Isotopic labeling, stereospecific

Part—VI has been solely devoted to Miscellaneous Assay Methods wherein radioimmunoassay (RIA) (Chapter 32) has been discussed extensively. Various arms of theoretical aspects viz., hapten determinants and purity importance of antigenic determinants and analysis of competitive antibody binding of isotopically labeled compounds. The applications of RIA in pharmaceutical analysis, such as morphine, hydromorphone and hydrocordone in human plasma clonazepam, flurazepam in human plasma chlordiazepoxide in plasma barbiturates, flunisolide in human plasma have been described elaborately. Lastly, the novel applications of RIA-techniques, combined RIA-technique-isotope dilution and stereospecificity have also been included to highlight the importance of RIA in the analytical armamentarium. 

Stereochemical probes of the specificity of substrates, products, and effectors in enzyme-catalyzed reactions, receptor-ligand interactions, nucleic acid-ligand interactions, etc. Most chirality probe studies attempt to address the stereospecificity of the substrates or ligands or even allosteric effectors. However, upon use of specific kinetic probes, isotopic labeling of achiral centers, chronfium-or cobalt-nucleotide complexes, etc., other stereospecific characteristics can be identified, aU of which will assist in the delineation of the kinetic mechanism as well as the active-site topology. A few examples of chirality probes include ... 

K. Yonaha u. K. Soda, Ad v. Biochem. Eng./Biotcchnol. 33,95 -130 (1986) . .Applications of Stereoselectivity of Enzymes Synthesis of Optically Active Amino Acids and a-Hydroxy Adds and Stereospecific Isotope Labeling of Amino Acids, Amines, and Coenzymes". 

As a whole, such high stereospecificity is difficult to attain by chemical catalysis, although asymmetric synthesis has developed markedly in recent years.80) Stereospecificity has been applied to the production of optically active compounds such as amino acids, and also to the synthesis of stereospecifically isotope-labeled compounds, the synthesis of which is not easily achieved by a chemical method (Table I. 3).75)... 

From solvolytic studies of isotopically labeled substrates it was shown that cyclopropyl-car biny 1-cyclobutyl interconversion is stereospecific . The stereospecific interconversion of cyclobutyl cations to the corresponding cyclopropylcarbinyl cation was also cleanly observed in superacid medium, and was used to prepare otherwise unstable cw-(a-methylcyclopropyl)carbinyl cation 17 Thus ionization of cw-2-chloro- or cw-3-chloro-l-methylcyclobutane in SbF5-S02ClF at -135 °C yielded the cw-isomer which rapidly rearranged irreversibly into the trans-isomer 18 at about -100 °C. The trans-isomer 18 is the only cation obtained when the preparation was carried out at -80 °C, or when prepared from the cyclopropylmethyl carbinoP (equation 24). 

This mechanism predicts that the hydrogen atom and cyanide group of HCN will be added across the same side of the carbon-carbon double bond and, consistent with this prediction, the addition of DCN see Isotopes Isotope Labeling) to cyclohexadiene (equation 9) has been shown to occur stereospecifically cis see Stereochemistry)) ... 

For acyclic alkenes, the facile rearrangement process obviates most possibilities of stereoselective transformation. The one exception is that of stereospecific isotopic labelling, for which hydrozirconation is a potentially powerful technique compounds of type Bu CHDCHDX (36), useful for NMR-based mechanistic studies, are conveniently obtained as in Scheme 9. This requires that four separate processes — hydrozirconation of alkyne, cleavage of alkeiiylzirconium, hydrozirconation of alkene and cleavage of alkylzirconium — all be completely stereospecific. 

The essentially complete assignment of H, 13C and 15N resonances of recombinant human ubiquitin were obtained by analysis of HNCA, HN(CO)CA, HNCO and HCCH-TOCSY spectra and reference to previously reported H (Di Stefano and Wand, 1987) and 15n (Schneider et al., 1992) resonance assignments (Wand et al., 1996). Stereospecific assignments of leucine 5-CH3 and valine y-CHj groups were obtained using the isotopic labeling strategy developed by Neri et al. (1990). This approach provided unequivocal prochiral... 

