OxyB is a cytochrome P450 enzyme that catalyzes the first phenol coupling reaction during the biosynthesis of vancomycin-like glycopeptide antibiotics. The phenol coupling reaction occurs on a linear peptide intermediate linked as a C-terminal thioester to a peptide carrier protein (PCP) domain within the multidomain glycopeptide nonribosomal peptide synthetase (NRPS). Using model peptides with the sequence (R)(NMe)Leu-(R)Tyr-(S)Asn-(R)Hpg-(R)Hpg-(S)Tyr-S-PCP and (R)(NMe)Leu-(R)Tyr-(S)Asn-(R)Hpg-(R)Hpg-(S)Tyr-(S)Dpg-S-PCP (where Hpg = 4-hydroxyphenylglycine, and Dpg = 3,5-dihydroxyphenylglycine), or containing (R)Leu instead of (R)(NMe)Leu, attached to recombinant PCPs derived from modules-6 and -7 in the vancomycin NRPS, we show that cross-linking of Hpg4 and Tyr6 by OxyB can occur in both hexapeptide- and heptapeptide-PCP conjugates. Thus, whereas OxyB may act preferentially on a hexapeptide still linked to the PCP-6 in NRPS subunit-2, it is possible that a linear heptapeptide intermediate linked to PCP-7 in NRPS subunit-3 may also be transformed into monocyclic product. For turnover, OxyB requires electrons, which in vitro can be supplied by spinach ferredoxin and E. coli flavodoxin reductase. Turnover is also dependent upon the presence of molecular oxygen. The model substrate (R)(NMe)Leu-(R)Tyr-(S)Asn-(R)Hpg-(R)Hpg-(S)Tyr-S-PCP is transformed into cross-linked product by OxyB with a kcat of 0.1 s-1 and Km in the range 4-13 muM. Equilibrium binding of this substrate to OxyB, monitored by UV-vis, is accompanied by a typical low-to-high spin state change in the heme, characterized with a Kd of 17 +/- 5 muM.
Oligosaccharides play important roles in cell-surface events through carbohydrate-protein and carbohydrate-carbohydrate interactions.[1] The chemical synthesis of structurally defined oligosaccharides would be highly desirable in structure-activity studies because oligosaccharides from natural sources can be produced in only limited qualities. Recent progress in oligosaccharide synthesis has resulted in a number of new and efficient glycosidation methodologies, which are amenable to the synthesis of protected oligosaccharides 1 by standardized and routine protocols.[2] However, deprotection of the protected oligosaccharides 1!2, including the cleavage of various O-protecting groups and the replacement of Nprotecting groups with N-acetyl groups is difficult to achieve by standardized protocols (Scheme 1). The complete deprotection of protected oligosaccharides frequently requires careful selection of the reaction solvents to prevent the partially deprotected intermediates from precipitating. Herein, we describe an efficient method for the deprotection of protected oligosaccharides based on a polymer-assisted strategy and its application to the synthesis of dimeric and trimeric Lewis X derivatives.Our polymer-assisted strategy for the deprotection of protected oligosaccharides 1 is illustrated in Scheme 1. The solid-supported protected oligosaccharide 3 linked through a tetrahydropyranyl (THP) linker was designed as a key intermediate. The solid-supported complex oligosaccharides would smoothly undergo deprotection because they are aggregated to only a very limited extent. A Birch reduction was adapted for removing the solid-supported benzyl ethers and esters on 3.[3] The THP linker would survive deprotection reactions and can be cleaved under mildly acidic conditions to release the fully deprotected oligosaccharide 2 without anomerization or cleavage of the glycosidic bonds. [4,5] The ease of handling of solid-supported compounds would be effective not only for the high-speed synthesis of a single target oligosaccharide but also for the deprotection of a protected oligosaccharide library.[6] The polymer-supported protected oligosaccharide 3 can be prepared using the following methodology: 1) acetal formation of the protected saccharides 1 with prelinker 4 containing a dihydropyranyl (DHP) moiety and an activated ester and 2) subsequent Scheme 1. Polymer-assisted strategy for deprotection of the protected oligosaccharides 1. Bn= benzyl, PNP = para-nitrophenyl, Phth = phthalyl, Troc = 2,2,2-trichloroethoxycarbonyl.Scheme 2. Reagents and conditions: a) LiAlH 4 , THF; b) tert-butyl bromoacetate, NaH, DMF; c) 1 n aq. NaOH, dioxane; d) 4-nitrophenol, EDCI, DIEA, CH 2 Cl 2 , 42 % from 6. DMF = N,N-dimethylformamide, EDCI = 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, DIEA = diisopropylethylamine.
We describe an efficient synthesis of H type 1 and 2 trisaccharides by one-pot glycosylation involving glycosidation of glycal epoxide.
