One-pot synthesis of sialo-containing glycosyl amino acids by use of an N-trichloroethoxycarbonyl-beta-thiophenyl sialoside

We describe an efficient synthesis of 2,6- and 2,3-sialyl T antigens linked to serine in a one-pot glycosylation. We first investigated the glycosidation of thiosialosides by varying the N-protecting group. Modification of the C-5 amino group of beta-thiosialosides into the N-9-fluorenylmethoxycarbonyl, N-2,2,2-trichloroethoxycarbonyl (N-Troc), and N-trichloroacetyl derivatives enhanced the reactivity of these compounds towards glycosidation. Addition of a minimum amount of 3 A molecular sieves was also effective in improving the yield of alpha-linked sialosides. Next, we conducted one-pot syntheses of the glycosyl amino acids by using the N-Troc sialyl donor. The N-Troc derivative can be converted into the N-acetyl derivative without racemization of the amino acids. Branched-type one-pot glycosylation, initiated by regioselective glycosylation of the 3,6-dihydroxy galactoside with the N-Troc-beta-thiophenyl sialoside, provided the protected 2,6-sialyl T antigen in good yield. Linear-type one-pot glycosylation, initiated by chemoselective glycosylation of galactosyl fluoride with the N-Troc-beta-thiophenyl sialoside, afforded the protected 2,3-sialyl T antigen in excellent yield. Both protected glycosyl amino acids were converted into the fully deprotected 2,6- and 2,3-sialyl T antigens linked to serine in good yields.     

1‐Picolinyl‐5‐azido Thiosialosides: Versatile Donors for the Stereoselective Construction of Sialyl Linkages

With the picolinyl (Pic) group as a C‐1 located directing group and N₃ as versatile precursor for C5‐NH₂, a novel 1‐Pic‐5‐N₃ thiosialyl donor was designed and synthesized, based on which a new sialylation protocol was established. In comparison to conventional sialylation methods, the new protocol exhibited obvious advantages, including excellent α‐stereoselectivity in the absence of a solvent effect, broad substrate scope encompassing the challenging sialyl 8‐ and 9‐hydroxy groups of sialic acid acceptors, flexibility in sialoside derivative synthesis, high temperature tolerance and easy scalability. In particular, the applicability to the synthesis of complex and bioactive N‐glycan antennae when combined with the MPEP glycosylation protocol via the “latent‐active” strategy has been shown. Mechanistically, the excellent α‐stereoselectivity of the novel sialylation protocol could be attributed to the dramatic electron‐withdrawing effect of the protonated Pic groups, which was supported by control reactions and DFT calculations.     

Solid-phase synthesis of O-sulfated glycopeptide by the benzyl-protected glycan strategy

To expand the repertoire of our benzyl-protection strategy for solid-phase glycopeptide synthesis, an O-sulfated glycopeptide was chosen as the synthetic target. Trisaccharyl serine derivatives (Galβ1-4-GlcNAcβ1-2-Manα1-3-Ser) carrying (4-methoxyphenyl)methyl (MPM) groups at either 3-O or 6-O of the Gal residue were prepared through three stereoselective glycosylations. Cleavage of MPM followed by reaction with Me₃N·SO₃ efficiently afforded 3-O- and 6-O-sulfo-glycoserines, respectively. A preliminary debenzylation study using the sulfated glycoserines revealed that the sulfate groups persisted under ‘low-acidity TfOH’ conditions, when using a limited amount of TfOH and extending the reaction period. The 3-O-sulfo-glycoserine was then introduced into an icosapeptide modeled after an α-dystroglycan fragment by a combination of automated and manual solid-phase peptide synthesis procedures. The synthesized glycopeptide was successfully debenzylated by the low-acidity TfOH cocktail with slight damage to the sulfate functionality.     

