A Design of Experiment (DoE) approach to optimise spray drying process conditions for the production of trehalose/leucine formulations with application in pulmonary delivery.

The present study evaluates the effect of L-leucine concentration and operating parameters of a laboratory spray dryer on characteristics of trehalose dry powders, with the goal of optimizing production of these powders for inhaled drug delivery. Trehalose/L-leucine mixtures were spray dried from aqueous solution using a laboratory spray dryer. A factorial design of experiment (DoE) was undertaken and process parameters adjusted were: inlet temperature, gas flow rate, feed solution flow rate (pump setting), aspiration setting and L-leucine concentration. Resulting powders were characterised in terms of particle size, yield, residual moisture content, and glass transition temperature. Particle size was mainly influenced by gas flow rate, whereas product yield and residual moisture content were found to be primarily affected by inlet temperature and spray solution feed rate respectively. Interactions between a number of different process parameters were elucidated, as were relationships between different responses. The leucine mass ratio influenced the physical stability of powders against environmental humidity, and a high leucine concentration (30% w/w) protected amorphous trehalose from moisture induced crystallization. High weight ratio of leucine in the formulation, however, negatively impacted the aerosol performance. Thus, in terms of L-leucine inclusion in a formulation designed for pulmonary delivery, a balance needs to be found between physical stability and deposition characteristics.

One-pot oligosaccharide synthesis exploiting solvent reactivity effects.

[reaction: see text] One-pot syntheses of trisaccharides have been accomplished simply by changing the solvent system between the two subsequent glycosylation reactions and utilizing the difference in glycosylation rate between different solvents. By tuning the reactivity of acceptors and donors and performing the first glycosylation in Et(2)O (low glycosylation rate) and the second in CH(2)Cl(2)/Et(2)O (higher glycosylation rate), trisaccharides were synthesized in high yields (76-84%).

Syntheses of deoxy analogues of methyl 3,6-di-O-alpha-D-mannopyranosyl-alpha-D-mannopyranoside for studies of the binding site of concanavalin A.

All of the monodeoxy analogues of methyl 3,6-di-O-alpha-D-mannopyranosyl-alpha-D-mannopyranoside have been synthesized together with two dideoxy (2,3'- and 4,3'-) analogues. The 2'- and 2"-deoxy functions were introduced by NIS-promoted couplings with 2,3,4-tri-O-acetyl-D-glucal as donor, followed by reduction of the resulting 2'- and 2"-iodo derivatives, whereas the 3'-, 3"-, 4'-, 4"-, 6'-, and 6"-deoxy functions were introduced by using known deoxyglycosyl chlorides as donors in coupling reactions promoted by silver trifluoromethanesulfonate. The 2- and 4-deoxy functions on the central mannose residue were introduced by displacement, using triiodoimidazole and triphenylphosphine, of a hydroxyl group by iodine on suitably protected derivatives followed by reduction of the resulting iodo analogues.

Synthesis of 2-(4-aminophenyl)ethyl 3-deoxy-5-O-(3,4,6-tri-O-beta-D- glucopyranosyl-alpha-D-glucopyranosyl)-alpha-D-manno-oct-2-ulopyrano sid onic acid, a highly branched pentasaccharide corresponding to structures found in lipopolysaccharides from Moraxella catarrhalis.

Syntheses of the pentasaccharide 2-(4-aminophenyl)ethyl 3-deoxy-5-O-(3,4,6- tri-O-beta-D-glucopyranosyl-alpha-D-glucopyranosyl)-alpha-D-manno-oct-2- ulopyranosidonic acid and of the tetrasaccharide 3,4,6-tri-O-beta-D-glucopyranosyl-alpha-D-glucopyranoside, both as its methyl and 2-(4-trifluoro-acetamidophenyl)ethyl glycoside, are described. These oligosaccharides correspond to structures found in the lipopolysaccharide of Moraxella catarrhalis and were needed for biological experiments aimed at producing antibodies against the bacteria. The best way to introduce the glucopyranosyl groups into the 3-, 4-, and 6-positions of the branched target compounds was found to be a one-step reaction using a 3,4,6-triol as acceptor and 2,3,4,6-tetra-O-benzoyl-D-glucopyranosyl bromide as donor in a silver trifluoromethanesulfonate-promoted coupling. The spacer arm, necessary for the formation of immunoactive glycoconjugates, was introduced into the glucose moiety via a dimethyl(methylthio)sulfonium trifluoromethanesulfonate-promoted reaction using the ethyl thioglucoside as donor, whereas for Kdo, the acetylated glycal derivative, methyl 4,5,7,8-tetra-O-acetyl-2,6-anhydro-3-deoxy-D-manno-oct-2-enonate, was used as donor and phenylselenyl trifluoromethanesulfonate as a stereocontrolling promoter.

