Structure and conformation of 1-beta-D-ribo furanosyl pyridin-2-one-5-carboxamide: an anti-inflammatory agent.

The pyrimidine nucleoside, 1-beta-D-ribofuranosyl pyridine-2-one-5-carboxamide, is an anti inflammatory agent used in the treatment of adjuvant-induced arthritis. It is the 2-one isomer of 1-beta-D-ribofuranosyl pyridine-4-one 5-carboxamide, an unusual nucleoside isolated from the urine of patients with chronic myelogenic leukemia and an important cancer marker. Crystals of 1-beta-D-ribofuranosyl pyridine-2-one-5-carboxamide are monoclinic, space group C2, with the cell dimensions a = 31.7920(13), b = 4.6872 (3), c = 16.1838(11), beta = 93.071(3) degrees , V = 2408.2(2) A(3), D(calc) = 1.496 mg/m(3) and Z = 8 (two molecules in the asymmetric unit). The structure was obtained by the application of direct methods to diffractometric data and refined to a final R value of 0.050 for 1669 reflections with I >or= 3sigma. The nucleoside exhibits an anti conformation across the glycosidic bond (chi(CN) = -15.5 degrees , -18.9 degrees ), a C3 '-endo C2 '-exo [(3)(2)T] ribose pucker and g(+) across the C(4 ')-C(5 ') exocyclic bond. The amino group of the carboxamide group is distal from the 2-one and lacks the intramolecular hydrogen bonding found in the related 2-one molecule. Nuclear magnetic resonance studies shows also an anti conformation across the glycosidic bond but the solution conformation of the furanose ring is not the same as that found in the solid state.

Thiophilic interaction chromatography of serum albumins.

An investigation of the binding of native and recombinant human serum albumin and bovine serum albumin on three thiophilic gels, PyS, 2S, and 3S was performed. In addition to these proteins, we studied serum albumins from several species such as goat, rabbit, guinea pig, rat, hamster, baboon, and pig. Our results reveal that recombinant human serum albumin (rHSA) binds completely to PyS whereas native human serum albumin and bovine serum albumin bind only partially to PyS. The binding affinities of rHSA, human serum albumin and bovine serum albumin to 2S and 3S gels are less than their binding to PyS. Serum albumins from goat, rabbit, guinea pig, rat, hamster, baboon, and pig bind much stronger to 3S gel than human and bovine serum albumins. The binding of pig and hamster serum albumins is stronger than that of rat, goat, baboon, and rabbit.

Thiophilic interaction chromatography of mammalian and avian transferrins.

Transferrins are a class of iron-binding proteins widely distributed in biological fluids. All transferrins possess two metal binding sites, each of which can bind a ferric iron. Transferrins play a major role in plasma iron transport and have anti-bacterial, anti-inflammatory, and immunological functions. Lactoferrin is an iron-binding bilobal protein of the transferrin family found in neutrophilic leukocytes and external secretion of mammals. In an earlier communication, we have shown that both human serum transferrin and lactoferrin bind to 3S-gel. Ovotransferrin, the major egg-white protein, is an avian transferrin. In this paper, the details of the binding of mammalian and avian transferrins to 2S gel is presented. Both, apo and holo ovotransferrin, bind to 2S T-gel. Holo and apo lactoferrin from other mammalian species such as cow, rabbit, dog, mouse, and rat bind to T-gel.

Thiophilic interaction chromatography (TIC) of amyloid-beta protein precursor.

Neuritic plaques, one of the diagnostic characteristics of an AD, contain extracellular deposits of amyloid-beta (Abeta) derived from amyloid-beta protein precursor (AbetaPP). The objective of this study was to extract AbetaPP out of HEK293 cells and to purify it. Two procedures were chosen for purification of AbetaPP: Thiophilic Interaction Chromatography (TIC) and molecular sieving. Using Superdex 75, Superose 12, and Fractogel gel matrices, AbetaPP was isolated on HPLC. The chromatograms illustrate the purification of AbetaPP. Our method describes a new and elegant way for the extraction and purification of AbetaPP from HEK293 cell lines using thiophilic interaction chromatography (TIC).

