Neutron Diffraction of Alpha,Beta and Gamma Cyclodextrins: Hydrogen Bonding Patterns

Abstract

Cyclodextrins (CD's) have proved useful as model systems for the study of hydrogen bonding. They are torus-shaped molecules composed of six(α), seven (β) or eight(γ) (1?4) linked glucoses. Because of their particular geometry, they are able to act as a “host” to form inclusion complexes with “guest” molecules very much like enzymes. Cyclodextrins have been shown to exert catalytic activity on suitable included-substrate molecules; they catalyze the hydrolysis of phenylacetates, of organic pyrophosphates and of penicillin derivatives. They also accelerate aromatic chlorinations and diazo coupling by means of their primary and/or secondary hydroxyl groups, so that the rates of hydrolysis are enhanced by up to a factor of 400. In order to understand the hydrogen bonding in these enzyme models, neutron diffraction data were collected to unambiguously determine the hydrogen atom positions, which could not be done from the x-ray diffraction data. α-CD has been shown to have two different structures with well-defined hydrogen bonds, one “tense” and the other “relaxed”. An “induced-fit”-like mechanism for α-CD complex formation has been proposed. Circular hydrogen bond networks have also been found for α-CD due to the energetically favored cooperative effect. β-CD with a disordered water structure possesses an unusual flip-flop hydrogen bonding system of the type O-H…H-O representing an equilibrium between two states: O-H…O?O…O. γ-CD with a disordered water structure similar to β-CD also possesses the flip-flop hydrogen bond. This study demonstrates that hydrogen bonds are operative in disordered systems and display dynamics even in the solid state.     

Ab initio study of the role of lysine 16 for the molecular switching mechanism of Ras protein p21

Quantum chemical computations using the ab initio molecular orbital (MO) method have been performed to investigate the molecular switching mechanism of Ras protein p21, which has an important role in intracellular signal cascades. Lys(16) was demonstrated to be crucial to the function of Ras p21, and the hydrolysis of GTP to GDP was found to be an one-step reaction. The potential energy barrier of this hydrolysis reaction from GTP to (GDP + P) was calculated to be approximately 42 kcal/mol. The role of GAP (GTPase-activating protein) was also discussed in terms of the delivery of the water molecules required for the hydrolysis.     

Water structural changes in the L and M photocycle intermediates of bacteriorhodopsin as revealed by time-resolved step-scan Fourier transform infrared (FTIR) spectroscopy

In previous Fourier transform infrared (FTIR) studies of the photocycle intermediates of bacteriorhodopsin at cryogenic temperatures, water molecules were observed in the L intermediate, in the region surrounded by protein residues between the Schiff base and Asp96. In the M intermediate, the water molecules had moved away toward the Phe219-Thr46 region. To evaluate the relevance of this scheme at room temperature, time-resolved FTIR difference spectra of bacteriorhodopsin, including the water O-H stretching vibration frequency regions, were recorded in the micro- and millisecond time ranges. Vibrational changes of weakly hydrogen-bonded water molecules were observed in L, M, and N. In each of these intermediates, the depletion of a water O-H stretching vibration at 3645 cm-1, originating from the initial unphotolyzed bacteriorhodopsin, was observed as a trough in the difference spectrum. This vibration is due to the dangling O-H group of a water molecule, which interacts with Asp85, and its absence in each of these intermediates indicates that there is perturbation of this O-H group. The formation of M is accompanied by the appearance of water O-H stretching vibrations at 3670 and 3657 cm-1, the latter of which persists to N. The 3670 cm-1 band of M is due to water molecules present in the region surrounded by Thr46, Asp96, and Phe219. The formation of L at 298 K is accompanied by the perturbations of Asp96 and the Schiff base, although in different ways from what is observed at 170 K. Changes in a broad water vibrational feature, centered around 3610 cm-1, are kinetically correlated with the L-M transition. These results imply that, even at room temperature, water molecules interact with Asp96 and the Schiff base in L, although with a less rigid structure than at cryogenic temperatures.     

Water structural changes in the bacteriorhodopsin photocycle: analysis by Fourier transform infrared spectroscopy.

