Solid-phase synthesis in a aqueous solution

Abstract

An aqueous solution of a protected amino acid comprising a Fmoc protected amino acid, a solubilising basic compound and water, wherein the solubilising basic compound is selected from an amine or prec

Description

SOLID-PHASE SYNTHESIS IN AQUEOUS SOLUTION This invention relates to a solid- phase synthesis method in aqueous solution for the production of an oligomer particularly a peptide or protein, using Fmoc-protected amino acids. The invention is particularly concerned with a method of synthesizing a peptide by dissolving Fmoc-protected amino acids in an aqueous solvent or phase and reacting the amino acids to form a peptide. Peptides and polypeptides are used in a large number of fields and in large quantities and have contributed enormously to the advance of chemical and biological science and have profoundly impacted the development of the modern pharmaceutical industry. Their high specificity and low toxicity profiles in humans and animals provides significant benefits compared to small molecule therapeutics. Growth in the peptide market is significantly outstripping that in the overall pharmaceutical market due to an increased number of therapeutic targets and improved delivery methodologies. Large numbers of therapeutic peptides are commercially available with hundreds in clinical phases and advanced pre-clinical stages. Worldwide sales are in the scale of tens of billions of dollars. Accordingly, there is a huge and growing world-wide demand for peptides The term “peptide” as used herein includes any organic compound which includes two or more amino acid residues bonded together by amide bonds, also known as peptide bonds, and are typically formed by a condensation reaction of a carboxyl group of one amino acid and an amine group of another amino acid and includes shorter peptide chains of amino acid residues having two or more amino acid residues including dipeptides and oligomers of amino acid residues having shorter peptide chains, and polypeptides having longer peptide chains of amino acid residues and fragments of peptides or polypeptides. The amino acid residues may be natural amino acids or artificial amino acids. Peptides may be made by a number of different processes including solution phase chemistry, solid phase chemistry and enzymatic chemistry or a combination of these methods. In these processes, protected amino acids are required for the synthesis of the peptides, such as urethane protected amino acids. Examples of the N-urethane protecting group include terf-butyloxycarbonyl (Boc), benzyloxycarbonyl (Z), ethanesulfonylethoxycarbonyl (Esc) and fluorenylmethyloxycarbonyl (Fmoc) with Fmoc being the most commonly used protecting group, especially for solid phase synthesis. Peptide synthesis has for many decades been carried out in an organic solvent as the protected amino acids typically employed, are insoluble in water. Organic solvents such as N,N’-dimethyl formamide (DMF), N,N’-dimethylacetamide (DMA), N-methylpyrrolidone (NMP) are well known for use in peptide synthesis. There are two common strategies for solid phase peptide synthesis. In one approach a difference in lability to acids of the Na protecting group and side chain protecting groups is utilised. The most commonly employed approach is termed Boc-chemistry. In another approach the Na protecting group is orthogonal to the side chain protection and the most common approach of this type uses the base labile Fmoc group as the Na protecting group. Other orthogonal protecting group strategies include Esc and Smoc (2,7-disulfo-9-fluorenylmethoxycarbonyl) protection. This invention is concerned with a solid- phase synthesis method in aqueous solution for the production of an oligomer, particularly a peptide or protein, using Fmoc protected amino acids. Peptides have traditionally been made predominantly by solid phase synthesis methodology for decades. In solid phase synthesis the carboxyl terminal of the growing peptide chain is anchored to a solid support by a cleavable linker and the peptide is assembled by sequential addition of protected amino acids to the amino acid chain anchored to the support. In solution phase peptide synthesis, the growing peptide chain is retained in solution in organic solvents and excess reagents or by-products are removed by extraction into an immiscible solvent. The concept of liquid phase synthesis on soluble polymers was originally introduced by Manfred Mutter et al in the 1970s (J.Am.Chem.Soc. 1974, 96, 23, 733-736). More recently this concept was further developed by Ajinomoto in AJIPHASE® technology and Bachem Ag with Molecular Hiving™ where a soluble anchor (or tag) is used to hold a peptide in solution in organic solvent. The peptide is grown by sequentially