Novel anti-inflammatory diketopiperazine alkaloids from the marine-derived fungus Penicillium brasilianum.

Diketopiperazine alkaloids have proven the most abundant heterocyclic alkaloids up to now, which usually process diverse scaffolds and rich biological activities. In our search for bioactive diketopiperazine alkaloids from marine-derived fungi, two novel diketopiperazine alkaloids, penipiperazine A (1) and its biogenetically related new metabolite (2), together with a known analogue neofipiperzine C (3), were obtained from the strain Penicillium brasilianum. Their planar structures and absolute configurations were elucidated by extensive spectroscopic analyses, 13C NMR calculation, Marfey's, ECD, and ORD methods. Compound 1 featured a unique 6/5/6/6/5 indole-pyrazino-pyrazino-pyrrolo system, and its plausible biogenetic pathway was also proposed. Additionally, compounds 1-3 have been tested for their inflammatory activities. 1 and 2 significantly inhibited the release of NO and the expression of related pro-inflammatory cytokines on LPS-stimulated RAW264.7 cells, suggesting they could be attracting candidate for further development as anti-inflammatory agent. KEY POINTS: • A novel diketopiperazine alkaloid featuring a unique 6/5/6/6/5 indole-pyrazino-pyrazino-pyrrolo system was isolated from the marine fungus Penicillium brasilianum. • The structure of 1 was elucidated by detailed analysis of 2D NMR data, 13C NMR calculation, Marfey's, ECD, and ORD methods. • Compounds 1 and 2 significantly inhibited the release of NO and the expression of related pro-inflammatory cytokines on LPS-stimulated RAW264.7 cells.

N-Desmethylmajusculamide B, a lipopeptide isolated from the Okinawan cyanobacterium Okeania hirsuta.

A new lipopeptide, N-desmethylmajusculamide B (1), was isolated from the Okinawan cyanobacterium Okeania hirsuta along with 2 known compounds majusculamide A (2) and majusculamide B (3). The planar structure of (1) was elucidated by a detailed analysis of mass spectrometry and nuclear magnetic resonance spectra. The absolute configurations of the amino acid residues were determined using Marfey's analysis. The configuration of C-16 in the α-methyl-ß-keto-decanoyl moiety was determined unambiguously to be S by conducting a semisynthesis of N-desmethylmajusculamide B from 3. The cytotoxicity against mouse L1210 leukemia cells was evaluated for majusculamides (1-3).

Absolute Stereochemistry Determination of Bioactive Marine-Derived Cyclopeptides by Liquid Chromatography Methods: An Update Review (2018-2022).

Cyclopeptides are considered as one of the most important classes of compounds derived from marine sources, due to their structural diversity and a myriad of their biological and pharmacological activities. Since marine-derived cyclopeptides consist of different amino acids, many of which are non-proteinogenic, they possess various stereogenic centers. In this respect, the structure elucidation of new molecular scaffolds obtained from natural sources, including marine-derived cyclopeptides, can become a very challenging task. The determination of the absolute configurations of the amino acid residues is accomplished, in most cases, by performing acidic hydrolysis, followed by analyses by liquid chromatography (LC). In a continuation with the authors' previous publication, and to analyze the current trends, the present review covers recently published works (from January 2018 to November 2022) regarding new cyclopeptides from marine organisms, with a special focus on their biological/pharmacological activities and the absolute stereochemical assignment of the amino acid residues. Ninety-one unreported marine-derived cyclopeptides were identified during this period, most of which displayed anticancer or antimicrobial activities. Marfey's method, which involves LC, was found to be the most frequently used for this purpose.

Expanding the utility of Marfey's analysis by using HPLC-SPE-NMR to determine the Cß configuration of threonine and isoleucine residues in natural peptides.

