Syntheses of beta-d-Galf-(1-->6)-beta-d-Galf-(1-->5)-d-Galf and beta-d-Galf-(1-->5)-beta-d-Galf-(1-->6)-d-Galf,trisaccharide units in the galactan of Mycobacterium tuberculosis

The galactofuran is a crucial constituent of the cell wall of mycobacteria. An efficient synthesis of the two trisaccharide units of the galactan is described. The strategy relies on the use of substituted d-galactono-1,4-lactones as precursors for the internal and the reducing galactofuranoses. Dec-9-enyl beta-d-Galf-(1-->6)-beta-d-Galf-(1-->5)-beta-d-Galf (2) and dec-9-enyl beta-d-Galf-(1-->5)-beta-d-Galf-(1-->6)-beta-d-Galf (9) so far reported as convenient substrates for the galactofuranosyl transferase, and possibly useful for immunological studies, were obtained by the trichloroacetimidate method of glycosylation.     

Direct chemical synthesis of the beta-D-mannans: the beta-(1-->2) and beta-(1-->4) series

The direct syntheses of a beta-(1-->2)-mannooctaose and of a beta-(1-->4)-mannohexaose are reported by means of 4,6-O-benzylidene-protected beta-mannosyl donors. The synthesis of the (1-->2)-mannan was achieved by means of the sulfoxide coupling protocol, whereas the (1-->4)-mannan was prepared using the analogous thioglycoside/sulfinamide methodology. In the synthesis of the (1-->4)-mannan, the glycosylation yields and stereoselectivities remain approximately constant with increasing chain length, whereas those for the (1-->2)-mannan consist of two groups with the formation of the tetra- and higher saccharides giving yields and selectivities consistently lower than those of the lower homologues. The decrease in yield after the trisaccharide in the (1-->2)-mannan synthesis is attributed to steric interference by the n-3 residue and is consistent with the collapsed, disordered structure predicted by early computational work. The consistently high yields and selectivities seen in the synthesis of the (1-->4)-mannan are congruent with the more open, ordered structure originally predicted for this polymer. The lack of order in the structure of the (1-->2)-mannan, as compared to the high degree of order in the (1-->4)-mannan, is also evident from a comparison of the NMR spectra of the two polymers and even from their physical nature: the (1-->2)-mannan is a gum and the (1-->4)-mannan is a high melting solid.     

Synthesis of alpha-D-Galp-(1-->3)-beta-D-Galf-(1-->3)-D-man,a terminal trisaccharide of Leishmania type-2 glycoinositolphospholipids

The synthesis of alpha-D-Galp-(1-->3)-beta-D-Galf-(1-->3)-D-Man, present in the type-2 glycoinositolphospholipids and in the core of the lipophosphoglycan of Leishmania, is described. The glycosyl aldonolactone approach, followed by reduction of the lactone with diisoamylborane, was utilized for the introduction of the internal galactofuranosyl unit and the trichloroacetimidate method for the O-glycosidation reaction. A high-yield synthesis of the beta-D-Galf-(1-3)-D-Man unit, also present in the lipopeptidophosphoglycan of Trypanosoma cruzi, is reported.     

Solution conformations of mycothiol bimane, 1-D-GlcNAc-alpha-(1 --> 1)-D-myo-Ins and 1-D-GlcNAc-alpha-(1 --> 1)-L-myo-Ins

