Amino acids, preparation racemization

Table 2 shows a list of collagen model peptides which have teen prepared. Many efforts have been made to prevent racemization. The polycondensation reaction seemed to be more sensitive to racemization than the coupling steps preparing the monomeric tripeptide. Therefore, the sequence of the monomer was selected with Gly or Pro at the C-terminal chain end, because racemization is mostly favored at the carboxy-activated amino acid, and these amino acids cannot racemize. 

Oshikawa, T. and Yamashita, M., Preparation of optically active (S)-2-ami-noalkylphosphonic acids from (S)-amino acids without racemization, Bull. Chem. Soc. Jpn., 63, 2728, 1990. 

The amino acids prepared by these methods are formed as racemic mixtures and are optically inactive. 

Preparative Methods substituted 2,3-methanoamino acids are difficult to prepare. Unfortunately, most of the reported syntheses give racemic materials whereas stereochemically pure compounds are required for studies of cyclopropane-based peptidomimetics. The only 2,3-methanologs of protein amino acids prepared in optically active form are ( )- and (Z)-cyclo-Phe and -Tyr, all four stereoisomers of cyc/o-Met, (Z)-cyclo-Arg and (25,35)-(Z)-cyc/o-Trp, although several routes to enantio-enriched 2,3-methanologs of simple nonproteogenic amino acids have been reported. " The most practical synthesis of the title compound is that based on a diastereoselective, rhodium-catalyzed cyclopropanation reaction. ... 

Peptide synthesis. Vinyl esters of amino acids, prepared by transesterification with vinyl acetate, have been used as activated esters in peptide synthesis. The coupling reaction is best carried out in ethyl cyanoacetate, for this solvent suppresses the formation of colored products derived from liberated acetaldehyde. Racemization appears to be slight. 

Unless a resolution step is included, the a-amino acids prepared by the synthetic methods just described are racemic. Optically active amino acids, when desired, may be obtained by resolving a racemic mixture or by enantioselective synthesis. A synthesis is described as enantioselective if it produces one enantiomer of a chiral compound in an amount greater than its mirror image. Recall from Section 7.9 that optically inactive reactants cannot give optically active products. Enantioselective syntheses of amino acids therefore require an enantiomerically enriched chiral reagent or catalyst at some point in... 

LEM systems have also been shown to be successful in separating commodity-type biochemicals such as propionic acid (10) and acetic acid (10,22) and have been used for the preparation of L-amino acids from racemic D,L mixtures by means of enzymatic hydrolysis of amino acid esters (23). In addition to biochemical separations, the work of Mohan and Li showed that enzymes could be encapsulated in liquid emulsion membranes with no deleterious effect on enzyme action (24). Later work by these authors indicated that encapsulated live cells could remain viable and function in the LEM interior phase for period as long as five days (25). 

The a-amino acids prepared by the synthetic methods just described are racemic unless a resolution step is included, enantiomerically enriched reactants are used, or the reaction is modified so as to become enantioselective. Considerable progress has been made in the last of these methods, allowing chemists to prepare not only L-amino acids, but also their much rarer D-enantiomers. We have already seen one example of this approach in the synthesis of the anti-parkinsonism drug L-dopa by enantioselective hydrogenation (see Section 14.14). A variation of the Strecker synthesis using a chiral catalyst has recently been developed that gives a-amino acids with greater than 99% enantioselectivity. 

Several methods are used for the synthesis of amino acids. A mqjor problem with any synthesis is preparing enantiopure products. Most of the syntheses shown here give racemic amino acids, but methods are known that produce amino acids highly enriched in one enantiomer (see Chapter 9). Enantioselective synthetic methods will not be discussed. A method used quite often to obtain an enantiopure amino acid prepares the racemic compound, followed by isolation of the 1-amino acid by resolution, as described in Chapter 9, Section 9.8. 

As pointed out previously, all of the amino acids prepared in this section are racemic. To obtain an enantiopure amino acid requires separation of the enantiomers via resolution. As discussed in Chapter 9 (Section 9.2), the physical properties of enantiomers are identical except for specific rotation. Because separation methods rely on differences in physical properties, this is a problem. It is overcome if the racemic amino acid mixture reacts with a reagent that has a stereogenic center. The resulting product will be a mixture of diastereomers, which have different physical properties and may be separated. 

