Synthesis and Evaluation of Fluoroalkyl Phosphonyl Analogues of 2- C-Methylerythritol Phosphate as Substrates and Inhibitors of IspD from Human Pathogens.

Targeting essential bacterial processes beyond cell wall, protein, nucleotide, and folate syntheses holds promise to reveal new antimicrobial agents and expand the potential drugs available for combination therapies. The synthesis of isoprenoid precursors, isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), is vital for all organisms; however, humans use the mevalonate pathway for production of IDP/DMADP while many pathogens, including Plasmodium falciparum and Mycobacterium tuberculosis, use the orthogonal methylerythritol phosphate (MEP) pathway. Toward developing novel antimicrobial agents, we have designed and synthesized a series of phosphonyl analogues of MEP and evaluated their abilities to interact with IspD, both as inhibitors of the natural reaction and as antimetabolite alternative substrates that could be processed enzymatically to form stable phosphonyl analogues as potential inhibitors of downstream MEP pathway intermediates. In this compound series, the S-monofluoro MEP analogue displays the most potent inhibitory activity against Escherichia coli IspD and is the best substrate for both the E. coli and P. falciparum IspD orthologues with a Km approaching that of the natural substrate for the E. coli enzyme. This work represents a first step toward the development of phosphonyl MEP antimetabolites to modulate early isoprenoid biosynthesis in human pathogens.

Synthesis of methylerythritol phosphate analogues and their evaluation as alternate substrates for IspDF and IspE from Agrobacterium tumefaciens.

The methylerythritol phosphate biosynthetic pathway, found in most Bacteria, some parasitic protists, and plant chloroplasts, converts D-glyceraldehyde phosphate and pyruvate to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), where it intersects with the mevalonate pathway found in some Bacteria, Archaea, and Eukarya, including the cytosol of plants. D-3-Methylerythritol-4-phosphate (MEP), the first pathway-specific intermediate in the pathway, is converted to IPP and DMAPP by the consecutive action of the IspD-H proteins. We synthesized five D-MEP analogues-D-erythritol-4-phosphate (EP), D-3-methylthrietol-4-phosphate (MTP), D-3-ethylerythritol-4-phosphate (EEP), D-1-amino-3-methylerythritol-4-phosphate (NMEP), and D-3-methylerythritol-4-thiolophosphate (MESP)-and studied their ability to function as alternative substrates for the reactions catalyzed by the IspDF fusion and IspE proteins from Agrobacterium tumefaciens, which covert MEP to the corresponding eight-membered cyclic diphosphate. All of the analogues, except MTP, and their products were substrates for the three consecutive enzymes.

[Screening of potential antibiotics, inhibitors of the nonmevalonate pathway of isoprenoid biosynthesis--2-C-methyl-D-erythritol-2,4-cyclodiphosphate derivatives].

The recently discovered nonmevalonate pathway of isoprenoid biosynthesis is a prospective target in screening of new antibiotics. Because of the absence of the pathway in the animal cells, the specific inhibitors of the pathway will be a new class of antibiotics against many pathogens (which cause, e.g., malaria, tuberculosis, etc), combining high efficiency and low toxicity. Several derivatives of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEC) were synthesized. 4-Phospho-methyl-D-erythritol-1,2-cyclophosphate, benzyl ether and benzyliden derivative of MEC inhibited the 14C-MEC incorporation into isoprenoids of chromoplasts from red pepper with IC50 of 1.7-5 MM. Some inhibition (about 10%) was also observed with the use of dimethyl ether and isopropyliden derivative of MEC.

Synthesis of 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate and kinetic studies of Mycobacterium tuberculosis IspF.

Many pathogenic bacteria utilize the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway for the biosynthesis of isopentenyl diphosphate and dimethylallyl diphosphate, two major building blocks of isoprenoid compounds. The fifth enzyme in the MEP pathway, 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (ME-CPP) synthase (IspF), catalyzes the conversion of 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate (CDP-ME2P) to ME-CPP with a corresponding release of cytidine 5-monophosphate (CMP). Because there is no ortholog of IspF in human cells, IspF is of interest as a potential drug target. However, study of IspF has been hindered by a lack of enantiopure CDP-ME2P. Herein, we report the first, to our knowledge, synthesis of enantiomerically pure CDP-ME2P from commercially available D-arabinose. Cloned, expressed, and purified M. tuberculosis IspF was able to utilize the synthetic CDP-ME2P as a substrate, a result confirmed by mass spectrometry. A convenient, sensitive, in vitro IspF assay was developed by coupling the CMP released during production of ME-CPP to mononucleotide kinase, which can be used for high throughput screening.

Dihydroxylation of 4-substituted 1,2-dioxines: a concise route to branched erythro sugars.

The synthesis of 2-C-branched erythritol derivatives, including the plant sugar (+/-)-2-C-methylerythritol 2, was achieved through a dihydroxylation/reduction sequence on a series of 4-substituted 1,2-dioxines 3. The asymmetric dihydroxylation of 1,2-dioxines was examined, providing access to optically enriched dihydroxy 1,2-dioxanes 4. The synthesized 1,2-dioxanes were converted to other erythro sugar analogues and tetrahydrofurans through controlled cleavage of the endoperoxide linkage.

