Pyridoxyl derivatives

Pyridoxal Derivatives. Various aldehydes of pyridoxal (Table 3) react with hemoglobin at sites that can be somewhat controlled by the state of oxygenation (36,59). It is thereby possible to achieve derivatives having a wide range of functional properties. The reaction, shown for PLP in Figure 3, involves first the formation of a Schiff s base between the amino groups of hemoglobin and the aldehyde(s) of the pyridoxal compound, followed by reduction of the Schiff s base with sodium borohydride, to yield a covalendy-linked pyridoxyl derivative in the form of a secondary amine. 

Proof that a lysine residue has been modified can be readily obtained because pyridoxyl derivatives of lysine possess characteristic white-blue fluorescence (Ronchi et al. 1969). In addition, they have a distinctive absorption maximum at 325 nm with of 9710 cm (Fisher et al. 1963). Finally, a radiochemical label can be introduced by reducing the pyridoxal-5-phosphate protein complex with tritium-labelled sodium borohydride. The peptide containing the derivatized lysine can therefore be detected either by fluorimetry, spectrophotometry or radiochemical techniques following routine procedures of proteolytic digestion and fractionation. Acid hydrolysis in 6 N HCl for 24 hr of peptides containing pyridoxal-5-phosphate lysine yields pyridoxyl-lysine since phosphate esters are readily hydrolyzed under these conditions. Pyridoxyl-lysine is eluted between lysine and histidine from a 55 cm column of Beckman 50 resin with 0.15 M citrate buffer pH 5.28. 

Pyridoxal and pyridoxal 5-phosphate are the only naturally occurring compounds which have been used as fluorescent labels. These compounds form Schiff bases with primary amino groups. The reduction of the C=N bond with sodium borohydride (NaBH4) leads to the formation of fluorescent pyridoxyl derivatives [260,261],... 

The reaction of pyridoxal with a primary amine and the reduction of the Schiff base to the pyridoxyl derivative are shown in Figure 11. As only primary amino groups form Schiff bases, the reaction should be specific for compounds with primary amino groups. However, proline and hydroxyproline react as well and, as noted [2611 even secondary amines can be brought to reaction, esjjecially in non-aqueous solvents. A ketoenimine is assumed to be an intermediate of the reaction sequence in this case. Histidine reacts first via the primary amino group to form a Schiff base, which is rearranged to a non-redudble imidazolopyridine derivative. 

Figure 11. Formation of a Schiff base with pyridoxal and its reduction to a pyridoxyl derivative.

Pyridoxyl derivatives are stable at 0 °C for some weeks if protected from light. Their fluorescence decays within minutes if the solutions are irradiated at the wavelength of the absorption maximum. 

One of the advantages of the pyridoxal method is the possibility of using sodium borotritide (NaBT4), which is available with a specific activity of about 116 Ci mmol Thus, radioactively labelled pyridoxyl derivatives can be obtained, allowing the detection of about 0.1 pmol of an amine or amino acid. 

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]. 

Method. Solutions of amino acids in phosphate buffer (pH 9.3) are mixed with an equal volume of freshly prepared 0.4 M pyridoxal solution (adjusted to pH 9.3) and permitted to stand at 8 °C for 30 min. (The molar ratio of pyridoxal to amino acid should be >75 1.) At this point, 1 ml of sodium tetrahydroborate solution (100 mg/ml in 0.1 N sodium hydroxide) is added and the contents are gently shaken. Excess of sodium tetrahydroborate is destroyed by addition of sufficient hydrochloric acid (pH 1-2) prior to column chromatography. The pyridoxal derivatives are separated on a column (100 X 0.6 cm) of Aminex A-5 ion-exchange resin (Bio-Rad) at a mobile phase flow-rate of 33 ml/h. The eluting solvents consist of 0.2 N buffers at pH 3.40,4.44 and 4.86 and a 0.35 N buffer at pH 5.86 (all of the buffers are sodium citrate). The separation of a number of pyridoxyl-... 

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. 

In principle, pyridoxyl amino acids and other PLP derivatives can be used to treat bacteria, protozoans, and cancer cells evaluating their efficacy in depressing growth. This may lead to... 

The coenzyme-enzyme Schiff base in aspartate aminotransferase was reduced with NaB H4, and the resulting derivative hydrolysed to give [4 - H]pyridoxyl-lysine. The latter on A-chlorination, followed by base catalysed dehydrohalogenation gave... 

Of the number of chromophoric derivatives of chiral amines for potential use in the establishment of their absolute configuration by BCD measurement only a few have proven to be generally useful. Of these, intensive investigation of the Af-salicylidene (Schiff base) derivatives of chiral primary amines, including unsubstituted and ring-substituted a- and S-arylalkylamines, a-amino acids, unsaturated and satnrated aliphatic and alicyclic amines, and amino sugars, has resulted in the formulation of the salicylidenamino chirality rule ° °. The application of this rule has recently been reviewed and has been successfully used for the establishment of absolute configuration of chiral primary amines in connection with other stereochemical studies. In related studies, the conformations of a series of pyridoxyl-L-a-amino acid Schiff bases were deduced from their CD spectra ... 

Pyridoxylamino acids exhibit spectral characteristics similar to those of pyridoxamine absorption maxima at 255 and 328 nm, and fluorescence emission at 400 nm. Fluorescence efficiency is pH-dependent, with maximum fluorescence at pH 5.28 (except for the histidine derivative, which shows maximum fluorescence at pH 12). Molar absorption coefficients and fluorescence efficiencies are, with few exceptions, the same for all pyridoxyl amino acids. Between 10 and lOOpmol of an amino acid can be determined in the effluent from an amino acid analyser [261]. 

The pyridoxyl residue increases the retention time, but most amino acid derivatives are eluted from the cation exchange column in the same order as the free amino acids. For continuous fluorescence measurement, one part of the effluent is mixed with 49 parts of sodium citrate buffer, pH 5.28. 

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