2-Amino-3-carbamoyl-5 hydrolysis

D. Enzymes Involved In Af-Carbamoyl Amino Acid Hydrolysis... 

An even more elegant approach for the production of D-phydroxyphenylglydne on an industrial scale uses foe bacterium. Agrobacterium radiobacter (Figure A8.8). The organism is able to produce both D-hydantoinase and a second enzyme, N-carbamoyl-D-amino acid aminohydrolase, which catalyse the hydrolysis of N-carbamoyl-D-amino add. 

The main application of the enzymatic hydrolysis of the amide bond is the en-antioselective synthesis of amino acids [4,97]. Acylases (EC 3.5.1.n) catalyze the hydrolysis of the N-acyl groups of a broad range of amino acid derivatives. They accept several acyl groups (acetyl, chloroacetyl, formyl, and carbamoyl) but they require a free a-carboxyl group. In general, acylases are selective for i-amino acids, but d-selective acylase have been reported. The kinetic resolution of amino acids by acylase-catalyzed hydrolysis is a well-established process [4]. The in situ racemization of the substrate in the presence of a racemase converts the process into a DKR. Alternatively, the remaining enantiomer of the N-acyl amino acid can be isolated and racemized via the formation of an oxazolone, as shown in Figure 6.34. 

In many cases, the racemization of a substrate required for DKR is difficult As an example, the production of optically pure cc-amino acids, which are used as intermediates for pharmaceuticals, cosmetics, and as chiral synfhons in organic chemistry [31], may be discussed. One of the important methods of the synthesis of amino acids is the hydrolysis of the appropriate hydantoins. Racemic 5-substituted hydantoins 15 are easily available from aldehydes using a commonly known synthetic procedure (Scheme 5.10) [32]. In the next step, they are enantioselectively hydrolyzed by d- or L-specific hydantoinase and the resulting N-carbamoyl amino acids 16 are hydrolyzed to optically pure a-amino acid 17 by other enzymes, namely, L- or D-specific carbamoylase. This process was introduced in the 1970s for the production of L-amino acids 17 [33]. For many substrates, the racemization process is too slow and in order to increase its rate enzymes called racemases are used. In processes the three enzymes, racemase, hydantoinase, and carbamoylase, can be used simultaneously this enables the production of a-amino acids without isolation of intermediates and increases the yield and productivity. Unfortunately, the commercial application of this process is limited because it is based on L-selective hydantoin-hydrolyzing enzymes [34, 35]. For production of D-amino acid the enzymes of opposite stereoselectivity are required. A recent study indicates that the inversion of enantioselectivity of hydantoinase, the key enzyme in the... 

Ammonia is highly toxic to animal tissues. In the urea cycle, ornithine combines with ammonia, in the form of carbamoyl phosphate, to form citrulline. A second amino group is transferred to citrulline from aspartate to form arginine—the immediate precursor of urea. Arginase catalyzes hydrolysis of arginine to urea and ornithine thus ornithine is regenerated in each turn of the cycle. 

The bacterial D-hydantoinase has been isolated as crystals from cells of Pseudomonas putida (= P. striata) (Table 1) [5]. Because the purified enzyme showed the highest activity and affinity toward dihydrouracil, the enzyme was identified as dihydropyrimidinase (EC. 3.5.2.2). Interestingly, the enzyme also attacked a variety of aliphatic and aromatic D-5-mono-substituted hydantoins, yielding the corresponding D-form of N-carbamoyl-a-amino acids. Thus, the enzyme can be used for the preparation of various D-amino acids. Under the conditions used for the enzymatic hydrolysis of hydantoin at pH 8 to 10, the L-isomers of the remaining hydantoins are racemized through base catalysis. Therefore, the racemic hydantoins can be converted quantitatively into N-carbamoyl-D-amino acids through this step. 