Expression and characterization of soluble 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase from bacterial pathogens.

Many bacterial pathogens utilize the 2-C-methyl-D-erythritol 4-phosphate pathway for biosynthesizing isoprenoid precursors, a pathway that is vital for bacterial survival and absent from human cells, providing a potential source of drug targets. However, the characterization of 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDP-ME) kinase (IspE) has been hindered due to a lack of enantiopure CDP-ME and difficulty in obtaining pure IspE. Here, enantiopure CDP-ME was chemically synthesized and recombinant IspE from bacterial pathogens were purified and characterized. Although gene disruption was not possible in Mycobacterium tuberculosis, IspE is essential in Mycobacterium smegmatis. The biochemical and kinetic characteristics of IspE provide the basis for development of a high throughput screen and structural characterization.

Synthesis and analysis of a fluorinated product analogue as an inhibitor for 1-deoxy-D-xylulose 5-phosphate reductoisomerase.

1-deoxy-D-xylulose 5-phosphate (DXP) reductoisomerase (DXR) is an NADPH-dependent enzyme catalyzing the rearrangement and reduction of DXP to methyl-D-erythritol 4-phosphate (MEP). Two mechanisms for this enzymatic reaction have been proposed, involving either an alpha-ketol rearrangement or a retroaldol/aldol rearrangement. In this study, a fluorinated product analogue, FCH(2)-MEP, was synthesized as a possible mechanism-based inactivator for DXR if the retroaldol/aldol mechanism is operative. FCH(2)-MEP was found to be a weak competitive inhibitor, and thus was unable to discriminate between the mechanisms. This result is due to the inability of the targeted enzyme, DXR, to oxidize FCH(2)-MEP to the aldehyde intermediate that is common to both mechanisms. While FCH(2)-MEP failed to act as a mechanism-based inactivator, the insight gained from this study will assist in the future design of inhibitors of DXR.

The dioxanone approach to (2S,3R)-2-C-methylerythritol 4-phosphate and 2,4-cyclodiphosphate, and various MEP analogues.

Efficient syntheses of the non-mevalonate pathway intermediates 2-C-methylerythritol 4-phosphate (MEP) and 2-C-methylerythritol 2,4-cyclodiphosphate (ME-2,4-cycloPP), as well as the parent tetrol 2-C-methylerythritol, in enantiopure form from (2S,4R)-cis-2-phenyl-4-tert-butyldimethylsilyloxy-1,3-dioxan-5-one are reported. The 2S configuration of the C-methyl group was installed by highly axial-face selective addition of CH3MgBr (20:1) to the chiral dioxanone carbonyl group. Primary selective mono-phosphorylation and 2,4-bis-phosphorylation, followed by desilation and hydrogenolysis to the free mono- and diphosphates, and, in the latter case, cyclization to form the eight-membered phosphoryl anhydride, afforded MEP and ME-2,4-cycloPP in good yields. The C2 epimeric analogues, 2-C-methylthreitol and its 4-phosphate, were accessed by LiAlH4 reduction of the cis,cis epoxide of (2S,4R)-4-tert-butyldimethylsilyloxymethyl-5-methylene-2-phenyl-1,3-dioxane, primary-selective phosphorylation, and cleavage of the silyl, benzylidene, and benzyl protecting groups. Regioselective cleavage of the acetal ring of 1,3-benzylidene 2-C-methylerythritol silyl ether by ozonolysis afforded a 1,2,3-triol 3-monobenzoate intermediate that was converted to the novel amino sugar, 1-amino-1-deoxy-2-C-methylerythritol.

Enantioselective synthesis of C3 fluoro-MEP.

The first enantioselective synthesis of C(3) fluoro-MEP is herein reported. The synthetic pathway developed takes advantage of a selective hydrofluorination of a 2,3-epoxy-1-alcohol to introduce the required tertiary fluoride unit.

Synthesis of new analogues of salacinol containing a pendant hydroxymethyl group as potential glycosidase inhibitors.

The synthesis of new analogues of the naturally occurring glycosidase inhibitor, salacinol, and its ammonium analogue, ghavamiol is described. These analogues contain an additional hydroxymethyl group at C-1, which was intended to form additional polar contacts within the active site of glycosidase enzymes. The target zwitterionic compounds were synthesized by means of nucleophilic attack at the least hindered carbon atom of 2,4-O-benzylidene-l (or d)-erythritol 1,3-cyclic sulfate by 2,5-anhydro-1,3:4,6-di-O-benzylidene-2,5-dideoxy-5-thio (or 1,5-imino)-l-iditol.

Asymmetric synthesis of four isomers of 2-C-trifluoromethylerythritol.

Optically active 2-C-trifluoromethylerythritols, analogues of 2-C-methylerythritol, which is a key intermediate in the biosynthesis of isoprenoid with a mevalonate-independent route, were conveniently synthesized from 1,1,1-trifluoro-2,3-epoxypropane.

[Dipentaerythritol in the synthesis of the new type of phospholipids].

