Methyl β-lactoside: 600-MHz H- and 75-MHz C-n.m.r. studies of H- and C-enriched compounds

Using UDP-d-galactose : 2-acetamido-2-deoxy-d-glucose 4-β-d-galactosyltransferase (EC 2.4.1.22), several methyl β-lactosides have been prepared with ²H- and/or ¹³C-enrichment at specific sites to facilitate study by ¹³C (75 MHz) and ¹H (600 MHz) n.m.r. spectroscopy. ¹³C-Chemical shift assignments were verified and the ¹H-spectrum of β-lactoside was fully assigned. Sites of enrichment were selected to permit all of the potential three-bond C-C and C-H couplings through the glycosidic bond to be obtained. Replacement of H-3 of the d-glucose residue of methyl β-lactoside with ²H allowed resolution of C-1–H-4′ coupling in the 600-MHz ¹H-spectrum. Single or multiple ¹³C-enrichment at C-1, C-2, C-3, C-1′, C-3′, and/or C-4′ in the disaccharide allowed observation of intra- and inter-residue couplings. ¹³C-Spin-lattice relaxation-times (T₁) are interpreted in terms of molecular motion in solution. The data suggest that methyl β-lactoside has an extended conformation with little rotation about the glycosidic bond. Inter-residue couplings are best explained by tortion angles of φ ~ 40° and ψ ~ 15°, indicating that the conformations of β-lactoside in solution and in the crystal are similar.     

Crystal structure of methyl 2,6-dichloro-2,6-dideoxy-3,4-O-isopropylidene-α-D-altropyranoside. A pyranoside system close to a skew-boat conformation

The crystal structure of methyl 2,6-dichloro-2,6-dideoxy-3,4-O-isopropylidene-α-D-altropyranoside (1) has been determined by X-ray diffraction. The compound crystallizes in the orthorhombic system, space group P2₁2₁2₁, with unit-cell dimensions a  7.932, b  8.133, and c  20.447 Å. The structure was solved by the heavy-atom method and refined by the least-squares technique to an R value of 0.047 by using 736 intensities measured on a diffractometer. The pyranoside ring is close to a skew-boat conformation, with C-2 and C-5 being maximally displaced from the least-squares plane through the remaining four atoms. The H-1H-2 dihedral angle of  158° is in agreement with the J_1,2 value of 4.5 Hz. Thus the solid-state conformation appears to correspond with the conformation in solution. The dioxolane ring is in a twist form, with O-4 and, C-8 puckered on opposite sides of the plane of the other ring atoms. The pyranose-ring substituents are in equatorial and pseudoequatorial orientations. The hydrogen atoms at C-3 and C-4 are in a cis arrangement. The orientations of both the methoxyl group and the chloromethyl group with respect to the ring are gauche—trans. The exocyclic anomeric C-1O-1 bond-distance (1.39 Å) is the shortest CO bond in the structure. The intracyclic CO bonds are significantly different, C-1O-5 being less than C-5O-5.     

Étude par r.m.n. de quatre disaccharides peracétylés sélectivement enrichis en 13C

Four peracetylated disaccharides ¹³C-labelled at the C-1′ position and having α-d-(1′→3), β-d-(1′→3), α-d-(1′→4), and β-d-(1′→4) linkages were prepared starting from the commercially available d-[1-¹³C]glucose. They were studied on the basis of their ³J_¹³CH coupling constants in relation with the conformation in solution of oligosaccharides as models for the corresponding polymer. A method of analysis of the n.m.r. spectra is described and the coupling constants J_{¹³C-1′H} given, particularly the ²J coupling (in the same cycle and with sign determination) and the ³J coupling (through the glycosidic bond). In that case, the values obtained give experimental information on the ψ angle values. They are compared with the known X-ray data for similar compounds.     

