DFT studies of the ionization of alpha and beta glycopyranosyl donors

Current attempts at mimicking the transition states (TSs) of glycosyl processing enzymes (GPEs) that proceed through TSs with a high degree of oxacarbenium ion formation suffer from a paucity of data about the conformations of such oxacarbenium ions. Because TSs are maxima, the current models based on minimized structures may need some refinement. As part of studies directed at optimizing chemical glycosylation the ionization of 3,4,6-tri-O-acetyl-alpha/beta-D-glucopyranosyl chlorides and triflates, 2,3,4,6-tetra-O-methyl-alpha/beta-D-glucopyranosyl fluorides, chlorides and triflates, 2,3,4,6-tetra-O-methyl-alpha/beta-D-mannopyranosyl fluorides, 2,3-di-O-methyl 4,6-O-benzylidene alpha/beta-D-mannopyranosyl triflates and 2,3-di-O-methyl 4,6-O-benzylidene alpha/beta-D-glucopyranosyl triflates was studied by a prototypic density functional theory (DFT) procedure. In all cases, the alpha-anomers ionized smoothly to 4H3 half chair conformations or adjacent envelopes. By contrast, all beta-anomers exhibited an abrupt conformational change before ionization was complete. The nature of the conformations sampled depends on both the leaving group and the protecting group. The methods presented can be readily adapted to the study of any GPE or chemical glycosylation and provide a method for initial evaluation of plausible TSs, which in turn can be used in mimetic design.     

O-2 Substituted pyranosyl oxacarbenium ions are C-2-O-2 2-fold rotors with a strong syn preference

The substituent at O-2 of glycopyranosides is known to have a pronounced effect on both the formation and the cleavage of glycosides at C-1. This is primarily attributed to stereoelectronic effects on the formation and stability of the related glycopyranosyl oxacarbenium ions. Previous QM studies of 2-O-methyl substituted manno and gluco configured pyranosyl oxacarbenium ions found a preference for the methyl carbon to be syn to the CH-2 methine. This study examines the conformational preference of variously substituted O-2 tetrahydropyranosyl oxacarbenium ions and confirms this syn preference. Neutral analogues are shown to have the expected 3-fold rotation whereas the charged species exhibit 2-fold rotation about C-2-O-2. Natural bond order (NBO) calculations suggest that the dominant stabilizing interaction is a unimodal O-2 lone pair to C-1-O-5 pi-bond hyperconjugative interaction. This syn conformational preference has important implications for mimics of glycopyranosyl oxacarbenium ion transition states. It also suggests a conformational based mechanism that can be exploited to tune the reactivity of glycopyranosyl donors in the glycosylation reaction.     

Synthesis of C-glycopyranosylphloroacetophenone derivatives and their anomerization facilitated by 1,3-diaxial interactions

The reaction of 2,3,4-tri-O-benzyl-6-deoxy-alpha-D-glucopyranosyl fluoride, 2,3,4,6-tetra-O-benzyl-alpha-D-allopyranosyl fluoride, and 2,3,4-tri-O-benzyl-alpha-L-fucopyranosyl fluoride with 2,4-di-O-benzylphloroacetophenone, in the presence of boron trifluoride diethyl etherate, afforded, respectively, the corresponding 3-C-beta-D-glycopyranosylphloroacetophenone derivatives exclusively in anomerically pure form. Alternatively, the reaction of 2,3,4,6-tetra-O-benzyl-alpha-D-gulopyranosyl fluoride with 2,4-di-O-benzylphloroacetophenone afforded both the 3-C-beta-D-gulopyranosylphloroacetophenone derivative (4C(1) conformation) as the major product and the 3-C-alpha-D-gulopyranosylphloroacetophenone derivative (1C(4) conformation) as the minor product under identical conditions. Including the previously prepared C-glycosylphloroacetophenone derivatives that contain 3-C-beta-D-glucosyl, 3-C-beta-D-xylosyl, 3-C-beta-2-deoxy-D-arabino-hexosyl, 3-C-beta-D-galactosyl, 3-C-beta-L-arabinosyl, and 3-C-alpha-L-arabinosyl moieties, the conformation is dictated primarily by the preference of the bulky aromatic aglycon to orient equatorially, due to the strong repulsion of the aglycon. The anomerization is directed secondarily by the presence of 1,3-diaxial interactions in the sugar moiety.     

