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Background and Objective: Many recent studies have focused on the development of methods to overcome the barriers faced by oral drug delivery. This study describes the synthesis of three glycolipid-based drug delivery systems designed to be associated with charged drugs to improve their oral bioavailability.Methods: D-Glucose was used as a scaffold to construct charged glycolipids with anionic and/or cationic properties. Three glycolipids with positive and negative functionalities were designed, synthesized by various carbohydrate, lipid and Bocchemistry methods. The products were purified by flash column chromatography and characterized by ESI-MS and NMR.Results: The first glycolipid was synthesized by complete lipidation of D-glucose using lipoamino acids with positively charged amino groups. The negatively charged glycolipid was obtained by coupling succinate bearing a free carboxylic group to lauroylated glucose. Combining both strategies, the target compound, which can bear either positive or negative charges, was prepared through the conjugation of succinate to the carbohydrate core followed by the addition of lipoamino acids. Separation of the compounds from impurities (not fully lapidated derivatives) by flash column chromatography proved to be challenging.Conclusion: Ensuring the purity of all semi-products used in each reaction was paramount to prevent complicated purification of the final compounds. Once the three glycolipids were carefully purified, protecting groups were cleaved to give cationic and anionic properties. These glycolipids can undergo complexation with charged drugs to improve their oral bioavailability. This system has the potential to serve as a universal template for oral drug delivery.

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.

Despite advances in the development of new vaccines, there are still some diseases with no vaccine solutions. Therefore, further efforts are required to more comprehensively discern the different antigenic components of these microorganisms on a molecular level. This review summarizes advancement in the development of new carbohydrate-based vaccines. Following traditional vaccine counterparts, the carbohydrate-based vaccines introduced a new approach in fighting infectious diseases. Carbohydrates have played various roles in the development of carbohydrate-based vaccines, which are described in this review, including carbohydrates acting as antigens, carriers or targeting moieties. Carbohydrate-based vaccines against infectious diseases, such as group A streptococcus, meningococcal meningitis and human immunodeficiency virus, are also discussed. A number of carbohydrate- based vaccines, such as Pneumovax 23, Menveo and Pentacel, have been successfully marketed in the past few years and there is a promising standpoint for many more to come in the near future.

Peptide-based vaccines are considered to be the next generation of modern immunizations, as they are safe, easy to produce and well-defined. However, due to their weak immunogenic effect, it is important to first develop an appropriate adjuvant for peptide-based vaccines.The aim of this work was to synthesize a series of four adjuvanting moieties as alkyne derivatives, incorporating dipalmitoyl serine (DPS), 1,3-diglyceride (DG), two hexadecane lipoamino acids (diLAA), and 2,3-dipalmitoyl-S-glycerylcysteine (Pam2Cys). Next aim was to synthesize and attach the azide derivative of biotinylated J14 peptide (model B-cell epitope) to the alkynes through copper- catalyzed alkyne-azide 1,3-dipolar cycloaddition (CuAAC) reaction. Final aim was to test the ability of the final biotin labeled conjugates to directly interact with in vitro expressed TLR2 and 8 using AlphaScreen proximity assay.All of the peptides were synthesized by manual stepwise solid phase peptide synthesis (SPPS) on rink amide MBHA resin using HATU/DIPEA Fmoc-chemistry. The target compounds were synthesized in a solution phase using CuAAC reaction.Pam2Cys analogue bound to TLR2 as expected. Analogues of DPS and C16-LAA showed also affinity to TLR2, while it did not bind to the control protein (TLR8), demonstrating ability of the DPS and C16-LAA to be recognized by TLR2.Four alkyne derivatives of lipids were successfully synthesized and coupled to a biotinylated J14 peptide to give a series of self-adjuvanting ligands. These ligands showed different affinity to TLR2 upon testing by AlphaScreen assay. The DPS derivative showed the most promising affinity in comparison to the standard TLR2 agonist, Pam2Cys.

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.

