WO2012101612A1 - Procédés de synthèse peptidique en phase solide de fmoc sur support biodégradable et utilisations desdits procédés - Google Patents

Procédés de synthèse peptidique en phase solide de fmoc sur support biodégradable et utilisations desdits procédés Download PDF

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Publication number
WO2012101612A1
WO2012101612A1 PCT/IB2012/050413 IB2012050413W WO2012101612A1 WO 2012101612 A1 WO2012101612 A1 WO 2012101612A1 IB 2012050413 W IB2012050413 W IB 2012050413W WO 2012101612 A1 WO2012101612 A1 WO 2012101612A1
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Prior art keywords
peptide
alginate
amino acid
linker
beads
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PCT/IB2012/050413
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English (en)
Inventor
Robert Marks
Khalil Abu-Rabeah
Hilla BEN-HAMO FADLON
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The National Institute for Biotechnology in the Negev Ltd.
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Publication of WO2012101612A1 publication Critical patent/WO2012101612A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C07K1/042—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers characterised by the nature of the carrier

Definitions

  • the invention in some embodiments, relates to the field of chemical synthesis and more particularly, but not exclusively, to an alternative method for solid phase peptide synthesis using biodegradable and biocompatible polymer beads, such as alginate or beads as a solid support.
  • Solid phase peptide synthesis is a method in which molecules are bound to a solid support, and synthesized step-by-step in a reactant solution, using linkers to bind intermediates to the support.
  • Linkers may be attached to the support by means of a spacer. Building blocks are protected at all reactive functional groups, in order to prevent polymerization of amino acids.
  • Protecting groups for amino groups may be 9-fluorenylmethyloxycarbonyl group (Fmoc) or t- butyloxycarbonyl (Boc). The Fmoc group is removed from the amino terminus with base while the Boc group is removed with acid.
  • the process involves repeated cycles of coupling-wash-deprotection-wash.
  • the free N- terminal amine of a solid-phase attached peptide is coupled to a single N-protected amino acid unit. This unit is then deprotected, revealing a new N-terminal amine to which a further amino acid may be attached.
  • the peptide remains covalently attached to the support until cleaved from it by a cleaving agent.
  • the synthesized peptide is thus 'immobilized' on the solid-phase and can be retained during repeated wash cycles, whereas liquid-phase reagents and by-products of synthesis are removed.
  • the solid support comprises an insoluble polymer, which may be in the form of a bead.
  • the physical properties of the solid support, and the applications to which it can be utilized, vary with the material from which the support is constructed, the amount of cross-linking, as well as the linker and handle being used. It is generally considered that supports should have the minimum amount of cross-linking to confer stability, resulting in a well-solvated system where solid-phase peptide synthesis can be carried out.
  • an efficient solid support includes that it must be physically stable and permit the rapid filtration of liquids, such as excess reagents; it must be inert to all reagents and solvents used during SPPS; it must swell extensively in the solvents used to allow for penetration of the reagents; and it must allow for the attachment of the first amino acid.
  • Known types of solid support include gel-type supports (such as polystyrene, polyacrylamide, polyethylene glycol); surface-type supports (including controlled pore glass, and highly cross-linked polystyrene) and gel-type polymers supported by rigid matrices. None of the materials which are currently used are biodegradable and biocompatible, or suitable for administration to a human or a non-human animal.
  • the present invention in at least some embodiments, relates to methods for solid phase peptide synthesis using biodegradable and biocompatible polymer beads (such as alginate, agar, carragenan, chitin, polyvinylpyrrolidine, hyaluronic acid, or polyurethanes beads) as a solid support.
  • biodegradable and biocompatible polymer beads such as alginate, agar, carragenan, chitin, polyvinylpyrrolidine, hyaluronic acid, or polyurethanes beads
  • the present invention provides a method for solid phase peptide synthesis which is devoid of at least some of the limitations of known methods.
  • the method of the present invention requires fewer steps than known methods, and may result in similar or greater yields, at similar or lower cost.
  • Peptides synthesized according to the methods of the present invention are particularly suitable for use as vaccines, as described in further detail below.
  • the solid support In standard methods of peptide synthesis, the solid support must be separated from the synthesized peptide upon completion of the synthetic process. According to the method of the present invention, the synthesized peptide is allowed to remain on the polymer support. Furthermore, the biodegradable and biocompatible polymer of the present invention may serve as a peptide carrier and an adjuvant, such that no additional carrier or adjuvant is required.
  • the method of the present invention comprises providing a biodegradable and biocompatible polymer bead; contacting the bead with a carboxylic group activator to form a linker; providing a plurality of amino acids; and coupling the amino acids on the bead comprising the linker.