Monovalent cation size and DNA conformational stability

The effect of monovalent cations on the thermal stability of a small model DNA hairpin has been measured by capillary electrophoresis, using an oligomer with 16 thymine residues as an unstructured control. The melting temperature of the model hairpin increases approximately linearly with the logarithm of increasing cation concentration in solutions containing Na(+), K(+), Li(+), NH(4)(+), Tris(+), tetramethylammonium (TMA(+)), or tetraethylammonium (TEA(+)) ions, is approximately independent of cation concentration in solutions containing tetrapropylammonium (TPA(+)) ions, and decreases with the logarithm of increasing cation concentration in solutions containing tetrabutylammonium (TBA(+)) ions. At constant cation concentration, the melting temperature of the DNA model hairpin decreases in the order Li(+) ～ Na(+) ～ K(+) > NH(4)(+) > TMA(+) > Tris(+) > TEA(+) > TPA(+) > TBA(+). Isothermal studies indicate that the decrease in the hairpin melting temperature with increasing cation hydrophobicity is not due to saturable, site-specific binding of the cation to the random coil conformation, but to the concomitant increase in cation size with increasing hydrophobicity. Larger cations are less effective at shielding the charged phosphate residues in B-form DNA because they cannot approach the DNA backbone as closely as smaller cations. By contrast, larger cations are relatively more effective at shielding the phosphate charges in the random coil conformation, where the phosphate-phosphate distance more closely matches cation size. Hydrophobic interactions between alkylammonium ions interacting electrostatically with the phosphate residues in the coil may amplify the effect of cation size on DNA thermal stability.     

The two-conformer hypothesis: 2,3,4,6-tetra-O-methyl-mannopyranosyl and -glucopyranosyl oxacarbenium ions

Computational chemistry can give information about the probable conformations of reactive intermediates that are difficult to determine experimentally. Based on density functional theory (DFT) calculations of tetra-O-methyl-D-mannopyranosyl and -glucopyranosyl oxacarbenium ions, two families of conformations, which we call B0 and B1, were found. For the manno configuration, a 4H3 and 3E almost isoenergetic pair were found, whereas for the gluco-configuration a 4H3 and 5S1 pair favouring 4H3 were calculated. These results corroborate earlier results and suggest that this two or more conformer hypothesis is general. Nucleophilic attack on these pairs of cations was modelled with methanol and led to four cases to consider namely alpha- or beta-attack on B0 or B1. The resulting complexes (G0, G1 and F0, F1) demonstrate facial selectivity. The relative energies of these complexes are dominated by intramolecular hydrogen bonding and the conformational consequences to the pyranose ring of changes in the C-5-O-5-C-1-C-2 torsion angle. Constrained variation of the nucleophilic oxygen (methanol) to C-1 distance shows that these ion dipole complexes are the only minima with this constraint.     

Guanine residues in d(T2AG3) and d(T2G4) form parallel-stranded potassium cation stabilized G-quadruplexes with anti glycosidic torsion angles in solution.

We report below on proton NMR studies of the G-quadruplex structure formed by the human telomere sequence d(T2AG3) and the tetrahymena telomere sequence d(T2G4) in K cation containing solution. We observe well-resolved proton NMR spectra corresponding to a G-quadruplex monomer conformation predominant at 50 mM K cation concentration and a G-quadruplex dimer conformation predominant at 300 mM K cation concentration. By contrast, d(T2AG3T) and d(T2G4T) form only the G-quadruplex monomer structures independent of K cation concentration as reported previously [Sen, D., & Gilbert, W. (1992) Biochemistry 31, 65-70]. We detect well-resolved resonances for the exchangeable guanine imino and amino protons involved in G-tetrad formation with the hydrogen-bonded and exposed amino protons separated by up to 3.5 ppm. The observed NOEs between the amino and H8 protons on adjacent guanines within individual G-tetrads support the Hoogsteen pairing alignment around the tetrad. The imino protons of the internal G-tetrads exchange very slowly with solvent H2O in the d(T2AG3) and d(T2G4) quadruplexes. The nature and intensity of the observed NOE patterns establish formation of parallel-stranded right-handed G-quadruplexes with all anti guanine glycosidic torsion angles. A model for the parallel-stranded G-quadruplex is proposed which is consistent with the experimental NOE data on the d(T2AG3) and d(T2G4) quadruplexes in solution.(ABSTRACT TRUNCATED AT 250 WORDS)     

Can the stereochemical outcome of glycosylation reactions be controlled by the conformational preferences of the glycosyl donor?

