Pyridoxal amino acid Schiff bases

A series of pyridoxal amino acid Schiff base complexes have been prepared in which Al is trigonally coordinated by N and two O atoms.415 These provide a model for the intermediate in the pyridoxal-catalyzed reactions of amino acids. Some Schiff base complexes produced in reactions of the bases with Al(OPri)3 have been assigned a structure with five-coordinate aluminum.416417... 

In the transamination reaction (shown in reaction scheme LVIII), a pyridoxal amino acid Schiff base chelate is first formed, and a shift of the hydrogen atom in the a-carbon takes place to give a tautomeric Schiff base, which finally undergoes hydrolytic cleavage. The result is a transamination reaction in which the amino acid is converted to a keto acid and the pyridoxal to pyridoxamine. In this type of reaction, the metal ion serves to maintain the planarity of the Schiff base chelate and exerts an electron-withdrawing action in the same direction as the heterocyclic ring (149). 

A modification of the pyridoxal—amino acid reaction (mentioned above) has been made for automatic analysis of amino acids by ligand-exchange chromatography [95]. This technique involves separation of the amino acids prior to fluorimetric reaction and determination. As the amino acids are eluted from the column, they are mixed with the pyridoxal-zinc(II) reagent to produce a highly fluorescent zinc chelate. Amounts of as low as 1 nmole of amino acid may be detected. The first reaction involved is the formation of the pyridoxyl-amino acid (Schiff base) as in Fig.4.46. The zinc then forms a chelate which probably has the structure shown in Fig. 4.48. 

The reaction mechanism consists of formation of a Schiff base by pyridoxal phosphate with a reactive amino group of the enzyme entry of glycine and formation of an enzyme-pyridoxal phosphate-glycine-Schiff base complex loss of a proton from the a carbon of glycine with the generation of a carbanion condensation of the carbanion with succinyl-CoA to yield an enzyme-bound intermediate (a-amino-yS-ketoadipic acid) decarboxylation of this intermediate to ALA and liberation of the bound ALA by hydrolysis. ALA synthesis does not occur in mature erythrocytes. 

In work aimed at elucidating the mode of pyridoxal mediated dephosphonylation of a-aminophosphonic acids, it was found that simple aminophosphonates reacted with pyridoxal to form Schiff bases, which complexed copper(II) ions, but did not react further. In contrast, o-hydroxyphenylphosphaglycine did react with pyridoxal at 40 °C with the formation of pyridoxamine, along with o-hydroxybenzoylphosphonic acid on the one hand (equation 45), and salicylaldehyde and H3PO4 (not shown) on the other. Apparently, the presence of the -hydroxy group is necessary for the success of the reaction, presumably by complexing the copper ion in the fashion indicated. The formation of 6>-hydroxybenzoylphosphonic acid illustrates the capability of a-aminophosphonic acids to participate in transamination (similarly to amino acids), while salicylaldehyde is the result of dephosphonylation (analogous to decarboxylation). 

Fig. 4-29. The role of Schiff base in nonenzymic transamination. 1 pyridoxal + amino acid in presence of catalyst (metal or enzyme) yields a Schiff base. 2 Hydrogen and double-bond shifts lead to the formation of an isomeric Schiff base. 2 The base splits and leads to the formation of the keto acid and pyridoxamine. 4 The reaction is reversible. In the reversible step, the amino group of pyridoxamine is transferred to the keto acceptor...

At pH values where the enzyme is bound to pyridoxal as a SchifF base (40), reaction with sodium borohydride destroys the activity of the enzyme by reducing the SchifF base to a pyridoxylamine (42). This observation gives strength to the hypothesis that the subsequent reaction of enzyme with an amino acid involves transamination. Skeletal muscle phosphorylase is an exception , indicating that this particular enzyme does not require a SchifF base linkage for its activity. Further research is required to determine whether the role of pyridoxal phosphate in this enzyme can be attributed simply to maintenance of the active site of the enzyme in the required conformation or whether a new set of catalytic properties must be ascribed to the vitamin Bg aldehyde. 

Amino acids react with pyridoxal to form Schiff bases. The luminescence of the Eu chelates of the latter have been studied by Zolin et al. (1975), who suggest this approach as a method for identification of the residue at the N-terminus of peptides and proteins. 

FIGURE 18.27 Pyridoxal-5-phosphate forms stable Schiff base adducts with amino acids and acts as an effective electron sink to stabilize a variety of reaction intermediates. 