  • the bead is prepared by reversibly cross-linking a solution of a biodegradable and biocompatible polymer, such as, for example, by adding a solution of calcium or magnesium to a solution of the biodegradable and biocompatible polymer, or optionally be adding the polymer solution to the solution of calcium or magnesium.
  • the cross-linking is reversible by addition of a chelating agent.
  • the carboxylic group activator is selected from the group consisting of DCC, HOBt and PyBOP, and mixtures thereof.
  • functional groups of amino acids are protected with Fmoc during coupling.
  • the functional groups are deprotected by use of a base, such as, for example, piperidine.
  • the method of the present invention may be used in the preparation of an immunogenic peptide, which may be further used as a vaccine.
  • the vaccine may optionally be administered orally (such as in the form of a tablet, optionally comprising an enteric coating) or subcutaneously.
  • the biodegradable and biocompatible polymer optionally and preferably functions as a protein carrier and adjuvant.
  • DMF dimethylformamide
  • DCM dichloromethane
  • NMM N-methyl morpholine
  • DCC dicyclohexylcarbodiimide
  • HOBt Hydroxy benzotriazole
  • NMP- 1 -methyl 2-pirolidinone ddw: double distilled water
  • TFA trifluoroacetic acid.
  • FIG. 1 shows the structure of alginate
  • FIG. 2 shows a representation of binding of alginate molecules by calcium
  • FIG. 3 shows an acetylated alginate monomer
  • FIG. 4 shows a procedure for the production of a linker for use in the process of the present invention
  • FIG. 5 shows a representation of uptake of antigen by M-cells
  • FIG. 6 shows a representation of uptake of antigens by dendritic cell projections
  • Figure 7 shows a representation of the gastrointestinal route taken by an orally administered vaccine formulation
  • FIG. 8 shows a representation of a portion of the small intestine, showing the M-cells and Peyer's patches
  • FIG. 9 shows the peak results after synthesis using the PyBOP method.
  • FIG. 10 shows the peak results after tripeptide synthesis.
  • Some embodiments of the invention relate to improved methods for solid phase peptide synthesis using biodegradable and biocompatible polymer beads, including but not limited to beads comprising alginate, agar, carragenan, and chitin, polyvinylpyrrolidine, hyaluronic acid, and polyurethanes (for example, those based on lysine diisocyanate).
  • the method of the present invention comprises providing a biodegradable and biocompatible polymer bead; activating a carboxylic group of the bead by contacting the bead with a carboxylic group activator to form a linker; providing a plurality of amino acids; and coupling the amino acids on the bead comprising the linker.
  • the biodegradable and biocompatible bead is prepared by reversibly cross-linking a solution of a biodegradable and biocompatible polymer, for example by addition of a calcium or magnesium solution to stimulate cross-linking.
  • the viscosity and shape of the beads formed can be controlled by modulating the concentration of cross-linker solution used.
  • the cross-linking is reversible by addition of a chelating agent.
  • a hydrogel is formed upon addition of a chelating agent.
  • the biodegradable and biocompatible polymer is alginate, which is a polymer occurring naturally in the cell walls of brown algae.
  • Alginates are chain-forming heteropolysaccharides made up of blocks of mannuronic acid and guluronic acid, having a structure as shown in Figure 1. Alginates are commercially available as sodium, potassium or alginate. According to at least some embodiments, the bead comprises alginate.
  • Alginate is a natural ionic polysaccharide, which is isolated from brown Algea, such as Laminaria hyperborean and lessonia, found in coastal waters around the globe, and some bacteria, such as Azotobacter vinelandii and Pseudomonas aruginosa. More specifically, alginate is a linear block copolymer composed of 1,4-linked ⁇ -D-mannuronic acid (M) and a-L-guluronic acid (G) residues. As used herein, the term "alginate” comprises alginate obtained from any suitable source, whether natural or synthetic.
  • Beads comprising alginate may optionally be prepared through gelation.
  • Alginate is able to gelate in the presence of aqueous divalent cations, such as Calcium, Strontium and Barium, optionally in the presence of a biocatalyst, including but not limited to a peptide or other organic material.
  • aqueous divalent cations such as Calcium, Strontium and Barium
  • Other ions such as Magnesium and Sodium
  • Alginate can also form an acid gel at pH below the pKa value of the uronic acid residues.
  • alginate beads prepared according to any suitable method may be employed.