Previous static and dynamical density functional theory studies of the 2,6-di-O-acetyl-3,4-O-isopropylidene-D-galactopyranosyl cations and their methanol adducts has led to an hypothesis that these cations exist in two families of conformers characterized as (2)S(O) and B(2,5), respectively. These families differ by ring inversion, each with its own reactivity. New calculations on the 2,6-di-O-acetyl-3,4-di-O-methyl-D-galactopyranosyl cation confirmed these trends. Removing the isopropylidene group allows more flexibility, but two families of conformers can be discerned with the monocyclic oxocarbenium ions in the E(3) conformation and the bicyclic dioxolenium ions in the (4)H(5) conformation. Attack on the beta-face of these monocyclic cations is favored by hydrogen bonding and the anomeric effect. The experimentally observed high beta-stereoselectivity of mannopyranosyl donors and high alpha-stereoselectivity of glucopyranosyl donors with the 4,6-O-benzylidene protecting groups can be rationalized assuming that the trans-fused 1,3-dioxane ring allows population of only one family of conformers. The combination of hydrogen bonding and conformational changes of the pyranose ring in response to the C-5[bond]O-5[bond]C-1[bond]C-2 torsion angle changes are identified as key factors in stereoselectivity. Based on these observations a strategy to design face discriminated glycosyl donors that exist predominantly in only one family of conformers is proposed.     

DFT studies of the role of C-2-O-2 bond rotation in neighboring-group glycosylation reactions

Although the synthetic utility of the 1,2-trans relationship of the products of neighboring group participation is well established, it is still common to find glycosylation reactions where the stereochemical purity of the products is not 100%. As part of an ongoing series of density functional theory (DFT) studies of the factors that affect glycosylation reactions which are aimed at allowing synthetic chemists to achieve such selectivities, the structures of four oxacarbenium ions and eight methanol complexes of these ions were optimized for the prototypical ions 2-O-acetyl-3,4,6-tri-O-methyl-D-gluco- (1) and mannopyranos-1-yl (2). These studies corroborate the two-conformer hypothesis and further demonstrate that glycopyranosyl oxacarbenium ions exhibit facial selectivity that depends on, besides the inherent steric and Van der Waals effects, the conformational effect associated with the change from sp(2) to sp(3) hybridization at C-1 during nucleophilic attack and H-bonding between the incoming nucleophile and the electronegative atoms of the electrophile. Further studies based on systematic C-2-O-2 bond rotations found TSs that connect the monocyclic ions with the bicyclic ions associated with neighboring-group participation. It was also possible to find two TSs that connect nucleophilic attack at C-1 with C-2-O-2 bond rotation ultimately leading to 1,2-trans O-glycosides, that is, the probable TS that determines the stereochemistry of neighboring-group participation. Both of these TSs exhibit intramolecular H-bonding, which is considered the first step in proton transfer. It is further hypothesized that this coupling of proton transfer and nucleophilic attack is integral to glycosylation. It is further hypothesized that in many cases analogous intermolecular H-bonding is also favorable with the most likely acceptor the anion that is ion-paired to the oxacarbenium ion. These general features are found for both 1 and 2, but characteristic features of each isomer are found that provide further insights into the origins of stereoselectivity.     

Guest-dependent conformation of 18-crown-6 tetracarboxylic acid: relation to chiral separation of racemic amino acids

(+)-18-crown-6 tetracarboxylic acid (18C6H(4)) has been used as a chiral selector for various amines and amino acids. To further clarify the structural scaffold of 18C6H(4) for chiral separation, single crystal X-ray analysis of its glycine(+) (1), H3O+ (2), H5O2+ (3), NH4+ (4), and 2CH3NH3+ (5) complexes was performed and the guest-dependent conformation of 18C6H(4) was investigated. The crown ether ring of 18C6H4 in 3, 4, and 5 took a symmetrical C2 or C2-like conformation, whereas that in 1 and 2 took an asymmetric C1 conformation, which is commonly observed in complexes with various optically active amino acids. The overall survey of the present and related complexes suggests that the molecular conformation of 18C6H4 is freely changeable within an allowable range, depending on the molecular shape and interaction mode with the cationic guest. On the basis of the present results, we propose the allowable conformational variation of 18C6H4 and a possible transition pathway from its primary conformation to the conformation suitable for chiral separation of racemic amines and amino acids.     

