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Summary Conversion of aldo to keto sugars by the metalloenzyme D-xylose isomerase (XI) is a multistep reaction that involves hydrogen transfer. We have determined the structure of this enzyme by neutron diffraction in order to locate H atoms (or their isotope D). Two studies are presented, one of XI containing cadmium and cyclic D-glucose (before sugar ring opening has occurred), and the other containing nickel and linear D-glucose (after ring opening has occurred but before isomerization). Previously we reported the neutron structures of ligand-free enzyme and enzyme with bound product. The data show that His54 is doubly protonated on the ring N in all four structures. Lys289 is neutral before ring opening and gains a proton after this; the catalytic metal-bound water is deprotonated to hydroxyl during isomerization and O5 is deprotonated. These results lead to new suggestions as to how changes might take place over the course of the reaction.

Structural features of clusters involving a metal ion (Li+, Na+, Be2+, Mg2+, Zn2+, Al3+, or Ti4+) surrounded by a total of 18 water molecules arranged in two or more shells have been studied using density functional theory. Effects of the size and charge of each metal ion on the organization of the surrounding water molecules are compared to those found for a Mg[H2O]62+• [H2O]12 cluster that has the lowest known energy on the Mg2+• [H2O]18 potential energy surface (Markham et al. in J Phys Chem B 106:5118–5134, 2002). The corresponding clusters with Zn2+ or Al3+ have similar structures. In contrast to this, clusters with a monovalent Li+ or Na+ ion, or with a very small Be2+ ion, differ in their hydrogen-bonding patterns and the coordination number can decrease to four. The tetravalent Ti4+ ionizes one inner-shell water molecule to a hydroxyl group leaving a Ti4+(H2O)5 (OH−) core, and an H3O+• • • H2O moiety dissociates from the second shell of water molecules. These observations highlight the influence of cation size and charge on the local structure of hydrated ions, the high-charge cations causing chemical changes and the low-charge cations being less efficient in maintaining the local order of water molecules.

The most stable is not necessarily the most easy to obtain! Two new stable crystal forms of the 120 year old compound sym-trinitrobenzene are reported. These elusive forms have been obtained by the use of an additive, trisindane, that can act as a mimic in the crystallization experiment.

Prostaglandin H2 synthase (EC 1.14.99.1) is an integral membrane enzyme containing a cyclooxygenase site, which is the target for the non-steroidal anti-inflammatory drugs, and a spatially distinct peroxidase site. Previous crystallographic studies of this clinically important drug target have been hindered by low resolution. We present here the 2.0 A resolution X-ray crystal structure of ovine prostaglandin H2 synthase-1 in complex with alpha-methyl-4-biphenylacetic acid, a defluorinated analog of the non-steroidal anti-inflammatory drug flurbiprofen. Detergent molecules are seen to bind to the protein's membrane-binding domain, and their positions suggest the depth to which this domain is likely to penetrate into the lipid bilayer. The relation of the enzyme's proximal heme ligand His388 to the heme iron is atypical for a peroxidase; the iron-histidine bond is unusually long and a substantial tilt angle is observed between the heme and imidazole planes. A molecule of glycerol, used as a cryoprotectant during diffraction experiments, is seen to bind in the peroxidase site, offering the first view of any ligand in this active site. Insights gained from glycerol binding may prove useful in the design of a peroxidase-specific ligand.

In this paper, we report the results of a study involving the coordination geometries of CuI, CuII, and CuIII crystal structures in the Cambridge Structural Database, and on Cu binding sites in proteins taken from the Protein Data Bank. The motifs used to bind two bridged Cu ions are also described. In addition, we report the results of ab initio molecular-orbital calculations performed on a variety of model CuI/CuII complexes (CuI/CuII⋅XnYm (X, Y=NH3, SH2); n+m=4; n=0–4) to provide data on the structural and energetic changes that occur in isolated complexes when the oxidation state of the Cu ion is changed from II to I while the coordination number is conserved. The use of such simple ligands in these calculations eliminates constraints on the geometric changes that may be imposed by more-complicated ligands.

