Enzymatic synthesis of cyclic amino acids by N-methyl-l-amino acid dehydrogenase from Pseudomonas putida

A new enzymatic system for the synthesis of enantiomerically pure cyclic amino acids (CAA) from the corresponding diamino acids or racemic CAA is described. α,ω-Diamino acids were oxidized to α-keto acids with amino acid oxidases (AAO). The α-keto acids were spontaneously transformed into cyclic imino acids in the reaction medium. The resulting imines were reduced to the l-form CAA with N-methyl-l-amino acid dehydrogenase (NMAADH) from Pseudomonas putida ATCC12633 using NADPH as a cofactor. l-Form CAA were also obtained from racemic CAA using d-amino-acid oxidase and NMAADH. Using this method, a new compound [1,4]-thiazepane-3-carboxylic acid (Fig. 1) was synthesized from aminopropylcystein.     

Synthesis of libraries of thiazole, oxazole and imidazole-based cyclic peptides from azole-based amino acids. A new synthetic approach to bistratamides and didmolamides

Treatment of a 1 : 1 mixture of the thiazole-based amino acids 8a and 8b with FDPP-i-Pr(2)NEt in CH(3)CN gave a mixture of the cyclic trimers 14, 15, 16 and 17 and the cyclic tetramers 19 and 23 in the ratio 2 : 7 : 5 : 8 : 1 : 1 and in a combined yield of 70%. Separate coupling reactions between the bisimidazole amino acid 45 and the thiazole/oxazole amino acids 43a and 42a in the presence of FDPP-i-Pr(2)NEt led to the bisimidazole based cyclic trimers 55 and 57 respectively (54-57%) and to the cyclic tetramer 56 (8-11%). Similar coupling reactions involving the bisthiazole and bisoxazole amino acids 49 and 47 with the imidazole/oxazole/thiazole amino acids 41a, 42a and 43a gave rise to the library of oxazole, thiazole and imidazole-based cyclic peptides 58, 59, 60, 61, 62, 63, 64 and 65. A coupling reaction between the bisthiazole amino acid 49 and the oxazole amino acid 73 led to an efficient (36% overall) synthesis of bistratamide H (67) found in the ascidian Lissoclinum bistratum. Coupling reactions involving oxazolines with thiazole amino acids were less successful. Thus, a coupling reaction between the phenylalanine-based oxazoline amino acid 71a and either the thiazole amino acid 8a or the bisthiazole amino acid 74 gave only a 2% yield of the cyclic hexapeptide didmolamide A (4) found in the ascidian Didemnum molle. Didmolamide B (68) was obtained in 9% yield from a coupling reaction between 74 and the phenylalanine threonine amino acid 72, using either FDPP or DPPA.     

Design and synthesis of chiral alpha,alpha-disubstituted amino acids and conformational study of their oligopeptides

alpha,alpha-Disubstituted amino acids are alpha-amino acids in which the hydrogen atom at the alpha-position of the L-alpha-amino acid is replaced with an alkyl substituent. The introduction of an alpha-alkyl substituent changes the properties of amino acids, with the conformational freedom of the side chain in the amino acids and the secondary structure of their peptides being especially restricted. The author developed a synthetic route of optically active alpha-ethylated alpha,alpha-disubstituted amino acids using chiral cyclic 1,2-diol as a chiral auxiliary. It was found that the preferred secondary structure of peptides composed of chiral alpha-ethylated alpha,alpha-disubstituted amino acids is a fully extended C5-conformation, whereas that of peptides composed of chiral alpha-methylated alpha,alpha-disubstituted amino acids is a 3(10)-helical structure. Also, a new chiral cyclic amino acid; (3S,4S)-1-amino-3,4-di(methoxy)cyclopentanecarboxylic acid {(S,S)-Ac5c(dOM)}, and a bicyclic amino acid; (1R,6R)-8-aminobicyclo[4.3.0]non-3-ene-8-carboxylic acid {(R,R)-Ab5,6= c}, in which the alpha-carbon atom is not the chiral center but chiral centers exist at the side-chain cycloalkane skeleton, were designed and synthesized. The (S,S)-Ac5c(dOM) hexa- and octapeptides preferentially formed left-handed (M) helices, in which the helical-screw direction is exclusively controlled by the side-chain chiral centers. Contrary to the left-handed helices of (S,S)-Ac(5)c(dOM) peptides, the (R,R)-Ab5,6= c hexapeptide formed both diastereomeric right-handed (P) and left-handed (M) helices, and the twelve chiral centers at the side chain showed no preference for helical-screw direction. Thus, the chiral environment at the side chain is important for the control of helical-screw direction. Furthermore, the author designed a new class of chiral cyclic alpha,alpha-disubstituted amino acids that have pendant chiral centers at the substituent of the delta-nitrogen atom. The synthetic route would provide various optically-active cyclic alpha,alpha-disubstituted amino acids bearing a pendant chiral moiety.     

