Advanced Marfey's Method Research Articles

Hantupeptin A, a Cytotoxic Cyclic Depsipeptide from a Singapore Collection of Lyngbya majuscula

Chemical investigation of the marine cyanobacterium Lyngbya majuscula from Pulau Hantu Besar, Singapore, has led to the isolation of a cyclodepsipeptide, hantupeptin A (1). The planar structure of 1 was assigned on the basis of extensive 1D and 2D NMR spectroscopic experiments. The absolute configuration of the amino and hydroxyl acid residues in the molecule was determined by application of the advanced Marfey method, chiral HPLC analysis, and Mosher's method. Hantupeptin A showed cytotoxicity to MOLT-4 leukemia cells and MCF-7 breast cancer cells with IC(50) values of 32 and 4.0 microM, respectively.

Pompanopeptins A and B, new cyclic peptides from the marine cyanobacterium Lyngbya confervoides

A new 3-amino-6-hydroxy-2-piperidone (Ahp) containing peptolide, pompanopeptin A ( 1), and a novel N-methyl-2-amino-6-(4′-hydroxyphenyl)hexanoic acid ( N-Me-Ahpha) containing cyclic pentapeptide connected with a sixth amino acid residue via a rare ureido linkage, pompanopeptin B ( 2), were isolated from the marine cyanobacterium Lyngbya confervoides collected from the southeastern coast of Florida. Their planar structures were determined by a combination of NMR spectroscopic analysis and mass spectrometry. The absolute configurations were established using advanced Marfey's method and chiral HPLC analysis of the chemical degradation products. Compound 1 selectively inhibited trypsin over elastase and chymotrypsin, with an IC 50 value of 2.4 μM; selectivity is conferred by an arginine residue in the cyclic core.

Lucentamycins A−D, Cytotoxic Peptides from the Marine-Derived Actinomycete Nocardiopsis lucentensis

Four new 3-methyl-4-ethylideneproline-containing peptides, lucentamycins A-D (1-4), have been isolated from the fermentation broth of a marine-derived actinomycete identified by phylogenetic methods as Nocardiopsis lucentensis (strain CNR-712). The planar structures of the new compounds were assigned on the basis of 1D and 2D NMR spectroscopic techniques, while the absolute configurations of the amino acid residues were determined by application of the advanced Marfey method. Lucentamycins A (1) and B (2) showed significant in vitro cytotoxicity against HCT-116 human colon carcinoma.

Novel Cyclic Peptide, Epichlicin, from the Endophytic Fungus,Epichloe typhina

The novel cyclic peptide, epichlicin, was isolated from Epichloe typhina, an endophytic fungus of the timothy plant (Phleum pretense L.). Its structure was determined by NMR studies and by mass spectrometry. Enantiomers of 3-amino tetradecanoic acid, a constituent amino acid of epichlicin, were synthesized as authentic standards. The stereochemistry of each amino acid was elucidated through a combination of the advanced Marfey method and chemical manipulation. Epichlicin showed inhibitory activity toward the spore germination of Cladosporium phlei, a pathogenic fungus of the timothy plant at an IC50 value of 22 nM.

Elution behavior of diaminopimelic acid and related diamino acids using the advanced Marfey's method

The advanced Marfey's method consists of a chromatography technique for the separation of amino acids into each enantiomer by derivatization with 1-fluoro-2,4-dinitrophenyl-5- l-leucinamide ( l-FDLA), and a detection method using liquid chromatography/mass spectrometry (LC/MS) which can determine the non-empirically the absolute configuration of various amino acids including the non-protein ones. However, this method has not been applied to the determination of the absolute configuration of an amino acid with a “meso” configuration such as diaminopimelic acid (A 2pm). In the present study, this method was successfully applied to determine the absolute configurations of diaminosuccinic acid (DAS), A 2pm, cystine (Cys), selenocystine (SeCys) and homocystine (HomoCys) using a racemization procedure and the dl-FDLA method, and the resulting elution behavior was summarized as follows: (1) the ll- and meso-isomers were eluted prior to the dd-isomer except for one case; (2) the ll- and meso-isomers are closely eluted and the elution was occasionally reversed; (3) the retention time for both the l- and d-derivatives of the meso-isomer was not changed; (4) the complementary use of the two solvent systems using CH 3CN and MeOH was effective to obtain a chromatogram with a high resolution; (5) the abnormality, such as the elution order and peak shape, was observed in the elution behavior of DAS.

