A new methodology for the synthesis of fluorinated exo-glycals and their time-dependent inhibition of UDP-galactopyranose mutase

Fluorinated carbohydrates constitute a very important class of mechanistic probes for glycosyl-processing enzymes. In this study, we describe the first synthesis of fluorinated and phosphonylated exo-glycals and their corresponding nucleotide sugars in the galactofuranose series. The synthetic protocol that we have developed is a Selectfluor-mediated fluorination/elimination sequence on phosphonylated exo-glycals, and it offers a new entry into fluorinated carbohydrate chemistry. The challenging E/Z stereochemical assignment of the resulting tetrasubstituted alkenes, which bear an alkoxy, an alkyl, a fluoro, and a phosphonyl group, has been achieved through NMR experiments. The corresponding (E)- and (Z)-nucleotide fluorosugars have been prepared and tested as inhibitors of UDP-galactopyranose mutase (UGM). UGM is a flavoenzyme that catalyzes the isomerization of uridine diphosphate(UDP)-galactopyranose into UDP-galactofuranose, a key step of the biosynthesis of important mycobacterial cell-wall glycoconjugates. The two diastereomeric molecules were found to display time-dependent inactivation of UGM, as expected from preliminary results using non-fluorinated exo-glycal nucleotides. The inhibitory properties of the two fluorinated molecules led us to suggest that the inactivation mechanism proceeds through two-electron processes, despite the presence of the flavin cofactor within the UGM catalytic site.     

Chemical probes of UDP-galactopyranose mutase

Many pathogenic prokaryotes and eukaryotes possess the machinery required to assemble galactofuranose (Galf)-containing glycoconjugates; these glycoconjugates can be critical for virulence or viability. Accordingly, compounds that block Galf incorporation may serve as therapeutic leads or as probes of the function of Galf-containing glycoconjugates. The enzyme UDP-galactopyranose mutase (UGM) is the only known generator of UDP-galactofuranose, the precursor to Galf residues. We previously employed a high-throughput fluorescence polarization assay to investigate the Klebsiella pneumoniae UGM. We demonstrate the generality of this assay by extending it to UGM from Mycobacterium tuberculosis. To identify factors influencing binding, we synthesized a directed library containing a 5-arylidene-2-thioxo-4-thiazolidinone core, a structure possessing features common to ligands for both homologs. Our studies offer a blueprint for identifying inhibitors of the growing family of UGM homologs and provide insight into UGM inhibition.     

Identification of inhibitors for UDP-galactopyranose mutase

The flavoenzyme uridine 5'-diphosphate (UDP)-galactopyranose mutase (UGM) plays a key role in the cell wall biosynthesis of many pathogens, including Mycobacterium tuberculosis. Using a synthetic fluorescent ligand, we screened 16 000 compounds in a fluorescence polarization assay. Effective inhibitors of UGM were identified.     

Inhibitors of UDP-galactopyranose mutase thwart mycobacterial growth

Galactofuranose (Galf) residues are fundamental components of the cell wall of mycobacteria. A key enzyme, UDP-galactopyranose mutase (UGM), that participates in Galf incorporation mediates isomerization of UDP-Galf from UDP-galactopyranose (UDP-Galp). UGM is of special interest as a therapeutic target because the gene encoding it is essential for mycobacterial viability and there is no comparable enzyme in humans. We used structure-activity relationships and molecular design to devise UGM inhibitors. From a focused library of synthetic aminothiazoles, several compounds that block the UGM from Klebsiella pneumoniae or Mycobacterium tuberculosis were identified. These inhibitors block the growth of M. smegmatis.     

Synthesis of acyclic galactitol- and lyxitol-aminophosphonates as inhibitors of UDP-galactopyranose mutase

UDP-galactopyranose mutase (UGM) catalyzes the isomerization of UDP-galactopyranose (UDP-Galp) into UDP-galactofuranose (UDP-Galf), an essential step of the mycobacterial cell wall biosynthesis. Acyclic alditol-aminophosphonates in the d-galactose and d-lyxose series were designed as mimics of high energy intermediates of the UGM catalyzed isomerization. Interestingly, the d-lyxitol-aminophosphonate displayed better inhibition properties than the d-galactitol-aminophosphonate.     

Mechanistic investigation of UDP-galactopyranose mutase from Escherichia coli using 2- and 3-fluorinated UDP-galactofuranose as probes.