Synthesis of orthogonally protected cyclic homooligomers from sugar amino acids

Two new families of orthogonally protected cyclic homooligomers with two to four sugar units were synthesized from pyranoid sugar amino acids. Cyclic oligomers composed of amide-linked sugar amino acids (1-3) were prepared by cyclization of linear oligomers of the novel orthogonally protected pyranoid sugar amino acid 12 using a solution-phase coupling method. These orthogonally protected cyclic molecules can be selectively or fully deprotected, affording the macrocycles ready to further functionalization. The straightforward reduction of the amide bonds in the cyclic oligomers 1-3 gave the corresponding amine-linked macrocycles 4-6. This kind of amine-linked carbohydrate-based cyclic oligomer has never been reported before. These flexible molecular receptors could be studied as molecular hosts for molecular, cationic, and anionic recognition. Conformational analysis by molecular modeling (AM1) showed that all of the deprotected cyclic trimers and tetramers preferred a (4)C(1) chair conformation with oxygen atoms of the sugar ring located on the interior of the cavity and the secondary hydroxyl groups outward. In the amide-linked macrocycles, all of the amide bonds are in s-trans conformation. The estimated size of the internal cavity is about 4.5 A for the cyclic trimer and 6.9 A for the cyclic tetramer. The amine-linked macrocycles displayed similar conformational behavior with a slight decrease in internal cavity.     

Stereoconversion of amino acids and peptides in uryl-pendant binol schiff bases

(S)-2-Hydroxy-2'-(3-phenyluryl-benzyl)-1,1'-binaphthyl-3-carboxaldehyde (1) forms Schiff bases with a wide range of nonderivatized amino acids, including unnatural ones. Multiple hydrogen bonds, including resonance-assisted ones, fix the whole orientation of the imine and provoke structural rigidity around the imine C==N bond. Due to the structural difference and the increase in acidity of the alpha proton of the amino acid, the imine formed with an L-amino acid (1-l-aa) is converted into the imine of the D-amino acid (1-D-aa), with a D/L ratio of more than 10 for most amino acids at equilibrium. N-terminal amino acids in dipeptides are also predominantly epimerized to the D form upon imine formation with 1. Density functional theory calculations show that 1-D-Ala is more stable than 1-L-Ala by 1.64 kcal mol(-1), a value that is in qualitative agreement with the experimental result. Deuterium exchange of the alpha proton of alanine in the imine form was studied by (1)H NMR spectroscopy and the results support a stepwise mechanism in the L-into-D conversion rather than a concerted one; that is, deprotonation and protonation take place in a sequential manner. The deprotonation rate of L-Ala is approximately 16 times faster than that of D-Ala. The protonation step, however, appears to favor L-amino acid production, which prevents a much higher predominance of the D form in the imine. Receptor 1 and the predominantly D-form amino acid can be recovered from the imine by simple extraction under acidic conditions. Hence, 1 is a useful auxiliary to produce D-amino acids of industrial interest by the conversion of naturally occurring L-amino acids or relatively easily obtainable racemic amino acids.     