All the methods of the preceding section produce amino acids in racemic form. However, we noted that most of the amino acids in natural polypeptides have the S configuration. Thus, many synthetic procedures—in particular, peptide and protein syntheses— require enantiomerically pure compounds. To meet this requiranent, either the racemic amino adds must be resolved (Section 5-8) or a single enantiomer must be prepared by enantioselective reactions. 

DL-[2,3- H]Ornithine (XI) was prepared from ethyl 2-aceta-mido-4-cyanobutyrate by tritiation and then reduction with tritium, deprotection, and back-exchange of labile tritium. The H-decoupled % NMR spectrum shows four main signals, two of which each comprise a pair of lines (see Figure 15). This is not an indication of double labeling because the spacings are unequal and the intensities are incorrect for coupled doublets. The line at 6 4.04 is from the 2-position and that at 6 3.03 is from the 5 position. The lines at 6 2.02 and 1.96 evidently arise from the diastereotopic tritons in the 3-position (the two 3-sites are nonequivalent by virtue of the 2-chirality that the amino acid is racemic is irrelevant). The pair of lines at 6 1.85 and 1.77 analogously arise from the 4-sites. There is a weak line at... 

Stereospecific nitrilases were used for the conversion of a-arninonitiiles to optically active L-amino acids (Fig. 4). In an early investigation, L-alanine was formed by an l-specific nitrilase from alginate-immobilized cells of Acinetobacter sp. APN [25]. A decrease of the enantioselectivity with the time was supposed to be caused by a racemase forming d- from L-alanine. The stereoinversion of racemic a-aminopropionitrile led to a conversion yield above 50%. Similar L-a-amino acid preparations showed no stereoinversion and additionally accumulated the D-amide due to the presence of a nitrile hydratase/ amidase system [26,27]. Additionally, a number of L-a-amino acids were synthesized by a 45-kDa monomeric nitrilase from R. rhodochrous PA-34 [28]. Remarkable in this case was the preferential hydrolysis of a-aminopropionitrile to D-alanine in contrast to the l-alanine formation by the Acinetobacter nitrilase (Fig. 4). 

Brevibacterium sp. R312, which was enriched on acetonitrile as sole nitrogen-source, contains two amidases a nonstereoselective, wide-spectrum acylamide amidohydrolase [73] and an enantioselective a-aminoamidase. In early reports of stereoselective nitrile bioconversions, the a-aminoamidase has been used to prepare optically active L-a-amino acids from racemic a-aminonitriles [26] and a-amino amides [74]. Around 50% L-a-amino... 

Enzymatic hydrolysis is also used for the preparation of L-amino acids. Racemic D- and L-amino acids and their acyl-derivatives obtained chemically can be resolved enzymatically to yield their natural L-forms. Aminoacylases such as that from Pispergillus OTj e specifically hydrolyze L-enantiomers of acyl-DL-amino acids. The resulting L-amino acid can be separated readily from the unchanged acyl-D form which is racemized and subjected to further hydrolysis. Several L-amino acids, eg, methionine [63-68-3], phenylalanine [63-91-2], tryptophan [73-22-3], and valine [72-18-4] have been manufactured by this process in Japan and production costs have been reduced by 40% through the appHcation of immobilized cell technology (75). Cyclohexane chloride, which is a by-product in nylon manufacture, is chemically converted to DL-amino-S-caprolactam [105-60-2] (23) which is resolved and/or racemized to (24)... 

A -Nitroso derivatives, prepared from secondary amines and nitrous acid, are cleaved by reduction (H2/Raney Ni, EtOH, 28°, 3.5 h CuCl/concd. HCl"). Since many V-nitroso compounds are carcinogens, and because some racemization and cyclodehydration of V-nitroso derivatives of V-alkyl amino acids occur during peptide syntheses, V-nitroso derivatives are of limited value as protective groups. 

Since the proline residue in peptides facilitates the cyclization, 3 sublibraries each containing 324 compounds were prepared with proline in each randomized position. Resolutions of 1.05 and 2.06 were observed for the CE separation of racemic DNP-glutamic acid using peptides with proline located on the first and second random position, while the peptide mixture with proline preceding the (i-alamine residue did not exhibit any enantioselectivity. Since the c(Arg-Lys-0-Pro-0-(i-Ala) library afforded the best separation, the next deconvolution was aimed at defining the best amino acid at position 3. A rigorous deconvolution process would have required the preparation of 18 libraries with each amino acid residue at this position. 