The factors that make ene and retro-ene reactions proceed are nicely illustrated by a synthesis of enantiopure, isotopically labeled acetic acid CH(D)(T)C02H, a useful compound for studying the mechanisms of enzyme-catalyzed reactions. One ene and one retro-ene reaction occur in this synthesis. The ene reaction is driven by formation of a new tr bond at the expense of a C=C tt bond the retro-ene reaction is driven by the formation of a C=0 77 bond. Note that both pericyclic reactions proceed stereospecifically, even at the very high temperatures required for them to proceed ... 

Both alkaloids have (+) and (-) forms but only the (-) hyoscyamine and (-) scopolamine are active. The biosynthetic pathway of tropane alkaloids, Fig. (1) is not totally understood, especially at the enzymatic level. Edward Leete has pioneered the biosynthetic studies of tropane alkaloid since 1950"s using whole plants and isotope labels [85-86]. The tropane alkaloid hyoscyamine is bioconverted by the enzyme H6H (hyoscyamine 6p-hydroxylase, EC 1.14.11.11) to scopolamine via 6p-hydroxyhyoscyamine. Hyoscyamine is the ester of tropine and (S)-tropic acid. The (S)-tropic acid moiety derives from the amino acid L-phenylalanine, while the bicyclic tropane ring derives from L-omithine primarily or L-arginine via tropinone. Tropinone is stereospecifically reduced to form either, tropine which is incorporated into hyoscyamine, or on the other hand into pseudotropine which proceeds to calystegines, a group of nortropane derivates that were first found in the Convolvulaceae family [87]. 

Buchwald, Friedman and Knowles succeeded in preparing 2,4-dinitrophenyl phosphate in which the three free oxygen atoms on phosphate were labeled stereospecifically with different isotopes of oxygen. Solvolysis of this compound in methanol and analysis of the methyl phosphate product showed that the reaction had proceeded with inversion of configuration at phosphorus (79). This remarkable experiment supports a concerted bimolecular displacement mechanism, with no metaphosphate intermediate, for the solvolysis of 2,4-dinitrophenyl phosphate in methanol. However, it does not rigorously exclude a stepwise mechanism in which a metaphosphate intermediate with a very short lifetime is formed and reacts with methanol faster than it rotates, and it does not provide direct evidence for a bimolecular, concerted reaction with solvent water. 

Kainosho et al.121 proposed a method termed Stereo-Array Isotope Labeling (SAIL). They used stereochemically and/or optimally enriched amino acids23,120 to label the protein. The method can decrease the resonance complexity and relaxation rate, and can increase the stereospecific assignment quality significantly. 

Our laboratory has studied the stereochemistry of methyl group formation in a number of a, 0 elimination reactions of amino acids catalyzed by pyridoxal phosphate enzymes. The reactions include the conversions of L-serine to pyruvate with tryptophan synthase 02 protein (78) and tryptophanase (79), of L-serine and l-tyrosine with tyrosine phenol-lyase (80), and l-cystine with S-alkylcysteine lyase (81). In the latter study, the stereospecific isotopically labeled L-cystines were obtained enzymatically by incubation of L-serines appropriately labeled in the 3-position with the enzyme O-acetyl serine sulfhy-drase (82). The serines tritiated in the 3-position were prepared enzymatically starting from [l-3H]glucose and [l-3H]mannose by a sequence of reactions of known stereochemistry (81). The cysteines were then incubated with 5-alkyl-cysteine lyase in 2H20 as outlined in Scheme 19. The pyruvate was trapped as lactate, which was oxidized with K2Cr202 to acetate for analysis. Similarly, Cheung and Walsh (71) examined the conversion of D-serine to pyruvate with... 

Simultaneously and independently, Cullis and Lowe developed a second general methodology for the synthesis of, 0, 0-labeled chiral phosphate monoesters (76, 77). This synthesis relies upon the synthesis of a cyclic hydrobenzoin triester of the alcohol or phosphoric acid followed by hydrogenolysis to liberate the isotopically labeled monoester product (Fig. 2). Hydrobenzoin, chiral by virtue of stereospecific labeling with 0 and 0, is the source of the two specified oxygen isotopes, and O is derived from H2 0 via P OCL. The reader is referred to the articles by Cullis and Lowe for details of the synthesis. 

