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Glycosylation of peptides is a promising strategy for modulating the physicochemical properties of peptide drugs and for improving their absorption through biological membranes., Glycosylation of peptides is a promising strategy for modulating the physicochemical properties of peptide drugs and for improving their absorption through biological membranes. This review highlights various methods for the synthesis of glycoconjugates and recent progress in the development of glycosylated peptide therapeutics. Furthermore, the impacts of glycosylation in overcoming the existing barriers that restrict oral and brain delivery of peptides are described herein.

Active immunisation against gonadotropin releasing hormone (GnRH) is a potential alternative to surgical castration. This study focused on the development of a GnRH subunit lipopeptide vaccine. A library of vaccine candidates that contained one or more (up to eight) copies of monomeric or dimeric GnRH peptide antigen, an adjuvanting lipidic moiety based on lipoamino acids, and an additional T helper epitope, was synthesised by solid phase peptide synthesis. The candidates were evaluated in vivo in order to determine the minimal components of this vaccine necessary to induce a systemic immune response. BALB/c mice were immunised with GnRH lipopeptide conjugates, co-administered with or without Complete Freund's Adjuvant, followed by two additional immunisations. Significant GnRH-specific IgG titres were detected in sera obtained from mice immunised with four of the seven lipopeptides tested, with an increase in titres observed after successive immunisations. This study highlights the importance of for epitope optimisation and delivery system design when producing anti-hapten antibodies in vivo. The results of this study also contribute to the development of future clinical and veterinary immunocontraceptives.

Glycosylation of therapeutic peptides has been reported to improve delivery and targeting of various vaccines and drugs to specific cells/tissues. However, chemical synthesis of complex oligosaccharide derivatives via conventional methods can be challenging due to the need for several orthogonal hydroxyl group protections. Liposaccharyl galactosyltransferase C, a naturally occurring glycosyltransferase enzyme from Neisseria meningitidis, was found to have the ability to transfer a galactosyl moiety to glyco(lipo)peptides. An enzymatic glycosylation of Leu-enkephalin glyco(lipo)peptides was developed and optimized in this study in order to prepare pain regulating peptides with potentially improved central nervous system delivery.

Peptide-based vaccine delivery can be hampered by rapid peptidase activity and poor inherent immunogenicity. The self-adjuvanting lipid core peptide system (LCP) has been shown to confer improved stability and immunogenicity on peptide epitopes of group A Streptococcus, Chlamydia, hookworm, and malaria pathogens. However, various diseases, including cancer, still require targeted delivery of their vaccine candidates. For this reason, we have selected two model peptides (ovalbumin CD4(+) and/or CD8(+) T cell epitopes), and incorporated two or four copies of either epitope into our LCP vaccine. Optimised glycosylation of ovalbumin peptides yielded 46 % when microwave-assisted double coupling with 2 eq of carbohydrate derivative, activated by N,N-diisopropylethylamine and (O-benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, was performed. All ovalbumin peptides were successfully synthesised and purified in 11-55 % yields by Fmoc- or Boc-chemistry using solid-phase peptide synthesis. The mannosylated ovalbumin peptides were nontoxic to human erythrocytes in haemolytic assay (2 % haemolysis) and showed increased (up to 20-fold) stability in plasma.

Liposomes and enzymes: Liposome formulations improved solubility of a lipid-modified lactose enkephalin and, when used in enzymatic transformation, led to a twofold increase in glycosylation in comparison to substrate solubilised in 5 % dimethyl sulfoxide (DMSO; see figure).

Four group A streptococcal glycolipopeptide vaccine candidates with different lipidic adjuvanting moieties were prepared and characterized. The immunogenicity of the compounds was evaluated by macrophage and dendritic cell uptake studies and by in vivo quantification of systemic IgG antibody by ELISA. Three of the candidates showed significant induction of the IgG response.

The development of techniques for efficient peptide production renewed interest in peptides as therapeutics. Numerous modifications for improving stability, transport and affinity profiles now exist. Several new adjuvant and carrier systems have also been developed, enhancing the immunogenicity of peptides thus allowing their development as vaccines. This review describes the established and experimental approaches for manufacturing peptide drugs and highlights the techniques currently used for improving their drug like properties.