Synthesis of a tri- and tetradeoxy analogue of methyl 3,6-di-O-alpha-D-mannopyranosyl-alpha-D-mannopyranoside for investigation of the binding site of various plant lectins.

The synthesis of a the 2,4,3'-trideoxy and 2,4,3',4'-tetradeoxy analogues of the trimannoside part of the core structure of N-linked glycoproteins, methyl 3,6-di-O-alpha-D-mannopyranosyl-alpha-D-mannopyranoside, is described. A 2,4-dideoxy (1-->6) linked disaccharide was used as a common intermediate acceptor, which was coupled with a 3-deoxy and a 3,4-dideoxy benzochlorosugar donor, the latter prepared from methyl alpha-D-mannopyranoside in five steps. Despite the acid-sensitive donors and acceptor, acceptable glycosylation yields were obtained of both the trideoxy- and the tetradeoxy trisaccharide using silver triflate as a promoter (65 and 51%, respectively). Deprotection in one step then gave the target products.

Synthesis of a hexasaccharide corresponding to part of the heptose-hexose region of the Salmonella Ra core, and a penta- and a tetra-saccharide that compose parts of this structure.

The synthesis of the hexasaccharide 2-(4-trifluoroacetamidophenyl)ethyl O-alpha-D-galactopyranosyl-(1-->3)-[O-alpha-D-galactopyranosyl-(1-->6)]- O-alpha-D-glucopyranosyl-(1-->3)-[O-L-glycero-alpha-D-manno-heptopyranos yl- (1-->7)]-O-L-glycero-alpha-D-manno-heptopyranosyl-(1-->3)-L-glycero-alph a-D- manno-heptopyranoside, corresponding to the heptose and part of the hexose region in the Salmonella Ra core, is described. Syntheses of the pentasaccharide 2-(4-trifluoroacetamidophenyl)ethyl O-alpha-D-galactopyranosyl-(1-->3)-O-alpha-D-glucopyranosyl-(1-->3)-[O-L - glycero-alpha-D-manno-heptopyranosyl-(1-->7)]-O-L-glycero-alpha-D-manno- heptopyranosyl-(1-->3)-L-glycero-alpha-D-manno-heptopyranoside and the tetrasaccharide 2-(4-trifluoroacetamidophenyl)ethyl O-alpha-D-glucopyranosyl-(1-->3)-[O-L-glycero-alpha-D-manno-heptopyranos yl- (1-->7)]-O-L-glycero-alpha-D-manno-heptopyranosyl-(1-->3)-L-glycero-alph a-D- manno-heptopyranoside are also described. Coupling of methyl 2,3,4,6-tetra-O-benzyl-1-thio-beta-D- glucopyranoside and methyl 2-O-benzyl-4,6-O-benzylidene-3-O-(2,3,4,6-tetra-O-benzyl-alpha-D- galactopyranosyl)-1-thio-beta-D-glucopyranoside to a triheptoside derivative with a free HO-3', using dimethyl(methylthio)sulfonium triflate and N-iodosuccinimide-silver triflate as promoters, gave the protected tetra- and penta-saccharide, respectively. Removal of the benzylidene group from the pentasaccharide followed by a regio- and stereo-selective coupling using halide-assisted conditions and 2,3,4,6-tetra-O-benzyl-alpha-D- galactopyranosyl bromide as donor gave the protected hexasaccharide. Deprotection then gave the target structures.

Synthesis of L-glycero-D-manno-heptopyranose-containing oligosaccharide structures found in lipopolysaccharides from Haemophilus influenzae.

Syntheses are described of the tetrasaccharide 2-(4-trifluoroacetamidophenyl)ethyl O-(beta-D-galactopyranosyl)-(1-->2)-O- (L-glycero-alpha-D-manno-heptopyranosyl)-(1-->2)-O-(L-glycero-alpha-D- manno-heptopyranosyl)-(1-->3)-L-glycero-alpha-D-manno-heptopyranoside (20) and the three trisaccharides 2-(4-trifluoroacetamidophenyl)ethyl O-(L-glycero-alpha-D-manno-heptopyranosyl)-(1-->2)-O-(L-glycero-alpha-D- manno-heptopyranosyl)-(1-->3)-L-glycero-alpha-D-manno-heptopyranoside (17), 2-(4-trifluoroacetamidophenyl)ethyl O-(beta-D-glucopyranosyl)-(1-->4)- O-(beta-D-glucopyranosyl)-(1-->4)-L-glycero-alpha-D-manno-heptopyrano side (5), and 2-(4-trifluoro-acetamidophenyl)ethyl O-(beta-D-galactopyranosyl)-(1-->4)-O-(beta-D-glucopyranosyl)-(1-->4)- L-glycero-alpha-D-manno-heptopyranoside (8), corresponding to structures found in the lipooligosaccharides of Haemophilus influenzae.

