Carbamoyl Amino Acids Research Articles

A Practical Method for Amino Acid Analysis by LC-MS Using Precolumn Derivatization with Urea.

Amino acid (AA) analysis is important in biochemistry, food science, and clinical medicine. However, due to intrinsic limitations, AAs usually require derivatization to improve their separation and determination. Here, we present a liquid chromatography-mass spectrometry (LC-MS) method for the derivatization of AAs using the simple agent urea. The reactions proceed quantitatively under a wide range of conditions without any pretreatment steps. Urea-derivatized products (carbamoyl amino acids) of 20 AAs exhibit better separation on reversed-phase columns and increased response in a UV detector compared to underivatized ones. We applied this approach to AA analysis in complex samples using a cell culture media as a model, and it showed potential for the determination of oligopeptides. This fast, simple, and inexpensive method should be useful for AA analysis in complex samples.

Urea-mediated warm ponds: Prebiotic formation of carbamoyl amino acids on the primordial Earth

The emergence of prebiotic peptides on the early Earth was a crucial step for the origin of life. Carbamoyl amino acids may have played a vital role in prebiotic peptide formation. Herein, we report carbamoyl amino acids obtained with nearly 100% yield under prebiotic conditions. The reaction mechanism involves nucleophilic substitution with the release of NH3 from urea, which was verified by 15N-labeling approach. Meanwhile, experiments indicated that carbamoyl amino acids can be also well-obtained under UV radiation in the urea pool, which provides a better condition for carbamoyl amino acids to form prebiotic peptides. The study implies that peptide precursors other than classical amino acids may have been present on the primitive Earth.

A novel, enantioselective, thermostable recombinant hydantoinase to aid the synthesis of industrially valuable non-proteinogenic amino acids

Overexpression of a novel hydantoinase (hyuH) from P. aeruginosa (MCM B-887) in E. coli yielded optically pure carbamoyl amino acids. The use of optically pure carbamoyl amino acids as substrates facilitates the synthesis of non-proteinogenic amino acids. The enzyme hyuH shared a maximum of 92 % homology with proven hydantoinase protein sequences from the GenBank database, highlighting its novelty. Expression of hydantoinase gene was improved by >150 % by overexpressing it as a fusion protein in specialized E. coli CODON + host cells, providing adequate machinery for effective translation of the GC-rich gene. The presence of distinct residues in the substrate binding and active site of MCM B-887 hydantoinase enzyme explained its unique and broad substrate profile desirable for industrial applications. The purified enzyme, with a specific activity of 53U/mg of protein, was optimally active at 42 °C and pH 9.0 with a requirement of 2 mM Mn2+ ions. Supplementation of 500 mM of Na-glutamate enhanced the thermostability of the enzyme by more than 200 %.

Myeloperoxidase-catalyzed oxidation of cyanide to cyanate: A potential carbamylation route involved in the formation of atherosclerotic plaques?

Protein carbamylation by cyanate is a post-translational modification associated with several (patho)physiological conditions, including cardiovascular disorders. However, the biochemical pathways leading to protein carbamylation are incompletely characterized. This work demonstrates that the heme protein myeloperoxidase (MPO), which is secreted at high concentrations at inflammatory sites from stimulated neutrophils and monocytes, is able to catalyze the two-electron oxidation of cyanide to cyanate and promote the carbamylation of taurine, lysine, and low-density lipoproteins. We probed the role of cyanide as both electron donor and low-spin ligand by pre-steady-state and steady-state kinetic analyses and analyzed reaction products by MS. Moreover, we present two further pathways of carbamylation that involve reaction products of MPO, namely oxidation of cyanide by hypochlorous acid and reaction of thiocyanate with chloramines. Finally, using an in vivo approach with mice on a high-fat diet and carrying the human MPO gene, we found that during chronic exposure to cyanide, mimicking exposure to pollution and smoking, MPO promotes protein-bound accumulation of carbamyllysine (homocitrulline) in atheroma plaque, demonstrating a link between cyanide exposure and atheroma. In summary, our findings indicate that cyanide is a substrate for MPO and suggest an additional pathway for in vivo cyanate formation and protein carbamylation that involves MPO either directly or via its reaction products hypochlorous acid or chloramines. They also suggest that chronic cyanide exposure could promote the accumulation of carbamylated proteins in atherosclerotic plaques.

