Structure and Mechanism of Human UDP-xylose Synthase: EVIDENCE FOR A PROMOTING ROLE OF SUGAR RING DISTORTION IN A THREE-STEP CATALYTIC CONVERSION OF UDP-GLUCURONIC ACID

UDP-xylose synthase (UXS) catalyzes decarboxylation of UDP-d-glucuronic acid to UDP-xylose. In mammals, UDP-xylose serves to initiate glycosaminoglycan synthesis on the protein core of extracellular matrix proteoglycans. Lack of UXS activity leads to a defective extracellular matrix, resulting in strong interference with cell signaling pathways. We present comprehensive structural and mechanistic characterization of the human form of UXS. The 1.26-Å crystal structure of the enzyme bound with NAD⁺ and UDP reveals a homodimeric short-chain dehydrogenase/reductase (SDR), belonging to the NDP-sugar epimerases/dehydratases subclass. We show that enzymatic reaction proceeds in three chemical steps via UDP-4-keto-d-glucuronic acid and UDP-4-keto-pentose intermediates. Molecular dynamics simulations reveal that the d-glucuronyl ring accommodated by UXS features a marked ⁴C₁ chair to B_O,3 boat distortion that facilitates catalysis in two different ways. It promotes oxidation at C₄ (step 1) by aligning the enzymatic base Tyr¹⁴⁷ with the reactive substrate hydroxyl and it brings the carboxylate group at C₅ into an almost fully axial position, ideal for decarboxylation of UDP-4-keto-d-glucuronic acid in the second chemical step. The protonated side chain of Tyr¹⁴⁷ stabilizes the enolate of decarboxylated C₄ keto species (²H₁ half-chair) that is then protonated from the Si face at C₅, involving water coordinated by Glu¹²⁰. Arg²⁷⁷, which is positioned by a salt-link interaction with Glu¹²⁰, closes up the catalytic site and prevents release of the UDP-4-keto-pentose and NADH intermediates. Hydrogenation of the C₄ keto group by NADH, assisted by Tyr¹⁴⁷ as catalytic proton donor, yields UDP-xylose adopting the relaxed ⁴C₁ chair conformation (step 3).     

Coordination isomers of biological iron transport compounds. III. (1) Transport of lambda-cis-chromic desferriferrichrome by Ustilago sphaerogena

Microbial iron transport studies of the structure and conformation dependent ferrichrome uptake system in Ustilago sphaerogena have been limited previously to kinetically labile metal ions such as the native ferrichrome complex and the aluminum(III) and gallium(III) analogs. Although two coordination isomers are possible (λ-cis amd δ-cis), no information can be obtained concerning their biological activity using kinetically labile complexes. In this report, both the ligand and chromic ion moieties of kinetically inert λ-cis-chromic [¹⁴C]-desferriferrichrome are shown to be taken up in Ustilago sphaerogena at rates comparable to that of ferrichrome. The λ-cis coordination isomer must be therefore at least one of the biologically active isomers and the transport system cannot rely on the rapid isomerization or dissociation of the labile ferric complex.     

Synthesis, crystal structure, and catalytic studies on dinuclear copper(II) mesocates

Imine based bis-bidentate ligands H₂-m-xysal, (L₁H₂); H₂-m-xysal-Cl, (L₂H₂); H₂-m-xysal-Br, (L₃H₂); H₂-m-xysal-OCH_3, (L₄H₂); H₂-m-xysal-(t-Bu)_2, (L₅H₂) were synthesized and characterized. These substituted 1,3-bis(hydroxylbenzyl)-diaminoxylene dianion ligands upon treating with copper(II) acetate in 2:2 equivalent of L:M ratio, resulted in a series of binuclear [Cu₂(m-xysal)₂] neutral complexes 1-5. The crystal structures determined for the complexes 1 and 2 indicate a dinuclear association. The CH?π interaction observed between the metal-chelate ring and the hydrogens associated with m-xylene spacer moiety being first in this series of complexes, is demonstrated to stabilize the helical conformation through intramolecular self assembly process. The position of the resonance on the EPR spectra and the absence of ΔMs = ±1 feature for the complexes 2, 3, and 5 obtained for room temperature solid state samples revealed that the metal centers though exist in the dinuclear unit, they are separated from each other and possess a non-interacting monomer-type metal-metal association. The Cu(II) centers in all these complexes possessing an intermediate geometry between tetrahedral and square planar, an appropriate catalytic study converting 4-nitrobenzaldehye to corresponding nitroaldol was carried out using complex 5.     