A series of anionic liposaccharide derivatives were synthesized in order to develop a system, which would have the capacity to act as an absorption enhancer and to improve oral bioavailability of drugs. The addition of a liposaccharide to a drug enhances drug stability against enzymatic degradation, while the lipophilicity can be controlled by variation of the lipid side chain. All liposaccharide derivatives were purified and fully characterized by nuclear magnetic resonance and high-resolution mass spectrometry. The thermodynamic profiles, critical aggregation concentrations and size of the synthesized liposaccharides were determined by isothermal titration microcalorimetry, transmission electron microscopy and dynamic light scattering. These liposaccharides formed nanoparticles with sizes below 100 nm.

The characterization of peptide-based drugs is essential to obtain information about their potential suitability. In this study, a therapeutic peptide epitope alone or in combination with a lipid or liposaccharide moiety were assessed to determine their particle sizes by diffusion ordered NMR spectroscopy, dynamic light scattering, and mathematical expressions. These methods were compared and their suitability for different types of peptides is discussed herein. When compared with the mathematical expressions, we found that the NMR method resulted in a particle size that was consistent with the radius of the peptide monomer. The dynamic light scattering method showed that when lipids were conjugated to the peptide epitope, the resulting particles had a larger sized distribution compared with the peptide alone. These experiments provided information which can be applied when formulating these peptides as drugs.

Lipidic α-amino acids (LAAs) have been described as non-natural amino acids with long saturated or unsaturated aliphatic chains. In the continuing prospect to discover anticancer agents from marine sources, we have obtained a mixture of two cytotoxic LAAs (1a and 1b) from the zoanthid Protopalythoa variabilis. The anti-proliferative potential of 14 synthetic LAAs and 1a/1b were evaluated on four tumor cell lines (HCT-8, SF-295, MDA-MB-435, and HL-60). Five of the synthetic LAAs showed high percentage of tumor cell inhibition, while 1a/1b completely inhibited tumor cell growth. Additionally, apoptotic effects of 1a/1b were studied on HL-60 cell line. 1a/1b-treated cells showed apoptosis morphology, loss of mitochondrial potential, and DNA fragmentation.

The incorporation of lipid moieties into synthetic peptide vaccines has been demonstrated to self-adjuvant otherwise poorly immunogenic peptides, whereas carbohydrates have emerged to be advantageous carriers for assembling these peptides. With the advent of an efficient native chemical ligation method, which is compatible with both peptides and carbohydrates, we have developed highly pure self-adjuvanting tetravalent group A streptococcal vaccine candidates assembled on carbohydrate templates. The utility of chemoselective ligation has overcome difficulties in the synthesis and purification of branched high molecular weight peptides. Circular dichroism measurements provided the evidence of α-helix formation of the assembled peptide epitopes, which may have impact on their immunogenicity.

Group A streptococcus is a Gram-positive bacteria that causes a range of infectious diseases. Targeting the bacteria, a new self-adjuvanting vaccine candidate, incorporating a carbohydrate carrier and an amino acid-based adjuvant, was synthesised utilising carbohydrate chemistry and solid-phase peptide synthesis procedures. Characterisation of the candidate was achieved using reverse-phase HPLC and electrospray ionisation mass spectrometry. The successful synthesis and characterisation of the vaccine candidate may contribute to the discovery of a therapeutically and clinically viable vaccine against group A streptococcus.

α-d-Galactosidases (α-d-galactoside galactohydrolase, E.C. 3.2.1.22) are exoglycosidases, which in vivo catalyze the hydrolysis of simple and complex oligo- and polysaccharides containing terminal α-d-galactosyl groups. A number of industrial applications of α-d-galactosidases are known, mainly in the sugar industry, pulp and paper industry or medicine. Although α-d-galactosidases occur in all living systems, microbial and possibly plant enzymes are preferred for practical applications. The regioselectivity of glycosidases is somewhat poor. Selective protection of oligosaccharides and development of new modified donors/acceptors is a promising method to overcome this problem. Genetic engineering has enabled the creation of glycosynthases from the hydrolytic glycosidases, which are highly complementary to glycosyltransferases. The present review summarizes recent advances in the application of α-d-galactosidases in synthetic and biotransformation applications.

Synthetic lipopeptide vaccines are being increasingly investigated mainly because of the advantages they offer over traditional vaccines, including safety of use in humans, high specificity in eliciting immune responses, greater purity and large scale/cost-effective production capacity. Moreover, a number of lipopeptide vaccines designed to possess self-adjuvanting properties have been developed and tested in vitro and in vivo. Producing high levels of serum-specific antibodies against incorporated peptide epitopes, they are showing their potential as effective vaccine candidates without the need for a co-administered adjuvant and/or carrier protein, often associated with undesirable effects in humans. This review presents recent insights on lipopeptide vaccine research and development, particularly on (1) the influence of the orientation of peptide epitopes and lipids on immune responses, (2) the use of carbohydrates for vaccine targeting, adjuvanting or as peptide epitope carriers, and (3) synthetic approaches to highly pure, multi-epitopic vaccine molecules using native chemical ligation techniques. Incorporation of different types of antigens within the same lipopeptide construct could provide a lipopeptide vaccine candidate suitable for safe and effective mucosal administration, which is a comfortable way of drug delivery.