Linear quantitation of Abeta aggregation using Thioflavin T: reduction in fibril formation by colostrinin.

Thioflavin T (ThT) fluorescence is a commonly used method to monitor Abeta protein fibril formation. This method is particularly attractive since ThT fluoresces only when bound to fibrils, the reaction is completed within 1min and ThT does not interfere with aggregation of Abeta fibrils. One of the drawbacks of this method is the lack of a strict quantitative relationship between ThT fluorescence and fibril content. It was observed that, when the same gram molecular weight of Abeta (1-40) is dissolved into varying amounts of base then placed into a constant volume of aqueous buffer, a non-linear fluorescent response is obtained. By maintaining a strict relationship between Abeta content and the volume of base, this anomalous result can be alleviated and a linear dose response curve is obtained at much lower Abeta concentrations than is typically observed. In addition, differences in Abeta batch to batch preparations are alleviated. It was previously reported that colostrinin (CLN), a proline-rich peptide derived from colostrum, reduces fibril content and protects neuroblastoma cells against Abeta peptide-induced toxicity. The newly developed ThT fluorescence protocol was used to quantify Abeta fibril content after treatment with CLN. We also demonstrate that CLN, can solubilize Abeta fibrils in a dose and time-dependent fashion.

Crystal structure, conformation, and absolute configuration of kanamycin A.

Kanamycin, an antibiotic complex produced by Streptomyces kanamycetius isolated from Japanese soil, was described by Okami and Umezawa as early as 1957 and consists of three components: Kanamycin A (the major component), B, and C. The disulfate salt of kanamycin A [4-O-(6-amino-6-deoxy-alpha-d-glucopyranosyl)-6-O-(3-amino-3-deoxy-alpha-d-glucopyranosyl)-2-deoxystreptamine] is a broad-spectrum antibiotic that is used to treat gonorrhea, salmonella, tuberculosis, and many other diseases. Crystals of kanamycin A monosulfate monohydrate obtained from water are triclinic, space group P1, with a=7.2294(14), b=12.4922(15), c=7.1168(9), alpha=94.74(1), beta=89.16(1), gamma=91.59(1), V=640.2(2)A(3), micro(CuKalpha)=18.4cm(-1), FW 600.6, D(calc)=1.558g/cm(3), CAD-4 diffractometric data (2693 reflections, 25543sigma(I)), structure by shelx-86 and refined by full-matrix least squares to a final R value of 0.038. The wrong conformer had an R value of 0.043. Both of the d-glucose moieties are attached to the deoxystreptamine by alpha linkages. This absolute configuration agrees with the earlier determination by both chemical and X-ray methods with photographic data. The (phi,psi) values for the glycosidic linkages are 101.6 degrees , -121.1 degrees , 106.3 degrees , and -140.4 degrees , respectively. Kanamycin interacts with the ribosomal S12 protein to stabilize the codon-anticodon binding between mRNA and the aminoacyl tRNA and inhibits the elongation of peptide chains through a series of reactions resulting in the prevention of ribosomes from moving along mRNA.

Thiophilic interaction chromatography of human transferrins.

Lactoferrin and serum transferrin, the iron-binding proteins, are widely distributed in biological fluids. Transferrins have many biological functions: anti-bacterial, anti-inflammatory, and immunoregulatory. A facile monitoring of the concentration of transferrins in biological fluids may have medical relevance. Thiophilic-interaction chromatography of transferrins may provide an efficient means for their quantitation in serum.

Investigation of human serum albumin (HSA) binding specificity of certain photosensitizers related to pyropheophorbide-a and bacteriopurpurinimide by circular dichroism spectroscopy and its correlation with in vivo photosensitizing efficacy.