A novel convergent synthetic strategy for the construction of multicomponent self-adjuvanting lipopeptide vaccines was developed. A tetraalkyne-functionalized glucose derivative and lipidated Fmoc-lysine were prepared by novel efficient and convenient syntheses. The carbohydrate building block was coupled to the self-adjuvanting lipidic moiety (three lipidated Fmoc-lysines) on solid support. Four copies of a group A streptococcal B cell epitope (J8) were then conjugated to the glyco-lipopeptide using a copper-catalyzed cycloaddition reaction. The approach was elaborated by the preparation of a second vaccine candidate which incorporated an additional promiscuous T-helper epitope.

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).

Since the invention of solid phase synthetic methods by Merrifield in 1963, the number of research groups focusing on peptide synthesis has grown exponentially. However, the original step-by-step synthesis had limitations: the purity of the final product decreased with the number of coupling steps. After the development of Boc and Fmoc protecting groups, novel amino acid protecting groups and new techniques were introduced to provide high quality and quantity peptide products. Fragment condensation was a popular method for peptide production in the 1980s, but unfortunately the rate of racemization and reaction difficulties proved less than ideal. Kent and co-workers revolutionized peptide coupling by introducing the chemoselective reaction of unprotected peptides, called native chemical ligation. Subsequently, research has focused on the development of novel ligating techniques including the famous click reaction, ligation of peptide hydrazides, and the recently reported α-ketoacid-hydroxylamine ligations with 5-oxaproline. Several companies have been formed all over the world to prepare high quality Good Manufacturing Practice peptide products on a multi-kilogram scale. This review describes the advances in peptide chemistry including the variety of synthetic peptide methods currently available and the broad application of peptides in medicinal chemistry.

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.

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.

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.

any drugs have limited penetration through the gastrointestinal tract and therefore cannot be administered orally. The aim of this study was to develop charged liposaccharide absorption enhancers to improve oral bioavailability of model drug tobramycin. In vitro experiments, haemolysis, and cytotoxicity tests confirmed that the synthesized liposaccharides were non-toxic at the concentrations used for oral administration. The partitioning coefficient of tobramycin was improved to an acceptable range for oral delivery (log P = 1-3), when it was mixed with anionic liposaccharides at molar ratio of 1:7.

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 nasal route as a site of vaccine delivery for both local and systemic effect is currently of considerable interest. The administration of vaccines to mucosal surfaces such as the nasopharynx associated lymphoid tissues confers many advantages since the nasal mucosa is a primary site through which most inhaled antigens are encountered. However, the success of intranasally delivered mucosal vaccines is limited by lack of effective vaccine formulations or delivery systems suitable for use in humans. This review provides a brief overview of the mucosal immune system at the nasal surface, enhancement techniques for induction of mucosal immune response after intranasal administration of particulate systems and an explanation of the inherent properties of polyacrylate polymer-based particulate systems that may facilitate mucosal immune responses.

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.

Group A streptococcus (GAS) is associated with many human diseases, ranging in severity from benign to life-threatening. A promising strategy for developing vaccines against GAS involves the use of carbohydrates as carriers for peptide antigens. This study describes the optimized synthesis of d-glucose and d-galactose derived carriers, bearing an adipate linker and four tert-butoxycarbonyl protected aminopropyl groups. Prophylactic GAS vaccine candidates were synthesized by conjugating multiple copies of a single GAS M protein derived peptide antigen (either J8 or J14) onto the carbohydrate carriers. These antigens contain peptide sequences, which are highly conserved and offer the potential to prevent infections caused by up to 70% of GAS strains. Lipophilic amino acids were also conjugated to the d-glucose anomeric carbon to produce a self-adjuvanting liposaccharide vaccine. High serum IgG antibody titers against each of the incorporated peptide epitopes were detected following subcutaneous immunization of B10.BR (H-2 (k)) mice with the liposaccharide vaccine candidates.

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.