五氯化磷辅助下氨基酸的自组装成肽反应研究

L-α-氨基酸和D-α-氨基酸可与五氯化磷直接发生磷酰化反应，随后自组装成多肽，但β-氨基酸不能成肽，DL-α-氨基酸成肽困难；在SOCl2存在下，α-氨基酸也不能成肽，用电喷雾质谱研究了氨基酸的自组装反应，反应过程中有五元环状的氨基酸五配位磷中间体生成，使用硅烷基保护的氨基酸，在^31PNMR中可观察到五配位磷中间体。     

Effects of hydrogen-bonding and stacking interactions with amino acids on the acidity of uracil

The effects of hydrogen-bonding interactions with amino acids on the (N1) acidity of uracil are evaluated using (B3LYP) density functional theory. Many different binding arrangements of each amino acid to three uracil binding sites are considered. The effects on the uracil acidity are found to significantly depend upon the nature of the amino acid and the binding orientation, but weakly depend on the binding site. Our results reveal that in some instances small models for the amino acids can be used, while for other amino acids larger models are required to properly describe the binding to uracil. The gas-phase acidity of uracil is found to increase by up to approximately 60 kJ mol(-1) due to discrete hydrogen-bonding interactions. Although (MP2) stacking interactions with aromatic amino acids decrease the acidity of uracil, unexpected increases in the acidity are found when any of the aromatic amino acids, or the backbone, hydrogen bond to uracil. Consideration of enzymatic and aqueous environments leads to decreases in the effects of the amino acids on the acidity of uracil. However, we find that the magnitude of the decrease varies with the nature of the molecule bound, as well as the (gas-phase) binding orientations and strengths, and therefore solvation effects should be considered on a case-by-case basis in future work. Nevertheless, the effects of amino acid interactions within enzymatic environments are as much as approximately 35 kJ mol(-1). The present study has general implications for understanding the nature of active site amino acids in enzymes, such as DNA repair enzymes, that catalyze reactions involving anionic nucleobase intermediates.     

Residual dipolar couplings in short peptides reveal systematic conformational preferences of individual amino acids

Residual dipolar couplings (RDCs) observed by NMR in solution under weak alignment conditions can monitor average net orientations and order parameters of individual bonds. By their simple geometrical dependence, RDCs bear particular promise for the quantitative characterization of conformations in partially folded or unfolded proteins. We have systematically investigated the influence of amino acid substitutions X on the conformation of unfolded model peptides EGAAXAASS as monitored by their (1)H(Nu)-(15)N and (1)H(alpha)-(13)C(alpha) RDCs detected at natural abundance of (15)N and (13)C in strained polyacrylamide gels. In total, 14 single amino acid substitutions were investigated. The RDCs show a specific dependence on the substitution X that correlates to steric or hydrophobic interactions with adjacent amino acids. In particular, the RDCs for the glycine and proline substitutions indicate less or more order, respectively, than the other amino acids. The RDCs for aromatic substitutions tryptophane and tyrosine give evidence of a kink in the peptide backbone. This effect is also observable for orientation by Pf1 phages and corroborated by variations in (13)C(alpha) secondary shifts and (3)J(HNH)(alpha) scalar couplings in isotropic samples. RDCs for a substitution with the beta-turn sequence KNGE differ from single amino acid substitutions. Terminal effects and next neighbor effects could be demonstrated by further specific substitutions. The results were compared to statistical models of unfolded peptide conformations derived from PDB coil subsets, which reproduce overall trends for (1)H(Nu)-(15)N RDCs for most substitutions, but deviate more strongly for (1)H(alpha)-(13)C(alpha) RDCs. The outlined approach opens the possibility to obtain a systematic experimental characterization of the influence of individual amino acid/amino acid interactions on orientational preferences in polypeptides.     