A series of pyropheophorbide-a and bacteriopurpurinimides were investigated to understand the correlation between HSA (site II) binding affinity and in vivo photosensitizing activity. In our study, photosensitizers that bound to site II of HSA produced a significant difference in the circular dichroism spectra of the corresponding complexes, especially at Soret band region of the photosensitizers. Our results suggest that CD spectroscopy of the photosensitizer-HSA complexes could be a valuable tool in screening new photosensitizers before evaluating them for in vivo efficacy.

Protective effect of colostrinin on neuroblastoma cell survival is due to reduced aggregation of beta-amyloid.

Colostrinin (CLN), a mixture of proline-rich polypeptides, has shown a stabilizing effect on cognitive function in Alzheimer's patients measured by the Alzheimer's disease Assessment Scale-cognitive (ADAS-cog) and in Instrumental Activities of Daily Living (ILDL) in recently conducted clinical trials. The aim of this study was to elucidate a possible mode of action of CLN in the treatment of Alzheimer's disease. Here, we report that CLN prevents the aggregation of beta-amyloid peptide Abeta (1-40) in vitro. The impact of CLN on the fibril formation was monitored by optical and electron microscopy. The electron micrographs illustrate that, at 25 microM, Abeta (1-40) peptides formed fibrils after 24-48 h of incubation. The presence of 0.25 microM CLN completely abolished the fibril formation. Abeta (1-40) peptides grow into dense fibers when examined at the 20th day. In the presence of CLN, however, the fibrils are much shorter and less dense. Addition of CLN as late as the 17th day can still dissolves the preformed fibrils. These observations were compared to the effect of CLN on the neurotoxic activity of beta-amyloid peptides in the cell culture model (SHSY-5Y). The beta-amyloid peptides were pre-incubated with CLN at various times and used to treat SHSY-5Y neuroblastoma cells for up to 4 days. The cytotoxic effect was monitored by trypan blue exclusion. We demonstrated that 24-48 h treatment was the onset of toxicity of 10-50 microM of beta-amyloid peptides. Pre-incubation of 0.0025-0.25muM of CLN with 25 microM of beta-amyloid peptides leads to near-complete abolition of cytotoxicity. Low doses of CLN (2.5 nM) can attain cytotoxic protection levels similar to those of highest doses (0.25 microM). Thus, the time course for the appearance of beta-amyloid fibrils coincides with that for cytotoxicity, and that the reduction of fibrils of beta-amyloid peptides by CLN is concomitant with the reduction of the cytotoxic effects of beta-amyloid on SHSY-5Y neuroblastoma cells. Our studies suggest that the neuroprotective effects exerted by CLN are related to the reduction of beta-amyloid fibrils.

Copper binding to prion octarepeat peptides, a combined metal chelate affinity and immunochemical approaches.

Based on the hypothetical proposal of Sulkowski [E. Sulkowski, FEBS Lett. 307 (2) (1992) 129] for the implication of transition metal ions in the structural changes/oligomerisation of normal cellular prion protein (PrPc) resulting in the pathological isoform (PrPsc), we focused our study on the octarepat domain of this protein which has been supposed to be the metal binding site. We have studied the copper binding to synthetic prion octarepeat peptides (PHGGGWGQ)n (n=1, 3, 6) using metal chelate and size-exclusion modes of chromatographies. This copper binding induces oligomerisation resulting in multiple aggregates. Moreover, heterogeneity of metal bound octarepeat oligomers by ESI-MS has been demonstrated. In addition, anti prion antibodies specific to the octarepeat region were used to discriminate between metal free and copper, nickel and zinc bound hexamer octarepeat peptide. Differential recognition of Cu(II) and Zn(II) bound complexes has been observed which signify differences in exposed epitopes of aggregated peptides.

Crystal structure and conformation of 5-fluorouridine: conformational preferences for 5-fluorinated pyranosides.