The effects of monovalent cations Li+, Na+, K+, NH4+, Rb+ and Cs+ on the solid and solution structures of the nucleic acid components. Metal ion binding and sugar conformation.

The interactions of the monovalent ions Li+, Na+, K+, NH4+, Rb+ and Cs+ with adenosine-5'-monophosphoric acid (H2-AMP), guanosine-5'-monophosphoric acid (H2-GMP) and deoxyguanosine-5'-monophosphoric acid (H2-dGMP) were investigated in aqueous solution at physiological pH. The crystalline salts M2-nucleotide.nH2O, where M = Li+, Na+, K+ NH4+, Rb+ and Cs+, nucleotide = AMP, GMP and dGMP anions and n = 2-4 were isolated and characterized by Fourier Transform infrared (FTIR) and 1H-NMR spectroscopy. Spectroscopic evidence showed that these ions are in the form of M(H2O)n+ with no direct metal-nucleotide interaction, in aqueous solution. In the solid state, Li+ ions bind to the base N-7 site and the phosphate group (inner-sphere), while the NH4+ cations are in the vicinity of the N-7 position and the phosphate group, through hydrogen bonding systems. The Na-nucleotides and K-nucleotides are structurally similar. The Na+ ions bind to the phosphate group of the AMP through metal hydration shell (outer-sphere), whereas in the Na2-GMP, the hydrated metal ions bind to the base N-7 or the ribose hydroxyl groups (inner-sphere). The Na2-dGMP contains hydrated metal-carbonyl and metal-phosphate bindings (inner-sphere). The Rb+ and Cs+ ions are directly bonded to the phosphate groups and indirectly to the base moieties (via H2O). The ribose moiety shows C2'-endo/anti conformation for the free AMP acid and its alkali metal ion salts. In the free GMP acid, the ribose ring exhibits C3'-endo/anti conformer, while a C2'-endo/anti sugar pucker was found in the Na2-GMP and K2-GMP salts and a C3'-endo/anti conformation for the Li+, NH4+, Rb+ and Cs+ salts. The deoxyribose has C3'-endo/anti conformation in the free dGMP acid and O4'-endo/anti in the Na2-dGMP, K2-dGMP and a C3'-endo/anti for the Li+, NH4+, Rb+ and Cs+ salts. An equilibrium mixture of the C2'-endo/anti and C3'-endo/anti sugar puckers was found for these metal-nucleotide salts in aqueous solution.     

Halogen-substituted (C-β-D-glucopyranosyl)-hydroquinone regioisomers: synthesis, enzymatic evaluation and their binding to glycogen phosphorylase

Electrophilic halogenation of C-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl) 1,4-dimethoxybenzene (1) afforded regioselectively products halogenated at the para position to the D-glucosyl moiety (8, 9) that were deacetylated to 3 (chloride) and 16 (bromide). For preparing meta regioisomers, 1 was efficiently oxidized with CAN to afford C-(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl) 1,4-benzoquinone 2 which, in either MeOH or H(2)O-THF containing few equivalents of AcCl, added hydrochloric acid to produce predominantly meta (with respect to the sugar moiety) chlorinated hydroquinone derivatives 5 and 18, this latter being deacetylated to 4. The deacetylated meta (4, 5) or para (3, 16) halohydroquinones were evaluated as inhibitors of glycogen phosphorylase (GP, a molecular target for inhibition of hepatic glycogenolysis under high glucose concentrations) by kinetics and X-ray crystallography. These compounds are competitive inhibitors of GPb with respect to α-D-glucose-1-phosphate. The measured IC(50) values (μM) [169.9±10.0 (3), 95 (4), 39.8±0.3 (5) 136.4±4.9 (16)] showed that the meta halogenated inhibitors (4, 5) are more potent than their para analogs (3, 16). The crystal structures of GPb in complex with these compounds at high resolution (1.97-2.05 ?) revealed that the inhibitors are accommodated at the catalytic site and stabilize the T conformation of the enzyme. The differences in their inhibitory potency can be interpreted in terms of variations in the interactions with protein residues of the different substituents on the aromatic part of the inhibitors.     