Reliable and sensitive analysis of amino acids in the peptidoglycan of actinomycetes using the advanced Marfey's method

For the chemotaxonomic classification of actinomycetes, we developed a new method for the detection of the 2, 6-diaminopimelic acid (A 2pm) stereoisomers and 3-hydroxy diaminopimelic acid (3-OH A 2pm) in the cell wall peptidoglycan of actinomycetes using “the advanced Marfey's method”, which consists of a chromatographic technique for the separation of amino acids into each of its enantiomers by derivatization with 1-fluoro-2, 4-dinitrophenyl-5- l-luecinamide ( l-FDLA) and d-FDLA, and a detection method using liquid chromatography/mass spectrometry (LC/MS). This method was successfully applied to determine the absolute configuration of the A 2pm and detect the 3-OH A 2pm included in the cell wall peptidoglycan of the standard strains. Because the procedure can be performed with very small amounts of an amino acid, it was possible to use one colony from an agar plate of the actinomycetes for the acid hydrolysis. In addition, the constituent amino acids and their absolute configurations in the cell wall of the actinomycetes could be simultaneously determined. Thus, a reliable, sensitive and rapid analytical method for amino acids including A 2pm in the peptidoglycan of the microorganisms was established.

Pleofungins, Novel Inositol Phosphorylceramide Synthase Inhibitors, from Phoma sp. SANK 13899

Pleofungins (formerly called F-15078) A, B, C and D, novel depsipeptide antifungal antibiotics, were found in a mycelium extract of the producing fungus, Phoma sp. SANK 13899. The structures of pleofungins A, B, C and D were elucidated mainly by various NMR studies. The absolute configurations of the amino acids and N-methyl amino acids of pleofungin A constituents in the hydrolysate were determined by the application of advanced Marfey's method in combination with gas chromatography/mass spectrometry analysis of their silylation products with N-methyl-N-(tert-butylsilyl)trifluoroacetamide. Two alpha-hydroxy acid constituents, alpha-hydroxyisocaproic acid and alpha-hydroxyisovaleric acid, were isolated from the hydrolysate and their stereochemistries were determined by their specific rotations.

Clonostachysins A and B, New Anti-dinoflagellate Cyclic Peptides from a Marine-derived Fungus

The two new anti-dinoflagellates, clonostachysins A and B, were obtained from a marine sponge derived fungus Clonostachys rogersoniana strain HJK9. Their chemical structures were determined by spectroscopic studies as highly N-methylated cyclic peptides of the nine amino acids. The absolute stereochemistry was elucidated by the advanced Marfey's method. Both clonostachysins A and B exhibited a selectively inhibitory effect on a dinoflagellate Prorocentrum micans at 30 microM, but had no effect on other microalgae and bacteria even at 100 microM.

Golmaenone, a New Diketopiperazine Alkaloid from the Marine‐Derived Fungus Aspergillus sp.

A new diketopiperazine alkaloid, golmaenone (1). and related alkaloids, neoechinulin A (2). and L-alanyl-L-tryptophan anhydride (3). have been isolated from the culture broth of the marine-derived fungus Aspergillus sp. The structure and absolute stereochemistry of the new compound (1). was assigned by spectroscopic methods and the advanced Marfey's method. Compounds 1 and 2 exhibited a significant radical scavenging activity against 1,1-diphenyl-2-picrylhydrazyl (DPPH) with IC(50) values of 20 and 24 microM, respectively, which are similar to the positive control, ascorbic acid (IC(50), 20 microM). Compounds 1 and 2 also showed an ultraviolet-A (UV-A) (320--390 nm) protecting activity with ED(50) values of 90 and 170 microM, respectively, which are more active than oxybenzone (ED(50), 350 microM) currently being used as sunscreen.

Scytalidamides A and B, New Cytotoxic Cyclic Heptapeptides from a Marine Fungus of the Genus Scytalidium.

Two new cyclic heptapeptides have been isolated from the culture broth of a marine fungus, Scytalidium sp., collected from the Bahamas. The planar structures of scytalidamides A (1) and B (2) were assigned on the basis of 1D and 2D NMR spectroscopic techniques, while the absolute configuration of the amino acid residues in both molecules was determined by application of the advanced Marfey's method. The absolute stereochemistry of the uncommon 3-methylproline moiety in scytalidamide B (2) was confirmed by isolation and CD measurements, as well as application of the advanced Marfey's method. Scytalidamides A (1) and B (2) showed moderate in vitro cytotoxicity toward HCT-116 human colon adenocarcinoma with IC(50) values of 2.7 and 11.0 microM, respectively.

Scytalidamides A and B, new cytotoxic cyclic heptapeptides from a marine fungus of the genus Scytalidium.

