Search

In a previous work, we defined a novel HIV-1 fusion inhibitor peptide (E1P47) with a broad spectrum of activity against viruses from different clades, subtypes, and tropisms. With the aim to enhance its efficacy, in the present work we address the design and synthesis of several peptide amphiphiles (PAs) based on the E1P47 peptide sequence to target the lipid rafts of the cell membrane where the cell–cell fusion process takes place. We report the synthesis of novel PAs having a hydrophobic moiety covalently attached to the peptide sequence through a hydrophilic spacer of polyethylene glycol. Characterization of self-assembly in condensed phase and aqueous solution as well as their interaction with model membranes was analyzed by several biophysical methods. Our results demonstrated that the length of the spacer of polyethylene glycol, the position of the peptide conjugation as well as the type of the hydrophobic residue determine the antiviral activity of the construct. Peptide amphiphiles with one alkyl tail either in C-terminus (C-PAmonoalkyl) or in N-terminus (N-PAmonoalkyl) showed the highest anti-HIV-1 activities in the cellular model of TZM-bl cells or in a preclinical model of the human mucosal tissue explants., Financial support from the Spanish Ministry of Economy, Industry and Competitiveness (MINECO, Spain) and the European Regional Development Fund (Grant RTI2018-094120-B-I00 to I.H.) is gratefully acknowledged. R. P. acknowledges financial support from Ministry of Economy, Industry and Competitiveness, Spain (Grant CTQ2017-88948-P) and the European Regional Development Fund (FEDER). We acknowledge Ignacio Pérez-Pomeda from the Cell Culture Core Facilities of IQAC for his participation in cellular assays and Dr. Yolanda Pérez from the Nuclear Magnetic Resonance Facility of IQAC for performing NMR analyses. Imma Carrera is acknowledged for helping in the surface tension measurements. Jaume Caelles, from the SAXS-WAXS service at IQAC, is acknowledged for technical assistance in the measurements of SAXS. SAXS experiments were performed at NCD beamline at ALBA Synchrotron with the collaboration of ALBA staff (special thanks to Juan Carlos Martínez).

© 2020 by the authors., The effective use of fusion inhibitor peptides against cervical and colorectal infections requires the development of sustained release formulations. In this work we comparatively study two different formulations based on polymeric nanoparticles and lipid vesicles to propose a suitable delivery nanosystem for releasing an HIV-1 fusion inhibitor peptide in vaginal mucosa. Polymeric nanoparticles of poly-d,l-lactic-co-glycolic acid (PLGA) and lipid large unilamellar vesicles loaded with the inhibitor peptide were prepared. Both formulations showed average sizes and polydispersity index values corresponding to monodisperse systems appropriate for vaginal permeation. High entrapment efficiency of the inhibitor peptide was achieved in lipid vesicles, which was probably due to the peptide’s hydrophobic nature. In addition, both nanocarriers remained stable after two weeks stored at 4 °C. While PLGA nanoparticles (NPs) did not show any delay in peptide release, lipid vesicles demonstrated favorably prolonged release of the peptide. Lipid vesicles were shown to improve the retention of the peptide on ex vivo vaginal tissue in a concentration sufficient to exert its pharmacological effect. Thus, the small size of lipid vesicles, their lipid-based composition as well as their ability to enhance peptide penetration on vaginal tissue led us to consider this formulation as a better nanosystem than polymeric nanoparticles for the sustained delivery of the HIV-1 fusion inhibitor peptide in vaginal tissues., This work was supported by grant RTI2018-094120-B-I00 from the Spanish Ministry of Economy, Industry and Competitiveness (MINECO) and the European Regional Development Fund.

Despite the great effort to decrease the HIV infectivity rate, current antiretroviral therapy has several weaknesses; poor bioavailability, development of drug resistance and poor ability to access tissues. However, molecules such as peptides have emerged as a new expectative to HIV eradication. The vaginal mucosa is the main spreading point of HIV. There are natural barriers such as the vaginal fluid which protects the vaginal epithelium from any foreign agents reaching it. This work has developed and characterized Nanoparticles (NPs) coated with glycol chitosan (GC), loaded with an HIV-1 inhibitor peptide (E2). In vitro release and ex vivo studies were carried out using the vaginal mucosa of swine and the peptide was determined by HPLC MS/MS validated method. Moreover, the peptide was labeled with 5(6)-carboxyfluoresceine and entrapped into the NPs to carried out in vivo studies and to evaluate the NPs penetration and toxicity in the vaginal mucosa of the swine. The mean size of the NPs, ξ and the loading percentage were fundamental features for to reach the vaginal tissue and to release the peptide within intercellular space. The obtained results suggesting that the fusion inhibitor peptides loaded into the NPs coated with GC might be a new way to fight the HIV-1, due to the formulation might reach the human epithelial mucosa and release peptide without any side effects.

Background: A slower progression of AIDS and increased survival in GBV-C positive individuals, compared with GBV-C negative individuals has been demonstrated; while the loss of GBV-C viremia was closely associated with a rise in mortality and increased progression of AIDS. Following on from the previous reported studies that support the thesis that GBV-C E2 interferes with HIV-1 entry, in this work we try to determine the role of the GBV-C E1 protein in HIV-1 inhibition. Methods: The present work involves the construction of several overlapping peptide libraries scanning the GBV-C E1 protein and the evaluation of their anti-HIV activity. Results: Specifically, an 18-mer synthetic peptide from the GBV-C E1 protein, E1(139-156), showed similar antiviral activity against HIVs from viruses from clades A, B, C, D and AE. Competitive ELISA using specific gp41-targeting mAbs, fluorescence resonance energy transfer as well as haemolysis assays demonstrated that this E1 peptide sequence interacts with the highly conserved N-terminal region of the HIV-1 gp41 (the fusion peptide) which is essential for viral entry. Conclusions: We have defined a novel peptide lead compound and described the inhibitory role of a highly conserved fragment of the E1 protein. General Significance: The results together allow us to consider the non-pathogenic E1 GBV-C protein as an attractive source of peptides for the development of novel anti-HIV therapies.

