Reversed-phase liquid chromatographic resolution of diastereomers of protein and non-protein amino acids prepared with newly synthesized chiral derivatizing reagents based on cyanuric chloride

Two new chiral monochloro-s-triazines (MCT) were synthesized [viz N-(4-chloro-6-piperidinyl-[1,3,5]-triazine-2-yl)-l-leucine amide and N-(4-chloro-6-piperidinyl-[1,3,5]-triazine-2-yl)-l-leucine) (CDR 1 and 2, respectively)] by the nucleophilic displacement of chlorine atoms in s-triazine moiety. One of the Cl atoms was replaced with piperidine, and the second Cl atom in the 6-piperidinyl derivative was replaced with amino acid amide (viz l-Leu–NH₂) and amino acid (l-Leu). These reagents were characterized and used as CDRs for chiral separation of protein and non-protein amino acids, and were separated on a reversed-phase C₁₈ column. The reaction conditions were optimized for the synthesis of diastereomers using one MCT reagent. The separation method was validated for limit of detection, linearity, accuracy, precision, and recovery.     

Application of amino acid amides as chiral auxiliaries in difluoro dinitro benzene and cyanuric chloride moieties for high-performance liquid-chromatographic enantioseparation of selenomethionine and its mixture with methionine and cysteine

l-Ala-NH₂, l-Val-NH₂, l-Leu-NH₂, and d-Phg-NH₂ were used as chiral auxiliaries to synthesize four chiral derivatizing reagents (CDRs) of each of the three categories, viz., difluoro dinitro benzene (DFDNB) based chiral variants, and cyanuric chloride (CC) based monochloro-s-triazine reagents (MCTs) and dichloro-s-triazine reagents (DCTs). DFDNB based chiral variants were synthesized by substituting one of the fluorine atoms of DFDNB with respective amino acid amides. The MCTs and DCTs were synthesized by substituting chlorine atom with aforesaid amino acid amide moieties in 6-methoxy dichloro-s-triazine and in CC, respectively. In total, 12 CDRs were characterized and used for microwave-assisted synthesis (45 s at 80% of 800 W using DFDNB-based chiral variants, 80 s at 90% of 800 W power using MCTs, and 50 s at 80% of 800 W power using DCTs) of diastereomers of (A) SeMet, and (B) mixture of (1) SeMet and Met, and (2) SeMet, Met, and Cys. The diastereomers were enantioseparated by reversed-phase high-performance liquid chromatography using gradient elution with mobile phases containing aq. TFA (0.1%)—MeCN in different compositions. The method was validated for accuracy, precision, and limit of detection.     

Chromatographic separation of enantiomers of non-protein alpha-amino acids after derivatization with Marfey's reagent and its four variants

Some non-protein α-amino acids were derivatized with 1-fluoro-2,4-dinitrophenyl-5-l-alaninamide (Marfey’s reagent, MR, FDNP-l-Ala-NH₂,) and four of its structural variants FDNP-l-Phe-NH₂, FDNP-l-Val-NH₂, FDNP-l-Leu-NH₂ and FDNP-l-Pro-NH₂. The resultant diastereomers were separated by normal and reversed phase thin layer chromatography (TLC) and reversed phase HPLC. In normal phase TLC, best resolution was obtained with solvent combination of phenol-water (3:1) while in reversed phase TLC mixtures of acetonitrile with triethylammonium phosphate buffer were found successful for resolution of diastereomers. The separation behavior of diastereomers prepared with different reagents was compared. The diastereomers of most of the amino acids prepared with FDNP-l-Leu-NH₂ were best separated while those prepared with FDNP-l-Pro-NH₂ failed to separate in most of the cases. The diastereomers were also separated on a reversed phase C₈ column with gradient elution using mixture of aqueous-trifluoroacetic acid (TFA) and acetonitrile and with detection at 340 nm. The effects of TFA concentration, flow rate and run time on HPLC separation were studied.     