Glycosyl azides are shown to be efficient donors for beta-galactosidases, beta-glucosidases and alpha-mannosidases. Only alpha-galactosidases do not cleave the respective glycosyl azide I and, moreover, they exhibit competitive inhibition (especially a.-galactosidase from Talaromyces flavus). High water solubility and ready synthesis of glycosyl azides enable transglycosylation reactions even with difficult acceptors like N-acetyl-D-mannosamine in good yields. The versatility of glycosyl azides was demonstrated in the synthesis of five disaccharides - two of them are described for the first time. All the reactions were highly regioselective, yielding beta(1 -> 6) isomers. beta-Galactosidase from E. coli proved to have the best synthetic capabilities. The present study shows that glycosyl azides are a valuable alternative to common p-nitrophenyl glycoside donors and in many synthetic reactions.

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 hydrophilic ion paring strategy (HIP) is a method explored to improve the cell/tissue uptake of poorly adsorbed drugs and to optimize their physico-chemical characteristics. In this context, we here describe the synthesis of some ion pairs of two model cationic antibiotics, erythromycin (ERY) and kanamycin A (KAN), with liposaccharides having different levels of lipophilicity and charge. The formation of drug-liposaccharide complexes was confirmed by Fourier transform infrared spectroscopy (FT-IR), differential scanning calorimetry (DSC) and powder X-ray diffraction (PXRD) analysis. The effect of the amphiphilic liposaccharide moieties on the antimicrobial activity of ERY and KAN was assessed by measuring the minimal inhibitory concentration (MIC) of the compounds against a panel of bacterial strains that were susceptible or resistant to the parent antibiotics. The ion pairing did not depress the in vitro antibiotic activity, although no lowering of MIC values was registered. The experimental findings would motivate the future investigation of this ion pairing strategy in drug design, for instance allowing improvement of the encapsulation efficiency of hydrophilic antibiotics in lipid-based nanocarriers, or changing their in vivo biodistribution and pharmacokinetic profile.

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.

Lectin–glycan interactions play a role in biological processes, host–pathogen interactions and in disease. A more detailed understanding of these interactions is not only useful for the elucidation of their biological function but can also be applied in immunology, drug development and delivery and diagnostics. We review some commonly used biophysical techniques for studying lectin–glycan interactions; namely: frontal affinity chromatography, glycan/lectin microarray, surface plasmon resonance, electrochemical impedance spectroscopy, isothermal titration calorimetry, fluorescent assays, enzyme linked lectin sorbent assay and saturation transfer difference nuclear magnetic resonance spectroscopy. Each method is evaluated on efficiency, cost and throughput. We also consider the advantages and limitations of each technique and provide examples of their application in biology, drug discovery and delivery, immunology, glycoprofiling and biosensing.

Immunocastration using gonadotropin-releasing hormone (GnRH)-based vaccines has been investigated in rams to reduce aggressive and sexual behaviour and to control meat quality. Despite considerable efforts, a practical GnRH vaccine has yet to be developed for rams. In the present study, a A GnRH-lipopeptide vaccine (GnRH-LP) including two copies of GnRH, 2-amino-d,l-hexadecanoic acid (C16), and a unique T helper epitope, was examined in rams. Rams received a primary and secondary vaccination of GnRH-LP without additional adjuvant (Group 1) or with the adjuvant AdjuVac™ (Group 2). In both Group 1 and 2 anti-GnRH antibody titres increased after secondary vaccination, however, the antibody titres were higher (p