Mixed monolayers of E2(279-298), a synthetic peptide belonging to the structural protein E2 of the GB virus C (GBV-C), formerly know as hepatitis G virus (HGV), and the phospholipids dipalmitoylphosphatidyl choline (DPPC) and dimiristoylphosphatidyl choline (DMPC),which differ in acyl chains length, were obtained at the A/W interface (monolayers of extension) in order to provide new insights on E2/phospholipids interaction. Analysis of the surface pressure-area isotherms, Brewster angle microscopy images, relative thickness, and mean areas per molecule has allowed us to establish the conditions under which the mixed components of the monolayer are miscible or immiscible and know how the level of the E2/phospholipid interaction varies with the composition of the mixed films, the surface pressure, and the hydrocarbon chains length of the phospholipids. The steric hindrance caused by the penetration of the polymer strands into the more or less ordered hydrocarbon chains of the phospholipids was suggested to explain the differences in the peptide interaction with the phospholipids studied. Therefore, the novelty of results obtained with the Langmuir film balance technique, supplemented with BAM images allow us to achieve a deeper understanding of the interaction.

Synthetic peptides derived from GB virus C (GBV-C) have previously been studied in our group for the development of new systems capable of diagnosing diseases caused by this humanotropic virus. We also recently described specific peptide domains of the E2 envelop protein of GBV-C that have the capacity to interfere with the HIV-1 fusion peptide, produce a notable decrease in cellular membrane fusion, and perturb HIV-1 infectivity in a dose-dependent manner. The present work discloses the design and synthesis of both linear and cyclic branched peptides based on a previously reported N-terminal sequence of the GBV-C E2 protein. Immunoassays and cell–cell fusion assays were performed to evaluate their diagnostic value to detect anti-GBV-C antibodies in HIV-1 patients, as well as their putative anti-HIV-1 activity as entry inhibitors. Our results showed that chemical modifications of the selected E2(7–26) linear peptide to afford cyclic architecture do not result in an enhanced inhibition of gp41 HIV-1-mediated cell–cell fusion nor improved sensitivity in the detection of GBV-C antibodies in HIV-1 co-infected patients. Thus, the ELISA data reinforce the potential utility of linear versions of the E2(7–26) region for the development of new peptide-based immunosensor devices for the detection of anti-GBV-C antibodies in HIV-1 co-infected patients. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.

(D-Met2,Pro5)-enkephalin-N-(β-D-galactopyranosyl)amide, a glycopeptide containing a galactose residue at the C-terminal position of the μ-selective enkaphalin analogue (D-Met2,Pro5)-enkaphalinamide, has been synthesized by the liquid phase procedure according to different strategies. Such analogue exhibited a great antinociceptive potency after intracerebroventricular injection in the “in vivo” tail immersion test when compared to the parent compound and the corresponding glucose derivative. Therefore, we suggest that the opioid receptors mediating analgesia, interact more efficiently with galactosyl rather than glucosyl residues. To confirm this hypothesis, we have synthesized a series of galactosyl conjugated (D-Met2,Pro5)-enkaphalins, containing up to three galactose residues linked to the oligopeptide backbone throughout a hydrophobic carbon c-6 spacer.

The GB virus C/hepatitis G virus (GBV C/HGV) is a Flaviviridae member that despite its non pathogenicity, has become of great interest given that it could inhibit the replication of the human immunodeficiency virus (HIV). Therefore, a better knowledge of the virus peptides involved in the cellular membrane fusion mechanism has become our aim. The selected peptide, named E2(347-363), corresponds to the GBV-C/HGV E2 protein and has been synthesized in order to study its interaction with in vitro membrane models. Two phospholipids, varying the charge and the unsaturations of the hydrocarbon chain have been chosen: 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt) (DOPG). For our porpoise, we have used the Langmuir monolayer technique and Brewster angle microscopy (BAM) to gain deeper insight into the peptide/lipid interactions. The results obtained allow us to argue in favour of considering E2(347-363) a success candidate for developing further experiments in order to determine its potential role in the GBV C/HGV virus/cell membrane fusion process.

The physicochemical characterization of the peptide sequence E1(145-162) corresponding to the structural protein E1 of the hepatitis G virus was done by studying its interaction with model membranes. Small unilamellar vesicles (SUVs) of dimyristoylphosphatidylglycerol or dimyristoylphosphatidylcholine were chosen as mimetic membranes. Peptide incorporation and location in the phospholipid bilayer was investigated by fluorescence anisotropy with SUVs labeled with diphenylhexatriene (DPH) or trimethylammonium-DPH. The addition of the peptide E1(145-162) showed significant changes in the anisotropy values of the probe located at the air/water interface. These results indicate that the peptide E1(145-162) preferably interacts with the lipid surface without penetrating inside the bilayer. A series of fluorescence experiments based on tryptophan peptide fluorescence were modeled by means of multivariate curve resolution-alternating least squares (MCR-ALS) algorithm to further study the peptide interaction with bilayers at different temperatures. The preliminary results obtained with MCR-ALS showed how the peptide concentration decay is directly linked to the appearance of a new specie, which corresponds to the lipid-peptide binding. These results provide useful information for the design of synthetic immunopeptides that can be incorporated into a liposomal system with potential to promote a direct delivery of the membrane-incorporated immunogen to the immunocompetent cells, thus increasing the immuno response from the host.

In this work, a peptide for ocular delivery (POD) and human immunodeficiency virus transactivator were conjugated with biodegradable poly(lactic-co-glycolic acid) (PGLA)- polyethylene glycol (PEG)-nanoparticles (NPs) in an attempt to improve ocular drug bioavail- ability. The NPs were prepared by the solvent displacement method following two different pathways. One involved preparation of PLGA NPs followed by PEG and peptide conjugation (PLGA-NPs-PEG-peptide); the other involved self-assembly of PLGA-PEG and the PLGA-PEG- peptide copolymer followed by NP formulation. The conjugation of the PEG and the peptide was confrmed by a colorimetric test and proton nuclear magnetic resonance spectroscopy. Flur- biprofen was used as an example of an anti-inflammatory drug. The physicochemical properties of the resulting NPs (morphology, in vitro release, cell viability, and ocular tolerance) were studied. In vivo anti-inflammatory effcacy was assessed in rabbit eyes after topical instillation of sodium arachidonate. Of the formulations developed, the PLGA-PEG-POD NPs were the smaller particles and exhibited greater entrapment effciency and more sustained release. The positive charge on the surface of these NPs, due to the conjugation with the positively charged peptide, facilitated penetration into the corneal epithelium, resulting in more effective preven- tion of ocular inflammation. The in vitro toxicity of the NPs developed was very low; no ocular irritation in vitro (hen’s egg test-chorioallantoic membrane assay) or in vivo (Draize test) was detected. Taken together, these data demonstrate that PLGA-PEG-POD NPs are promising vehicles for ocular drug delivery., This work was supported by the Cooperation Research Program CSIC-CITMA and a project (MAT2011-26994) funded by the Spanish Ministry of Science and Technology. AV is a recipient of a PhD grant from the CSIC. The authors thank Nacho Pérez of the IQAC-CSIC for performing the cytotoxicity assays.