Aqueous-phase quantitative NMR determination of amino acid enantiomer ratio by 13C-NMR using chiral neodymium shift reagent

A neodymium-(S)-PDTA (PDTA = N,N,N′,N′-tetrakis[(hydroxycarbonyl)methyl]-1,2-diaminopropane) complex was found exceptionally useful in the quantitative determination of enantiomer ratios of water-soluble natural amino acids by ¹³C-NMR. The method is demonstrated on mixtures of l- and d-enantiomers of various amino acids. The interactions of the chiral shift reagent with the amino acid molecules were rationalized by molecular orbital calculations.     

Chemotaxis and transport of amino acids in Allomyces arbuscula

Among a number of amino acids tested, l-lysine and l-arginine are the principal attractants in the chemotaxis of the zygotes of Allomyces arbuscula. The reaction can be stimulated to a greater or lesser extent by a number of compounds chemically related to l-leucine. No relationship between transport of attracting amino acids and their effect on chemotaxis has been found.     

Effect of amino acids on growth ofSphaerotilus discophorus

The effect of casein hydrolysate, of mixtures of amino acids and of individual amino acids on the growth of 4 strains ofSphaerotilus discophorus was determined. Growth was virtually completely inhibited by 1.0% Bacto Casamino Acids, 0.54% simulated casein hydrolysate and 0.2% of a uniform mixture of 18 amino acids. The latter were prepared withl amino acids except thatdl-serine,dl-valine anddl-threonine were present in the uniform amino acid mixture.Experiments designed to test the toxicity of the 18 individual amino acids at 0.018 – 0.36% concentration indicated that arginine, glutamic acid, leucine, lysine and proline were non-toxic. However, aspartic acid and methionine were moderately toxic; growth was greatly repressed at a concentration of 0.36%. The remaining 11 amino acids which included alanine, cystine, glycine, tyrosine, histidine, isoleucine, phenylalanine, serine, threonine, tryptophane and valine were the most toxic of the group. They prevented growth partially or completely, at a concentration of 0.18% or 0.36%.dl-Serine anddl-valine were especially toxic and prevented growth at a concentration of 0.018%. The toxicity of the individuall-amino acids can account for the toxicity of Casamino Acids and simulated casein hydrolysate. l-Methionine or cyanocobalamin (vitamin B₁₂) is required for the growth ofS. discophorus. Alsod- anddl-methionine can replace cyanocobalamin although they completely repress growth when used at the relatively high concentration of 200 µg per ml of medium.     

Uptake of amino acids by actidione-treated yeast cells

Uptake of glyeine,l-cysteine,l-leucine,l-methionine,l-aspartic acid andl-lysine was investigated in resting cells ofSaccharomyces cerevisiae treated with 0.3mm actidione for blocking protein synthesis. The amino acids were taken up against substantial concentration gradients (up to nearly 1,000∶1 for μm l-cysteine and glycine). They were present in the free form inside the cells. Their unidirectional transmembrane fluxes were under a negative feedback control by the intracellular concentration of the amino acid involved. The amino acids tested apparently employed more than one transport agéncies for their membrane passage, the half-saturation constants being 6.2–7.7×10⁻⁴ m for glycine, 2.5×10⁻⁴ m forl-cysteine, 6×10⁻⁵ and 4×10⁻⁴ m forl-lysine, 3×10⁻⁵ and 6×10⁻⁴ m forl-methionine, 7–18×10⁻⁵ and 1.6×10⁻³ m forl-aspartic acid and 6×10⁻⁵ and 2×10⁻³ m forl-leucine. The specificities of the transport systems are overlapping but there emerges a wide-affinity transport system for glycine, alanine, leucine, methionine, serine, cysteine, phenylalanine, aspartic acid, asparagine, glutamic acid and tryptophan (and possibly for other amino acids), and more specific systems for each of the following: glycine, lysine, methionine, histidine, arginine, and aspartic and glutamic acids. Proline had the peculiar effect of stimulating the transport of all the amino acids tested. The amino acids apparently interacted in the uptake not only by competition for the binding site but also by allotopic inhibition (e.g.l-cysteine) and possibly stimulation (l-proline). The initial rate of uptake of amino acids and their steady-state level of distribution were characterized by identical activation energies: 7.5 kcal/mole forl-lysine, 6.9 kcal/mole forl-aspartic acid, and 13.2 kcal/mole for glycine.     