The Prebiotic C-Terminal Elongation of Peptides Can Be Initiated by N-Carbamoyl Amino Acids.

The formation of peptides upon 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC)-promoted activation of N-carbamoylamino acids (CAA), was considered in the scope of our recent works on carbodiimide promoted C-terminus elongation of peptides in a prebiotic context. Thus EDC promoted activation of CAA derivatives of Tyr(Me) or Ala in dilute aqueous medium pH 5.5-6.5 in the presence of excess of AA, resulted in peptide formation by C-terminus activation/elongation. Kinetic results similar to those of EDC-mediated activation of N-acyl-AA lead us to postulate the formation of a 2-amino-5(4H)-oxazolone intermediate by cyclization of the activated CAA, in spite of the absence of epimerization occurred at CAA residues. Thus, in a prebiotic context, CAA may have played a similar role as N-acyl-AA in the initiation of C-terminus peptide elongation.

Process parameter optimization for hydantoinase-mediated synthesis of optically pure carbamoyl amino acids of industrial value using Pseudomonas aeruginosa resting cells

Hydantoinase-mediated enzymatic synthesis of optically pure carbamoyl amino acids was investigated as an environmentally friendly, energy-efficient alternative to the otherwise energy-intensive, polluting chemical synthesis. Hydantoinase-producing bacterial strain was identified as Pseudomonas aeruginosa by 16S rRNA gene sequencing and biochemical profiling using the BIOLOG Microbial Identification System. Hydantoinase activity was assessed using hydantoin analogs and 5-monosubstituted hydantoins as substrates in a colorimetric assay. The hydantoinase gene was PCR amplified using gene-specific primers and sequenced on an automated gene analyzer. Hydantoinase gene sequence of P. aeruginosa MCM B-887 revealed maximum homology of only 87% with proven hydantoinase gene sequences in GenBank. MCM B-887 resting cells converted >99% of substrate into N-carbamoyl amino acids under optimized condition at 42°C, pH 8.0, and 100mM substrate concentration in <120min. Hydantoin hydrolyzing activity was D-selective and included broad substrate profile of 5-methyl hydantoin, 5-phenyl hydantoin, 5-hydroxyphenyl hydantoin, o-chlorophenyl hydantoin, as well as hydantoin analogs such as allantoin, dihydrouracil, etc. MCM B-887 resting cells may thus be suitable for bio-transformations leading to the synthesis of optically pure, unnatural carbamoyl amino acids of industrial importance.

Dimeric and monomeric surfactants derived from sulfur-containing amino acids

Anionic urea-based dimeric (gemini) surfactants derived from the amino acids L-cystine, D-cystine and DL-cystine, as well as monomeric surfactants derived from L-cysteine, L-methionine and L-cysteic acid were synthesized and their solution properties characterized by electrical conductivity, equilibrium surface tension, and steady-state fluorescence spectroscopy techniques. The geminis studied showed the lowest critical micelle concentration (cmc) values, however the monomeric cysteine counterpart exhibited superior efficiency in lowering surface tension, an unusual finding that can be attributed to the free sulfhydryl group. Chirality seems to play a role in the surface active properties of the gemini surfactants, but not on micelle formation. All the surfactants studied showed a higher preference for adsorption at the air/water interface rather than to form micelles, a fact that may be related to the urea moiety. The polarity of the interfacial region, measured with the solvatochromic probe E(T)(30) (Reichardt's betaine dye), was similar to sodium dodecyl sulphate (SDS) micelles.

Investigation of N‐carbamoylamino acid nitrosation by {NO + O2} in the solid‐gas phase. Effects of NOx speciation and kinetic evidence for a multiple‐stage process