Synthesis of the Lewis b hexasaccharide and squarate acid-HSA conjugates thereof with various saccharide loadings.

The Lewis b hexasaccharide, alpha-L-Fucp-(1 --> 2)-beta-D-Galp-(1 --> 3)-[alpha-L-Fucp-(1 --> 4)]-beta-D-GlcpNAc-(1 --> 3)-beta-D-Galp-(l --> 4)-beta-D-Glcp, has been synthesised using a convergent synthesis. Starting from ethyl 4,6-O-benzylidene-2-deoxy-2-phthalimido-1-thio-beta-D-glucopyranoside, a thioglycoside tetrasaccharide donor block, was constructed through two orthogonal glycosylations with glycosyl bromide donors. First, the galactose moiety was introduced using silver triflate as a promoter and then the two fucose residues under halide-assisted conditions. Finally, this tetrasaccharide was linked to a spacer-equipped 3I,4I-diol lactose acceptor in a DMTST-promoted coupling to give, after deprotection, the Lewis b hexasaccharide as its 2-aminoethyl spacer-equipped derivative. This was coupled to human serum albumin (HSA), using the squarate ester methodology, in various saccharide-protein ratios, to give neoglycoconjugates with different saccharide loadings in about 50%) efficiency.

Synthesis of colitose-containing oligosaccharide structures found in polysaccharides from Vibrio cholerae O139 synonym Bengal using thioglycoside donors.

The syntheses of the two colitose-containing trisaccharides 8-methoxycarbonyloctyl (3,6-dideoxy-alpha-L-xylo-hexopyranosyl)-(1-->2)-beta-D-galactopyranosyl -(1-->3)-2-acetamido-2-deoxy-beta-D-glucopyranoside and 8-methoxycarbonyloctyl beta-D-galactopyranosyl-(1-->3)-[(3,6- dideoxy-alpha-L-xylo-hexopyranosyl)-(1-->4)]-2-acetamido-2-deoxy-beta-D- glucopyranoside, and tetrasaccharide 8-methoxycarbonyloctyl (3,6-dideoxy-alpha-L-xylo-hexopyranosyl)-(1-->2)-beta-D-galactopyranosyl - (1-->3)-[(3,6-dideoxy-alpha-L-xylo-hexopyranosyl)-(1-->4)]-2-acetamido-2 -deoxy- beta-D-glucopyranoside are described. The oligosaccharides correspond to structures found in the capsular polysaccharide and the lipopolysaccharide of Vibrio cholerae O139 and also to lipopolysaccharide structures of E. coli O55 and Salmonella greenside. The colitose residues were introduced via dimethyl(methylthio)sulfonium trifluoromethanesulfonate promoted glycosylations using colitose thioglycosides as glycosyl donors.

Interactions of five D-mannose-specific lectins with a series of synthetic branched trisaccharides.

The interaction of a series of synthetic, branched trisaccharides with five D-mannose-specific lectins was studied by precipitation-inhibition assay. The branched methyl alpha-D-mannotrioside, alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-alpha-D-Man pOMe, the best inhibitor of the Con A-Dextran interaction, was 42 times more potent than alpha-D-ManpOMe, and 3-6 times more potent than the two trisaccharides substituted with D-glucosyl groups, and 8-15 times those with D-galactosyl groups. Surprisingly, methyl O-alpha-D-mannopyranosyl-(1----3)-alpha-D-mannopyranoside was bound to Con A 8-fold more avidly than methyl alpha-D-mannopyranoside. However, the related pea lectin (PSA) was singularly different from Con A in its carbohydrate-binding activity, showing no significantly enhanced binding to any of the sugars examined. The trisacchrides containing terminal, nonreducing, (1----3)-linked alpha-D-mannopyranosyl groups, i.e., alpha-D-Manp-(1----3)-[alpha-D-Glep-(1----6)]alpha-D-Manp OMe, alpha-D-Manp-(1----3)]-alpha-D-Galp-(1----6)]-alpha-D-ManpOMe++ +, and alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-alpha-D-Man pOMe, were the best inhibitors of the snowdrop lectin (GNA)-D-mannan precipitation system. On the other hand, all branched trisaccharides exhibited very similar inhibitory potencies toward the daffodil lectin (NPA)-D-mannan interaction, whereas alpha-D-Manp-(1----3)-[alpha-D-Galp-(1----6)]-alpha-D-ManpOMe++ + and alpha-D-Manp-(1----3)-[alpha-D-Manp-(1----6)]-alpha-D-Man pOMe were somewhat better inhibitors than the other branched trisaccharides of the amaryllis lectin (HHA)-D-mannan precipitation reaction. (ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis of methyl 3-O-(alpha-D-glucopyranosyl)-7-O-(L-glycero-alpha-D- manno-heptopyranosyl)-L-glycero-alpha-D-manno-heptopyranoside.