Synthesis of novel water-soluble linear and heterocyclic phosphino amino acids from 2-phosphinophenols or 2-phosphinophenolethers, formaldehyde and amino acids

Acyclic and cyclic amino acid derivatives of 2-phosphinophenols have been synthesised by reaction of primary phosphinophenols (4-R-2-H₂PC₆H₃OH; R=H, Me, OMe) 1a–c with formaldehyde and amino acids (o- and p-aminobenzoic acid, -lysine) via in situ formed hydroxymethyl species 2a–c. Condensation reactions with glycine did not afford defined products except when the methoxymethyl and tetrahydropyranyl ethers of 1d,e were used instead of the hydroxy compounds. o-Aminobenzoic acid gives rise to linear bis(o-carboxyphenylaminomethyl)phosphines 3a–e. p-Aminobenzoic acid, dependent on the molar ratio, affords bis(p-carboxyphenylaminomethyl)phosphines 4a,d as well as eight-membered heterocyclic 1,5,3,7-diazadiphosphacyclooctanes 5a–e. The aliphatic amino acids glycine and -lysine form six-membered heterocyclic 1,3,5-diazaphosphorinanes 6d and 7a–e, respectively, in presence of excess formaldehyde. -lysine differs from glycine by reaction at the terminal amino group. The structures of the compounds have been elucidated by multinuclear NMR spectroscopy. The salts of the phosphino amino acids are soluble in water. Water solubility increases with the number of hydrophilic groups, i.e. free phenols are more soluble than their ethers. Ligand concentrations in water from 0.1 to 1 M were observed.     

An expedient route for the reduction of carboxylic acids to alcohols employing 1-propanephosphonic acid cyclic anhydride as acid activator

A simple and efficient method for the synthesis of alcohols from the corresponding carboxylic acids is described. Activation of carboxylic acid with 1-propanephosphonic acid cyclic anhydride (T3P) and subsequent reduction using NaBH₄ yield the alcohol in excellent yields with good purity. Reduction of several alkyl/aryl carboxylic acids and N^α-protected amino acids/peptide acids as well as N^β-protected amino acids was successfully carried out to obtain corresponding alcohols in good yields. All the products were fully characterized by ¹H NMR and mass spectral analyses. The procedure is mild, simple and the isolation of the products is easy.     

N-Methylation and N,N-dimethylation of amino acids. An artifact production in the analysis of organic acids using diazomethane as derivatizing agent

In the fractions of the methyl esters of urinary organic acids seventeen N-methylated or N,N-dimethylated amino acid methyl esters are identified by gas chromatography-mass spectrometry. It is shown for twelve amino acids that their amino group reacts with diazomethane to form these derivatives. Using deuterated reagents, in particular deuterated diazomethane, in the sample preparation procedure during the organic acid analysis, it is shown that the N-methylated and N,N-dimethylated amino acids are artifacts from diazomethane and are not biochemical N-methylation products.     

Capillary gas chromatographic analysis of amino acids in geological environments with electron capture detection

Studies have been performed on the analysis of 21 amino acids using a fused-silica open tubular (FSOT) capillary column, and electron-capture detection (ECD) or flame-ionization detection (FID). It was shown with the N(O)-heptafluorobutyryl (HFB) amino acid isobutyl esters that the ECD response was several hundred times more sensitive than the FID response. The relative standard deviation (RSD) of retention relative to norleucine is determined with the ECD. RSD values for all N(O)-HFB amino acid isobutyl esters are relatively small (≦ 0.5%). This method has been successfully applied to trace analysis of most of the amino acids from fossil brachiopods and black shales.     

Creation of hydrogen bonded 1D networks by co-crystallization of N,N'-bis(2-pyridyl)aryldiamines with dicarboxylic acids

The preparation and crystal structures of fourteen complexes of N,N'-bis(2-pyridyl)aryldiamines with dicarboxylic acids and two complexes with squaric acid are reported. The recognition between the carboxylic acids and the 2-aminopyridine units occurs through the formation of the cyclic R(2)2 (8) hydrogen bond motif, whereas squaric acid creates the analogous R(2)2 (9) motif. In the 1:1 complexes the cyclic motifs generate infinite hydrogen-bonded 1D networks with the alternating component molecules. These networks are further organised into densely packed layers assembled through weaker C-H...O interactions. Analysis of the intermolecular interactions in these complexes led us to the synthesis of N,N'-bis(2-pyridyl)-2,2'-oxybis(aminobenzene) (5) which acts as a tritopic receptor of the carboxylic group and forms exclusively 2:1 complexes with dicarboxylic acids.     

