Tryptophan isotopic labelled

Blocher, M., Hitz, T., and Luisi, P. L. (2001). Stereoselectivity in the oligomerization of racemic Tryptophan A-Carboxyanhydride (NCA-Trp) as determined by isotopic labelling and mass spectrometry. Helv. Chim. Acta, 84, 842-8. 

There are recognized at present three naturally occurring members of this group, cinchonamine, quinamine, and conquinamine, all minor alkaloids of certain Cinchona and Memijia species. The elucidation of their structures led to the suggestion that the quinoline moiety of the major bases, e.g., cinchonine and quinine, of these plants was probably derived from tryptophan via an indolic precursor. It has since been demonstrated from the results of feeding experiments with isotopically labeled tryptophan that this amino acid really can serve as a precursor of various indole alkaloids (1) as well as of quinine (2). The details of these processes are not yet known but probably involve an intermediate(s) related to cinchonamine (2, 3, 6). 

In summary, after feeding the stable isotope labeled compounds to V. hilgendotfii, LC/ESI-TOF-MS analyses of Cypridina luciferin extracted from the specimens strongly suggested that three amino acids of L-tryptophan, L-arginine and L-isoleucine participate in biosynthesis of Cypridina luciferin (Scheme 1). 

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... 

Amino Acids and Peptides. - Wasserman s method of one-carbon homologation of carboxylic acids to give a-ketocarboxylates involves reaction with cyanomethylenetriphenyl-phosphorane followed by ozone (Scheme 24) and has been used as a key step in a chemo-enzymatic synthesis of isotopically labelled L-valine, L-isoleucine, and o/fo-isoleucine. Alkylation of the carbanion derived from the imino-substituted methylphosphonate diphenyl ester (186) with indol-3-ylmethyl bromide followed by appropriate deprotection has been used to prepare the phosphonate analogue (187) of tryptophan (Scheme 25). The deprotected analogue (188) and derived peptides show activity as inhibitors of chymotrypsin. Two approaches to solid phase Wadsworth-Enunons reactions which have applications in combinatorial chemistry have been reported. In one diethylphosphonoacetamide is bound to PEG-PAL resin via a peptide link, while... 

Brevianamide.—Echinulin (109) is elaborated in Aspergillus amstelodami along a pathway which includes (110) and (111). The structurally related metabolite, brevianamide A (112), isolated from Penicillium brevicompactum, appears to be derived in a similar way. Radioactive mevalonate, proline, and tryptophan gave labelled brevianamide A. Incorporation of cyclo-L-[methylene- C]-tryptophyl-L-[5- H]proline [as (113)] without change in isotope ratio indicated that this precursor was utilized intact for brevianamide A production. As further evidence of its role in brevianamide A biosynthesis (113) has been isolated from P. brevicompactum cultures. By analogy with echinulin biosynthesis, (114) could lie between (113) and brevianamide A (112). Its isolation" (from A. ustus) provides support for this suggestion. 

Proton ENDOR is a useful probe of the origin of the EPR signals from organic-based radicals. In a series of cw ENDOR studies including samples with isotopically labeled amino acids, proton ENDOR was used to identify the amino acid radical in cytochrome-c peroxidase (CcP) as a tryptophan species. Similarly, isotopic labeling was used to identify the tyrosyl... 

Different approaches have been carried out to investigate the biosynthesis of EAs. Early feeding experiments with isotope-labeled precursors, e.g., [ C]-L- tryptophan 1, to rye ears infected with C. purpurea or to fermentation cultures of different Claviceps species proved its incorporation into 2 [100-102]. Further biochemical experiments with crude and purified enzyme extracts fi om producers revealed the determinant step in the biosynthesis of EAs by the key enzyme 4-dimethylallyltransferase (4-DMATS) [103, 104], i.e., the prenylation of 1 at C-4 position [17, 104, 105]. 

Tryptophan is a precursor of ergot alkaloids in Claviceps and in some cases plays also the role of an inducer and derepressor (Bu Lock and Barr, 1968 Vining, 1970 Robbers et al., 1972). Isotopically labelled tryptophan shown a high level of incorporation into ergot alkaloids (41%) in the strain of P. sizovae indicating that it is also here a direct precursor Kozlovsky et al. (1985). In Claviceps not only L- but also D-tryptophan are utilised for the ergot alkaloid biosynthesis (Robbers et al., 1972 Floss, 1976). The effect of both L- and D-tryptophan and also of their analogue, D,L-6-methyltryptophan on the alkaloid... 