The title trisaccharide glycoside, which is related to part of the core region of the lipopolysaccharide from Salmonella, and the disaccharide glycosides methyl 3-O-alpha-D-glucopyranosyl-L-glycero-alpha-D-manno-heptopyranoside and methyl 7-O-L-glycero-alpha-D-manno-heptopyranosyl-L-glycero-alpha-D-manno- heptopyranoside have been synthesised. Methyl 2,3,4-tri-O-benzyl-L-glycero-alpha-D-manno- heptopyranoside, obtained via a one-carbon elongation at C-6 of methyl 2,3,4-tri-O-benzyl-alpha-D-manno- hexodialdo-1,5-pyranoside, was used as precursor both for the heptosyl donor and acceptor.

Synthesis of an artificial antigen that corresponds to a disaccharide repeating unit of the capsular polysaccharide of Haemophilus influenzae type d. A facile synthesis of methyl 2-acetamido-2-deoxy-beta-D-mannopyranoside.

The synthesis is described of p-nitrophenyl 2-acetamido-3-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-2-deoxy-beta -D- mannopyranosiduronic acid, corresponding to the disaccharide repeating unit of the capsular polysaccharide of Haemophilus influenzae type d, which, after conversion of the p-nitro- into a p-amino-phenyl residue, may be attached to a protein to make an artificial antigen for immunological studies. The synthesis incorporates a facile route to the 2-acetamido-2-deoxy-beta-D-mannopyranosyl unit.

Synthesis of 2-(4-trifluoroacetamidophenyl)ethyl O-(L-glycero-alpha-D-manno-heptopyranosyl)-(1----7)-O-(L-glycero-alpha- D-manno- heptopyranosyl)-(1----3)-L-glycero-alpha-D-manno-heptopyranoside, corresponding to the heptose region of the Salmonella Ra core structure.

The title trisaccharide was synthesized from methyl 2,3,4-tri-O-benzyl-L-glycero-alpha-D-manno-heptopyranoside by acetolysis, followed by conversion into ethyl thioglycosides and also glycosyl bromides, which were both used in glycosylation reactions. In glycosylations using thioglycosides as glycosyl donors, N-iodosuccinimide-silver triflate and dimethyl(methylthio)sulfonium triflate were used as promoters, and in glycosylations with glycosyl bromides silver triflate was used. The protecting groups introduced into intermediates during the synthesis of the title trisaccharide were designed to allow later glycosylation at O-3' to give larger oligosaccharide fragments of the Salmonella LPS core region, and also to allow the introduction of phosphate groups at O-4 and O-4', a structural element that is suggested to be present in the Ra core.

Synthesis and conformational and NMR studies of alpha-D-mannopyranosyl and alpha-D-mannopyranosyl-(1----2)-alpha-D-mannopyranosyl linked to L-serine and L-threonine.

alpha-D-Mannopyranosyl and alpha-D-mannopyranosyl-(1----2)-alpha-D- mannopyranosyl linked to L-serine and L-threonine have been synthesised as model substances for the linkage region in certain O-linked glycoproteins. Metropolis Monte Carlo simulations were performed with a modified version of the GESA program, to yield theoretical NOEs and interatomic distances as ensemble-average values, and these were compared with results from steady-state NOE experiments. The NOEs were determined as ensemble-average and as global minimum values. NMR chemical shift differences, obtained for signals of the glycopeptides relative to those of the respective monomers, were interpreted in terms of short inter-residue atomic distances as found within the global minima, and on the basis of averaged distances derived from Monte Carlo simulations.