New Optically Active tert-Butylarylthiophosphinic Acids and Their Selenium Analogues as the Potential Synthons of Supramolecular Organometallic Complexes: Syntheses and Crystallographic Structure Determination.

The aim of the research described in this publication is two-fold. The first is a detailed description of the synthesis of a series of compounds containing a stereogenic heteroatom, namely the optically active P-stereogenic derivatives of tert-butylarylphoshinic acids bearing sulfur or selenium. The second is a detailed discussion dedicated to the determination of their structures by an X-ray analysis. Such a determination is needed when considering optically active hetero-oxophosphoric acids as new chiral solvating agents, precursors of new chiral ionic liquids, or ligands in complexes serving as novel organometallic catalysts.

Enzymatic Approach to the Synthesis of Enantiomerically Pure Hydroxy Derivatives of 1,3,5-Triaza-7-phosphaadamantane.

A series of enantiomerically pure derivatives of 6-(1-hydroxyalkyl)-1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decane 5 were successfully synthesized for the first time. A series of hydrolytic enzymes was applied in a stereoselective acetylation performed under kinetic resolution conditions. Although the secondary alcohols: α-aryl-hydroxymethyl-PTA (phosphines) 5b-d', PTA-oxides 8b-d', and PTA-sulfides 9b-d' were found to be totally unreactive in the presence of all the enzymes and various conditions applied, the primary alcohols, i.e., the hydroxymethyl derivatives PTA oxide 8a and PTA sulfide 9a, were successfully resolved into enantiomers with moderate to good enantioselectivity (up to 95%). The absolute configurations of the products were determined by an X-ray analysis and chemical correlation.

Absolute Configuration and Conformation of (-)-R-t-Butylphenylphosphinoamidate: Chiroptical Spectroscopy and X-ray Analysis.

The absolute configuration and conformations of (-)-tert-butylphenylphosphinoamidate were determined using three different chiroptical spectroscopic methods, namely vibrational circular dichroism (VCD), electronic circular dichroism (ECD), and optical rotatory dispersion (ORD). In each of the spectroscopic methods used, experimental data for the (-)-enantiomer of tert-butylphenylphosphinoamidate were measured in the solution phase. Using the concentration-dependent experimental infrared spectra, the existence of dimers in the solution was investigated, and the monomer-dimer equilibrium constant was determined. Concomitant quantum mechanical predictions of the VCD, ECD, and ORD for monomeric tert-butylphenylphosphinoamidate were carried out using density functional theory (DFT) calculations using the B3LYP functional and the 6-31G(d), 6-311G(2d,2p) and aug-cc-pVDZ basis sets. Similar predictions for dimeric tert-butylphenylphosphinoamidate were also obtained using the B3LYP/6-31G(d) method. A comparison of theoretically predicted data with the corresponding experimental data led to the elucidation of the absolute configuration as (-)-(R)-tert-butylphenylphosphinoamidate with one predominant conformation in the solution. This conclusion was independently supported by X-ray analysis of the complex with (+)-R-2,2'-dihydroxy-1,1'-binaphthol ((+)-R- BINOL).

Diastereoisomerically Pure, (S)-O-1,2-O-isopropyli dene-(5-O-α-d-glucofuranosyl) t-butanesulfinate: Synthesis, Crystal Structure, Absolute Configuration and Reactivity.

The reaction of t-butylmagnesium chlorides with diastereomerically pure (R)-1,2-O-isopropylidene-3,5-O-sulfinyl-α-d-glucofuranose (R)-4 was found to be stopped at the stage of the corresponding, diastereoisomerically pure 1,2-O-isopropylidene-(5-O-α-d-glucofuranosyl) t-butanesulfinate (S)-10 for which the crystal structure and the (S)-absolute configuration was determined by X-ray crystallography. Comparison of the absolute configurations of the starting sulfite (R)-4, and t-butanesulfinate (S)-10 (which crystallizes in the orthorhombic system, space group P212121, with the single compound molecule present in the asymmetric unit), clearly indicates that the reaction of nucleophilic substitution at the stereogenic sulfur atom in the sulfite (R)-4 occurs with the full inversion of configuration via the trigonal bipyramidal sulfurane intermediate 4c in which both the entering and leaving groups are located in apical positions.