Carbon-13 as a tool for the study of carbohydrate structures,conformations and interactions

The application of ¹³C-NMR spectroscopy to problems involving the structures and interactions of carbohydrates is described. Both ¹³C-enriched and natural abundance compounds were used and some advantages of the use of the stable isotope are described. Carbon-carbon and carbon-proton coupling constants obtained from 1-¹³ C enriched carbohydrates were employed in the assignment of their chemical shifts and to establish solution conformation. In all cases studied thus far, C-3 couples to C-1 only in the β-anomers while C-5 couples to C-1 only in the α-anomers. C-6 and C-2 always couple to C-1 in both anomeric species. The alkaline degradation of glucose [1-¹³ C] to saccharinic acids was followed by ¹³C-NMR. The conversion of glucose [1-¹³ C] to fructose-1,6-bisphosphate [1,6-¹³ C] by enzymes of the glycolytic pathway was shown as an example of the use of ¹³C-enriched carbohydrates to elucidate biochemical pathways. In a large number of glycosyl phosphates the ³¹P to H-1 and ³¹P to C-2 coupling constants demonstrate that in the preferred conformation the phosphate group lies between the O-5 and the H-1 of the pyranose ring. The influence of paramagnetic Mn²⁺ ions on the proton decoupled ¹³C-NMR spectra of uridine diphosphate N-acetylglucosamine indicates that the Mn²⁺ interacts strongly with the pyrophosphate moiety and with the carbonyl groups of the uracil and N-acetyl groups.     

The ectomycorrhizal fungus Tricholoma matsutake biosynthesizes benzoic acid and benzaldehyde independently

Metabolism of benzoic acids and benzaldehydes are crucial to produce hormones, defense compounds and attractants for pollinators in plants. Tricholoma matsutake contains benzoic acid and benzaldehyde, but their roles have not been fully studied. First we conducted tracer experiments to gain insight into benzoic acid and benzaldehyde biosynthesis in T. matsutake. ¹³C and ²H were incorporated into benzoic acid from uniformly ¹³C- and ²H- labelled l-phenylalanine and (E)-cinnamate 1?d after supplementation of the precursors without any substitution of ¹³C and ²H. In contrast, no ¹³C and ²H were incorporated into benzaldehyde from these precursors 10?d after the supplementation. The results indicate that T. matsutake has a metabolic pathway to biosynthesize benzoic acid from l-phenylalanine and (E)-cinnamate in which benzaldehyde is not a metabolic intermediate. However, 30?d after the supplementation of ¹³C- and ²H- labelled l-phenylalanine, ¹³C and ²H were incorporated into all the carbon and hydrogen atoms of benzaldehyde. In addition, ²H was not incorporated into benzaldehyde from exogenously supplemented ²H-labelled benzoic acid. This result indicates that T. matsutake has a metabolic pathway to biosynthesize benzaldehyde from l-phenylalanine in which benzoic acid is not an intermediate. Taken together, our results strongly suggest that T. matsutake biosynthesizes benzoic acid and benzaldehyde in separate pathways.     

Enzyme models. The attachment of N-methylhydroxamic acid to cyclohexaamylose. Its reactivity and specificity with phenyl esters

The N-methylacetohydroxamic acid group has been introduced into cyclohexaamylose by the following sequence of reactions: (1) carboxymethylation of cyclohexaamylose by iodoacetic acid, (2) methylation of carboxymethylcyclohexaamylose with diazomethane, and (3) reaction of the carboxymethylcyclohexaamylose methyl ester with N-methylhydroxylamine to form the N-methylacetohydroxamic acid-substituted cyclohexaamylose. By employing purification procedures involving ionexchange chromatography, the synthesis yielded a mono-substituted cyclohexaamylose-N-methylacetohydroxamic acid with selective modification of the C-2, C-3 hydroxyl group side of the cyclohexaamylose ring.p-Nitrophenyl acetate and 2-hydroxy-5-nitro-α-toluenesulfonic acid sultone react 20- and 70-fold faster with cyclohexaamylose-N-methylacetohydroxamic acid than with N-methylmethoxyacetohydroxamic acid. Cyclohexaamylose-N-methylacetohydroxamic acid also displays a marked kinetic stereospecificity for p-nitroover m-nitrophenyl acetate (whereas cyclohexaamylose itself exhibits the reverse stereospecificity). These reactions were shown to be competitively inhibited by cyclohexanol. This evidence indicates that cyclohexaamylose-N-methylacetohydroxamic acid binds the substrate in a reversible complexation step prior to nucleophilic attack and thus is an enzyme model.     

