Synthesis, biological activity and molecular modelling studies of shikimic acid derivatives as inhibitors of the shikimate dehydrogenase enzyme of Escherichia coli.

Shikimic acid (SA) pathway is the common route used by bacteria, plants, fungi, algae, and certain Apicomplexa parasites for the biosynthesis of aromatic amino acids and other secondary metabolites. As this essential pathway is absent in mammals designing inhibitors against implied enzymes may lead to the development of antimicrobial and herbicidal agents harmless to humans. Shikimate dehydrogenase (SDH) is the fourth enzyme of the SA pathway. In this contribution, a series of SA amide derivatives were synthesised and evaluated for in vitro SDH inhibition and antibacterial activity against Escherichia coli. All tested compounds showed to be mixed type inhibitors; diamide derivatives displayed more inhibitory activity than synthesised monoamides. Among the evaluated compounds, molecules called 4a and 4b were the most active derivatives with IC50 588 and 589 µM, respectively. Molecular modelling studies suggested two different binding modes of monoamide and diamide derivatives to the SDH enzyme of E. coli.

Ribosome binding site libraries and pathway modules for shikimic acid synthesis with Corynebacterium glutamicum.

BACKGROUND: The shikimic acid (SA) pathway is a fundamental route to synthesize aromatic building blocks for cell growth and metabolic processes, as well as for fermentative production of various aromatic compounds. Genes encoding enzymes of SA pathway are not continuous on genome and they are differently regulated. RESULTS: In this study, efforts were made to construct continuous genetic modules of SA pathway that are regulated by a same Ptac promoter. Firstly, aro genes [aroG (NCgl2098), aroB (NCgl1559), aroD (NCgl0408) and aroE (NCgl1567)] from Corynebacterium glutamicum and ribosome binding site (RBS) libraries that were tailored for the above genes were obtained, and the strength of each RBS in the 4 libraries was quantified. Secondly, 9 genetic modules were built up from the RBS libraries, a previously characterized ribozyme insulator (RiboJ) and transcriptional promoter (Ptac) and terminator, and aroG, aroB, aroD and aroE. The functionality and efficiency of the constructed genetic modules were evaluated in C. glutamicum by determination of SA synthesis. Results showed that C. glutamicum RES167ΔaroK carrying a genetic module produced 4.3 g/L of SA, which was 54 folds higher compared to that of strain RES167ΔaroK (80 mg/L, without the genetic module) during fermentation in 250-mL flasks. The same strain produced 7.4, and 11.3 g/L of SA during 5-L batch and fed-batch fermentations, respectively, which corresponding to SA molar yields of 0.39 and 0.24 per mole sucrose consumption. CONCLUSION: These results demonstrated that the constructed SA pathway modules are effective in increasing SA synthesis in C. glutamicum, and they might be useful for fermentative production of aromatic compounds derived from SA pathway.

Synthesis of marine natural product (-)-pericosine E.

The first synthesis of (-)-pericosine E (6), a metabolite of the Periconia byssoides OUPS-N133 isolated originally from the sea hare Aplysia kurodai, has been achieved. Efficient and regioselective synthetic procedures for the synthesis of key intermediates, anti- and syn-epoxides 9 and 10, were developed using an anti-epoxidation of diene 12 with TFDO and a bromohydrination of 12 with NBS in CH(3)CN/H(2)O (2:3), respectively. In addition, comparison of the specific optical rotations between synthetic 6 and natural 6 elucidated that the naturally preferred enantiomer of pericosine E had the same absolute configuration as (-)-6 synthesized from chlorohydrin (-)-8 and anti-epoxide (+)-9.

A C2-symmetric chiral pool-based flexible strategy: synthesis of (+)- and (-)-shikimic acids, (+)- and (-)-4-epi-shikimic acids, and (+)- and (-)-pinitol.