Mycothiol is an abundant small molecular weight thiol found only in actinomycetes, which include mycobacteria. Mycothiol biosynthetic and detoxification enzymes are novel and unique to actinomycetes, thereby representing potential antimycobacterial targets. To better guide inhibitor design, we have determined by NMR the solution conformations of mycothiol bimane (MSmB) and the pseudodisaccharide 1-D-GlcNAc-alpha-(1 --> 1)-D-myo-Ins (D-GI), molecules that represent the natural substrates for the mycothiol-dependent detoxification enzyme mycothiol-S-conjugate amidase (MCA) and the mycothiol biosynthetic enzyme D-GlcNAc-alpha-(1 --> 1)-D-myo-Ins deacetylase (AcGI deacetylase), respectively. Comparison of the mean structure of MSmB and the energy-minimized structures of two competitive spiroisoxazoline-containing MCA inhibitors shows striking similarities between these molecules in the region of the scissile amide bond of MSmB and provides structural evidence that those inhibitors are substrate mimics. Owing to our earlier finding that AcGI deacetylase will not deacetylate the unnatural isomer 1-d-GlcNAc-alpha-(1 --> 1)-L-myo-Ins (L-GI), the solution conformation of L-GI was also determined. The interglycosidic bond angles for all three compounds are comparable. When considered together with the observation that a simplified cyclohexyl thioglycoside mycothiol analogue is a good substrate for MCA, it appears that the stereochemistry of the inositol ring is critical for deacetylase function, superceding the importance of the full complement of hydroxyl groups on the "nonreducing" ring.     

Synthesis of (1-->4)-linked 2-deoxy-2-fluoroglucose oligomers. 1

[reaction: see text] Thioglycosides of natural monosaccharides are readily converted into their corresponding chlorides by diphenylchlorosulfonium chloride. This reagent can likewise effect the conversion of the more stable 4-chlorophenylthio 2-deoxy-2-fluoroglucose derivatives into chloride glycosyl donors. On the basis of this activation strategy, it was possible to assemble unnatural oligosaccharides composed of 2-fluorodeoxy sugars.     

Synthesis of the core tetrasaccharide of Trypanosoma cruzi glycoinositolphospholipids: Manp(alpha1-->6)-Manp(alpha1-->4)-6-(2-aminoethylphosphonic acid)-GlcNp(alpha1-->6)-myo-Ins-1-PO4

[structure: see text] Synthesis of the core tetrasaccharide Manp(alpha1-->6)-Manp(alpha1-->4)-6-(2-aminoethylphosphonic acid)-GlcNp(alpha1-->6)-myo-Ins-1-PO4, found in glycoinositolphospholipids of Trypanosoma cruzi parasites, is described. The key building block, 6-O-(2-azido-3-O-benzyl-6-O-((2-benzyloxycarbonylaminoethyl)phosphonic acid benzyl ester)-2-deoxy-alpha-D-glucopyranosyl)-1-di-O-benzylphosphoryl-4,5-O-isopropylidene-2,3-O-(D-1,7,7-trimethyl[2,2,1]bicyclohept-6-ylidene)-D-myo-inositol, was synthesized using a partially protected glucosyl D-camphorinositolphosphate and a (2-benzyloxycarbonylaminoethyl)phosphonic acid derivative in a regioselective phosphonate esterfication. Elongation with ethyl 2-O-benzoyl-3,4,6-tri-O-benzyl-alpha-D-mannopyranosyl-(1-->6)-2,3,4-tri-O-benzyl-1-alpha-D-thiomannopyranoside using dimethyl(methylthio)sulfonium trifluoromethanesulfonate gave a fully protected tetrasaccharide which was successfully deprotected subsequently with sodium methoxide, sodium in liquid ammonia, and aq hydrochloric acid to give title compound.     

Application of the anomeric samarium route for the convergent synthesis of the C-linked trisaccharide alpha-D-Man-(1-->3)-[alpha-D-Man-(1-->6)]-D-Man and the disaccharides alpha-D-Man-(1-->3)-D-Man and alpha-D-Man-(1-->6)-D-Man

Studies are reported on the assembly of the branched C-trisaccharide, alpha-D-Man-(1-->3)-[alpha-D-Man-(1-->6)]-D-Man, representing the core region of the asparagine-linked oligosaccharides. The key step in this synthesis uses a SmI(2)-mediated coupling of two mannosylpyridyl sulfones to a C3,C6-diformyl branched monosaccharide unit, thereby assembling all three sugar units in one reaction and with complete stereocontrol at the two anomeric carbon centers. Subsequent tin hydride-based deoxygenation followed by a deprotection step produces the target C-trimer. In contrast to many of the other C-glycosylation methods, this approach employes intact carbohydrate units as C-glycosyl donors and acceptors, which in many instances parallels the well-studied O-glycosylation reactions. The synthesis of the C-disaccharides alpha-D-Man-(1-->3)-D-Man and alpha-D-Man-(1-->6)-D-Man is also described, they being necessary for the following conformational studies of all three carbohydrate analogues both in solution and bound to several mannose-binding proteins.     