The determination of amino acid chirality in natural peptides is typically addressed by Marfey's analysis. This approach relies on the complete hydrolysis of the peptide followed by the reaction of the resulting amino acid pool with Marfey's reagent, a chiral derivatizing agent which turns amino acid enantiomers into diastereomeric pairs which can be resolved by conventional reversed-phase HPLC. However, for certain amino acids possessing a second chiral centre at Cß, the discrimination between the two possible epimers may still be challenging due to the lack of chromatographic resolution. Such is the case of isoleucine and threonine which can also be found in natural nonribosomal peptides as their allo-diastereomers. We describe a new approach based on the extension of Marfey's analysis using HPLC-SPE-NMR to sort out this challenge. Marfey's derivatives of these epimeric amino acids at Cß can be differentiated by their distinct NMR spectra. Thus, simple comparison of the NMR spectra of trapped HPLC peaks with the corresponding spectra of standards enables the unambiguous assignment of the absolute configuration at the second chiral centre in such cases. The general applicability of this approach is showcased for two model cyclic peptides bearing L-Ile and L-Thr.

Kribbellichelins A and B, Two New Antibiotics from Kribbella sp. CA-293567 with Activity against Several Human Pathogens.

Current needs in finding new antibiotics against emerging multidrug-resistant superbugs are pushing the scientific community into coming back to Nature for the discovery of novel active structures. Recently, a survey of halophilic actinomyectes from saline substrates of El Saladar del Margen, in the Cúllar-Baza depression (Granada, Spain), led us to the isolation and identification of 108 strains from the rhizosphere of the endemic plant Limonium majus. Evaluation of the potential of these strains to produce new anti-infective agents against superbug pathogens was performed through fermentation in 10 different culture media using an OSMAC approach and assessment of the antibacterial and antifungal properties of their acetone extracts. The study allowed the isolation of two novel antibiotic compounds, kribbellichelin A (1) and B (2), along with the known metabolites sandramycin (3), coproporphyrin III (4), and kribelloside C (5) from a bioassay-guided fractionation of scaled-up active extracts of the Kribbella sp. CA-293567 strain. The structures of the new molecules were elucidated by ESI-qTOF-MS/MS, 1D and 2D NMR, and Marfey's analysis for the determination of the absolute configuration of their amino acid residues. Compounds 1-3 and 5 were assayed against a panel of relevant antibiotic-resistant pathogenic strains and evaluated for cytotoxicity versus the human hepatoma cell line HepG2 (ATCC HB-8065). Kribbellichelins A (1) and B (2) showed antimicrobial activity versus Candida albicans ATCC-64124, weak potency against Acinetobacter baumannii MB-5973 and Pseudomonas aeruginosa MB-5919, and an atypical dose-dependent concentration profile against Aspergillus fumigatus ATCC-46645. Sandramycin (3) confirmed previously reported excellent growth inhibition activity against MRSA MB-5393 but also presented clear antifungal activity against C. albicans ATCC-64124 and A. fumigatus ATCC-46645 associated with lower cytotoxicity observed in HepG2, whereas Kribelloside C (5) displayed high antifungal activity only against A. fumigatus ATCC-46645. Herein, we describe the processes followed for the isolation, structure elucidation, and potency evaluation of these two new active compounds against a panel of human pathogens as well as, for the first time, the characterization of the antifungal activities of sandramycin (3).

Co-occurring Congeners Reveal the Position of Enantiomeric Amino Acids in Nonribosomal Peptides.

The absolute configuration of the constituent amino acids in microbial nonribosomal peptides is typically determined by Marfey's method after total hydrolysis of the peptide. A challenge to structure elucidation arises when both d and l enantiomeric configurations of an amino acid are present. Determining the actual position of each amino acid enantiomer within the peptide sequence typically requires laborious approaches based on peptide partial hydrolysis or even total synthesis of the possible diastereomers. Herein, an alternative solution is discussed based on the homogeneous backbone chirality that governs all peptides biosynthesized by a common nonribosomal peptide synthetase. The information on configuration provided by Marfey's analysis of co-occurring minor congeners can reveal unequivocally the stereochemical sequence of the whole peptide family.

