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 oligosaccharide and glycopeptide synthesis using glycosynthases

Enzymatic approaches for the preparation of oligosaccharides are interesting alternatives to traditional chemical synthesis, the main advantage being the regio- and stereoselectivity offered without the need for protecting groups. The use of solid-phase techniques offers easy workup procedures and the prospect of automatability. Here, we report the first application of glycosynthases to solid-phase oligosaccharide synthesis by use of the 51 kDa serine and glycine mutants of Agrobacterium sp. beta-glucosidase, Abg E358S and E358G. Acceptors were linked to PEGA resin through a backbone amide linker (BAL), and using these mutated enzymes, a galactose moiety was transferred from a donor sugar, alpha-D-galactosyl fluoride, with high efficiency (>90%) together with excellent recovery of material. Furthermore, it was demonstrated that a resin-bound model glycopeptide was also an acceptor for the glycosynthase.     

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.     

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.     

Solid-phase synthesis of peptide and glycopeptide thioesters through side-chain-anchoring strategies

An efficient new strategy for the synthesis of peptide and glycopeptide thioesters is described. The method relies on the side-chain immobilization of a variety of Fmoc-amino acids, protected at their C-termini, on solid supports. Once anchored, peptides were constructed using solid-phase peptide synthesis according to the Fmoc protocol. After unmasking the C-terminal carboxylate, either thiols or amino acid thioesters were coupled to afford, after cleavage, peptide and glycopeptide thioesters in high yields. Using this method a significant proportion of the proteinogenic amino acids could be incorporated as C-terminal amino acid residues, therefore providing access to a large number of potential targets that can serve as acyl donors in subsequent ligation reactions. The utility of this methodology was exemplified in the synthesis of a 28 amino acid glycopeptide thioester, which was further elaborated to an N-terminal fragment of the glycoprotein erythropoietin (EPO) by native chemical ligation.     

Solid-phase synthesis of thioether-linked glycopeptide mimics for application to glycoprotein semisynthesis

[reaction: see text]. Glycoproteins are particularly suited to protein semisynthesis since homogeneous samples for biological analyses are not readily available using traditional recombinant techniques. Here we apply glycosyl iodoacetamides, normally used for the modification of bacterially derived proteins, to solid-phase glycopeptide synthesis. This provides access to glycopeptide alpha-thioesters, which may lend themselves to the semisynthesis of homogeneous N-linked glycoprotein mimics and novel glycopeptide libraries.     

Solid-phase synthesis of core 3 and core 6 O-glycan-linked glycopeptides by benzyl-protection method

Core 3 and core 6 O-glycoamino acids were prepared in a protected form suited for Fmoc solid-phase peptide synthesis (SPPS). An N-trichloroacetyllactosamine derivative (2) was used as a highly β-selective glycosyl donor in 3-O-glycosylation of acceptors 3/4 and in 6-O-glycosylation of acceptors 5/6. Zn reduction of trisaccharides 7/8 and 13/14 was followed by acetylation to readily transform trichloroacetamido and azido groups to acetamido groups. Selective deprotection by Pd(0)-catalysis afforded core 3 O-glycan building blocks 11/12 and core 6 O-glycan building blocks 17/18. Usefulness of these building blocks for SPPS was demonstrated by the syntheses of the core 3-linked MUC2 tandem repeat glycopeptide and the core 6-linked glycopeptide segment of MUC6. The synthetic glycopeptides detached from the resin were debenzylated under the ‘low-acidity TfOH’ conditions.     

Solid-phase synthesis of 1,5-disubstituted 2-aryliminoimidazolidines

The solid-phase synthesis of 1,5-disubstituted 2-aryliminoimidazolidines, starting from resin-bound N-acylated amino acid amides, is described. Exhaustive reduction of resin-bound acylated amino acid amides with borane-THF afforded the corresponding disecondary amines. Further reaction with arylisothiocyanates in the presence of mercuric chloride (HgCl(2)) yielded the corresponding resin-bound 1,5-disubstituted 2-aryliminoimidazolidines. Cleavage of the product from the resin using HF/anisole (95/5) for 1.5 h at 0 degrees C gave the desired products in good yield and purity. The preparation of a large combinatorial library of such compounds is also discussed.     

