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Streptomyces lunaelactis strains have been isolated from moonmilk deposits, which are calcium carbonate speleothems used for centuries in traditional medicine for their antimicrobial properties. Genome mining revealed that these strains are a remarkable example of a Streptomyces species with huge heterogeneity regarding their content in biosynthetic gene clusters (BGCs) for specialized metabolite production. BGC 28a is one of the cryptic BGCs that is only carried by a subgroup of S. lunaelactis strains for which in silico analysis predicted the production of nonribosomal peptide antibiotics containing the non-proteogenic amino acid piperazic acid (Piz). Comparative metabolomics of culture extracts of S. lunaelactis strains either holding or not holding BGC 28a combined with MS/MS-guided peptidogenomics and 1H/13C NMR allowed us to identify the cyclic hexapeptide with the amino acid sequence (D-Phe)-(L-HO-Ile)-(D-Piz)-(L-Piz)-(D-Piz)-(L-Piz), called lunaemycin A, as the main compound synthesized by BGC 28a. Molecular networking further identified 18 additional lunaemycins, with 14 of them having their structure elucidated by HRMS/MS. Antimicrobial assays demonstrated a significant bactericidal activity of lunaemycins against Gram-positive bacteria, including multi-drug resistant clinical isolates. Our work demonstrates how an accurate in silico analysis of a cryptic BGC can highly facilitate the identification, the structural elucidation, and the bioactivity of its associated specialized metabolites.

Summary Currently, piperazic acid is chemically synthesized using ecologically unfriendly processes. Microbial synthesis from glucose is an attractive alternative to chemical synthesis. In this study, we report the production of L‐piperazic acid via microbial fermentation with the first engineered fungal strain of Aureobasidium melanogenum; this strain was constructed by chassis development, genetic element reconstitution and optimization, synthetic rewiring and constitutive genetic circuit reconstitution, to build a robust L‐piperazic acid synthetic cascade. These genetic modifications enable A. melanogenum to directly convert glucose to L‐piperazic acid without relying on the use of either chemically synthesized precursors or harsh conditions. This bio‐based process overcomes the shortcomings of the conventional synthesis routes. The ultimately engineered strain is a very high‐efficient cell factory that can excrete 1.12 ± 0.05 g l‐1 of L‐piperazic acid after a 120‐h 10.0‐l fed‐batch fermentation; this is the highest titre of L‐piperazic acid reported using a microbial cell factory., L‐piperazic acid via microbial fermentation with the first engineered fungal strain of Aureobasidium melanogenum was achieved. This strain was constructed by chassis development, genetic element reconstitution and optimization, synthetic rewiring, and constitutive genetic circuit reconstitution to build a robust L‐piperazic acid synthetic cascade. The ultimately engineered strain has the highest titer of L‐piperazic acid of 1.12 ± 0.05 g l‐1 ever reported.

Covering: up to the end of 2018 Piperazic acid is a cyclic hydrazine and a non-proteinogenic amino acid found in diverse non-ribosomal peptide (NRP) and hybrid NRP-polyketide (PK) structures. Piperazic acid was first identified as a residue in the monamycins in 1959. Since then, the piperazic acid residue has been found in >30 families of natural products, representing >140 compounds. Many of these compounds have potent biological activity, ranging from anti-malarial to anti-apoptotic to anti-bacterial activity, although high toxicity often accompanies this potent biological activity. Recently, we identified a piperazate synthase, responsible for N-N bond formation to give piperazic acid. Here, we review piperazic acid-containing natural products discovered from 1959 to 2018, with an emphasis on the biosynthetic routes to these natural products.

The culture broth of endophytic Streptomyces sp. AB100, isolated from the shoots of medicinal plant Atropa belladonna (L.) was investigated for the presence of antibacterial compounds. After initial testing followed by bioactivity-guided fractionation, six new piperazic acid (PA)-containing congeners of two known peptides, JBIR-39 and JBIR-40, were identified by HR-MS/MS and NMR analyses. Only the dehydroxylated hexapeptidic derivatives with unusual incorporation of four PA moieties exhibited weak antibacterial activity against Gram-positive test organism Bacillus subtilis. A 16S rDNA-based phylogenetic tree of known Streptomyces spp. producing PA-containing hexapeptides isolated from different habitats and endophyte Streptomyces AB100 showed considerable diversity, suggesting that these metabolites may play an important environmental role beyond their antibacterial activity.