Biosynthesis of aromatic amino acids in Nocardia sp. 239: effects of amino acid analogues on growth and regulatory enzymes

Summary Further steps required for overproduction of aromatic amino acids by a mutant strain of Nocardia sp. 239 (Noc 87-13), unable to grow on l-phenylalanine as a sole carbon and energy source, were investigated. A number of analogues of the aromatic amino acids displayed severe inhibitory effects on the activities of regulatory enzymes in the biosynthetic pathway and growth of the organism in glucose mineral medium. l-Tryptophane analogues strongly inhibited 3-deoxy-d-arabino-heptulosonate 7-phosphate (DAHP) synthase activity. l-Tyrosine analogues especially inhibited DAHP synthase and chorismate mutase, whereas l-phenylalanine analogues strongly inhibited chorismate mutase and prephenate dehydratase activity. Addition of the aromatic amino acids and their precursors chorismate, 4-hydroxyphenylpyruvate, phenylpyruvate and anthranilate, to the medium counteracted the growth inhibitory effect of specific analogues. The data indicate that ortho- (OFP) and para-fluoro-d,l-phenylalanine (PFP), and l-phenylalanine amide, are the most suitable analogues for the isolation of feedback-inhibition-insensitive prephenate dehydratase mutants. Attempts to isolate l-tyrosine and l-trytophane auxotrophic mutants were only successful in the latter case, resulting in the selection of a stable anthranilate synthase-negative mutant (Noc 87-13-14). Uptake of aromatic amino acids in Nocardia sp. 239 most likely involves a common transport system. This necessitates the use of anthranilate, rather than l-trytophane, as a supplement during the isolation of l-tyrosine auxotrophic and OFP- and/or PFP-resistant mutant derivative strains of Noc 87-13-14. Offprint requests to: L. Dijkhuizen     

Protease-catalysed coupling of N-protected amino acids and peptides with 4-aminoantipyrine

The enzymatic synthesis of N-protected l-aminoacyl- and l-peptidyl-antipyrine amides was accomplished by proteases from different classes. Serine and cysteine proteases proved to be suitable tools for the production of amino acids and peptides conjugated to 4-aminoantipyrine, whereas metalloproteases do not seem to be very qualified for accepting this nucleophile. The product yields were optimised by applying ample opportunities of medium engineering, e.g. aqueous-organic, biphasic, suspension and solid-to-solid reaction systems. Thus, yields up to 100% could be obtained. The products were purified and characterised by polarimetry and NMR spectroscopy. These results broaden the common knowledge of the catalytic potential of proteases, in particular with regard to the suitability of a special heterocyclic 1,2-amino ketone as a nucleophile for the biocatalytic amidation of amino acids and peptides.     

Stimulation of L-asparaginase production inEscherichia coli by organic and amino acids

The effect of 18 amino acids and 7 organic acids on the production ofl-asparaginase EC-2 by a strain ofEscherichia coli in a chemically defined medium was investigated under moderate aeration. All the amino acids and some of the organic acids stimulated the enzyme production. The specific activity without stimulants was about 0.16 nkat per mg dry weight, with stimulants it lay between 1 and 6 nkat per mg dry weight but withl-leucine andl-methionine the values were 12 nkat and 17 nkat per mg, respectively. When two organic or amino acids were added simultaneously at concentrations that were suboptimal for stimulation, the stimulating effects were cumulative in most cases. When cells were grown under conditions approaching anaerobiosis, the specific activity reached, even in the absence of stimulants, values as high as 5 nkat per mg; under these conditions, a further substantial increase in specific activity was only caused byl-leucine andl-methionine. Stimulating effects ofdl-lactate and of some amino acids were also found in other strains ofEscherichia coli. The ability to grow on a medium withl-asparagine as the sole source of both nitrogen and carbon was found in two strains; growth took place even when there was no measurable activity ofl-asparaginase EC-2.     