Pyridoxal phosphate mainly serves as coenzyme in the amino acid metabolism and is covalently bound to its enzyme via a Schiff base. In the enzymatic reaction, the amino group of the substrate and the aldehyde group of PLP form a Schiff base, too. The subsequent reactions can take place at the a-, (3-, or y-carbon of the respective substrate. Common types of reactions are decarboxylations (formation of biogenic amines), transaminations (transfer of the amino nitrogen of one amino acid to the keto analog of another amino acid), and eliminations. 

In 1983, Yamada et al. developed an efficient method for the racemization of amino acids using a catalytic amount of an aliphatic or an aromatic aldehyde [50]. This method has been used in the D KR of amino acids. Figure 4.25 shows the mechanism of the racemization of a carboxylic acid derivative catalyzed by pyridoxal. Racemization takes place through the formation of Schiff-base intermediates. 

Another interesting example is SHMT. This enzyme catalyzes decarboxylation of a-amino-a-methylmalonate with the aid of pyridoxal-5 -phosphate (PLP). This is an unique enzyme in that it promotes various types of reactions of a-amino acids. It promotes aldol/retro-aldol type reactions and transamination reaction in addition to decarboxylation reaction. Although the types of apparent reactions are different, the common point of these reactions is the formation of a complex with PLP. In addition, the initial step of each reaction is the decomposition of the Schiff base formed between the substrate and pyridoxal coenzyme (Fig. 7-3). 

The role of Schiff bases formed between pyridoxal phosphate and amino acid residues as intermediate products in many enzymatic reactions is well known and documented. NMR is an excellent tool for studies of the enzymatic processes involving Schiff bases formation. 

Penicillamine reacts with pyridoxal-5-phosphate to form a thiazolidine derivative, and is able to displace many amino acids from their Schiff base complexes, forming stable compounds of this type. The reactivity of the thiol group of penicillamine is less than that of cysteine, probably because of steric hindrance by the adjacent methyl groups of penicillamine, which in consequence is less rapidly oxidized in vivo [7]. 

Identification of pyridoxal phosphate as coenzyme suggested the aldehyde group on pyridoxine might form an intermediate Schiff s base with the donor amino acid. Pyridoxamine phosphate thus formed would in turn donate its NH2 group to the accepting a-ketonic acid, a scheme proposed by Schlenk and Fisher. 15N-labeling experiments and, later, the detection of the Schiff s base by its absorption in UV, confirmed the overall mechanism. Free pyridoxamine phosphate however does not participate in the reaction as originally proposed. Pyridoxal phosphate is invariably the coenzyme form of pyridoxine. 

The Schiff base can undergo a variety of reactions in addition to transamination, shown in Fig. 6.4 for example, racemization of the amino acid via the a-deprotonated intermediate. Many of these reactions are catalyzed by metal ions and each has its equivalent nonmetallic enzyme reaction, each enzyme containing pyridoxal phosphate as a coenzyme. Many ideas of the mechanism of the action of these enzymes are based on the behavior of the model metal complexes. 

Mechanistically, transamination by the free coenzyme proceeds through a number of discrete steps as illustrated in Fig. 3. The first step of the process, aldi-mine formation (Fig. 3, Step I), is common to all pyridoxal-dependent reactions. The rate and extent of this reaction are influenced by factors including reactant concentrations, the nature of the amino acid, pH, and solvent. However, it is important to realize that the coenzyme itself facilitates Schiff base formation in... 

Pyridoxal phosphate (4) is the most important coenzyme in amino acid metabolism. Its role in transamination reactions is discussed in detail on p. 178. Pyridoxal phosphate is also involved in other reactions involving amino acids, such as decarboxylations and dehydrations. The aldehyde form of pyridoxal phosphate shown here (left) is not generally found in free form. In the absence of substrates, the aldehyde group is covalently bound to the e-amino group of a lysine residue as aldimine ( Schiffs base ). Pyridoxamine phosphate (right) is an intermediate of transamination reactions. It reverts to the aldehyde form by reacting with 2-oxoacids (see p. 178). 

In the absence of substrates, the aldehyde group of pyridoxal phosphate is covalently bound to a lysine residue of the transaminase (1). This type of compound is known as an aldimine or Schiffs base. During the reaction, amino acid 1 (A, la) displaces the lysine residue, and a new aldimine is formed (2). The double bond is then shifted by isomerization. 

