Biosynthesis of the Ansamycins

The ansamycin antibiotics represent a new class of natural products, combining an aromatic nucleus with a branching aliphatic side chain, and considerable interest has centered on their formation in nature. Their common structural features suggest immediately a similar biosynthetic pathway. Moreover, the streptovaricins, rifamycins, tolypo-micin B and halomicin B all have the same number of carbon atoms in the ansa ring (C17), while geldanamycin and the maytansinoids also have the same number of carbon atoms (C15). Thus far, biosynthetic 

Extensive and often difficult chemical degradations were required to determine the distribution of isotopes when C-labeled radiotracers were used in early biosynthetic studies 13,19,56,89). The more recent use of cmr spectroscopy employing the pulsed Fourier transform method has made the study of the ansamycins easier, especially that of their aromatic chromophores, which are not easily degraded. Carbon-13 occurs in 1.1 % natural abundance and natural abundance cmr spectra of many ansamycins have been reported, as described in Section IV. When highly enriched C-labeled compounds are used as precursors in ansamycin-producing cultures, cmr data reveal the extent and site of incorporation of C-labeled precursors 106). 

No intermediates have been observed thus far for streptovaricin (or any other ansamycin) between the primary metabolite and an assembled unit containing all of the skeletal carbon atoms of the antibiotic. However, considerable evidence is accumulating on the sequence of events which transpire following assemblage of the carbon skeleton. This evidence is derived from the identification of numerous compounds related to the streptovaricins but differing from them in details of oxygenation pattern or methylation (23, 91), and from experiments interconnecting radio-actively labeled streptovaricins (89). 

A final intermediate isolated, protostreptovaricin V, is like the others but lacks one aliphatic C-methyl group. The missing methyl presumably has been lost by decarboxylation and thus seems likely to be the C-IO methyl, which appears as a carboxyl in the streptovaricins, or the C-14 

Radioactive streptovaricin D was prepared biosynthetically by administering [m //i3 /- C]methionine to a growing culture of S. spectabilis 89). The streptovaricin D isolated was then incubated in a growing culture after two hours, labeling was found in streptovaricin C as well 

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Ning, S., Muller, R.,Yu,T.W.,Taylor, M., Hoffmann, D., Kim, C. G., Zhang, X., Hutchinson, C. R., Floss, H. G., Biosynthesis of the ansamycin antibiotic rifamycin deductions from the molecular analysis of the rif biosynthetic gene cluster of Amycolatopsis mediterranei S699, Chem Biol. 1998, 5, 69-79. 

A description of the biosynthesis of the ansamycins can be divided into two parts, namely, the biosynthesis of the chromophoric units and the biosynthesis of the ansa moieties. It was established that the chromophoric moiety of both the benzenoid and the naphthalenoid ansamycins consists of a AW-C7N unit as the biosynthetic precursor. On the other hand, the ansa chains of these ansamycins are derived from polyketide units composed of such as acetic (C2 unit) and propionic (C3 unit) moieties through the polyketide biosynthesis pathway. The biosynthesis of the ansamycins has been reviewed [20,22]. 

As a prelude to the description of the biosynthesis of the ansamycins, the biosynthesis of the AW-C7N unit will be discussed, followed by a description of the biosynthesis of the ansa moiety. 

With respect to the biosynthesis of the ansamycins, the incorporation of [l- C]glucose and [l- Cjglycerate into the chromophoric moiety of rifamycin S was investigated [176]. 

The biosynthetic precursor of the chromophoric moiety was studied vigorously by several research groups. As a result it was established that during the biosynthesis of the ansamycins, a W-C7N unit (Fig. 4) was incorporated into the chromophores of both the benzenoid ansamycins, such as geldanamycin (85) [177, 178] and ansamitocin P-3 (81) [179], and also the naphthalenoid ansamycins, such as the rifamycins [22,180-182] and actamycin (69) [183]. 

Amino-5-hydroxybenzoic Acid - A Key Intermediate in the Biosynthesis of the Ansamycin and Mitomycin Antibiotics. - It has been previously established that the ansa chain of rifamycin S (52) and part of the naphthaquinone moiety is polyketide in origin, being derived from acetate and propionate units, as illustrated schematically in Scheme 5. Other ansamycin antibiotics, such as geldanamycin and actamycin, have similar biosynthetic origin. However, the source of the CyN unit remained an enigma. It was suggested by Hornemann in 1974 that the CyN unit in the... 

The ansa-chain of the ansamycins streptovaricins (4), rifamycins (263), geldanamycin (4), and herbimycin (32) has been shown to be polyketide in origin, being made up of propionate and acetate units with the 0-methyl groups coming from methionine. The remaining aromatic C N portion of the ansamacroHdes is derived from 3-amino-5-hydroxybenzoic acid (264—266) which is formed via shikimate precursors. Based on the precursors of the rifamycins and streptovaricins isolated from mutant bacteria strains, a detailed scheme for the biosynthesis of most of the ansamacroHdes has been proposed (95,263). 

Investigations of the biosynthesis of the rifamycins, streptovaricins and geldanamycin proved that the ansa chain of the ansamycins is synthesized as proposed by Woodward. The isolation of the precursors rifamycin W, damavaricin C and D and the protostreptovaricins, together with the fact that rifamycin B and tolypomycin Y are cosynthesized by Streptomyces tolypophorus26 make it probable that the rifamycins, streptovaricins and tolypomycin Y have a common progenitor. 