Hauser, K., Krejtschi, C., Huang, R., Wu, L., Keiderling, T.A. Site-specific relaxation kinetics of a tryptophan zipper hairpin peptide using tanpaature-jump IR spectroscopy and isotopic labeling. J. Am. Chem. Soc. 130,2984—2992 (2008)... 

Hu, X.H., Spiro, T.G. Tyrosine and tryptophan structure markers in hemoglobin ultraviolet resonance Raman spectra mode assignments via subunit-specific isotope labeling of recombinant protein. Biochemistry 36, 15701-15712 (1997)... 

While studies in the intact animal with isotopically-labeled compounds have contributed important information to the knowledge of tryptophan metabolism, the most pertinent information has been derived from studies with enz3ones. These results of these studies are discussed in the following sections. 

D-tryptophan was a direct precursor. The use of various isotopically labelled -tryptophans had demonstrated that the carbon C-2 of the indole ring of this precursor is converted into the carbon C-2 of the pyrrole ring in the product, as the amino-nitrogen is then oxidized to the nitro-group. Tritium from C-2 of the side-chain of L-tryptophan, but not D-tryptophan, is retained during the biosynthesis. ... 

Biosynthesis of some classes of terpene indole alkaloids is well understood. In certain cases, many of the enzymes that are responsible for biosynthesis have been cloned and mechanistically studied. In other cases, biosynthesis pathway is only proposed based on the results of feeding studies with isotopically labeled substtates and from the structures of isolated biosynthetic intermediates. All terpene indole alkaloids are derived from tryptophan and the iridoid terpene secologanin (Fig. 14.11). Tryptophan decarboxylase, a pyridoxal-dependent enzyme [29], converts tryptophan to tryptamine [30]. The following strictosidine synthase-catalyzed Mannich reaction connects ttyptamine and secologanin to yield strictosidine [31]. The Apocynaceae, Loganiaceae, Rubiaceae, and Nyssaceae families of plants each produce terpene indole alkaloids with dramatically diverse structures [32-34]. The mechanisms and control of... 

Incorporation studies with isotopes showed that when anthranijate was converted to tryptophan, the carboxyl group df anthranilate was lost as carbon dioxide, but the nitrogen was retained. Because the enzymes in the tryptophan biosynthetic pathway have only a limited specificity, it was possible to substitute 4-methyl-anthranilate in E. coli extracts that could convert anthranilate to indole. This nonisotope label was conserved during the conversion to yield 6-methyl indole. 

However, recently it has proved possible to positively identify tryptophan radicals in cytochromec peroxidase[147] and tyrosine radicals in ribonucleotide reductase, prostaglandin H synthase and photosystem II of chloroplasts [148], This has been achieved by a combination of the techniques discussed already, but with the powerful, additional non-invasive tool of isotopic substitution. As deuterons (5=1) give different splitting than protons (S = 1/2), substituting different labelled amino-acid residues into the enzyme should reveal the nature of the radical-containing residue. This is easily achieved in an auxotrophic mutant that requires this amino acid to be supplied in the medium. The specific residue can then be identified by site-directed mutagenesis of the evolutionary conserved amino-acid residues [108,149-151]. 

Isotopic experiments (763) with tryptophan labeled with N and deuterium in the indole ring have shown that quinolinic acid nitrogen is probably entirely derived from the indole nitrogen of tryptophan, and that scission of the benzene ring probably occurs between carbons 3 and 4. Presumably, therefore, the hydroxyanthranilic acid is converted to intermediate A without participation of a catechol-type intermediate, and it is possible that the phosphate-bond energy of hydroxyanthranilic acid phosphate (if this is in fact an intermediate) may contribute to the transformation. It is known... 

Labeled amino acides. [DsJ-L-Tryptophan and [DsJ-tryptamin were prepared by the deuterium-exchange method as previously described. [ CsJ-L-Isoleucine and [ N2]-L-arginine were obtained from Cambridge Isotope Laboratories and Spectra Stable Isotopes, respectively. 

ESI-TOF-MS analyses for the stable iostope labeled compounds. To confirm the isotopic purity of the labeled compounds, [DsJ-L-tryptophan, [Dsl-tryptamine, [ CfiJ-L-isoleucine and [ NaJ-L-arginine were analyzed by ESI-TOF-MS (Table 2). 

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