The galactofuranose moiety found in many surface constituents of microorganisms is derived from UDP-D-galactopyranose (UDP-Galp) via a unique ring contraction reaction catalyzed by UDP-Galp mutase. This enzyme, which has been isolated from several bacterial sources, is a flavoprotein. To study this catalysis, the cloned Escherichia coli mutase was purified and two fluorinated analogues, UDP-[2-F]Galf (9) and UDP-[3-F]Galf (10), were chemically synthesized. These two compounds were found to be substrates for the reduced UDP-Galp mutase with the Km values determined to be 65 and 861 microM for 9 and 10, respectively, and the corresponding kcat values estimated to be 0.033 and 5.7 s(-1). Since the fluorine substituent is redox inert, a mechanism initiated by the oxidation of 2-OH or 3-OH on the galactose moiety can thus be firmly ruled out. Furthermore, both 9 and 10 are poorer substrates than UDP-Galf, and the rate reduction for 9 is especially significant. This finding may be ascribed to the inductive effect of the 2-F substituent that is immediately adjacent to the anomeric center, and is consistent with a mechanism involving formation of oxocarbenium intermediates or transition states during turnover. Interestingly, under nonreducing conditions, compounds 9 and 10 are not substrates, but instead are inhibitors for the mutase. The inactivation by 10 is time-dependent, active-site-directed, and irreversible with a K(I) of 270 microM and a k(inact) of 0.19 min(-1). Since the K(I) value is similar to Km, the observed inactivation is unlikely a result of tight binding. To our surprise, the inactivated enzyme could be regenerated in the presence of dithionite, and the reduced enzyme is resistant to inactivation by these fluorinated analogues. It is possible that reduction of the enzyme-bound FAD may induce a conformational change that facilitates the breakdown of the putative covalent enzyme-inhibitor adduct to reactivate the enzyme. It is also conceivable that the reduced flavin bears a higher electron density at N-1, which may play a role in preventing the formation of the covalent adduct or facilitating its breakdown by charge stabilization of the oxocarbenium intermediates/transition states. Clearly, this study has led to the identification of a potent inactivator (10) for this enzyme, and study of its inactivation has also shed light on the possible mechanism of this mutase.     

Synthesis and analysis of substrate analogues for UDP-galactopyranose mutase: implication for an oxocarbenium ion intermediate in the catalytic mechanism

[reaction: see text] UDP-D-galactofuranose (2), which is essential for both cell growth and virulence in many pathogenic microorganisms, is converted from UDP-D-galactopyranose (UDP-Galp, 1) by the flavin adenine dinucleotide (FAD)-dependent enzyme UDP-galactopyranose mutase (UGM). Here, we report the synthesis of UDP-GalOH (13) and show it as an inhibitor for UGM with a binding affinity similar to that of 1. These results are more consistent with a mechanism involving an oxocarbenium ion intermediate in UGM catalysis.     

Efficient exact-exchange time-dependent density-functional theory methods and their relation to time-dependent Hartree-Fock

A recently introduced time-dependent exact-exchange (TDEXX) method, i.e., a response method based on time-dependent density-functional theory that treats the frequency-dependent exchange kernel exactly, is reformulated. In the reformulated version of the TDEXX method electronic excitation energies can be calculated by solving a linear generalized eigenvalue problem while in the original version of the TDEXX method a laborious frequency iteration is required in the calculation of each excitation energy. The lowest eigenvalues of the new TDEXX eigenvalue equation corresponding to the lowest excitation energies can be efficiently obtained by, e.g., a version of the Davidson algorithm appropriate for generalized eigenvalue problems. Alternatively, with the help of a series expansion of the new TDEXX eigenvalue equation, standard eigensolvers for large regular eigenvalue problems, e.g., the standard Davidson algorithm, can be used to efficiently calculate the lowest excitation energies. With the help of the series expansion as well, the relation between the TDEXX method and time-dependent Hartree-Fock is analyzed. Several ways to take into account correlation in addition to the exact treatment of exchange in the TDEXX method are discussed, e.g., a scaling of the Kohn-Sham eigenvalues, the inclusion of (semi)local approximate correlation potentials, or hybrids of the exact-exchange kernel with kernels within the adiabatic local density approximation. The lowest lying excitations of the molecules ethylene, acetaldehyde, and pyridine are considered as examples.     

Investigation of binding of UDP-Galf and UDP-[3-F]Galf to UDP-galactopyranose mutase by STD-NMR spectroscopy, molecular dynamics, and CORCEMA-ST calculations