Synthesis of various sulfoforms of the trisaccharide beta-d-GlcpA-(1-->3)- beta-d-Galp-(1-->3)-beta-d-Galp-(1-->OMP) as probes for the study of the biosynthesis and sorting of proteoglycans

A straightforward preparation of various sulfoforms of the trisaccharide 4-methoxyphenyl O-(sodium beta-d-glucopyranosyluronate)-(1-->3)-( beta-d-galactopyranosyl)-(1-->3)-beta-d-galactopyranoside (1), namely its 6a- and 4a-monosulfate, 6b- and 4b-monosulfate and 6a,6b-disulfate derivatives, is reported for the first time. These compounds, which are partial structures of the linkage region of proteoglycans, will serve as probes for the study of the biosynthesis and sorting of these macromolecules. A key trisaccharide derivative, in which the two similar d-Gal units were differentiated at C-4,6 with 4,6-benzylidene and 4,6-di-tert-butylsilylene acetals, respectively, was used as a common intermediate. Both acetal groups showed excellent orthogonality, and allowed the preparation of all target compounds in high yield. Noteworthy is the possibility to prepare the 6a- and 6b-monosulfated and the 6a,6b-disulfated species through a one-pot regioselective procedure starting from a tetrol precursor.     

Inner-valence photoionization of O((1)D): experimental evidence for the 2s(2)2p(4)((1)D)-->2s(1)2p(5)((1)P) transition

Photoionization and autoionization of electronically excited atomic oxygen O((1)D) are investigated in the energy range between 12 and 26 eV using tunable laser-produced plasma radiation in combination with time-of-flight mass spectrometry. A broad, asymmetric, and intense feature is observed that is peaking at 20.53+/-0.05 eV. It is assigned to the 2s(2)2p(4)((1)D)-->2s(1)2p(5)((1)P) transition, which subsequently autoionizes by a Coster-Kronig transition, as predicted by the previous theoretical work [K. L. Bell et al., J. Phys. B 22, 3197 (1989)]. Specifically, the energy of the unperturbed transition occurs at 20.35+/-0.07 eV. Its shape is described by a Fano profile revealing a q parameter of 4.25+/-0.8 and a width of gamma=2.2+/-0.15 eV. Absolute photoionization cross section sigma is derived, yielding sigma=22.5+/-2.3 Mb at the maximum of the resonance. In addition, weak contributions to the O((1)D) yield from dissociative ionization originating from molecular singlet oxygen [O(2)((1)Delta(g))] are identified as well. Possible applications of the 2s(2)2p(4)((1)D)-->2s(1)2p(5)((1)P) transition as a state-selective and sensitive probe of excited oxygen in combination with photoionization mass spectrometry are briefly discussed.     

Convergent synthesis of a beta-(1-->3)-mannohexaose

An as yet unknown beta-(1-->3)-mannohexaose has been synthesized by a block route involving the coupling of two trisaccharides. Comparison of three closely related attempted mannohexaose syntheses reinforces the influence of subtle matching and/or mismatching interactions on the outcome of convergent oligosaccharide synthesis.     

