Exploration of the use of an acylsulfonamide safety-catch linker for the polymer-supported synthesis of hyaluronic acid oligosaccharides

The synthesis of hyaluronic acid oligosaccharides on polyethylene glycol (PEG) using an acylsulfonamide linker has been explored. Hyaluronic acid is a challenging synthetic target that usually involves the condensation of highly disarmed glucuronic acid building blocks. Amine-ended PEG monomethyl ether was efficiently functionalized with a hydroxyl-terminated acylsulfonamide linker. Suitably protected d-glucosamine (GlcN) and d-glucuronic acid (GlcA) monosaccharide building blocks were coupled to the polymer acceptor using the trichloroacetimidate glycosylation method. The sulfonamide safety-catch linker enables simultaneous cleavage of the monosaccharide from the polymer and orthogonal functionalization for further (bio)-conjugation of the sugar sample. Subsequent glycosylation of PEG-bound glycosyl acceptor to generate hyaluronic acid oligosaccharide chain failed. Model glycosylation experiments in solution and on soluble support using the same unreactive acceptors and donors allows for the synthesis of an orthogonally protected hyaluronic acid disaccharide and suggest that the encountered difficulties could be attributed to the presence of the N-acylsulfonamide.     

Solid phase synthesis of peptide-selenoesters

The synthesis of proteins by native chemical ligation greatly enhances the application of chemistry to complex molecules such as proteins. The essential building blocks for this approach traditionally have been peptide-thioester segments that are linked chemoselectively in consecutive reactions. By using peptide selenoesters instead of thioesters, the ligation rate can be significantly accelerated permitting couplings at difficult sites and potentially enabling new ligation strategies. To facilitate the routine synthesis of selenoester peptides, a general and straightforward procedure has been developed that generates a suitably functionalized resin from which the desired selenoester peptide can be readily synthesized. This simple approach utilizes readily available and cheap chemical agents and enables production of peptide selenoesters of excellent quality in short time and with high recovery. In addition, the stability of peptide selenoesters was examined under different native chemical ligation conditions and compared to thioesters. Selenoesters are slightly more reactive and more susceptible to hydrolysis and aminolysis than thioesters but sufficiently stable under mildly acidic conditions (pH 6.5). Under these conditions, rapid selenoester-mediated ligation is kinetically favoured.     

Cation exchange assisted binding-elution strategy for enzymatic synthesis of human milk oligosaccharides (HMOs)

A cation exchange assisted binding-elution (BE) strategy for enzymatic synthesis of human milk oligosaccharides (HMOs) was developed. An amino linker was used to provide the cation ion under acidic condition which can be readily bound to cation exchange resin and then eluted off by saturated ammonium bicarbonate. Ammonium bicarbonate in the collections was easily removed by vacuum evaporation. This strategy circumvented the incompatible issue between glycosyltransferases and solid support or large polymers, and no purification was needed for intermediate products. With current approach, polyLacNAc backbones of HMOs and fucosylated HMOs were synthesized smoothly.     

Solid-phase route to Fmoc-protected cationic amino acid building blocks

Diamino acids are commonly found in bioactive compounds, yet only few are commercially available as building blocks for solid-phase peptide synthesis. In the present work a convenient, inexpensive route to multiple-charged amino acid building blocks with varying degree of hydrophobicity was developed. A versatile solid-phase protocol leading to selectively protected amino alcohol intermediates was followed by oxidation to yield the desired di- or polycationic amino acid building blocks in gram-scale amounts. The synthetic sequence comprises loading of (S)-1-(p-nosyl)aziridine-2-methanol onto a freshly prepared trityl bromide resin, followed by ring opening with an appropriate primary amine, on-resin N(β)-Boc protection of the resulting secondary amine, exchange of the N(α)-protecting group, cleavage from the resin, and finally oxidation in solution to yield the target γ-aza substituted building blocks having an Fmoc/Boc protection scheme. This strategy facilitates incorporation of multiple positive charges into the building blocks provided that the corresponding partially protected di- or polyamines are available. An array of compounds covering a wide variety of γ-aza substituted analogs of simple neutral amino acids as well as analogs displaying high bulkiness or polycationic side chains was prepared. Two building blocks were incorporated into peptide sequences using microwave-assisted solid-phase peptide synthesis confirming their general utility.     

