Aldehyde Affinity Labels

Two aldehydic nucleotide derivatives have found use as affinity labels. The magnesium salt of (64), formed by oxidation of ATP with periodate, is a competitive inhibitor of pyruvate carboxylase with respect to [Mg. ATP2-],100 and (65), obtained from the / -anomer of 5-formyluridine-5 -triphosphate on treatment with alkali, is a non-competitive and reversible inhibitor of DNA-dependent RNA polymerase from E. coli.101 In each case, addition of borohydride gives stoicheiometric covalent linkage of the nucleotide to the enzyme, with irreversible inactivation. It is thought that condensation with lysine occurs to give a Schiff s base intermediate, which undergoes subsequent reduction. 

Reactive in affinity label cf. glycer-aldehyde 3-phos-phate dehydrogenase, p. 281. 

Pyridoxal.—Oxidation of eo-hydroxypyridoxal methyl hemiacetal (4) by manganese dioxide followed by phosphorylation of the intermediate dialdehyde with polyphosphoric acid affords (5) (Scheme 1). The latter should be a useful reagent for affinity labelling enzymes requiring pyridoxal phosphate as cofactor since there are two reactive aldehyde groups in the molecule. Pyridoxal phosphate itself can be used as a probe for the investigation of the active sites of enzymes because of its reaction with lysine residues and its ability to act as... 

In many of the examples cited of affinity labeling of enzymes by periodate-oxidized nucleotides, it has been assumed that the reaction involved formation of a Schiff base with an enzymic lysine, as in Fig. 3a,b. However, in very few papers has direct evidence been presented supporting the existence of a Schiff base intermediate. Lowe and Beechey (83), after examining in detail the structure of periodate-oxidized ATP, concluded that in aqueous solution there is little free aldehyde rather, the compound exists predominantly as an equilibrium mixture of three dialdehyde monohydrates (cyclic hemiacetals) and a dihydrate. The presence of cyclic hemiacetals may account for the ability of periodate-oxidized NADP and NADPH to function as coenzymes in several enzymic reactions (e.g., 78, 81). In many cases, the product of the covalent reaction of an enzyme and periodate-oxidized nucleotide may be a dihydroxymorpholino derivative (Fig. 3c), which is similar to the cyclic hemiacetals observed in aqueous solu-... 

Oxidation of ADP using periodate, followed by partial reduction with boro-hydride, affords a mixture of the fully reduced bis(hydroxymethyl) product and the semialdehydic reduction intermediate. By dint of comparison with model compounds using n.m.r. spectrometry, it has been shown that the first reduction step takes place at C-3, to give (107) (which will prefer to cyclize to the hemiacetal). It is not clear why preferential reduction at C-3 occurs either the aldehydic group at this position is more reactive per se, or it is more accessible to borohydride. It seems likely that (107) and its analogues could prove to be useful new affinity labels. 

An essential lysine residue in the active center of the catalytic subunit of DNA-dependent RNA polymerase can be affinity labeled with methylthioinosinedicarboxaldehyde (MMPR-OP) The c-amino group of the lysine forms a Schiff base with an aldehyde group of MMPR-OP, which can be converted to the stable amine by mild reduction with sodium borohydride. The [ S] MMPR-OP can be prepared easily for radioactive affinity labeling of the enzyme. 

For the latter CNS receptor ligands, different types of chelators have been introduced to the basic receptor binding framework. Originally a bidentate Schiff base was introduced by the reaction of an aliphatic amine with pyridine-aldehyde. Labelling occurred at low concentration and the radio conjugate was physiologically stable. Although receptor affinity was fully retained, brain uptake was low [67]. ACp was introduced at the same position and, as described earlier, and receptor affinity was perfectly retained. No brain uptake data are... 

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