Synthesis of 4,5-dideoxy-4-C-[(R,S)-phenylphosphinyl]-d-ribo- and l-lyxo-furanose and their 1,2,3-triacetates

2,3-O-Isopropylidene-d-ribose diethyl dithioacetal, prepared from d-ribose, was converted in three steps into the corresponding dimethyl acetal, which was monotosylated at O-5, and the ester oxidized at C-4 with pyridinium chlorochromate; addition of methyl phenylphosphinate to the resulting pentos-4-ulose derivative then provided (4R,S)-4,5-anhydro-2,3-O-isopropylidene-4-C-[(R,S)-(methoxy)phenylphosphinyl]-d-erythro-pentose dimethyl acetal. Hydrogenation of this compound in the presence of Raney Ni, followed by reduction with SDMA, hydrolysis, and acetylation, yielded the title compounds (seven kinds), the structures of which were established on the basis of their 400-MHz, ¹H-n.m.r. and mass spectra. A general dependence of the ²J_PH and ³J_PH values on the OPCH and PCCH dihedral angles provided an effective method for the assignment of the configurations and conformations of these 4-deoxy-4-phosphinyl-pentofuranoses.     

Infrared-spectral characteristics of some acetylated,anomeric glycosides

Barker and co-workers had described the C-1-H deformation bands in the ranges 844 ±8 cm^?1 and 891 ±7 cm^?1 as characteristic bands for the α and β anomers, respectively, of hexo- and pento-pyranoses and -pyranosides, and their derivatives. Later, Audichya and co-workers reported the presence of the 844 ±8-cm^?1 band for both anomers of some aryl d-glucoside derivatives, making the applicability of the earlier findings doubtful. Examination by us of the i.r. spectra of some aryl glycoside derivatives suggested that the origin of the band at 844 ±8 cm^?1 for the β anomers of the p-substituted-aryl glycoside derivatives studied by Audichya et al. could be a CH, out-of-plane deformation-mode of the substituted aromatic ring. Also, their further claim of a characteristic band in the region 961-957 cm^?1 for α anomers is shown to be of little diagnostic value. The relative intensities of bands in the COC stretching region, 1100-1000 cm^?1, and a band near 300 cm^?1 in the COC deformation region, found only for the β anomers, are shown to be helpful in differentiating the anomers of some peracetylated alkyl and aryl glycosides.     

NMR studies on UO22+ complexes with pyridoxal

The interaction of pyridoxal with dioxouranium(VI) acetate was studied by ¹H and ¹³C NMR measurements in D₂O and CD₃OD.The results indicate that the preferred bonding site is the C-3O^? donor, and the major species obtained under the experimental conditions used is the equimolar complex.     

1H, 13C and 199Hg NMR characteristics of new amine-mercuric chloride complexes

Reduction of some N-alkylimines has been achieved with NaBH₄ to give the corresponding secondary amines with high yields (85–95%). These amines were characterized on the bases of their ¹H and ¹³C NMR spectra. The reaction of these amines with mercuric chloride to afford the corresponding complexes was found to occur through a weak dative bond between the nitrogen lone pair of electrons and the mercury atom to form HgCl₂L₂ complexes. The ¹H, ¹³C and ¹⁹⁹Hg NMR chemical shifts have been obtained as well as ¹J(¹³CH) and ²J(¹³CH) coupling constants. Labelling with nitrogen-15 revealed that there is a weak coupling between the nitrogen and the ¹⁹⁹Hg.     