Involvement of the H3O+-Lys-164 -Gln-161-Glu-345 charge transfer pathway in proton transport of gastric H+,K+-ATPase

Gastric H(+),K(+)-ATPase is shown to transport 2 mol of H(+)/mol of ATP hydrolysis in isolated hog gastric vesicles. We studied whether the H(+) transport mechanism is due to charge transfer and/or transfer of hydronium ion (H(3)O(+)). From transport of [(18)O]H(2)O, 1.8 mol of water molecule/mol of ATP hydrolysis was found to be transported. We performed a molecular dynamics simulation of the three-dimensional structure model of the H(+),K(+)-ATPase alpha-subunit at E(1) conformation. It predicts the presence of a charge transfer pathway from hydronium ion in cytosolic medium to Glu-345 in cation binding site 2 (H(3)O(+)-Lys-164 -Gln-161-Glu-345). No charge transport pathway was formed in mutant Q161L, E345L, and E345D. Alternative pathways (H(3)O(+)-Gln-161-Glu-345) in mutant K164L and (H(3)O(+)-Arg-105-Gln-161-Gln-345) in mutant E345Q were formed. The H(+),K(+)-ATPase activity in these mutants reflected the presence and absence of charge transfer pathways. We also found charge transfer from sites 2 to 1 via a water wire and a charge transfer pathway (H(3)O(+)-Asn-794 -Glu-797). These results suggest that protons are charge-transferred from the cytosolic side to H(2)O in sites 2 and 1, the H(2)O comes from cytosolic medium, and H(3)O(+) in the sites are transported into lumen during the conformational transition from E(1)PtoE(2)P.     

Transition state analysis for human and Plasmodium falciparum purine nucleoside phosphorylases

Recent studies have shown that Plasmodium falciparum is sensitive to a purine salvage block at purine nucleoside phosphorylase (PNP) and that human PNP is a target for T-cell proliferative diseases. Specific tight-binding inhibitors might be designed on the basis of specific PNP transition state structures. Kinetic isotope effects (KIEs) were measured for arsenolysis of inosine catalyzed by P. falciparum and human purine nucleoside phosphorylases. Intrinsic KIEs from [1'-(3)H]-, [2'-(3)H]-, [1'-(14)C]-, [9-(15)N]-, and [5'-(3)H]inosines were 1.184 +/- 0.004, 1.031 +/- 0.004, 1.002 +/- 0.006, 1.029 +/- 0.006, and 1.062 +/- 0.002 for the human enzyme and 1.116 +/- 0.007, 1.036 +/- 0.003, 0.996 +/- 0.006, 1.019 +/- 0.005, and 1.064 +/- 0.003 for P. falciparum PNPs, respectively. Analysis of KIEs indicated a highly dissociative D(N)A(N) (S(N)1) stepwise mechanism with very little leaving group involvement. The near-unity 1'-(14)C KIEs for both human and P. falciparum PNP agree with the theoretical value for a 1'-(14)C equilibrium isotope effect for oxacarbenium ion formation when computed at the B1LYP/6-31G(d) level of theory. The 9-(15)N KIE for human PNP is also in agreement with theory for equilibrium formation of hypoxanthine and oxacarbenium ion at this level of theory. The 9-(15)N KIE for P. falciparum PNP shows a constrained vibrational environment around N9 at the transition state. A relatively small beta-secondary 2'-(3)H KIE for both enzymes indicates a 3'-endo conformation for ribose and relatively weak hyperconjugation at the transition state. The large 5'-(3)H KIE reveals substantial distortion at the 5'-hydroxymethyl group which causes loosening of the C5'-H5' bonds during the reaction coordinate.     

