X-ray structures of new dipeptide taste ligands

The molecular basis of sweet taste was investigated by carrying out the crystal state conformational analysis by X-ray diffraction of the following dipeptide taste igands:N-3,3-dimethylbutyl-aspartyl-phenylalanine methyl ester, I (N-DMB-Asp-Phe-OMe), its sodium salt (N-DMB-Asp-Phe-ONa), II , aspartyl-D -2-aminobutyric acid-(S)-α-ethylbenzylamide, III (Asp-D -Abu-(S)-α-ethylbenzylamide), aspartyl-N′-((2,2,5,5-tetramethylcyclopentanyl)-carbonyl)-(R)-1,1-diamino-ethane, IV (Asp-(R)-gAla-TMCP), and aspartyl-D -valine-(R)-α-methoxymethylbenzyl amide, V (Asp-D -Val-(R)-α-methoxymethylbenzylamide). With the exception of the sodium salt II , all compounds are sweet-tasting, showing in some cases considerable potency enhancement with respect to sucrose. The results of this study confirm the earlier model that an ‘L-shape’ molecular array is essential for eliciting sweet taste for dipeptide-like ligands. In addition, it was established that (i) substitution of the N-terminal group does not inhibit sweet taste, if its zwitterionic character is maintained; (ii) a hydrophobic group located between the stem and the base of the L-shape could be responsible for sweetness potency enhancement, as found in I, III and IV ; in fact, the extraordinary potency of the N-alkylated analogue I would support a model with an additional hydrophobic binding domain above the base of the ‘L’; (iii) removal of the methyl ester at the C-terminus of compound I with the salt formation gives rise to the tasteless compound II ; (iv) for the first time all possible side-chain conformers (g⁻,g⁺andt) for the N-substituted aspartyl residue were observed; and (v) a retro-inverso modification, incorporated at position 2 of the dipeptide chain, confers greater flexibility to the molecule, as demonstrated by the contemporary presence of six conformationally distinct independent molecules in the unit cell and yet sweet taste properties are maintained, as found in IV . © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Platinum-assisted preparation of PS ligands containing chiral centres; X-ray crystal structure of a dithiophosphinite platinum complex

The design and synthesis of some ligands containing P(III) bonded to sulfur (thiophosphinites) and chiral centres is described in this paper.Their complexes with platinum (II), [PtCl₂L], (L = bidentate dithiophosphinite) have been prepared and characterised and it has been shown that in many cases, the coordination to platinum protects these ligands from decomposition processes operated by moisture and oxygen. The first example of X-ray crystal structure of a platinum coordinated dithiophosphinite is described for complex cis-[PtCl₂L], [L = meso-2,3-bis(diphenylthiophosphinito)-dimethyl-succinate], 4a.     

A family of titanium (IV) alkoxo complexes with N,O and O,O chelating ligands. Crystal structure of [Ti(O-i-Pr)2{2-(−)-menthoxo-pyridine}2]

In view of the wide applicability and versatility of titanium based Lewis acids in selective organic synthesis including asymmetric synthesis, we have synthesized a family of mono and polyatomic titanium derivatives. The polymetallic complexes prepared are bridged by pyridimine, quinone and triazine based ligands. The synthesis of [{Ti(O-i-Pr)₃(Oddbf)}₂] (1), [Ti(O-i-Pr)₂(Oddbf)₂] (2), [{Ti(O-i-Pr)₂(Oddbf)(OMent)}₂] (3) (ddbfO = 2,3-dihydro-2,2-dimethyl-benzofuranoxo; MentO = (1R,2S,5R)-(−)-menthoxo), [{Ti(O-i-Pr)₃(OMenpy)}₂] (4), [Ti(O-i-Pr)₂(OMenpy)₂] (5) (MenpyO = (1S,2S,5R)-(−)-menthoxo-pyridine); [{(Ti(OR)₃)₂L}_n] (RO = isopropoxo, (1R,2S,5R)-(−)-menthoxo) (6-11) and [{(Ti(O-i-Pr)₃)₃L}_n] (12) was accomplished from a Lewis acid such as Ti(O-i-Pr)₄, [{Ti(O-i-Pr)₃(OMent)}₂] or [Ti(OMent)₄] and chelating ligands (ddbfOH = 2,3-dihydro-2,2-dimethyl-benzofuranol; MenpyOH = (1R,2S,5R)-(−)-5-methyl-2-isopropyl-1-(2′-pyridinyl)cyclohexan-1-ol; LH₂ = 4,6-dihydroxy-2,5-diphenyl-pyrimidine, 2,4-dihydroxy-5,6-dimethyl-pyrimidine, 5,8-dihydroxy-1,4-napthoquinone, 2,5-dihydroxy-1,4-benzoquinone and LH₃ = cyanuric acid) that provide a rigid framework for the metal centre. The molecular structure of 5 has been determined by single crystal X-ray diffraction studies.     