A hydration study of (1-->4) and (1-->6) linked alpha-glucans by comparative 10 ns molecular dynamics simulations and 500-MHz NMR

The hydration behavior of two model disaccharides, methyl-alpha-D-maltoside (1) and methyl-alpha-D-isomaltoside (2), has been investigated by a comparative 10 ns molecular dynamics study. The detailed hydration of the two disaccharides was described using three force fields especially developed for modeling of carbohydrates in explicit solvent. To validate the theoretical results the two compounds were synthesized and subjected to 500 MHz NMR spectroscopy, including pulsed field gradient diffusion measurements (1: 4.0. 10(-6) cm(2). s(-1); 2: 4.2. 10(-6) cm(2). s(-1)). In short, the older CHARMM-based force field exhibited a more structured carbohydrate-water interaction leading to better agreement with the diffusional properties of the two compounds, whereas especially the alpha-(1-->6) linkage and the primary hydroxyl groups were inaccurately modeled. In contrast, the new generation of the CHARMM-based force field (CSFF) and the most recent version of the AMBER-based force field (GLYCAM-2000a) exhibited less structured carbohydrate-water interactions with the result that the diffusional properties of the two disaccharides were underestimated, whereas the simulations of the alpha-(1-->6) linkage and the primary hydroxyl groups were significantly improved and in excellent agreement with homo- and heteronuclear coupling constants. The difference between the two classes of force field (more structured and less structured carbohydrate-water interaction) was underlined by calculation of the isotropic hydration as calculated by radial pair distributions. At one extreme, the radial O em leader O pair distribution function yielded a peak density of 2.3 times the bulk density in the first hydration shell when using the older CHARMM force field, whereas the maximum density observed in the GLYCAM force field was calculated to be 1.0, at the other extreme.     

Block synthesis of tetra- and hexasaccharides (beta-D-glycero-D-manno-Hep p-(1-->4)-[alpha-l-Rha p-(1-->3)-beta-D-glycero-D-manno-Hep p-(1-->4)]n-alpha-L-Rha p-OMe (n = 1 and 2)) corresponding to multiple repeat units of the glycan from the surface-layer glycoprotein from Bacillus thermoaerophilus

A fully stereocontrolled block synthesis of the title tetra- and hexasaccharides has been achieved taking advantage of the ability of the 4,6-O-benzylidene acetal to control the stereochemistry of the beta-D-glycero-D-mannoheptopyranoside unit and of a 2,3-O-diphenylmethylene acetal to install the alpha-L-rhamnopyranosidic linkages. Comparison of the spectral data for the hexasaccharide with that of the natural isolate confirms the structure of this very unusual and structurally challenging glycan.     

Chemoenzymatic synthesis of n-hexyl and O-beta-D-xylopyranosyl-(1-->6)-beta-D-glucopyranosides

Direct beta-glucosidation between 1,6-octanediol (5) and D-glucose (3) using the immobilized beta-glucosidase (EC 3.2.1.21) from almonds with the synthetic prepolymer ENTP-4000 gave a mono-beta-glucoside (6) in 61.4% yield, which was converted into the n-hexyl beta-D-glucopyranoside (1) by means of a chemoenzymatic method. The coupling of the n-hexyl beta-D-glucopyranoside congener (13) and 2,3,4-tri-O-acetyl-beta-D-xylosyl congener (14), followed by deprotection, afforded the synthetic n-hexyl O-beta-D-xylopyranosyl-(1-->6)-beta-D-glucopyranoside (2), which was identical to the natural 2 with respect to the spectral data and specific rotation.     

A conformational dynamics study of alpha-l-Rhap-(1-->2)[alpha-l-Rhap-(1-->3)]-alpha-l-Rhap-OMe in solution by NMR experiments and molecular simulations