Assignment of downfield proton resonances in purine nucleoside phosphorylase immucillin-H complex by saturation-transferred NOEs

Purine nucleoside phosphorylase (PNP) catalyzes N-ribosidic bond phosphorolysis in 6-oxypurine nucleosides and deoxynucleosides to form purine and alpha-D-phosphorylated ribosyl products. The transition state has oxacarbenium ion character with partial positive charge near C-1', ionic stabilization from the nearby phosphate anion, and protonation at N-7 of the purine. Immucillin-H (ImmH) has a protonated N-7 and resembles the transition-state charge distribution when N-4' is protonated to the cation. It binds tightly to the PNPs with a K(d) value 56 pM for human PNP. Previous NMR studies of PNP.ImmH.PO(4) have shown that the N-4' of bound ImmH is a cation and is postulated to have a significant contribution to its tight binding. Several unassigned downfield proton resonances (>11 ppm) are specific to the PNP.ImmH.PO(4) complex, suggesting the existence of strong hydrogen bonds. In this study, two of the proton resonances in this downfield region have been assigned. Using (15)N-7-labeled ImmH, a resonance at 12.5 ppm has been assigned to N-7H. The N-7H resonance is shifted downfield by only approximately 1 ppm from its position for ImmH free in aqueous solution, consistent with only a small change in the hydrogen bonding on N-7H upon binding of ImmH to PNP. In contrast, the downfield resonance at 14.9 ppm in the PNP.ImmH.PO(4) complex is assigned to N-1H of ImmH by using saturation-transferred NOE measurements on the PNP.ImmH complex. The approximately 4 ppm downfield shift of the N-1H resonance from its position for ImmH free in solution suggests that the hydrogen bonding to the N-1H in the complex has a significant contribution to the binding of ImmH to PNP. The crystal structure shows Glu201 is in a direct hydrogen bond with N-1H and to O-6 through a water bridge. In the complex with 6-thio-ImmH, the N-1H resonance is shifted further downfield by an additional 1.5 ppm to 16.4 ppm, but the relative shift from the value for 6-thio-ImmH free in solution is the same as in the ImmH complex. Since the binding affinity to hPNP for 6-thio-ImmH is decreased 440-fold relative to that for ImmH, the loss in binding energy is primarily due to the hydrogen bond energy loss at the 6-thiol.     

The DNA structure responds differently to physiological concentrations of K(+) or Na(+)

The influence of monovalent cations on DNA conformation and readout is an open question. This NMR study of DNA with either Na(+) or K(+) at physiological concentrations shows that the nature of the cation affects the (31)P chemical shifts (deltaP) and the sequential distances H2'(i)-H6/8(i+1), H2"(i)-H6/8(i+1), and H6/8(i)-H6/8(i+1). The deltaP and distance variations ascertain that the nature of the cation affects the DNA overall structure, i.e. both the conformational equilibria between the backbone BI (epsilon-zeta <0 degrees ) and BII (epsilon-zeta >0 degrees ) states and the helical parameters, via their strong mechanical coupling. These results reveal that Na(+) and K(+) interactions with DNA are different and sequence-dependent. These ions modulate the overall intrinsic properties of DNA, and possibly its packaging and readout.     

Metal binding to heparin monosaccharides: D-glucosamine-6-sulphate, D-glucuronic acid, and L-iduronic acid

In order to ascertain which residues in heparin may be responsible for its metal binding capacities we have investigated metal binding to some of its component monosaccharides by 1H and 13C NMR. The diamagnetic Zn ion and the paramagnetic Ni ion were used as probes. 4-Methylumbelliferyl-2-deoxy-2-acetamido-6-O-sulpho-D-glucosamine was used as a model for O-sulphates. Only weak interactions with the sulphate group were found. The 4C1 ring conformation of sodium methyl-beta-D-glucopyranosiduronate was not perturbed by binding to its carboxylate and little evidence exists for chelation. By contrast, the ring conformation of the sodium methyl-alpha-L-idopyranosiduronate is affected by the addition of Zn greater than Pb greater than Cd greater than Ca much greater than K ions. The sodium salt is suggested to be an equilibrium mixture of the 2SO and 1C4 ring conformations. Cation binding to the carboxylate group shifts this equilibrium towards the 1C4 conformation and suggests additional binding to O5 or, less likely, O4. This effect appears to be electrostatic in nature, as excess Na and protonation produce similar shifts. Lead complexation is different from the other ions and suggests some covalent character. The control of the ring conformation of iduronic acid by metal ions may have biological implications for the action of heparin and heparin-like compounds.     