Use of fluorobenzoyl protective groups in synthesis of glycopeptides: beta-elimination of O-linked carbohydrates is suppressed

Fluorobenzoyl groups have been investigated as alternatives to acetyl and benzoyl protective groups in carbohydrate and glycopeptide synthesis. D-Glucose and lactose were protected with different fluorobenzoyl groups and then converted into glycosyl bromides in high yields (>80% over two steps). Glycosylation of protected derivatives of serine with these donors gave 1,2-trans glycosides in good yields (approximately 60--70%) and excellent stereoselectivity without formation of ortho esters. The resulting glycosylated amino acid building blocks were then used in solid-phase synthesis of two model O-linked glycopeptides known to be unusually sensitive to beta-elimination on base-catalyzed deacylation. When either a 3-fluoro- or a 2,5-difluorobenzoyl group was used for protection of each of the two model glycopeptides the extent of beta-elimination decreased from 80% to 10% and from 50% to 0%, respectively, as compared to when using the ordinary benzoyl group. Fluorobenzoyl groups thus combine the advantages of the benzoyl group in formation of glycosidic bonds (i.e., high stereoselectivity and low levels of ortho ester formation) with the ease of removal characteristic of the acetyl group.     

Efficient microwave-assisted tandem N- to S-acyl transfer and thioester exchange for the preparation of a glycosylated peptide thioester

A peptide carrying a mercaptomethylated proline derivative at the C-terminus was prepared by solid-phase peptide synthesis (SPPS) and converted to the thioester of 3-mercaptopropionic acid (MPA) by aqueous MPA under microwave irradiation conditions. This post-SPPS thioesterification reaction was successfully applied to the synthesis of a glycopeptide thioester composed of 25 amino acid (AA) residues, which was then used for the preparation of a 61-AA glycopeptide by the thioester condensation method.     

Solution- and solid-phase synthesis of natural product-like tetrahydroquinoline-based polycyclics having a medium size ring

A solid-phase synthesis of tetrahydroquinoline-derived polycyclic 4, having a medium size ring with an enamide functionality, was achieved from tetrahydroquinoline derivative 3 in five steps with overall 40-45% yield. An enantiopure, tetrahydroquinoline-derived beta-amino ester, 1, was converted into compound 2 that has a free phenolic hydroxyl group as an anchoring site for solid-phase synthesis. The solid-phase worked well for this sequence, in which the synthesis of the unsaturated eight-membered enamide lactam was obtained by a ring-closing metathesis approach. Compound 4 is a novel, natural product-like polycyclic derivative that could further be utilized in library generation for developing small molecule chemical probes.     

A stereocontrolled construction of 2-azido-2-deoxy-1,2-cis-α-galactosidic linkages utilizing 2-azido-4,6-O-benzylidene-2-deoxygalactopyranosyl diphenyl phosphates: stereoselective synthesis of mucin core 5 and core 7 structures

TMSOTf-promoted glycosidation of 2-azido-4,6-O-benzylidene-2-deoxygalactosyl diphenyl phosphates with fluorenylmethoxycarbonyl (Fmoc)-protected serine and threonine derivatives in THF/Et₂O (1:1) gave glycosyl amino acids in high yields and with excellent levels of α-selectivity (α/β=94:6–95:5). The synthetic utility of the present glycosidation method was demonstrated by a stereoselective synthesis of mucin-type glycopeptide core 5 and core 7 building blocks, which are suitable for Fmoc-based solid-phase synthesis of O-glycopeptides.     

Facile synthesis of unsaturated pyroglutaminol derivatives

A novel approach to the synthesis of unsaturated pyroglutaminol derivatives was developed. Condensation of a protected serine derivative with Meldrum’s acid followed by decarboxylative cyclization afforded a tetramic acid, which was then converted to a hydroxylactam using sodium borohydride. Iodine displacement of the hydroxyl group induced spontaneous elimination to afford unsaturated pyroglutaminol derivatives in good yields.     