Decarbamoylation to D-amino acid was performed by treating the N-carbamoyl-D-amino acid with equimolar nitrite under acidic conditions [6]. But now, this step can also be carried out enzymatically. Recently, Shimizu and co-workers found a novel enzyme, D-decarbamoylase (IV-carbamoyl- n-amino acid amidohydrolase), which stereospecifically hydrolyzes JV-carbamoyl-D-amino acids, in several bacteria [7, 8], For example, Blastobacter sp. A17p-4 was found to produce D-decarbamoylase together with D-hydantoinase [8]. Therefore, a sequence of two enzyme-catalyzed reactions, the D-stereospecific hydrolysis of DL-5-(p-hydroxyphenyl) hydantoin and subsequent hydrolysis of the D-carbamoyl derivative to D-p-hydroxyphenylglycine, is possible (Fig. 1). Based on these results, a new commercial process for the production of D-p-hy-droxyphenylglycine has been developed [9]. 

Many kinds of enzymes with different substrate specificities are involved in hydantoin hydrolysis. Ogawa et al. [10] found two hydantoin-hydrolyzing enzymes in Blastobacter sp. A17p-4. These enzymes were purified to homogeneity and characterized (Table 1). One hydrolyzed dihydropyrimidines and 5-monosubstituted hydantoins to the corresponding AT-carbamoyl amino acids. Since the hydrolysis of 5-substituted hydantoins by this enzyme was D-stereo-specific, this enzyme was identified as D-hydantoinase, which is identical with dihydropyrimidinase. The other one preferably hydrolyzed cyclic imide compounds such as glutarimide and succinimide more than cyclic ureide compounds such as dihydrouracil and hydantoin. Because there have been no reports on enzymes which show same substrate specificity as this enzyme, it is considered to be a novel enzyme, which should be called imidase [10]. 

Above all in the hydrolysis of iV-carbamoyl amino acid, three types of enzymes with different substrate specificity function (Fig. 3). 

CO2 promotes the reaction of unreactive nitrile groups at moderate pH but cannot be truly considered as a catalyst since it is not released at the end of the reaction (unless the hydantoin undergoes further hydrolysis) unlike carbonyl compounds in a-aminonitrile hydration (Sect. 2.1.3). However, hydantoins can be considered as AA precursors through a two-step hydrolysis. The first step leads to an N-carbamoyl amino acid (CAA). The rate of this HCT-catalyzed step has been examined by Taillades and co-workers [43], extending the earlier work of Blagoeva et al. [49]. 

Fig. 5 The Primary Pump, a peptide-based protometabolism scenario [146] involving the following steps I amino acid N-carbamoylation II concentration through drying III NO -mediated CAA activation IV dissolution through watering (by e.g. sea water) V NCA reaction in aqueous phase (Va NCA hydrolysis Vb condensation with AA or peptide) VI slow hydrolysis of peptide bonds VII a-carbon epimerization (Vila of amino acid and CAA VNb of peptide residues). Additional steps corresponding to peptide N-carbamoylation/nitrosation have not been mentioned for the sake of clarity. It is worth mentioning that although the N-carbamoylation of peptides renders them unre-active towards NCA, this is reverted by NOx-mediated nitrosation [197], thus keeping peptides within the polymerization process...

The enzymes of the nucleic acid metabolism are used for several industrial processes. Related to the nucleobase metabolism is the breakdown of hydantoins. The application of these enzymes on a large scale has recently been reviewed [85]. The first step in the breakdown of hydantoins is the hydrolysis of the imide bond. Most of the hydantoinases that catalyse this step are D-selective and they accept many non-natural substrates [78, 86]. The removal of the carbamoyl group can also be catalysed by an enzyme a carbamoylase. The D-selective carbamoylases show wide substrate specificity [85] and their stereoselectivity helps improving the overall enantioselectivity of the process [34, 78, 85]. Genetic modifications have made them industrially applicable [87]. Fortunately hydantoins racemise readily at pH >8 and additionally several racemases are known that can catalyze this process [85, 88]. This means that the hydrolysis of hydantoins is always a dynamic kinetic resolution with yields of up to 100% (Scheme 6.25). Since most hydantoinases are D-selective the industrial application has so far concentrated on D-amino acids. Since 1995 Kaneka Corporation has produced 2000 tons/year of D-p-hydroxyphenylglycine with a D-hydantoinase, a d-carbamoylase [87] and a base-catalysed racemisation [85, 89]. 