Équilibres conformationnels de glucides au niveau de liaisons σ SP-SP: Partie V. Dérivés 2,3,-O-isopropylidène de pyranosides hybrides sp en C-4. Comparaison avec leurs analogues saturés

The conformation in solution of derivatives of methyl hexopyranosides has been studied by n.m.r. The esters of methyl 2,3-O-isopropylidene-α-D-manno- and -talopyranosides as well as their 4-deoxy-4-C-methyl analog having a manno configuration exist mainly in a flattened (^4,0F) chair conformation (⁴C₁). The presence in the talo epimer of the 4-deoxy-4-C-methyl analog of the bulky methyl group on the endo side of the bicyclic system results in a skew form (³S₁). The methyl 4-deoxy-2,3-O-isopropylidene-4-C-methylene-α-D-lyxo-hexopyranosides monosubstituted at C-4′ adopt, in solution, a conformation close to ³S₁, whichever their configuration (cis or trans) at the double bond, as indicated by their allylic coupling constants.     

Stathmin and Interfacial Microtubule Inhibitors Recognize a Naturally Curved Conformation of Tubulin Dimers

Tubulin is able to switch between a straight microtubule-like structure and a curved structure in complex with the stathmin-like domain of the RB3 protein (T₂RB3). GTP hydrolysis following microtubule assembly induces protofilament curvature and disassembly. The conformation of the labile tubulin heterodimers is unknown. One important question is whether free GDP-tubulin dimers are straightened by GTP binding or if GTP-tubulin is also curved and switches into a straight conformation upon assembly. We have obtained insight into the bending flexibility of tubulin by analyzing the interplay of tubulin-stathmin association with the binding of several small molecule inhibitors to the colchicine domain at the tubulin intradimer interface, combining structural and biochemical approaches. The crystal structures of T₂RB3 complexes with the chiral R and S isomers of ethyl-5-amino-2-methyl-1,2-dihydro-3-phenylpyrido[3,4-b]pyrazin-7-yl-carbamate, show that their binding site overlaps with colchicine ring A and that both complexes have the same curvature as unliganded T₂RB3. The binding of these ligands is incompatible with a straight tubulin structure in microtubules. Analytical ultracentrifugation and binding measurements show that tubulin-stathmin associations (T₂RB3, T₂Stath) and binding of ligands (R, S, TN-16, or the colchicine analogue MTC) are thermodynamically independent from one another, irrespective of tubulin being bound to GTP or GDP. The fact that the interfacial ligands bind equally well to tubulin dimers or stathmin complexes supports a bent conformation of the free tubulin dimers. It is tempting to speculate that stathmin evolved to recognize curved structures in unassembled and disassembling tubulin, thus regulating microtubule assembly.     