  • insoluble alginate beads are produced by dissolving alginate, preferably sodium alginate, in water to give an alginate solution, followed by adding the alginate solution to a solution of a cross-linker, such as calcium, Barium or Strontium solution, for example calcium sulphate or calcium chloride, preferably in a dropwise manner.
  • a cross-linker such as calcium, Barium or Strontium solution, for example calcium sulphate or calcium chloride
  • the alginate is cross linked by divalent calcium, Barium or Strontium ions, which are positively charged, and therefore attracted to the partially polar groups on the alginate.
  • This cross linking is reversible by removal of the calcium or magnesium.
  • calcium ions may be removed by using calcium chelator (EDTA for example) such that it is possible to reverse the beads to a produce a gel form.
  • Cross-linking of alginate by calcium is schematically shown in Figure 2.
  • the beads so produced may optionally be washed with DMF, and water removed, for example by shaking.
  • the hydrophobicity of the alginate is increased by acetylation, wherein the alginate hydroxyl group connects to an acetyl group.
  • the new group on the alginate is less hydrophilic, (more hydrophobic), therefore the mass transfer from the organic phase to the bead is improved.
  • the beads dissolve better in the organic phase, thereby decreasing any steric effects, such that a growing peptide chain will be exposed to the next amino acid and the yield increased.
  • An acetylated alginate monomer is shown in Figure 3.
  • a process for preparation of a linker is shown in Figure 4, wherein the process comprises providing an alginate carboxylic group, and activating the carboxylic group such that the Boc- ethylene-diamine linker is connected by its amine group.
  • the Boc group is then removed, exposing the free amine (which was protected in the side reaction), to reaction with a subsequent amino acid.
  • coupling of the amino acids on the bead comprises activation of the amino acids in order to speed up the reaction.
  • the carboxylic group activator for preparation of the linker and/or for activation of amino acids is a carbodiimide, such as dicylohexylcarbodiimide (DCC) or diisopropylcarbodiimide (DIC), or a triazolol, such as 1-hydroxy-benzotriazole (HOBt), 1- hydroxy-7-aza-benzotriazole (HOAt), ethyl 2-cyano-2-(hydroxyimino)acetate, 2-(lH- benzotriazol-l-yl)-l,l,3,3,-tetramethyluronium hexafluorophosphate (HBTU), 2-(lH-7- Azabenzotriazol-l-yl)— 1,1,3, 3-tetramethyl uronium hexafluorophosphate Methanaminium, 0-(6- Chloro-l-hydrocibenzotriazol-l-yl)- -1,1,3,3
  • the activating reagent is DCC, optionally together with N- hydroxysuccinimide (NHS) or HOBt, or mixtures thereof. In some embodiments, the activator is a mixture of DCC and HOBt. In some embodiments, wherein the linker is prepared in an organic phase, the activating reagent is PyBOP or HBTU. In some embodiments, wherein the linker is prepared in an aqueous phase, the activating agent is l-ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDC) or NHS.
  • EDC l-ethyl-3-(3- dimethylaminopropyl)carbodiimide
  • the amino acid functional groups are protected with Fmoc in order to prevent polymerization.
  • the method further comprises deprotection by use of a mild base, such as, for example, piperidine in DMF, in order to remove the Fmoc group to expose the a-amino group for reaction with an incoming activated amino acid.
  • the exposed amine is neutral, such that no further neutralization step is required.
  • the deprotection step is monitored in order to improve amino acid coupling. Monitoring may optionally comprise measurement of the amount of Fmoc release in the deprotection step. Fmoc has a maximum peak at 301 nm, which enables the release of Fmoc to be easily monitored by ultraviolet assay, in order to optimize the coupling method best suited to each amino acid, such as by changing the alginate molecular weight, bead density and size, selection of activating agents and reaction conditions (such as time).
  • the method of the present invention further comprises cleaving or otherwise separating the peptide from the bead, such that the peptide may optionally revert to an unbound state in which it is not attached to the bead.
  • the peptide is cleaved from the bead by use of a cleaving agent, such as, for example, trifluoracetic acid.
  • the peptide is characterized by at least one of a ninhydrin test and amino acid analysis.
  • the ninhydrin test otherwise known as the Kaiser test, detects free amines, wherein ninhydrin reacts with free amine to produce a deep blue color, indicating that N groups are unprotected and ready for coupling.
  • a Kaiser test uses 2,2-Dihydroxyindane-l,3- dione(ninhydrin), an indicator for primary amines, to monitor the reaction. Ninhydrin changes colors due to a reaction with primary amine from yellow to blue, therefore right after the coupling reaction the ninhydrin should be negative (yellow), but after deprotection it should be positive (dark blue).