The Fourier transform infrared difference spectra between light-adapted bacteriorhodopsin (BR) and its photointermediates, L and M, were analyzed for the 3750-3450-cm-1 region. The O-H stretching vibrational bands were identified from spectra upon substitution with 2H2O. Among them, the 3642-cm-1 band of BR was assigned to water by substitution with H2(18)O. By a comparison with the published infrared spectra of the water in model systems [Mohr, S.C., Wilk, W.D., & Barrow, G.M. (1965) J. Am. Chem. Soc. 87, 3048-3052], it is shown that the O-H bonds of the water in BR interact very weakly. Upon formation of L, the interaction becomes stronger. The O-H bonds of the protein side chain undergo similar changes. On the other hand, M formation further weakens the interaction of the same water molecules in BR. The appearance of a sharp band at 3486 cm-1, which was assigned tentatively to the N-H stretching vibration of the peptide bond, is unique to L. The results suggest that the water molecules are involved in the perturbation of Asp-96 in the L intermediate and that they are exerted from the protonated Schiff base which changes position upon the light-induced reaction.     

Computational investigation of a new ion-pair receptor for calix[4]pyrrole

Theoretical studies of a new ion-pair receptor, meso-octamethylcalix[4]pyrrole (OMCP), and its interactions with the halide anions F(-), Cl(-), and Br(-) and the cesium halides CsF, CsCl, and CsBr have been performed. Geometries, binding energies, and binding enthalpies were evaluated with the restricted hybrid Becke three-parameter exchange functional (B3LYP) method using the 6-31+G(d) basis set and relativistic effective core potentials. The optimized geometric structures were used to perform natural bond orbital (NBO) analysis. The two typical types of hydrogen bonds, N-H…X(-) and C-H…X(-), were investigated. The results indicate that hydrogen bonding interactions are dominant, and that the halide anions (F(-), Cl(-), and Br(-)) offer lone pair electrons to the σ*(N-H) or σ*(C-H) antibonding orbitals of OMCP. In addition, electrostatic interactions between the lone pair electrons of the halide anion and the LP* orbitals of Cs(+) as well as cation-π interactions between the metal ion and π-orbitals of the pyrrole rings have important roles to play in the Cs(+)?OMCP?X(-) complexes. The current study further demonstrates that this easy-to-make OMCP host compound functions as not only an anion receptor but also an ion-pair receptor.     

Design, synthesis and pharmacological evaluation of spirocyclic σ(1) receptor ligands with exocyclic amino moiety (increased distance 1)

Various pharmacophore models for potent σ(1) ligands specify a basic amino group flanked by two different hydrophobic regions in defined distances to the basic amine (distance 1 and distance 2, respectively). According to these models distance 1 of the potent spirocyclic σ(1) ligand 1 is too short. In order to find a new class of more potent σ(1) ligands and to verify the distance hypothesis of the pharmacophore models spirocyclic compounds 2 with an exocyclic amino group were designed and synthesized. The secondary amines 8 and 9 with N-benzyl residues are >100-fold less potent than the spirocyclic piperidine 1. However, the tertiary methylamines trans-11 and cis-11 represent potent σ(1) ligands with K(i)-values of 43 and 24 nM, respectively. Whereas one large benzyl moiety is required for high σ(1) receptor binding, a second large N-substituent is not tolerated by the σ(1) receptor protein. As a rule, cis-configured diastereomers with a longer distance 1 (predominantly 7.16-7.23 ?) show higher σ(1) affinities than their trans-configured counterparts (distance 1 is predominantly 5.88-6.26 ?).     

Frontier molecular orbital analysis of Cu(n)-O(2) reactivity

Frontier molecular orbital (FMO) theory coupled with density functional calculations has been applied to investigate the chemical reactivity of three key bioinorganic Cu(n)-O(2) complexes, the mononuclear end-on hydroperoxo-Cu(II), the side-on bridged mu-eta(2):eta(2)-O(2)(2-) Cu(II)(2) dimer and the bis-mu-oxo Cu(III)(2) dimer. Two acceptor orbitals (sigma* and pi*) of each complex and two types of donating substrates (sigma-substrate, phosphine; pi-substrate, alkylbenzene) are considered in the electrophilic attack mechanism. The angular dependences of different reaction pathways are determined using FMO theory and the angular overlap model. Including steric effects, the sigma*/sigma and pi*/pi pathways are found more reactive than the corresponding cross sigma*/pi and pi*/sigma pathways which have poor donor-acceptor orbital overlaps in the sterically constrained substrate access region.     

Theoretical investigation on the covalence in AgRnX and XAgRn (X = F – I)

CCSD(T) calculations were performed to investigate the stabilities and interaction mechanisms of the AgRnX and XAgRn (X?=?F – I) series. Dissociation energies and frontier orbital properties demonstrate an increased trend of stabilities. Ag spd hybrids and Rn/X sp hybrids come into the σ_Ag-Rn and σ_Ag-X bonding orbital. The nature of Ag-Rn, Ag-X and Rn-X interactions were investigated by atoms in molecules (AIM) theory. The negative energy density and positive Laplacian values, as well as small electron densities at bond critical points (BCPs), characterize the moderate strength with partial covalence of interactions. BCP properties (?G/V and G/ρ), electron density deformations and natural resonance theory (NRT) results display increased covalence down the periodic table.     

