin or mucosal microbiota. We here investigated the mechanism of action of epilancin 15X. The compound is bactericidal against Staphylococcus carnosus as well as Bacillus subtilis and appears to kill these bacteria by membrane disruption. Structure-activity relationship studies using engineered analogs show that its conserved positively charged residues and dehydroamino acids are important for bioactivity, but the N-terminal lactyl group is tolerant of changes. Epilancin 15X treatment negatively affects fatty acid synthesis, RNA translation, and DNA replication and transcription without affecting cell wall biosynthesis. The compound appears localized to the surface of bacteria and is most potent in disrupting the membranes of liposomes composed of negatively charged membrane lipids in a lipid II independent manner. Epilancin 15X does not elicit a LiaRS response in B. subtilis but did upregulate VraRS in S. carnosus. Treatment of S. carnosus or B. subtilis with epilancin 15X resulted in an aggregation phenotype in microscopy experiments. Collectively these studies provide new information on epilancin 15X activity.

Structure and Function of a Class III Metal-Independent Lanthipeptide Synthetase.

In bacteria, Ser/Thr protein kinase-like sequences are found as part of large multidomain polypeptides that biosynthesize lanthipeptides, a class of natural products distinguished by the presence of thioether cross-links. The kinase domain phosphorylates Ser or Thr residues in the peptide substrates. Subsequent β-elimination by a lyase domain yields electrophilic dehydroamino acids, which can undergo cyclase domain-catalyzed cyclization to yield conformationally restricted, bioactive compounds. Here, we reconstitute the biosynthetic pathway for a class III lanthipeptide from Bacillus thuringiensis NRRL B-23139, including characterization of a two-component protease for leader peptide excision. We also describe the first crystal structures of a class III lanthipeptide synthetase, consisting of the lyase, kinase, and cyclase domains, in various states including complexes with its leader peptide and nucleotide. The structure shows interactions between all three domains that result in an active conformation of the kinase domain. Biochemical analysis demonstrates that the three domains undergo movement upon binding of the leader peptide to establish interdomain allosteric interactions that stabilize this active form. These studies inform on the regulatory mechanism of substrate recognition and provide a framework for engineering of variants of biotechnological interest.

Genome Mining of Myxopeptins Reveals a Class of Lanthipeptide-Derived Linear Dehydroamino Acid-Containing Peptides from Myxococcus sp. MCy9171.

Myxobacteria exhibit a substantial capacity to produce bioactive natural products. The biosynthetic potential of ribosomally synthesized and post-translationally modified peptides (RiPPs) from myxobacteria remains largely underexplored. In our study, we identified a novel lanthipeptide-like biosynthetic pathway, mcy from Myxococcus sp. MCy9171, which was reconstituted in E. coli and in vitro proteolysis. Structural elucidation demonstrated that a series of dehydroamino acids were installed by an orphan McyB dehydratase onto the five McyA core peptides, named myxopeptins. Interestingly, compared with the canonical biosynthetic machinery of class I lanthipeptides, neither Cys residues existed in the diverse core regions, nor any LanC cyclase homologue was encoded in the mcy pathway. Thus, we propose myxopeptins as members of a new subclass of RiPPs, named lanthipeptide-derived linear dehydroamino acid-containing peptides (LDPs), which contain dehydrated amino acids as the class-defining post-translational modifications. Furthermore, sequence similarity network (SSN) analysis revealed the wide distribution of the biosynthetic potential of LDPs in various microbial phyla, implying a co-evolutionary scenario between the precursor peptide and class I lanthipeptide biosynthetic enzymes.

Iron-Catalyzed Synthesis of α-Azido α-Amino Esters via the Alkylazidation of Alkenes.

An iron-catalyzed alkylazidation of dehydroamino acids using peroxides as alkyl radical precursors is described. Non-natural azidated amino esters bearing an α-alkyl chain could be obtained in 18-94% yields using TMSN3 as an azide source. The obtained α-alkyl-α-azide α-amino esters could be further functionalized through cycloaddition or azide reduction with amide couplings to afford aminal-type peptides, α-triazolo amino acids, and tetrahydro-triazolopyridine, showing the great versatility of this now easily accessible class of amino acids.

Engineering of Nisin as a Means for Improvement of Its Pharmacological Properties: A Review.

Lantibiotics are believed to have a conceivable potential to be used as therapeutics, especially against clinically resistant bacterial strains. However, their low solubility and poor stability under physiological conditions limit their availability for clinical studies and further pharmaceutical commercialization. Nisin is a readily available and cheap lanthipeptide and thus serves as a good model in the search for the tools to engineer lantibiotics with improved pharmacological properties. This review aims to address technologies that can be applied to alter and enhance the antimicrobial activity, antibacterial spectrum and physicochemical properties (solubility, solution stability and protease resistance) of nisin. There are basically two general means to obtain nisin analogs-protein engineering and chemical functionalization of this antibiotic. Although bioengineering techniques have been well developed and enable the creation of nisin mutants of variable structures and properties, they are lacking spectacular effects so far. Chemical modifications of nisin based on utilization of the reactivity of its free amino and carboxylic moieties, as well as reactivity of the double bonds of its dehydroamino acids, are in their infancy.