We applied native chemical ligation (NCL) method to the synthesis of highly pure lipid-core peptide (LCP) vaccines to attach various peptide epitopes. lit the case of the synthesis of LCP vaccine with two different peptide epitopes, LCP moieties having two free Cys and two protected Cys derivatives (S-acetantidemethyl-Cys, (Cys(Acm)), N-methylsulfonylethyloxycarbonyl-Cys (Msc-Cys), or 1,3-thiazolidine-4-carboxylic) acid (Thz)) on oligolysine branches were prepared lit order to couple two different epitopes by stepwise NCL. It was found that the difficulty tit NCL of first two peptide antigen was associated with the steric hindrance. Using Thz instead of Cys(Acm) and Msc-Cys was important to reduce the steric hindrance and improve NCL yield. (C) 2008 Wiley Periodicals, Inc.

An extracellular alpha-d-galactosidase from Talaromyces flavus CCF 2686 with extremely broad and unusual acceptor specificity is produced exclusively in the presence of the specific inducer--6-deoxy-D-glucose (quinovose). The procedure for the preparation of this very expensive substance has been modified and optimized. Surprisingly, any of other common alpha-D-galactosidase inducers or substrates, e.g., D-galactose, melibiose and raffinose, did not stimulate its production. The crude alpha-D-galactosidase preparation was purified by anion-exchange chromatography and three isoenzymes with different substrate specificities were identified. The main isoenzyme (alphaGal1) was further purified by cation-exchange chromatography and fully characterized. When compared with other alpha-galactosidases and also with other isoenzymes produced by T. flavus, it showed a markedly different regioselectivity and also negligible hydrolytic activity towards melibiose. Moreover, it was active on polymeric substrates (locust bean gum, guar gum) and significantly inhibited by alpha-D-galactopyranosyl azide, D-galactose, D-xylose, melibiose, methyl alpha- and beta-D-galactopyranoside and lactose.

Aim: Systematically evaluate lipid core peptide vaccine delivery platforms to identify core features promoting strong CD8+ T-cell responses. Materials & methods: Three different self-adjuvanting lipid core peptide nanovaccines each comprising four copies of the dominant ovalbumin CD8+ T-cell epitope and varying in the utilization of a polylysine or glucose core with 2-amino-hexadecanoic acid (C16) or 2-amino-dodecanoic acid (C12) lipids were synthesized. Vaccines were tested for ability to induce CD8+ T-cell responses and inhibit tumor growth in vivo. Results: The construct utilizing C12 lipids and polylysine core induced very robust effector T cells shown to have in vivo effector capability as demonstrated by in vivo cytotoxicity and ability to inhibit tumor growth as well as modulation of dendritic cell activation. Conclusion: The C12 polylysine platform was an effective configuration for induction of potent CD8+ T-cell responses.

A self-adjuvanting vaccine candidate comprising four copies of luteinizing hormone releasing hormone (LHRH), a galactose carrier/core and lipoamino acid based adjuvant was synthesized in 21% yield by a solid phase peptide synthesis, carbohydrate and Boc-chemistry methods.

The transglycosylation potential of the extracellular α-d-galactosidase from the filamentous fungus Talaromyces flavus CCF 2686, chosen as the best enzyme from the screening, was investigated using a series of sterically hindered alcohols (primary, secondary and tertiary) as galactosyl acceptors. Nine alkyl α-d-galactopyranosides derived from the following alcohols – tert-butyl alcohol, 2-methyl-2-butyl alcohol, 2-methyl-1-propyl alcohol, 2,2,2-trifluoroethyl alcohol, 2-propyn-1-ol, n-pentyl alcohol, 3,5-dihydroxybenzyl alcohol, 1-phenylethyl alcohol and 1,4-dithio-dl-threitol – were prepared on a semi-preparative scale. This demonstrates a broad synthetic potential of the T. flavus α-d-galactosidase that has not been observed with another enzyme tested. Moreover, this enzyme exhibits good transglycosylation yields (6–34%). The enzymatic synthesis of tert-butyl α-d-galactopyranoside by transglycosylation was studied in detail.