The peptide corresponding to the sequence (279-298) of the Hepatitis G virus (HGV/GBV-C) E2 protein was synthesized, and surface activity measurements, pi-A compression isotherms, and penetration of E2(279-298) into phospholipid monolayers spread at the air-water interface were carried out on water and phosphate buffer subphases. The results obtained indicated that the pure E2(279-298) Langmuir monolayer exhibited a looser packing on saline-buffered than on pure water subphase and suggest that the increase in subphase ionic strength stabilizes the peptide monolayer. To better understand the topography of the monolayer, Brewster angle microscopy (BAM) images of pure peptide monolayers were obtained. Penetration of the peptide into the pure lipid monolayers of dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) and into mixtures of dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) at various initial surface pressures was investigated to determine the ability of these lipid monolayers to host the peptide. The higher penetration of peptide into phospholipids is attained when the monolayers are in the liquid expanded state, and the greater interaction is observed with DMPC. Furthermore, the penetration of the peptide dissolved in the subphase into these various lipid monolayers was investigated to understand the interactions between the peptide and the lipid at the air-water interface. The results obtained showed that the lipid acyl chain length is an important parameter to be taken into consideration in the study of peptide-lipid interactions.

The interaction with phospholipid bilayers of two synthetic peptides with sequences corresponding to a segment next to the native N-terminus and an internal region of the E2 structural hepatitis G virus (HGV/GBV-C) protein [E2(7–26) and E2(279–298), respectively] has been characterized. Both peptides are water soluble but associate spontaneously with bilayers, showing higher affinity for anionic than zwitterionic membranes. However, whereas the E2(7–26) peptide is hardly transferred at all from water to the membrane interface, the E2(279–298) peptide is able to penetrate into negatively charged bilayers remaining close to the lipid/water interface. The nonpolar environment clearly induces a structural transition in the E2(279–298) peptide from random coil to α-helix, which causes bilayer perturbations leading to vesicle permeabilization. The results indicate that this internal segment peptide sequence is involved in the fusion of HGV/GBV-C to membrane.

11 pages, 7 figures, 4 tables.-- PMID: 15381310 [PubMed].-- Printed version published Oct 1, 2004., The physicochemical properties of three peptides belonging to the β-interferon (β-IFN) molecule, -β-IFN(13–20), β-IFN(40–47) and β-IFN(109–116)-, which have been described to be antigenic epitopes of the neutralising antibodies responsible of the failure of the Multiple Sclerosis therapy, and their palmitoylated derivatives were analysed. Peptides were synthesised by solid-phase methodologies and characterized by amino acid analysis, analytical high-performance liquid chromatography and electrospray mass spectrometry. The activity of free and derivatized peptides was determined. In order to know how the synthesised peptides were able to interact with membrane models, studies of kinetics of penetration at constant area and compression isotherms were carried out. Moreover, differential scanning calorimetry (DSC) was used to investigate the thermotropic phase properties of binary mixtures of dipalmitoylphosphatidylcholine (DPPC) or dipalmitoylphosphatidylglicerol (DPPG) with the peptides.

In the present work, the physicochemical characterization of a hepatitis G virus (HGV) synthetic peptide by means of two different techniques was studied: lipid monolayers and differential scanning calorimetry (DSC). We have studied the surface activity of E1(53-66), and its palmitoylated derivative, PalmE1(53-66), and the interactions of the last one with dipalmiltoyl phosphatidylcholine (DPPC), and dipalmitoyl phosphatidylglycerol (DPPG) monolayers. Moreover, DSC was used to study the interaction of PalmE1(53-66) with multilamellar liposomes composed of DPPC.

The synthesis by solid-phase methodologies of peptides belonging to structural and non-structural proteins of GB virus C as well as its N-alpha-acylation with myristate and palmitate fatty acids is described. To explore the peptide-lipid interactions we have used liposomes composed of dipalmitoylphosphatidylcholine as model membranes and complementary spectroscopic and calorimetric techniques. Our results show that structural and more clearly the structural lipophilic peptide sequences incorporated into lipid bilayers perturb the packing of lipids and affect their thermotropic properties, more than the non-structural selected sequence. However, the binding of the synthetic sequences to lipid membranes occurred without any restructuration of the peptides.

The NS3 (513–522) fragment of the hepatitis G virus (HGV) was manually synthesised by solid phase synthesis following Fluoren-9-ylmethoxycarbonyl (Fmoc)/tBut strategy. It was characterised by amino acid analysis, analytical HPLC and FAB-mass spectrometry. It showed strong hydrophilic properties as predicted by applying the Hopp and Woods scale. In an effort to reduce these properties to improve its affinity for lipidic membrane models as lipid monolayers and liposomes, the palmitoyl derivative of the peptide was synthesised. The lipophilic derivative was obtained by following the same strategy stated above and final acylation with palmitic acid. Surface activity measurements show that palmitoyl–NS3 (513–522), in contrast with the parent peptide, is able to modify the surface activity of water-forming monomolecular films. The result of the study indicates that saturation of superficial pressure is gained proportionally to the increased amounts of injected palmitoyl–NS3 (513–522).

The development of peptide fusion inhibitors based on short synthetic peptides represents a promising option in the fight against HIV-1 infection, especially in individuals infected with multiresistant HIV-1 strains. GBV-C has the beneficial effect of retarding the progression of AIDS in people who are co-infected with both the GBV-C and HIV viruses. In previous works, the E1(22–39) GBV-C sequence (E1P8lin) was found to be capable of inhibiting the interaction of HIV-1 FP with bilayers and its cyclic analogue (E1P8cyc) showed a higher anti-HIV-1 activity. In the present work, in an attempt to gain a better understanding of the interaction of E1P8 peptides with HIV-1 FP, we analyzed direct interactions between peptides at the molecular level. Our results support that E1P8cyc might be more potent at blocking HIV-1 entry than E1P8lin as a consequence of the structure induced in the complex formed with HIV-1 FP, which is able to modify the conformation adopted by this functional domain of the HIV-1 gp41 protein in target cell membranes.