Via combination of a novel acid-promoted rearrangement of acetal functionality with the controlled installation of the epoxide unit to create the pivotal epoxide intermediates in enantiomerically pure form, a simple, concise, flexible, and readily scalable enantiodivergent synthesis of (+)- and (-)-shikimic acids and (+)- and (-)-4-epi-shikimic acids has emerged. This simple strategy not only provides an efficient approach to shikimic acids but also can readily be adopted for the synthesis of (+)- and (-)-pinitols. These concise total syntheses exemplify the use of pivotal allylic epoxide 14 and its enantiomer ent-14. A readily available inexpensive C2-symmetric L-tartaric acid (7) served as key precursor. In general, the strategy here provides a neat example of the use of a four-carbon chiron and offers a good account of the synthesis of functionalized cyclohexane targets.

Toward synthesis of carbasugars (+)-gabosine C, (+)-COTC, (+)-pericosine B, and (+)-pericosine C.

Asymmetric total synthesis of (+)-gabosine C, (+)-pericosine B, and (+)-pericosine C has been reported from readily available d-(-)-isoascorbic acid and d-ribose involving Grubbs ring closing metathesis, Morita-Baylis-Hillman (MBH) reaction, and Luche reduction.

Synthesis of the anti-influenza agent (-)-oseltamivir free base and (-)-methyl 3-epi-shikimate.

A new enantioselective synthesis of the anti-influenza agent (-)-oseltamivir free base (7.1% overall yield; 98% ee) and (-)-methyl 3-epi-shikimate (16% overall yield; 98% ee) has been described from readily available raw materials. Sharpless asymmetric epoxidation and diastereoselective Barbier allylation of an aldehyde are the key reactions employed in the incorporation of chirality, while the cyclohexene carboxylic ester core was constructed through a ring closing metathesis reaction.

Production of shikimic acid.

Shikimic acid is a key intermediate for the synthesis of the antiviral drug oseltamivir (Tamiflu®). Shikimic acid can be produced via chemical synthesis, microbial fermentation and extraction from certain plants. An alternative production route is via biotransformation of the more readily available quinic acid. Much of the current supply of shikimic acid is sourced from the seeds of Chinese star anise (Illicium verum). Supply from star anise seeds has experienced difficulties and is susceptible to vagaries of weather. Star anise tree takes around six-years from planting to bear fruit, but remains productive for long. Extraction and purification from seeds are expensive. Production via fermentation is increasing. Other production methods are too expensive, or insufficiently developed. In the future, production in recombinant microorganisms via fermentation may become established as the preferred route. Methods for producing shikimic acid are reviewed.

Facile carbohydrate-based stereocontrolled divergent synthesis of (+)-pericosines A and B.

An isomer-divergent synthesis of naturally occurring pericosines A and B is described starting from a known D-ribose derived ene-diol in 35% and 41% overall yields respectively of which the latter is the best synthetic method reported for pericosine B. The key features of this synthesis include the stereoselective NHK vinylation of the terminal aldehyde to the versatile diolefinic chiral intermediate and elegant conversions of the same to the corresponding final products via RCM (Ring Closing Metathesis).

Glycomimetic building blocks: a divergent synthesis of epimers of shikimic acid.

A divergent synthesis of (-)-4-epi-shikimic acid was developed. This route features a one-pot zinc-mediated reductive ring opening of an arabinofuranose followed by a Barbier reaction and culminates in a ring-closing metathesis. Functionalization of (-)-4-epi-shikimic acid via conjugate addition of a thiol occurs in high diastereoselectivity to afford a product with the features of fucosylated glycans.

Stereostructure reassignment and determination of the absolute configuration of pericosine D(o) by a synthetic approach.

A combination of chemical synthesis and NMR methods was used to reassign the structure of pericosine D(o) (8), a cytotoxic marine natural product produced by the fungus Periconia byssoides OUPS-N133 that was originally derived from the sea hare Aplysia kurodai. Chemical synthesis was used to prepare pericoisne D(o) (8) from a known chlorohydrin that was in turn derived from (-)-quinic acid. The absolute configuration of natural pericosine D(o) (8) was determined to be methyl (3R,4S,5S,6S)-6-chloro-3,4,5-trihydroxy-1-cyclohexene-1-carboxylate. HPLC analyses using a chiral-phase column indicated that pericosine D(o) (8) exists in an enantiomerically pure form in nature.