A careful choice of the reaction conditions (solvent, enzyme, acylating agent) allowed an efficient regioselective acylation of N-acetylhexosamines. 6-O-Acyl derivatives of 2-acetamido-2-deoxy- d -glucopyranose (GlcNAc), 2-acetamido-2-deoxy- d -galactopyranose (GalNAc) and 2-acetamido-2-deoxy- d -mannopyranose (ManNAc) have been isolated from regioselective esterifications catalysed by the protease subtilisin in CH3CN–DMSO 8:2 in good yields.

Group A streptococcus (GAS; Streptococcus pyogenes), known as the ‘flesh-eating bacterium’, is a human bacterial pathogen that normally causes benign infections (e.g. sore throat and pyoderma), but is also responsible for severe invasive infections (e.g. ‘flesh-eating’ disease and toxic shock syndrome), heart disease, and kidney failure. A safe commercial GAS vaccine is yet to be developed. Individual GAS antigens demonstrate potential universal expression across all GAS serotypes (>200 known), with dramatically reduced concern for autoimmune complications, and compelling efficacy in preclinical testing in mice. In this study, we developed a stepwise conjugation strategy, copper-catalysed alkyne–azide cycloaddition reaction (CuAAC), followed by mercapto–maleimide conjugation, to synthesise a multiantigenic, self-adjuvanting, peptide-based vaccine candidate against GAS. This multiantigenic vaccine includes two GAS antigens, J8 and NS1, a T-helper epitope, PADRE, and a self-adjuvanting moiety, dipalmitoyl serine.

Glycosylation of biopharmaceuticals can mediate cell specific delivery by targeting carbohydrate receptors. Additionally, glycosylation can improve the physico-chemical (drug-like) properties of peptide based drug candidates. The main purpose of this study was to examine if glycosylation of the peptide enkephalin could facilitate its binding to the carbohydrate receptor, asialoglycoprotein. Firstly, we described the one-pot enzymatic galactosylation of lactose modified enkephalin in the presence of uridine-5'-diphosphogalactose 4-epimerase and lipopolysaccharyl alpha-1,4-galactosyltransferase. Stability experiments using human plasma and Caco-2 cell homogenates showed that glycosylation considerably improved the stability of enkephalin (at least 60% remained stable after a 2 hr incubation at 37 degrees C). In vitro permeability experiments using Caco-2 cells revealed that the permeability of mono-and trisaccharide conjugated enkephalins was 14 and 28 times higher, respectively, than that of enkephalin alone (Papp 3.1x10(-8) cm/s). By the methods of surface plasmon resonance and molecular modeling, we demonstrated that the enzymatic glycosylation of enkephalin enabled binding the asialoglycoprotein receptor. The addition of a trisaccharide moiety to enkephalin improved the binding of enkephalin to the asialoglycoprotein receptor two fold (K-D=91 mu M). The docking scores from molecular modeling showed that the binding modes and affinities of the glycosylated enkephalin derivatives to the asialoglycoprotein receptor complemented the results from the surface plasmon resonance experiments.

Research related to peptide, vaccine, and gene delivery has grown exponentially over the last decade. In this review, we discuss the development of delivery systems for peptides, gene and vaccine products. Special focus is given to different lipidation and glycosylation strategies to improve the metabolic stability and membrane permeability of therapeutics, and their targeting to specific sites. The synthetic methods for preparation of the systems are also described. (C) 2009 Wiley Periodicals, Inc. Med Res Rev, 31, No. 4, 520-547, 2011

Carbohydrates, as carriers, providing numerous attachment points for the conjugation of peptide antigens and their optimal orientation for the recognition by cells of the immune system, reducing degradation of the attached peptide antigens and many other advantages make carbohydrate-based vaccine highly promising approach. Multiple copies of a single group A streptococcal (GAS) M protein derived specific peptide antigens (J8 or J14) were coupled onto carbohydrate cores (D-glucose and D-galactose) linked to lipophilic amino acids to produce a self-adjuvanting liposaccharide vaccine against GAS strains. In vivo experiments showed high serum IgG antibody titers against each of the incorporated peptide epitopes, J8 or J14.