In the present work the physicochemical characterization of the (306-324) synthetic peptide sequence from human fiiaggrin protein and its palmitoilated derivative was carried out. Monomolecular layers of dipalmitoilphosphatidilcholine (DPPC) were used to study lipidtpeptide interactions. Measurements of surface activity, compression isotherms and DPPC miscibility are reported.

Synthetic peptide vaccines are safer and cheaper than classical ones, but immunoresponse is usually lower. The early interaction of hepatitis A virus (HAV) with cells during the infection and immunological response processes is not as well-known as for other picornavirus. However, it seems that electrostatic interactions have an important role in the fusion of HAV with cell membranes. In order to gain more understanding in these processes, in the present work, we have studied the interactions of AcVP3110, a synthetic HAV peptide, an acetylated amidated derivative of the epitope VP3(110–121), with a neutral phospholipid (dipalmitoyl phosphatidylcholine), an anionic phospholipid (dipalmitoyl phosphatidylglycerol), and a cationic lipid (stearylamine), using a monolayer technique. AcVP3110 showed higher surface activity than the parent peptide, probably owing to its acyl moiety. Studies of the kinetics at constant area of the peptide injected under monolayers of the lipids assayed showed only slight differences depending on the charge of the lipid. On the other hand, when compression isotherms of these lipids on a subphase containing the peptide were performed, major interactions were found for stearylamine monolayers. Owing to the negative net charge of AcVP3110, it is quite clear that electrostatic forces do have a role in the interaction of this synthetic peptide with membranes, as has been seen with complete HAV.

In the present work the pattern of miscibility between a hepatitis A virus (HAV) synthetic peptide (AcVP3110) with monolayers composed of lipids of different charge was studied. Zwitterionic (dipalmitoyl phosphatidylcholine (DPPC)), anionic (dipamitoyl phosphatidylglycerol (DPPG)), and cationic (stearylamine (SA)) lipids were studied at the air/water interface by compression isotherms. AcVP3110, a putative HAV synthetic vaccine candidate, formed stable monolayers through compression, being predominantly correlated with β-sheet conformations. Both DPPC and DPPG peptide mixtures with the peptide presented positive deviations from ideality. This behavior reveals the presence of repulsive forces between these lipids and AcVP3110, which were expected especially with DPPG, owing to the fact that both lipid and peptide have negative net charge. Mixed monolayers with SA showed low deviations from ideality, which were positive or negative depending on the amount of peptide present in the monolayer. All this suggests that in addition to playing a role in hydrophobic interactions, electrostatic forces have a major role in miscibility between AcVP3110 and membrane lipids. This is in agreement with what has been found for complete hepatitis A virus, where ionizable groups in the surface of the virus or cell play an important role in the attachment and endocytosis processes. Moreover, amidation and acetylation of the VP3(110–121) peptide sequence do not modify the recognition of this continuous epitope by anti-HAV antibodies.

The present study was undertaken to examine the conformation by Circular Dichroism spectroscopy of the (440-460): AIAYYRGKDSSIIKDGDLVVC peptide sequence belonging to the non-structural protein (NS3) of hepatitis G virus (HGV). The conformation of free HGV-NS3(440-460) and two lipophilic derivates at the N- terminus with palmitic and miristic acids respectively, was studied at different concentrations in HEPES as well as in the presence of the most commonly used agents for stabilizing conformations (TFE,HFIP).

The synthesis of hydrophobic peptide derivatives related to the laminin sequence [YIGSR(NH(2))] is described. Hydrophobicity is achieved by the attachment of decanoic, myristic, or stearic acids to the amino terminal end of the peptide. Moreover, a cholesterol residue was also introduced as succinimidoyl-cholesteryl moiety at the same position. These peptidic compounds are designed to be inserted into lipid bilayers to prepare, what can be considered as, immunoliposomes to target these vesicles to tumor cells. Physicochemical aspects related to their surface activity, insertion into lipid layers, spreadibility, formation of aggregates, and haemolytic activity have been studied as a previous step in the selection of the most convenient derivative. The results obtained indicate that these peptide derivatives show a high tendency to form aggregates in aqueous media, this fact reducing their interaction with lipid mono- and bilayers. The most suitable derivatives for interacting with liposomes are myristoyl and decanoyl. Copyright 2001 Academic Press.

Mixed monolayers of an acylated derivative of hepatitis A synthetic peptide VP3(110-121) with neutral, cationic or anionic lipids were spread at the air/water interface. Deviations from ideality as well as thermodynamic values were calculated at different surface pressures using the free-excess energy, the interaction parameter and the enthalpy. The miscibility at the collapse point was also checked. Maximum deviations from ideality were found for mixtures containing the anionic lipid phosphatidylglycerol (PG), and it seems that the monolayer composition is not stable through compression, as the peptide is ejected from the film. Films containing neutral [dipalmitoylphosphatidylcholine (DPPC)] or cationic [stearylamine (SA)] lipids showed more regular behaviour. As the peptide has a net negative charge it is probable that electrostatic interactions are in part responsible of the good miscibility of palmitoyl VP3(110-121) with SA. In order to prepare liposomes containing palmitoyl VP3(110–121), lipids such as SA or DPPC/SA will be a more suitable choice than anionic lipids such as PG.

The synthesis on solid phase of a peptide sequence (GGRGRS) related to an integrin adhesion site as well as the preparation of some hydrophobic derivatives is described. The incorporation of these peptides to the surface of liposomes was carried out either through the NGPE (N-glutaryl dipalmitoyl phosphatidyl choline) carboxyl-group or mixing hydrophobic peptide derivatives with lipids since the beginning of the process. The influence of these factors on the entrapment yield of 5-FUR (5-fluorouridine) was determined. Best results, calculated as percentage of drug encapsulation, were obtained when the peptide was linked to preformed liposomes via an NGPE-amide bond. On the contrary, the presence of these hydrophobic peptides on the bilayers decreases the overall yield of encapsulation of 5-FUR. Nevertheless, considering drug/lipid relationship and scaling-up requirements it seems that the use of myristoyl peptide derivative should be the procedure of choice. Physicochemical studies carried out with the peptides indicated that the presence of hydrophobic moieties linked to the parent peptide increases the tendency to self aggregation as detected through fluorescence studies using DPH (1, 6 diphenyl hexatriene) as marker, reducing in this way the efficiency of incorporation of hydrophobic peptides to the surface of liposomes.