However, the use of a HPLC separation step enabled a remarkable acceleration of the deconvolution process. Instead of preparing all of the sublibraries, the c(Arg-Lys-O-Pro-O-P-Ala) library was fractionated on a semipreparative HPLC column and three fractions as shown in Fig. 3-2 were collected and subjected to amino acid analysis. According to the analysis, the least hydrophobic fraction, which eluted first, did not contain peptides that included valine, methionine, isoleucine, leucine, tyrosine, and phenylalanine residues and also did not exhibit any separation ability for the tested racemic amino acid derivatives (Table 3-1). 

First, they compared CSPs 1 and 3 prepared by the two-step solid-phase methodology with their commercially available counterparts (CSPs 2 and 4) obtained by direct reaction of the preformed selector with a silica support. Although no exact data characterizing the surface coverage density for these phases were reported, all of the CSPs separated all four racemates tested equally. These results shown in Table 3-3 subsequently led to the preparation of a series of dipeptide and tripeptide CSPs 5-10 using a similar synthetic approach. Although the majority of these phases exhibited selectivities lower or similar to those of selectors built around a single amino acid (Table 3-3), this study demonstrated that the solid-phase synthesis was a... 

Amino acid separations represent another specific application of the technology. Amino acids are important synthesis precursors - in particular for pharmaceuticals -such as, for example, D-phenylglycine or D-parahydroxyphenylglycine in the preparation of semisynthetic penicillins. They are also used for other chiral fine chemicals and for incorporation into modified biologically active peptides. Since the unnatural amino acids cannot be obtained by fermentation or from natural sources, they must be prepared by conventional synthesis followed by racemate resolution, by asymmetric synthesis, or by biotransformation of chiral or prochiral precursors. Thus, amino acids represent an important class of compounds that can benefit from more efficient separations technology. 

Two methods are used in practice to obtain enantiomerically pure amino acids. One way is to resolve the racemic mixture into its pure enantiomers (Section 9.8). A more direct approach, however, is to use an enantioselective synthesis to prepare only the desired 5 enantiomer directly. As discussed in the Chapter 19 Focus Oil, the idea behind enantioselective synthesis is to find a chiral reaction catalyst that will temporarily hold a substrate molecule in an unsymmetrical environment. While in that chiral environment, the substrate may be more... 

Another approach for the synthesis of enantiopure amino acids or amino alcohols is the enantioselective enzyme-catalyzed hydrolysis of hydantoins. As discussed above, hydantoins are very easily racemized in weak alkaline solutions via keto enol tautomerism. Sugai et al. have reported the DKR of the hydantoin prepared from DL-phenylalanine. DKR took place smoothly by the use of D-hydantoinase at a pH of 9 employing a borate buffer (Figure 4.17) [42]. 

For successful DKR two reactions an in situ racemization (krac) and kinetic resolution [k(R) k(S)] must be carefully chosen. The detailed description of all parameters can be found in the literature [26], but in all cases, the racemization reaction must be much faster than the kinetic resolution. It is also important to note that both reactions must proceed under identical conditions. This methodology is highly attractive because the enantiomeric excess of the product is often higher than in the original kinetic resolution. Moreover, the work-up of the reaction is simpler since in an ideal case only the desired enantiomeric product is present in the reaction mixture. This concept is used for preparation of many important classes of organic compounds like natural and nonnatural a-amino acids, a-substituted nitriles and esters, cyanohydrins, 5-alkyl hydantoins, and thiazoUn-5-ones. 

The Gly74Cys mutant was prepared via PCR using the plasmid that contains the gene-coding native AMDase. Although the change in amino acid is drastic, the mutant still exhibited some activity. As expected, the products were nearly racemic, if not entirely, in the case of the two substrates mentioned above. These results demonstrate that this position is effective to give a proton to the intermediate of the reaction. 

Another -activation of amino acids for peptide synthesis is achieved by preparing sulfenamides from sulfenylimidazoles. A sulfenylimidazole is formed in situ from the sulfenyl chloride (prepared from the disulfide and chlorine) and imidazole, which reacts further with an amino acid ester to give a sulfenamide in high yield. Conversion of such sulfenamides with IV-acyl amino acids by means of triphenylphosphine affords dipeptides with racemization of less than 0.5%.[481... 

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