Following an environmental carcinogen N2-dG adduct through replication: elucidating blockage and bypass in a high-fidelity DNA polymerase

We have investigated how a benzo[a]pyrene-derived N²-dG adduct, 10S(+)-trans-anti-[BP]-N²-dG ([BP]G*), is processed in a well-characterized Pol I family model replicative DNA polymerase, Bacillus fragment (BF). Experimental results are presented that reveal relatively facile nucleotide incorporation opposite the lesion, but very inefficient further extension. Computational studies follow the possible bypass of [BP]G* through the pre-insertion, insertion and post-insertion sites as BF alternates between open and closed conformations. With dG* in the normal B-DNA anti conformation, BP seriously disturbs the polymerase structure, positioning itself either deeply in the pre-insertion site or on the crowded evolving minor groove side of the modified template, consistent with a polymerase-blocking conformation. With dG* in the less prevalent syn conformation, BP causes less distortion: it is either out of the pre-insertion site or in the major groove open pocket of the polymerase. Thus, the syn conformation can account for the observed relatively easy incorporation of nucleotides, with mutagenic purines favored, opposite the [BP]G* adduct. However, with the lesion in the BF post-insertion site, more serious distortions caused by the adduct even in the syn conformation explain the very inefficient extension observed experimentally. In vivo, a switch to a potentially error-prone bypass polymerase likely dominates translesion bypass.     

Proline-glutamate chimera’s side chain conformation directs the type of β-hairpin structure

Our aim was to study the impact of two proline chimeras, containing a glutamic acid side chain in cis- or trans-configuration, on secondary structure formation. We further investigated to what extent the configuration of the side chain contributes to the overall peptide conformation. We used a 10 residue peptide (IYSNPDGTWT) that forms a β-hairpin in water. The turn-forming proline was substituted with either a cis- or trans-proline-glutamic acid chimera, resulting in the peptides IYSNP ^{cis -E} DGTWT (P1_P ^cis-E ) and IYSNP ^{trans -E} DGTWT (P1_P ^trans-E ). We studied the conformation of the modified peptides by circular dichroism (CD) and NMR-spectroscopy, and SEC/static light scattering (SLS) analysis. NMR analysis reveals that the modified peptides maintain the β-hairpin conformation in aqueous solution. At 5 °C and pH 4.3, the peptide (P1_P ^cis-E ) was found to adopt two coexisting β-hairpin conformations (2:2 β-hairpin, and 3:5 β-hairpin). In contrast to that, the peptide (P1_P ^trans-E ) adopts a 2:2 β-hairpin that exists in equilibrium with a 4:4 β-hairpin conformation. The adoption of ordered β-hairpin structures for both modified peptides could be confirmed by CD spectroscopy, while SEC/SLS analysis showed a monomeric oligomerization state for all three investigated peptides. With the combination of several NMR methods, we were able to elucidate that even small alterations in the side chain conformation of the proline-glutamate chimera (cis or trans) can significantly influence the conformation of the adopted β-hairpin.     

Design, synthesis and coordination chemistry of sidearm substituted bisoxazoline ligands

We describe herein the design, synthesis and coordination chemistry of a novel ligand motif that combines a bisoxazoline with a third flexible chelating substituent (“tail”). Four such ligands (6-9) were synthesized from phenylglycine in 4-5 steps each in ca. 20% overall yield. Coordination of 6 and 7 to [Re(CO)₄Cl]₂ yielded crystals suitable for X-ray analysis. Likewise, crystals were obtained from coordination of 6 to FeCl₂. The solid state structures of the resulting complexes (10-12) reveal κ² binding of the bisoxazoline motif; however, the structures of complexes 10 and 12 show that the “tail” is poised above the metal center, demonstrating the potential for alternate binding modes in solution. The complexes exhibit a relatively planar 6-membered chelate structure, in contrast to the boat conformation typical of trispyrazolylborate complexes.     

Synthesis,characterization and in vitro anti-neoplastic activity of gypsogenin derivatives