The development of 16 self-adjuvanting group A streptococcal vaccine candidates, composed of (i) a universal helper T-cell epitope (P25), (ii) a target GAS B-cell epitope (J14), and (iii) a lipid moiety, is described. Systemic J14-specific IgG antibodies were detected following subcutaneous immunization of BALB/c (H-2 (d)) mice with each construct without the need for an additional adjuvant. The effect of changing the order of P25, J14, and lipid moiety attachment or incorporation of P25 and J14 into a lipid-core peptide system on antibody titers was assessed. The point of lipid moiety attachment had the greatest influence on systemic J14-specific IgG antibody titers. Overall, the best vaccines featured a C-terminal lipid moiety, conjugated through a lysine residue to P25 at the N-terminus, and J14 on the lysine side chain.

Development of a synthetic vaccine against group A streptococcal infection is increasingly paramount due to the induction of autoimmunity by the main virulent factor – M protein. Peptide vaccines, however, are generally poorly immunogenic, necessitating administration with carriers and adjuvants. One of the promising approaches to deliver antigenic peptides is to assemble peptides on a suitable template which directs the attached peptides to form a well defined tertiary structure. For self-adjuvanting human vaccines, the conjugation of immunostimulatory lipids has been demonstrated as a potentially safe method. This study describes the design and optimized synthesis of two lipopeptide conjugated carbohydrate templates and the assembling of peptide antigens. These lipopeptide–carbohydrate assembled multivalent vaccine candidates were obtained in high yield and purity when native chemical ligation was applied. Circular dichroism studies indicated that the template-assembled peptides form four α-helix bundles. The developed technique extends the use of carbohydrate templates and lipopeptide conjugates for producing self-adjuvanting and topology-controlled vaccine candidates.

The scabies mite, Sarcoptes scabiei var. hominis, infests human skin, causing allergic reactions and facilitating bacterial infection by Streptococcus sp., with serious consequences such as rheumatic fever and rheumatic heart disease. To identify a possible drug target or vaccine candidate protein, we searched for homologues of the group 3 allergen of house dust mites, which we subsequently identified in a cDNA library. The native protein, designated Sar s 3, was shown to be present in the mite gut and excreted in fecal pellets into mite burrows within the upper epidermis. The substrate specificity of proteolytically active recombinant rSar s 3 was elucidated by screening a bacteriophage library. A preference for substrates containing a RS(G/A) sequence at the P1-P2′ positions was revealed. A series of peptides synthesized as internally quenched fluorescent substrates validated the phage display data and high performance liquid chromatography/mass spectrometry analysis of the preferred cleaved substrate and confirmed the predicted cleavage site. Searches of the human proteome using sequence data from the phage display allowed the in silico prediction of putative physiological substrates. Among these were numerous epidermal proteins, with filaggrin being a particularly likely candidate substrate. We showed that recombinant rSar s 3 cleaves human filaggrin in vitro and obtained immunohistological evidence that the filaggrin protein is ingested by the mite. This is the first report elucidating the substrate specificity of Sar s 3 and its potential role in scabies mite biology.

Background The Gram-positive bacterium Staphylococcus saprophyticus is the second most frequent causative agent of community-acquired urinary tract infections (UTI), accounting for up to 20% of cases. A common feature of staphylococci is colonisation of the human skin. This involves survival against innate immune defenses including antibacterial unsaturated free fatty acids such as linoleic acid which act by disrupting bacterial cell membranes. Indeed, S. saprophyticus UTI is usually preceded by perineal skin colonisation. Results In this study we identified a previously undescribed 73.5 kDa cell wall-anchored protein of S. saprophyticus, encoded on plasmid pSSAP2 of strain MS1146, which we termed S . s aprophyticus surface protein F (SssF). The sssF gene is highly prevalent in S. saprophyticus clinical isolates and we demonstrate that the SssF protein is expressed at the cell surface. However, unlike all other characterised cell wall-anchored proteins of S. saprophyticus, we were unable to demonstrate a role for SssF in adhesion. SssF shares moderate sequence identity to a surface protein of Staphylococcus aureus (SasF) recently shown to be an important mediator of linoleic acid resistance. Using a heterologous complementation approach in a S. aureus sasF null genetic background, we demonstrate that SssF is associated with resistance to linoleic acid. We also show that S. saprophyticus strains lacking sssF are more sensitive to linoleic acid than those that possess it. Every staphylococcal genome sequenced to date encodes SssF and SasF homologues. Proteins in this family share similar predicted secondary structures consisting almost exclusively of α-helices in a probable coiled-coil formation. Conclusions Our data indicate that SssF is a newly described and highly prevalent surface-localised protein of S. saprophyticus that contributes to resistance against the antibacterial effects of linoleic acid. SssF is a member of a protein family widely disseminated throughout the staphylococci.