  • ninhydrin may optionally be replaced with bromphenol blue.
  • Bromophenol is an indicator which changes from blue to yellow on protonation. It binds to unacylated resin-bound amino groups staining the resin blue, as the coupling reaction proceeds, the blue coloration fading to yellow, indicating the reaction progress.
  • Amino acid analysis provides the ratio between the different amino acids, wherein the peptide is hydrolyzed by a strong acid and heat to its amino acid derivatives, then separated on a column and the amino acid ratio determined. Terminal residue analysis may also optionally be performed, for example through Edman degradation or Sanger analysis as is known in the art.
  • the peptide may be characterized by one or more of the following tests:
  • Trp As the first amino acid a mixture of agents that cut at the carboxylic side of Trp can be used, thereby releasing the peptides. The peptide mixture can then be separated in a column and peptides analyzed by MS;
  • fluorescence polarization immunoassay for detection of the peptide on the bead
  • bead slicing and permeability estimation comprising histological slicing of the beads and amine detection by a fluorescent marker for the amine, which can provide an estimate of the diffusion of the reactants and depth of reaction.
  • alginate can be used as a vaccine carrier, protecting the vaccine from the vigorous conditions of the digestion system, and in addition, alginate has been reported to have adjuvant properties but it is not immunogenic in itself, which means that it induces the immune system against the antigen but not against itself.
  • the peptide synthesized through SPPS on the alginate bead is an immunogenic peptide.
  • the peptide is not cleaved from the alginate bead, in contrast to art known SPPS methods (which do not use alginate or other biocompatible materials).
  • art known SPPS methods 99% of the peptides grow inside the interior of the solid support which is not biocompatible; hence these peptides must be cleaved from the support and attached to a carrier for vaccine use.
  • the alginate bead can protect the peptide inside the resin interior from the conditions of the digestion system and preventing it from leaking, insuring that all peptides reach their destination.
  • Alginate has swelling abilities, a characteristic that is very important for SPPS resins, as noted above. The swelling allows for better diffusion of the reagents, enabling the growing peptide to have more space inside the resin internal area. This combination of alginate as a SPPS resin and carrier- adjuvant for oral vaccination can lower the costs of vaccine production, among many other advantages.
  • the peptide synthesized is a cholera toxin B subunit derived peptide, preferably having a length of 15 amino acids.
  • an immunogenic peptide such as cholera toxin B subunit derived peptide, prepared in accordance with the principles of the present invention may be used as a vaccine, for either human or non-human animal use, by oral or subcutaneous administration.
  • the methods of the present invention and products thereof are particularly useful in preparation of vaccines, since the polymer with the newly-synthesized peptide attached may be used directly as a vaccine, or may be used following reversal of the polymer to a hydrogel, or as a combination of beads and hydrogel.
  • the vaccine comprises a formulation comprising the biocompatible and biodegradable polymer in the form of a hydrogel, to which the synthesized immunogenic peptide is attached.
  • the vaccine formulation is administered orally, such that it enters the gastrointestinal tract.
  • the vaccine is preferably provided in the form of an enteric-coated tablet in order to provide protection against the low pH of stomach fluids and gastric enzymes.
  • the enteric coating comprises at least one pH dependent polymer, such as, for example, cellulose acetate phthalate (CAP); hydroxypropyl methylcellulose phthalate (HPMCP); polyvinyl acetate phthalate; cellulose acetate trimellitate; polymethacrylic acid methyl methacrylate or ethyl methacrylate, such as the various types of Eudragit; and hydroxypropyl methylcellulose acetate succinate (HPMCAS).
  • pH dependent polymer such as, for example, cellulose acetate phthalate (CAP); hydroxypropyl methylcellulose phthalate (HPMCP); polyvinyl acetate phthalate; cellulose acetate trimellitate; polymethacrylic acid methyl methacrylate or ethyl methacrylate, such as the various types of Eudragit; and hydroxypropyl methylcellulose acetate succinate (HPMCAS).
  • CAP cellulose acetate phthalate
  • HPMCP hydroxypropyl methylcellulose
  • the vaccine formulation Upon reaching the small intestine the vaccine formulation is released from the tablet and the immunogenic peptide may be taken up by the Peyer' s patches. These are aggregations of lymphoid tissue, covered by an epithelium which contains specialized cells called microfold cells (M-cells) which sample antigen directly from the lumen of the gastrointestinal tract. The peptides are then screened and recognized as a foreign body, such that antibody production is elicited. M- cell uptake of macromolecules is depicted in Figure 5.
  • Figure 5A presents an 'antigen-eye' view of the luminal surface of intestinal ephithelium
  • Figure 5B is a cross-representation through the epithelium.