The crystal structures of the molecular complexes of sym-trinitrobenzene (TNB) with acridine, 1,10-phenanthroline and phenazine are discussed. In all three cases, the structures are held together by C–H⋯O and π⋯π interactions, and TNB forms hydrogen bonded dimers and tapes that are not found in its native crystal structure. Acridine and 1,10-phenanthroline yield 1 ∶ 1 complexes that have very similar structures. The related molecule phenazine, however, gives a different 2 ∶ 3 complex in which guest exchange, as seen in other recently reported examples, is not observed. A case is made for the reporting of such ‘low yield’ supramolecular reactions because they still provide valuable information about the packing characteristics of organic molecules.

The binding of two metal ions that are in close proximity in proteins is examined using a combination of (1) crystallographic structural database analyses and (2) density functional theory calculations on model complexes. Divalent magnesium and manganese ions are the focus of the present study. It is found that in all proteins in the Protein Databank that have two closely positioned magnesium or manganese ions, these metal ions are generally bridged by at least one negatively charged oxygen-containing group—carboxylate, phosphate, or sulfate. This group transfers (negative) electron density to the metal ions and this helps to reduce electrostatic repulsion in the region. The geometry of the two-metal complex appears to depend on the nature of the negatively charged group between them. When a single oxygen atom is also in a bridging position, the two metal ions are found to be closer together than when only a carboxylate group binds them together. This suggests that this bridging oxygen atom may be negatively charged, e.g., a hydroxide ion rather than a water molecule. Details of the geometry of such bridges and the relevant motifs that are found in crystal structures are described.

1,4-Dimethylbenzo[c]phenanthrene (1,4-DMBcPh) is the dimethylated analogue of the benzo[c]phenanthrene (BcPh), where one of its two methyl groups resides in the highly congested fjord-region. A comparative X-ray crystallographic analysis, described herein, shows that BcPh is distorted out-of-plane so that there is an angle of 27° between the outermost rings. The additional methyl groups in 1,4-DMBcPh increase this nonplanarity to an angle of 37°. This methyl group-induced disruption of planarity results in P and M enantiomers of 1,4-DMBcPh, and this helicity is observed in a pronounced manner in its putative metabolites, the dihydrodiol and diol epoxide. Synthetically, photochemical cyclization offers convenient access to 1,4-DMBcPh as well as its metabolites. For this, Wittig reaction of 2,5-dimethylbenzyltriphenylphosphonium chloride and 2-naphthaldehyde provided a cis/trans mixture of alkenes which, when subjected to photolysis, provided 1,4-DMBcPh. Despite the high steric congestion in the fjord-regio...

The generally higher melting-point of a para-disubstituted benzene relative to the corresponding meta-isomer has been ascribed to the fact that, being more symmetrical, it can pack more tightly. Exceptionally, it was observed that whereas m-anisylpinacolone melts at 58.0 °C, the para-isomer melts lower at 39.5 °C. In this work we have attempted to understand this apparent anomaly. The crystal structures of both isomers were determined and the packing analysed. Energy calculations of the static structures and molecular dynamics (MD) simulations at temperatures just below the respective melting-points were performed. The structure analyses indicate that the intermolecular contacts are comparably weak in the two cases, and do not appear to be the direct cause of the melting-point difference. Thermal motion analysis, packing energies and MD simulations on minicrystals indicate the importance of both enthalpic and entropic factors in the melting behaviour of the two isomers. The higher melting point of the meta-isomer could originate from both a smaller ΔSf and higher ΔHf relative to the para-isomer. Copyright © 2000 John Wiley & Sons, Ltd.