LC/MS-based chemical profiling of a ginseng farm soil-derived actinomycete strain, Streptomyces sp. BYK1371, enabled the discovery of two new cyclic heptapeptides, depsidomycins B and C (1 and 2), each containing two piperazic acid units and a formyl group at their N-terminus. The structures of 1 and 2 were elucidated by a combination of spectroscopic and chemical analyses. These new compounds were determined to possess d-leucine, d-threonine, d-valine, and S-piperazic acid based on the advanced Marfey’s method and a GITC (2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl isothiocyanate) derivatization of their hydrolysates, followed by LC/MS analysis. Depsidomycins B and C displayed significant antimetastatic activities against metastatic breast cancer cells (MDA-MB-231).

Soliseptide A (1), a cyclic hexapeptide possessing piperazic acid groups, together with two known azalomycin derivatives (2 and 3) were isolated from Streptomyces solisilvae HNM30702. Their structures were determined through spectroscopic methods and single crystal X-ray diffraction analysis. Soliseptide A (1) possessed a cyclic hexapeptide core featured with two piperazic acid units rarely discovered in nature, and exhibited weak antibacterial and antiviral activities. Besides, compounds 2 and 3 displayed significant fungicidal effects.

Synthesis of five-, six-, seven-, eight-, and nine-membered cyclic α-hydrazino acids from a common starting material ‘diethylmalonate’ with 26, 16, 34, 13.5, and 13.33% overall yields is described. Sequential allylation or homoallylation and electrophilic amination followed by cyclization gave the desired rings. The methyl esters of eight- and nine-membered rings were synthesized by RCM and the corresponding free acids were generated after hydrolysis in the presence of 1 M BBr3 solution in DCM.

The short-step synthesis of the unsubstituted, 5-hydroxy- and 5-chloropiperidazine-3-carboxylic acids using an aza Diels-Alder reaction between the 1,3-diene and azodicarboxylate was described. This synthetic methodology could be used for the preparation of the optically active piperazic acid in a 35% overall yield.

A short and flexible route for the synthesis of 1,2-diisopropyl-3-methyl-(3 S,4 R,5 R)-4,5-dihydroxyhexahydro-1,2,3-pyrid-azinetricarboxylate, from readily available acrolein, is described. This approach involves an asymmetric α-hydrazination, dihydroxylation and intramolecular cyclisation as key steps.

Piperazic acid and its congeners are constituents of many structurally complex natural products. Their conformational rigidity and other advantageous physicochemical properties have motivated the incorporation of these non-proteinogenic amino acids into pharmaceutical agents and drug leads. This review provides an overview of recently published research on piperazic acids, their origins, chemical syntheses, and applications in constrained, biologically active peptides and peptidomimetics.

Titanium tetrachloride-mediated aza Diels–Alder cycloaddition using a chiral diene and an aza dienophile proceeds in highly diastereoselective manner to form a dehydropiperazic acid derivative in high yield, and diastereoselectivity of the reaction depends on the quantity of titanium tetrachloride.

A general two-step preparation of enantiopure N(alpha),N(beta)-orthogonally diprotected alpha-hydrazino acids 1 is developed on a multigram scale. The key reaction is the efficient electrophilic amination of N-benzyl amino acids 6 with N-Boc-oxaziridine 7 and accommodates various functional groups encountered in side chains of amino acids. The cyclic 2,3,4,5-tetrahydro-3-pyridazine carboxylic acid (piperazic acid) derivatives 2 and 3 or the cyclic 3,4-dihydro-3-pyrazolecarboxylate 4 are conveniently prepared from glutamic acid or aspartic acid via orthogonally diprotected alpha-hydrazino acids 1m and 1n.

The synthesis of piperazic acid derivatives via a novel multicomponent reaction of tetrahydropyridazines, isocyanides, and carboxylic acids is presented.

Peptides containing N2-acyl piperazic or 1,6-dehydropiperazic acids can be formed efficiently via a novel multicomponent reaction of 1,4,5,6-tetrahydropyridazines, isocyanides, and carboxylic acids. Remarkably, the reaction's induced intramolecularity can enable the regiospecific formation of products with N2-acyl piperazic acid, which counters the intrinsic and troublesome propensity for piperazic acids to react at N1 in acylations. The utility of the methodology is demonstrated in the synthesis of the bicyclic core of the interleukin-1β converting enzyme inhibitor, Pralnacasan.

A convenient one-pot bis-protection of [U- 15 N]-hydrazine using Boc-ON is described, the product being oxidised efficiently to di-tert-butyl [U- 15 N]-azodicarboxylate which is converted into [U- 15 N]-(S)-piperazic acid using known methodology.