In principle, isotopic labeling ought to permit the neutral products of a cationic reaction to be deduced from mass spectrometric measurements alone. As an example, the elimination reaction drawn in Scheme 1 yields a mixture of cis and trans alkenes when the alkyl group has >3 carbons. If the reaction passes through transition state drawn in Scheme 1 (called a syn-transition state), the ratio of alkene isomers from the stereospecifically deuterated precursors in Scheme 4 should correspond to the ratio of PhOH " to PhOD " observed in the mass spectrum. 

The [t 4j + t 2j] cycloaddition of alkenes and dienes is a very useful method for forming substituted cyclohexenes. This reaction is known as the Diels-Alder (abbreviated D-A in this chapter) reaction. The transition structure for a concerted reaction requires that the diene adopt the s-cis conformation. The diene and substituted alkene (called the dienophile) approach each other in approximately parallel planes. This reaction has been the object of extensive mechanistic and computational study, as well as synthetic application. For most systems, the reactivity pattern, regioselectivity, and stereoselectivity are consistent with a concerted process. In particular, the reaction is a stereospecific syn (suprafacial) addition with respect to both the alkene and the diene. This stereospecificity has been demonstrated with many substituted dienes and alkenes and also holds for the simplest possible example of the reaction, ethene with butadiene, as demonstrated by isotopic labeling. ... 

Stereospecific reduction at the Re and Si faces using the isotopically labelled reagent, NaB H4 or NaB H4, will produce the (R)- and (S)-enantiomers respectively (Fig. 47). The contribution of the ETH group was not merely the recognition of this principle, but more importantly the development of a protocol [101] for the configurational analysis of the resulting species of types (R)-3 and (5)-3 (Fig. 47). 

The 2-lerl-prenyl group itself stems from the mevalonate pathway and is introduced in a non-stereospecific manner, as shown by isotope labelling studies [164], The order of oxidations, of Diels-Alder reactions and of the tcrt-prenyl migration is still a subject of research on the individual alkaloids. 

As we examine transformations 159,160 (Figure 17.38), 161,162 (Fig. 17.39) and 163,164 (Figure 17.40), we notice that stereoselectivity manifests itself at several levels - two levels for 159, four levels for 160, three levels for 161, four levels for 162, three levels for 163, and, five levels for 164. In ncsie of these cases, is numerical identity between % stereospecificity and % stereoselectivity expected (except by accident). It is interesting to note that, in transformation 160, one may also define a racemate excess (re), re = (%E +% 3)g - (%E +% 3)b - as a measure of the excess of the (same) racemate formed in pathway "a" over that formed through pathway "b". The quantitative experimental determination of "re" would require judicious isotopic labelling. 

Lehmann, W.D. Theobald, N. Fischer, R. Heinrich, H.C. Stereospecificity of phenylalanine plasma kinetics and hydroxylation in man following oral application of a stable isotope-labelled pseudo-racemic mixture of l- and D-phenylalanine. Clinica. Chimica. Acta 1983, 128, 181-198. 

The high stereospecificity of decarboxylations catalysed by a series of bactericil decarboxylases was first demonstrated by Hanke [10a], who carried out the reactions in H20, isolated the corresponding chiral [l- H,]amines and showed that they were enantiomerically pure. Since then the steric courses of a number of decarboxylases have been more fully defined. Most of these act on L-amino acids and by appropriate isotopic labelling these have all been shown to proceed with overall retention at C (see Fig. 5). 

The stereospecificity is not limited to reactions for which the steric course is evident from the chemical configuration of substrate and product. Even at centers that are not chiral, the reactions are in many cases stereospecific (stereochemically cryptic reactions). The steric course of these reactions may be deduced by stereospecific isotope labeling of the substrates, transforming the reaction center into a chiral center of discernible stereochemical behavior. 

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