Solid-phase synthesis of conformationally constrained peptidomimetics based on a 3,6-disubstituted-1,4-diazepan-2,5-dione core

Starting from a Cl-trytyl linked hydroxylamine, a hydroxamic dipeptide having serine in the second position was prepared by using DMTMM as the coupling agent. Mitsunobu cyclization carried out under microwave heating gave very good yields of a 3,6-disubstituted-perhydro-diazepin-2,5-dione. This heterocycle can be used as a new platform for combinatorial chemistry or as a constraint to rigidify a small peptide.     

Solid-phase synthesis of novel trimers containing a phenylstatine core and analysis by high-resolution magic angle spinning

Here we describe a multistep solid-phase synthetic approach for the addition of amino acid residues to both the C- and N-termini of a phenylstatine core, yielding a library aimed at the development of structure-activity relationships in the S2 and S2' regions of the aspartyl proteases. Optimization of the synthetic strategy was performed on the basis of the in situ analysis of the compounds bound to the solid support through high-resolution magic angle spinning NMR Spectroscopy (HR-MAS NMR).     

Solid-phase organometallic synthesis

A solid-phase synthesis approach for a class of molybdenum carbonyl complexes has been developed. The system can be used to perform metal-complexation, ligand substitution reactions and oxidative eliminations on the solid phase and to cleave the final complexes under mild and selective conditions. Comparison is made to corresponding soluble complexes and liquid-phase reactions.     

Solid-phase template-directed synthesis of a [2]rotaxane using a solid-phase stopper

The first synthesis of a rotaxane by solid phase chemistry has been achieved, using the resin bead as a 'Mega' stopper during the synthesis. One of the advantages of this methodology over traditional solution routes include the ability to use mass action to drive the chemistry, without complicating the purification process.     

Solid-phase synthesis of N-acyl-N′-carbamoylguanidines

Amino acids immobilized on polystyrene-Wang or Rink amide resin were reacted with p-nitrophenyl chloroformate to give an activated urethane that was displaced by S-methylisothiourea. Following N-acylation with an acid chloride, the thiomethyl group was displaced by primary or secondary amines with the aid of mercury (II) chloride to yield the unsymmetrically substituted title compounds after resin cleavage.     

Solid-phase synthesis of a cyclodepsipeptide: cotransin

The first solid-phase synthesis of cotransin--a cyclic depsipeptide having high pharmacological potential--was achieved, by a proper choice of coupling reagents and use of either TBAF or DBU for Fmoc removal to suppress the otherwise dominating, sequence-derived diketopiperazine formation. Starting the assembly from C-terminal lactic acid allowed fast and epimerization-free cyclization in solution. Novel conditions for orthogonal use of the Fmoc/Bsmoc-protection system were discovered, and an unexpected nucleophilic behavior of DBU was observed.     

Solid-phase synthesis of a tyrphostin ether library

The solid-phase synthesis of a 4500-member (30 x 15 x 10) tyrphostin library is demonstrated utilizing the Irori-directed sorting system. Fmoc-protected PL-Rink resin was used as the solid support. After Fmoc-deprotection, aryl aldehydes were attached to the resin through reductive amination. Acylation of the resulting secondary amines with cyanoacetic acid was followed by a Knoevenagel condensation with phenolic aldehydes. Mitsunobu coupling of primary alcohols to the resin-bound phenols yielded the final library of compounds 1.     

Solid-phase synthesis of isoxazolines

The polymer-supported synthesis of isoxazolines is described via nitrile oxide intermediates, starting from primary nitroalkanes in a one-pot process.     

Solid-phase synthesis of beta-mannosides

The linkage of S-phenyl 2,3-di-O-benzyl-alpha-D-thiomannopyranoside to a cross-linked polystyrene support in the form of its 4,6-O-polystyrylborinate ester is described. The activation of this polymer-supported mannosyl donor is achieved at -60 degrees C in dichloromethane in the presence of 2,4,6-tri-tert-butylpyrimidine with the combination 1-benzenesulfinyl piperidine and trifluoromethanesulfonic anhydride. Addition of the donor alcohol at -60 degrees C followed by warming to room temperature and subsequent cleavage from the resin by gentle heating in aqueous acetone yields anomerically pure 2,3-di-O-benzyl-beta-D-mannopyranosides in excellent yield. Successful, diastereoselective coupling is demonstrated with a range of primary, secondary, and tertiary glycosyl acceptors, including typical carbohydrates and threonine derivatives.