The conformational preference of alpha-l-Rhap-(1-->2)[alpha-l-Rhap-(1-->3)]-alpha-l-Rhap-OMe in solution has been studied by NMR spectroscopy using one-dimensional (1)H,(1)H T-ROESY experiments and measurement of trans-glycosidic (3)J(C,H) coupling constants. Molecular dynamics (MD) simulations with a CHARMM22 type of force field modified for carbohydrates were performed with water as the explicit solvent. The homonuclear cross-relaxation rates, interpreted as effective proton-proton distances, were compared to those obtained from simulation. Via a Karplus torsional relationship, (3)J(C,H) values were calculated from simulation and compared to experimental data. Good agreement was observed between experimental data and the MD simulation, except for one inter-residue T-ROE between protons in the terminal sugar residues. The results show that the trisaccharide exhibits substantial conformational flexibility, in particular along the psi glycosidic torsion angles. Notably, for these torsions, a high degree of correlation (77%) was observed in the MD simulation revealing either psi(2)(+) psi(3)(+) or psi(2)(-)psi(3)(-) states. The simulations also showed that non-exoanomeric conformations were present at the phi torsion angles, but to a limited extent, with the phi(3) state populated to a larger extent than the phi(2) state. Further NMR analysis of the trisaccharide by translational diffusion measurements and (13)C T(1) relaxation experiments quantified global reorientation using an anisotropic model together with interpretation of the internal dynamics via the "model-free" approach. Fitting of the dynamically averaged states to experimental data showed that the psi(2)(+)psi(3)(+) state is present to approximately 49%, psi(2)(-) psi(3)(-) to approximately 39%, and phi(3) (non-exo) to approximately 12%. Finally, using a dynamic and population-averaged model, (1)H,(1)H T-ROE buildup curves were calculated using a full relaxation matrix approach and were found to be in excellent agreement with experimental data, in particular for the above inter-residue proton-proton interaction between the terminal residues.     

High resolution vacuum ultraviolet emission spectrum of D(2) from 78 to 103 nm: The D (1)Pi(u)-->X (1)Sigma(g) (+) and D(') (1)Pi(u) (-)-->X (1)Sigma(g) (+) band systems

The emission spectrum of the D(2) molecule has been studied at high resolution in the vacuum ultraviolet region 78.5-102.7 nm. A detailed analysis of the two D (1)Pi(u)-->X (1)Sigma(g) (+) and D(') (1)Pi(u) (-)-->X (1)Sigma(g) (+) electronic band systems is reported. New and improved values of the level energies of the two upper states have been derived with the help of the program IDEN [V. I. Azarov, Phys. Scr. 44, 528 (1991); 48, 656 (1993)], originally developed for atomic spectral analysis. A detailed comparison is made between the observed energy levels and solutions of coupled equations using the newest ab initio potentials by Wolniewicz and co-workers [J. Chem. Phys. 103, 1792 (1995); 99, 1851 (1993); J. Mol. Spectros. 212, 208 (2002); 220, 45 (2003)] taking into account the nonadiabatic coupling terms for the D (1)Pi(u) state with the lowest electronic states B (1)Sigma(u) (+), C (1)Pi(u), and B(') (1)Sigma(u) (+). A satisfactory agreement has been found for most of the level energies belonging to the D and D(') states. The remaining differences between observation and theory are probably due to nonadiabatic couplings with other higher electronic states which were neglected in the calculations.     

Quasiclassical trajectory simulations of OH(v) + NO2 --> HONO2* --> OH(v') + NO2: capture and vibrational deactivation rate constants

Quasiclassical trajectory calculations are used to investigate the dynamics of the OH(v) + NO(2) --> HONO(2) --> OH(v') + NO(2) recombination/dissociation reaction on an analytic potential energy surface (PES) that gives good agreement with the known structure and vibrational frequencies of nitric acid. The calculated recombination rate constants depend only weakly on temperature and on the initial vibrational energy level of OH(v). The magnitude of the recombination rate constant is sensitive to the potential function describing the newly formed bond and to the switching functions in the PES that attenuate inter-mode interactions at long range. The lifetime of the nascent excited HONO(2) depends strongly not only on its internal energy but also on the identity of the initial state, in disagreement with statistical theory. This disagreement is probably due to the effects of slow intramolecular vibrational energy redistribution (IVR) from the initially excited OH stretching mode. The vibrational energy distribution of product OH(v') radicals is different from statistical distributions, a result consistent with the effects of slow IVR. Nonetheless, the trajectory results predict that vibrational deactivation of OH(v) via the HONO(2) transient complex is approximately 90% efficient, almost independent of initial OH(v) vibrational level, in qualitative agreement with recent experiments. Tests are also carried out using the HONO(2) PES, but assuming the weaker O-O bond strength found in HOONO (peroxynitrous acid). In this case, the predicted vibrational deactivation efficiencies are significantly lower and depend strongly on the initial vibrational state of OH(v), in disagreement with experiments. This disagreement suggests that the actual HOONO PES may contain more inter-mode coupling than found in the present model PES, which is based on HONO(2). For nitric acid, the measured vibrational deactivation rate constant is a useful proxy for the recombination rate, but IVR randomization of energy is not complete, suggesting that the efficacy of the proxy method must be evaluated on a case-by-case basis.     