One of the most widely used enzymatic methods for D-amino acid production is the hydantoinase process [4]. The great advantage of this process is that, potentially, any optically pure D-amino acid can be obtained using the corresponding substrate from a wide spectrum of D,L-5-monosubstituted hydantoins, which are readily accessible by chemical synthesis [5]. In this cascade of reachons the chemically synthesized D,L-5-monosubstituted hydantoin ring is first hydrolyzed by a stereoselective hydantoinase enzyme (D-hydantoinase). Further hydrolysis of the resulting N-carbamoyl D-amino acid to the free D-amino acid is catalyzed... 

Like the synthesis of carbamoyl phosphate, this reaction requires ATP and uses glutamine as the source of the amino group. The reaction proceeds through an analogous mechanism in which the 0-4 atom is phosphorylated to form a reactive intermediate, and then the phosphate is displaced by ammonia, freed from glutamine by hydrolysis. CTP can then be used in many biochemical processes, including RNA synthesis. 

Amino-2prepared from 2-amino-3-cyanopyrazine 1-oxide by reflux with acetic acid-acetic anhydride followed by ready deacetylation by refluxing in methanol (538), and in a similar manner 3-amino-2-ethoxycarbonyl-5-hydroxypyrazine has been prepared from 2-amino-3-ethoxycarbonylpyrazine 1 -oxide through 3-acetamido-2-ethoxycarbonyl-5 ydroxy-pyrazine (538), and 2-amino-3-carbamoyl-6-hydroxy-5-methylpyrazine from 2-amino-3-cyano-5-methylpyrazine 1-oxide (538). The preparation of 24 ydroxy-6-methoxycarbonylpyrazine (10) has been claimed from 3-methoxycarbonylpyrazine 1-oxide with acetic anhydride followed by hydrolysis (1057) [cf. Nov Cek et al. (839), who claim it to be the 5-isomer, and Foks (744)]. 

Acylaminopyrazines have been deacylated by hydrolysis in acid, or alkali, by methanolysis, or by hydrazinolysis (in propan-2-ol) under a variety of conditions to give the corresponding amino compound unless otherwise specified in the following examples 2-acetamido-3-acetoxymethylpyrazine (28) (dilute acetic acid at reflux for 6h) (1075) 2-acetamido-5-chloro-3-amidinocarbamoylpyrazine (5% hydrochloric acid-acetic acid at 100°) (150) 2-acetamido-3-A(-(benzimidoyl)-carbamoyl-5-chloropyrazine (5% hydrochloric acid at room temperature) (150) ... 

Carboxypyrazine A -oxides have been prepared by hydrolysis of carbamoyl- and alkoxycarbonylpyrazine A(-oxides as follows (reagent and conditions) 2-carbamoyl-pyrazine 1-oxide (10% NaOH/reflux/12h) (838) 3-carbamoylpyrazine 1-oxide (10% NaOH/reflux/30 min) (1266, cf. 838) 3-A(-acetylcarbamoylpyrazine 1-oxide (10% NaOH/heat) (1057) 3-morpholinocarbonylpyrazine 1-oxide (18% HQ/reflux/ 8h) (870) 2-hydroxy-5-methoxycarbonylpyrazine 1-oxide 2.5N NaOH/20-25°/ 20min) (739) 3-hydroxy-5-methoxycarbonylpyrazine 1-oxide (KOH/22 /2h gave 3-carboxy-5-hydroxypyrazine 1-oxide, which interfered with the growth of Streptococcus faecium Escherichia coli at 6 x lO and 4 x 10" M, respectively) (1035) 2-amino-3-benzyloxycarbonyl-5-methyIpyrazine 1-oxide 2N NaOH/reflux/ 30min) (365c) and 2-amino-5-chloro-3-methoxycarbonylpyrazine 1-oxide 2.5N NaOH/heat) (876,1222). 

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