Shikimic acid labeled

A number of new syntheses of labeled intermediates of the shikimate pathway have also been published. These include syntheses of shikimic acid labeled in a variety of different positions. Particularly elegant is a synthesis of chorismic acid labeled stereospecially with tritium in either the E or the Z position of the enolpyruvyl side-chain. The synthesis is shown in Figure 9. Following elaboration of the enolpyruvyl side chain, bromination and dehydrobromination produces a -bromo-enolpyruvyl side chain in which the bromine can be selectively replaced by hydrogen without affecting the stereochemistry by reduction with a Zn/Ag couple. Tritium can be introduced into the position by carrying out the reduction in tritiated water and into... 

Theoretically, many of the above discrepancies could be settled by experiments with carboxyl-labeled shikimic acid because this functional group would be lost in the formation of phenylalanine, but retained in the case of a direct conversion to gallic acid. Only ambiguous evidence was obtained, however, from such efforts (10), and it was concluded that at least two pathways for gallic acid biosynthesis must exist (14), with the preferential route depending on leaf age and plant species investigated (15,16). 

The ester-substituted complex (34) has been used in synthesis of (+)- and (-)-shikimic acid, an important intermediate in the biosynthesis of aromatic compounds, as well as stereospecifically deuterium labeled shikimic acid.60 Addition of hydroxide anion to (+)-(34) gives the diene complex (+)-(182),... 

Streptonigrin.—Details of a study of the biosynthesis of streptonigrin (139) that had earlier been published in preliminary form (cf. Vol. 9, p. 24 Vol. 10, p. 23) are now available in full papers.51 52 In essence, the new results are that labelled anthranilic acid was not incorporated into streptonigrin (139),51 that l-rather than D-tryptophan was a precursor, and that label from C-7a in tryptophan (94) appeared, it was deduced, at C-8 in (139).52 The exclusive labelling of C-8 by tryptophan indicates that rings A and B do not derive from this amino-acid. These rings do not derive from phenylalanine and tyrosine, and negative results have been obtained with shikimic acid due, at the least, to poor cellular uptake.51... 

Exploration of geldanomycin (150) biosynthesis with D-[6-13C]glucose has revealed that the C7N unit of this antibiotic has a similar origin (labelling of C-17 and C-21 cf. nybomycin above), although shikimic acid (144) itself was a poor precursor.133 Similar observations were made on shikimic acid incorporation in the... 

Shihunine.—Preliminary results139 indicated that the orchid alkaloid shihunine (153) was derived from (152), an important intermediate in naphthoquinone biosynthesis.140 Further details are now available in a full paper.141 The intact incorporation of (152) is affirmed by the observation that (152), labelled with 13C at C-l, was an efficient and specific precursor for (153). [l-14C]Acetate was examined as a shihunine precursor and was found only to label C-5. This is consistent with the expected formation of (152) from shikimic acid (144) and a-ketoglutarate (151),140 the latter gaining acetate label in its carboxy-groups through the tricarboxylic acid cycle. 

In the presence of unlabeled D-glucose, there was no significant incorporation of labeled acetate, pyruvate, and formate into shikimic acid. Variously labeled D-glucose gave the distribution of activities shown in Fig. 1. Only D-glucose labeled in Cl, C2, equally in C3 and C4, or C6 (abbreviated G-1, G-2, G-3,4, and so on) were available for trial. The large deficiencies which occurred in Cl and C5 of shikimate were, therefore, assigned to G-5. The relative contributions of G-3 and G-4 were unknown, but could be... 

This enzymically prepared product was, therefore, tested with bacterial extracts. Almost quantitative conversion to shikimate took place. Furthermore, D-olheptulose diphosphate labeled in carbon atoms 4,5,6, and 7 with C, prepared from uniformly labeled D-oZiro-heptulose 7-phosphate and unlabeled n-fructose diphosphate (see Fig. 3), was converted to shikimate labeled exclusively in carbon atoms 3,4,5, and 6. It is clear, however, that a cyclization of the intact carbon-chain of D-aZtriose phosphate isomerase, carbon atoms 1,2, and 3 of the heptulose diphosphate would be derived from G-(l,6), G-(2,5), and G-(3,4), respectively. Carbon atoms 7,1, and 2 of shikimate, as discussed previously (see Fig. 1), are derived from the reverse sequence, namely, G-(3,4), G-(2,5), and G-(l,6). Apparently, carbon atoms 1,2, and 3 of the heptulose diphosphate become detached, and their order is reversed, before their incorporation into shikimic acid. 

The known intermediates and reactions, considered above, indicate that the conversion of shikimic acid to the aromatic amino acids takes place without rearrangement of the carbon atoms of the ring. Some studies of the incorporation of labeled precursors into the aromatic amino acids have been entirely in accord with this conclusion other studies have not. This discrepancy has been briefly reviewed, but not resolved. 