The carcinogenicity of benzo[a]pyrene (BaP) derivatives varies with the substituents they contain. The effects of these substituents on the geometric structures of the PAHs have been examined by X-ray diffraction studies. Data presented include bond lengths and angles. deviations from planarity in the bay region, and non-bonded H˙˙˙˙H distances. Structures in the sequence BaP, 11-methylBaP and dibenzo[a, l]pyrene illustrate the increased nonplanarity of the PAHs as the carcinogenicity increases. For comparison, the geometry of 7,12-dimethylbenz[a]anthracene (DMBA) is also discussed. The structures of a diol epoxide of 5,6-dimethylchrysene and the product of the reaction of the diol epoxide of 5-methylchrysene with methanol are also presented. These show how the bay-region methyl group affects the structure, and presumably also the reactivity of the diol epoxide.

Divalent manganese, magnesium, and zinc fill unique roles in biological systems, despite many apparently similar chemical properties. A comparison of the liganding properties of divalent manganese, magnesium, and zinc has been made on the basis of data on crystal structures (from the Cambridge Structural Database and the Protein Databank) and molecular orbital and density functional calculations. The distribution of coordination numbers for divalent manganese in crystal structure determinations, and the identities of ligands, have been determined from analyses of data derived from the structural databases. Enthalpy and free energy changes for processes such as loss of water or ionization of water from hydrated cations have been evaluated from computational studies. The energy penalty for changing the hexahydrate of divalent manganese to a pentahydrate with one water molecule in the second coordination shell is intermediate between the high value for magnesium and the low value for zinc. The preferred coor...

The three-dimensional structure of a dihydrodiol epoxide of 5, 6-dimethylchrysene was elucidated by X-ray diffraction techniques. The effects of the steric overcrowding by the 5-methyl group in the bay region of this compound are described. The carbon atom of the 5-methyl group is found to lie out of the plane of the aromatic system, thereby avoiding the nearer C-H group of the epoxide ring; this C-H hydrogen atom is pushed in the opposite direction. As a result, the molecule is distorted so that the relative orientations of the epoxide group and the aromatic ring systems are very different for the diol epoxides of (nearly planar) benzo[a]pyrene (studied by Neidle and co-workers) and (distorted) 5, 6-dimethylchrysene (described here). The main effect of the 5-methyl group is to change the relative angle between the epoxide-bearing ring (the site of attack when the diol epoxide acts as an alkylating agent) and the aromatic ring system (which is presumed to lie partially between the DNA bases in the DNA adduct that is about to be formed). This may favor some specific alkylation geometry.

The three-dimensional structure of the carcinogenic nicotine-derived nitrosaminoketone, 4-(methyl-nitrosamino)-l-(3-pyridyl)-l-butanone, has been determined by X-ray crystallographic techniques. The molecule is essentially planar except for the methylnitrosamine group which is oriented at a dihedral angle of 68.7° to the pyridine ring. Molecules pack by way of —H⋯O interactions that involve the —NNO group.

The effects of a metal ion-bound carboxylate group on the acidity of a water molecule bound to the same cation have been assessed by ab initio molecular orbital calculations. In the hexahydrate Mg[H2O]62+ the free energy required to deprotonate one coordinated water molecule is only 40% of that required to deprotonate a free water molecule, indicating that the presence of the magnesium ion facilitates the ionization of water. However, if one of the water molecules in this hexahydrate Mg[H2O]62+ is replaced by a carboxylate ligand, the energy required to dissociate a proton from a metal ion-bound water molecule is increased by approximately 80 kcal/mol and is intermediate between the energy required to deprotonate one water molecule in Mg[H2O]62+ and that for a free water molecule. This effect of the carboxylate group on the pKa of metal ion-bound water appears to be primarily the result of a reduction of the net positive charge of the overall Mg[H2O]52+−(RCOO-) complex rather than any changes in the elect...