N-Acyl derivatives of piperazic acids display an unusual degree of conformational rigidity. As a consequence, α-N-coupling of a piperazic acid of given configuration (d or l) with an α-aminoacyl unit of opposite configuration (l or d) produces a “heterochiral” dipeptide that exists in a conformation conducive to the formation of a peptide turn. Piperazic acids and related compounds may thus be regarded as conformationally rigid analogues of proline, and because they are readily available in either antipodal form by synthesis, they should become of great interest in the study of peptide turns.

The preparation and SAR of several piperazic acid-based stromelysin (MMP-3) inhibitors is presented. The standard P3′ methyl amide substituent can be replaced by other carboxy based substituents and maintain good binding affinity. Removal of a hydrogen-bond acceptor results in a 30-fold decrease in activity.

Since their first discovery in 1959, natural products containing the piperazic acid motif have been isolated from a variety of sources and exhibit diverse biological activity profiles. This review provides information about their isolation and biological activities, and presents an overview of recent total syntheses of these molecules.

Since their first discovery in 1959, natural products containing the piperazic acid motif have been isolated from a variety of sources and exhibit diverse biological activity profiles. This review provides information about their isolation and biological activities, and presents an overview of recent total syntheses of these molecules.

Streptomyces sp. Sp080513SC-24 was isolated from a marine sponge, Haliclona sp., which is inhabited by diverse Actinobacteria , and then its culture was comprehensively searched for secondary metabolites. New two hexapeptides, JBIR-39 (1), and -40 (2), were isolated from the fermentation broth of Sp080513SC-24. The structures of 1 and 2 were elucidated on the basis of 1D and 2D NMR spectroscopy and MS analyses.

N-Acyl derivatives of piperazic acids display an unusual degree of conformational rigidity. As a consequence, α-N-coupling of a piperazic acid of given configuration (d or l) with an α-aminoacyl unit of opposite configuration (l or d) produces a “heterochiral” dipeptide that exists in a conformation conducive to the formation of a peptide turn. Piperazic acids and related compounds may thus be regarded as conformationally rigid analogues of proline, and because they are readily available in either antipodal form by synthesis, they should become of great interest in the study of peptide turns.

The short-step synthesis of the unsubstituted, 5-hydroxy- and 5-chloropiperidazine-3-carboxylic acids using an aza Diels-Alder reaction between the 1,3-diene and azodicarboxylate was described. This synthetic methodology could be used for the preparation of the optically active piperazic acid in a 35% overall yield.

A novel cyclic peptide antibiotic, pargamicin A was isolated from the culture broth of an actinomycete strain. The producing organism, designated ML1-hF4, was identified as a member of the genus Amycolatopsis. Pargamicin A was identified as a novel cyclic hexapeptide antibiotic containing piperazic acid by various spectroscopic analyses. Pargamicin A showed potent antibacterial activity against Staphylococcus aureus strains including MRSA and Enterococcus faecalis/faecium strains including VRE.

An alternative method for the synthesis of unnatural cyclic amino acid equivalent from the simple cyclic hydrazine via oxidation and cyanation was developed.

A preparation of l-(benzyloxycarbonyl)hexahydro 3-pyridazinecarboxylic acid in five steps (73% overall yield) from 4-phenylurazole is described.

Diels–Alder reactions between penta-2,4-dienoic acid (and substituted analogues) and 4-phenyl-1,2,4-triazoline-3,5-dione (or 1,2,4-triazoline-3,5-dione) give adducts which, after hydrogenation and hydrolysis, yield the piperazic acid residues identified in the hydrolysates of the monamycin series of cyclohexadepsipeptide antibiotics.

Monamycin, a crystalline antibacterial preparation isolated from cultures of Streptomyces jamaicensis, consists of a mixture of fifteen cyclohexadepsipeptides, six of which contain chlorine. Each molecular species consists of one residue of α-hydroxy-acid (either L-2-hydroxy-3-methylbutanoic acid or L-2-hydroxy-3-methylpentanoic acid) and five residues of α-amino-or imino-acid. The latter consist of N-methyl-D-leucine, L-proline (or trans-4-methyl L-proline), D-isoleucine (or D-valine), (3R)-piperazic acid [or (3R,5S)-5-chloropiperazic acid, or (3R)-4,5 (or 5,6)-dehydropiperazic acid], and (3S,5S)-5-hydroxypiperazic acid. The name piperazic acid is assigned to hexahydropyridazine-3-carboxylic acid.

Two imino-acids isolated from acid hydrolysates of monamycin, have been established as (3R,5S)-5-chloropiperazic acid and (3S,5S)-5-hydroxypiperazic acid through degradation and the analysis of 1H n.m.r. spectra (piperazic acid is hexahydropyridazine-3-carboxylic acid).