Two new cyclic heptapeptides have been isolated from the culture broth of a marine fungus, Scytalidium sp., collected from the Bahamas. The planar structures of scytalidamides A (1) and B (2) were assigned on the basis of 1D and 2D NMR spectroscopic techniques, while the absolute configuration of the amino acid residues in both molecules was determined by application of the advanced Marfey's method. The absolute stereochemistry of the uncommon 3-methylproline moiety in scytalidamide B (2) was confirmed by isolation and CD measurements, as well as application of the advanced Marfey's method. Scytalidamides A (1) and B (2) showed moderate in vitro cytotoxicity toward HCT-116 human colon adenocarcinoma with IC(50) values of 2.7 and 11.0 microM, respectively.

A new antifouling hexapeptide from a Palauan sponge, Haliclona sp.

Bioassay-guided fractionation of an extract of the sponge Haliclona sp. provided a known hexapeptide, waiakeamide (1), and a new sulfone derivative (2). The structures of hexapeptides 1 and 2 were elucidated by extensive NMR analyses and the advanced Marfey's method with LC/MS. These compounds showed repellent activity against the blue mussel, Mytilus edulis galloprovincialis.

Simultaneous detection and determination of the absolute configuration of thiazole-containing amino acids in a peptide

For the simultaneous detection and determination of the absolute configuration of a thiazole-containing (Tzl-) amino acid in a peptide, we have developed a reliable method using the ‘advanced Marfey's method’, which includes HPLC with a rational guideline, a sensitive derivatizing reagent, 1-fluoro-2,4-dinitrophenyl-5- l-leucinamide ( l-FDLA), and a racemization procedure using dl-FDLA for determination of the absolute configuration of constituent amino acids in a peptide. Tzl-amino acids could be directly detected in the hydrolysate by this method, although they were racemized under ordinary hydrolysis conditions. In order to depress the racemization, the flash hydrolysis was introduced. As a result, the flash hydrolysis for 1 h was sufficient to detect each constituent amino acid, and it was possible to identify the original peak. Consequently, the absolute configuration of microcyclamide ( 1 ) possessing Tzl-amino acids was determined by the advanced Marfey's method combined with flash hydrolysis. Additionally, this method was successfully applied to the simultaneous detection and determination of the absolute configuration of two other naturally occurring peptides, waiakeamide ( 2 ) and goadsporin ( 3 ). The established method with the flash hydrolysis had an additional advantage in that labile amino acids, such as tryptophan and methionine sulfoxide, during acid hydrolysis can be detected in the intact form.

Goadsporin, a chemical substance which promotes secondary metabolism and Morphogenesis in streptomycetes. II. Structure determination.

The structure of goadsporin was determined by using spectroscopic techniques. NMR analysis revealed that goadsporin consists of 19 amino acids, two of which are dehydroalanines (Deala), and six of which are cyclized to oxazoles (Oxz) and thiazoles (Thz) by dehydrative cyclization and dehydrogenation from serine, threonine and cysteine. NMR analysis established seven partial structures, and their sequence was determined by CID-MS/MS. Negative mode FAB-MS/MS gave product ions arising from charge-remote fragmentation that allowed determination of the sequence of the amino acid components as AcNH-Ala-MeOxz-Val-Deala-MeOxz-Ile-Leu-Thz-Ser-Gly-Gly-MeOxz-Leu-Deala-Oxz-Ala-Gly-Thz-Val-OH. The chiral amino acids were determined by the advanced Marfey's method to have L-configurations.

New cyclic depsipeptide antibiotics, clavariopsins A and B, produced by an aquatic hyphomycetes, Clavariopsis aquatica. 2. Structure analysis.

The structures of new cyclic decadepsipeptides, clavariopsins A and B, were determined to be cyclo[-(R)-2-hydroxyisovaleryl-L-pipecoyl-L-MeVal-L-Val-L-MeAsp-L-MeIle-L-MeIle-Gly L-MeVal-L-Tyr(OMe)-] and cyclo[-(R)-2-hydroxyisovaleryl-L-pipecolyl-L-Val-L-Val-L-MeAsp-L-Melle-L-MeIle-Gly-L-MeVal-L-Tyr(OMe)-], respectively, by spectroscopic analyses, especially using 2D NMR techniques. The absolute stereochemistry was elucidated by the advanced Marfey's method and chiral HPLC analysis.

Beta-cyanoalanine production by marine bacteria on cyanide-free medium and its specific inhibitory activity toward cyanobacteria.