In this study we aimed to address the poor drug-like properties of Gonadotropin-Releasing Hormone (GnRH) peptide through modification with lipids and carbohydrates.GnRH peptide was conjugated to 2-amino-D,L-octanoic acid (C8) and 2-amino-D,L-dodecanoic acid (C12) in monomer and dimer, along with (6-9) or without (2-5 and 11) a glucose moiety. Peptides were tested for their biological activity using different tumour cell lines. The toxicity of the constructs was evaluated in peripheral blood mononuclear cells (PBMC).All (glyco)lipopeptides showed improved metabolic stability in Caco-2 cell homogenates. Those with single lipid moiety (2, 4 and 8) exhibited prodrug-like properties. Permeability across Caco-2 cell monolayers was enhanced in the dimer C8-modified (glyco)lipopeptide (3) and the lipopeptide with C12 inserted mid-sequence (11). Most of the constructs showed moderate-to-high antiproliferative activity against GnRH-receptor positive DU145 and OVCAR-3 cells (up to 60%). Compound 11 was the most effective with IC50 = 26.4 ± 1.07 μg.ml(-1), which was comparable to triptorelin (25.1 ± 1.14 μg.mL(-1)). The sensitivity of OVCAR-3 cells to the effect of all analogues except for 11 decreased significantly in estrogen-reconstituted media. Only compounds 2, 4, 5 and 8 showed a steroid-dependent effect in DU145 cells. No compounds exhibited significant toxicity on PBMCs.These results indicated lipidation and glycosylation improves the druggability of GnRH and could lead to an increased direct antitumour activity in some hormone dependent and independent reproductive cancers.

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.

Since the invention of solid phase synthetic methods by Merrifield in 1963, the number of research groups focusing on peptide synthesis has grown exponentially. However, the original step-by-step synthesis had limitations: the purity of the final product decreased with the number of coupling steps. After the development of Boc and Fmoc protecting groups, novel amino acid protecting groups and new techniques were introduced to provide high quality and quantity peptide products. Fragment condensation was a popular method for peptide production in the 1980s, but unfortunately the rate of racemization and reaction difficulties proved less than ideal. Kent and co-workers revolutionized peptide coupling by introducing the chemoselective reaction of unprotected peptides, called native chemical ligation. Subsequently, research has focused on the development of novel ligating techniques including the famous click reaction, ligation of peptide hydrazides, and the recently reported α-ketoacid-hydroxylamine ligations with 5-oxaproline. Several companies have been formed all over the world to prepare high quality Good Manufacturing Practice peptide products on a multi-kilogram scale. This review describes the advances in peptide chemistry including the variety of synthetic peptide methods currently available and the broad application of peptides in medicinal chemistry.

The aim of this study was to synthesize charged amphoteric molecules, which after complexation with poorly bioavailable drugs would have the potential to improve their oral uptake. Novel anionic liposaccharide derivatives containing d-glucose and lipoamino acids were synthesized by solution phase peptide synthesis. High sensitivity isothermal titration microcalorimetry was used to determine the critical aggregation concentration and the thermodynamic profiles. Hemolytic and cytotoxic activities of the liposaccharides were studied and they revealed that the liposaccharides were non-toxic at the concentration used for oral administration. Mixing a model drug, tobramycin, with the liposaccharide containing two lipids formed aggregates around 200 nm, which increased tobramycin partitioning between n-octanol/water. The results suggested that the studied liposaccharide with two lipids was safe to apply biologically and may have an absorption enhancing activity on hydrophilic, orally poorly available drugs.

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

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

Although most commercial vaccines are delivered by injection, there is an increasing interest in needle-free vaccine delivery for reasons including the ability to elicit immune responses at mucosal surfaces, ease of administration, and the ability to administer vaccines without the need for trained medical professionals. This review summarizes strategies and technologies that are being used to improve oral vaccine absorption. Peptides and proteins, which comprise important vaccine components, exhibit unfavorable physicochemical properties including degradation in the gastrointestinal tract, and poor transport across the intestinal wall, which hinder oral vaccine development. Approaches to overcome these obstacles aim to provide new vaccines and delivery systems that are capable of eliciting protective immune responses, and are making an impact on current vaccine development.

Development of mucosal vaccine delivery system is an important area for improving public health. Oral vaccines have large implications for rural and remote populations since the access to trained medical staff to administer vaccines by injection is limited. New mucosal vaccine strategies are focused on development of non-replicating subunit vaccines, DNA, plant, and other types of recombinant vaccines. The conjugation of lipids to peptide antigens is one approach which enables the production of highly customized all-in-one self-adjuvanting vaccines. Lipid-modified peptide vaccines have been successfully investigated in humans and demonstrated to be potent and more importantly very safe.