Gypsogenin (L¹; 3-hydroxy-23-oxoolean-12-en-28-oic acid), a natural saponin, was isolated from the boiling water extract of Gypsophila arrostii roots. In addition, the derivatives gypsogenin thiosemicarbazone (L²; 23-[(aminocarbonothioyl)hydrazono]-3-hydroxolean-12-en-28-oic acid) and gypsogenin thiosemicarbazone glyoxime (L³H₂; (3β)-3-hydroxy-23-[({[(1Z,2E)-N-hydroxy-2-(hydroxyimino)ethanimidoyl]amino}carbonothioyl)hydrazono] olean-12-en-28-oic acid) as well as the Cu(II) and Co(II) complexes of L³H₂ were prepared. The structures were established on NMR analysis (¹H, ¹³C NMR, HMBC, HMQC, and NOESY), FT-IR and completed by analysis of LC/MS. Furthermore, the antiproliferative effects of the Co(II) and Cu(II) complexes of the gypsogenin derivatives were assayed in human promyelocytic leukemia (HL 60) cells. These complexes were found to be potent anticancer agents with concentrations that inhibited 50% of proliferation (I_pC₅₀) between 5 μM and 40 μM. Cell death was distinguished by HO/PI double staining. The Co(II) complex of L³H₂ has shown approximately %50 apoptotic effect at 10 μM concentration. Paclitaxel has been used as positive control.     

Exploration of Insulin Amyloid Polymorphism Using Raman Spectroscopy and Imaging

We aimed to investigate insulin amyloid fibril polymorphism caused by salt effects and heating temperature and to visualize the structural differences of the polymorphisms in situ using Raman imaging without labeling. The time course monitoring for amyloid formation was carried out in an acidic condition without any salts and with two species of salts (NaCl and Na₂SO₄) by heating at 60, 70, 80, and 90°C. The intensity ratio of two Raman bands at 1672 and 1657 cm⁻¹ due to antiparallel β-sheet and α-helix structures, respectively, was revealed to be an indicator of amyloid fibril formation, and the relative proportion of the β-sheet structure was higher in the case with salts, especially at a higher temperature with Na₂SO₄. In conjunction with the secondary structural changes of proteins, the S-S stretching vibrational mode of a disulfide bond (∼514 cm⁻¹) and the ratio of the tyrosine doublet I₈₅₀/I₈₂₆ were also found to be markers distinguishing polymorphisms of insulin amyloid fibrils by principal component analysis. Especially, amyloid fibrils with Na₂SO₄ media formed the gauche-gauche-gauche conformation of disulfide bond at a higher rate, but without any salts, the gauche-gauche-gauche conformation was partially transformed into the gauche-gauche-trans conformation at higher temperatures. The different environments of the hydroxyl groups of the tyrosine residue were assumed to be caused by fibril polymorphism. Raman imaging using these marker bands also successfully visualized the two- and three- dimensional structural differences of amyloid polymorphisms. These results demonstrate the potential of Raman imaging as a diagnostic tool for polymorphisms in tissues of amyloid-related diseases.     

Synthesis and properties of 3′-C-methylnucleosides and their phosphoric esters

3′-C-Methyluridine and 3′-C-methylcytidine were synthesized in 11 steps starting from d-glucose. The conformation of 3′-C-methylnucleosides was studied in solution and in the crystal by using the techniques of c.d., ¹H-n.m.r. spectroscopy, and X-ray diffraction analysis. 3′-C-Methyluridine 2′,3′-cyclophosphate was prepared, and its hydrolysis with nucleases was studied. 3′-C-Methyluridine 5′- mono- and 5′-tri-phosphate were also synthesized.     

Highly constrained dipeptoid analogues containing a type II′ β-turn mimic as novel and selective CCK-A receptor ligands

Conformationally constrained dipeptoid analogues containing the type II′ β-turn mimic (2S,5S,11bR)-2-amino-3-oxohexahydroindolizino[8,7-b]indole-5-carboxylate framework in place of the α-MeTrp residue, show high binding affinity and selectivity for CCK-A receptors, suggesting that a turn-like conformation could contribute to the bioactive conformation at this CCK receptor subtype.     