Synthesis of N,N-Ac,Boc laurylthio sialoside and its application to O-sialylation

The combination of the 5-N-tert-butoxycarbonyl (Boc) group of laurylthio sialoside and cyclopentyl methyl ether (CPME) as a solvent enhanced the reactivity and α-selectivity of the sialyl donor during sialylation. Selective deprotection of the N-Boc group of sialoside, including an acid-sensitive isopropylidene function, was successfully achieved by Yb(OTf)₃-SiO₂. Transformation of N,N-Ac,Boc into an N-acetylglycolyl group of sialoglycoside was easily performed via selective N-deacylation of the mixed Ac-N-Boc carbamate, subsequent Boc group removal, and acylation.     

7-Deazaguanosine: Synthesis of an oligorbonucleotide building block and disaggregation of the U-G-G-G-G-U G4 structure by the modified base

Building blocks derived from 7-deazaguanosine (c⁷G, 1 ) were prepared for solid-phase oligoribonucleotide synthesis. Compound 1 was converted into the isobutyurl derivative 2b and the (dimethylamino)methylidene compound 3 (Scheme 1). After tritylation (→ 4a , b ), silylation was studied with regard to regioselectivity. It was found that the triisopropylsilyl group in combination with the (dimethylamino)methylidene residue gave the highest 2′ -selectivity (→ 5e ). The 2′ -O -silyl derivative 5e was reacted with PCl₃ affording the 3′ -phosphonate 7 which was used in solid-phase oligoribonucleotide synthesis. Oligonucleotides derived from U-G-G-G-G-U with an increasing number of c⁷G residues instead of G were synthesized. Aggregation was studied by polyacrylamidegel electrophoresis and CD Spectroscopy. Disaggregation of the G₄-structure of U-G-G-G-G-U was observed when c⁷G replaced G, demonstrating that guanine N(7) participates in the aggregation process.     

Solid-phase synthesis of core 2 O-linked glycopeptide and its enzymatic sialylation

The core 2-type tetrasaccharide building blocks (1a/1b) for solid-phase synthesis of glycopeptide were synthesized via stereoselective glycosylation of the disaccharyl Ser/Thr (3a/3b) with a glycosyl fluoride (2) carrying the 2-trichloroacetamido group that was readily converted into a 2-acetamido group by reduction. A segment of glycoprotein leukosialin (215-224) was synthesized by the solid-phase protocol, the building block (1b) being utilized. Cleavage of the synthetic glycopeptide from resin was effected with reagent K and subsequent treatment of the product with a cocktail for the ‘low-acidity TfOH’ facilitated complete removal of the benzyl groups with minimum loss of glycosidic linkages. To the deprotected glycopeptide (21), were enzymatically introduced N-acetylneuraminic acid (sialic acid) residues in remarkably high efficiency by using the specific sialyltransferases.     

Solid-phase synthesis of glycopeptide carrying a tetra-N-acetyllactosamine-containing core 2 decasaccharide

A novel synthesis of tetralactosaminyl O-glycoamino acid is described. The stereoselective assemblage of a lactosaminyl unit was performed by 2-trichloroacetamido group-assisted β-glycosylation. Initial investigation into the synthesis of decasaccharyl threonine 2 showed limited success because of the low yield in the step concerning the removal of 4-O-chloroacetyl groups. In contrast, 4-O-benzylated decasaccharyl threonine 50 was efficiently synthesized from key LacNAc derivative 35 carrying a 3-O-allyl protecting group at the Gal residue by reiterative glycosylation using the (N-phenyl)trifluoroacetimidate method. Decasaccharide 50 was used as a building block in the solid-phase synthesis of a MUC1-related glycopeptide. Synthetic glycopeptide was obtained through two acidic processes: cleavage from resin with reagent K at a lowered temperature and debenzylation with a diluted cocktail of low-acidity TfOH. Desired glycopeptide 54 was isolated as the major product, while a series of the saccharide-shortened minor products were generated due to the acid-labile property of the β-GlcNAc glycosidic linkages.     