The interaction of the multiple antigenic peptide MAP4VP3 with lipid membranes has been studied by spectroscopic techniques. MAP4VP3 is a multimeric peptide that corresponds to four units of the sequence 110-121 of the capsid protein VP3 of hepatitis A virus. In order to evaluate the electrostatic and hydrophobic components on the lipid-peptide interaction, small unilamelar vesicles of different compositions, including zwitterionic dipalmitoylphosphatidylcholine (DPPC), anionic dipalmitoylphosphatidylcholine/phatidylinositol (DPPC:PI 9:1), and cationic dipalmitoylphosphatidylcholine/stearylamine (DPPC:SA 9.5:0.5), were used as membrane models. Intrinsic tryptophan fluorescence changes and energy transfer experiments show that MAP4VP3 binds to all three types of vesicles with the same stoichiometry, indicating that the electrostatic component of the interaction is not important for binding of this anionic peptide. Steady-state polarization experiments with vesicles labeled with 1,6-diphenyl-1,3,5-hexatriene or with 1-anilino-8-naphtalene sulphonic acid indicate that MAP4VP3 induces a change in the packing of the bilayers, with a decrease in the fluidity of the lipids and an increase in the temperature of phase transition in all the vesicles. The percentage of lipid exposed to the bulk aqueous phase is around 60% in intact vesicles, and it does not change upon binding of MAP4VP3 to DPPC vesicles, indicating that the peptide does not alter the permeability of the membrane. An increase in the amount of lipid exposed to the aqueous phase in cationic vesicles indicates either lipid flip-flop or disruption of the vesicles. Binding to DPPC vesicles occurs without leakage of entrapped carboxyfluorescein, even at high mol fractions of peptide. However, a time-dependent leakage is seen with cationic DPPC/SA and anionic DPPC/PI vesicles, indicating that the peptide induces membrane destabilization and not lipid flip-flop. Resonance energy transfer experiments show that MAP4VP3 leakage from cationic vesicles is due to membrane fusion, whereas leakage from anionic vesicles is not accompanied by lipid mixing. Results show that MAP4VP3 interacts strongly with the lipid components of the membrane, and although binding is not of electrostatic nature, the bound form of the peptide has different activity depending on the membrane net charge; thus, it is membrane disruptive in cationic and anionic vesicles, whereas no destabilizing effect is seen in DPPC vesicles.

The reactivities of two panels of anti-HAV human sera from geographically distinct areas (Chile and Spain) to synthetic peptides from the VP1, VP2 and VP3 hepatitis A virus capsid proteins were examined by an enzyme-linked immunosorbent assay (ELISA) procedure. Two and four branched multiple antigenic peptides (MAPs) and palmitoylated peptides were compared with free synthetic sequences for the detection of IgM anti-HAV antibodies in the two panels of human sera. Our results showed that acute hepatitis A patient sera recognized preferentially homogeneous two branched MAPs and palmitic acid conjugated peptides. The palmitoyl-derived VP3(110–121) peptide and the corresponding dimeric MAP were the most sensitive and appropriate for serological studies of HAV-infected patients by ELISA, sensitivity and specificity being higher than 90% and 95%, respectively. These peptide-based tests open up new avenues in the development of peptide-based immunosorbent assays for the detection of acute HAV disease.

Shorter analogues of a continuous epitope of hepatitis A virus, VP3(110–121) peptide, failed to react with convalescent sera, indicating the importance of the entire peptide in the epitope structure. To better understand the influence of the structural properties of this 12-mer peptide epitope on its biological activity, the interaction of smaller peptide analogues with phospholipid biomembrane models was investigated by a combination of spectroscopic and biophysical techniques. In this article we describe our findings concerning the surface activity and the interaction of peptides with simple mono- and bilayer membranes composed of a zwitterionic phospholipid (dipalmitoyl phosphatidylcholine, DPPC), an anionic phospholipid (dipalmitoyl phosphatidylglicerol, DPPG), or a DPPC/DPPG mixture. The results indicate that the net negative charge of the peptide is in some way responsible of the specific interactions between VP3(110–121) and membrane phospholipids, and necessary to induce β-type conformations upon vesicle interaction. © 2000 John Wiley & Sons, Inc. Biopoly 54: 477–488, 2000

Interaction studies of three different presentations of a continuous B epitope of Hepatitis A virus linked to a synthetic T epitope related to Hepatitis B virus with monolayers composed of dipalmitoylphosphatidylcholine (DPPC) have been carried out. Surface activity of the peptides was determined as a function of its bulk concentration in an aqueous solution. The maximum pressure achieved at saturation, the molecular area and the surface excess of the different synthetic peptides were determined. The different physicochemical behaviour of the constructs was dependent on the relative position of the B and T epitopes. The ability to insert into DPPC monolayers showed that the highest interaction with phospholipid monolayers corresponded to the peptide with the T-cell epitope located at the C-terminus.

Previous work from our group showed that the entrapment of HAV-related synthetic peptides into multilamellar liposomes yielded satisfactory immunoresponses when administered to mice. In the present work we report investigative results for several liposome formulations of a 20-mer peptide related to VP3 capsid protein of HAV. In this sense, the recently introduced surface plasmon resonance technique has been applied to compare the different strategies of association between the synthetic peptide and phospholipid vesicles and to demonstrate that no significant alteration in antigenicity is produced when the peptide sequence is covalently coupled to the surface of small unilamellar vesicles. In addition, conformational data obtained by the circular dichroism technique have shown a decrease in the helical contribution of peptide-once linked to phospholipid; probably this change is due to the restriction introduced at the N-terminus of the sequence when coupled to the derivatized phospholipids at the surface of vesicle bilayers.