Svalbamides A and B, Pyrrolidinone-Bearing Lipodipeptides from Arctic Paenibacillus sp.

Two new secondary metabolites, svalbamides A (1) and B (2), were isolated from a culture extract of Paenibacillus sp. SVB7 that was isolated from surface sediment from a core (HH17-1085) taken in the Svalbard archipelago in the Arctic Ocean. The combinational analysis of HR-MS and NMR spectroscopic data revealed the structures of 1 and 2 as being lipopeptides bearing 3-amino-2-pyrrolidinone, d-valine, and 3-hydroxy-8-methyldecanoic acid. The absolute configurations of the amino acid residues in svalbamides A and B were determined using the advanced Marfey's method, in which the hydrolysates of 1 and 2 were derivatized with l- and d- forms of 1-fluoro-2,4-dinitrophenyl-5-alanine amide (FDAA). The absolute configurations of 1 and 2 were completely assigned by deducing the stereochemistry of 3-hydroxy-8-methyldecanoic acid based on DP4 calculations. Svalbamides A and B induced quinone reductase activity in Hepa1c1c7 murine hepatoma cells, indicating that they represent chemotypes with a potential for functioning as chemopreventive agents.

Assessment and application of Marfey's reagent and analogs in enantioseparation: a decade's perspective.

Of the various methods available for high-performance liquid chromatography separation of enantiomers (of e.g. amino acids and amino group containing compounds) by the pre-column derivatization approach, use of Marfey's reagent has been most successful with continued application since its introduction in 1984. The reagent is prepared from difluoro dinitro benzene by nucleophilic substitution of one of its F atoms by l-alanine amide. There is flexibility to prepare several chiral variants (by substituting the F atom with different chiral auxiliaries) and to tailor the hydrophobicity and resolution, ultimately, of the diastereomeric derivatives. The present paper assesses and reviews applications of Marfey's reagent and its chiral variants (i.e. other FDNP reagents) for enantioseparation of certain amino group containing drugs/amino acids, and to provide some case studies on enantiomeric separations that are important for the pharmaceutical industry. Various explanations for separation mechanism and elution order using FDNP reagents are included and the question of the configuration of the corresponding enantiomer using an indirect approach has also been addressed.

New Scabimycins A-C Isolated from Streptomyces acidiscabies (Lu19992).

Peptide natural products displaying a wide range of biological activities have become important drug candidates over the years. Microorganisms have been a powerful source of such bioactive peptides, and Streptomyces have yielded many novel natural products thus far. In an effort to uncover such new, meaningful compounds, the metabolome of Streptomyces acidiscabies was analyzed thoroughly. Three new compounds, scabimycins A-C (1-3), were discovered, and their chemical structures were elucidated by NMR spectroscopy. The relative and absolute configurations were determined using ROESY NMR experiments and advanced Marfey's method.

Unique Polyhalogenated Peptides from the Marine Sponge Ircinia sp.

Two new bromopyrrole peptides, haloirciniamide A (1) and seribunamide A (2), have been isolated from an Indonesian marine sponge of the genus Ircinia collected in the Thousand Islands (Indonesia). The planar structure of both compounds was assigned on the basis of extensive 1D and 2D NMR spectroscopy and mass spectrometry. The absolute configuration of the amino acid residues in 1 and 2 was determined by the application of Marfey's method. Compound 1 is the first dibromopyrrole cyclopeptide having a chlorohistidine ring, while compound 2 is a rare peptide possessing a tribromopyrrole ring. Both compounds failed to show significant cytotoxicity against four human tumor cell lines, and neither compound was able to inhibit the enzyme topoisomerase I or impair the interaction between programmed cell death protein PD1 and its ligand, PDL1.