Synthesis,crystal structure,and conformation of 3-O-(6-O-acetyl-2,3-anhydro-4-deoxy-α-l-ribo-hexopyranosyl)-1,2:5,6-di-O-isopropylidene-α-d-glucofuranose

3-O-(6-O-Acetyl-2,3-anhydro-4-deoxy-α-l-ribo-hexopyranosyl)-1,2:5,6-di-O-isopropylidene-α-d-glucofuranose has been synthesised and its monocrystal investigated by X-ray diffraction methods. The compound crystallises in the orthorhombic system, space group P2₁2₁2₁, with cell constants a = 8.790(7), b = 11.678(4), and c = 21.457(10) Å. The intensity data were collected with a four-circle CAD-4 diffractometer. From a total of 1684 intensities, 1275 were of I > 2σ_I. The structure was solved by direct methods and refined by the full-matrix, least-squares procedure, resulting in R 0.057. The 4-deoxy-2,3-anhydropyranose ring is characterised by a sofa conformation (₅E), the 1,2-O-isopropylidene ring has a hybrid conformation (E + T), and the 5,6-O-isopropylidene and the α-d-glucofuranose rings have twist (T) conformations. The φ and ψ torsion angles for the glycosidic linkage are 54(4)° and 29(4)°, respectively.     

Enantioselective binding of chiral 1,14-dimethyl[5]helicene–spermine ligands with B- and Z-DNA

Duplex DNA adopts a right-handed B-DNA conformation under physiological conditions. Z-DNA, meanwhile, has a left-handed helical structure and is in equilibrium with right-handed B-DNA. We recently reported that the bisnaphthyl maleimide–spermine conjugate (1) induced a B- to Z-DNA transition with high efficiency at low salt concentrations. It was also found that the bisnaphthyl ligand (1) spontaneously transformed into the corresponding [5]helicene derivative (2). Because [5]helicene 2 can potentially be chiral and because the chiral discrimination of B- and Z-DNA is also of interest, we became interested in whether enatiomerically pure [5]helicene–spermine conjugates might discriminate the chirality of B- or Z-DNA. In this study, we have demonstrated an efficient synthesis of chiral DNA-binding ligands by the conjugation of a [5]helicene unit with a spermine unit. These chiral helicene ligands exhibited recognition of B- and Z-DNA, with (P)-3 displaying preference for B-DNA and (M)-3 for Z-DNA. The characteristic features of the helicene–spermine ligands developed in this study include two points: the cationic spermine portion produces electrostatic interactions along the phosphate backbone of the minor groove, and the helicene forms complexes in an end-stacking mode. Such binding modes, together with the thermodynamic parameters, account for the mode of chiral recognition of (P)- and (M)-3 for B- and Z-DNA.     

RNase H mediated cleavage of RNA by cyclohexene nucleic acid (CeNA)

Cyclohexene nucleic acid (CeNA) forms a duplex with RNA that is more stable than a DNA–RNA duplex (ΔT_m per modification: +2°C). A cyclohexenyl A nucleotide adopts a 3′-endo conformation when introduced in dsDNA. The neighbouring deoxynucleotide adopts an O4′-endo conformation. The CeNA:RNA duplex is cleaved by RNase H. The V_max and K_m of the cleavage reaction for CeNA:RNA and DNA:RNA is in the same range, although the k_cat value is about 600 times lower in the case of CeNA:RNA.     

Selective benzoylation of some N-acetyl-N-aryl-β-D-xylopyranosylamines

Selectivity in the benzoylation of N-acetyl-N-p-methoxyphenyl-, -p-bromophenyl-, and -p-chlorophenyl-β-D-xylopyranosylamines has been demonstrated. The structures of the products was shown by using periodate oxidation and n.m.r. spectroscopy. The relative reactivity of the hydroxyl groups was HO-3 ≈ HO-4 > HO-2. The β-D-xylopyranosylamine derivatives were shown to possess the ⁴C¹ conformation.     