  • enterocytes Most nutrient-absorbing enterocytes have special membranes covered with microvilli!, glycocalyx and mucopolysaccharide gels.
  • Goblet cells (G) secrete mucouos in tandem with protective ion and fluid secretion by enterocytes.
  • M-cells are interspersed between enterocytes in the follicle-associated epithelium. Projections from dendritic cells cross intercellular junctions to span the luminal and sub-epithelial spaces. For simplicity, this representation ignores the complicated architecture of the gut wall.
  • Soluble antigens may be taken up by projections that dendritic cells extend into the intestinal lumen, as represented in Figure 6.
  • Figure 8 is a representation of a portion of the small intestine, showing the M-cells and Peyer's patches.
  • the methods of the present invention and products thereof are also particularly useful in preparation of vaccines since in contrast to standard methods which require a step of conjugation to a protein carrier following peptide purification by HPLC, the biocompatible polymer acts as a carrier.
  • a carrier with adjuvant properties may be used.
  • Alginate is particularly useful as the biodegradable and biocompatible polymer, since it is known to act as an adjuvant to enhance the effect of an immunogenic protein and to act as a peptide carrier.
  • a vaccine comprising a cholera toxin B subunit derived peptide.
  • the methods of the present invention do not require and preferably do not include a purification step following synthesis of the peptide, therefore an overall higher peptide load may be required in order to compensate for loss of material due to formation of a certain amount of peptides which are shorter than those required, as compared to standard synthesis methods.
  • the overall cost of the methods of the present invention is not higher than that of standard methods, since cost efficiency is increased due to the fact that no purification step is used; no separate protein carrier is required and hence also no process of linking the peptide to a carrier; and no external adjuvant is required.
  • the SPPS reactors were purchased from Torviq (Minnesota, USA).
  • the AAA method was Waters Acc Q Tag.
  • ⁇ -NMR Proton nuclear magnetic resonance
  • FTIR Fourier transform infrared spectroscopy
  • the spectrophotometer used was Ultrospec 2100 pro.
  • the Syringe pump used for the bead preparation was Kd-Scientific.
  • Alginate beads were produced by dripping the alginate solution into a 1M calcium chloride solution. For each SPPS batch, 0.0075 grams of dry alginate was used (1.5ml of alginate solution).
  • the beads were then transferred into an SPPS syringe reactor and washed with DMF. The washing process was repeated a number of times and the syringe placed on a shaker after each wash, in order to remove any remnants of water.
  • aqueous solution of sodium alginate (M/G ratio of 1.56, molecular mass ranging between 80-120kDa) was prepared by dissolving the alginate in distilled water (dw). The suspension was stirred overnight (overnight) in a rotary shaker at room temperature (RT).
  • alginate beads For the preparation of alginate beads, a sodium alginate solution was placed in a syringe which was pressed using a syringe pump through a needle (32G). The beads' size was controlled by applying a coaxial stream. Droplets of alginate solution were immediately immersed into a CaCl 2 aqueous solution (1 M) leading to bead formation. The beads were kept for 30 minutes in the solution. The beads' diameter was measured using a ruler, and averaged about 1mm.
  • the beads made from 2 ml alginate solution, were filtered and placed in a reactor. Beads were washed twice with DMF (2ml, Mercury), the washing procedure including filtering out the CaCl 2 aqueous solution, adding 2ml DMF and putting the reactor on a shaker for 15 minutes at room temperature. Beads were kept in 3ml DMF at 4°C for preservation.
  • DMF 2ml, Mercury
  • Alginate carboxylic groups were activated by contacting the alginate beads produced in Example 1 with 0.3mmol DCC, 0.3mmol HOBt in the syringe reactor for 3 hours. 3mmol of Boc-ethylene-diamine was then added to the reactor, and the beads were shaken for an additional 3 hours. The procedure was repeated twice. The beads were then washed three times with DMF.
  • the mixture was set aside for 1.5 hours and the tube was then spun down in order to remove dicyclohexylurea (DCU) as a white precipitate.
  • DCU dicyclohexylurea
  • the supernatant containing the active ester was added to the syringe reactor, and was shaken for 4 hours. This amino acid activation process was repeated once, with shaking overnight. The next day, the beads were washed 5 times with DMF. Following each wash, the syringe was shaken for 10 minutes.