AbstractNitrosation of N‐carbamoylamino acids (CAA) by gaseous NO + O2, an interesting synthetic pathway to amino acid N‐carboxyanhydrides (NCA), alternative to the phosgene route, was investigated on N‐carbamoyl‐valine either in acetonitrile suspension or solventless conditions, and compared to the classical nitrosating system NaNO2 + CF3COOH (TFA), the latter being quite less efficient in terms of either rate, stoichiometric demand, or further tractability of the product. The rate and efficiency of the NO + O2 reaction mainly depends on the O2/NO ratio. Evaluation of the contribution of various nitrosating species (N2O3, N2O4, HNO2) through stoichiometric balance showed the reaction to be effected mostly by N2O3 for O2/NO ratios below 0.3, and by N2O4 for O2/NO ratios above 0.4. The relative contribution of (subsequently formed) HNO2 always remains minor. Differential scanning calorimetry (DSC) monitoring of the reaction in the solid phase by either HNO2 (from NaNO2 + TFA), gaseous N2O4 or gaseous N2O3, provides the associated rate constants (ca. 0.1, 2 and 108 s−1 at 25°C, respectively), showing that N2O3 is by far the most reactive of these nitrosating species. From the DSC measurement, the latent heat of fusion of N2O3, 2.74 kJ · mol−1 at −105 °C is also obtained for the first time. The kinetics was investigated under solventless conditions at 0°C, by either quenching experiments or less tedious, rough calorimetric techniques. Auto‐accelerated, parabolic‐shaped kinetics was observed in the first half of the reaction course, together with substantial heat release (temperature increase of ca. 20°C within 1–2 min in a 20‐mg sample), followed by pseudo‐zero‐order kinetics after a sudden, important decrease in apparent rate. This kinetic break is possibly due to the transition between the initial solid‐gas system and a solid‐liquid‐gas system resulting from water formation. Overall rate constants increased with parameters such as the specific surface of the solid, the O2/NO ratio, or the presence of moisture (or equivalently the hydrophilicity of the involved CAA), however without precise relationship, while the last two parameters may directly correlate to the increasing acidity of the medium. Copyright © 2007 John Wiley &amp; Sons, Ltd.

The Peptide Formation Mediated by Cyanate Revisited. N-Carboxyanhydrides as Accessible Intermediates in the Decomposition of N-Carbamoylamino Acids

Similar to many ureas, N-carbamoylamino acids were shown to be hydrolyzed in aqueous solution through elimination mechanisms at close to neutral pH, the nucleophilic attack of water being a minor process. Two competing elimination mechanisms can take place involving either cyanate or isocyanate transient intermediates. Peptide formation was observed and attributed to the latter pathway through the intermediacy of amino acid N-carboxyanhydride (NCA). Eventually, cyanate and its precursors (including urea) unexpectedly behave as amino acid activating agents because of their ability in amino acid carbamoylation. Owing to its ability to generate a background prebiotic production of NCAs on the primitive Earth, this reaction is suggested to have contributed to the origin of life process.

Uremia and insulin resistance: N-carbamoyl-asparagine decreases insulin-sensitive glucose uptake in rat adipocytes

In uremia, urea-derived cyanate reacts with amino groups irreversibly forming carbamoyl amino acids (C-AA) and carbamoyl proteins. Carbamoylated molecules can affect binding and trafficking and alter metabolic pathways. The C-AA role in insulin-sensitive glucose transport has not been explored and may contribute to insulin resistance in uremia. Insulin-stimulated glucose uptake by cultured rat adipocytes was measured using both 3-minute and 3-second assays. Adipocytes were incubated for 24 hours in medium containing 0.5 micromol/mL of 15 different C-AA. 125I-insulin binding studies were done. C-asparagine in plasma from 10 uremic patients on continuous ambulatory peritoneal dialysis (CAPD) was measured using high-performance liquid chromatography (HPLC). Insulin-sensitive glucose uptake was reduced 34% by N-carbamoyl-l-asparagine, (N-C-Asn), in a dose-dependent manner with a half-maximally effective concentration of 0.15 micromol/mL. Fourteen other N-carbamoyl-amino acids as well as 0.5 micromol/mL of asparagine did not affect insulin sensitive glucose uptake. N-C-Asn, l-asparagine, and the other N-carbamoyl amino acids (0.5 micromol/mL) had no effect on basal glucose uptake. These data suggest that that N-C-Asn affects the insulin sensitive glucose transporter system. 125I-insulin binding studies demonstrated that N-C-Asn did not alter insulin binding. Glucose uptake measured using a 3-second assay showed that the glucose affinity of the transporter and glucose phosphorylation were not affected. In uremic patients managed by CAPD, the mean free N-C-Asn plasma level was 1.33 micromol/mL. These data suggest that N-C-Asn concentration may contribute to the insulin resistance seen in uremia.

A New Biocatalytic Route to Enantiopure N‐Carbamoyl Amino Acids by Fast Enzyme Screening.