Sugar complexation with calcium ion. Crystal structure and FT-IR study of a hydrated calcium chloride complex of D-ribose

The single crystal structure of CaCl(2).C(5)H(10)O(5).3H(2)O was determined with M(r)=315.16, a=7.537(3), b=11.426(5), c=15.309(6) A, beta=90 degrees, V=1318.3(9) A(3), P2(1)2(1)2(1), Z=2, mu=0.71073 A and R=0.0398 for 2322 observed reflections. The ribose moiety of the complex exists as a furanose with alpha-D configuration. All five oxygen atoms of the ribose molecule are involved in calcium binding. Each calcium ion is shared by two such sugar molecules, coordinating through O(1), O(2), O(3) of one molecule and O(4) and O(5) of the other. The C-C, O-H, C-O and C-O-H vibrations are shifted and the relative intensities changed in the complex IR spectrum, corresponding to the changes in bond distances and angles of the sugar structure. All the hydroxyl groups, water molecules and chloride ions are involved in forming an extensive hydrogen-bond network of O-H...Cl...O-H structure, and the chloride ions play an important role in the crystal packing.     

Chemical synthesis of lipids and the origin of life

Keeping in mind the importance of amphiphilic lipids for the formation of semipermeable membranes, a review summary of the sources of appropriate precursors, and chemical reactions for the abiotic synthesis of lipids is presented here within the framework of the theory of chemical evolution. It covers the presence in different cosmic environments of precursors for the formation of the biochemical molecules necessary for the emergence of life on Earth. It starts (1) with a short introduction. Then the following matters are briefly reviewed: (2) The circumstellar and interstellar molecules, some of which, could generate straight chain fatty acids through C₉. (3) The possible reactions of hydrogenation and hydrolysis of cyanopolyynes which in the presence first of hydrogen and then liquid water could lead to the formation of aliphatic acids. (4) The composition of comets, where the preliminary analysis by mass spectrometry indicate straight chain hydrocarbons through only C₅. (5) The organic compounds in carbonaceous chondrites where aliphatic acids through C₁₂ have been identified, although the branched chain isomers are abundant. (6) The synthesis of some biochemical compounds, such as amino acids present in carbonaceous chondrites, which were probably formed by condensation of presolar precursors, aldehydes and ketones, with HCN in the presence of ammonia and liquid water in the meteorite parent body. The isotopic evidence seems to support this interpretation. (7) The formation of the Earth-Moon system by the catastrophic impact of a Mars-size body with the proto-Earth. (8) The subsequent capture of cometary water, organic and inorganic compounds, which must have led to a very reactive primitive Earth's atmospheric environment. The cometary iron-nickel grains could have catalyzed the formation of fatty acids by Fischer-Tropsch reactions. (9) The laboratory synthesis of straight chain fatty acids from C₅ through C₂₀ by Fischer-Tropsch processes. The amounts are usually in excess of the yields of aliphatic hydrocarbons. The chemical synthesis of glycerophospholipids including phosphatidylcholine. (10) The formation of liposomes, primarily, from phosphatidylcholine and the encapsulation within them of biopolymers. (11) Speculations on protocellular models of increasing complexity based on liposomes enclosing catalytic biomolecules. (12) Finally, some of the important problems remaining to be solved concerning the experimental approach to the study of the origin of life are briefly considered. It is hoped that in the next century, significant advances will be made in our understanding of the origin of life on Earth.     

Non-canonical nucleotide exchange catalyzed by Escherichia coli DNA-polymerase I and the two-center model of the mechanism of action of this enzyme