Nucleophilic Substitution at Tetracoordinate Sulfur. Kinetics and Mechanism of the Chloride-Chloride Exchange Reaction in Arenesulfonyl Chlorides: Counterintuitive Acceleration of Substitution at Sulfonyl Sulfur by ortho-Alkyl Groups and Its Origin.

The chloride-chloride exchange reaction in arenesulfonyl chlorides was investigated experimentally and theoretically by density functional theory (DFT) calculations. The second order rate constants and activation parameters of this identity reaction were determined for 22 variously substituted arenesulfonyl chlorides using radio-labeled Et4N36Cl. The chloride exchange rates of 11 sulfonyl chlorides bearing para-and meta-substituents (σ constants from -0.66 to +0.43) in the aromatic ring followed the Hammett equation with a ρ-value of +2.02. The mono- and di-ortho-alkyl substituted sulfonyl chlorides exhibit an enhanced reactivity although both inductive and steric effects lower the reaction rate. The DFT calculations of their structures together with X-ray data showed that an increased reactivity is mainly due to a peculiar, rigid, strongly compressed and sterically congested structure. The DFT studies of the title reaction revealed that it proceeds via a single transition state according to the SN2 mechanism. The analogous fluoride exchange reaction occurs according to the addition-elimination mechanism (A-E) and formation of a difluorosulfurandioxide intermediate. The reliability of the calculations performed was supported by the fact that the calculated relative rate constants and activation parameters correlate well with the experimental kinetic data.

Crystal and molecular structure of hexagonal form of lipase B from Candida antarctica.

During crystallization screenings of commercially available hydrolytic enzymes, the new, hexagonal crystal form of CAL-B, has been discovered and hereby reported. The NAG molecules, which were closing the glycosylation site in the orthorhombic form, in hexagonal structure make the glycosylation site open. It is unknown whether the opening and closing of the glycosylation site by the 'lid' NAG molecules, could be related to the opening and closing of the active center of the enzyme upon substrate binding and product release.

Crystallographic and molecular dynamics simulation analysis of Escherichia coli dihydroneopterin aldolase.

BACKGROUND: Dihydroneopterin aldolase (DHNA) catalyzes the conversion of 7,8-dihydroneopterin to 6-hydroxymethyl-7,8-dihydropterin and also the epimerization of DHNP to 7,8-dihydromonapterin. Previously, we determined the crystal structure of Staphylococcus aureus DHNA (SaDHNA) in complex with the substrate analogue neopterin (NP). We also showed that Escherichia coli DHNA (EcDHNA) and SaDHNA have significantly different binding and catalytic properties by biochemical analysis. On the basis of these structural and functional data, we proposed a catalytic mechanism involving two proton wires. RESULTS: To understand the structural basis for the biochemical differences and further investigate the catalytic mechanism of DHNA, we have determined the structure of EcDHNA complexed with NP at 1.07-Å resolution [PDB:2O90], built an atomic model of EcDHNA complexed with the substrate DHNP, and performed molecular dynamics (MD) simulation analysis of the substrate complex. EcDHNA has the same fold as SaDHNA and also forms an octamer that consists of two tetramers, but the packing of one tetramer with the other is significantly different between the two enzymes. Furthermore, the structures reveal significant differences in the vicinity of the active site, particularly in the loop that connects strands ß3 and ß4, mainly due to the substitution of nearby residues. The building of an atomic model of the complex of EcDHNA and the substrate DHNP and the MD simulation of the complex show that some of the hydrogen bonds between the substrate and the enzyme are persistent, whereas others are transient. The substrate binding model and MD simulation provide the molecular basis for the biochemical behaviors of the enzyme, including noncooperative substrate binding, indiscrimination of a pair of epimers as the substrates, proton wire switching during catalysis, and formation of epimerization product. CONCLUSIONS: The EcDHNA and SaDHNA structures, each in complex with NP, reveal the basis for the biochemical differences between EcDHNA and SaDHNA. The atomic substrate binding model and MD simulation offer insights into substrate binding and catalysis by DHNA. The EcDHNA structure also affords an opportunity to develop antimicrobials specific for Gram-negative bacteria, as DHNAs from Gram-negative bacteria are highly homologous and E. coli is a representative of this class of bacteria.