The principles of crystal engineering have been used to design a family of structures with potential as octupolar nonlinear optical (NLO) materials. The major aim in such an exercise, a carry-over of molecular symmetry into the crystal, is possible with a retrosynthetic approach. An appropriate choice of precursor trigonal molecules leads from the concept of the dimeric Piedfort unit. The crystal structures and NLO properties of a series of 2,4,6-triaryloxy-1,3,5-triazines, 1−6, are reported. These compounds consistently form quasi-trigonal or trigonal networks that are two-dimensionally noncentrosymmetric. Substitutional variations on the phenyl moieties that were expected to maintain or to perturb this trigonal network have been explored. Molecular nonlinearities have been measured by Harmonic Light Scattering (HLS) experiments. Among the compounds studied, 2,4,6-triphenoxy-1,3,5-triazine, 1 adopts a noncentrosymmetric crystal structure with a measurable SHG powder signal. All these crystal structures a...

Mono- and bi-cyclic 1,3-diazetidin-2-ones (aza-β-lactams) are synthesised and evaluated as non-natural analogues of β-lactams. The aza-β-lactams are designed on the principle that their reaction with active site serine hydroxy will form a carbamoyl–enzyme intermediate that is sluggish to hydrolysis. The synthesis of racemic mono- and bi-cyclic aza-β-lactams is carried out starting from pyrimidinone 18 which is transformed to the densely functionalised substrate 20. The chemical reactivity of tricarbonyl 20 for selective functional group manipulation was first assessed and then it was transformed to amino alcohol 24. Cyclisation of 24 affords aza-carbapenams and its homologation followed by aldol cyclisation provides access to aza-carbacephams. The X-ray structures of aza-carbapenam 35 and aza-carbacepham 42 suggest that the structural requirements for biological activity in β-lactams are fulfilled. An unexpected ozonolysis product, phenol 52 resolves spontaneously during crystallisation and its crystal structure was also determined. The biological activity of the novel mono- and bi-cyclic aza-β-lactams was evaluated with potent gram-positive bacterial strain, Bacillus subtilis and compared with β-lactam antibiotics, ampicillin and penicillin G. Of the 19 aza-β-lactams tested, eight compounds show inhibition better than the standards while another eight are of comparable activity. This study shows that aza-β-lactams represent a novel and non-natural lead towards serine peptidase inhibitors.

The coordination geometry of divalent calcium ions has been investigated by analyses of the crystal structures of small molecules containing this cation that are found in the Cambridge Structural Database, protein crystal structures in the Protein Databank, and by ab initio molecular orbital calculations on hydrated structures of the form Ca[H2O]n2+·mH2O, in which there are n water molecules in the first coordination shell and m water molecules in the second coordination shell (hydrogen bonded to water molecules in the first shell). Calcium ions in crystal structures generally bind to oxygen atoms in ligands (rather than any other element), and their preferred coordination numbers range from 6 to 8. In protein crystal structures the tendency of calcium to bind water molecules is less than for magnesium (1.5 versus 2.2 water molecules on the average per metal ion site, respectively). The ratio of bidentate to monodentate binding of calcium ions to carboxylate groups is similar for small molecules and prote...

The time-of-flight neutron Laue technique has been used to determine the location of hydrogen atoms in the enzyme D-xylose isomerase (XI). The neutron structure of crystalline XI with bound product, D-xylulose, shows, unexpectedly, that O5 of D-xylulose is not protonated but is hydrogen-bonded to doubly protonated His54. Also, Lys289, which is neutral in native XI, is protonated (positively charged), while the catalytic water in native XI has become activated to a hydroxyl anion which is in close proximity to C1 and C2, the molecular site of isomerization of xylose. These findings impact our understanding of the reaction mechanism.