Different biosynthetic pathways of the pyrimidine moiety of thiamin in procaryotes and eucaryotes

[¹⁴C]Formate is incorporated into the C-2 of the pyrimidine moiety of thiamin by Escherichia coli and Salmonella typhimurium. In Saccharomyces cerevisiae, it is incorporated into C-4. Radioactive carbons of [1-¹⁴C]glycine and [2-¹⁴C]glycine are incorporated by S. typhimurium into the C-4 and C-6 of the pyrimidine, respectively, but not by S. cerevisiae. These facts suggest that procaryotes and eucaryotes have different biosynthetic pathways for pyrimidine. In this study, the procaryotes tested incorporated [¹⁴C]formate into the C-2 and the eucaryotes incorporated it into the C-4 of the pyrimidine.     

The synthesis and N.M.R. spectroscopy of derivatives of 6-amino-6-deoxy-D-galactose-6-15N

Derivatives of 6-amino-6-deoxy-D-galactose-6-¹⁵N have been synthesized by reaction of the 6-deoxy-6-iodo (1) or 6-O-p-tolylsulfonyl derivative of 1,2:3,4-di-O-isopropylidene-α-D-galactopyranose with potassium phthalimide-¹⁵N. The reaction of 1 also yielded an elimination product, 6-deoxy-1,2:3,4-di-O-isopropylidene-β-L-arabino-hex-5-enopyranose. The structures of the 6-amino-6-deoxy-D-galactose derivatives and their precursors were characterized by proton- and ¹³C-n.m.r. spectroscopy, with confirmation of the ¹³C assignments by selective proton decoupling. Selective broadening of the C-1, C-4, C-5, and C-6 resonances of 6-amino-6-deoxy-1,2:3,4-di-O-isopropylidene-α-D-galactopyranose by low concentrations of cupric ion was observed, and studied by computerized measurements of the ¹³C linewidths. The application of this broadening to ¹³C-spectral assignments of amino sugar derivatives is indicated.     

Synthesis,characterization and antitumor activity of a series of N-substituted iminodiacetato(diammine) platinum(II) complexes

A series of water-soluble N-substituted iminodiacetato (diammine)platinum(II) complexes [Pt(NRIDA)(NH₃)₂] have been synthesized and characterized by measurement of physical properties (conductivity and pH) and by various spectroscopic techniques (infrared, ¹H and ¹³C{¹H} nuclear magnetic resonance). The iminodiacetate ligand is coordinated to platinum through an O,N linkage. The results obtained suggest that these complexes are relatively stable for more than 24 h in aqueous solution. Preliminary in vitro and in vivo screening test for antitumor activity of these complexes against L1210 murine leukemia were performed. Many of complexes had acceptable in vitro cytotoxicity, but none displayed a significant level of in vivo antitumor efficacy.     

Different rotamer populations around the C-5C-6 bond for α- and β-d-galactopyranosides through the combined interaction of the gauche and anomeric effects: A 300-MHz 1H-n.m.r. and mndo study

A 300-MHz ¹H-n.m.r. study of methyl 2,3,4-tri-O-methyl-ga- (1) and β-d-galactopyranoside-6-(dimethyl phosphate) (3), using various solvents, shows that the gauche (gg) rotamer populations about the C-5C-6 bond are are the same in all solvents, whereas those of the gauche(trans) (gt) and trans(gauche) (tg, O-5 and O-6 trans) rotamers are solvent dependent. The tg population increases with decreasing polarity of the solvent, which is attributed to an increased electrostatic repulsion between O-5 and O-6 in apolar solvents. The tg population of 3 is larger than that of 1 and the same difference is observed in the corresponding compounds (2 and 4) which have a trigonal-bipyramidal five-coördinated phosphorus (P^v) at position 6 and which have a higher electron density at O-6. These differences in rotamer populations are due to an effect additional to that of the coulombic effect between O-5 and O-6. That these differences are caused by a combination of the gauche and anomeric effects is supported by the finding that the tg population increases with increasing pK_a of the group at C-1. The results of the n.m.r. measurements (in CCl₄) are reproduced fairly accurately by MNDO calculations on model systems. The solvent dependence of the rotamer population around the C-5ẋC-6 bond is a good criterion for the assignment of the H-6S,6R resonances since, for galactopyranosides, J_5,6S increases and J_5,6R decreases as the polarity of the solvent decreases.     