Crystallization and preliminary X-ray crystallographic analysis of arylesterase from Pseudomonas fluorescens

Large crystals of arylesterase from Pseudomonas fluorescens have been grown at room temperature using ammonium sulfate as a precipitant. They grow to dimensions of 0.7 × 0.7 × 0.6 mm³ within a month. The crystals belong to the trigonal space group P3₁ (or P3₂), with unit cell dimensions of a= 147.12 Å and c= 131.08 Å. The asymmetric unit seems to contain six molecules of dimeric aryles-terase, with corresponding crystal volume per protein mass (VM ) of 2.53 ų/Da and solvent fraction of 51.5% by volume. The crystals diffract to at least 2.2 Å Bragg spacing when exposed to X-rays from a rotating-anode source. X-ray data have been collected to 2.9 Å Bragg spacing from native crystals. © 1993 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray crystallographic analysis of chitinase from barley seeds

Chitinase from barley seeds has been crystallized at room temperature using polyethylene glycol as precipitant. The crystal is monoclinic, belonging to the space group P2₁, with unit cell parameters of a = 69.43 Å, b = 44.55 Å, c = 81.41 Å, and β = 111.95 Å. The asymmetric unit seems to contain two molecules of chitinase with a corresponding crystal volume per protein mass (V_M) of 2.25 ų/Da and a solvent content of 45% by volume. The crystal diffracts to at least 2.0 Å with X-rays from a rotating anode source and is very stable in the X-ray beam. X-ray data have been collected to better than 2.2 Å Bragg spacing from a native crystal. © 1993 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray crystallographic analysis of ImmE7 protein of colicin E7

The ImmE7 protein, which can bind specifically to the DNase colicin E7 and neutralize its bactericidal activity, has been purified and crystallized in two different crystal forms by vapor diffusion method. The orthorhombic crystals belong to space group I222 or I2₁2₁2₁ and have unit cell dimensions a = 75.1 Å, b = 50.5 Å, and c = 45.4 Å. The second form is monoclinic space group P2₁ with ceil dimensions a = 29.3 Å, b = 102.7 Å, c = 53.0 Å and β = 91.5°. The orthorhombic crystals diffract to 1.8 Å resolution, and are suitable for high-resolution X-ray analysis. © 1995 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray crystallographic analysis of phosphoribulokinase from Rhodobacter sphaeroides.

A recombinant form of Rhodobacter sphaeroides phosphoribulokinase (PRK), expressed in Escherichia coli and isolated by affinity chromatography, was crystallized by the sitting drop vapor diffusion technique using NH4H2PO4 (pH 5.6) as the precipitating agent. PRK crystallizes in the cubic space group P432, with unit cell parameters a = b = c = 129.55 A. Based on the assumption of one 32-kDa monomer per asymmetric unit, the Vm value is 2.83 A3/Da. The octameric molecular symmetry is consistent with two planar tetramers stacked in a nearly eclipsed arrangement. A native data set has been collected to 2.6 A resolution.     

Crystallization and preliminary X-ray crystallographic analysis of arylesterase from Vibrio mimicus.

Single crystals of arylesterase (EC 3.1.1.2) from Vibrio mimicus have been obtained from ammonium sulfate as a precipitant at room temperature for 2 months. The present crystals diffract up to 2.2 A resolution and belong to monoclinic space group P2(1). The cell dimensions are a = 55.65(1) A, b = 53.46(1) A, c = 65.79(1) A, and beta = 106.54(1) degrees. There are two molecules of molecular weight 22 kDa in an asymmetric unit with a solvent content of 43%.     

Crystallization and preliminary X-ray crystallographic analysis of phospholipid transfer protein from maize seedlings

Phospholipid transfer protein from maize seedlings has been crystallized using trisodium citrate as precipitant. The crystal belongs to the orthorhombic space group P2₁2₁2₁ with unit cell dimensions of a = 24.46 Å, b = 49.97 Å, and c = 69.99 Å. The presence of one molecule in the asymmetric unit gives a crystal volume per protein mass (V_m) of 2.36 Å ³/Da and a solvent content of 48% by volume. The X-ray diffraction pattern extends at least to 1.6 Å Bragg spacing when exposed to both CuK_α and synchrotron X-rays. A set of X-ray data to approximately 1.9 Å Bragg spacing has been collected from a native crystal. © 1994 Wiley-Liss, Inc.     