Branched-chain polypeptides based on a polylysine backbone were synthesized for use as delivery systems for biologically active molecules. To develop a new immunoadjuvant system for hepatitis A virus (HAV) antigens, a peptide fragment (110-121) of the VP3 hepatitis A protein has been linked to cationic and amphoteric polymeric polypeptides. In the present paper, we describe the physicochemical characterization of three branched polypeptide-[HAV-VP3(110-121) peptide] constructs, where the peptide is attached to the polymer side chains by disulfide linkage. The surface activity of these three conjugates has been studied as a function of time and concentration in the subphase. Moreover, its insertion into phospholipid DPPC (dipalmitoylphosphatidylcholine) monolayers has also been determined. The results show that these constructs are surface active and can insert into the lipid monolayers. AK construct is slightly more active than EAK and SAK constructs both in the presence and absence of DPPC monolayers. This behavior suggests that construct disposition at interfaces is mainly dependent on nonsubstituted side chains. Fluorescence polarization studies, performed with DPPC vesicles saturated with either DPH (1,6-diphenyl1,3,5-hexatriene) or ANS (1-anilino-8-naphthalenesulfonic acid), indicate a strong rigidifying effect of the three constructs on the polar heads and alkyl chains of bilayers.

The surface properties of a laminin related decapeptide were determined using Langmuir films. Moreover, its interaction with phospholipids was studied using both DPPC mono- and bi-layer liposomes as membrane models. Changes in fluidity of the bilayers induced by this peptide were determined by means of polarizable probes such as 8-anilino-1-naphthalene sulfonic acid (ANS) and 1,3,5-diphenylhexatriene (DPH). Changes in permeability of liposomes were studied through CF latency. The results of surface properties and liposome interactions suggest that this peptide forms some type of aggregate in water solution that competes with its incorporation at a water–air interface and with its binding to bilayers.

The present study was undertaken to examine the structural features that may be important to explain the immunogenicity of the (110-121) peptide sequence (FWRGDLVFDFQV) of VP3 capsid protein of hepatitis A virus. A conformational analysis of the preferred conformations by CD and molecular mechanics was carried out. Present results suggest that the interaction with liposomes as biomembrane model induces and stabilizes the amphipathic beta-structure of the peptide. To study the contribution of amino acid replacements at the RGD tripeptide as well as the influence of the peptide chain length on peptide conformation, solid-phase peptide synthesis of several peptide analogs was carried out and the peptide conformation was studied using CD spectroscopy. The results show that the RGD sequence is necessary to induce the beta-structure in the presence of liposomes.

The synthesis of three cyclic peptides related to an active sequence of Laminin is described. These molecules have no, one, or two stearoyl residues linked to the amino terminal end of the sequence. The physicochemical properties of these peptides as well as their interactions with dipalmitoylphosphatidylcholine molecules ordered in mono- and bilayers are described. Properties based on aqueous solutions of peptides show maximum activity for the analogue with one stearoyl residue. But properties measured from organic solutions of peptides (compression isotherms), as well as those in water but at high temperatures, gave as the most surface active the distearoyl derivative. This behavior is interpreted as a strong tendency of this highly hydrophobic molecule to form aggregates (probably micelles), very stable in aqueous media. Thus, the monostearoyl-substituted peptide seems to be the most adequate for efficient insertion into mono- and bilayers.

The conjugation of the [Abu 105,109 ] VP3(101-121) peptide sequence from the VP3 capsid protein of hepatitis A virus to synthetic branched polypeptide carriers is described. The establishment of either a disulfide or an amide bond between the peptide and the carrier molecules is reported. The interaction of VP3 conjugates with dipalmitoylphosphatidylcholine (DPPC) mono- and bilayers was studied, and the influence of the different hydrophilicities and peptide orientations on the conjugates is discussed. Results showed an increased interaction with biomembrane models due to the conjugation ofVP3 peptide to the described macromolecular structures.

The interaction of the synthetic multiple antigenic peptide construct MAP 4 -VP1(11-25) with lipid membranes was studied by a combination of spectroscopic and biophysical techniques. MAP 4 -VP1 showed an important surface activity when spread in an air–water interface, indicative of an amphipatic conformation. Since this peptide has a net anionic charge, we studied the degree of interaction with model membranes of zwitterionic dipalmitoylphosphatidylcholine (DPPC), anionic phosphatidylinositol (PI and DPPC/PI 9:1), or cationic stearylamine (SA and DPPC/SA 9:1). MAP 4 -VP1 was readily soluble in aqueous buffer, yet spontaneously inserted from an aqueous subphase into cationic and zwitterionic monolayers up to limiting pressures of 20–30 mN/m. The induced surface pressure increases were in the order cationic > zwitterionic > anionic monolayers. This indicates that the interaction is mainly driven by electrostatic forces, although there is an important hydrophobic component, responsible for the penetration in monolayers of neutral DPPC. Interaction of MAP 4 -VP1 with unilamellar vesicles of DPPC, DPPC/SA (9:1), and DPPC/PI (9:1) labeled with 1,6-diphenyl-1,3,5-hexatriene (DPH) or with 1-anilino-8-naphthalene sulfonic acid (ANS) was determined by changes in fluorescence polarization as a function of temperature. Results showed that the presence of the positively charged SA in the membrane strongly enhances the incorporation of MAP 4 -VP1 and that the peptide increases the fluidity of the bilayer and decreases the temperature of the phase transition.

The influence of PEG in the physicochemical stability of liposomes has been determined. Vesicles composed of PC/PG/Ch/DX (A), PC/PG/Ch/DX/PE-PEG (B), PC/PG/Ch/DX/PE-PEG/NGPE/E(8) (C), and PC/PG/Ch/...

A peptide with the amino acid sequence of 96–107 of Hepatitis A virus capsid protein VP2 was synthesized as such (FR2), as stearoyl derivative (stearoyl-FR2), and as a multiple antigen derivative (MAP- FR2) by standard solid phase procedures. The products were characterized by HPLC, mass spectrometry, and amino acid analysis. Their surface properties were studied using Langmuir films. Free peptide as well as its derivatives showed a high surface activity giving surface pressure increases in a concentration dependent way. FR2 molecules reached the air/water interface within few minutes. In contrast, stearoyl-FR2 and MAP4-FR2 showed an induction time concentration dependence, suggesting the presence of aggregates. These products were also able to insert into dipalmitoyl phosphatidyl choline (DPPC), dipalmitoyl phosphatidyl ethanolamine (DPPE) and dipalmitoyl phosphatidyl glycerol (DPPG) monolayers spread at 5, 10, and 20 mN m−1. Miscibility studies of the peptides with phospholipids showed no deviation with respect to ideality, which suggests the presence of weak molecular interactions. Finally, FR2 was incorporated into liposomes in order to study the immunogeneicity and surface activity of these vesicles.