Solid-phase synthesis of second-generation polyproline dendrimers

Second-generation dendrimers have been prepared on solid phase by successive additions of branched polyproline building blocks starting from two different branching units anchored to the solid support. The preparation of Pro-rich building blocks was carried out by stepwise solid-phase synthesis and their iterative addition was performed by a convergent approach, also using solid-phase synthesis. cis-4-Amino-L-proline and imidazolidine-2-carboxylic acid were used as branching units due to their structural resemblance to proline. The optimized strategy allowed the target compounds to be obtained with high purities without the need for purification steps.     

A Dde resin based strategy for inverse solid-phase synthesis of amino terminated peptides, peptide mimetics and protected peptide intermediates.

This report describes a Dde resin based attachment strategy for inverse solid-phase peptide synthesis (ISPPS). This attachment strategy can be used for the synthesis of amino terminated peptides with side chains and the carboxyl terminus either protected or deprotected. Amino acid t-butyl esters were attached through their free amino group to the Dde resin. The t-butyl carboxyl protecting group was removed by 50% TFA, and inverse peptide synthesis cycles performed using an HATU/TMP based coupling method. Protected peptides were cleaved from the resin with dilute hydrazine. Side chain protecting groups could then be removed by treatment with TFMSA/TFA. The potential of this approach was demonstrated by the synthesis of several short protected and unprotected peptides in good yield and with low epimerization. Its potential for peptide mimetic synthesis was demonstrated by the synthesis of two peptide trifluoromethylketones.     

Preparation of tri(ethylene glycol) grafted core‐shell type polymer support for solid‐phase peptide synthesis

A core‐shell type polymer support for solid‐phase peptide synthesis has been developed for high coupling efficiency of peptides and versatile applications such as on‐bead bioassays. Although various kinds of polymer supports have been developed, they have their own drawbacks including poor accessibility of reagents and incompatibility in aqueous solution. In this paper, we prepared hydrophilic tri(ethylene glycol) (TEG) grafted core‐shell type polymer supports (TEG SURE) for efficient solid‐phase peptide synthesis and on‐bead bioassays. TEG SURE was prepared by grafting TEG derivative on the surface of AM PS resin via biphasic diffusion control method and subsequent acetylation of amine groups which are located at the core region of AM PS resin. The performance of TEG SURE was evaluated by synthesizing several peptides. Three points can be highlighted: (1) easy control of loading level of TEG, (2) improved efficiency of peptide synthesis compared with the conventional resins, and (3) applicability of on‐bead bioassays.     

New Allyl Ester Linker and Solid-phase Block Synthesis of the Serglycin Core Region

The prototype glycopeptidyl fragments of serglycin, a proteoglycan with the characteristic peptide sequence of repeating L-seryl-L-glycine, were synthesized by a convergent method involving block condensation on a solid support. In order to facilitate detachment of the protected glycopeptides from the resin, a new allyl ester type of linker, which is cleavable by Pd(0)-catalysis, was designed and used in combination with the commercial acid-labile Sieber amide resin for the solid-phase synthesis. Glycopeptide blocks consisting of [O-(2,3,4-tri-O-acetyl-D-xylosyl)-L-seryl-L-glycine]n (n=1-8) were produced in good yields. Block condensation in a solution was also successful to synthesize up to the hexadecapeptide (n=8).     

Synthesis of building blocks of human milk oligosaccharides. Fucosylated derivatives of the lacto- and neolacto-series

The synthesis of protected fucosylated derivatives of a Galβ(1→3)GlcNAc and of lactosamine Galβ(1→4)GlcNAc building blocks contained in human milk oligosaccharides is described. Both chemical and enzymatic methods have been exploited for selective protection of the disaccharide. Fucosylation of the appropriate derivatives allowed an easy and relatively short access to different products from common precursors.     

