Configuration Of Constituent Amino Acids Research Articles

Androsamide, a Cyclic Tetrapeptide from a Marine Nocardiopsis sp., Suppresses Motility of Colorectal Cancer Cells.

A cyclic tetrapeptide, androsamide (1), was isolated from a marine actinomycete of the genus Nocardiopsis, strain CNT-189. The planar structure of 1 was assigned by the interpretation of 1D and 2D NMR spectroscopic data. The absolute configurations of constituent amino acids of 1 were determined by application of the Marfey's and advanced Marfey's methods. Androsamide (1) strongly suppressed the motility of Caco2 cells caused by epithelial-mesenchymal transition.

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.

Abnormal elution behavior of ornitine derivatized with 1-fluoro-2,4-dinitrophenyl-5-leucinamide in advanced Marfey’s method

During the course of establishing the advanced Marfey’s method that has been developed to non-empirically determine the absolute configuration of constituent amino acids in a peptide using LC–MS, we encountered the “ornitine mystery” in the di-DLA (2,4-dinitrophenyl-5-leucinamide) derivative such that the elution order of ornitine (Orn) was opposite ( d→ l) in spite of their relatively long retention time. In order to resolve this problem, the elution behavior of several mixed DLA and DPEA (2,4-dinitrophenyl-5-phenylethylamine) derivatives with different absolute configurations was carefully observed during HPLC. The length of the methylene chain in basic amino acids was obviously critical for this behavior, because Dab (2,4-diamino- n-butyric acid) and lysine (Lys) did not exhibit this abnormality. The presence of the carboxyamide moiety at the ω position was also essential for this phenomenon, because it was never observed in the DPEA derivatives at the ω position. Furthermore, it was found that the following combination of absolute configurations of Orn and DLA at the ω position only induced this abnormality: d-Orn and l-DLA, and l-Orn and d-DLA. This suggested that the structural interaction such as hydrogen bonding between the carboxyamide of DLA at the ω position and carboxylic acid at the α position in these derivatives reduced their retention power on the reversed-phase column.

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.

A Nonempirical Method Using LC/MS for Determination of the Absolute Configuration of Constituent Amino Acids in a Peptide:  Elucidation of Limitations of Marfey's Method and of Its Separation Mechanism

As the first step in establishing our proposed method, the advanced Marfey's method, which is planned for the simultaneous determination of the absolute configuration of amino acids in a peptide, we applied Marfey's method to commercially available amino acids, and the separation behavior was examined in detail. Although good resolution of the diastereomeric pair of an individual amino acid was obtained for all amino acids tested and the applicability of the method was confirmed, the (1-fluoro-2,4-dinitrophenyl)-5-l-alaninamide (FDAA) derivative of the l-amino acid was not always eluted prior to its corresponding d-amino acid derivative. Because this proposed method relies on the elution order of a derivatized amino acid with FDAA to determine its absolute configuration, its separation mechanism was carefully investigated using UV and NMR spectral techniques. The results suggested that the resulting conformations of the l- and d-amino acid derivatives are stable and that the resolution between the l- and d-amino acid derivatives is due to the difference in their hydrophobicity, which is derived from the cis- or trans-type arrangement of two more hydrophobic substituents at both α-carbons of an amino acid and l-alanine amide, so that the FDAA derivative of the cis (Z)-type arrangement interacts more strongly with ODS silica gel and has a longer retention time than that of the trans (E)-type arrangement. Therefore, the l-amino acid derivative is usually eluted from the column before its corresponding d-amino acid derivative in Marfey's method. According to this separation mechanism, the elution order of a desired amino acid can be elucidated from the average retention time of the l- and d-amino acid derivatives, and the dl-serine and -asparagine derivatives are critical for Marfey's method.

Application of d,l-FDLA derivatization to determination of absolute configuration of constituent amino acids in peptide by advanced Marfey's method

A derivatization procedure including d,l-FDLA was developed for the advanced Marfey's method which is an unempirical method for the determination of the absolute configuration of an amino acid. The procedure was successfully applied to the determination of the absolute configuration of Ahp, one of constituent amino acids in aeruginopeptin 228-A ( 1).

A method usingL/CMS for determination of absolute configuration of constituent amino acids in peptide --- advanced Marfey's method ---

A method including Marfey's method and frit-FAB or ESIL/CMS was developed for the determination of the absolute configuration of constituent amino acids in peptides. It was successfully applied to anabaenopeptins produced by the cyanobacterium Anabaena flos-aquae NRC 525-17.

The structure of cerexin B (studies on antibiotics from the genus Bacillus. XVII).

The structure of cerexin B was examined. The constitutent fatty acids were elucidated by gas chromatography and mass spectrometry to be beta-hydroxy isodecanoic acid, beta-hydroxy decanoic acid, beta-hydroxy isoundecanoic acid and beta-hydroxy anteisoundecanoic acid. The configurations of constituent amino acids were determined as asparagine (2D, 1L), valine (D), phenylalanine (D), allo-threonine (D), tryptophan (D), and allo-isoleucine (D) from their optical activities. Treatment with conc. hydrochloric acid cleaved at the gamma-hydroxylysine residue to give two peptide fragments, one of which (the N-terminal side) was then deacylated with Polymyxin Acylase. Their amino acid sequences were examined by EDMAN degradation. From the results and analogy to cerexin A, the structure of cerexin B was deduced.