Hepatitis A virus (HAV) is composed mainly of three structural capsid proteins: VP1, VP2 and VP3. Our group has reported the synthesis and the immunogenic evaluation of VP3 (110-121) peptide sequence. In the present work, in order to stimulate a T-cell immune response, we have selected the HAV-VP3 (102-121) peptide which has maximum amphipathicity. Its synthesis was carried out manually in the solid phase and semipreparative HPLC was used for purification of the crude peptide. Finally the purified peptide was characterized by analytical HPLC, amino acid analysis and MS. A palmitoyl derivative of VP3 (102-121) was synthesized to modify the hydrophobicity of the peptide. Both free and lipophilically derivatized peptides were incorporated into multilamelar liposomes. Physicochemical studies of the HAV-related peptides described above were carried out using monolayers as membrane models. Compression isotherms, surface activity and penetration kinetics into dipalmitoylphosphatidylcholine monolayers were determined. Moreover, changes in the fluidity of bilayers induced by these peptides were determined by means of polarizable probes such as 8-anilino-1-naphthalenesulfonic acid and 1,6-diphenyl-1,3,5-hexatriene. The integrity of the membranes has also been ascertained with the carboxyfluorescein.

The synthesis on a solid support of a dodecapeptide containing a HAV-VP2 exposed sequence is described. The surface activity of this peptide as well as its miscibility and interactions with dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylethanolamine (DPPE), and phosphatidylinositol (PI) were determined. Interaction energies calculated applying the Gibbs equation gave very small values, thus suggesting either ideal miscibility or weak interactions. In all cases the electrical charge in DPPE and PI monolayers seems to create a high electrostatic potential, decreasing its interaction with any external molecule. This peptide was able to accommodate a certain amount of anilinonaphthalenesulfonate molecules, showing an intermediate hydrophobic character. Moreover, after incubation with saturated liposomes, the peptide showed a clear rigidifying effect at the level of polar heads, especially at temperatures under the transition temperature.

The synthesis of a VP3(110–121)-HAV peptide was carried out by solid phase methodology. A physicochemical study on the interaction of this peptide with phospholipids involving lipid mono- and bilayers is described. The surface activity of the peptide is indicative of a medium hydrophobic character suggesting the potential ability of this peptide to associate with lipids. Moreover, the presence of the peptide in the incubation media of dipalmitoylphosphatidylcholine (DPPC) and DPPC/DPPG liposomes saturated with anilinonaphthalene sulphonate (ANS) or diphenylhexatriene (DPH) show a strong influence in the bilayers fluidity determined by polarization fluorescence. The presence of lipids in the gel state has a strong influence on the polarity of a Trp environment, inducing a blue shift and fluorescence intensity increases. No interaction was detected when measurements were carried out at temperatures above T c .

A peptide construct containing 4 peptide chains corresponding to VP1(11–25) linked to a lysine core was synthesized by solid phase methodology. The surface activity and the ability of this peptide to interact with monolayers of dipalmitoylphos-phatidylcholine (DPPC), phosphatidylinositol (PI) and stearylamine (SA) was studied using Langmuir-Blodgett films. Interactions between the MAP 4 -VP1 and bilayers were determined by polarization fluorescence studies. The presence of the peptide decreases the transition temperature of DPPC/DPH liposomes thus indicating the insertion of the MAP into the bilayer core. In contrast ANS-DPPC liposomes were not affected by the presence of the peptide constructs.

Four peptide sequences corresponding to the E1 protein of GBV-C: NCCAPEDIGFCLEGGCLV (P7), APEDIGFCLEGGCLVALG (P8), FCLEGGCLVALGCTICTD (P10) and QAGLAVRPGKSAAQLVGE (P18) were studied as they were capable of interfering with the HIV-1 fusion peptide (HIV-1 FP). In this work, the surface properties of the E1 peptide sequences are investigated and their physicochemical characterization is done by studying their interaction with model membranes; moreover, their mixtures with HIV-1 FP were also studied in order to observe whether they are capable to modify the HIV-1 FP interaction with model membranes as liposomes or monolayers. Physicochemical properties of peptides (pI and net charge) were predicted showing similarities between P7 and P8, and P10 and HIV-1 FP, whereas P18 appears to be very different from the rest. Circular dichroism experiments were carried out showing an increase of the percentage of α-helix of P7 and P8 when mixed with HIV-1 FP corroborating a conformational change that could be the cause of their inhibition ability. Penetration experiments show that all the peptides can spontaneously insert into phospholipid membranes. Analysis of compression isotherms indicates that the peptides interact with phospholipids and the E1 peptides modify the compression isotherms of HIV-1 FP, but there is one of the peptides that excelled as the best candidate for inhibiting the activity of HIV-1 FP, P7, and therefore, that could be potentially used in future anti-HIV-1 research.

Here we used solid-phase methods to prepare oligonucleotides carrying fibrin/ filaggrin citrullinated peptides. Post-synthetic conjugation protocols were successfully applied for the synthesis of oligonucleotides carrying small peptides. A stepwise protocol using acid treatment for the final deprotection allowed the preparation of polypyrimidine oligonucleotides carrying longer and arginine-rich peptides. An ELISA-based test using the oligonucleotide- citrullinated peptide conjugates was developed for the detection of anti-citrullinated protein/peptide antibodies in human serum from rheumatoid arthritis patients., The authors thank Raimon Sanmartí (Arthritis Unit, Hospital Clinic of Barcelona) for providing the serum samples. This work was supported by the Spanish Ministry of Education (CTQ2010-20541-C03-01), La Marató de TV3 Foundation (030331), the EECC (FUNMOL, FP7-NMP-213382-2), and the Generalitat de Catalunya (2009/SGR/208). CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008–2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III with assistance from the European Regional Development Fund.