The hypertrehalosaemic neuropeptide conformational twins of cicadas consist of only L-amino acids: are they cis-trans isomers?

It is known for almost 25 years that the corpora cardiaca (neurosecretory glands) of cicadas synthesize two isobaric peptides with hypertrehalosaemic activity denominated Placa-HrTH-I and II. Both decapeptides have the same amino acid sequence (pGlu-Val-Asn-Phe-Ser-Pro-Ser-Trp-Gly-Asn amide) and mass, but differ in their chromatographic retention time. The slightly more hydrophobic peptide, Placa-HrTH-II, co-elutes with the synthetic peptide of the same sequence and is less active in biological assays than Placa-HrTH-I. Ion mobility separation in conjunction with high-resolution mass spectrometry detected the differing structural feature between both peptides in the region Pro6-Ser7-Trp8. Here, it was shown that Placa-HrTH-I co-eluted with a synthetic peptide containing D-Pro in position 6, while dextrorotatory amino acid residues in positions 7 and 8 could be excluded in this way. Amino acid hydrolysis followed by chiral analysis using a relative of Marfey's reagent was then used to validate the presence of D-Pro in Placa-HrTH-I. Interestingly, this experiment unambiguously proved both the absence of D-Pro and the presence of L-Pro in Placa-HrTH-I. Racemization as a reason for the structural differences of the twin adipokinetic hormones was hence ruled out and cis-trans isomerism as the likely alternative came into focus. It remains to be investigated if Pro6 in cis-conformation is indeed present and responsible for the increased bioactivity of Placa-HrTH-I.

Bacicyclin, a new antibacterial cyclic hexapeptide from Bacillus sp. strain BC028 isolated from Mytilus edulis.

A new cyclic hexapeptide, cyclo-(Gly-Leu-Val-IIe-Ala-Phe), named bacicyclin (1), was isolated from a marine Bacillus sp. strain associated with Mytilus edulis. The sequences of the amino acid building blocks of the cyclic peptide and its structure were determined by 1D- and 2D-NMR techniques. Marfey's analysis showed that the amino acid building blocks had L-configuration in all cases except for alanine and phenylalanine, which had D-configuration. Bacicyclin (1) exhibited antibacterial activity against the clinically relevant strains Enterococcus faecalis and Staphylococcus aureus with minimal inhibitory concentration values of 8 and 12 µM, respectively. These results demonstrate the potential of marine bacteria as a promising source for the discovery of new antibiotics.

Marine Natural Peptides: Determination of Absolute Configuration Using Liquid Chromatography Methods and Evaluation of Bioactivities.

Over the last decades, many naturally occurring peptides have attracted the attention of medicinal chemists due to their promising applicability as pharmaceuticals or as models for drugs used in therapeutics. Marine peptides are chiral molecules comprising different amino acid residues. Therefore, it is essential to establish the configuration of the stereogenic carbon of their amino acid constituents for a total characterization and further synthesis to obtain higher amount of the bioactive marine peptides or as a basis for structural modifications for more potent derivatives. Moreover, it is also a crucial issue taking into account the mechanisms of molecular recognition and the influence of molecular three-dimensionality in this process. In this review, a literature survey covering the report on the determination of absolute configuration of the amino acid residues of diverse marine peptides by chromatographic methodologies is presented. A brief summary of their biological activities was also included emphasizing to the most promising marine peptides. A case study describing an experience of our group was also included.

Gaussian and linear deconvolution of LC-MS/MS chromatograms of the eight aminobutyric acid isomers.