UDP-galactopyranose mutase (UGM) is the key enzyme involved in the biosynthesis of Galf. UDP-Galp and UDP-Galf are two natural substrates of UGM. A protocol that combines the use of STD-NMR spectroscopy, molecular modeling, and CORCEMA-ST calculations was applied to the investigation of the binding of UDP-Galf and its C3-fluorinated analogue to UGM from Klebsiella pneumoniae. UDP-Galf and UDP-[3-F]Galf were bound to UGM in a manner similar to that of UDP-Galp. The interconversions of UDP-Galf and UDP-[3-F]Galf to their galactopyranose counterparts were catalyzed by the reduced (active) UGM with different catalytic efficiencies, as observed by NMR spectroscopy. The binding affinities of UDP-Galf and UDP-[3-F]Galf were also compared with those of UDP-Galp and UDP by competition STD-NMR experiments. When UGM was in the oxidized (inactive) state, the binding affinities of UDP-Galf, UDP-Galp, and UDP-[3-F]Galf were of similar magnitudes and were lower than that of UDP. However, when UGM was in the reduced state, UDP-Galp had higher binding affinity compared with UDP. Molecular dynamics (MD) simulations indicated that the "open" mobile loop in UGM "closes" upon binding of the substrates. Combined MD simulations and STD-NMR experiments were used to create models of UGM with UDP-Galf and UDP-[3-F]Galf as bound ligands. Calculated values of saturation-transfer effects with CORCEMA-ST (complete relaxation and conformational exchange matrix analysis of saturation transfer) were compared to the experimental STD effects and permitted differentiation between two main conformational families of the bound ligands. Taken together, these results are used to rationalize the different rates of catalytic turnover of UDP-Galf and UDP-[3-F]Galf and shed light on the mechanism of action of UGM.     

Efficient synthesis of a configurationally stable l-serinal derivative

An efficient synthesis of a configurationally stable l-serinal derivative 8 was achieved using an N-hydroxymethyl group in about 50% overall yield in four steps from l-serine. Not more than 1% racemization was observed during the preparation of 8. Its enantiomeric integrity was maintained for at least 15 days at room temperature, and it was stable on silica gel. The orthogonal protective groups of 8 would make it a useful chiral synthon.     

Efficient synthesis of scutellarein

Scutellarein is a component of Scutellaria, recently known as a potent cytotoxic agent on human leukaemia cells. The aim of this study was the synthesis of scutellarein and its methylated derivative. The new features are the innovating method to afford flavones from flavanones and the A-ring regioselective bromination step that lead to the target molecule by a facile and high-yielding pathway.     

Diversity-oriented synthesis of a drug-like system displaying the distinctive N-->C=O interaction

This study describes the syntheses and characterization of two hydrazino ureas. These fold into a six-membered ring by virtue of the infrequently observed (delta+)N-->C=O (delta-) interaction when solvated by polar protic media. The highly polar functional group resulting from this interaction is hypothesized to especially reproduce electronic but also steric features of the transition states of peptide hydrolysis. The urea moiety constitutes an additional key element of modern HIV-1 protease (HIV PR) inhibitors and is meant to interact with the enzyme flaps. We have developed an efficient, convergent synthetic route to enantiopure compounds that uses CDI to couple two independent building blocks, one derived from amino acids and the other one from easily accessible hydrazines. It is thus amenable to rapid generation of diversity in order to screen for novel HIV PR inhibitors. A complete study using one- and two-dimensional NMR as well as UV spectroscopy confirmed the sole existence of the cyclic constitution of target compounds 7 and 8 in methanol. Total reversal to the linear aldehydic form is observed upon passage to apolar media.     

Efficient, diastereoselective chemical synthesis of a beta-mannopyranosyl phosphoisoprenoid

[reaction: see text] Tetrabutylammonium benzyl dihydrophytylphosphate was coupled to S-phenyl 2,3-di-O-benyl-4, 6-O-benzylidene-1-thio-alpha-D-mannopyranoside S-oxide on activation with triflic anhydride in toluene at -78 degrees C to give the corresponding beta-mannosyl phosphate in 56% yield with no detectable formation of the alpha-anomer. Treatment with sodium in liquid ammonia then afforded the unprotected beta-mannosyl phosphoisoprenoid.     

Efficient synthesis of substituted dihydrotetraazapentacenes

We describe a versatile and very efficient synthesis of previously unknown substituted 5,14-dihydro-5,7,12,14-tetraazapentacenes (DHTAPs). A structural study by NMR spectroscopy showed that the conjugated pi-system of the pentacyclic skeleton rearranges depending on the electronic effect of the substituent(s).     

Efficient synthesis of cyclopropylhydrazine salts

An efficient procedure for the synthesis of cyclopropylhydrazine in the form of its salts is reported. The copper salt-catalyzed addition of cyclopropylboronic acid to the azo group of di-tert-butyl azodicarboxylate and subsequent deprotection gave the cyclopropylhydrazine salts in high yields.     

Efficient synthesis of spiroisoxazoline oxindoles

The synthesis of spiroisoxazoline oxindoles containing ester groups at position 4′ and aromatic or ester groups at position 3′ of the isoxazoline ring is reported. The compounds were synthesized in yields up to 94% by 1,3-dipolar cycloaddition of 3-methylene indolin-2-ones and chlorooximes in the presence of triethylamine or zinc.     

Efficient BOP-mediated synthesis of fulgimides

A mild and efficient method for the synthesis of fulgimides is presented in which the peptide coupling reagent BOP is employed for dehydratation of fulgenic acid monoamides (succinamic acids). The disclosed method proved to be superior to those described in the literature.