In screening the culture broth of marine bacteria collected at Yap (Micronesia), Palau (Belau), and Okinawa (the southwest islands of Japan) for antimicroalgal activity, 37 out of 2,594 bacterial isolates tested were found to produce anticyanobacterial substances against Oscillatoria amphibia NIES-361. One strain, C-979, identified as a Vibrio sp., was selected and cultured in 2.4 liters of marine broth 2216 to identify the bioactive compound produced by the strain. The purified very hydrophilic compound (16.4 mg) was determined to be beta-cyano-L-alanine (L-CNAla) by instrumental analyses and the application of the advanced Marfey method. L-CNAla did not inhibit the growth of bacteria, yeast, or eukaryotic microalgae, but some cyanobacteria were found to be sensitive to L-CNAla at a concentration of 0.4 to 25 microg/ml. The effect of L-CNAla on some other environmental organisms, including invertebrates and a macroalgae, is discussed. CNAla production in marine broth was examined by thin-layer chromatography for the 37 bacterial isolates which produced an anticyanobacterial substance. The broth of 36 of these strains contained CNAla, suggesting the wide distribution of CNAla production by marine bacteria. This is the first report on bacteria that produce CNAla without a supply of the cyanide ion in the medium.

Nostophycin, a Novel Cyclic Peptide from the Toxic Cyanobacterium Nostoc sp. 152

For the elucidation of the biosynthetic relationship between nontoxic and hepatotoxic peptides produced by cyanobacteria, we investigated the secondary metabolites from the toxic cyanobacterium Nostoc sp. strain 152, which produces microcystins. A novel cyclic peptide, nostophycin, possessing weakly cytotoxic activity was isolated together with microcystins from this strain. Nostophycin is composed of six amino acids, d-glutamine, glycine, l-phenylalanine, d-allo-isoleucine, 2 mol of l-proline, and a novel β-amino acid moiety, (2S,3R,5R)-3-amino-2,5-dihydroxy-8-phenyloctanoic acid (Ahoa). The sequence of the constituent amino acids in nostophycin and its structure were determined by 2D-NMR techniques and MS/MS experiments, and the advanced Marfey's method has been used to simultaneously determine their absolute configurations including the β-amino acid moiety. The stereochemistries at the C-2 and C-5 positions of Ahoa were determined by the 1H NMR spectral analysis of its lactone derivative with the acety...

Further application of advanced Marfey's method for determination of absolute configuration of primary amino compound

Abstract The advanced Marfey's method was applied to the determination of the absolute configuration of primary amino compounds and its utility was extended from amino acids to primary amino compounds. This method was successfully applied to the characterization of not only constituent amino acids but also amino compounds in microginin and nostophycin produced by cyanobacteria.

A Nonempirical Method Using LC/MS for Determination of the Absolute Configuration of Constituent Amino Acids in a Peptide: Combination of Marfey's Method with Mass Spectrometry and Its Practical Application

The “advanced Marfey's method” proposed in our preceding paper has been developed to nonempirically determine the absolute configuration of constituent amino acids in a peptide using liquid chromatography/mass spectrometry (LC/MS). For the establishment of this method, we had to resolve the following three problems: (1) elucidation of the limitation of Marfey's method, which is chosen as the chromatography technique, and its separation mechanism, because this proposed method relies on the elution order of an amino acid derivatized with 1-fluoro-2,4-dinitrophenyl-5-l-alaninamide (l-FDAA) to determine its absolute configuration; (2) how to effectively combine Marfey's method with mass spectrometry in order to detect and identify a desired amino acid without a standard sample; and (3) how to obtain the corresponding enantiomer from either the l- or d-amino acid in a peptide sample. In a preceding paper, we investigated problem 1 and finally described the rational application guideline for Marfey's method to elucidate the elution order of a desired amino acid according to the proposed separation mechanism. In this paper, we further investigated the two remaining problems. Because the sensitivity of the amino acids derivatized with the original derivatizing reagent, l-FDAA, was poor for LC/MS analysis using any interfaces due to their possible thermal instability and low hydrophobicity, we chose electrospray ionization and frit-fast atom bombardment (Frit-FAB) as the interface and developed 1-fluoro-2,4-dinitrophenyl-5-l-leucinamide (l-FDLA) instead of l-FDAA as a new derivatizing reagent in order to combine Marfey's method with mass spectrometry. Furthermore, we introduced a racemization procedure using 1-fluoro-2,4-dinitrophenyl-5-dl-leucinamide (dl-FDLA), the “dl-FDLA derivatization”, instead of the conventional chemical racemization for obtaining the corresponding enantiomer from either the l- or d-amino acid. Thus, we have established a nonempirical method using LC/MS, the “advanced Marfey's method”. The method was successfully applied to the characterization of constituent amino acids in microcystin LR produced by cyanobacteria.

Comparative study of toxic and non-toxic cyanobacterial products: Novel peptides from toxic Nodularia spumigena AV1

Two types of novel peptides, cyclic peptides, nodulapeptins A ( 1) and B ( 2) and linear peptides, spumigins A∼C ( 3∼6), were isolated together with nodularin from toxic Nodularia spumigena AV1. Their structures were determined by 2D-NMR techniques, the advanced Marfey's method and MS/MS experiments.