Sandeep Handa

3-Amino-5-hydroxybenzoic Acid Synthase, the Terminal Enzyme in the Formation of the Precursor of mC7N Units in Rifamycin and Related Antibiotics

The biosynthesis of ansamycin antibiotics, like rifamycin B, involves formation of 3-amino-5-hydroxybenzoic acid (AHBA) by a novel variant of the shikimate pathway. AHBA then serves as the starter unit for the assembly of a polyketide which eventually links back to the amino group of AHBA to form the macrolactam ring. The terminal enzyme of AHBA formation, which catalyzes the aromatization of 5-deoxy-5-amino-3-dehydroshikimic acid, has been purified to homogeneity from Amycolatopsis mediterranei, the encoding gene has been cloned, sequenced, and overexpressed in Escherichia coli. The recombinant enzyme, a (His)6 fusion protein, as well as the native one, are dimers containing one molecule of pyridoxal phosphate per subunit. Mechanistic studies showed that the enzyme-bound pyridoxal phosphate forms a Schiff’s base with the amino group of 5-deoxy-5-amino-3-dehydroshikimic acid and catalyzes both an α,β-dehydration and a stereospecific 1,4-enolization of the substrate. Inactivation of the gene encoding AHBA synthase in theA. mediterranei genome results in loss of rifamycin formation; production of the antibiotic is restored when the mutant is supplemented with AHBA.

Evaluating precursor-directed biosynthesis towards novel erythromycins through in vitro studies on a bimodular polyketide synthase

BACKGROUND Modular polyketide synthases (PKSs) catalyse the biosynthesis of complex polyketides using a different set of enzymes for each successive cycle of chain extension. Directed biosynthesis starting from synthetic diketides is a potentially valuable route to novel polyketides. We have used a purified bimodular derivative of the erythromycin-producing polyketide synthase (DEBS 1-TE) to study chain extension starting from a variety of diketide analogues and, in some cases, from the alternative acyl-CoA thioester substrates. RESULTS Chain initiation in vitro by DEBS 1-TE module 2 using a synthetic diketide analogue as a substrate was tolerant of significant structural variation in the starter unit of the synthetic diketide, but other changes completely abolished activity. Interestingly, a racemic beta-keto diketide was found to be reduced in situ on the PKS and utilised in place of its more complex hydroxy analogue as a substrate for chain extension. The presence of a diketide analogue strongly inhibited chain initiation via the loading module. Significantly higher concentrations of diketide N-acetylcysteamine analogues than their corresponding acyl-CoA thioesters are required to achieve comparable yields of triketide lactones. CONCLUSIONS Although a broad range of variation in the starter residue is acceptable, the substrate specificity of module 2 of a typical modular PKS in vitro is relatively intolerant of changes at C-2 and C-3. This will restrict the usefulness of approaches to synthesise novel erythromycins using synthetic diketides in vivo. The use of synthetic beta-keto diketides in vivo deserves to be explored.

Biosynthesis of tetronasin: Part 6. Preparation of structural analogues of the diketide and triketide biosynthetic precursors to tetronasin

Abstract The preparation of three analogues of the putative diketide biosynthetic precursor ( 2 ) and eight analogues of the putative triketide biosynthetic precursor ( 3 ) of the acyl tetronic acid ionophore tetronasin, as N-acetylcysteamine thioesters ( 4 ), ( 5 ), ( 6 ), ( 12 ), ( 13 ), ( 14 ), ( 15 ), ( 22 ), ( 23 ), ( 24 ) and ( 25 ) is described. Five examples are 19 F-labelled; a new, enantiospecific method for the creation of a fluorinated quaternary α-centre is presented.

Cascade radical cyclisations leading to steroid ring constructions. Regio- and stereo-chemical studies using ester- and fluoro-alkene substituted polyene acyl radical intermediates

A study of the factors affecting the regio- and stereo-selective outcomes of consecutive 6-endo-trig cyclisations of polyene acyl-radical intermediates, leading to decalone, perhydrophenanthrone, and steroid ring constructions, has been carried out. Thus, whereas the E-substituted diene selenyl ester 7 underwent sequential cyclisations in the presence of Bu3SnH-AIBN leading to the trans-decalone 8 exclusively, the corresponding Z- and E-isomers of the methoxycarbonyl-substituted diene 16, under similar conditions, gave rise to a 2∶1 mixture of the trans- and cis-decalones 17a and 17b respectively in 62–73% yield. Cyclisation of the triene selenoate 30 led to a single tricyclic product in 57% yield whose cis,syn,trans relative stereochemistry 32 was established by X-ray diffraction analysis. When solutions of the trienyne selenoates 41a–c in benzene were treated with Bu3SnH–AIBN they each underwent cascades of three 6-endo-trig followed by a 5-exo-dig cyclisation leading to the full steroid ring systems 42, 45, and 47 respectively in 20–40% yields. The stereochemistries of the major steroid diastereoisomers resulting from 41a and 41c were established as trans,anti,trans,anti,cis, e.g.47, following X-ray crystallographic analysis of the corresponding dione 44 produced from 42d and 47 after ozonisation. In each of the cyclisations leading to 42 and 45 varying amounts of other bicyclic products tentatively assigned as 43 and 46 respectively, resulting from a competing radical pathway involving first a 10-endo-trig macrocyclisation of the corresponding acyl radical intermediate onto the C9–C10 olefin in 43/46, followed by a 5-exo-trig cyclisation of the resulting radical intermediate onto the proximal C13–C14 double bond, were produced concurrently. Finally, when the fluoro-alkene selenoate 56 was treated with Bu3SnH–AIBN, a complex mixture of polycyclic products resulted, from which only the indanone 57 could be separated and characterized. The origins of the differing regio- and stereo-selective outcomes in the aforementioned radical cascades are briefly considered.

Biosynthesis of tetronasin: Part 2 identification of the tetraketide intermediate attached to the polyketide synthase

Abstract Incorporation experiments with deuterium labelled N-acetyl cysteamine analogues ( 4a ) and ( 4b ) of the proposed enzyme-bound tetraketide precursor ( 4 ) show that the acyl residue is incorporated intact into tetronasin ( 6 ). Equivalent experiments with the deuterium labelled N-acetyl cysteamine analogues ( 7 ), ( 8 ) and ( 9 ), all diastereoisomers of ( 4 ), result in no intact incorporation into ( 6 ).

Biosynthesis of tetronasin: Part 5. Novel fluorinated and non-fluorinated analogues of tetronasin via intact incorporation of di-, tri- and tetraketide analogue precursors

Abstract Incorporation experiments with both fluorine-labelled and unlabelled N-acetylcysteamine analogues ( 6 ), ( 7 ), ( 8 ), ( 9 ), ( 11 ), ( 13 ), ( 14 ) and ( 17 ) of the proposed di-, tri- and tetraketide precursors showed that the acyl residues are incorporated intact into tetronasin ( 1 ). Equivalent experiments with N-acetylcysteamine analogues ( 12 ), ( 15 ), ( 16 ) and ( 18 ) resulted in no detected intact incorporation into ( 1 ).

A new approach to steroid ring construction based on a novel radical cascade sequence

A new approach to steroid ring construction based on sequential cascade 6-endo-trig cyclisation/macrocyclisation/transannulation reactions and exemplified in the synthesis of the cis, anti, cis, anti, cis tetracycle 17 from 15, is presented.