Solid phase synthesis of a functionalized bis-peptide using "safety catch" methodology

In 1962, R.B. Merrifield published the first procedure using solid-phase peptide synthesis as a novel route to efficiently synthesize peptides. This technique quickly proved advantageous over its solution-phase predecessor in both time and labor. Improvements concerning the nature of solid support, the protecting groups employed and the coupling methods employed over the last five decades have only increased the usefulness of Merrifield's original system. Today, use of a Boc-based protection and base/nucleophile cleavable resin strategy or Fmoc-based protection and acidic cleavable resin strategy, pioneered by R.C. Sheppard, are most commonly used for the synthesis of peptides(1). Inspired by Merrifield's solid supported strategy, we have developed a Boc/tert-butyl solid-phase synthesis strategy for the assembly of functionalized bis-peptides(2), which is described herein. The use of solid-phase synthesis compared to solution-phase methodology is not only advantageous in both time and labor as described by Merrifield(1), but also allows greater ease in the synthesis of bis-peptide libraries. The synthesis that we demonstrate here incorporates a final cleavage stage that uses a two-step "safety catch" mechanism to release the functionalized bis-peptide from the resin by diketopiperazine formation. Bis-peptides are rigid, spiro-ladder oligomers of bis-amino acids that are able to position functionality in a predictable and designable way, controlled by the type and stereochemistry of the monomeric units and the connectivity between each monomer. Each bis-amino acid is a stereochemically pure, cyclic scaffold that contains two amino acids (a carboxylic acid with an α-amine)(3,4). Our laboratory is currently investigating the potential of functional bis-peptides across a wide variety of fields including catalysis, protein-protein interactions and nanomaterials.     

利用重组大肠杆菌进行寡糖合成的研究进展

随着更多寡糖生物学活性的阐明,寡糖合成研究已成为糖生物学研究的热点之一,其中,以重组大肠杆菌作为酶盒或生物反应器,利用Leloir途径合成寡糖的方法,是近年来发展起来的一类重要的寡糖生物合成技术,并取得了较多的进展。将从细菌糖基转移酶的表达和鉴定、糖核苷酸的供给和寡糖的合成途径等几个方面,关注利用细菌功能尤其是利用重组大肠杆菌合成寡糖的研究进展,并分析各技术手段的优缺点及其应用前景。     

Iterative one-pot syntheses of chitotetroses

Rapid syntheses of chitotetrose derivatives were achieved in good yields using the newly developed reactivity independent iterative one-pot strategy. The protective groups on donors and acceptors were independently evaluated allowing matching of the two partners in glycosylation. No anomeric reactivity adjustments or intermediate purification were necessary thus significantly improving the overall synthetic efficiency. Only near stoichiometric amounts of building blocks were required for the assembly of target molecules further highlighting the potential of the iterative one-pot method in complex oligosaccharide synthesis.     

Convenient strategies for the synthesis of 1,4-phenylene spaced sugars

Two synthetic strategies have been developed for the synthesis of spaced sugars with lipophilic 1,4-phenylene core. A building block combining the usefulness of Weinreb amide functionality and modified Julia olefination reaction has been explored towards this objective. This building block offers complete flexibility in attaching any desired sugar derivative across phenylene spacer via C-C bond formation. The other strategy uses carbohydrate based building blocks for the synthesis of symmetrical as well as unsymmetrical 1,4-phenylene spaced sugars. This is the first report for the synthesis of 1,4-phenylene spaced sugars via C-C bond formation.     

Bilberry xyloglucan--novel building blocks containing beta-xylose within a complex structure

Bilberries are known to have one of the most complex xyloglucan structures described in the plant kingdom until now. To characterise this structure, xyloglucans were enzymatically degraded and the oligosaccharides obtained were analysed. More than 20 different building blocks were found to make up the xyloglucan polymer. Bilberry xyloglucan was of XXXG-type, but some XXG-type oligomers were present, as well. The building blocks contain galactose-xylose (L) and fucose-galactose-xylose (F) side chains. In both side chains, the galactose units can be acetylated. In addition, beta-xylose-alpha-xylose (U) side chains were shown. This U chain was present in three building blocks described before (XUXG, XLUG and XUFG) and four novel blocks that have not been described in the literature previously: XUG, XUUG, XLUG and XXUG.     