The first representatives of phospholipids of new types were synthesized on the basis of dipentaerythritol, 2,2'-[oxybis(methylen)]bis(hydroxymethyl))-1,3-propandiol. The starting polyol was phosphorylated with hexaethylphosphoric triamide to biphosphite, which was converted into thio- and selenophosphates by treatment with sulfur or selenium. The phosphoacetals were transformed into thio- and selenoanalogues of phospholipids by direct acylation with higher fatty acid chlorides. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2005, vol. 31, no. 6; see also http://www.maik.ru.

Synthesis and evaluation of 1-deoxy-D-xylulose 5-phosphoric acid analogues as alternate substrates for methylerythritol phosphate synthase.

[structure: see text] Four deoxyxylulose phosphate (DXP) analogues were synthesized and evaluated as substrates/inhibitors for methylerythritol phosphate (MEP) synthase. In analogues CF(3)-DXP (1), CF(2)-DXP (2), and CF-DXP (3), the three methyl hydrogens at C1 of DXP were sequentially replaced by fluorine. In the fourth analogue, Et-DXP (4), the methyl group in DXP was replaced by an ethyl moiety. Analogues 1, 2, and 4 were not substrates for MEP synthase under normal catalytic conditions and were instead modest inhibitors with IC(50) values of 2.0, 3.4, and 6.2 mM, respectively. In contrast, 3 was a good substrate (k(cat) = 38 s(-)(1), K(m) = 227 muM) with a turnover rate similar to that of the natural substrate. These results are consistent with a retro-aldol/aldol mechanism rather than an alpha-ketol rearrangement for the enzyme-catalyzed conversion of DXP to MEP.

Synthesis of enantiopure 2-C-methyl-D-erythritol 4-phosphate and 2,4-cyclodiphosphate from D-arabitol.

[reaction: see text] Two key intermediates of the newly discovered mevalonate-independent pathway for isoprenoid biosynthesis were prepared. Optically pure 2-C-methyl-D-erythritol 4-phosphate and 2,4-cyclodiphosphate were chemically synthesized from D-arabitol using a convenient benzylidene and tert-butyldimethylsilyl protection of polyhydroxylated intermediates. The new scheme offers a straightforward route to analogues and labeled forms.

Synthesis of 2-methyl-D-erythritol via epoxy ester-orthoester rearrangement.

The biomimetic epoxy ester[bond]orthoester rearrangement has been applied to a new synthesis of 2-methyl-D-erythritol, a branched five-carbon sugar of importance to the deoxyxylulose pathway of isoprenoid biosynthesis. The intermediate orthoacetate is one of the few [2.2.1]-orthoesters to have been reported. Labeling studies with O-18 indicated that this reaction proceeds exclusively via a 5-exo cyclization. NMR analysis of chiral esters indicated an ee of 87% for the starting epoxide and an ee of 86% for the product. This route represents a rapid and convenient method for the synthesis of 2-methyl-D-erythritol and is expected to be useful for generating isotopically labeled intermediates for biochemical studies.

Synthesis of 4-diphosphocytidyl-2-C-methyl-D-erythritol and 2-C-methyl-D-erythritol-4-phosphate.

2-C-Methyl-D-erythritol 4-phosphate (MEP, 2) and 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDPME, 3) are metabolites in the MEP pathway for biosynthesis of isoprenoid compounds in bacteria, plant chloroplasts, and algae. The free phosphoacid of 2 was prepared from benzyloxyacetone in five steps with an overall yield of 27% and an enantiomeric ratio (er) of 75:25. Following titration to the corresponding tributylammonium salt, 2 was coupled to cytidine 5'-monophosphate using a protocol originally developed for synthesis of base-sensitive nucleoside diphosphate sugars to give 3 in 40% yield, following purification by size exclusion chromatography.

Synthesis of 2-C-methyl-D-erythritol 2,4-cyclopyrophosphate.

[reaction: see text] The synthesis of 2-C-methyl-D-erythritol 2,4-cyclopyrophosphate, a biochemical intermediate in the deoxyxylulose pathway of isoprenoid biosynthesis, was accomplished in four steps. Bisphosphorylation of 2-C-methyl-D-erythritol 1,3-diacetate, followed by carbodiimide cyclization and deprotection, led to the title compound in 42% overall yield.

A combined X-ray and NMR study of borate esters of furanoidic cis-1,2-diols.

Crystals of K[B(AnErytH(-2)2] x 2 H2O (AnEryt = 1,4-anhydroerythritol) form from aqueous alkaline solutions containing a double molar amount of diol over borate. The spiro-type monoanions are the main borate species in the mother liquors of crystallisation according to 11B and 13C NMR spectroscopy. Ribofuranosides form analogous borate esters using their 1,4-anhydroerythritol core. Crystals of Na[B(Me beta-D-Ribf 2,3H(-2))2] x 2 H2O were grown from alkaline methyl beta-D-ribofuranoside solutions that had attacked boron-containing Duran vessels. NMR spectra show closely resembling borate-ester speciation in solutions of diols with the 1,4-anhydroerythritol core--1,4-anhydroerythritol itself, methyl beta-D-ribofuranoside and guanosine.