Analysis of amino acids without derivatization in barley extracts by LC-MS-MS

A method has been developed for quantification of 20 amino acids as well as 13 (15)N-labeled amino acids in barley plants. The amino acids were extracted from plant tissues using aqueous HCl-ethanol and directly analyzed without further purification. Analysis of the underivatized amino acids was performed by liquid chromatography (LC)-electrospray ionization (ESI) tandem mass spectrometry (MS-MS) in the positive ESI mode. Separation was achieved on a strong cation exchange column (Luna 5micro SCX 100A) with 30 mM ammonium acetate in water (solvent A) and 5% acetic acid in water (solvent B). Quantification was accomplished using d (2)-Phe as an internal standard. Calibration curves were linear over the range 0.5-50 microM, and limits of detection were estimated to be 0.1-3.0 microM. The mass-spectrometric technique was employed to study the regulation of amino acid levels in barley plants grown at 15 degrees C uniform root temperature (RT) and 20-10 degrees C vertical RT gradient (RTG). The LC-MS-MS results demonstrated enhanced concentration of free amino acids in shoots at 20-10 degrees C RTG, while total free amino acid concentration in roots was similarly low for both RT treatments. (15)NO(3) (-) labeling experiments showed lower (15)N/(14)N ratios for Glu, Ser, Ala and Val in plants grown at 20-10 degrees C RTG compared with those grown at 15 degrees C RT.     

Effect of acids and bases on the solubility of amino acids

The effect of acids and bases on the solubility of amino acids at 298.2 K, within the pH range, (pH from 2 to 10) and out of the pH range, was determined. The amino acids considered were glycine, dl-alanine, dl-valine and dl-serine. The acids used were HCl and HNO₃ and the bases NaOH and KOH. It was observed that in the pH range (pH from 2 to 10) there is no significant difference in the solubility of dl-alanine in the presence of different ions such as Na⁺ or K⁺ at high pH, and Cl⁻ or NO₃⁻ at low pH values. However, at higher concentrations of the acids or the bases, there is an effect of the counterions in solubilities. For all the amino acids considered here the solubility was higher in nitric acid, as compared to hydrochloric acid, and higher in potassium hydroxide than in sodium hydroxide.     

Characterisation of dissolved combined amino acids in marine waters

Dissolved combined amino acids (DCAA) are important constituents of the dissolved organic nitrogen (DON) pool in marine environments, although little is known about their sources, dynamics and sinks. The DCAA pool consists of various compounds including proteins and peptides, proteins linked to sugars and amino acids adsorbed to humic and fulvic acids, clays and other materials. The proportions of each of these components and the extent to which they are used by microplankton living within the photic zone are not known. An investigation was carried out, using (15)N isotope dilution techniques, to determine the concentration and composition of dissolved amino acid pools in the marine environment. A near-shore seawater sample was collected and split into fractions to determine the concentrations of dissolved free amino acids (DFAA), DCAA and a <3 kDa dissolved peptide fraction (DPEP; obtained by ultrafiltration). DCAA and DPEP fractions were hydrolysed to yield free amino acids and all samples were analysed by gas chromatography/mass spectrometry (GC/MS) as isobutyloxycarbonyl/tert-butyldimethylsilyl derivatives. The DFAA was the smallest fraction representing approximately 1% of total dissolved amino acids. The majority of DCAA was contained in the low molecular weight DPEP fraction (90%) and was probably as a result of release from phytoplankton and degradation by heterotrophic bacteria.     