Isomeric molecules present a challenge for analytical resolution and quantification, even with MS-based detection. The eight aminobutyric acid (ABA) isomers are of interest for their various biological activities, particularly γ-aminobutyric acid (GABA) and the d- and l-isomers of ß-aminoisobutyric acid (ß-AIBA; BAIBA). This study aimed to investigate LC-MS/MS-based resolution of these ABA isomers as their Marfey's (Mar) reagent derivatives. HPLC was able to separate three Mar-ABA isomers l-ß-ABA (l-BABA), and l- and d-α-ABA (AABA) completely, with three isomers (GABA, and d/l-BAIBA) in one chromatographic cluster, and two isomers (α-AIBA (AAIBA) and d-BABA) in a second cluster. Partially separated cluster components were deconvoluted using Gaussian peak fitting except for GABA and d-BAIBA. MS/MS detection of Marfey's derivatized ABA isomers provided six MS/MS fragments, with substantially different intensity profiles between structural isomers. This allowed linear deconvolution of ABA isomer peaks. Combining HPLC separation with linear and Gaussian deconvolution allowed resolution of all eight ABA isomers. Application to human serum found a substantial level of l-AABA (13 µM), an intermediate level of l-BAIBA (0.8 µM), and low but detectable levels (<0.2 µM) of GABA, l-BABA, AAIBA, d-BAIBA, and d-AABA. This approach should be useful for LC-MS/MS deconvolution of other challenging groups of isomeric molecules.

Diketopiperazines from Costa Rican endolichenic fungus Colpoma sp. CR1465A.

Three new diketopiperazines (1-3), cyclo(l-Pro-d-trans-Hyp) (1), cyclo(l-Pro-d-Glu) (2), and cyclo(d-Pro-d-Glu) (3) and five known diketopiperazines (4-8) were isolated from the endolichenic fungus Colpoma sp. CR1465A identified from the Costa Rican plant Henriettea tuberculosa (Melatomataceae). The structures of the new compounds 1-3 were elucidated using a combination of extensive spectroscopic analyses, including 2D NMR and HR-MS, and their absolute configurations were determined by a combination of NOESY analysis and Marfey's method. Cyclo(l-Pro-d-allo-Thr) (4) was recently isolated from a South China Sea marine sponge Callyspongia sp., but its NMR spectroscopic data were not reported, and cyclo(l-Pro-l-Asp) (5) was previously reported but only as a synthetic product. The NMR data assignments of compounds 4 and 5 are reported for the first time. All of the isolated compounds were tested for antifungal and antimicrobial properties.

Amino Acid-Derived Metabolites from the Ascidian Aplidium sp.

Four new iodobenzene-containing dipeptides (1-4), a related bromotryptophan-containing dipeptide (5), and an iodophenethylamine (6) were isolated from the ascidian Aplidium sp. collected off the coast of Chuja-do, Korea. The structures of these novel compounds, designated as apliamides A-E (1-5) and apliamine A (6) were determined via combined spectroscopic analyses. The absolute configuration of the amino acid residue in 1 was determined by advanced Marfey's analysis. Several of these compounds exhibited moderate cytotoxicity and significant inhibition against Na+/K+-ATPase (4).

A new trichostatin analog from Streptomyces sp. CPCC 203909.

A new trichostatin analog (1) and two known analogs (2, 3) have been isolated from the rice fermentation of the Streptomyces sp. CPCC 203909. Their structures were determined by spectroscopic and chemical methods. The absolute configurations of 1 were assigned by Marfey's method, combined with comparing the NMR and circular dichroism spectroscopic data of 2 and 3. Compound 1 showed cytotoxicity against human embryonic kidney 293 cell line with IC50 value of 39.2 µM.

Stereoselective Amine-omics Using Heavy Atom Isotope Labeled l- and d-Marfey's Reagents.

