Enzymes stereochemistry determination

Use of Enzymes in Determining the Stereochemistry at a Chiral Methylene Group... 

As described above, the stereochemical course of the reaction was proven to be accompanied by inversion of configuration. The most probable explanation is that the substrate adopts a planar conformation at some stage of the reaction, and the chirality of the product is determined by the face of this intermediate that is approached by a proton. If this assumption is correct and the conformation of the substrate in the active site of the enzyme is restricted in some way, the steric bulk of the o-substituents will have some effect on the reactivity. Thus, studies of the o-substituted compounds will give us information on the stereochemistry of the intermediates. 

The conversion includes at least three enzymic reactions.169-171 In the first stage, which requires pyridoxamine 5 -phosphate as a cofactor,171,172 dehydration of 7b occurs through intermediate formation of the Schiff base.173 Reduction of the resulting, unsaturated derivative with NADPH, the mechanism of which is not completely clear,174 leads to CDP-3,6-dideoxy-D-eryf/iro-hexos-4-ulose,169 and, in the third stage, further reduction of the latter at C-4 of the hexosyl group produces the derivatives of paratose or abequose the stereochemistry of the reaction is determined by the source of the enzyme.168 The tyvelose derivative is formed as a result of enzymic epimerization at C-2 of the hexosyl group in CDP-paratose.175... 

With the stereochemistry of the citrate lyase reaction determined, that of the Si citrate synthetase (the common enzyme) was established as shown in Fig. 70. Condensation of (J )-acetic-d, t acid (configuration known by synthesis) with oxalo-acetate gives what turns out to be mainly (2S,3/ )-citric-2-d,2-/ acid (112).41 When this acid is then cleaved with citrate lyase, the major product is (/ )-acetic-d, t acid, as established by the malate synthetase/fumarase diagnosis. It follows that both the Si-citrate synthetase and citrate lyase reactions must involve the same stereochemical course. Since that of the lyase reaction is inversion (vide supra), that of the Si synthetase reaction must be inversion also. And since the overall stereochemical result shown in Fig. 70 is not dependent on the magnitude of the... 

The alternative approach to the determination of the stereochemistry of hydroxylation )3 to the nitrogen occurring in the biosynthesis of haemanthamine involved the synthesis of stereospecifically labeled tyrosine. Catalytic hydrogenation of suitable acylaminocinnamic acids labeled with isotopic hydrogen in fi position proceeds stereoselectively to furnish an equimolecular mixture of L-(/3i -3H]-and D-[j8 -3H]tyrosine (419) and (420). Enzymic resolution of the racemic amino acid yielded the L and d isomers. The two optically active forms of tyrosine were... 

Overton and coworkers discovered a leucine 2,3-aminomutase in plant tissue cultures of Andrographis paniculata that converts (S)-leucine in (R)-f-leucine [43] (Scheme 1.6.10). The enzyme activity was investigated in cell free extracts by incubation with (S)-[U-14C]leucine and by measuring the radioactivity of the methyl ester camphanamide derivatives of the reaction mixtures by radio-GC. The stereochemistry of the /i-am i no acid was determined by radio-GC comparison of the enzyme reaction product as methyl ester camphanamide derivative with an authentic sample. The enzyme is not dependent on cobalamin, because addition of intrinsic factor does not induce its inhibition. 

Exact localization of the hydrogen isotope is usually required, and it is inadvisable to rely on the method of synthesis, as the label is not always situated entirely at the position predicted.69 In many mechanistic and biological studies with enzymes, the position of the label alters during the reaction, and, in others, where a proton isotope from the solvent is incorporated, it is important to localize the incorporated isotope, and, if it is at a methylene group, to determine its stereochemistry. 

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