In a number of nonenzymatic reactions catalyzed by pyridoxal, a metal ion complex is formed—a combination of a multivalent metal ion such as cupric oi aluminum ion with the Schiff base formed from the combination of an amino acid and pyridoxal (I). The electrostatic effect of the metal ion, as well as the electron sink of the pyridinium ion, facilitates the removal of an a -hydrogen atom to form the tautomeric Schiff base, II. Schiff base II is capable of a number of reactions characteristic of pyridoxal systems. Since the former asymmetric center of the amino acid has lost its asymmetry, donation of a proton to that center followed by hydrolytic cleavage of the system will result in racemic amino acid. On the other hand, donation of a proton to the benzylic carbon atom followed by hydrolytic cleavage of the system will result in a transamination reaction—that is, the amino acid will be converted to a keto acid and pyridoxal will be converted to pyridoxamine. Decarboxylation of the original amino acid can occur instead of the initial loss of a proton. In either case, a pair of electrons must be absorbed by the pyridoxal system, and in each case, the electrostatic effect of the metal ion facilitates this electron movement, as well as the subsequent hydrolytic cleavage (40, 43). 

Several nickel(II) complexes have been reported with Schiff bases derived from the condensation of salicylaldehyde and various amino acids. The structures of the complexes were investigated by means of electronic and NMR spectra as well as X-ray crystallography.2336-2341 Recently the X-ray structure of complex (321), prepared by the reaction of pyridoxal-HCl, o-phospho-DL-threonine and N NOsVfiHaO at pH 5, has been reported.2341... 

The principles of the above reactions form the basis of a series of important metabolic interconversions involving the coenzyme pyridoxal phosphate (structure 2.41). This condenses with amino acids to form a Schiff base (structure 2.42). The pyridine ring in the Schiff base acts as an electron sink which very effectively stabilizes a negative charge. 

As discussed in Chapter 2, section C2, pyridoxal phosphate condenses with amino acids to form a Schiff base (structure 8.44). Each of the three groups around the chiral carbon at the top of structure 8.44 may be cleaved to give an anion that is stabilized by delocalization of the electrons over the 7r orbitals. 

NMR studies have been carried out on Schiff bases derived from pyridoxal phosphate and amino acids, since they have been proposed as intermediates in many important biological reactions such as transamination, decarboxylation, etc.90 The pK.d values of a series of Schiff bases derived from pyridoxal phosphate and a-amino adds, most of which are fluorinated (Figure 11), have been derived from H and19F titration curves.91 The imine N atom was found to be more basic and more sensitive to the electron-withdrawing effect of fluorine than the pyridine N atom. Pyridoxal and its phosphate derivative are shown in Figure 12a. The Schiff base formation by condensation of both with octopamine (Figure 12b) in water or methanol solution was studied by 13C NMR. The enolimine form is favoured in methanol, while the ketoamine form predominates in water.92... 

Figure It Schiff base derived from pyridoxal phosphate and a-amino acids...

It is well over 40 years since Pfeiffer discovered that certain reactions of a-amino acid esters, in particular, ester exchange, racemization and oxygenation, are effected very readily when their Schiff bases with salicylaldehyde are complexed to a transition metal ion (most notably Cu11). The Schiff bases result from a condensation reaction between a reactive carbonyl group and the amino group of the amino acids. Snell and his co-workers43 were also one of the first to point out that similar reactions also occurred if pyridoxal was used instead of salicylaldehyde, and that there is a close analogy with pyridoxal phosphate-promoted enzymic reactions of a-amino acid metabolism. Since then much work has been due on these and other similar systems and their reactivities. 

Structures of catalytic intermediates in pyridoxal-phosphate-dependent reactions. The initial aldimine intermediate resulting from Schiff s base formation between the coenzyme and the a-amino group of an amino acid (a). This aldimine is converted to the resonance-stabilized... 

Schiff base formation can have a considerable effect on both the position and degree of activation of the coordinated amino acid. The Schiff bases derived from amino acids and pyridoxal have attracted considerable attention due to the biochemical significance of vitamin B6 and the realization that many of the enzymic reactions involving B6 could be brought about in the absence of enzyme by using pyridoxal and various metal ions.444,445,461 4 2,342... 

Amino acids undergo a condensation reaction with pyridoxal in alkaline medium to form a Schiff base which can be converted into stable pyridoxyl-amino acids by catalytic reduction or by reduction with sodium tetrahydroborate. The reactions involved are illustrated in Fig. 4.46. The resulting derivatives can be detected in quantities as low as 5-10"10 moles by fluorescence at 332 nm (excitation) and 400 nm (emission). Column chromatography may be used to separate die pyridoxyl-amino acid derivatives [93,94]. 

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