Crystals of 5-fluorouridine (5FUrd) have unit cell dimensions a = 7.716(1), b = 5.861(2), c = 13.041(1)A, alpha = gamma = 90 degrees, beta = 96.70 degrees (1), space group P2(1), Z = 2, rho obs = 1.56 gm/c.c and rho calc = 1574 gm/c.c The crystal structure was determined with diffractometric data and refined to a final reliability index of 0.042 for the observed 2205 reflections (I > or = 3sigma). The nucleoside has the anti conformation [chi = 53.1(4) degrees] with the furanose ring in the favorite C2'-endo conformation. The conformation across the sugar exocyclic bond is g+, with values of 49.1(4) and -69.3(4) degrees for phi(theta c) and phi (infinity) respectively. The pseudorotational amplitude tau(m) is 34.5 (2) with a phase angle of 171.6(4) degrees. The crystal structure is stabilized by a network of N-H...O and O-H...O involving the N3 of the uracil base and the sugar 03' and 02' as donors and the 02 and 04 of the uracil base and 03' oxygen as acceptors respectively. Fluorine is neither involved in the hydrogen bonding nor in the stacking interactions. Our studies of several 5-fluorinated nucleosides show the following preferred conformational features: 1) the most favored anti conformation for the nucleoside [chi varies from -20 to + 60 degrees] 2) an inverse correlation between the glycosyl bond distance and the chi angle 3) a wide variation of conformations of the sugar ranging froni C2'-endo through C3'-endo to C4'-exo 4) the preferred g+ across the exocyclic C4'-C5' bond and 5) no role for the fluorine atom in the hydrogen bonding or base stacking interactions.

Crystal and molecular structure and absolute configuration of lincomycin hydrochloride monohydrate.

Lincomycin is a broad-spectrum antibiotic synthesized by Streptomyces lincolnensis that is particularly active against Gram-positive bacteria. It is widely used in human and veterinary applications. The crystal structure of lincomycin has been undertaken with a view to obtain the conformational and structural features of the drug in order to afford a comparison of its structural features with other aminoglycoside antibiotics. We report here the details of its structural and conformational features as determined by single-crystal X-ray crystallography. Crystals of lincomycin hydrochloride are orthorhombic, space group P2(1)2(1)2, with the cell dimensions a=18.5294(3) Angstroms, b=20.5980(4) Angstroms, c=6.17380(10) Angstroms, V=2356.35(7) Angstroms3. The structure was solved using X-ray diffraction data and refined to a final R-value of 0.0391 for 2321 reflections (I > or = 2sigma). The absolute configuration was established using the anomalous dispersion of the sulfur and chlorine atoms in the structure. The molecule consists of an amino acid linked by an amide group to a monosaccharide of galactose stereochemistry. A network of hydrogen-bonds stabilizes the crystal structure.

Immobilized metal-ion affinity chromatography of human antibodies and their proteolytic fragments.

Immobilized metal-ion affinity chromatography (IMAC) performed with four different transition metal ions: copper(II), nickel(II), zinc(II) and cobalt(II), was used to study the adsorption properties of human polyclonal gamma-globulines (IgG), Cohn II-III fractions, and their pepsin cleaved fragments: Fab'2 and F'c. In each case, digested products showed lower affinity for metal ions, as well by decreasing pH elution as by competition with imidazole. An explanation was proposed by the presence of a histidine (His) cluster in the F'c domain of IgGs, identified by computer calculation (accessible surface area (ASA) determination) as the more probable His 433-x-His 435 sequence presented in the CH3 domain of human IgG heavy chain. As shown by IMAC and electrophoresis, F'c and undigested IgG have higher affinity for transition metal ions than Fab'2 fragments and could be then separated in one step by IMAC. When chelated Zn(II) or Co(II) are used as ligands, the Fab'2 fragment could be easily recovered under mild conditions (pH 7) in the non-retained fraction. This approach could be used as a powerful alternative to conventional protein A/G methods for the commercial preparation of non immunogen active Fab'2 fragments.