Insight into the Structure of Victorin, the Host-Selective Toxin from the Oat Pathogen Cochliobolus victoriae. Studies of the Unique Dehydroamino Acid β-Chlorodehydroalanine.

Victorins, a family of peptide toxins, produced by the fungal pathogen Cochliobolus victoriae and responsible for disease of some oat varieties, contain a β-chlorodehydroalanine residue, ΔAla(βCl). To determine the conformational properties of this unique dehydroamino acid, a series of model compounds was studied using X-ray, NMR, and FT-IR methods, supported by theoretical calculations. The ΔAla(βCl) geometrical isomers differ in conformational profile. The isomer Z prefers the helical conformation α (φ, ψ = -61°, -24°), PPII type conformation β (φ, ψ = -47°, 136°), and semiextended conformation β2 (φ, ψ = -116°, 9°) in weakly and more polar solutions. The isomer E prefers mainly the extended conformation C5 (φ, ψ = -177°, 160°), but with an increase of the environment polarity also conformations β (φ, ψ = -44°, 132°) and α (φ, ψ = -53°, -39°). In the most stable conformations the N-H···Cl hydrogen bond (5γ) occurs, created between the chlorine atom of the side chain and the N-H donor of the flanking amide group. The method of synthesis of the β-chlorodehydroalanine residue is proposed, by chlorination of dehydroalanine and then the photoisomerization from the isomer Z to E. The presented results indicate that the assignment of the geometrical isomer of the ΔAla(βCl) residue in naturally occurring victorins still remains an open question, despite being crucial for biological activity.

Association of Radical Chemistry with LanD Flavoprotein Activity for C‐Terminal Macrocyclization of a Ribosomal Peptide by Formation of an Unsaturated Thioether Residue

AbstractLanD flavoproteins catalyze oxidative decarboxylation of the C‐terminal Cys residue of a peptide to produce an enethiol. This enethiol is highly reactive and can be coupled with an upstream dehydroamino acid through Michael addition to form S‐[2‐aminovinyl](3‐methyl)cysteine, an unsaturated thioether residue known to be characteristic of an array of C‐terminally macrocyclized, ribosomally synthesized and posttranslationally modified peptides (RiPPs). Based on a two‐stage bioinformatics mining of posttranslational modifications (PTMs) related to C‐terminal Cys processing, we report herein that LanD activity can couple with radical S‐adenosylmethionine chemistry to provide a new unsaturated thioether residue, S‐[2‐aminovinyl]‐3‐carbamoylcysteine, by conjugating the resultant enethiol with Cβ of the Asn residue in the C‐terminal NxxC motif of a peptide for macrocyclization. This study furthers our understanding of the variety of PTMs involved in creating the structure diversity of macrocyclic RiPPs.

Rhodium‐Catalyzed C−H Alkylation for the Stereoselective Synthesis of β,β‐Disubstituted Dehydroamino‐Acid Motifs

AbstractIn this article, we describe the stereoselective formation of β,β‐disubstituted dehydroamino acids (ΔAAs) in peptides using a cationic rhodium catalyst and methyl trifluoroborate salt. Using this approach, an alkyl group was directly installed on a β‐monosubstituted ΔAA in the peptide chain and both isomers of dehydroisoleucine, an important yet usually inaccessible ΔAA, were successfully obtained.

Electrosynthesis of Protected Dehydroamino Acids

A NaCl-mediated electrochemical oxidation of amino acid carbamates (R1 = Boc, Cbz) afforded α-methoxylated α-amino acids. Subsequent acid-catalyzed elimination delivered valuable dehydroamino acid derivatives. The simplicity of our setup using graphite-electrodes was showcased, producing N-Boc-ΔAla-OMe on a decagram scale.

Synthesis of Alkyl Fluorides and Fluorinated Unnatural Amino Acids via Photochemical Decarboxylation of α-Fluorinated Carboxylic Acids

Leveraging α-fluoroalkyl or fluorobenzyl radicals to introduce fluorinated motifs allows for the rapid preparation of fluorine-containing building blocks. Herein, we report the generation of α-fluoroalkyl or fluorobenzyl radicals from readily available α-fluorocarboxylic acids under mild reaction conditions and their utilization in the Giese-type addition on Michael acceptors and dehydroamino acids, resulting in the preparation of mono- and difluorinated Michael addition adducts and unnatural fluorinated amino acids.