Secondary transport of amino acids in Nitrosomonas europaea

Nitrosomonas europaea is capable of incorporating exogenously supplied amino acids. Studies in whole cells revealed that at least eight amino acids are actively accumulated, probably by the action of three different transport systems, each with high affinity ( molar range) for several amino acids. Evidence for the action of secondary mechanisms of transport was obtained from efflux, counterflow and exchange experiments. More detailed information was obtained from studies in liposomes in which solubilized integral membrane proteins of N. europaea were incorporated. Uptake of l-alanine in these liposomes could be driven by artificially imposed pH gradients and electrical potentials, but not by chemical sodium-ion gradients. These observations indicate that l-alanine is transported by a H⁺/alanine symport system. The ecological significance of secondary amino acid transport systems in autotrophic ammonium-oxidizing bacteria is discussed.     

3-Ketoglutarate generation in pancreatic B-cell mitochondria regulates insulin secretory action of amino acids and 2-keto acids

The various neutral amino acids and aliphatic 2-keto acids exhibit differential effects on insulin secretion. The common denominator for all these effects is the 2-ketoglutarate generation in the pancreatic B-cell mitochondria. The neutral amino acidsl-leucine andl-norvaline and the aliphatic ketomonocarboxylic acids 2-ketoisocaproate, 2-ketocaproate, 2-ketovalerate, and 2-keto-3-methylvalerate all stimulate insulin secretion and increase 2-ketoglutarate generation in pancreatic B-cell mitochondria through activation of glutamate dehydrogenase and transamination withl-glutamate andl-glutamine, respectively. The neutral amino acidsl-valine,l-norleucine, andl-alanine and the aliphatic 2-keto acids 2-ketoisovalerate and pyruvate do not stimulate insulin secretion and do not increase 2-ketoglutarate generation in pancreatic B-cell mitochondria. Inhibition of 2-keto acid induced insulin secretion byl-valine andl-isoleucine is accompanied by reduced 2-ketoglutarate generation in pancreatic B-cell mitochondria. Thus intramitochondrial 2-ketoglutarate generation in pancreatic B-cells may regulate the insulin secretory potency of amino acids and 2-keto acids.     

Transport of glutamine inXenopus laevis oocytes: Relationship with transport of other amino acids

Summary We have investigated transport of the amino acid glutamine across the surface membranes of prophase-arrestedXenopus laevis oocytes. Glutamine accumulation was linear with time for 30 min; it was stereospecific with aK _m of 0.12±0.02mm andV _max of 0.92±0.17 pmol/oocyte · min forl-glutamine. Transport ofl-glutamine was Na⁺-dependent, the cation not being replaceable with Li⁺, K⁺, choline, tris(hydroxymethyl)-aminomethane (Tris), tetramethylammonium (TMA) or N-methyld-glucamine NMDG); external Cl^– appeared to be necessary for full activation of Na⁺-dependent glutamine transport. Two external Na⁺ may be required for the transport of one glutamine molecule.l-glutamine transport (at 50 m glutamine) was inhibited by the presence of other amino acids:l-alanine,d-alanine,l-leucine,l-asparagine andl-arginine (about 60% inhibition at 1mm);l-histidine,l-valine and glycine (25 to 40% inhibition at 1mm);l-serine,l-lysine,l-phenylalanine andl-glutamate (45 to 55% inhibition at 10mm). N-methylaminoisobutyric acid (meAIB) had no effect at 10mm, but 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) inhibited Na⁺/glutamine transport by about 50% at 10mm.l-glutamine was a competitive inhibitor of the Na⁺-dependent transport ofl-alanine,d-alanine andl-arginine; this evidence is consistent with the existence of a single system transporting all four amino acids. Glutamine uptake in oocytes appears to be catalyzed by a transport system distinct from the cotransport Systems A, ASC, N and Gly, although it resembles System B^0,+.     