Synthesis and in vitro antitumor activity of novel iridium(III) complexes with enantiopure C2-symmetrical vicinal diamine ligands

Four novel iridium(III) complexes with enantiopure C₂-symmetrical vicinal diamine ligands were designed, synthesized, and characterized by FT-IR, NMR, and MS. The cytotoxicities of all of the complexes against the human solid tumor cell lines A2780, A549, KB, and MDA-MB-231 were evaluated. Both R,R-configured complexes (R,R)-5a and (R,R)-5b exhibited more potent or similar activity compared with oxaliplatin, whereas their corresponding (S,S)-isomers (S,S)-5a and (S,S)-5b were found to be mostly inactive. As indicated by the activation of caspase-3, the cleavage of PARP, and the upregulation of p53, the preliminary mechanism studies revealed that the mode of cell death initiated by (R,R)-5a in A2780 cells was predominantly p53-mediated apoptosis. In addition, the structure of (R,R)-5a was unambiguously confirmed through single crystal X-ray structure determination.     

Divanadium(V) complexes with 4-R-benzoic acid (1-methyl-3-oxo-butylidene)-hydrazides: Syntheses, structures and properties

In acetonitrile, reactions of bis(acetylacetonato)oxidovanadium(IV) ([VO(acac)₂]) with 4-R-benzoylhydrazine in 1:1 mole ratio provide coordinatively symmetrical complexes (1-5) of the {OV(μ-O)VO}⁴⁺ motif in 40-47% yields. On the other hand, in methanol the same reactants provide complexes (6-10) containing the {OV(μ-OMe)₂VO}⁴⁺ core in 37-50% yields. In both series of complexes, the ligand is the O,N,O-donor deprotonated Schiff base system 4-R-benzoic acid (1-methyl-3-oxo-butylidene)-hydrazide formed by template condensation of acac⁻ with 4-R-benzoylhydrazine (R = H, Cl, OMe, NO₂ and NMe₂). All the complexes have been characterized by elemental analysis, magnetic and spectroscopic (IR, UV-Vis and NMR) measurements. Molecular structures of three representative complexes (4, 6 and 7) have been determined by X-ray crystallography. In each complex, the dianionic planar ligand is coordinated to the metal centre via the enolate-O, the imine-N and the O-atom of the deprotonated amide functionality. Cyclic voltammetric measurements in dichloromethane revealed that complexes 1-5 are redox inactive, while complexes 6-10 display a metal centred reduction in the potential range −0.06 to 0.0.32 V (versus Ag/AgCl).     

Complexes containing the (R,R)-N,N-bis(2-hydroxy-3-functionalised-benzylidene)-1,2-diaminocyclohexane ligand: synthesis and X-ray analyses of titanium chloro and oxo derivatives

Titanium complexes based on the (R,R)-N,N^′-bis(2-hydroxy-3-functionalised-benzylidene)-1,2-diaminocyclohexane ligand and containing either chloro or bridged oxo co-ligands have been prepared, subjected to single crystal X-ray analysis and examined as pre-catalysts for the asymmetric phospho-aldol (PA) reaction. Catalysis does take place although at a much slower rate than with related aluminium complexes and then only to afford racemic products; significant observations that lead to an important design point in PA pre-catalysts.     

Synthesis and X-ray structure of a C5-C4-linked glucofuranose-oxazolidin-2-one

The formation of (4R)-4-carbamoyl-4-[(4R)-3-O-benzyl-1,2-O-isopropylidene-β-l-threofuranos-4-C-yl]-oxazolidin-2-one instead of expected imidazolidin-2,4-dione (hydantoin) derivative from 5-amino-5-cyano-5-deoxy-3-O-benzyl-1,2-O-isopropylidene-α-d-glucofuranose or 3-O-benzyl-1,2-O-isopropylidene-α-d-xylo-hexofuranos-5-ulose under Bucherer-Bergs reaction conditions is reported. Single crystal X-ray diffraction data revealed that ³T₄ is the prefered conformation for the furanose ring, while E₂ and ²T₁ conformations are adopted by the 1,3-dioxolane and 2-oxazolidinone five-membered rings, respectively.