Synthesis of Glycosylated Amino Acids for Use in Solid Phase Glycopeptide Synthesis,Part 2: N-(9-Fluorenylmethyloxycarbonyl)-3-0-[2,4,6-TRI-0-Acetyl-3-0-(2,3,4-TRI-0-Acetyl-α-D-Xylopyranosyl)-β-D-Glucopyranosyl]-L-Serine

Abstract

FMOC-serine phenacylester was glycosylated with a derivative of the disaccharide α-D-Xylp-(1→3)-β-D-Glcp, and the product was treated with zinc to remove the phenacyl group. The title derivative is useful for synthesis of blood clotting factor IX glycopeptide fragments by the solid-phase approach.     

Solid-phase synthesis of O-linked glycopeptide analogues of enkephalin

The synthesis of 18 N-alpha-FMOC-amino acid glycosides for solid-phase glycopeptide assembly is reported. The glycosides were synthesized either from the corresponding O'Donnell Schiff bases or from N-alpha-FMOC-amino protected serine or threonine and the appropriate glycosyl bromide using Hanessian's modification of the Koenigs-Knorr reaction. Reaction rates of D-glycosyl bromides (e.g., acetobromoglucose) with the L- and D-forms of serine and threonine are distinctly different and can be rationalized in terms of the steric interactions within the two types of diastereomeric transition states for the D/L and D/D reactant pairs. The N-alpha-FMOC-protected glycosides [monosaccharides Xyl, Glc, Gal, Man, GlcNAc, and GalNAc; disaccharides Gal-beta(1-4)-Glc (lactose), Glc-beta(1-4)-Glc (cellobiose), and Gal-alpha(1-6)-Glc (melibiose)] were incorporated into 22 enkephalin glycopeptide analogues. These peptide opiates bearing the pharmacophore H-Tyr-c[DCys-Gly-Phe-DCys]- were designed to probe the significance of the glycoside moiety and the carbohydrate-peptide linkage region in blood-brain barrier (BBB) transport, opiate receptor binding, and analgesia.     

Pyrrolo-dC oligonucleotides bearing alkynyl side chains with terminal triple bonds: synthesis, base pairing and fluorescent dye conjugates prepared by the azide-alkyne "click" reaction

5-(Octa-1,7-diynyl)-2'-deoxyuridine was converted into the furano-dU derivative 7 by copper-catalyzed cyclization; the pyrolodC-derivative 3 was formed upon ammonolysis. The bicyclic nucleosides 3 and 7 as well as the corresponding non-cyclic precursors 4 and 6 all containing terminal C[triple bond]C bonds were conjugated with the non-fluorescent 3-azido-7-hydroxycoumarin 5 employing the copper(I)-catalyzed Huisgen-Sharpless-Meldal cycloaddition "click reaction". Strongly fluorescent 1H-1,2,3-triazole conjugates (30-33) are formed incorporating two fluorescent reporters-the pyrdC nucleoside and the coumarin moiety. Oligonucleotides incorporating 6-alkynyl and 6-alkyl 7H-pyrrolo[2,3-d]pyrimidin-2(3H)-one nucleosides (3 and 2f) have been prepared by solid-phase synthesis using the phosphoramidite building blocks 10 and 13 ; the pyrrolo-dC oligonucleotides are formed during ammonia treatment. The duplex stability of oligonucleotides containing 3 and related derivatives was studied. Oligonucleotides with terminal triple bonded nucleosides such as 3 are more stabilizing than those lacking a side chain with terminal unsaturation; open-chain derivatives (4) are even more efficient. The click reaction was also performed on oligonucleotides containing the pyrdC-derivative and the fluorescence properties of nucleosides, oligonucleotides and their coumarin conjugates were studied.     