Miscellaneous Alkaloids. Shikimic acid (57) is a precursor of antliranilic acid (28) and, in yeasts and Escherichia coli (a bacterium), antliranilic acid (o-aminobenzoic acid) is known to serve as a precursor of tryptophan (26). A similar but yet unknown path is presumed to operate in higher plants. Nonetheless, antliranilic acid itself is recognized as a precursor to a number of alkaloids. Tims damascenine [483-64-7] (134), C10H13NO3, from the seed coats of Tdigella damascena has been shown (95) to incorporate labeled antliranilic acid when unripe seeds of the plant are incubated with labeled precursor. 

Phenazines.—Shikimic acid (134) is clearly implicated as a precursor for microbial phenazines, e.g. iodinin (135), and it can act as the sole source of the carbon skeleton. Essential proof that two molecules of shikimic acid are involved in phenazine biosynthesis was provided when it was shown that on incorporation of DL-[1,6- C2, 2- H]shikimic acid [as (134)] into iodinin (135) in Brevibacterium iodinum, some (7.5%) of the molecules of (135) produced were dideuteriated. [The shikimic acid was incorporated with the usual high efficiency (similar values for and H) and the deuterium label was confined to the expected positions (see below).]... 

It follows from this result and the C labelling studies " that phenazine biosynthesis proceeds from two shikimic acid units linked as in (136) or (137). By determining that the sites of deuterium labelling in the iodinin (135) derived from... 

The specific and proximate precursor of the mCyN unit in ansamycin polyketides is 3-amino-5-hydroxybenzoic acid 59 (AHBA) [94]. The biosynthesis of AHBA has recently been described by Floss and co-workers from the initial branch point of the shikimic acid pathway prior to 3-deoxy-D-flra/jzno-heptulo-sonic acid 7-phosphate (DAHP) [95]. The pathway shown in Scheme 25 was delineated by feedings of the proposed AHBA precursors, in labelled forms, to cell-free extracts of both the rifamycin B producer A. mediterranei S699 and the ansatrienin A producer S. collinus Tul892. In these experiments each of the compounds 61-64 was converted into AHBA with generally increasing efficiency. Most importantly the shikimate pathway compound DAHP cannot replace phosphoenolpyruvate 61 and erythrose 4-phosphate 60, or aminoDAHP 62 as the precursor of AHBA 59. 

Further information was gleaned from feeding [l- C]glucose. ° This compound was found to label only C-1 and C-10 of the C7N unit, a labelling pattern which is similar to that observed in shikimic acid (191) formed from [1- C]glucose. Neither shikimic acid, however, nor the labelled aromatic amino-acids tested were found to be precursors for the C7N unit of rifamycin S. This does not, of course, exclude earlier intermediates on the shikimate pathway and it was suggested that 3-dehydroquinate (189) or 3-dehydroshikimate (192) may be the key intermediate in the biosynthesis of this unit in rifamycin S (193). 

Claisen rearrangement of chorismic acid 1 to prephenic acid 2 (Scheme 1), which is catalyzed by the enzyme chorismate mutase, can be considered as the key step in the biosynthesis of aromatic compounds, that is the so-called shikimic acid pathway. The chair-like transition state geometry 3 was proved by double isotope-labeling experiments [2]. However, in the laboratory this particular reaction can be accelerated not only by enzymes but also by catalytic antibodies [3]. For the generation of such antibodies haptenes such as 4 were used, that is, molecules whose structure is very similar to the transition state of the particular reaction and which are tightly bound by the antibody. 

The enantioselective synthesis of (-i-)-shikimic acid and (-i-)-5-ep/-shikimic acid by an asymmetric Diels-Alder reaction of (5)-a-sulfmylacrylates with furan was reported. In the Diels-Alder reaction between acryloyloxazolidinone and furan catalyzed by a chiral cationic Cu(II) complex a high enantioselectivity was observed <97TL57>. The synthesis of ubiquinones-3 specifically labelled with C at the C(5)- or C(6)-positions starting with 2,5-bis(trimethylsilyloxy)-3-methylfuran and (2- C)-2-bromo-l,2-dichloroethene was reported <97T2505>. Diels-Alder reactions of 5-triisopropylsilyl-2-vinylfuran and 2-triethylsilyl-4-vinylfuran with acetylenic and olefinic dienophiles occur with high site specificity in the extraannular mode to produce trialkylsilylbenzofuran derivatives <97H(45)1795>. 

The studies of the origin of GHB in A. bisporus demonstrated the involvement of the shikimate-chorismate pathway (Scheme 102). Labeling experiments showed an efficient incorporation of H- and C-labeled shikimic acid 439,440) and C-labeled chorismic acid 441) into the 4-hydroxyaniline moiety of GHB. It was also demonstrated that in the biochemical shikimate-4-hydroxyaniline conversion in the mushroom, amination occurred at the 4 position of one of the carboxylic acid intermediates [initially assumed to be shikimic acid 439)]. Additionally, the p-aminobenzoic acid, which proved to be 441) the precursor of 4-hydroxyaniline, underwent a decarboxylative hydroxylation catalyzed by a FAD-dependent monooxygenase 4-aminobenzoate hydroxylase in the presence of NAD(P)H and O2. This enzyme from A. bisporus was recently purified to homogeneity by Tsuji et al. 442). 

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