Characterization of an NH-pi interaction in Co(III) ternary complexes with aromatic amino acids

The NH-pi interaction has been detected in the crystal structures of Co(III) ternary complexes with N,N-bis(carboxymethyl)-(S)-phenylalanine (BCMPA) and aromatic amino acids including (S)-phenylalanine ((S)-Phe), (R)-phenylalanine ((R)-Phe), and (S)-tryptophan ((S)-Trp)). Additionally, this interaction has been studied in solution for Co(III) ternary complexes with BCMPA or NTA (NTA = nitrilotriacetic acid) and several amino acids (AA) by means of electronic absorption, circular dichroism (CD), and (1)H NMR spectroscopies. The CD intensities of the Co(III) complexes with aromatic amino acids measured in the d-d region ( approximately 20.5 x 10(3) cm(-)(1)) are significantly decreased in ethanol solutions relative to water. Analogous complexes with aliphatic amino acids do not exhibit this solvent effect. The (1)H NMR spectra of the Co(III) complexes with aromatic amino acids measured in DMSO-d(6) exhibit upfield shifts of the NH peaks compared with those with aliphatic amino acids, which suggest a shielding effect due to the aromaticity. The upshift values coincide with those experimentally evaluated from the crystal structures. The magnitude of the upfield shifts agrees well with Hammett's rule, indicating that the increase of pi-electron densities on the aromatic rings leads attractive NH-pi interaction that exerts a larger shielding effect for the NH protons. In ligand-substitution reactions of the carbonatocobalt(III) complexes with amino acids, the yields of those with aromatic amino acids are higher than the yields obtained for complexes with aliphatic amino acids. This observation is discussed in connection with the important contribution of the NH-pi interaction as one of the promotion factors in the reaction.     

Assignment of the configurations of the amino acids in peptidic siderophores

Pyoverdins and azotobactins contain beta-hydroxyaspartic acid, N delta-hydroxyornithine, citrulline and homoserine, in addition to the common protein amino acids. Configuration assignment of all of these was achieved by acid hydrolysis of the peptide, derivatization of the constituent amino acids to the N-pentafluoropropionyl amino acid esters and gas chromatographic separation of the stereoisomers on capillaries coated with Chirasil-Val. This approach is straightforward for the protein amino acids, but the less common amino acids are either partially degraded during acid hydrolysis or their derivatives exhibit unfavourable gas chromatographic properties. By judicious combination of partial and total hydrolysis and dual derivatization, these problems may be overcome.     

Cholic acid-based fluorescent probes for enantioselective recognition of trifunctional amino acids

The ditopic fluorescent photoinduced electron transfer (PET) amino acid sensory probes and were designed and synthesized from cholic acid. To confer the probes with specific binding ability, an amidothiourea moiety and a cyclic diamino-chiral receptive site were introduced on the C17 side chain and the C7 and C12 hydroxyl pendants, respectively. In acetonitrile, the probes demonstrated differential binding toward trifunctional amino acids like serine, lysine, threonine and tyrosine against other simple amino acids. Enantioselectivities (KD/KL) of up to 8.9 and sensitivities in the micromolar range with the probes were observed for trifunctional amino acids.     

N-phosphoryl amino acids and peptides. Part II: Fast atom bombardment mass spectrometry of N-di-isopropyloxyphosphoryl and N-dibutyloxyphosphoryl amino acids

The positive ion fast atom bombardment mass spectra of N-di-isopropyloxyphosphoryl (Dipp) and N-dibutyloxyphosphoryl (Dbp) amino acids or amino acid methyl esters are presented; and according to the observation of the metastable ions and the high-resolution accurate mass measurement their fragmentation patterns are postulated. Both types of compounds were found to undergo similar fragmentations to produce (HO)₂P(O)N?CH? R + H⁺, in most cases as the most abundant fragment ion of structural significance. An intrinsical difference between the two types of compounds is that N-Dippamino acids appear to favour the successive losses of two molecules of propylene, while the loss of HCOOH seems to be preferred by the Dbp amino acids. For those compounds containing an extra functional group on the side chain of amino acids such as serine or glutamic acid some other type of fragmentation was observed besides the normal phenomenon.