Abstract The enantioselective enzymatic deamidation of ( rac )- N -carbamoyl amino acid amides (Cbm-AA-NH 2 ) to enantiopure ( L )- N -carbamoyl amino acids (Cbm-AA-OH) is described for the first time. Via fast screening methods of biocatalysts several proteases like Chirazyme P1, Chirazyme P2 and Subtilisin were identified, which give conversions of up to 47% and >98% ee. This conversion is most productive on aliphatic and primary amino acids.

Carbamoylation of amino acids and proteins in uremia

Cyanate spontaneously transformed from urea increases as renal function decreased. Acting as a potential toxin, the active form of cyanate, isocyanic acid, carbamoylates amino acids, proteins, and other molecules, changing their structure, charge, and function. The resulting in vivo carbamoylation can modify the molecular activity of enzymes, cofactors, hormones, low-density lipoproteins, antibodies, receptors, and transport proteins. Antibodies specific for epsilon-amino-carbamoyl-lysine (homocitrulline) located carbamoylated proteins in situ in neutrophils, monocytes, and erythrocytes. Carbamoylated proteins were found in renal tissue from uremic patients but not in normal transplanted kidneys. The irreversible reaction with cyanate converts free amino acids (F-AAs) to carbamoyl-amino acids (C-AAs). The Carbamoylation Index (CI), C-AA/F-AA, quantifies the decrease of the F-AA pool for each essential amino acid. C-AAs contribute, in part, to malnutrition of uremia. C-AAs interfered with protein synthesis to lower 14C hemoglobin synthesis in human reticulocytes and osteocalcin synthesis in rat osteosarcoma-derived tissue culture. Insulin-sensitive glucose uptake was decreased 33% in cultured rat adipocytes by alpha-amino-carbamoyl-asparagine. alpha-Amino carbamoylation occurs primarily in F-AA, while epsilon-amino carbamoylation of lysine in protein occurs continuously during the protein life span. Protein catabolism releases epsilon-amino-carbamoyl-lysine. Quantitation of alpha versus epsilon carbamoylation may yield a more sensitive measurement of protein intake versus protein catabolism, and could be useful in decisions concerning the time to initiate dialysis or subsequent changes in dialysis prescription. Carbamoylated molecules can block, enhance, or be excluded from metabolic pathways, thereby influencing the fate of noncarbamoylated molecules. Although not an "all-or-none" phenomenon, urea-derived cyanate and its actions are contributing causes of toxicity in uremia.

Enzymatic synthesis and subsequent racemization rates determination of optically active d-5-phenylhydantoin and d-5- hydroxylphenylhydantoin

A process using immobilized d-hydantoinase for the preparation of optically active hydantoins was developed. At basic pH, the racemic hydantoin was hydrolyzed into d -N- carbamoyl-amino acid. The equilibrium constant of the hydrolysis reaction increased with pH. At acidic pH, the d -N- carbamoyl-amino acid was favorably converted to d-hydantoin. Optically active d-phenylhydantoin ( d-PH) and d -p hydroxyphenylhydantoin ( d-pHPH) were prepared directly from the racemic hydantoins d, l-PH and d, l-pHPH, respectively via corresponding d -N- carbamoylamino acid in one pot reaction just by adjusting the reaction pH from 8.5 to 5.5. The d-PH and d - p HPH obtained in this mild enzymatic reaction were nearly optically uniform. The racemization rate of these hydantoins increased with pH and temperature. It was more strongly affected at basic than acidic pH. The racemization rate of p HPH was slightly higher than that of PH.

Essential carbamoyl-amino acids formed in vivo in patients with end-stage renal disease managed by continuous ambulatory peritoneal dialysis: Isolation, identification, and quantitation

Carbamoyl-amino acids (C-AA) are formed by reaction of amino acids with cyanate, which is spontaneously formed from urea at body temperature and pH. In vivo derivatized C-AA are not measured by the usual amino acid analysis methods, which require a free amino group for derivatization. Free-amino acids (F-AA) but no C-AA were found in the postabsorptive plasma of eight normal persons with blood urea nitrogen (BUN) levels ranging from 9 to 16 mg/dl. In a longitudinal study of postprandial plasma (n = 43), essential amino acids, both C-AA and F-AA, were isolated and quantified by reverse-phase high-pressure liquid chromatography in six patients with end-stage renal disease who were managed by continuous ambulatory peritoneal dialysis. The mean BUN was 61 mg/dl (range, 36 to 79 mg/dl). In uremia, removal of F-AA from the essential amino acid pool to form C-AA is measured by the ratio of C-AA to F-AA (carbamoylation index (CI)). Using the mean value for each essential amino acid, the CIs were as follows: leucine, 4; valine, 3.3; isoleucine, 11.4; threonine, 9; lysine, 2; methionine, 3.5; histidine, 3.5; phenylalanine, 0.5; and tyrosine, 1.3. Carbamoylation of F-AA may account, in part, for the lower than normal levels of F-AA in patients with uremia. The derivatized amino group of C-AA interferes with formation of a peptide bond in protein synthesis, which requires an underivatized amino acid. A decrease in the F-AA pool available for protein synthesis and anabolism in the presence of C-AA may provide additional contributing factors for the development of malnutrition in uremia.