Limited in vitro efficacy of CYP17A1 inhibition on human castration resistant prostate cancer

Although accumulating evidence indicates high expression of CYP17A1(P45017A1) allows castration resistant prostate cancer (CRPC) to maintain high intratumoral androgen levels, the potential P45017A1 activity has not been characterized yet. The aim of this study was to examine the potential CYP17A1 activity including 17α-hydroxylase and 17,20-lyase activities in human CRPC and the effect of a CYP17A inhibitor. We used three human CRPC cell lines: C4-2 and C4-2AT6 which was established from C4-2 under androgen ablation conditions for 6 months, and PC3. To ascertain the potential CYP17A1 activity, we cultured with the steroid precursors: ¹³C-[2,3,4]-progesterone (13C-Prog), and analyzed the sequential biosynthesis ¹³C-[2,3,4]-17-hydroxyprogesterone (13C-17OHP) and ¹³C-[2,3,4]-androstenedione(13C-Adione) by liquid chromatography/mass spectrometry (LC/MS/MS).The C4-2AT6 cells showed significantly higher CYP17A1 expression than C4-2 cells (p < 0.001). LC/MS/MS analysis enabled us to detect the 13C-17-OHP and 13C-A-dione in these cell lines. The concentration ratio of 13C-Adione/13C-17OHP (Adione–17OHP ratio), which is thought to reflect the differences between 17-hydroxylase and 17,20-lyase activities, was then determined. The Adione–17OHP ratio in C4-2AT6 cells was significantly higher than that of C4-2 cells (p < 0.001). Abiraterone were able to inhibit the CYP17A activities, although abiraterone did not have anti-proliferative effects on C4-2 and C4-2AT6 cells at clinically achievable concentrations of <1000 nM in vitro. The present study clearly demonstrates CRPC have the dual activities of CYP17A1 mediated by 17-hydroxylase activity and 17,20-lyase activity. Abiraterone doesn’t have an in vitro anti-proliferative efficacy in CRPC cells, suggesting limited efficacy in vitro.     

5-deoxystansioside,an iridoid glucoside from Tecoma stans

In addition to plantarenaloside and stansioside, a new iridoid glucoside with a formyl group at C-4 has been isolated from Tecoma stans. The new glucoside was shown to be 5-deoxystansioside by ¹³C NMR and ¹H NMR spectroscopy.     

Acid dissociation of cyclooctaamylose

Acid dissociation constants of aqueous cyclooctaamylose (8-Cy) have been determined at 15–45°C by pH potentiometry. Standard enthalpies and entropies of dissociation are derived from the temperature dependences of these pK_a's. These results are compared to corresponding measurements of aqueous cyclohexaamylose and cycloheptaamylose, and the observed trends are interpreted in terms of complexation of cycloamylose with hydroxide ion. ¹³C-nmr spectral measurements are reported for 8-Cy in 99.8% D₂O solution, and assignments of the observed lines are made with the help of deuterium-induced differential isotopic shift experiments.     

