Racemization of the Amino Acid Substrate

Amino acid racemases have long been known to be important in bacterial metabolism, because several u-amino acids are required for the synthesis of cell wall mucopolysaccharides. u-Serine is found in relatively large amounts in mammalian brain, where it acts as an agonist of the N-methyl-n-aspartate (NMDA) glutamate receptor. Serine racemase has been purified from rat brain and cloned fromhuman brain (Wolosker et al., 1999 De Miranda et al., 2000). 

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Racemization of the Amino Acid Substrate Deprotonation of the a-carbon of the amino acid leads to tautomerization of the Schiff base to the quinonoid ketimine, as shown in Figure 9.2. The simplest reaction that the ketimine can undergo is reprotonation at the now symmetrical a-carbon. This is not a stereospecific process therefore, displacement of the substrate by the reactive lysine residue results in the racemic mixture of d- and L-amino acid. 

Pyridoxal 5 -phosphate dependent enzymes constitute an important class of proteins involved predominately in amino acid metabolism. The PLP-cofactor is capable of catalyzing a variety of reactions at the a-, [3-, and/or y-carbons of amino acid substrates. These reactions include tranamination, racemization, decarboxylation, and aldoyltic cleavage reactions at the a-carbon and elimina-tion/substitution reactions at either the 3-, or y-position of the amino acid substrate (67-74) The chemical properties of the cofactor (67-71) are responsible for the great diversity of reactions catalyzed by PLP, while reaction specificity is ultimately determined by the active site environment imposed by the surrounding apo-protein to which the cofactor is covalendy bound (69). 

SHMT also catalyzes racemization of alanine and transamination of both its enantiomers. The particular reaction catalyzed by SHMT is mainly determined by the structure of the amino acid substrate. In the case of serine or glycine, the true substrates, SHMT does not catalyze any of the alternate reactions. The currently accepted model attributes this reaction specificity to the existence of open and closed active-site conformations. The physiological substrates generate the closed conformation, whereas alternate substrates react while the enzyme remains in the open conformation, which permits reaction paths leading to decarboxylation, transamination, and racemization. ... 

In each of these transformations, one of the bonds to the cr-carbon of the amino acid substrate is broken in the first step of the reaction. Decarboxylation breaks the bond joining the carboxyl group to the cr-carbon transamination, racemization, and o , 8-elimination break the bond joining the hydrogen to the cr-carbon and —Cp bond cleavage breaks the bond joining the R group to the a-carbon. 

The reaction mechanism for glutamate racemase has been studied extensively. It has been proposed that the key for the racemization activity is that the two cysteine residues of the enzyme are located on both sides of the substrate bound to the active site. Thus, one cysteine residue abstracts the a-proton from the substrate, while the other detivers a proton from the opposite side of the intermediate enolate of the amino acid. In this way, the racemase catalyzes the racemization of glutamic acid via a so-called two-base mechanism (Fig. 15). 

Many of the amino acids originally tested by Krebs were racemic mixtures. When naturally occurring L-amino acids became available the oxidase was found to be sterically restricted to the unnatural, D series. [D-serine occurs in worms free and as D-phosphoryl lombricine (Ennor, 1959)]. It could not therefore be the enzyme used in the liver to release NH3 in amino acid metabolism. D-amino acid oxidase was shown by Warburg and Christian (1938) to be a flavoprotein with FAD as its prosthetic group. A few years later Green found an L-amino acid oxidase in liver. It was however limited in its specificity for amino acid substrates and not very active—characteristics which again precluded its central role in deamination. 

An elegant four-enzyme cascade process was described by Nakajima et al. [28] for the deracemization of an a-amino acid (Scheme 6.13). It involved amine oxidase-catalyzed, (i )-selective oxidation of the amino acid to afford the ammonium salt of the a-keto acid and the unreacted (S)-enantiomer of the substrate. The keto acid then undergoes reductive amination, catalyzed by leucine dehydrogenase, to afford the (S)-amino acid. NADH cofactor regeneration is achieved with formate/FDH. The overall process affords the (S)-enantiomer in 95% yield and 99% e.e. from racemic starting material, formate and molecular oxygen, and the help of three enzymes in concert. A fourth enzyme, catalase, is added to decompose the hydrogen peroxide formed in the first step which otherwise would have a detrimental effect on the enzymes. 