Axially chiral Ni(II) complexes of α‐amino acids: Separation of enantiomers and kinetics of racemization

Herein we present design, synthesis, chiral HPLC resolution, and kinetics of racemization of axially chiral Ni(II) complexes of glycine and di‐(benzyl)glycine Schiff bases. We found that while the ortho‐fluoro derivatives are configurationally unstable, the pure enantiomers of corresponding axially chiral ortho‐chloro‐containing complexes can be isolated by preparative HPLC and show exceptional configurational stability (t_1/2 from 4 to 216 centuries) at ambient conditions. Synthetic implications of this discovery for the development of new generation of axially chiral auxiliaries, useful for general asymmetric synthesis of α‐amino acids, are discussed.     

Enantiomeric derivatization for biomedical chromatography

Derivatization reactions aimed at creating the basis for the chromatographic resolution of biologically and pharmaceutically important enantiomers are reviewed, with emphasis on the literature published in the last 10 years. Three main aspects of chiral derivatization are discussed. (a) Enantiomers containing suitable functional groups (amino, carboxyl, hydroxyl, epoxy, etc.) are transformed into covalently bonded diastereomeric derivatives using homochiral derivatizing agents. The diastereomers formed (esters, amides, urethanes, urea and thiourea, etc., derivatives) can be separated on achiral stationary phases. The derivatization reactions often afford further advantages, such as the improvement of chromatographic properties and the detectability of the solutes using UV and fluorimetric detectors. (b) Covalent but achiral derivatization is often necessary even with the use of chiral stationary phases enabling in principle direct enantioseparations (Pirkle-type columns, cyclodextrin-bonded phases, glycoprotein column and functionalized cellulose columns). The main goals of these derivatization reactions (which are analogous to those discussed above), are to introduce functional groups into the molecule of the enantiomers that improve the possibilities for chiral interactions or block functional groups to avoid non-specific interactions. (c) In the broader sense, the dynamic formation of diastereomers using chiral mobile phase additives (cyclodextrins, various reagents to form diastereomeric ion pairs, adducts, mixed metal complexes) can also be considered to be chiral derivatization reactions and is therefore briefly discussed also.     

Regulation of pteridine biosynthesis and aromatic amino acid hydroxylation inDrosophila melanogaster

The relationship between high dietary levels of aromatic amino acid and regulation of pteridines inDrosophila eyes was examined by measuring changes in pool levels of six pterins in the wild type and mutants and amino acid pool levels in flies that carry mutations for pteridine biosynthesis. The effect upon relative viability and developmental times was also analyzed; relative viability was affected byl-phenylalanine,l-tryptophan, andl-tyrosine in decreasing order and thed-amino acids had little or no effect. The changes in concentration of biopterin, dihydrobiopterin, pterin, sepiapterin, drosopterins, and isoxanthopterin showed a characteristic pattern of increased and/or decreased amounts in response to each of the threel-amino acids. Pterin was regularly increased, and isoxanthopterin decreased.l-Tyrosine caused a 2.1-fold increase in dihydrobiopterin, the largest increase found in this study;l-tryptophan also caused dihydrobiopterin to increase butl-phenylalanine did not. Of 18 eye-color mutants examined, 2 were found to contain high levels of phenylalanine and/or tyrosine,Pu ² andHn ^r3. These two mutants, along withpr ^c4 cn/pr ^m2b cn, were shown to be very sensitive to dietaryl-phenylalanine, indicating that having low levels of certain pteridines makes them susceptible to toxic effects of these amino acids. Therefore, high levels of aromatic amino acids can perturb the balance among pteridine pools, and low levels of some pteridines in mutants are correlated with the inability to withstand the toxic effects of phenylalanine. From the patterns of change in the pteridines we suggest that tetrahydropterin may also be a cofactor for hydroxylation of phenylalanine, along with tetrahydrobiopterin.This work was sponsored in part by a grant from the U.S.-Spain Joint Committee for Scientific and Technological Cooperation.     