A convergent synthesis of core 2 branched sialylated and sulfated oligosaccharides.

A convergent pathway for the syntheses of core 2 oligosaccharide analogues 1 and 2, and a natural form sialylated and sulfated hexasaccharide 3 was developed. Construction of pentasaccharides 24, 27 and hexasaccharide 28 was achieved by complete regioselective glycosylation of the 6-OH in the acceptors 5, 7 and 8, respectively, owing to the much higher reactivity of the primary hydroxyl group over the secondary axial hydroxyl group in these structures. Stereoselective sialylation was accomplished using donor 10 with defined configuration established through X-ray crystallographic analysis. Target oligosaccharides 1-3 were then obtained by the systematic deprotection of intermediates 24, 27 and 29. With these target oligosaccharides 1-3 obtained, biological evaluations of these molecules as enzyme substrates was undertaken and selectin binding studies are planned.

A novel synthetic route to fused propenochlorin and benzochlorin photodynamic therapy probes.

Reaction of meso-(2-formylvinyl)octaethylporphyrin with (CH3)3SiCN-Cu(OTf)2 produced unexpected 10(3)-trimethylsiloxyl and 10(3)-hydroxyl fused propenochlorins which, in H2SO4, underwent subsequent migration of the 8-ethyl group to the 10(3)-position of the exocyclic benzene ring to form a novel benzochlorin.

Synthesis, structure, and conformation of anti-tumor agents in the solid and solution states: hydroxyl derivatives of Ftorafur.

The pyrimidine antimetabolite Ftorafur [FT; 5-fluoro-1-(tetrahydro-2-furyl)uracil] has shown significant antitumor activity in several adenocarcinomas with a spectrum of activity similar to, but less toxic than, 5-fluorouracil (5-FU). It is considered as a prodrug that acts as a depot form of 5-FU, and hence the two drugs exhibit a similar spectrum of chemotherapeutic activity. Ftorafur is metabolized in animals and humans when hydroxyl groups are introduced into the tetrahydrofuran moiety. These metabolites are also thought to be as active as ftorafur but less toxic than 5-FU. Hydroxyl derivatives: 2'-hydroxyftorafur (III), 3'-hydroxyftorafur (IV) and 2',3'-dihydroxyftorafur (II) were synthesized and X-ray and NMR studies of these hydroxyl derivatives were undertaken in our laboratories to study the structural and conformational features of Ftorafur and its metabolites in the solid and solution states. X-ray crystallographic investigations were carried out with data collected on a CAD-4 diffractometer. The structures were solved and refined using the SDP crystallographic package of Enraf-Nonius on PDP 11/34 and Microvax computers. All of the compounds studied had the base in the anti conformation. The glycosidic torsion angles varied from -20 to 60 degrees. There is an inverse correlation between the glycosyl bond distances and the chi angle. Molecules with a lower chi angle have a larger bond distance and vice versa. The sugar rings show a wide variation of conformations ranging from C2'-endo through C3'-endo to C4'-exo. The crystal structures are stabilized by hydrogen bonds involving the base nitrogen atom N3 and the hydroxyl oxygen atoms of the sugar rings as donors and the keto oxygens O2 and O4 of the base and the hydroxyl oxygen atoms O2' and O3' as acceptors. The NMR studies were carried out on Brüker 400 and 600 MHz instruments. Simulated proton spectra were obtained through Laocoon, and pseudorotational parameters were solved by Pseurot. Presence of syn or anti forms was demonstrated with the use of NOE experiments. The glycosyl conformations in solution vary more widely than in the solid state. The conformations of the sugar molecules are in agreement with the values obtained in the solid state. The studies of the structure and conformation in the solid and solution states give a model for the Ftorafur molecule that could be used in structure, function and biological activity correlation studies.