In a different approach, fluorescence-based DNA microarrays are utilized (88). In a model study, chiral amino acids were used. Mixtures of a racemic amino acid are first subjected to acylation at the amino function with formation of A-Boc protected derivatives. The samples are then covalently attached to amine-functionalized glass slides in a spatially arrayed manner (Fig. 10). In a second step, the uncoupled surface amino functions are acylated exhaustively. The third step involves complete deprotection to afford the free amino function of the amino acid. Finally, in a fourth step, two pseudo-Qn nX. om.Qx c fluorescent probes are attached to the free amino groups on the surface of the array. An appreciable degree of kinetic resolution in the process of amide coupling is a requirement for the success of the ee assay (Horeau s principle). In the present case, the ee values are accessible by measuring the ratio of the relevant fluorescent intensities. About 8000 ee determinations are possible per day, precision amounting to +10% of the actual value ((S(S). Although it was not explicitly demonstrated that this ee assay can be used to evaluate enzymes (e.g., proteases), this should in fact be possible. So far this approach has not been extended to other types of substrates. 

In most cases of formation of peptides containing d-amino acid, the L form of the amino acid is the substrate for the incorporating enzyme. In contrast, the free D-amino acid is ordinarily a poor substrate for the incorporation reaction. Whether the racemization occurs on the enzyme or afterwards remains to be determined in most cases. In the case of the D-valyl residue formed in penicillin, a tripeptide derivative containing L-valine is an intermediate, and the conversion is thought to occur by way of an a,/3-dehydro form of the valyl residue. 

A modification of this reaction concerns the availability of the keto acid substrate. To circumvent its complicated lengthy chemical synthesis, 2-keto-6-hydro-xyhexanoic acid was synthesized by treatment of racemic 6-hydroxynorleucine with D-amino acid oxidase and catalase (Fig. 37). The production of racemic 6-hydroxynorleucine occurs by hydrolysis from 5-(4-hydroxybutyl)hydantoin. d-Amino acid oxidase converts the D-enantiomer of racemic 6-hydroxynorleucine to the corresponding ketoacid which is reductively aminated to l 6-hydroxynorleucine by GluDH. 

It is evident that natural proteins are not a primary source of large amounts of amino acids, despite the fact that many of the acids are commercially significant chemicals and a few are commodity chemicals. The technical difficulties just alluded to include undesirable distributions of the amino acids in natural proteins, the sensitivity of proteins and amino acids to chemical hydrolysis conditions, racemization, the multiplicity of the product acids and the often low concentration of the desired acid or acids in the hydrolysate, and the consequent separation problems. Microbial synthesis of specific amino acids from biomass substrates or biomass-derived intermediates often has substantial advantages over thermochemical processing methods and is used for the commercial production of several of the amino acids. This is discussed in more detail in the next section. 

An amino acid racemase which shows very broad substrate specificity was discovered in Pseudomonas striata (= Ps. putida), purified, and characterized1 91. The enzyme catalyzes racemization of various amino acids except aromatic and acidic... 

L-Phe-OCH3 is the by-product formation of p-aspartame. This isomer is of bitter taste and has to be completely removed from the a-isomer. The advantages of the enzymatic route are (i) No P-isomer is produced, (ii) the enzyme is completely stereoselective, so that racemic mixtures of the substrate or the appropiate enantiomer of the amino acid can be used, (iii) no racemization occurs during synthesis and (iv) the reaction takes place in aqueous media under mild conditions. 

The most versatile of the coenzymes is perhaps pyridoxal phosphate (PEP). The PEP containing enzymes catalyze a wide variety of reactions such as racemization, transamination, [3- and a-decarboxylation, and interconversion of side chains. The first step of all these reactions is the transition between an internal aldimine intermediate to an external aldimine intermediate, which involves the condensation of PEP with an external amino acid substrate to form a Schiff base. The internal aldimine intermediate can then either undergo a-decarboxylation to convert the amino acid substrate into amines and aldehydes, or lose the a-hydrogen... 