Synthesis of alpha-(2-->5)Neu5Gc oligomers

A facile synthesis of the sialic acid oligomers alpha-(2-->5)Neu5Gc (1) is presented. Monosaccharides 2-4 with suitable functionality were used as the building blocks. After selective removal of the paired carboxyl and amine protecting groups, the fully protected oligomers were assembled through consecutive coupling of the building blocks by well established peptide coupling techniques. By this approach, fully protected oligomers as large as an octasaccharide were synthesized. Deprotection of these fully protected oligomers was conducted in two steps (LiCl in refluxing pyridine and 0.1 n NaOH) to afford the desired products in high yield. Enzymatic degradation of the octamer with neuraminidase, monitored by capillary electrophoresis (CE), was also accomplished. The stepwise exo-cleavage adducts were all well separated and identified in the CE spectrum. The strategy described here for solution-phase synthesis also provides the basis for future solid-phase synthesis of poly-alpha-(2-->5)Neu5Gc.     

Spin-orbit effect in the energy pooling reaction O2(a 1Delta)+O2(a 1Delta)-->O2(b 1Sigma)+O2(X 3Sigma)

Five-dimensional nonadiabatic quantum dynamics studies have been carried out on two new potential energy surfaces of S(2)((1)A(')) and T(7)((3)A(")) states for the title oxygen molecules collision with coplanar configurations, along with the spin-orbit coupling between them. The ab initio calculations are based on complete active state second-order perturbation theory with the 6-31+G(d) basis set. The calculated spin-orbit induced transition probability as a function of collision energy is found to be very small for this energy pooling reaction. The rate constant obtained from a uniform J-shifting approach is compared with the existing theoretical and experimental data, and the spin-orbit effect is also discussed in this electronic energy-transfer process.     

Inelastic scattering matrix elements for the nonadiabatic collision B(2P1/2)+H2(1Sigmag+,j)<-->B(2P3/2)+H2(1Sigmag+,j')

Inelastic scattering matrix elements for the nonadiabatic collision B(2P1/2)+H2(1Sigmag+,j)<-->B(2P3/2)+H2(1Sigmag+,j') are calculated using the time dependent channel packet method (CPM). The calculation employs 1 2A', 2 2A', and 1 2A" adiabatic electronic potential energy surfaces determined by numerical computation at the multireference configuration-interaction level [M. H. Alexander, J. Chem. Phys. 99, 6041 (1993)]. The 1 2A' and 2 2A', adiabatic electronic potential energy surfaces are transformed to yield diabatic electronic potential energy surfaces that, when combined with the total B+H2 rotational kinetic energy, yield a set of effective potential energy surfaces [M. H. Alexander et al., J. Chem. Phys. 103, 7956 (1995)]. Within the framework of the CPM, the number of effective potential energy surfaces used for the scattering matrix calculation is then determined by the size of the angular momentum basis used as a representation. Twenty basis vectors are employed for these calculations, and the corresponding effective potential energy surfaces are identified in the asymptotic limit by the H2 rotor quantum numbers j=0, 2, 4, 6 and B electronic states 2Pja, ja=1/2, 3/2. Scattering matrix elements are obtained from the Fourier transform of the correlation function between channel packets evolving in time on these effective potential energy surfaces. For these calculations the H2 bond length is constrained to a constant value of req=1.402 a.u. and state to state scattering matrix elements corresponding to a total angular momentum of J=1/2 are discussed for j=0<-->j'=0,2,4 and 2P1/2<-->2P1/2, 2P3/2 over a range of total energy between 0.0 and 0.01 a.u.