Chemoenzymatic synthesis of carbasugars (+)-pericosines A-C from diverse aromatic cis-dihydrodiol precursors.

cis-Dihydrocatechols, derived from biological cis-dihydroxylation of methyl benzoate, iodobenzene and benzonitrile, using the microorganism Pseudomonas putida UV4, were converted into pericosines A, C, and B, respectively. This approach constitutes the shortest syntheses, to date, of these important natural products with densely packed functionalities.

Metabolic engineering for the production of shikimic acid in an evolved Escherichia coli strain lacking the phosphoenolpyruvate: carbohydrate phosphotransferase system.

BACKGROUND: Shikimic acid (SA) is utilized in the synthesis of oseltamivir-phosphate, an anti-influenza drug. In this work, metabolic engineering approaches were employed to produce SA in Escherichia coli strains derived from an evolved strain (PB12) lacking the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS-) but with capacity to grow on glucose. Derivatives of PB12 strain were constructed to determine the effects of inactivating aroK, aroL, pykF or pykA and the expression of plasmid-coded genes aroGfbr, tktA, aroB and aroE, on SA synthesis. RESULTS: Batch cultures were performed to evaluate the effects of genetic modifications on growth, glucose consumption, and aromatic intermediate production. All derivatives showed a two-phase growth behavior with initial high specific growth rate (mu) and specific glucose consumption rate (qs), but low level production of aromatic intermediates. During the second growth phase the mu decreased, whereas aromatic intermediate production reached its maximum. The double aroK- aroL- mutant expressing plasmid-coded genes (strain PB12.SA22) accumulated SA up to 7 g/L with a yield of SA on glucose of 0.29 mol/mol and a total aromatic compound yield (TACY) of 0.38 mol/mol. Single inactivation of pykF or pykA was performed in PB12.SA22 strain. Inactivation of pykF caused a decrease in mu, qs, SA production, and yield; whereas TACY increased by 33% (0.5 mol/mol). CONCLUSIONS: The effect of increased availability of carbon metabolites, their channeling into the synthesis of aromatic intermediates, and disruption of the SA pathway on SA production was studied. Inactivation of both aroK and aroL, and transformation with plasmid-coded genes resulted in the accumulation of SA up to 7 g/L with a yield on glucose of 0.29 mol/mol PB12.SA22, which represents the highest reported yield. The pykF and pykA genes were inactivated in strain PB12.SA22 to increase the production of aromatic compounds in the PTS- background. Results indicate differential roles of Pyk isoenzymes on growth and aromatic compound production. This study demonstrated for the first time the simultaneous inactivation of PTS and pykF as part of a strategy to improve SA production and its aromatic precursors in E. coli, with a resulting high yield of aromatic compounds on glucose of 0.5 mol/mol.

Facile and efficient synthesis of naturally occurring carbasugars (+)-pericosines A and C.

An efficient synthesis of antitumor marine natural product (+)-pericosine A was achieved from (-)-quinic acid in 11.7% overall yield, which is 20 times better than our previously reported synthesis. The crucial steps of this synthesis include the regio- and stereoselective bromohydrination of an unstable diene and the ring opening of an epoxide. This synthetic route was applicable to a synthesis of (+)-pericosine C and also to a synthesis of (-)-pericosine C.

Synthesis of (-)-pericosine B, the antipode of the cytotoxic marine natural product.

The stereoselective synthesis of (-)-pericosine B, which is the antipode of the cytotoxic metabolite of the fungus Periconia byssoides OUPS-N133 separated from the sea hare, was accomplished in 9 steps in 12% total yield from (-)-quinic acid, together with the synthesis of its epimer. Every crucial step of this total synthesis, including ring opening of a beta-epoxide and NaBH4 reduction of an unstable beta,gamma-unsaturated enone, proceeded with excellent stereoselectivity.

