Amino acid Schiff base formation

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 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. 

Formaldehyde fixes proteins in tissue by reacting with basic amino acids— such as lysine,5 7—to form methylol adducts. These adducts can form crosslinks through Schiff base formation. Both intra- and intermolecular cross-links are formed,8 which may destroy enzymatic activity and often immunoreactiv-ity. These formaldehyde-induced modifications reduce protein extraction efficiency and may also lead to the misidentification of proteins during proteomic analysis. 

Electrophilic site for Schiff base formation with reactive amines such as a-amino acids. Corresponding transaminated species, pyridoxamine, reacts with electrophilic carbonyl compounds... 

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... 

In the case of non-protein amino acid-derived alkaloids, the second obligatory intermedia is derived from the obligatory intermedia enzymatically and by the Schiff base formation as, for example, in the hygrine pathway. The second obligatory intermedia is, in this case, the A-methyl-A pyrrolinium cation. 

Figure 5.10 Agar overlay screening procedure to screen microbial populations for hydantoinase activity (Morin, Hummel and Kula, 1986). A screen for dihydropyiidinase activity based on Schiff base formation with PDMB (upper panel) led to the identification of numerous strains with D-hydantoinase activity, which in combination with a D-carbamoylase is employed to produce D-amino acids.

Schiff base formation occurs normally with 3-aminopyridine (69CR(C)(269)1319> and aldehydes, but imines of 2-aminopyridine, though isolable, are less stable and bis(pyridyl-amino) compounds (e.g. 93) are readily formed. 9-Aminoacridine fails to react with benzal-dehyde. Nitrosobenzenes condense with aminopyridines in alkali (but not in acetic acid) to give azopyridines (Scheme 82). 

In addition to the above-mentioned reactions, metal complexes catalyze decarboxylation of keto acids, hydrolysis of esters of amino acids, hydrolysis of peptides, hydrolysis of Schiff bases, formation of porphyrins, oxidation of thiols, and so on. However, polymer-metal complexes have not yet been applied to these reactions. 

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... 

Amino acids and their derivatives undergo a wide range of reactions, e.g. racemization, peptide bond formation, ester hydrolysis, aldol-type condensation, Schiff base formation and redox reactions, which are catalyzed by coordination to a metal centre. A number of reviews are available which cover some of these reactions.48,69,70... 

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... 

Since the Schiff base formation is reversible, it should be reduced by sodium borohydride for the fixation of the label. The rate of the reduction of the Schiff base becomes slow as the number of the phosphate groups of the label increases. However, except for adenylate kinase, the NP -PL bound to the proteins were easily fixed by borohydride reduction. After reductive fixation, labeled proteins are cleaved by appropriate methods. The labeled lysine is cleaved by neither trypsin nor lysyl endopeptidase. There are at least three ways to detect the labeled peptide during isolation 1) use of radioactive reagent, 2) use of radioactive sodium borohydride for reduction of the Schiff base, and 3) use of fluorescence derived from the pyridoxyl moiety of the reagent (excitation at 295 nm and emission at 390 nm at acidic pH). The labeled lysyl residue is not positively identified in the amino acid sequence analysis. However, the presence of the label in the peptide isolated can be confirmed by the presence of pyridoxyl lysine in the amino acid analysis. 

Benzodiazocine 264 was prepared through a 4-component Ugi reaction including a primary amine tethered to a BOC-protected internal amino nucleophile, followed by a postcondensation base-catalyzed cyclization. Thus, 2 equiv of aldehyde 262 were employed to promote Schiff base formation and a one-pot, double scavenging protocol with immobilized tosylhydrazine and di-isopropylethylamine removed both the excess aldehyde and any unreacted acid 261. The intermediate 263 was then subjected to treatment with TFA, followed by proton scavenging with resin bound morpholine, to promote cyclization to afford the eight-membered ring (Scheme 47) <2001TL4963>. 

Figure 7.1 Schiff base formation between acetaldehyde and an amino acid.

Due to their increased reactivity toward nucleophiles, dipeptide a-phenacyl esters are prone to piperazine-2,5-dione (DKP) formation. Coupling of amino acid phenacyl esters with an activated carboxy component for dipeptide synthesis often results in substantial cyclization of the amino acid phenacyl esters via Schiff base formation (Scheme... 

FIGURE 9.35 Point of attachment of pyridoxal phosphate to a residue of lysine of Bg-re-quiring enzymes. Pyridoxal phosphate can form a Schiff base with a specific residue of lysine that resides near the active site of the enzyme. This Schiff base strengthens the binding of the cofactor to the enz)rme, but its bond must be reversed to allow Schiff base formation with an incoming amino acid substrate. 

Control of the degree of alkylation of an amino acid is difficult. Alkylation can lead to /V-mono- and di-alkyl derivatives, or betaines RIN+—(CR R2—) C( >2, when an alkyl halide is used. Many practical devices can be employed to get the mono-alkylated compound. Schiff-base formation with an aldehyde, followed by reduction, is a standard route. 

The decarboxylation is more enigmatic. Although models for Schiff base formation and transamination are common, models for decarboxylation are rare. If PLP is mixed with an amino acid in aqueous solution, then Schiff base formation will occur, followed by slow transamination. No decarboxylation is observed. If an a-methyl amino acid is used, thus preventing transamination, slow decarboxylation is observed, but only at temperatures above 100°C (106). Thus, the problem in enzymic decarboxylation is to catalyze a very slow reaction and avoid a much more favorable reaction. 

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