  • This linker was synthesized as follows. Briefly, a solution of di-tert-butyl dicarbonate (2.45 g, 0.011 mol) in dioxane (30 ml) was added over a period of 21 ⁇ 2 hours to a solution of 1,2- diaminoethane (5.25g, 0.087mol) in dioxane (30 ml). The mixture was stirred for 22 hours at room temperature. The dioxane was then evaporated in vacuum, and 50 ml of doubly deionized water (ddw) was added. The insoluble bis-substituted diamine was removed by filtration using a Biichner funnel. The filtrate was extracted with DCM (3x150 ml). The DCM layer was separated, dried over Na 2 S04 filtered and evaporated to dryness, providing 1.06gr (60%) as an oil, verified by ⁇ -NMR.
  • the linker was coupled to the beads as described below.
  • the linker was coupled with EDC, in two solvent systems; DMF/ddw (1 : 1) and 0.1M 2- morpholinoethanesulfonic acid (MES) buffer pH 6.
  • EDC 0.2 mmol, 31mg
  • Sulfo-NHS O.lmmol, 21.7 mg
  • NHS was added instead of sulfo-NHS.
  • the reaction was shaken for 90 minutes followed by the addition of EDA-Boc linker (0.5 mmol, 79 ⁇ ) and kept shaking for an additional 6 hours at room temperature.
  • the resin was then washed twice with the reaction solvent to remove the urea by-products. The procedure was repeated, this time the reaction was kept shaking overnight.
  • the linker was also coupled with HBTU. DIEA (0.4 mmol, 69.5 ⁇ ) and HBTU (0.2 mmol, 75.8mg) were added to the reactor for activation of the alginate carboxylic groups. 3 ml of fresh DMF were added, the activation was left for 30 minutes on the shaker before adding the EDA-Boc linker (0.5 mmol, 79.1 ⁇ ). The pH was adjusted with DIEA to be approximately 8, the reactor was then shaken for 90 minutes. The resin was washed with DMF once and the procedure repeated.
  • the linker was also coupled to the beads using HBTU.
  • DIEA 0.4 mmol, 69.5 ⁇
  • HBTU 0.2 mmol, 75.8mg
  • EDA-Boc linker 0.5 mmol, 79.1 ⁇
  • the pH was adjusted with DIEA to be approximately 8, the reactor was then shaken for 90 minutes.
  • the resin was washed with DMF once and the procedure repeated.
  • the linker was also coupled to the beads using PyBOP. Unlike HBTU coupling, here, the activation was done in the presence of the amine (see 1.1.4.3). For activation, 2ml of DMF, NMM (0.3 mmol, 33 ⁇ ) ,PyBOP (0.2 mmol, 104 mg) and the linker (0.5 mmol, 79.1 ⁇ ) were added to the reactor containing beads. The pH was adjusted with NMM to be approximately 8, the reactor was shaken for 90 minutes at room temperature. The resin was washed with DMF once and the procedure repeated itself.
  • the reaction was shaken for 90 minutes, centrifuged at 2,000 rounds per minute (RPM) for 10 minutes, the upper solvent (without the urea precipitate) was added to the reactor containing the beads that were kept shaking for an additional 6 hours at room temperature, followed by washing the resin twice with the reaction solvent to remove the urea by-products. The procedure was repeated, this time the reaction was kept shaking overnight at room temperature.
  • RPM rounds per minute
  • DIEA (0.4mmol, 69.5 ⁇ 1) and HBTU (0.2mmol, 75.8mg) were added to a tube containing the amino acids (0.21 mmol) solvated in DMF (3 ml). The reaction was left shaking at room temperature, the mixture was added to the reactor containing the beads. The reaction pH was checked to be around 8, and DIEA was added as needed. Once adjusted, the reactor was shaken for 90 minutes. The resin was washed with DMF once and the procedure repeated.
  • NMM (0.3 mmol, 33 ⁇ ) ,PyBOP (0.2 mmol, 104 mg) and the amino acid (0.3 mmol) were added to the reactor containing the swollen beads in 2ml DMF. The pH was checked to be around 8, and NMM was added as needed. Once adjusted, the reactor was shaken for 90 minutes at room temperature. The resin was washed with DMF once and the procedure repeated.
  • the solvents were removed from the reactor containing the beads, and cold HC1 was then added (4 ml) to the reactor and it was shaken for 15 minutes at room temperature.
  • the acid was filtered and DMF was then added, the reactor was vortexed and left on the shaker for 15 minutes at room temperature, and the DMF step wash was repeated one more time.
  • the alginate beads were tested using the Kaiser test, a negative (yellow) result was expected, the washing procedure repeated until the results were negative.
  • the alg-EDA-Wang resin was synthesized (HMPA linker was purchased from Iris- biotechGmbH, Germany) to test the peptide created in the new assays created for peptide synthesis on an alginate resin.
