Aspartic acid/asparagine mixtures

The abbreviations of the essential 20 amino acids are listed in Table 12-5 these abbreviations do not need to be defined. In analytical situations, undifferentiated mixtures of aspartic acid/asparagine (Asx, B) or glutamic acid/gluta-mine (Glx, Z) may occur. 

The amide bonds in peptides and proteins can be hydrolyzed in strong acid or base Treatment of a peptide or protein under either of these conditions yields a mixture of the constituent amino acids. Neither acid- nor base-catalyzed hydrolysis of a protein leads to ideal results because both tend to destroy some constituent ammo acids. Acid-catalyzed hydrolysis destroys tryptophan and cysteine, causes some loss of serine and threonine, and converts asparagine and glutamine to aspartic acid and glutamic acid, respectively. Base-catalyzed hydrolysis leads to destruction of serine, threonine, cysteine, and cystine and also results in racemization of the free amino acids. Because acid-catalyzed hydrolysis is less destructive, it is often the method of choice. The hydrolysis procedure consists of dissolving the protein sample in aqueous acid, usually 6 M HC1, and heating the solution in a sealed, evacuated vial at 100°C for 12 to 24 hours. 

Four mechanisms have been advanced for the prebiotic formation of amino acids. The first involves a cyanohydrin (reaction 2) and a related route (reaction 3) can be invoked to account for the presence of hydroxy acids. These particular reactions have been studied in considerable detail both kinetically and in terms of thermodynamic quantities.347 An alternative route (4) involves the hydrolysis of a-aminonitriles, which are themselves formed directly in anhydrous CH4/NH3 mixtures.344 Cyanoacetylene, formed in CH4/N2 irradiations,349 yields significant amounts of asparagine and aspartic acids (reaction 5). Finally, a number of workers336,350"354 have proposed that HCN oligomers, especially the trimer aminoacetonitrile and the tetramer diaminomaleonitrile, could have been important precursors for amino acid synthesis. Reaction mixtures involving such species have yielded up to 12 amino acids. Table 11 indicates the range of amino acids produced in these kinds of sparking syntheses. Of some interest is the fact that close parallels between these kinds of experiments and amino acid contents of carbonaceous chondrite meteorites exist.331,355,356... 

Figure 6.8 Experimental variation of the retention of 23 phenylthiohydantoin (PTH) derivatives of amino acids with mobile phase composition in RPLC. Mobile phase mixtures of acetonitrile and 0.05M aqueous sodium nitrate buffer (pH — 5.81). All mobile phases contain 3% THF. Stationary phase ODS silica. Solutes D = aspartic acid C-OH = cysteic acid E = glutamic acid N = asparagine S = serine T = threonine G = glycine H = histidine Q = glutamine R = arginine A = alanine METS = methionine sulphone ABA = a-aminobutyric acid Y = tyrosine P = proline V = valine M = methionine NV = norvaline I = isoleucine F = phenylalanine L = leucine W = tryptophan K = lysine. Figure taken from ref. [610]. Reprinted with permission.

As mentioned in Section 1.1, the first diazotization of amines, followed by dediazoniation, was carried out by Piria in 1848, well before Griess discovered and isolated aromatic diazo compounds (1858). Piria added an impure HNO3 —HCl solution to a mixture of asparagine and aspartic acid in water and obtained malic acid (7-1). It was not possible for Piria, however, to realize that the primary reaction products were diazonium ions. Yet, Piria s process was one of the few types of reaction via aliphatic diazonium ions that became important for synthetic purposes, after Ingold s group (Brewster et al., 1950) discovered that a-amino acids undergo clean retentive deamination (see Sect. 7.7). 

Amino acids mixtures stimulate aflatoxin biosynthesis, particularly asparagine and aspartic acid. Proline has been reported to stimulate conidial germination in a culture that was probably A. parasiticus, although reported as A. flavus (14). These investigators reported the greatest toxin formation with a mixture of amino acids followed by a mixture of proline plus glutamate or proline plus aspartate. The effect in the latter instances may have been due to the effect on conidial germination rather than a direct effect on aflatoxin biosynthesis. 