Preparation and calculated conformations of the 2'-, 3'-, 4'-, and 6'-deoxy, 3'-O-methyl, 4'-epi, and 4'- and 6'-deoxy-fluoro derivatives of methyl 4-O-alpha-D-galactopyranosyl-beta-D-galactopyranoside (methyl beta-D-galabioside)

The glycosyl chlorides of the 3-O-methyl (6) and 4-deoxy-4-fluoro (8) O-benzylated derivatives of D-galactopyranose and 2,3,4,6-tetra-O-benzyl-D-glucopyranose were condensed with methyl 2,3,6-tri-O-benzoyl-beta-D-galactopyranoside to give, after deprotection, the 3'-O-methyl (23), 4'-deoxy-4'-fluoro (25), and 4'-epi (27) derivatives, respectively, of methyl beta-D-galabioside (1). The glycosyl fluorides of 2,3,4-tri-O-benzyl-D-fucopyranose and the 3-deoxy (12) and 4-deoxy (16) O-benzylated derivatives of D-galactopyranose were condensed with methyl 2,3,6-tri-O-benzyl-beta-D-galactopyranoside (21), to give, after deprotection, the 6'-deoxy (31), 3'-deoxy (34), and 4'-deoxy (37) derivatives of 1, respectively. The 2'-deoxy (41) derivative of 1 was prepared by N-iodosuccinimide-induced condensation of 3,4,6-tri-O-acetyl-D-galactal and 21 followed by deprotection. Treatment of methyl 2,3,6-tri-O-benzoyl-4-O-(2,3-di-O-benzoyl-alpha-D-galactopyranosyl)-beta -D- galactopyranoside with Et2NSF3 (DAST), followed by deprotection, provided the 6'-deoxy-6'-fluoro (46) derivative of 1. Molecular mechanics calculations yielded conformations for 23, 25, 27, 31, 34, 37, 41, and 46 with small deviations from the calculated conformation for 1 (phi H/psi H: -40 degrees/-6 degrees).     

Identification of mixed di-cation forms of G-quadruplex in solution

Multinuclear NMR study has demonstrated that G-quadruplex adopted by d(G3T4G4) exhibits two cation binding sites between three of its G-quartets. Titration of tighter binding K+ ions into the solution of d(G3T4G4)2 folded in the presence of 15NH4+ ions uncovered a mixed mono-K+-mono-15NH4+ form that represents intermediate in the conversion of di-15NH4+ into di-K+ form. Analogously, 15NH4+ ions were found to replace Na+ ions inside d(G3T4G4)2 quadruplex. The preference of 15NH4+ over Na+ ions for the two binding sites is considerably smaller than the preference of K+ over 15NH4+ ions. The two cation binding sites within the G-quadruplex core differ to such a degree that 15NH4+ ions bound to the site, which is closer to the edge-type loop, are always replaced first during titration by K+ ions. The second binding site is not taken up by K+ ion until K+ ion already resides at the first binding site. Quantitative analysis of concentrations of the three di-cation forms, which are in slow exchange on the NMR time scale, at 12 K+ ion concentrations afforded equilibrium binding constants. K+ ion binding to sites U and L within d(G3T4G4)2 is more favorable with respect to 15NH4+ ions by Gibbs free energies of approximately -24 and -18 kJ mol(-1) which includes differences in cation dehydration energies, respectively.     

The biomimetic [Cr3O(O2CCH2CH3)6(H2O)3]+ decreases plasma insulin, cholesterol, and triglycerides in healthy and type II diabetic rats but not type I diabetic rats

The in vivo effects of administration of the synthetic, functional biomimetic cation [Cr(3)O(O(2)CCH(2)CH(3))(6)(H(2)O)(3)](+) to healthy and type I and type II diabetic model rats are described. In contrast to current chromium-containing nutrition supplements, which only serve as sources of absorbable chromium, the trinuclear cation has been shown in in vitro assays to interact with the insulin receptor, activating its kinase activity, presumably by trapping the receptor in its active conformation. Thus, treatment of rats with the trinuclear cation would be expected to result in changes in lipid and carbohydrate metabolism related to insulin action. After 24 weeks of intravenous administration (0-20 micro g Cr/kg body mass), the cation results in a concentration-dependent lowering of levels of fasting blood plasma LDL cholesterol, total cholesterol, triglycerides, and insulin and of 2-h plasma insulin and glucose levels after a glucose challenge; these results confirm a previous 12-week study examining the effect of the synthetic cation on healthy rats and are in stark contrast to those of administration of other forms of Cr(III) to rats, which have no effect on these parameters. The cation has little, if any, effect on rats with STZ-induced diabetes (a type I diabetes model). However, Zucker obese rats (a model of the early stages of type II diabetes) after 24 weeks of supplementation (20 micro g/kg) have lower fasting plasma total, HDL, and LDL cholesterol, triglycerides, and insulin levels and lower 2-h plasma insulin levels. The lowering of plasma insulin concentrations with little effect on glucose concentrations suggests that the supplement increases insulin sensitivity.     