Metabolism of amino acid amides in Pseudomonas putida ATCC 12633

The metabolism of the natural amino acid l-valine, the unnatural amino acids d-valine, and d-, l-phenyglycine (d-, l-PG), and the unnatural amino acid amides d-, l-phenylglycine amide (d-, l-PG-NH₂) and l-valine amide (l-Val-NH₂) was studied in Pseudomonas putida ATCC 12633. The organism possessed constitutive l-amidase activities towards l-PG-NH₂ and l-Val-NH₂, both following the same pattern of expression, suggesting the involvement of similarly regulated enzymes, or a common enzyme. Quite surprisingly, growth in mineral media with l-PG-NH₂ resulted in variable, long lag phases of growth and strongly reduced l-amidase activities. Conversion of d-PG-NH₂ into d-PG and l-PG also occurred and could be attributed to the presence of an inducible d-amidase and the racemization of the amino acid amide in combination with l-amidase activity, respectively. The further degradation of l-PG and d-PG involved constitutive l-PG aminotransferase and inducible d-PG dehydrogenase activities, respectively, both with a high degree of enantioselectivity. Amino acid racemase activity for d- and l-PG was not detected. Correspondence to: L. Dijkhuizen     

Determination of the chirality of amino acid residues in the course of subtractive Edman degradation of peptides.

A chiral reagent, 1-fluoro-2,4-dinitro-5-L-alanine, was synthesized for the analysis of enantiomeric mixtures of amino acids after precolumn derivatization. The resulting diastereomers can be separated and quantitated by microbore RP-HPLC. These derivatives are relatively stable under the conditions used for acid hydrolysis of peptide bonds. Thus, this reagent was included in the protocol of a subtractive Edman degradation procedure of peptides to determine the sequence position of amino acid residues with concomitant identification of their chirality at a nanomolar level.     

Changes in free amino acids in the brain during embryonic development in layer and broiler chickens

Developmental changes in the levels of the excitatory amino acids l-glutamate (Glu) and l-Aspartate (Asp) and inhibitory amino acids glycine (Gly) and γ-amino butyric acid (GABA), as well as taurine and its related amino acids l-methionine (Met), l-cysteine (Cys) and l-serine (Ser) in the brain and pectoralis muscle at various embryonic stages and hatch in broiler and layer type chickens were determined. Brain concentrations of Asp, GABA and taurine were higher than those in the muscle, but the difference in the two types was small. The concentrations of the precursors of taurine including Met, Cys and Ser were lower than that of taurine. In conclusion, the synthesis of some amino acids and their metabolites such as Asp, GABA and taurine in the chick embryo is very high in order to support brain development.     

S-methyl thioesters are produced from fatty acids and branched-chain amino acids by brevibacteria: focus on L-leucine catabolic pathway and identification of acyl-CoA intermediates

Despite their importance as potent odors that contribute to the aroma of numerous cheeses, S-methyl thioesters formation pathways have not been fully established yet. In a first part of our work, we demonstrated that Brevibacterium antiquum and Brevibacterium aurantiacum could produce S-methyl thioesters using short-chain fatty acids or branched-chain amino acids as precursors. Then, we focused our work on l-leucine catabolism using liquid chromatography tandem mass spectrometry and gas chromatography-mass spectrometry analyses coupled with tracing experiments. For the first time, several acyl–CoAs intermediates of the l-leucine to thioesters conversion pathway were identified. S-methyl thioisovalerate was produced from l-leucine, indicating that this amino acid was initially transaminated. Quite interestingly, data also showed that other S-methyl thioesters, e.g., S-methyl thioacetate or S-methyl thioisobutyrate, were produced from l-leucine. Enzymatic and tracing experiments allowed for postulating catabolic pathways leading to S-methyl thioesters biosynthesis.