Fig. 2. The elution pattern of a standard mixture of OPA-derivatized primary amines, separated on a 5 (Jim Nucleosil C-18 column (200 X 4.6 mm id). The flow-rate was 1 mL/min employing the indicated gradient of metlianol and Na phosphate buffer (50 mA4, pH 5.25). Each peak represents 39 pmol except for those indicated below. 1, glutathione 2, cysteic acid 3, O-phosphoserine (19.5 pmol) 4, cysteine sulfinic acid 5, aspartic acid 6, asparagine (19.5 pmol) 7, glutamic acid 8, histidine 9, serine 10, glutamine 11, 3-methyl-histidine 12, a-aminoadipic acid (9.8 pmol) 13, citrulline (9.8 pmol) 14, carnosine 15, threonine,glycine 16, O-phosphoethanolamine 17, taurine (19.5 pmol) 18, p-alanine (19.5 pmol) 19, tyrosine 20, alanine 21, a-aminoisobutyric acid 22, aminoisobutyric acid 23, y-amino-ii-butyric acid 24, p-amino-u-butyric acid 25, a-amino-butyric acid 26, histamine 27, cystathione (19.5 pmol) 28, methionine 29, valine 30, phenylalanine 31, isoleucine 32, leucine 33, 5-hydroxytryptamine (5-H i ) 34, lysine. The chromatographic system consisted of a Varian LC 5000 chromatograph and a Schoeffel FS 970 fluorimeter.

Removal of the Fmoc group from the N-terminus of the resin-bound peptide chain is normally achieved by treating the peptidyl resin with 20-50% piperidine in DMF. The reaction is typically complete within 4-10 min, depending on the nature of the peptide being synthesized. With peptides containing aspartic acid and asparagine, inclusion of 0.1 M HOBt in the deprotection mixture has been found to be partially effective in suppressing aspartimide formation (18). 

With aspartate (Asp, D) in hand, the conversion to asparagine (Asn, N) has been shown to be straightforward. As expressed in US. Patent 5326908, when a slurry of aspartic acid (Asp, D) in methanol (CH3OH) and sulfuric acid (H2SO4) is prepared, the 4-carboxylate (that furthest from the amino group) is preferentially methylated and, without isolation, treatment of the reaction mixture with at least a fivefold excess of ammonia (NH3) produces asparagine (Asn, N). After the removal of excess ammonia, adjustment of the pH to the isoelectric point of the amino add (pi 5.4) yielded a crystalline material (Scheme 12.38). 

There are many examples of nitrilase-catalyzed reactions in which amides form a considerable amount of the reaction products, such as the transformations of acrylonitrile analogs and a-fluoroarylacetonitriles by nitrilase 1 from Arabidopsis thaliana [17], the conversion of p-cyano-L-alanine into a mixture of L-asparagine and L-aspartic acid by nitrilase 4 from the same organism [18] or the transformations of mandelonitrile by nitrilase from Pseudomonas jhiorescens [19] or some fungi [8], Moreover, formamide is the only product of the cyanide transformation by cyanide hydratase. Therefore, this enzyme was classified as a lyase (EC 4.2.1.66), although it is closely related to nitrilases, as far as its aa sequence and reaction mechanism are concerned [3]. 

Isolation, l-Aspartic acid is readily prepared from natural aspara ne in nearly the theoretical yield by Vickery and Pucher s (827) modification of Schiff s (680) method. A mixture containing 3.9 ml. of 3.5 N HCl per g. of natural asparagine monohydrate is refluxed for 3 hours, 3JS N NH4OH is added to bring the solution to pH 3, and 2 volumes of 95% ethanol are added. The resulting crystalline product is recrystallised from boiling water. 

The amino-acid composition usually is obtained by complete acid hydrolysis of the peptide into its component amino acids and analysis of the mixture by ion-exchange chromatography (Section 25-4C). This procedure is complicated by the fact that tryptophan is destroyed under acidic conditions. Also, asparagine and glutamine are converted to aspartic and glutamic acids, respectively. 

Figure 9.44 Chromatogram of assay of asparagine synthetase. Peaks 1, o-phthaldialdehyde aspartate 2, glutamate 3, asparagine 4, glutamine. A mixture of 0.025 mAf of each amino acid was made and 20 yu.L injected. (From Unnithan et al., 1984.)...

These expressions are applicable to drugs and drug-related ionizable substances, such as asparagine, aspartic add, caffeine, leucine, nalidixic acid, paracetamol, salicylic acid, and sulfanilamide. However, the log-linear expression (8.17) or the first two terms on the right-hand side of Equation 8.18 and even its modificafion in terms of the complete Equation 8.18 cannot model cases where the solubility curve exhibits a maximum as is observed in many cases, a problem solved when fluctuation theory is employed in the calculations as pointed out by Ruckenstein and Shulgin, [57]. If the solute is rather poorly soluble in the solvents and their mixtures, then solute-solute interactions can be ignored and the mutual interactions of the solvent components can then be treated either as ideal or as non-ideal. The molar volume of the binary solvent mixture is expressed as VJ +s) = where c is an empirical parameter that in... 

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