Chemical and Chemoenzymatic Synthesis of Peptide and Protein Therapeutics Conjugated with Human N-Glycans.

Glycosylation is one of the most ubiquitous post-translational modifications. It affects the structure and function of peptides/proteins and consequently has a significant impact on various biological events. However, the structural complexity and heterogeneity of glycopeptides/proteins caused by the diversity of glycan structures and glycosylation sites complicates the detailed elucidation of glycan function and hampers their clinical applications.

Better Peptides via Chemical Glycosylation: Somatostatin Analogues Having a Human Complex-Type N-Glycan with Improved Drug Properties.

Somatostatin (somatotropin release-inhibiting factor, SRIF) is a growth hormone inhibitory factor in the form of a 14- or 28-amino acid peptide. SRIF affects several physiological functions through its action on five distinct SRIF receptor subtypes (sst1-5). Native SRIF has only limited clinical applications due to its rapid degradation in plasma.

Spontaneously Cleavable Glycosylated Linker Capable of Extended Release of Its Conjugated Peptide.

Reversibly glycosylated conjugates were developed by adding complex-type N-linked oligosaccharides to peptides through self-cleavable linkers with the aim of increasing the solubility and stability of the peptides in plasma. The amino or carboxyl group of the peptide was connected to a glycosylated Ascendis or ester/thioester-type linker, respectively. Use of the linkers enabled extended release of the peptides depending on the pH and temperature of the buffer according to a first order reaction, and their cleavage rate was also affected by the structure of the peptide-linker coupling.

Chemoenzymatic synthesis and Fcγ receptor binding of homogeneous glycoforms of antibody Fc domain. Presence of a bisecting sugar moiety enhances the affinity of Fc to FcγIIIa receptor.

Structurally well-defined IgG-Fc glycoforms are highly demanded for understanding the effects of glycosylation on an antibody's effector functions. We report in this paper chemoenzymatic synthesis and Fcγ receptor binding of an array of homogeneous IgG-Fc glycoforms. The chemoenzymatic approach consists of the chemical synthesis of defined N-glycan oxazolines as donor substrates, the expression of the Fc domain in a CHO cell line in the presence of an α-mannosidase inhibitor kifunensine, and an endoglycosidase-catalyzed glycosylation of the deglycosylated Fc domain (GlcNAc-Fc homodimer) with the synthetic glycan oxazolines.

Endo-beta-N-acetylglucosaminidase-catalyzed polymerization of beta-Glcp-(1-->4)-GlcpNAc oxazoline: a revisit to enzymatic transglycosylation.

An alternative synthesis of beta-Glcp-(1-->4)-GlcpNAc oxazoline is described, and its enzymatic reaction with the endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) was re-investigated. Under normal transglycosylation conditions with a catalytic amount of enzyme, Endo-A showed only marginal activity for transglycosylation with the disaccharide oxazoline, consistent with our previous observations. However, when used in a relatively large quantity, Endo-A could promote the transglycosylation of the disaccharide oxazoline to a GlcpNAc-Asn acceptor.

Introducing N-glycans into natural products through a chemoenzymatic approach.

The present study describes an efficient chemoenzymatic method for introducing a core N-glycan of glycoprotein origin into various lipophilic natural products. It was found that the endo-beta-N-acetylglucosaminidase from Arthrobactor protophormiae (Endo-A) had broad substrate specificity and can accommodate a wide range of glucose (Glc)- or N-acetylglucosamine (GlcNAc)-containing natural products as acceptors for transglycosylation, when an N-glycan oxazoline was used as a donor substrate. Using lithocholic acid as a model compound, we have shown that introduction of an N-glycan could be achieved by a two-step approach: chemical glycosylation to introduce a monosaccharide (Glc or GlcNAc) as a handle, and then Endo-A catalyzed transglycosylation to accomplish the site-specific N-glycan attachment.

Expeditious chemoenzymatic synthesis of homogeneous N-glycoproteins carrying defined oligosaccharide ligands.