New enantiomeric fluorine-containing derivatives of sulforaphane: synthesis, absolute configurations and biological activity.

Three pairs of enantiomers of the unknown sulforaphane analogs bearing organofluorine substituents bonded to the sulfinyl sulfur atom and having different number of methylene groups in the central carbon chain were synthesized and fully characterized, including determination of their absolute configurations. All the new compounds were tested in vitro for their cytotoxicity against melanoma cells to show increased activity in comparison with the natural sulforaphane. The influence of the particular structural changes in the molecule on the cytotoxicity is discussed.

Structure and activity of Yersinia pestis 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase as a novel target for the development of antiplague therapeutics.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is a key enzyme in the folate-biosynthetic pathway and is essential for microorganisms but absent from mammals. HPPK catalyzes Mg(2+)-dependent pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP). Previously, three-dimensional structures of Escherichia coli HPPK (EcHPPK) have been determined at almost every stage of its catalytic cycle and the reaction mechanism has been established. Here, the crystal structure of Yersinia pestis HPPK (YpHPPK) in complex with HP and an ATP analog is presented together with thermodynamic and kinetic characterizations. The two HPPK molecules differ significantly in a helix-loop area (alpha2-Lp3). YpHPPK has lower affinities than EcHPPK for both nucleotides and HP, but its rate constants for the mechanistic steps of both chemical transformation and product release are comparable with those of EcHPPK. Y. pestis, which causes plague, is a category A select agent according to the Centers for Disease Control and Prevention (CDC). Therefore, these structural and biochemical data are valuable for the design of novel medical countermeasures against plague.

Structural basis for the aldolase and epimerase activities of Staphylococcus aureus dihydroneopterin aldolase.

Dihydroneopterin aldolase (DHNA) catalyzes the conversion of 7,8-dihydroneopterin (DHNP) to 6-hydroxymethyl-7,8-dihydropterin (HP) and the epimerization of DHNP to 7,8-dihydromonopterin (DHMP). Although crystal structures of the enzyme from several microorganisms have been reported, no structural information is available about the critical interactions between DHNA and the trihydroxypropyl moiety of the substrate, which undergoes bond cleavage and formation. Here, we present the structures of Staphylococcus aureus DHNA (SaDHNA) in complex with neopterin (NP, an analog of DHNP) and with monapterin (MP, an analog of DHMP), filling the gap in the structural analysis of the enzyme. In combination with previously reported SaDHNA structures in its ligand-free form (PDB entry 1DHN) and in complex with HP (PDB entry 2DHN), four snapshots for the catalytic center assembly along the reaction pathway can be derived, advancing our knowledge about the molecular mechanism of SaDHNA-catalyzed reactions. An additional step appears to be necessary for the epimerization of DHMP to DHNP. Three active site residues (E22, K100, and Y54) function coordinately during catalysis: together, they organize the catalytic center assembly, and individually, each plays a central role at different stages of the catalytic cycle.

Is the critical role of loop 3 of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase in catalysis due to loop-3 residues arginine-84 and tryptophan-89? Site-directed mutagenesis, biochemical, and crystallographic studies.