  • HMPA linker was purchased from Iris- biotechGmbH, Germany
  • the wang linker was attached to the alg-EDA bead through an amide bond using the same procedure described above for amino acid coupling.
  • the remaining procedures, including peptide synthesis, were performed as described above.
  • the Fmoc groups were removed as described herein.
  • the beads were washed twice with DMF for 15 minutes each time.
  • 3 ml of Piperedine/DMF solution (1 :4 v/v) were added to the reactor containing the beads.
  • the reactor was left on the shaker at room temperature for 10 minutes, washed once with DMF, and Piperedine/DMF solution was added once more for 15 minutes.
  • a sample was taken for a Kaiser test, a positive (blue) result was expected, and the washing procedure was repeated until positive results.
  • the beads were washed twice with DMF for 15 minutes and shaken at room temperature each time.
  • a general washing procedure which was also used after synthesis was complete, was performed as follows. The beads were washed, twice with cold HC1 solution (5%, 4°C), vigorously shaken with a vortex apperatus, and the reactor containing the beads was left on the shaker for 10 minutes at room temperature.
  • the peptide needs to be cleaved from the resin, including for performing various analytic methods to determine the extent of peptide synthesis.
  • 2ml of a mixture containing 90% TFA, 5% ddw (double distilled water) and 5% triethylsilane was added and the syringe shaken for 2 hours.
  • the liquid was drained.
  • the beads were washed and shaken for 10 minutes with the following solvents: 3 times with DMF, 3 times with DMF/ddw (1: 1 volume ratio) and 3 times with ddw.
  • the liquid was drained and fresh ddw was added.
  • the syringe containing the beads was left on a shaker for 24 hours, and the ddw replaced by fresh ddw.
  • the dipeptide Lys-Ala was successfully synthesized using the above described linker and the above described coupling methods. It was proved that the peptide was successfully synthesized by performing AAA (amino acid analysis).
  • AAA amino acid analysis
  • the peptide was hydrolyzed with 6 molar (M) hydrochloric acid at 120°C for at least 12 hours to liberate the amino acids.
  • M molar
  • the hydrolysis was performed inside a sealed, evacuated tube to minimize the destruction of amino acids, which are sensitive to oxygen.
  • the amino acids were subjected to HPLC separation, and then derivatized for detection at 254 nm. In addition, Gin and Asn are converted into Glu therefore only 16 of the amino acids are routinely detectable using this method (due to the hydrolysis effect).
  • the results of AAA are presented as a HPLC plot, peak areas versus amount for standard amino acids.
  • Figure 9 shows the peak results after synthesis using the above described PyBOP method (similar results were obtained using DCC/ HOBt, HBTU/DIEA and EDC/NHSS); circles indicate Lys and Ala.
  • Each amino acid is recognized by its typically relative time (RT). From the amount (pmol) of each amino acid in the peptide, the ratio between them can be calculated.
  • the results for synthesis with PyBOP provided a ratio of Ala/Lys of 1.14:1 which is close to unity.
  • the tripeptide Gly-Lys-Ala was successfully synthesized using the above described linker and the above described coupling method with the DCC/HOBt method. It was proved that the peptide was successfully synthesized by performing AAA as described above.
  • Figure 10 shows the peak results after synthesis.
  • the AAA results showed that the ratio of the amino acids Gly,Lys,Ala was 1 :1.4:2.3 respectively. This was a logical ratio because as we progressed in the reaction the next amino acid would have more steric hindered, the yield decreases and therefore the amount of each amino acid decreased with every step.
  • the peptide CTP has 15 amino acids, with a sequence of VEVPGSQHIDSQKKA. It was synthesized using PyBOP and HBTU chemistry as described above, the beads were then disassembled, dialyzed and tested by Edman degradation. The results are shown in Table 1 below. The PyBOP synthesized peptide matched 12 out of the 15 amino acids, while the HBTU peptide matched 8 amino acids out of the 15 amino acids. However, it is possible that the last amino acids did not match because of failure of the Edman degradation technique to successfully detect these last amino acids; hence it is highly likely that the 15 amino acid peptide was in fact successfully synthesized.
  • Table 2 shows the AAA summary results of CTP3, synthesized using PyBOP chemistry, showing the number of times each amino acid appears in the sequence and the analysis ratio of the amounts according to the AAA.
  • AAA measures the amounts of Glu and Gin together.
  • the AAA results show the amounts of the different amino acids.
  • the calculated ratio between the amino acids should be proportional to the number of times they appear in the sequence.
