Stereospecificity of ketoreductase domains of the 6-deoxyerythronolide B synthase

6-Deoxyerythronolide B synthase (DEBS) is a modular polyketide synthase (PKS) responsible for the biosynthesis of 6-dEB (1), the parent aglycone of the broad spectrum macrolide antibiotic erythromycin. Individual DEBS modules, which contain the catalytic domains necessary for each step of polyketide chain elongation and chemical modification, can be deconstructed into constituent domains. To better understand the intrinsic stereospecificity of the ketoreductase (KR) domains, an in vitro reconstituted system has been developed involving combinations of ketosynthase (KS)-acyl transferase (AT) didomains with acyl-carrier protein (ACP) and KR domains from different DEBS modules. Incubations with (2S,3R)-2-methyl-3-hydroxypentanoic acid N-acetylcysteamine thioester (2) and methylmalonyl-CoA plus NADPH result in formation of a reduced, ACP-bound triketide that is converted to the corresponding triketide lactone 4 by either base- or enzyme-catalyzed hydrolysis/cyclization. A sensitive and robust GC-MS technique has been developed to assign the stereochemistry of the resulting triketide lactones, on the basis of direct comparison with synthetic standards of each of the four possible diasteromers 4a-4d. Using the [KS][AT] didomains from either DEBS module 3 or module 6 in combination with KR domains from modules 2 or 6 gave in all cases exclusively (2R,3S,4R,5R)-3,5-dihydroxy-2,4-dimethyl-n-heptanoic acid-delta-lactone (4a). The same product was also generated by a chimeric module in which [KS3][AT3] was fused to [KR5][ACP5] and the DEBS thioesterase [TE] domain. Reductive quenching of the ACP-bound 2-methyl-3-ketoacyl triketide intermediate with sodium borohydride confirmed that in each case the triketide intermediate carried only an unepimerized d-2-methyl group. The results confirm the predicted stereospecificity of the individual KR domains, while revealing an unexpected configurational stability of the ACP-bound 2-methyl-3-ketoacyl thioester intermediate. The methodology should be applicable to the study of any combination of heterologous [KS][AT] and [KR] domains.     

Formal synthesis of amphidinin B

An efficient, convergent, and highly stereoselective formal synthesis of amphidinin B (1) is reported herein. In Amphidinin B both C10–C21 (4) and C1–C9 (5) fragments were derived from geraniol 6 and mono-PMB ether of 1,4-butane diol 7 in 19 and 9 steps, respectively. The key steps involved in this synthesis are Sharpless asymmetric epoxidation, Evans aldol, Julia olefination, oxa-Michael, Keck allylation, Mannich reaction, Evans asymmetric alkylation, and Yamaguchi esterification.     

Formal synthesis of tirandamycin B

A formal synthesis of tirandamycin B is described. The key intermediate is synthesized by Marshall allenyl zinc method, litiofuran coupling, and Achmatowicz reaction to construct the bicyclic core of tirandamycin B.     

Formal synthesis of (+)-didemniserinolipid B

The formal synthesis of (+)-didemniserinolipid B is achieved using Weinreb reaction, Yadav’s chiral propargyl alcohol protocol and hydrolytic ketal formation as the key steps.     

Formal synthesis of schulzeines B and C

A formal synthesis of schulzeines B and C, marine natural products with inhibitory effect against α-glucosidase, has been achieved. The key reactions of the synthesis are N-acyliminium ion cyclization, Sharpless asymmetric dihydroxylation, olefin cross metathesis, and asymmetric allylboration.     

Formal total synthesis of (+)-didemniserinolipid B

The formal total synthesis of (+)-didemniserinolipid B, a marine tunicate possessing a 6,8-dioxabicyclo[3.2.1]octane framework, was accomplished starting from l-(+)-tartaric acid. The key transformations in the synthesis include the elaboration of a γ-hydroxy-amide readily obtained by desymmetrization of tartaric acid bis-amide via the controlled addition of a Grignard reagent followed by stereoselective reduction of the resulting ketone.     