Monopalmityloxy shikimic acid: enzymatic synthesis and anticoagulation activity evaluation.

The monopalmityloxy shikimic acids have been synthesized from shikimic acid and palmitic acid catalyzed by Novozym 435 in 2-methyl-2-butanol. The anticoagulation activity in vivo via oral administration of monopalmityloxy shikimic acid has been evaluated through arteriovenous shunt model of rats and through the determination of thrombin time, prothrombin time, and activated partial thromboplastin time via rats. After reaction, the solid shikimic acid has been observed to dissolve in the reaction system completely. The subsequent high-performance liquid chromatography-mass spectroscopy analysis showed that the monopalmityloxy shikimic acids, as the only products, had been formed and the overall conversion rate was over 70%. The result showed that it had anti-thrombosis activity, could prolong the coagulating time and bleeding time in vivo, and lengthen the coagulating time in vitro. Compared with control group, the differences of the treatment group and aspirin group of rats are significant (P < 0.05) for prothrombin time and thrombin time, and very significant (P < 0.01) for activated partial thromboplastin time. It suggested that the product had the anticoagulation activity. The mechanism might be the co-action of the inhibition of intrinsic coagulation and the inhibition of extrinsic coagulation, and the inhibiting effect on intrinsic pathway is stronger than that on extrinsic pathway.

Differential inhibition of class I and class II 5-enolpyruvylshikimate-3-phosphate synthases by tetrahedral reaction intermediate analogues.

The shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase or EPSPS) is best known as the target of the herbicide glyphosate. EPSPS is also considered an attractive target for the development of novel antibiotics since the pathogenicity of many microorganisms depends on the functionality of the shikimate pathway. Here, we have investigated the inhibitory potency of stable fluorinated or phosphonate-based analogues of the tetrahedral reaction intermediate (TI) in a parallel study utilizing class I (glyphosate-sensitive) and class II (glyphosate-tolerant) EPSPS. The (R)-difluoromethyl and (R)-phosphonate analogues of the TI are the most potent inhibitors of EPSPS described to date. However, we found that class II EPSPS are up to 400 times less sensitive to inhibition by these TI analogues. X-ray crystallographic data revealed that the conformational changes of active site residues observed upon inhibitor binding to the representative class I EPSPS from Escherichia coli do not occur in the prototypical class II enzyme from Agrobacterium sp. strain CP4. It appears that because the active sites of class II EPSPS do not possess the flexibility to accommodate these TI analogues, the analogues themselves undergo conformational changes, resulting in less favorable inhibitory properties. Since pathogenic microorganisms such as Staphylococcus aureus utilize class II EPSPS, we conclude that the rational design of novel EPSPS inhibitors with potential as broad-spectrum antibiotics should be based on the active site structures of class II EPSP synthases.

First total synthesis of antitumor natural product (+)- and (-)-pericosine A: determination of absolute stereo structure.

The first total synthesis of (+)- and (-)-pericosine A has been achieved, enabling the revision and determination of the absolute configuration of this antitumor natural product as methyl (3S,4S,5S,6S)-6-chloro-3,4,5-trihydroxy-1-cyclohexene-1-carboxylate. Every step of this total synthesis proceeded well with excellent stereoselectivity. Structures of the intermediates in crucial steps were confirmed by detailed 2D NMR analysis.

Mycobacterial shikimate pathway enzymes as targets for drug design.

The aetiological agent of tuberculosis (TB), Mycobacterium tuberculosis, is responsible for millions of deaths annually. The increasing prevalence of the disease, the emergence of multidrug-resistant strains, and the devastating effect of human immunodeficiency virus co-infection have led to an urgent need for the development of new and more efficient antimycobacterial drugs. Since the shikimate pathway is present and essential in algae, higher plants, bacteria, and fungi, but absent from mammals, the gene products of the common pathway might represent attractive targets for the development of new antimycobacterial agents. In this review we describe studies on shikimate pathway enzymes, including enzyme kinetics and structural data. We have focused on mycobacterial shikimate pathway enzymes as potential targets for the development of new anti-TB agents.