Deletion mutagenesis, biochemical, and X-ray crystallographic studies have shown that loop 3 of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) is required for the assembly of the active center, plays an important role in the stabilization of the ternary complex of HPPK with MgATP and 6-hydroxymethyl-7,8-dihydropterin (HP), and is essential for catalysis. Whether the critical functional importance of loop 3 is due to the interactions between residues R84 and W89 and the two substrates has been addressed by site-directed mutagenesis, biochemical, and X-ray crystallographic studies. Substitution of R84 with alanine causes little changes in the dissociation constants and kinetic constants of the HPPK-catalyzed reaction, indicating that R84 is not important for either substrate binding or catalysis. Substitution of W89 with alanine increases the K(d) for the binding of MgATP by a factor of 3, whereas the K(d) for HP increases by a factor of 6, which is due to the increase in the dissociation rate constant. The W89A mutation decreases the rate constant for the chemical step of the forward reaction by a factor of 15 and the rate constant for the chemical step of the reverse reaction by a factor of 25. The biochemical results of the W89A mutation indicate that W89 contributes somewhat to the binding of HP and more significantly to the chemical step. The crystal structures of W89A show that W89A has different conformations in loops 2 and 3, but the critical catalytic residues are positioned for catalysis. When these results are taken together, they suggest that the critical functional importance of loop 3 is not due to the interactions of the R84 guanidinium group or the W89 indole ring with the substrates.

Noncatalytic assembly of ribonuclease III with double-stranded RNA.

Ribonuclease III (RNase III) represents a family of double-stranded RNA (dsRNA) endonucleases. The simplest bacterial enzyme contains an endonuclease domain (endoND) and a dsRNA binding domain (dsRBD). RNase III can affect RNA structure and gene expression in either of two ways: as a dsRNA-processing enzyme that cleaves dsRNA, or as a dsRNA binding protein that binds but does not cleave dsRNA. We previously determined the endoND structure of Aquifex aeolicus RNase III (Aa-RNase III) and modeled a catalytic complex of full-length Aa-RNase III with dsRNA. Here, we present the crystal structure of Aa-RNase III in complex with dsRNA, revealing a noncatalytic assembly. The major differences between the two functional forms of RNase III.dsRNA are the conformation of the protein and the orientation and location of dsRNA. The flexibility of a 7 residue linker between the endoND and dsRBD enables the transition between these two forms.

Reaction trajectory of pyrophosphoryl transfer catalyzed by 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase.

6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the Mg(2+)-dependent pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP). The reaction follows a bi-bi mechanism with ATP as the first substrate and AMP and HP pyrophosphate (HPPP) as the two products. HPPK is a key enzyme in the folate biosynthetic pathway and is essential for microorganisms but absent from mammals. For the HPPK-catalyzed pyrophosphoryl transfer, a reaction coordinate is constructed on the basis of the thermodynamic and transient kinetic data we reported previously, and the reaction trajectory is mapped out with five three-dimensional structures of the enzyme at various liganded states. The five structures are apo-HPPK (ligand-free enzyme), HPPK.MgATP(analog) (binary complex of HPPK with its first substrate) and HPPK.MgATP(analog).HP (ternary complex of HPPK with both substrates), which we reported previously, and HPPK.AMP.HPPP (ternary complex of HPPK with both product molecules) and HPPK.HPPP (binary complex of HPPK with one product), which we present in this study.

Essential roles of a dynamic loop in the catalysis of 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the transfer of pyrophosphoryl group from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP) following an ordered bi-bi mechanism with ATP as the first substrate. The rate-limiting step of the reaction is product release, and the complete active center is assembled and sealed only upon the binding of both ATP and HP. The assembly of the active center involves large conformational changes in three catalytic loops, among which loop 3 undergoes the most dramatic and unusual changes. To investigate the roles of loop 3 in catalysis, we have made a deletion mutant, which has been investigated by biochemical and X-ray crystallographic analysis. The biochemical data showed that the deletion mutation does not have significant effects on the dissociation constants or the rate constants for the binding of the first substrate MgATP or its analogues. The dissociation constant of HP for the mutant increases by a factor of approximately 100, which is due to a large increase in the dissociation rate constant. The deletion mutation causes a shift of the rate-limiting step in the reaction and a decrease in the rate constant for the chemical step by a factor of approximately 1.1 x 10(5). The crystal structures revealed that the deletion mutation does not affect protein folding, but the catalytic center of the mutant is not fully assembled even upon the formation of the ternary complex and is not properly sealed. The results together suggest that loop 3 is dispensable for the folding of the protein and the binding of the first substrate MgATP, but is required for the assembling and sealing of the active center. The loop plays an important role in the stabilization of the ternary complex and is critical for catalysis.