Studies using 14C-labelled precursors and C-l3 carbon magnetic resonance have suggested that the biosynthesis of the benzenic ansamycin geldanamycin follows essentially the same pathway as that of the rifamycins and streptovaricins50. Geldanamycin is composed of 3 acetate and four propionate units which are attached to a C7N unit in the same direction of growth as is found in rifamycins and streptovaricins. The incorporation of three acetate units into the ansa chain, as opposed to two in the naphthalenic ansamycins, excludes the existance of a common precursor. 

Nothing is known about the biosynthesis of the maytansine group, the only ansamycins so far identified as being of plant origin. Their similarity to the bacterial metabolite geldanamycin has led to the hypothesis that despite their occurrence in plants, microorganisms might be involved in their production. 

It was also found that when [r- C]-AHBA (118) was administered to a streptovaricin C (44) producing culture, C-21 (the quinone methide carbonyl at 188.3 ppm) of 44 was specifically labelled (Fig. 5) [93], and the existence of 118 itself in the fermentation broth was reported [186,187]. It was also reported that the biosynthesis of the naphthalenoid ansamycin antibiotic actamycin (69) was markedly increased by the... 

The biosynthesis of the rifamycins, including the fact that AHBA (118) was identified as the starter unit for the ansamycins, has been reviewed [22]. 

Regarding the biosynthesis of the benzenoid ansamycin antibiotic ansamitocin P-3 (81), it was reported that the C-NMR signal of the benzyl carbon of ansamitocin was enhanced by feeding [1 -13C]-AHBA (118) [179]. 

As mentioned in the introduction to this chapter, ansamycin antibiotics consist of a chromophoric unit and an ansa moiety. The main portion of the chromophore is derived from a m-CyN unit, and the biosynthesis of this unit was described above. The biosynthesis of the ansa moiety (ansa chain) will be discussed below. 

Because the biosynthesis of the w-CyN unit was discussed previously, only the biosynthesis of the ansa moiety of the benzenoid ansamycin antibiotics with a C15 ansa moiety and the benzenoid ansamycin antibiotics with a C17 ansa moiety will be discussed here. 

Funayama et al. reported the possibility for the application of their model to the stereochemistry and biosynthesis of the Imown benzenoid ansamycin antibiotics [202]. According to the analysis, herbimycin A (86) seemed to possess the same biosynthetic building units as those of... 

An ansamycin antibiotic produced by Streptomyces spectabilis with antibacterial (against tuberculosis pathogens), antiviral, and antitumor activities. S. occurs as a multi-component mixture of up to ten individual compounds (S. A to G, J, K, and U) with S. C (methyl streptovaricate, C4oH5,NO,4, Mr 769.84, amorphous, mp. 189-191 °C, [a][) 4-602°) as the main component. The S. are inhibitors of bacterial RNA-polyme-rase as well as the reverse transcriptase of oncogenic viruses. The biosynthesis of the aromatic core branches off from the shikimic acid pathway while the alkyl chain is formed on the polyketide pathway. 

As implied above, there are two kinds of ansamycins, one that possesses a benzene ring and another that possesses a naphthalene ring in the structure these are known as the benzenoid and naphthalenoid ansamycins, respectively. The m-CyN unit is involved in the biosyntheses of the chromo-phores of both these ansamycins, and the polyketide biosynthetic pathway is also concerned in the biosynthesis of the ansa chains of both types of ansamycin and a part of the chromophore of the naphthalenoid ansamycins. 

Regarding the biosynthesis of the ansa chain and part of the chromophore of rifamycin S, it was shown that two molecules of acetic acid and eight molecules of propionic acid were incorporated, as shown in the figure. During the biosynthesis, the methyl moiety (C-34) at the sixth propionic acid residue (C-27, C-28, and C-34) was eliminated, and the bond between C-29 and C-12 at the seventh propionic acid (C-12, C-13, and C-29) was cleaved and an oxygen was inserted [8,9]. The other part of the naphthalene nucleus was shown to be derived from an m-CyN unit. The more detailed biosynthesis of various naphthalenoid ansamycins will be described later (Section 13.4). 

The origin of the chromophore moiety of these benzenoid ansamycins is the m-CyN unit, and no unit(s) derived from the polyketide pathway are concerned in the biosynthesis of the chromophore, unlike the naphthale-noid ansamycins. Therefore, in the benzenoid ansamycins, it can be more clearly seen how the m-CyN unit is incorporated into the chromophore. This matter will be discussed in Section 13.4. 

THE BIOSYNTHESIS-STEREOCHEMISTRY MODEL (CELMER S MODEL) OF THE ANSAMYCINS... 

The syntheses of [5- H] labelled 5-epishikimic acid and [6 H] labelled shikimic acid have been achieved from L-shikimic acid. The synthesis of several deuterated and "C derivatives of shikimic acid has been described in order to study the biosynthesis of ansatrienin (a member of the ansamycin antibiotics). These derivatives were also used in feeding experiments to determine the biosynthesis of the cyclohexanecarboxylic acid starter unit of ca-cyclohexyl fatty acids in Alicyclobacillus acidocaldarius. ... 

Kim C-G, Kirschning A, Bergon P, Zhou P, Su E, Sauerbrei B, Ning S, Ahn Y, Breuer M, Leistner E, Floss HG (1996) Biosynthesis of 3 -Amino-5 -hydroxybenzoic Acid, the Precursor of mCyN Units in Ansamycin Antibiotics. J Am Chem Soc 118 7486... 

Streptovaridns and Mitomycins.—Consideration of the structures deduced for metabolites with the streptovaricin skeleton which lack in particular the extensive oxygenation of say streptovaricin D, has allowed a reasonable sequence of events for streptovaricin biosynthesis to be proposed. (The streptovaridns belong to the class of ansamydns. For earlier reviews on ansamycin biosynthesis see ref. 5, p. 52, and ref. 6, p. 45.)... 

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. 

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