Natural dolapyrrolidone: Isolation and absolute stereochemistry of a substructure of bioactive peptides

During the course of our chemical analysis of the hydrophilic fractions from marine cyanobacterium Moorena producens, we have isolated natural dolapyrrolidone (Dpy, 1 ), a natural pyrrolidone derived from phenylalanine, for the first time as a single compound. Compound 1 , with an (S)-l absolute stereochemistry, was previously identified as a substructure that is common among several bioactive natural peptides. Surprisingly, the absolute stereochemistry of the isolated natural 1 , determined through total synthesis, was (R)-d . This result was unambiguously determined by HPLC analysis using a chiral stationary column by comparing the retention times of the natural 1 and authentic samples of synthetic enantiomers. To verify the unexpected result, the absolute stereochemistry of the natural 1 was confirmed by X-ray crystallographic analysis of Pt-complex derivative using the synthetic enantiomer.     

Early transition metal complexes of dinucleating Pacman ligands: X-ray crystal structures of mixed-valence V/V complexes

The use of a Schiff-base calixpyrrole (L) as a dinucleating ligand for early transition metals is described. Salt elimination reactions between the crystallographically-characterised [K₄(THF)₃(PhMe)(L)] and titanium(III) and vanadium(III) halides form the new dinuclear complexes [(MCl)₂(L)] (M = Ti, V). Adventitious, and partial, oxidation of [(VCl)₂(L)] resulted in the formation of the unusual mixed-valence vanadyl complexes [(VO)(S)(VCl)(L)] (S = THF or pyridine), which both adopt desired Pacman geometries in the solid state in which the oxo ligand is accommodated within the dinuclear molecular cleft.     

Crystallization and preliminary X-ray crystallographic analysis of squalene-hopene cyclase from Alicyclobacillus acidocaldarius.

The membrane-associated protein squalene-hopene cyclase from Alicyclobacillus acidocaldarius was overexposed in Escherichia coli and purified by ion exchange and gel permeation chromatography. Crystals of three interrelated forms were grown by vapor diffusion under identical conditions. The crystals diffract to about 2.3 A resolution, but they are unstable in the X-ray beam. An interpretable heavy-atom derivative was obtained.     

Preliminary crystallographic analysis of class 3 rat liver aldehyde dehydrogenase

NAD-linked aldehyde dehydrogenases (A1DH) (EC 1.2.1.3) catalyze the irreversible oxidation of a wide variety of aldehydes to their respective carboxylic acids. Crystals of a class 3 AIDH (from an Escherichia coli expression system) suitable for X-ray analysis have been obtained. These crystals, which can be grown to a size of 0.8 x 0.3 x 0.2 mm, diffract to 2.5 A resolution. Analysis of the diffraction pattern indicates that the crystals belong to the monoclinic space group P21, with cell parameters a = 65.11 A, b = 170.67 A, c = 47.15 A, and beta = 110.5 degrees. Assuming one dimer per asymmetric unit, the value Vm is calculated to be 2.45 and the solvent content of the crystal is estimated to be 50%. A self-rotation function study produced significant rotation peaks (58% of the origin) on the kappa = 180 section at psi = 90 degrees and phi = 71 degrees and 341 degrees, indicating that the pseudo-dimer axis is (or is very nearly) perpendicular to the b-axis.     

Novel trigonal prismatic iron complexes of expanded hexadentate Jäger type ligands: synthesis and X-ray analysis

The mononuclear iron complexes Fe2a and Fe2b of expanded hexadentate Jäger type ligands H₃2a and H₃2b with [N₄O₂]-donorset were synthesized via reaction of the free ligands with iron(II) acetate. The identity of both compounds was proven by mass spectrometry and micro analysis. Furthermore crystals suitable for X-ray structure determination were obtained in case of Fe2a. The X-ray investigation reveals that this compound is a hexacoordinated iron complex with a rather rare trigonal prismatic geometry of the coordination polyeder: in this complex all six coordination sites are occupied by the donor atoms of the ligand exclusively, which was observed for the first time in iron complexes of Jäger type ligands. Electronic properties of Fe2b were characterized using EPR-spectroscopy and temperature dependent susceptibility measurements, indicating a high-spin Fe(III) complex.     

Crystallization and preliminary X-ray crystallographic studies of UDP-N-acetylenolpyruvylglucosamine reductase.

The overexpression and purification of the second enzyme in Escherichia coli peptidoglycan biosynthesis, UDP-N-acetylenolpyruvylglucosamine reductase (MurB), provided sufficient protein to undertake crystallization and X-ray crystallographic studies of the enzyme. MurB crystallizes in 14-20% PEG 8000, 100 mM sodium cacodylate, pH 8.0, and 200 mM calcium acetate in the presence of its substrate UDP-N-acetylglucosamine enolpyruvate. Crystals of MurB belong to the tetragonal space group P4(1)2(1)2 with a = b = 49.6 A, c = 263.2 A, and alpha = beta = gamma = 90 degrees at -160 degrees C and diffract to at least 2.5 A. Screening for heavy atom derivatives has yielded a single site that is reactive with both methylmercury nitrate and Thimerosal.     

Crystallization and preliminary X-ray crystallographic analysis of the 38-kDa immunodominant antigen of Mycobacterium tuberculosis.