Synthesis of alpha-galactosylated fragments related to the core-structure of the GPI anchor of Trypanosoma brucei

A series of octyl glycosides di- to tetrasaccharides related to the GPI anchor of Trypanosoma brucei was prepared. Treatment of octyl 2-O-benzoyl-4,6-O-(1,1,3,3-tetraisopropyl-1,3-disiloxane-1,3 -diyl)-alpha-D-mannopyranoside with ethyl 2,3,4,6-tetra-O-benzyl-1-thio-beta-D-galactopyranoside under activation with bromine and silver trifluoromethanesulfonate afforded the alpha-linked disaccharide octyl 2-O-benzoyl-3-O-(2,3,4,6-tetra-O-benzyl-alpha-D-galactopyranosyl)-4,6-O- (1,1,3,3-tetraisopropyl-1,3-disiloxane-1,3-diyl)-alpha -D-mannospyranoside, the siloxane ring of which was regioselectively opened with a HF-pyridine complex to give the disaccharide acceptor octyl 3-O-(2,3,4,6-tetra-O-benzyl-alpha-D-galactopyranosyl)-2-O-benzoyl-4-O-(3 -fluoro-1,1,3,3-tetraisopropyl-1,3-disiloxane-3-yl)-alpha-D- mannopyranoside (4). Mannosylation of 4 with benzobromomannose (7), followed by fluoride catalyzed desilylation gave the trisaccharide octyl 2-O-benzoyl-6-O-(2,3,4,6-tetra-O-benzoyl-alpha-D-mannopyranosyl)-3-O-(2, 3,4,6-tetra-O-benzyl-alpha-D-galactopyranosyl)-alpha-D-mannospyranosi de, which was deblocked via the deacylated intermediate octyl 3-O-(2,3,4,6-tetra-O-benzyl-alpha-D-galactopyranosyl)-6-O-(alpha-D-manno pyranosyl)-alpha-D-mannospyranoside to afford the octyl glycoside trisaccharide octyl 3-O-(alpha-D-galactopyranosyl)-6-O-(alpha-D-mannopyranosyl)-alpha-D-m annospyranoside. Glycosylation of 4 with 3,4,6-tri-O-acetyl-2-O-(2,3,4,6-tetra-O-benzoyl-alpha-D-mannopyranosyl)- alpha-D-mannopyranosyl trichloroacetimidate resulted in the tetrasaccharide octyl 2-O-benzoyl-4-O-(1-fluoro-1,1,3,3-tetraisopropyl-1,3-disiloxane -3-yl)-3-O-(2,3,4,6-tetra-O-benzyl-alpha-D-galactopyranosyl)-6-O-[2-O -(2,3,4,6-tetra-O-benzoyl-alpha-D-mannopyranosyl)-3,4,6-tri-O-acetyl-alp ha-D-mannopyranosyl]-alpha-D-mannospyranoside, sequential desilylation, deacylation and debenzylation, respectively, of which via the intermediate octyl 2-O-benzoyl-3-O-(2,3,4,6-tetra-O-benzyl-alpha-D-galactopyranosyl)-6-O-[2 -O-(2,3,4,6-tetra-O-benzoyl-alpha-D-mannopyranosyl)-3,4,6-tri-O-acetyl-a lpha-D-mannopyranosyl]-alpha-D-mannospyranoside afforded the octyl glycoside tetrasaccharide octyl 3-O-(alpha-D-galactopyranosyl)-6-O-[2-O-(alpha-D-mannopyranosyl)-alpha-D -mannopyranosyl]-alpha-D-mannospyranoside.