Synthesis and spectroscopic properties of CpRuCl(R-DAB(4e)) and CpRuCo(CO)3(R-DAB(6e)). Part XVII. X-ray structure determination of CpRuCo(CO)3(t-Bu-DAB(6e))

CpRuCl(PPh₃)₂ reacted with excess R-DAB in refluxing toluene to give CpRuCl(R-DAB(4e)) (1a: R = i-Pr; 1b: R = t-Bu; 1c: R = neo-Pent; 1d: R =p-Tol). ¹H NMR and ¹³C NMR spectroscopic data indicated that in these complexes the R-DAB ligand is bonded in a chelating 4e coordination mode.Reaction of 1a and 1b with one equivalent of [Co(CO)₄]⁻ afforded CpRuCo(CO)₃(R-DAB(6e)) (2a: R = i-Pr; 2b: R = t-Bu). The structure of 2b was determined by a single crystal X-ray structure determination. Crystals of 2b are monoclinic, space group P2₁/n, with four molecules in a unit cell of dimensions: a = 16.812(4), b = 12.233(3), c = 9.938(3) Å and β = 105.47(3)°. The structure was solved via the heavy atom method and refined to R = 0.060 and R_w = 0.065 for the 3706 observed reflections. The molecule contains a RuCo bond of 2.660(3) Å and a cyclopentadienyl group that is η⁵-coordinated to ruthenium [RuC(cyclopentadienyl) = 2.208(3) Å (mean)]. Two carbonyls are terminally coordinated to cobalt (CoC(1) = 1.746(7) and CoC(2) = 1.715(6) Å) while the third is slightly asymmetrically bridging the RuCo bond (RuC(3) = 2.025(6) and CoC(3) = 1.912(6) Å). The RuC(3)O(3) and CoC(3)O(3) angles are 138.4(5)° and 136.5(5)°, respectively. The t-Bu-DAB ligand is in the bridging 6e coordination mode: σ-N coordinated to Ru (RuN(2) = 2.125(4) Å), μ₂-N′ bridging the RuCo bond and η²-CN coordinated to Co (RuN(1) = 2.113(5), CoN(1) = 1.941(4) and CoC(4) = 2.084(5) Å). The η²-CN′ bonded imine group has a bond length of 1.394(7) Å indicating substantial π-backbonding from Co into the anti-bonding orbital of this CN bond.¹H NMR spectroscopy indicated that 2a and 2b are fluxional on the NMR time scale. The fluxionality of 6e bonded R-DAB ligands is rarely observed and may be explained by the reversible interchange of the σ-N and η²-CN′ coordinated imine parts of the R-DAB ligand.     

31 P and 13 C nmr measurements of a fructose-1-phosphate, Mn (II) and fructose 1,6-diphosphatase complex

Evidence for a ternary FDPase-Mn(II)-fructose-1-P complex is derived from ³¹P and ¹³C nmr measurements. Preliminary estimates of phosphorus and carbon to Mn(II) distances suggest that the oxygen at C-1 is coordinated to the metal ion in both the binary and ternary complex.     

Synthesis of branched-chain,pyrrolidino-sugar derivatives related to apiose

3-C-(Acetamidomethyl)-1,2-O-isopropylidene-β-l-threofuranose (4) and the 3-acetate (5) have been prepared in high yields from mono-O-isopropylidene-d-apiose [3-C-(hydroxymethyl)-1,2-O-isopropylidene-β-l-threofuranose] (1). Acid-catalyzed methanolysis of 4 caused migration of the isopropylidene group and the formation of methyl 4-acetamido-4-deoxy-3-C-(hydroxymethyl)-2,3-O-isopropylidene-β-d-erythrofuranoside (8) in 25% yield. The major product (45%) from the acetolysis of 4 was also a pyrrolidine derivative, namely, 4-acetamido-3-C-(acetoxymethyl)-1-O-acetyl-4-deoxy-2,3-O-isopropylidene-β-d-erythrofuranose (10). Acetolysis of 5 removed the isopropylidene group and gave four acetylated pyrrolidines (isomeric at C-1 and C-2). Conditions which resulted in minimal epimerization at C-2 were established, and the major isomers 12 and 13 were isolated in reasonable yields. ¹H- and ¹³C-n.m.r. data for equilibrium solutions of the pyrrolidines, and for intermediates 1-5, are given.