The electrophilic activation of terminal alkynes by arene-ruthenium(II) catalysts has provided selective access to enol esters. Enol esters are much more reactive than alkyl esters and have been used in a variety of reactions. In the past decade, Dixneuf and co-workers have developed selective approaches to the Markovnikov and antz-Markovnikov addition of carboxylic acids across alkynes by employing different arene-ruthenium(II) catalysts [48,53,54]. Of special interest is the synthesis of AT-Boc-protected 1-alanine isopropenyl ester 110 from N-Boc-l-alanine 108 and propyne 109 mediated by (Ty -cymene)RuCl2(PPh3) complex 107 (Scheme 30) [53]. Addition of the amino acid 108 to the propyne 109 proceeded exclusively in the Markovnikov sense and without accompanying racemization of the substrate. 

Like homogeneous catalysis, the removal of a-hydrogen of the amino acid fragment by OH ions, the local concentration of which is apparently high in the polymer phase, is probably the rate-determining step of heterogeneous racemization. Under similar conditions, the rate of a-amino acid racemization decreases in the sequence Ala = Ser>Phe>Nva>Lys>Val, and correlates with the rate of substrate racemization in the presence of Schiff bases and transamination of amino acids by pyridoxal phosphate. 

There are considerable differences in the hydrolysis rates of different amino acids. If the rate is too low for practical purposes, then the chloroacetyl derivatives of the racemates can be applied as substrates instead of the acetyl derivatives. Of course, it is often worthwhile to recover the unchanged D-acylamino acid and hydrolyze it with aqueous acid to produce the D-enanthiomer of the amino acid. The unnatural D isomers are frequently used as building components in studies of structure-activity relationships, in the preparation of hormone analogs resistant to the action of proteolytic enzymes and in the synthesis of microbial peptides. 

Heteroatoms like sulfur, nitrogen, and oxygen are accepted in alkyl or (hetero)aryl substituents as well as alkenyl and alkynyl substituents (Scheme 4) [21,22]. Only for methionine amide and homomethionine amide is a slightly lower stereoselectivity observed, probably caused by (enzymatic or chemical) racemization of the L-acid under the basic reaction conditions. Cyclic amino acid amides such as proline amide and piperidine-2-carboxyamide can also be resolved. For these substrates product inhibition is observed, which leads to a decrease in enzyme activity with advancing conversion. 

We prepared the (5)-enantiomer of cericlamine via enzymatic resolution of a-methyl-3,4-dichlorophenylalanine amide (28) and subsequent reduction of the amino acid as described in Scheme 11 [61]. Racemic a-methyl-3,4-dichlorophenylalanine amide (28) is prepared by phase transfer-catalyzed benzylation of the benzaldehyde Schiff base of alanine amide (27) followed by acidic workup. Since this substrate is nearly insoluble in water, the enzymatic resolution using the amidase from O. anthropi NCIMB 40321 is performed at pH 5.3. At this pH there is a sufficient amount of the substrate in solution and still approximately 50% of the amidase activity left to allow the enzymatic hydrolysis... 

Resolution of Racemic Amines and Amino Acids. Acylases (EC3.5.1.14) are the most commonly used enzymes for the resolution of amino acids. Porcine kidney acylase (PKA) and the fungaly3.spet i//us acylase (AA) are commercially available, inexpensive, and stable. They have broad substrate specificity and hydrolyze a wide spectmm of natural and unnatural A/-acyl amino acids, with exceptionally high enantioselectivity in almost all cases. Moreover, theU enantioselectivity is exceptionally good with most substrates. A general paper on this subject has been pubUshed (106) in which the resolution of over 50 A/-acyl amino acids and analogues is described. Also reported are the stabiUties of the enzymes and the effect of different acyl groups on the rate and selectivity of enzymatic hydrolysis. Some of the substrates that are easily resolved on 10—100 g scale are presented in Figure 4 (106). Lipases are also used for the resolution of A/-acylated amino acids but the rates and optical purities are usually low (107). 

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. 

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