Dynamic roles of arginine residues 82 and 92 of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase: crystallographic studies.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the pyrophosphoryl transfer from ATP to 6-hydroxymethyl-7,8-dihydropterin (HP), the first reaction in the folate biosynthetic pathway. Arginine residues 82 and 92, strictly conserved in 35 HPPK sequences, play dynamic roles in the catalytic cycle of the enzyme. At 0.89-A resolution, two distinct conformations are observed for each of the two residues in the crystal structure of the wild-type HPPK in complex with two HP variants, two Mg(2+) ions, and an ATP analogue. Structural information suggests that R92 first binds to the alpha-phosphate group of ATP and then shifts to interact with the beta-phosphate as R82, which initially does not bind to ATP, moves in and binds to alpha-phosphate when the pyrophosphoryl transfer is about to occur. The dynamic roles of R82 and R92 are further elucidated by five more crystal structures of two mutant proteins, R82A and R92A, with and without bound ligands. Two oxidized forms of HP are observed with an occupancy ratio of 0.50:0.50 in the 0.89-A structure. The oxidation of HP has significant impact on its binding to the protein as well as the conformation of nearby residue W89.

Catalytic roles of arginine residues 82 and 92 of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase: site-directed mutagenesis and biochemical studies.

The roles of a pair of conserved positively charged residues R82 and R92 at a catalytic loop of Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) have been investigated by site-directed mutagenesis and biochemical analysis. In the structure of HPPK in complex with ATP and a 6-hydroxymethyl-7,8-dihydropterin (HP) analogue, the guanidinium group of R82 forms two hydrogen bonds with the alpha-phosphate and that of R92 two hydrogen bonds with the beta-phosphate. In the structure of HPPK in complex with alpha,beta-methyleneadenosine triphosphate (AMPCPP, an ATP analogue) and HP, the guanidinium group of R82 has no direct interaction with AMPCPP and that of R92 forms two hydrogen bonds with the alpha-phosphate. Substitution of R82 with alanine caused a decrease in the rate constant for the chemical step by a factor of approximately 380, but there were no significant changes in the binding energy or binding kinetics of either substrate. Substitution of R92 with alanine caused a decrease in the rate constant for the chemical step by a factor of approximately 3.5 x 10(4). The mutation caused no significant changes in the binding energy or binding kinetics of MgATP. It did not cause a significant change in the binding energy of HP either but caused a decrease in the association rate constant for the binding of HP by a factor of approximately 4.5 and a decrease in the dissociation rate constant by a factor of approximately 10. The overall structures of the ternary complexes of both mutants were very similar to the corresponding structure of wild-type HPPK as described in the companion paper. The results suggest that R82 does not contribute to the binding of either substrate, and R92 is dispensable for the binding of MgATP but plays a role in facilitating the binding of HP. Both R82 and R92 are important for catalysis, and R92 plays a critical role in the transition state stabilization.

Chemical transformation is not rate-limiting in the reaction catalyzed by Escherichia coli 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase.

6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase (HPPK) catalyzes the transfer of pyrophosphate from ATP to 6-hydroxymethyl-7,8-dihydropterin (HMDP). Because HPPK is essential for microorganisms but is absent from human and animals, the enzyme is an excellent target for developing antimicrobial agent. Thermodynamic analysis shows that Mg(2+) is important not only for the binding of nucleotides but also for the binding of HMDP. Transient kinetic analysis shows that a step or steps after the chemical transformation are rate-limiting in the reaction catalyzed by HPPK. The pre-steady-state kinetics is composed of a burst phase and a steady-state phase. The rate constant for the burst phase is approximately 50 times larger than that for the steady-state phase. The latter is very similar to the k(cat) value measured by steady-state kinetics. A set of rate constants for the individual steps of the HPPK-catalyzed reaction has been determined by a combination of stopped-flow and quench-flow analyses. These results form a thermodynamic and kinetic framework for dissecting the roles of active site residues in the substrate binding and catalysis by HPPK.