A new C(1)-auxiliary for anomeric stereocontrol in the synthesis of alpha-sialyl glycosides

The installation of the novel N,N-dimethylglycolamide ester auxiliary onto the C(1)-position of protected neuraminic acid donors allows for the exploitation of C(1)-neighboring group participation to generate sialoside conjugates with good to excellent alpha-selectivity under a variety of sialylation protocols, including those that would otherwise lead to nonselective or beta-selective sialoside products.     

A solution- and solid-phase approach to tetrahydroquinoline-derived polycyclics having a 10-membered ring

With the goal of library generation using a polycyclic derivative 5 having an enamide functional group, a simple and practical, enantioselective synthesis of tetrahydroquinoline derivative 2 was achieved. The phenolic hydroxyl group in compound 2 was utilized as an anchoring site for solid-phase synthesis. The ring closing metathesis approach yielded the desired polycyclic product 5 on solid phase in five steps (overall 40% yield). Compound 5 is a novel scaffold for the library generation of natural product-like polycyclics having a functionalized medium ring for obtaining a new class of small molecules to be utilized as chemical probes.     

Selectin Receptors 4: Synthesis of Tetrasaccharides Sialyl Lewis A and Sialyl Lewis X Containing A Spacer Group1,2

ABSTRACT

Synthesis of two isomeric tetrasaccharides, namely Neu5Acα(2→3)Galβ(1→3)[Fucα(1→4)GlcNAcβ (sLe^a) and Neu5Acα(2→3)Galβ(1→4)[Fucα(1→3)]GlcNAcβ (sLe^x) as 3-aminopropyl glycosides is described. Preparation of these compounds was performed by sialylation of selectively protected trisaccharides Le^a and Le^x which contain three unsubstituted OH groups at positions 2, 3 and 4 of Gal residue. Glycosylation of Le^x trisaccharide with ethylthio sialoside under promotion by NIS and TfOH in acetonitrile was effective and regio- and stereoselective to give sLe^x derivative in 81% yield. In contrast, sialylation of the Lc^a acceptor was accompanied by a variety of undesirable by-processes, namely. N-thioethylation of the GlcNAc residue, β-sialylation, and lactonisation. In order to improve the yield of sLc^a tetrasaccharide the glycosylation of Le^a acceptor by sialyl donors of ethyl and phenyl thioglycoside (promoted by NIS-TfOH or NBS-Bu₄NBr), xanthate (promotion by NIS-TfOH mixture or MeOTf) and phosphite (promoted by TMSOTf) types was also studied. Among the reactions investigated the glycosylation by phenyl thioglycoside sialoside promoted by NIS-TfOH gives the best yield (39%) of sLe^a tetrasaccharide product.     

Synthesis of [5'-13C]ribonucleosides and 2'-deoxy[5'-13C]ribonucleosides

The present efficient synthesis of [5'-13C]ribonucleosides and 2'-deoxy[5'-13C]ribonucleosides is characterized by the synthesis of the D-[5-13C]ribose derivative as an intermediate via the Wittig reaction of 4-aldehydo-D-erythrose dialkyl acetals with Ph3P13CH3I-BuLi to introduce the 13C label at the 5-position of a pentose. This was followed by the highly diastereoselective osmium dihydroxylation for the preparation of 2,3-di-O-benzyl-D-[5-13C]ribose dialkyl acetal and the cyclization from D-[5-13C]ribose dialkyl acetal derivatives to the alkyl D-[5-13C]ribofuranoside derivative by the use of LiBF(4). The obtained D-[5-13C]ribose derivative was converted into [5'-13C]ribonucleosides and subsequently into the corresponding 2'-deoxynucleosides.     

Combinatorial synthesis of heterocycles: solid-phase synthesis of 2-amino-4(1H)-quinazolinone derivatives

A new solid-phase synthesis of various substituted 2-amino-4(1H)-quinazolinones from a resin bound amine component is described. The amine was readily converted to the corresponding polymer bound S-methylthiopseudourea. Condensation with different substituted isatoic anhydrides afforded 2-amino-4(1H)-quinazolinone derivatives. The method is amenable for combinatorial library generation.