Biological amines and amino acids play essential roles in many biochemical processes. The chemical complexity of biological samples is challenging, and the selective identification and quantification of amines and amino acid stereoisomers would be very useful for amine-focused "amino-omics" studies. Many amines and amino acids are chiral, and their stereoisomers cannot be resolved on achiral media without chiral derivatization. In prior studies, we demonstrated the use of Marfey's reagent─a chiral derivatization reagent for amines and phenolic OH groups─for the LC-MS/MS resolution and quantification of amines and amino acid stereoisomers. In this study, a heavy atom isotope labeled Marfey's reagent approach for the stereoselective detection and quantification of amines and amino acids was developed. Heavy (13C2) l-Marfey's (Hl-Mar) and heavy (2H3) d-Marfey's (Hd-Mar) were synthesized from 13C2-l-Ala and 2H3-d-Ala, respectively. Both light and heavy Marfey's reagents were used to derivatize standard amine mixtures, which were analyzed by LC-QToF-HRMS. Aligned peak lists were comparatively analyzed by light vs heavy Mar mass differences to identify mono-, di-, and tri-Marfey's adducts and then by the retention time difference between l- and d-Mar derivatives to identify stereoisomers. This approach was then applied to identify achiral and chiral amine and amino acid components in a methicillin-resistant Staphylococcus aureus (MRSA) extract. This approach shows high analytical selectivity and reproducibility.

Nocaviogua A and B: two lipolanthines from root-nodule-associated Nocardia sp.

Nocaviogua A (1) and B (2), two lipolanthines featuring a non-canonical avionin (Avi)-containing macrocycle and a long acyl chain, were identified from the mutualistic actinomycete Nocardia sp. XZ19_369, which was isolated from the nodules of sea buckthorn collected in Tibet. Their planar structures were elucidated via extensive analyses of 1D and 2D NMR, as well as HRMS data. The absolute configurations were fully elucidated by advanced Marfey's analysis and GIAO NMR calculations, representing the first time that the configurations of this family of lipolanthines have been determined. Nocaviogua A (1) exhibited weak cytotoxicity against human chronic uveal melanoma cells (UM92-1), non-small cell lung cancer (NCI-H2170), and breast cancer (MDA-MB-231). Our work provides valuable information on this burgeoning class of lipolanthines for further investigations.

Analysis of D-amino acids secreted from murine islets of Langerhans using Marfey's reagent and reversed phase LC-MS/MS.

D-amino acids (D-AAs) are important signaling molecules due to their ability to bind ionotropic N-methyl-D-aspartate receptors. D-serine (D-Ser), D-alanine (D-Ala), and D-aspartate (D-Asp) have been found individually in the endocrine portion of the pancreas, the islets of Langerhans, and/or their secretions. However, there has been no report of a comprehensive assessment of D-AAs in islet secretions. To evaluate the release of these compounds, the effectiveness of both 1-(9-fluorenyl)-ethyl chloroformate (FLEC reagent) and 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (Marfey's reagent, MR) in separation of D/L-AA enantiomeric pairs in islet-specific buffers were evaluated. MR-derivatized D/L AAs showed greater than baseline resolution (Rs ≥ 1.5) of 13 enantiomeric pairs when using a non-linear gradient and an acidic mobile phase system, while FLEC-derivatized AAs exhibited limited resolution on both biphenyl and C18 columns. The optimized MR method yielded highly reproducible separations with retention times less than 1% RSD. Excellent linearity between the analyte concentrations and response (R2 > 0.98) were obtained, with less than 15% RSD for all analyte responses. Most analytes had an LOD at or below 100 nM, except for L-Ala (200 nM). The optimized MR method was used to quantify D-AAs in secretions of 150 murine islets after incubation in 3- and 20-mM glucose. In response to both solutions, D-Ser and D-glutamine were tentatively identified via comparison of retention time and quantifier-to-qualifer ion ratios with standards, and from spiking experiments. Both were secreted in low quantities which did not differ significantly in either low (D-Ser: 44 ± 2 fmol islet-1h-1; D-Gln: 300 ± 100 fmol islet-1h-1) or high (D-Ser: 23 ± 1 fmol islet-1h-1; D-Gln: 120 ± 50 fmol islet-1h-1) glucose across 3 biological replicates. The method developed is robust and can be applied to further examine the release of D-AAs and their potential roles in islet physiology.