Oligosaccharides play important roles in cell-surface events through carbohydrate-protein and carbohydrate-carbohydrate interactions.[1] The chemical synthesis of structurally defined oligosaccharides would be highly desirable in structure-activity studies because oligosaccharides from natural sources can be produced in only limited qualities. Recent progress in oligosaccharide synthesis has resulted in a number of new and efficient glycosidation methodologies, which are amenable to the synthesis of protected oligosaccharides 1 by standardized and routine protocols.[2] However, deprotection of the protected oligosaccharides 1!2, including the cleavage of various O-protecting groups and the replacement of Nprotecting groups with N-acetyl groups is difficult to achieve by standardized protocols (Scheme 1). The complete deprotection of protected oligosaccharides frequently requires careful selection of the reaction solvents to prevent the partially deprotected intermediates from precipitating. Herein, we describe an efficient method for the deprotection of protected oligosaccharides based on a polymer-assisted strategy and its application to the synthesis of dimeric and trimeric Lewis X derivatives.Our polymer-assisted strategy for the deprotection of protected oligosaccharides 1 is illustrated in Scheme 1. The solid-supported protected oligosaccharide 3 linked through a tetrahydropyranyl (THP) linker was designed as a key intermediate. The solid-supported complex oligosaccharides would smoothly undergo deprotection because they are aggregated to only a very limited extent. A Birch reduction was adapted for removing the solid-supported benzyl ethers and esters on 3.[3] The THP linker would survive deprotection reactions and can be cleaved under mildly acidic conditions to release the fully deprotected oligosaccharide 2 without anomerization or cleavage of the glycosidic bonds. [4,5] The ease of handling of solid-supported compounds would be effective not only for the high-speed synthesis of a single target oligosaccharide but also for the deprotection of a protected oligosaccharide library.[6] The polymer-supported protected oligosaccharide 3 can be prepared using the following methodology: 1) acetal formation of the protected saccharides 1 with prelinker 4 containing a dihydropyranyl (DHP) moiety and an activated ester and 2) subsequent Scheme 1. Polymer-assisted strategy for deprotection of the protected oligosaccharides 1. Bn= benzyl, PNP = para-nitrophenyl, Phth = phthalyl, Troc = 2,2,2-trichloroethoxycarbonyl.Scheme 2. Reagents and conditions: a) LiAlH 4 , THF; b) tert-butyl bromoacetate, NaH, DMF; c) 1 n aq. NaOH, dioxane; d) 4-nitrophenol, EDCI, DIEA, CH 2 Cl 2 , 42 % from 6. DMF = N,N-dimethylformamide, EDCI = 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, DIEA = diisopropylethylamine.
A u t o m a t e d P a r a l l e l S y n t h e s i s o f a P r o t e c t e d O l i g o s a c c h a r i d e L i b r a r yAbstract: An efficient synthesis of a protected dimeric Lewis X epitope by two sequential one-pot glycosylations is described. Combinatorial synthesis of the dimeric Lewis X epitope derivatives by the one-pot glycosylation was accomplished utilizing an automated synthesizer to provide 12-protected oligosaccharides.Oligosaccharides on cell surface play important roles in many biological processes. 3 Most biologically active oligosaccharides are rare, and are difficult to purify. Additionally, their structural diversity based upon stereo-and regioisomers makes it difficult to determine their structure in comparison with that of oligopeptides and oligonucleotides. Therefore, the chemical synthesis of such oligosaccharides would strongly assist one in the elucidation of their structure-activity relationships. Recent developments of the chemical synthesis of oligosaccharide involving automated solid-phase synthesis, allows the high speed synthesis of a single target oligosaccharide. 4,5 Furthermore, combinatorial chemistry enables one to synthesize oligosaccharide libraries containing tri-or tetrasaccharides. 6,7,8d However, preparation of biologically active and complex natural product-based oligosaccharide libraries is still difficult to accomplish.One-pot glycosylation, involving sequential activation of glycosyl donors in a single vessel, is effective not only for the high speed synthesis of a single target oligosaccharide, but also for the parallel synthesis of oligosaccharide libraries. 7c,8,9 We have investigated the one-pot glycosylation based on the chemoselective activation of glycosyl donors attached with different leaving groups with appropriate activators, 9 and have already reported a one-pot sixstep synthesis of a di-branched heptasaccharide composed of a b(1,6) linked pentasaccharide backbone using seven independent building blocks. 9f If the requisite manipulations in the one-pot glycosylation are adaptable to an automated synthesizer, it would be an attractive way to synthesize structurally complex oligosaccharides. Scheme 1 Strategy for the one-pot synthesis of dimeric Lewis X derivative 1.
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