Synthesis and carbohydrate binding studies of fluorescent α-amidoboronic acids and the corresponding bisboronic acids

Fluorescent boronic acids are very useful for the design and synthesis of carbohydrate sensors. In an earlier communication, we first described the effort of developing water soluble fluorescent α-amidoboronic acids, which change fluorescence upon sugar binding. In this report, we describe a general method of functionalizing such boronic acids and their applications in the preparation of bis-α-amidoboronic acids with significantly enhanced binding for oligosaccharides as compared to their monoboronic acid counterparts. The advantages of good water solubility, easy modification to generate diversity, and modularity in synthesis will make α-amidoboronic acids very useful building blocks for future synthesis of boronic acid-based fluorescent sensors.     

Synthesis of a shark repellent peptide toxin, Pardaxin (16-33) on a highly flexible polymer support: CLPSER

A high swelling resin, CLPSER has been developed and utilized for the solid phase synthesis of Pardaxin, which is an 18-residue peptide. The resin was characterized by gel phase (13)C NMR, IR and SEM. The utility of the new polymer support in polypeptide synthesis was further established by the comparative synthesis of pardaxin with commercially available Merrifield resin. The MALDI TOF MS, amino acid analysis and the HPLC revealed the superior quality of CLPSER.     

Enzymatic preparation of heparin disaccharides as building blocks in glycosaminoglycan synthesis.

Pharmaceutical heparin and heparan sulfate, isolated from a side-stream of a commercial heparin manufacturing process, have been enzymatically depolymerzed with heparin lyases obtained from Flavobacterium heparinun. Heparin afforded a trisulfated disaccharide product that was recovered from the reaction mixture using gel permeation chromatography. Heparan sulfate afforded unsulfated disaccharide that was conveniently recovered from the product mixture by ion exchange chromatography. Both disaccharides were obtained in gram amounts at 90% or higher purity. Both enzymatically prepared disaccharides were chemically protected to prepare building blocks required for the future chemical synthesis of therapeutically valuable heparin oligosaccharides.     

Enhancing solid phase synthesis by a noncovalent protection strategy-efficient coupling of rhodamine to resin-bound peptide nucleic acids.

Resins for solid-phase synthesis can affect coupling efficiencies by interacting with reactants. We have observed that polyethylene glycol-polystyrene (PEG-PS) solid support absorbs added activated fluorophores, preventing efficient labeling of peptide nucleic acids (PNAs). We now report that addition of an inexpensive unactivated fluorophore blocks the resin and allows efficient labeling. This protection strategy may have general benefits for peptide and combinatorial synthesis.     

Non-polymerase-cycling-assembly-based chemical gene synthesis: strategies, methods, and progress

Chemical gene synthesis is a powerful tool for basic biological research and biotechnology applications. During the last 30 years, major advances have been made in the chemical synthesis of DNA sequences ranging from fragments of <1 kb to multi-gene sequences of >30 kb. There is a need for simple, reproducible, less error-prone and cost-effective methods that guarantee successful synthesis of the desired genes and are amenable to automation. Many polymerase chain reaction (PCR)-based and non-polymerase-cycling-assembly (PCA)-based strategies have been developed for chemical gene synthesis. The PCR-based method has been the subject of several recent reviews. Here, we provide an overview of the progress in non-PCA-based chemical gene synthesis using different strategies and methods, including enzymatic gene synthesis, annealing and ligation reaction, simultaneous synthesis of two genes via a hybrid gene, shotgun ligation and co-ligation, insertion gene synthesis, gene synthesis via one strand of DNA, template-directed ligation, ligase chain reaction, microarray-mediated gene synthesis, Blue Heron solid support technology and Sloning building block technology. The fundamental principle underlying each strategy, an example where applicable, and the advantages and disadvantages are discussed. The emphasis is on discussion of the most recent technologies and their potential applications, particularly for microarray-based genomics research.     

Synthesis of oligosaccharides by bacterial enzymes

Many human pathogens initiate disease by utilizing their microbial adhesin proteins to attach to glycoconjugates on host cell mucosal surfaces. Soluble oligosaccharides of identical or similar structure to these naturally occurring ligands can both prevent bacterial attachment as well as mediate the release of attached bacteria. Since it has not been possible to isolate large quantities of these compounds, we have developed enzyme-based technologies to synthesize several relevant human oligosaccharides. Using cloned bacterial glycosyltransferases, we can synthesize several hundred grams of these oligosaccharides at a time. The availability of these large quantities will allow these compounds to be tested as anti-adhesive pharmaceutical agents as well as lead to expanded practical applications.