Formal total synthesis of salicylihalamides A and B

An efficient synthesis of the macrolactone 3 of the salicylihalamides in 10 linear steps from alkene 6 is described. The key steps involved a Stille coupling between the chiral stannane 5 and benzyl bromide 4, which produced alkene 15 in good yield, and subsequent base-induced macrolactonization then gave compound 3. Macrolactone 3 was then converted into the known salicylihalamide A intermediate 18 in a three-step sequence. Compound 3 was also converted into another known salicylihalamide A and B intermediate 23 in a five-step sequence.     

Formal total synthesis of aspergillides A and B

The formal total synthesis of the cytotoxic 14-membered macrolides, aspergillides A and B is described. A combination of a chiron approach and an asymmetric synthesis is adopted for the synthesis of the target macrolides. The required 2,6-syn and 2,6-anti tetrahydropyrans were constructed via a tandem Sharpless asymmetric epoxidation and 6-exo cyclization on δ-hydroxy allylic alcohols, as the key steps. The requisite chiral synthon was prepared from l-ascorbic acid.     

Extender unit and acyl carrier protein specificity of ketosynthase domains of the 6-deoxyerythronolide B synthase

Polyketide synthases (PKSs) catalyze the production of numerous biologically important natural products via repeated decarboxylative condensation reactions. Modular PKSs, such as the 6-deoxyerythronolide B synthase (DEBS), consist of multiple catalytic modules, each containing a unique set of covalently linked catalytic domains. To better understand the engineering opportunities of these assembly lines, the extender unit and acyl carrier protein (ACP) specificity of keto synthase (KS) domains from modules 3 and 6 of DEBS were analyzed. These studies were undertaken with a newly developed didomain [KS][AT] construct, which lacks its own ACP domain and can therefore be interrogated with homologous or heterologous ACP or acyl-ACP substrates. By substituting the natural methylmalonyl extender unit with a malonyl group, a modest role was demonstrated for the KS in recognition of the nucleophilic substrate. The KS domain from module 3 of DEBS was found to exhibit a distinct ACP-recognition profile from the KS domain of module 6. On the basis of the above kinetic insights, a hybrid module was constructed ([KS3][AT3][KR5][ACP5][TE]) which displayed substrate recognition and elongation capabilities consistent with the natural module 3 protein. Unlike module 3, however, which lacks a ketoreductase (KR) domain, the hybrid module was able to catalyze reduction of the beta-ketothioester product of chain elongation. The high expression level and functionality of this hybrid protein demonstrates the usefulness of kinetic analysis for hybrid module design.     

Structural and mechanistic analysis of protein interactions in module 3 of the 6-deoxyerythronolide B synthase

We report the 2.6 A X-ray crystal structure of a 190 kDa homodimeric fragment from module 3 of the 6-deoxyerthronolide B synthase covalently bound to the inhibitor cerulenin. The structure shows two well-organized interdomain linker regions in addition to the full-length ketosynthase (KS) and acyltransferase (AT) domains. Analysis of the substrate-binding site of the KS domain suggests that a loop region at the homodimer interface influences KS substrate specificity. We also describe a model for the interaction of the catalytic domains with the acyl carrier protein (ACP) domain. The ACP is proposed to dock within a deep cleft between the KS and AT domains, with interactions that span both the KS homodimer and AT domain. In conjunction with other recent data, our results provide atomic resolution pictures of several catalytically relevant protein interactions in this remarkable family of modular megasynthases.     

Formal synthesis of (±)-indolizidine 209B

A new method for the synthesis of cis-C-8 methylated indolizidines and quinolizidines has been developed. Straightforward transformations including an isomerization to the trans configuration led to a formal synthesis of (±)-indolizidine 209B.     

Formal total synthesis of hemibrevetoxin B by a convergent strategy

Concise construction of the trans-fused 7/7/6/6 tetracyclic ether part of hemibrevetoxin B (1) was achieved by a convergent strategy based on coupling reaction of an acyl anion equivalent, reductive cyclization of an α,ε-dihydroxyketone, and introduction of a methyl group at the central ring junction by the Nicolaou method. The resultant tetracyclic ether was transformed into the known intermediate, which was already converted to 1 by the Yamamoto group, thereby completing the formal total synthesis of 1.     