Amino acid-derived piperidides as novel CCK B ligands with anxiolytic-like properties

The development of a novel series of carbamoylamino acid benzoylpiperidides as CCK B ligands is described. Selected members of the series antagonized CCK 8-induced calcium mobilization and showed efficacy in the mouse elevated-plus maze, a measure of potential anxiolytic activity.

Chiral analysis of synthetic peptides: high performance liquid chromatography of diastereoisomeric carbamoyl esters derived from N-terminal cleavage

A cleavage routine for chiral analysis has revealed that the N-terminal residue in model dipeptides, after derivatisation with a chiral isocyanate reagent, can be cleaved off as the N-carbamoyl ester under relatively mild conditions (MeOH/SOCl2/60 °C for 3 h) free from inherent problems of racemisation. The diastereoisomeric carbamoyl amino acid esters produced can be separated and identified by HPLC. The methodology has been optimised for a number of representative amino acids, including tyrosine, histidine, methionine and tryptophan which have side-functions capable of complicating the procedure.

Fast atom bombardment and tandem mass spectrometry of N-carbamoylamino acids in urine from patients with urea cycle disorders

Seven synthetic carbamoylamino acids (CAA) as tert-butyldimethylsilyl (TBDMS) derivatives were examined by fast atom bombardment (FAB) mass spectrometry and FAB tandem mass spectrometry (MSMS). In FAB-MS and FAB MSMS, the spectra of protonated molecules for CAA provide specific cleavages involving the TBDMS carbamoyl moiety. Further, it is demonstrated that urine samples from patients with ornithine carbamoyl transferase deficiency give collision-induced dissociation mass spectra that are consistent with such spectra obtained using synthetically prepared CAA.

Oxidations of some α-amino acids under mitsunobu reaction conditions

Reactions of α-amino acids with azodicarboxylates and Ph 3P results in oxidation at the α-carbon. N-acyl or carbamoyl amino acid esters give azodicarobxylate adducts, whereas free α-amino acid esters are converted to the corresponding α-keto esters.

Chromium(III) Complexes with Amino Acids. III. Chromium(III) Complexes with Acidic, Basic and Carbamoyl Amino Acids

Abstract Syntheses by both the isothermal matrix in a solid state and by the usual solution method have been tried for chromium (III) complexes with l- and Dl-asparagine, l- and Dl-aspartic acid, l- and Dl-glutamine, l- and Dl-glutamic acid, l- and Dl-lysine, and l-ornithine. [Cr(l-aspNH2)3]·2H2O, [Cr(Dl-aspNH2)3]·3H2O, (NH4)2[Cr(l-asp)2][Cr(d-asp)2]·4H2O, and (+)[Cr(l-lys)2(NH3)(H2O)]Cl·4H2O were obtained by the matrix method in the solid state, while [Cr(l-aspNH2)3]·2H2O, [Cr(Dl-aspNH2)3]·2H2O, and (+)[Cr(l-lys)2(NH3)(H2O)]Cl·4H2O were prepared by the usual solution method. The preparation of chromium(III) complexes with other amino acids was not successful. A new type of complex of chromium(III) containing terdentate Dl-aspartic acid and a new mixed chromium(III) complex containing three different ligands (l-lysine, ammine, and aqua) were prepared.

Studies on Latent Derivatives of Aminoethanethiols as Potentially Selective Cytoprotectants. VI. Synthesis of N-(2-Mercaptoethyl)carbamoylamino Acids

Studies on Latent Derivatives of Aminoethanethiols as Potentially Selective Cytoprotectants. VI. Synthesis of N-(2-Mercaptoethyl)carbamoylamino Acids