The 38-kDa lipoprotein is one of the most potent cell surface immunogens of Mycobacterium tuberculosis in antibody-and T cell-mediated reactions. Using a pure recombinant form of the protein, we have recently shown that it binds phosphate much like that of the phosphate-binding protein (M(r) = 34.4 kDa) that is localized in the periplasm of Escherichia coli and is involved as an initial receptor for active transport of phosphate. The purified 38-kDa protein has been crystallized in 2 forms that are suitable for high-resolution structural analyses. One form belongs to the monoclinic space group P2(1) with unit cell dimensions of a = 67.42 A, b = 113.38 A, c = 42.68 A, and beta = 108.53 degrees. The other is of orthorhombic space group P2(1)2(1)2 with a = 125.46 A, b = 72.27 A, and c = 73.43 A. Both crystal forms diffract to about 2 A resolution on a fine focus rotating anode.     

Synthesis of new platinum(II) complexes containing hybrid thioether-pyrazole ligands: Structural analysis by H and C{H} NMR spectroscopy and X-ray crystal structures

Treatment of the ligands 1,8-bis(3,5-dimethyl-1-pyrazolyl)-3,6-dithiaoctane (bddo), 1,9-bis(3,5-dimethyl-1-pyrazolyl)-3,7-dithianonane (bddn), and 1,6-bis(3,5-dimethyl-1-pyrazolyl)-2,5-dithiahexane (bddh) with several platinum starting materials as K₂PtCl₄, PtCl₂, [PtCl₂(CH₃CN)₂] and [PtCl₂(PhCN)₂] was developed under different conditions. The reactions did not yield pure products. The ratio of the NSSN, NS, SS, NN, and 2NS isomers has been calculated through NMR experiments. Treatment of the mixtures of complexes with NaBPh₄ affords [Pt(NSSN)](BPh₄)₂ (NSSN = bddo, bddn). These Pt(II) complexes have been characterised by elemental analyses, conductivity measurements, IR and ¹H and ¹³C NMR spectroscopy. The X-ray structures of the complexes [Pt(NSSN)](BPh₄)₂ (NSSN = bddo, bddn) have also been determined. In these complexes, the metal atom is tetracoordinated by the two azine nitrogen atoms of the pyrazole rings and two thioether sulfur atoms. When the [Pt(NSSN)](BPh₄)₂ (NSSN = bddo, bddn) complexes were heated under reflux in a solution of Et₄NBr in CH₂Cl₂/CH₃OH (1:1), a mixture of isomers was obtained.     

Preliminary X-ray crystallographic analysis of Tritrichomonas foetus inosine-5′-monophosphate dehydrogenase

Inosine-5′-monophosphate dehydrogenase (IMPDH) from the protozoan parasite Tritrichomonas foetus has been expressed in E. coli and crystallized. Crystals were grown to 0.1 mm in each dimension in 18 to 72 h using ammonium sulfate and low-molecular-weight polyethylene glycols. The crystals belong to the cubic space group P432 with unit cell edge = 157.25 Å. The enzyme is a homotetramer with each monomer having a molecular weight of 55,534 Da. There is one monomer per asymmetric unit, based on a volume/mass ratio of 2.7 ų/Da and self-rotation analysis. The crystals are adequately stable to allow a complete data set to be collected from a single crystal. Complete native data sets have been collected to 2.3 Å resolution at 4°C using synchrotron radiation. High-quality complete data extending to 3.0 Å resolution have been collected from crystals of four putative derivatives, and the data appear to be isomorphous with that of the native crystals in each case. Efforts to solve the derivatives for use in MIR phasing are underway. © 1995 Wiley-Liss, Inc.     

Development of CNS multi-receptor ligands: Modification of known D2 pharmacophores

Several known D₂ pharmacophores have been explored as templates for identifying ligands with multiple binding affinities at dopamine and serotonin receptors considered as clinically relevant receptors in the treatment of neuropsychiatric diseases. This approach has resulted in the identification of ligands that target multiple CNS receptors while avoiding others associated with deleterious effects. In particular, compounds 11, 15 and 22 may have potential for further development as antipsychotic agents as they favorably interact with the clinically relevant receptors including D₂R, 5-HT_1AR, and 5-HT₇R. We have also identified the pair of compounds 11 and 10 as high affinity D₂R ligands with and without SERT binding affinities, respectively. These differential binding profiles endow the pair with the potential for evaluating SERT contributions to antipsychotic drug activity in animal behavioral models. In addition, compound 11 has no significant affinity for 5-HT_2CR and binds only moderately to the H₁R, suggesting it may not induce weight gain or sedation when used clinically. Taken together, compound 11 displays an interesting pharmacological profile that necessitates the evaluation of its functional and in vivo effects in animal models which are currently ongoing.