It is shown, that DNA hydrolysis catalyzed by E. coli DNA polymerase I is inhibited, when a reaction mixture contains one type of deoxynucleoside 5'-triphosphate (dNTP). When the reaction mixture contains [32P]dNTP, then [32P] is incorporated into DNA and v. v. (32P) from DNA is transferred into dNTP. The nucleotide exchange between DNA and dNTP in the assay mixture is observed only in the case, when the chemical nature of nucleotide residue of dNTP and that of the 3'-terminus of DNA is the same. Analysis of products of DNA hydrolysis in the presence of one type of dNTP using electrophoresis in polyacrylamide gel shows that most of the DNA molecules are terminated at the 3'-termini by the dNMP residue of the same chemical nature as the dNTP in the assay mixture. However, in some cases DNA molecules contain one additional nucleotide residue. This phenomenon can be explained by incorporation of one additional dNMP residue originating from dNTP only in those cases, when a non-typical base pairing of this nucleotide residue with a template residue readily takes place. The above-mentioned facts can be interpreted within the model for DNA hydrolysis with involvement of two intermediate covalent forms of dNMP residues with DNA polymerase I; one dNMP-intermediate should be placed at the elongation center and the other--at the hydrolysis center. The DNA hydrolysis by 3'----5' exonuclease activity of DNA polymerase I proceeds through these two covalent forms. DNA polymerases alpha from calf thymus and T4 phage do not catalyze the nucleotide exchange between DNA and dNTP from the reaction media.     

Why is Mg2+ necessary for specific cleavage of the terminal phosphoryl group of ATP?

The mechanism of specific cleavage of the terminal phosphoryl group in hydrolysis of ATP, and the role of Mg2+ in the hydrolysis were studied by ab initio molecular orbital calculations. The tetravalent anion of methyl triphosphate was used as a model of the ATP anion, and its electronic structures were determined as a function of the distance between Mg2+ and its beta-phosphoryl group. We found that the closer location of Mg2+ to the beta-phosphoryl group than to the alpha- or gamma-phosphoryl group was effective in weakening the P-O bond at which the cleavage of ATP catalyzed by most enzymes takes place. Moreover, the orbital coefficient of the frontier electron of P gamma, which is related to the nucleophilic reaction, was shown to increase greatly with increasing interaction between Mg2+ and the beta-phosphoryl group.     

Proton inventory of the water-catalyzed hydrolysis of p-nitrotrifluoroacetanilide

A proton inventory study was made of the water-catalyzed hydrolysis of p-nitrotrifluoro-acetanilide at pH 4.0, 70°C. Multiple protons in the transition state were demonstrated; although two or three protons were shown to be involved, they were not distinguishable. Neither imidazolyl cation nor acetic acid catalyzed the water-catalyzed hydrolysis. The water-catalyzed hydrolysis proceeds through acid catalysis by a water molecule of the breakdown of the tetrahedral intermediate between the anilide and another water molecule. Acid catalysis by the first water molecule is probably assisted by proton transfer from the second water molecule.     

Catalytically important ionizations along the reaction pathway of yeast pyrophosphatase

Five catalytic functions of yeast inorganic pyrophosphatase were measured over wide pH ranges: steady-state PP(i) hydrolysis (pH 4. 8-10) and synthesis (6.3-9.3), phosphate-water oxygen exchange (pH 4. 8-9.3), equilibrium formation of enzyme-bound PP(i) (pH 4.8-9.3), and Mg(2+) binding (pH 5.5-9.3). These data confirmed that enzyme-PP(i) intermediate undergoes isomerization in the reaction cycle and allowed estimation of the microscopic rate constant for chemical bond breakage and the macroscopic rate constant for PP(i) release. The isomerization was found to decrease the pK(a) of the essential group in the enzyme-PP(i) intermediate, presumably nucleophilic water, from >7 to 5.85. Protonation of the isomerized enzyme-PP(i) intermediate decelerates PP(i) hydrolysis but accelerates PP(i) release by affecting the back isomerization. The binding of two Mg(2+) ions to free enzyme requires about five basic groups with a mean pK(a) of 6.3. An acidic group with a pK(a) approximately 9 is modulatory in PP(i) hydrolysis and metal ion binding, suggesting that this group maintains overall enzyme structure rather than being directly involved in catalysis.     