Novel octaketide macrolides related to 6-deoxyerythronolide B provide evidence for iterative operation of the erythromycin polyketide synthase

BACKGROUND: The macrolide antibiotic erythromycin A, like other complex aliphatic polyketides, is synthesised by a bacterial modular polyketide synthase (PKS). Such PKSs, in contrast to other fatty acid and polyketide synthases which work iteratively, contain a separate set or module of enzyme activities for each successive cycle of polyketide chain extension, and the number and type of modules together determine the structure of the polyketide product. Thus, the six extension modules of the erythromycin PKS (DEBS) together catalyse the production of the specific heptaketide 6-deoxyerythronolide B. RESULTS: A mutant strain of the erythromycin producer Saccharopolyspora erythraea, which accumulates the aglycone intermediate erythronolide B, was found unexpectedly to produce two novel octaketides, both 16-membered macrolides. These compounds were detectable in fermentation broths of wild-type S. erythraea, but not in a strain from which the DEBS genes had been specifically deleted. From their structures, both of these octaketides appear to be aberrant products of DEBS in which module 4 has 'stuttered', that is, has catalysed two successive cycles of chain extension. CONCLUSIONS: The isolation of novel DEBS-derived octaketides provides the first evidence that an extension module in a modular PKS has the potential to catalyse iterative rounds of chain elongation like other type I FAS and PKS systems. The factors governing the extent of such 'stuttering' remain to be determined.     

Formal synthesis of dysiherbaine

A formal synthesis of dysiherbaine was achieved from d-mannitol using Grignard addition on chiral imine, RCM and Michael addition as key steps.     

Formal synthesis of cortistatins

A unified strategy toward the asymmetric facile construction of the [6.7.6.5]oxapentacyclic skeleton of cortistatins is reported, featuring intramolecular Diels-Alder (IMDA), oxidative dearomatization, and an oxy-Michael addition reaction.     

Formal synthesis of fumonisin B1, a potent sphingolipid biosynthesis inhibitor

The formal total synthesis of the important toxin fumonisin B₁ is achieved from simple raw materials in a convergent manner. The key functionalities are derived from MacMillan α-hydroxylation, sharpless asymmetric dihydroxylation and ring-closing metathesis.     

Versatile synthesis of 6-substituted 8-deazapteridine-2,4-diamines. Formal total synthesis of 8,10-dideazaminopterin

A new synthesis of 4-amino-4-deoxy-8,10-dideazapteroic acid ( 11d ) and 6-substituted and 5,6-anellated 8-deazapteridine-2,4-diamines, 10a, 10d, 25 , is described. Starting from keteneaminals 1 or 12 and enaminones 4 or β-aminoketone 17 the title compounds can be prepared via functional group transformation of 2-amino-3-nitropyridines 5 or nicotinate 13a yielding 3-amino-α-picolinonitriles 9 which are cyclocondensed with guanidine.     

Formal synthesis of leustroducsin B via Reformatsky/Claisen condensation of silyl glyoxylates

A formal synthesis of leustroducsin B has been completed. The synthesis relies upon a recently developed Reformatsky/Claisen condensation of silyl glyoxylates and enantioenriched β-lactones that establishes two of the molecule's three core stereocenters and permits further elaboration to an intermediate in Imanishi's synthesis via reliable chemistry (Prasad reduction, asymmetric pentenylation, Mitsunobu inversion).     

Formal total synthesis of fostriecin

[reaction: see text] Addition of magnesium anion 4 (M = MgBr, R(1) = TES) to ketone 6 (R(2) = PMB) at -78 degrees C in THF proceeded under chelation control to provide alcohol 7, which possesses the full set of the chiral centers of fostriecin. Subsequently, 7 was transformed successfully to the known key intermediate 2.