1,4-Difluorobenzo[c]phenanthrene (1,4-DFBcPh) and its putative metabolites, the dihydrodiol and diol epoxides, have been synthesized and structurally characterized, and the extent of DNA binding by the metabolites has been assessed. 1,4-DFBcPh and 1,4-difluoro-10-methoxybenzo[c]phenanthrene were prepared by photochemical cyclization of appropriate naphthylphenylethylenes. The dihydrodiol was synthesized from 1,4-difluoro-10-methoxybenzo[c]phenanthrene, and the diol epoxides were diastereoselectively synthesized from the dihydrodiol. Interesting differences were noted in 1H NMR spectra of the series 1 (syn) diol epoxides of benzo[c]phenanthrene (BcPh) and 1,4-DFBcPh; the BcPh diol epoxide displays a quasi-diequatorial orientation of the hydroxyl groups, but in the 1,4-DFBcPh case these are diaxially disposed. This difference probably stems from the presence of the fjord-region fluorine atom in 1,4-DFBcPh. A through-space, fjord-region H-F coupling has also been observed for 1,4-DFBcPh and its derivatives. Comparative X-ray crystallographic analyses of BcPh and 1,4-DFBcPh and their dihydrodiols show that introduction of fluorine increases the molecular distortion by about 6-7 degrees . As a guide to estimating the molecular distortion and its effects, and for comparison with the X-ray structures in known cases, optimized structures of BcPh, 1,4-DFBcPh, and 1,4-DMBcPh (the dimethyl analogue) as well as their dihydrodiols and diol epoxides were computed. Relative aromaticities of these compounds were assessed by nucleus-independent chemical shift calculations, and 13C NMR chemical shifts were computed by gauge-inducing atomic orbital calculations. 1,4-DFBcPh and its dihydrodiol were subjected to metabolism, and the amount of DNA binding in human breast cancer MCF-7 cells was assessed. The extent of DNA binding was then compared with that for BcPh and its dihydrodiol and the potent carcinogen benzo[a]pyrene. The 1,4-DFBcPh series 2 (anti) diol epoxide-derived DNA adducts were also compared with those arising from intracellular oxidation of the dihydrodiol with subsequent DNA binding. These experiments showed that increased molecular distortion decreased metabolic activation to the terminal metabolites but that diol epoxide metabolites that are formed are the DNA-damaging species.

Time-of-flight neutron diffraction has been used to locate hydrogen atoms that define the ionization states of amino acids in crystals of d -xylose isomerase. This enzyme, from Streptomyces rubiginosus , is one of the largest enzymes studied to date at high resolution (1.8 Å) by this method. We have determined the position and orientation of a metal ion-bound water molecule that is located in the active site of the enzyme; this water has been thought to be involved in the isomerization step in which d -xylose is converted to d -xylulose or d -glucose to d -fructose. It is shown to be water (rather than a hydroxyl group) under the conditions of measurement (pH 8.0). Our analyses also reveal that one lysine probably has an −NH 2 -terminal group (rather than NH 3 + ). The ionization state of each histidine residue also was determined. High-resolution x-ray studies (at 0.94 Å) indicate disorder in some side chains when a truncated substrate is bound and suggest how some side chains might move during catalysis. This combination of time-of-flight neutron diffraction and x-ray diffraction can contribute greatly to the elucidation of enzyme mechanisms.

The metalloenzyme D-xylose isomerase forms well ordered crystals that diffract X-rays to ultrahigh resolution (

With the aim of understanding the transfer of molecular threefold symmetry into supramolecular systems, the crystal structures of 2,4,6-tris(5-chloro-3-pyridyloxy)-1,3,5-triazine, 1,3,5-tris(3-methylphenyl) benzene and 1,3,5-tripyrrolylbenzene are described. In all three cases, C-3 molecular symmetry is carried over into hexagonal crystal packing. This is rationalized in terms of the supramolecular or void symmetry and the relevant intermolecular interactions.

Conformational flexibility in a symmetrical tris-phenol leads to close packed structures that are also characterised by an extended though finite cooperative chain of hydrogen bonds.

The crystal structures of the toluene and chlorobenzene solvates of 2,3,7,8-tetraphenyl-1,9,10-anthyridine are nearly identical save for differences in the mode of solvent inclusion; these differences have an important bearing on the nature of the C–H···π interactions in these structures.