The synthesis, on a solid support, of a Hepatitis A viral protein peptide, HAV-VP3 (61-78), is described. The interaction of the peptide with, dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylcholine (DOPC), and distearoylphosphatidylcholine (DSPC) has been studied, using liposomes and monomolecular layers as biomembrane models. Changes in fluidity of the bilayers induced by this peptide were determined by means of polarizable probes such as 8-anilino-1-naphthalenesulfonic acid (ANS) and 1,6-diphenyl-1,3,5-hexatriene (DPH). In this way liposomes can be used as carriers for small immunogenic peptides

Transferrin was chemically modified by covalent linkage of cholesteryloxycarbonyl-6-aminohexanoic acid to the lysine amino groups. Cholesteryloxycarbonyl-6-aminohexanoic acid was prepared from cholesteryl chlorocarbonate and 6-aminohexanoic acid. This derivative was attached by an amide bond to the free amino groups of transferrin. The level of derivatization was quantified by amino acid analysis and determination of free amino groups by the trinitrobenzene sulfonic acid method. The physicochemical characteristics of native and derivatized transferrin were studied by several methods. Surface activity was determined by the pressure increases at the air/water interface. Surface hydrophobicity was checked by anilino naphthalene sulfonic acid titration. Lipid-protein interactions were determined by intrinsic fluorescence changes, with the bilayers in the fluid and gel state. The results obtained show that derivatization renders the protein highly hydrophobic. Nevertheless, the surface activity or the interaction with lipids do not correlate with the hydrophobicity of the molecule, thus suggesting the formation of some type of internal micelles or a strongly packed structure.

The aim of this study was to identify proteins that could inhibit the activity of the peptide sequence representing the N-terminal of the surface protein gp41 of HIV, corresponding to the fusion peptide of the virus (HIV-1 FP). To do this we synthesized and studied 58 peptides corresponding to the envelope protein E1 of the hepatitis G virus (GBV-C). Five of the E1 synthetic peptides: NCCAPEDIGFCLEGGCLV (P7), APEDIGFCLEGGCLVALG (P8), FCLEGGCLVALGCTICTD (P10), QAGLAVRPGKSAAQLVGE (P18) and AQLVGELGSLYGPLSVSA (P22) were capable of inhibiting the leakage of vesicular contents caused by HIV-1 FP. A series of experiments were carried out to determine how these E1 peptides interact with HIV-1 FP. Our studies analyzed the interactions with and without the presence of lipid membranes. Isothermal titration calorimetry revealed that the binding of P7, P18 and P22 peptides to HIV-1 FP is strongly endothermic, and that binding is entropy-driven. Gibbs energy for the process indicates a spontaneous binding between E1 peptides and HIV-1 FP. Moreover, confocal microscopy of Giant Unilamellar Vesicles revealed that the disruption of the lipid bilayer by HIV-1 FP alone was inhibited by the presence of any of the five selected peptides. Our results highlight that these E1 synthetic peptides could be involved in preventing the entry of HIV-1 by binding to the HIV-1 FP. Therefore, the continued study into the interaction between GBV-C peptides and HIV-1 FP could lead to the development of new therapeutic agents for the treatment of AIDS., This study was supported by project CTQ2006-15396-C02-02/01-BQU from the Secretaría de Estado de Investigación, Ministerio de Ciencia e Innovación, Dirección General de Programas y transferencia de conocimiento, Subdirección General de Proyectos de Investigación (Spain), and supported in part by NIH grant GM 068619. M.J. Sánchez-Martín is a recipient of an FPI program pre-doctoral grant. The authors are members of the consolidated research group by the Generalitat de Catalunya: Peptides and Proteins: physicochemical studies (2005SGR00278). The authors thank Dr. Marta Taulés Marín, of the Confocal Microscopy and Cellular Micromanipulation Unit, and Dr. Rafel Prohens López of the Fine Chemistry Unit, from the Barcelona Science Park, for heir invaluable assistance.

The interaction of gentamicin (G), kanamycin (K), spectinomycin (Sp), streptomycin (St) and a polymixin, colistin (C), with lipids was studied by using liposomes and monomolecular layers as membrane models. The lipids used were phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylinositol disphosphate (PIP 2 ), gangliosides (Gan) and cholesterol (Chol). The results show that only colistin is able to induce the leakage of entrapped carboxyfluorescein (CF) from the liposomes after incubation. Moreover, this interaction is not clearly related to a single type of phospholipid, is dependent on the PL/colistin ratio and is slightly modified by the pH or the temperature of the incubation media. The interactions of these molecules with the polar heads of phospholipids were studied using 1-anilino-8-napthalenesulphonic acid (ANS) as fluorescent probe. The polarization values indicate that these antibiotics induce rigidification of the membrane. Monolayers having the same lipid composition of liposomes were spread on aqueous subphases and pressure increases, after injection of drug molecules, were recorded. In this set of experiments all molecules exhibit interaction, colistin and kanamycin being those with the maximum activity. Moreover, no specific differences could be assigned to the different lipids used except for kanamycin and gangliosides. The mathematical equations of the penetration kinetics for all the assays carried out were determined. The Linewe aver-Burk equation gave the best fit to the experimental values, the regression coefficient being higher than 0.98 in all cases. Compression isotherms of the same phospholipid mixtures were recorded, the antibiotic molecules being dissolved in the subphase. The compressibility and areaJmolecule were slightly affected by the presence of aminoglycosides in the aqueous phase. In contrast, the presence of colistin induced expansion of the monolayer especially at low pressures, thus indicating the existence of interactions. Comparing these results with those in the literature, it appears clear that the membrane model used exerts a strong influence on the results obtained.

Peptides belonging to the envelope protein of HBV [Tyr148]S(139–148) and preS(120–145) have been synthesized using the continuous-flow fluoren-9-ylmethoxycarbonyl (Fmoc)-polyamide solid phase methodology. Furthermore, the synthesis of a new series of hydrophobic derivatives involving Nα-acylation of both peptides with stearic and cholanic acid as well as the introduction of the synthetic B-cell and macrophage activator Pam3-Cys-Ser-Ser is also described. Benzotriazol-1-yloxytris(pyrrolidino)phosphonium hexafluorophosphate (PyBOP) and benzotriazol-1-yloxytris(dimethylamino) phosphonium hexafluorophosphate (BOP) proved to be convenient reagents to promote the coupling of these lipid moieties to peptides attached to Kieselguhr-supported polyacrylamide resins. Some synthetic aspects concerning reaction conditions and the use of different scavengers at the cleavage stage are discussed. Finally, a cyclic derivative of the S peptide was obtained through a disulphide bond formation.