An efficient chemoenzymatic method for the construction of homogeneous N-glycoproteins was described that explores the transglycosylation activity of the endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) with synthetic sugar oxazolines as the donor substrates. First, an array of large oligosaccharide oxazolines were synthesized and evaluated as substrates for the Endo-A-catalyzed transglycosylation by use of ribonuclease B as a model system. The experimental results showed that Endo-A could tolerate modifications at the outer mannose residues of the Man3GlcNAc-oxazoline core, thus allowing introduction of large oligosaccharide ligands into a protein and meanwhile preserving the natural, core N-pentasaccharide (Man3GlcNAc2) structure in the resulting glycoprotein upon transglycosylation.

Enhanced amplification of polymerase chain reaction by addition of cosolutes derived from a cellular compatible solute.

We designed and synthesized artificial compatible solutes which can not only decrease the melting temperature of DNA duplexes dependent of their chemical structures but also improve the amplification of highly stable genome DNA sequence.

Enzymatic copolymerization to hybrid glycosaminoglycans: a novel strategy for intramolecular hybridization of polysaccharides.

Hybrid glycosaminoglycans (GAGs) having an intramolecularly hybridized structure of hyaluronan-chondroitin (3a) and hyaluronan-chondroitin 4-sulfate (3b) have been synthesized via enzymatic copolymerization catalyzed by hyaluronidase (HAase). N-Acetylhyalobiuronate (GlcAbeta(1-->3)GlcNAc)-derived oxazoline (1) was copolymerized with N-acetylchondrosine (GlcAbeta(1-->3)GalNAc)-derived oxazoline (2a) by HAase catalysis at pH 7.5 and 30 degrees C, giving rise to copolymer 3a with Mn 7.

Hyaluronidase-catalyzed copolymerization for the single-step synthesis of functionalized hyaluronan derivatives.

Hyaluronidase-catalyzed copolymerization was carried out with monomer combinations of 2-methyl (1a)/2-vinyl (1b), 2-methyl (1a)/2-ethyl (1c), 2-methyl (1a)/2-n-propyl (1d), and 2-vinyl (1b)/2-ethyl (1c) oxazoline derivatives of hyalobiuronate [GlcAbeta(1-->3)GlcN]. All copolymerization reactions proceeded successfully in a regio and stereoselective manner, giving rise to hyaluronan derivatives bearing different N-acyl groups at the C2 position of the glucosamine unit in the polymer chain. The composition of the N-acyl groups was controlled by varying the comonomer feed ratio.

A hyaluronidase supercatalyst for the enzymatic polymerization to synthesize glycosaminoglycans.

Hyaluronidase (HAase) catalyzes multiple enzymatic polymerizations with controlling regio- and stereoselectivity perfectly. This behavior, that is, the single enzyme being effective for multireactions and retaining the enzyme catalytic specificity, is not usual, and hence, HAase is a supercatalyst. Various sugar oxazoline monomers prepared based on the concept "transition-state analogue substrate" were successfully polymerized and copolymerized with HAase catalysis, yielding natural and unnatural glycosaminoglycans.

Bottom-up synthesis of hyaluronan and its derivatives via enzymatic polymerization: direct incorporation of an amido functional group.

This paper reports the synthesis of hyaluronan (HA) and its derivatives via the hyaluronidase-catalyzed polymerization of 2-substituted oxazoline derivative monomers designed as "transition-state analogue substrates". Polymerization of 2-methyl oxazoline monomer from N-acetylhyalobiuronate (GlcAbeta(1-->3)GlcNAc) effectively proceeded at pH 7.5 and 30 degrees C, giving rise to synthetic HA (natural type) in an optimal yield of 78% via ring-opening polyaddition under total control of regioselectivity and stereochemistry.

Enzymatic glycosidation of sugar oxazolines having a carboxylate group catalyzed by chitinase.

Enzymatic glycosidation using sugar oxazolines 1-3 having a carboxylate group as glycosyl donors and compounds 4-6 as glycosyl acceptors was performed by employing a chitinase from Bacillus sp. as catalyst. All the glycosidations proceeded with full control in stereochemistry at the anomeric carbon of the donor and regio-selectivity of the acceptor.