Total Synthesis of Complex Biosynthetic Late-Stage Intermediates and Bioconversion by a Tailoring Enzyme from Jerangolid Biosynthesis.

A highly convergent access to the late-stage biosynthetic intermediates projerangolid and jerangolid E is presented, and its utility is demonstrated by the synthesis of novel non-natural jerangolid derivatives. The key steps are fragment couplings by Julia-Kocienski olefination and olefin cross metathesis, as well as a stereoselective tetrahydropyran formation by intramolecular oxa-Michael addition. Bioconversion experiments with the tailoring O-methyltransferase JerF confirmed its proposed biosynthetic role and revealed relaxed substrate specificity of this enzyme as well as tolerance to organic cosolvents.

Characterisation of the Broadly-Specific O-Methyl-transferase JerF from the Late Stages of Jerangolid Biosynthesis.

We describe the characterisation of the O-methyltransferase JerF from the late stages of jerangolid biosynthesis. JerF is the first known example of an enzyme that catalyses the formation of a non-aromatic, cyclic methylenolether. The enzyme was overexpressed in E. coli and the cell-free extracts were used in bioconversion experiments. Chemical synthesis gave access to a series of substrate surrogates that covered a broad structural space. Enzymatic assays revealed a broad substrate tolerance and high regioselectivity of JerF, which makes it an attractive candidate for an application in chemoenzymatic synthesis with particular usefulness for late stage application on 4-methoxy-5,6-dihydro-2H-pyran-2-one-containing natural products.

Synthesis of complex intermediates for the study of a dehydratase from borrelidin biosynthesis.

Herein, we describe the syntheses of a complex biosynthesis-intermediate analogue of the potent antitumor polyketide borrelidin and of reference molecules to determine the stereoselectivity of the dehydratase of borrelidin polyketide synthase module 3. The target molecules were obtained from a common precursor aldehyde in the form of N-acetylcysteamine (SNAc) thioesters and methyl esters in 13 to 15 steps. Key steps for the assembly of the polyketide backbone of the dehydratase substrate analogue were a Yamamoto asymmetric carbocyclisation and a Sakurai allylation as well as an anti-selective aldol reaction. Reference compounds representing the E- and Z-configured double bond isomers as potential products of the dehydratase reaction were obtained from a common precursor aldehyde by Wittig olefination and Still-Gennari olefination. The final deprotection of TBS ethers and methyl esters was performed under mildly acidic conditions followed by pig liver esterase-mediated chemoselective hydrolysis. These conditions are compatible with the presence of a coenzyme A or a SNAc thioester, suggesting that they are generally applicable to the synthesis of complex polyketide-derived thioesters suited for biosynthesis studies.

Rieske Oxygenase-Catalyzed Oxidative Late-Stage Functionalization during Complex Antifungal Polyketide Biosynthesis.

Rieske oxygenases (ROs) from natural product biosynthetic pathways are a poorly studied group of enzymes with significant potential as oxidative functionalization biocatalysts. A study on the ROs JerL, JerP, and AmbP from the biosynthetic pathways of jerangolid A and ambruticin VS-3 is described. Their activity was successfully reconstituted using whole-cell bioconversion systems coexpressing the ROs and their respective natural flavin-dependent reductase (FDR) partners. Feeding authentic biosynthetic intermediates and synthetic surrogates to these strains confirmed the involvement of the ROs in hydroxymethylpyrone and dihydropyran formation and revealed crucial information about the RO's substrate specificity. The pronounced dependence of JerL and JerP on the presence of a methylenolether allowed the precise temporal assignment of RO catalysis to the ultimate steps of jerangolid biosynthesis. JerP and AmbP stand out among the biosynthetic ROs studied so far for their ability to catalyze clean tetrahydropyran desaturation without further functionalizing the formed electron-rich double bonds. This work highlights the remarkable ability of ROs to highly selectively oxidize complex molecular scaffolds.

In vitro studies of maleidride-forming enzymes.

In vitro assays of enzymes involved in the biosynthesis of maleidrides from polyketides in fungi were performed. The results show that the enzymes are closely related to primary metabolism enzymes of the citric acid cycle in terms of stereochemical preferences, but with an expanded substrate selectivity. A key citrate synthase can react both saturated and unsaturated acyl CoA substrates to give solely anti substituted citrates. This undergoes anti-dehydration to afford an unsaturated precursor which is cyclised in vitro by ketosteroid-isomerase-like enzymes to give byssochlamic acid.

Molecular basis of methylation and chain-length programming in a fungal iterative highly reducing polyketide synthase.

Exchange of 32 different sub-fragments of the C-methyltransferase (C-MeT), pseudo-ketoreductase (ΨKR) and ketoreductase (KR) catalytic domains of the tenellin iterative Type I polyketide synthase non ribosomal peptide synthetase (PKS-NRPS) TENS by homologous fragments from the desmethylbassianin (DMBS) and militarinone (MILS) PKS-NRPS led to the creation of chimeric synthetases in which programming fidelity was altered, resulting in the production of mixtures of products with different methylation patterns and chain lengths. Swap of KR domain subfragments with the homologous fragments from the KR of the heptaketide militarinone synthetase resulted in the synthesis of penta, hexa and heptaketides. The results of these and previous experiments are rationalised by considering the existence of competition for acyl-carrier protein (ACP) bound substrates between different catalytic domains of the PKS. In particular, competition between the C-MeT and ketoreductase domains (KR) can account for methylation programming, and competition between the KR and the off-loading NRPS accounts for chain-length selectivity.

Acupuncture in the treatment of alcohol withdrawal symptoms: a randomized, placebo-controlled inpatient study.

Thirty-four alcoholics were treated with acupuncture to the ear and the body in a randomized single-blind placebo-controlled design over 14 days. Orthodox points and placebo needles to orthodox points were used daily for a total of 10 treatments starting on the first day of admission as add-on therapy to standard medication with carbamazepine. The primary outcome was the Clinical Institute Withdrawal Assessment (CIWA-Ar-scale) assessed on days 1-6, 9 and 14. No initial differences were found regarding sociodemographic data, drinking history and alcohol-related data, indicating successful randomization. Longitudinal analysis of the Clinical Institute Withdrawal Assessment (CIWA-Ar-scale) data showed that patients assigned to acupuncture had a general tendency towards better outcome results and significantly fewer withdrawal symptoms on day 14 (Wilcoxon-W=177.500, Z=-2.009, p = 0.045). No significant differences were found in the Beck Depression Inventory (BDI), State-Trait Anxiety Inventory (STAI X1 and X2) and Eigenschaftswoerterliste (EWL S60). We conclude that acupuncture as an adjunctive treatment to carbamazepine medication shows promise for the treatment of alcohol withdrawal symptoms. Further investigation of this treatment modality appears to be warranted.