Thermodynamic constraints shape the structure of carbon fixation pathways

Thermodynamics impose a major constraint on the structure of metabolic pathways. Here, we use carbon fixation pathways to demonstrate how thermodynamics shape the structure of pathways and determine the cellular resources they consume. We analyze the energetic profile of prototypical reactions and show that each reaction type displays a characteristic change in Gibbs energy. Specifically, although carbon fixation pathways display a considerable structural variability, they are all energetically constrained by two types of reactions: carboxylation and carboxyl reduction. In fact, all adenosine triphosphate (ATP) molecules consumed by carbon fixation pathways - with a single exception - are used, directly or indirectly, to power one of these unfavorable reactions. When an indirect coupling is employed, the energy released by ATP hydrolysis is used to establish another chemical bond with high energy of hydrolysis, e.g. a thioester. This bond is cleaved by a downstream enzyme to energize an unfavorable reaction. Notably, many pathways exhibit reduced ATP requirement as they couple unfavorable carboxylation or carboxyl reduction reactions to exergonic reactions other than ATP hydrolysis. In the most extreme example, the reductive acetyl coenzyme A (acetyl-CoA) pathway bypasses almost all ATP-consuming reactions. On the other hand, the reductive pentose phosphate pathway appears to be the least ATP-efficient because it is the only carbon fixation pathway that invests ATP in metabolic aims other than carboxylation and carboxyl reduction. Altogether, our analysis indicates that basic thermodynamic considerations accurately predict the resource investment required to support a metabolic pathway and further identifies biochemical mechanisms that can decrease this requirement.     

Application of phosphate-backbone-modified oligonucleotides in the studies on EcoRI endonuclease mechanism of action.

Chemical synthesis of oligodeoxyribonucleotides modified at a preselected internucleotide bond by the replacement of one of the two "nonbridging" oxygens by a sulfur atom or an ethoxy group yields model substrates for studies on DNA-protein interactions. Chromatographic (RP-HPLC) separation of the diastereomers of oligonucleotides containing EcoRI canonical sequence together with the assignment of the substituent orientation in the DNA molecule allowed study of the stereochemical aspects of DNA-EcoRI endonuclease interactions. The DNA segment involved in interactions between EcoRI protein and phosphate groups appeared to be larger than its canonical sequence, ...GAATTC..., and was extended to the nonamer. The modification of certain internucleotide bonds within this nonamer caused significant or complete protection against the nucleolytic action of EcoRI and, in some cases, manifested the diastereoselectivity of the enzyme. On the basis of the results of EcoRI-catalyzed hydrolysis of stereodefined phosphorothioate and phosphotriester substrates, we propose a model to explain this phenomenon at the molecular level.     

Modeling studies of potato nucleoside triphosphate diphosphohydrolase NTPDase1: an insight into the catalytic mechanism

Nucleoside triphosphate diphosphohydrolase--NTPDase1 (apyrase, EC 3.6.1.5) was modeled based on sequence homology. The single polypeptide chain of apyrase is folded into two domains. The putative catalytic site with the apyrase conserved regions (ACR 1-5) is located between these two domains. Modeling confirmed that apyrase belongs to the actin superfamily of proteins. The amino acids interacting with the nucleoside triphosphate substrate and probably involved in the catalyzed hydrolysis were identified. The proposed two-step catalytic mechanism of hydrolysis involves Thr127 and Thr55 as potential nucleophilic factors responsible for the cleavage of the Pgamma and Pbeta anhydride bonds, respectively. Their action seems to be assisted by Glu170 and Glu78 residues, respectively. The presence of two nucleophiles in the active site of apyrase explains the differences in the hydrolytic activity between apyrases and other enzymes belonging to the NTPDase family.     

A comparative theoretical study for the methanol dehydrogenation to CO over Pt3 and PtAu2 clusters

The density functional theory (DFT) calculations are carried out to study the mechanism details and the ensemble effect of methanol dehydrogenation over Pt(3) and PtAu(2) clusters, which present the smallest models of pure Pt clusters and bimetallic PtAu clusters. The energy diagrams are drawn out along both the initial O-H and C-H bond scission pathways via the four sequential dehydrogenation processes, respectively, i.e., CH(3)OH → CH(2)OH → CH(2)O → CHO → CO and CH(3)OH → CH(3)O → CH(2)O → CHO → CO, respectively. It is revealed that the reaction kinetics over PtAu(2) is significantly different from that over Pt(3). For the Pt(3)-mediated reaction, the C-H bond scission pathway, where an ensemble composed of two Pt atoms is required to complete methanol dehydrogenation, is energetically more favorable than the O-H bond scission pathway, and the maximum barrier along this pathway is calculated to be 12.99 kcal mol(-1). In contrast, PtAu(2) cluster facilitates the reaction starting from the O-H bond scission, where the Pt atom acts as the active center throughout each elementary step of methanol dehydrogenation, and the initial O-H bond scission with a barrier of 21.42 kcal mol(-1) is the bottom-neck step of methanol decomposition. Importantly, it is shown that the complete dehydrogenation product of methanol, CO, can more easily dissociate from PtAu(2) cluster than from Pt(3) cluster. The calculated results over the model clusters provide assistance to some extent for understanding the improved catalytic activity of bimetal PtAu catalysts toward methanol oxidation in comparison with pure Pt catalysts.