  • the ratio was proportional besides two exceptions; a high ratio of the amino acid Ala, and a small ratio of Ser.
  • the high amount of Ala might be because it is the first amino acid to be synthesized, therefore subjected to less steric hindrance.
  • the small ratio of Ser is caused by its degradation under the method's vigorous conditions. Concerning these assumptions, the results indicate that the peptide synthesis succeeded.
  • Example 14 Immunization with a cholera toxin B subunit derived peptide.
  • Peroral immunization of rabbits Control samples of sera are taken prior to immunization. Cimetidine is injected to neutralize the gastric acid of the stomach, and the animals are gavaged with 5% sodium bicarbonate and then with the antigenic peptide, such as the above described CTP3, dissolved or suspended in 5% sodium bicarbonate. In order to induce hypoperistalsis, either paregoric or loperamide drugs are given to cause a reduction of the normal peristalitic mechanism by inhibiting intestinal motility. Blood is collected at different times following immunization.
  • Direct ELISA Antibodies from serum are detected using a standard ELISA with cholera toxin used for capturing antigen, and an anti rabbit IgG/IgM or IgA conjugated to HRP is used for detection.
  • mucosal physiological fluids such as milk and intestinal fluids may be tested for assessment of vaccine potential.
  • V. cholerae Maintenance of bacterial strains: V. cholerae is grown in lysogeny broth and preserved for long term storage by adding 15% glycerol to an overnight culture.
  • Ligated ileal loop assay A piece of small bowel of anesthetized, vaccinated and unvaccinated, rabbits that were put on a liquid diet is exposed and spacer loops are tied. V.cholerae (or PBS as control) is injected into the large loops then a second tie is made to isolate the site of injection. Rabbits are sacrificed 18-20 hours later, and the loops are tested for their fluid accumulation and contents.

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Abstract

La présente invention concerne des procédés améliorés de synthèse peptidique en phase solide au moyen de billes biodégradables biocompatibles, ainsi que des utilisations desdits procédés.
PCT/IB2012/050413 2011-01-30 2012-01-30 Procédés de synthèse peptidique en phase solide de fmoc sur support biodégradable et utilisations desdits procédés WO2012101612A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018113802A1 (fr) 2016-12-22 2018-06-28 Contipro A.S. Préparation médicale comprenant un support à base d'hyaluronane insoluble dans l'eau conjugué à des acides aminés ou des peptides, son procédé de préparation et son utilisation
CN112011161A (zh) * 2020-09-07 2020-12-01 河南应用技术职业学院 一种复合生物可降解合成高分子材料及其加工方法

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Publication number Priority date Publication date Assignee Title
CA2126132A1 (fr) * 1994-06-17 1995-12-18 Witold Neugebauer Synthese de peptide sur chitosane

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CA2126132A1 (fr) * 1994-06-17 1995-12-18 Witold Neugebauer Synthese de peptide sur chitosane

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MORGAN SUZANNE M ET AL: "Alginates as drug carriers: Covalent attachment of alginates to therapeutic agents containing primary amine groups", INTERNATIONAL JOURNAL OF PHARMACEUTICS, ELSEVIER BV, NL, vol. 122, no. 1-2, 1 January 1995 (1995-01-01), pages 121 - 128, XP002508776, ISSN: 0378-5173, DOI: 10.1016/0378-5173(95)00059-R *
NEUGEBAUER W ET AL: "PEPTIDE SYNTHESIS ON CHITIN", INTERNATIONAL JOURNAL OF PEPTIDE AND PROTEIN RESEARCH, MUNKSGAARD, COPENHAGEN, DK, vol. 47, no. 4, 1 April 1996 (1996-04-01), pages 269 - 275, XP000582513, ISSN: 0367-8377 *
PALMIERI G ET AL: "Peptide immobilization on calcium alginate beads: applications to antibody purification on assay", JOURNAL OF CHROMATOGRAPHY B : BIOMEDICAL APPLICATIONS, ELSEVIER SCIENCE PUBLISHERS, NL, vol. 664, no. 1, 3 February 1995 (1995-02-03), pages 127 - 135, XP004043714, ISSN: 0378-4347, DOI: 10.1016/0378-4347(94)00353-7 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018113802A1 (fr) 2016-12-22 2018-06-28 Contipro A.S. Préparation médicale comprenant un support à base d'hyaluronane insoluble dans l'eau conjugué à des acides aminés ou des peptides, son procédé de préparation et son utilisation
CN112011161A (zh) * 2020-09-07 2020-12-01 河南应用技术职业学院 一种复合生物可降解合成高分子材料及其加工方法

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