adding protected amino acids to the growing peptide chain. DMF has widespread use in peptide synthesis and is known to present serious environmental and health hazards including liver and developmental effects and reproductive harm. In 2023, The European Commission published a regulation restricting the amount of DMF that can be used in many industrial applications in the Ell with a view to lowering the levels of DMF at workplaces to reduce the risks to workers associated with use of DMF. The European Commission is also considering projects to control usage of other solvents such as restrictions of dichloromethane and NMP, both of which may be used in peptide synthesis. However, organic solvents are generally undesirable on environmental and public health and safety grounds and the large and growing demand for peptide products places ever greater need on developing alternative synthesis processes, large quantities and concerns about public health and safety. The environmental impact of using organic solvents may be significant and highly undesirable due to the need to manage waste streams containing such solvents and avoid such materials passing into water courses. Often, such waste streams containing organic solvents are processed by incineration, itself an environmentally undesirable process. Solid phase synthesis typically involves stepwise assembly of the peptide bound on an insoluble solid support in a mechanically agitated reaction vessel. Each amino acid is added by repetitive cycles of amino acylation and N-terminal deprotection. At the end of assembly, the peptide-resin is subjected to acid cleavage to release the crude peptide for purification and characterization prior to use. Fmoc-amino acids with suitably protected side chains are prepared on a multi-tonne scale and used to produce the majority of peptide-based drugs, currently in production. Fmoc-amino acids are also used routinely in the discovery and development of therapeutic and cosmetic peptides on a small scale using automated, semi-automated and manual peptide synthesis. A pressing and major need exists to reduce the reliance on organic solvents in of peptide synthesis. We have now devised a method of providing a N-alpha-urethane Fmoc-protected amino acid, dissolved in an aqueous solution, particularly in water, by providing a salt of the protected amino acid with a solubilizing basic amine. The salt is soluble in aqueous solution and enables the protected amino acids to be used in further synthetic processes including assembling peptides in water which are anchored to a soluble anchor or tag without needing to use exclusively an organic solvent such as DMF. In a first aspect, the invention provides a method of producing an aqueous solution of an Na-Fmoc-protected amino acid comprising contacting a protected amino acid with a solubilizing basic compound selected from an amine, a quaternary ammonium compound, a precursor of an amine, a precursor of a quaternary ammonium compound and mixtures thereof in an aqueous solvent to produce a dissolved salt of the protected amino acid. In a second aspect, the invention provides an aqueous solution comprising an Na-Fmoc-protected amino acid and a solubilizing basic compound selected from an amine, a quaternary ammonium compound, a precursor of an amine, a precursor of a quaternary ammonium compound and mixtures thereof, and water. The invention also provides for the use of an aqueous solution comprising an Na-Fmoc-protected amino acid according to the invention in the production of a peptide or a peptidomimetic. As employed herein, the terms ‘soluble’, ‘solubilized’, ‘dissolved’ and similar iterations refer to a visually clear homogenous mixture where the solute cannot be separated from the solvent by mechanical means. The dissolved an Na-Fmoc-protected amino acid may be employed in the synthesis of peptides in aqueous solid phase synthesis without needing to use exclusively an organic solvent such as DMF. In a third aspect, the invention provides a method of synthesising a peptide or a peptidomimetic comprising: i) providing an Na-Fmoc protected amino acid dissolved in an aqueous solvent according to the first aspect of the invention; ii) optionally adding the dissolved Na-Fmoc-protected amino acid to a solid support containing a terminal amine in order to ion-exchange with the solid support to form a salt of the Na-Fmoc-protected amino acid with the terminal amine on the solid support and removing Fmoc from the protected amino acid; iii) providing at least one further dissolved Na-Fmoc protected amino acid or Fmoc protected peptide and reacting the further dissolved Na-Fmoc protected amino acid with the de-protected amino acid to form a peptide bond; and iv) optionally deprotecting the Na-Fmoc protected amino acid in step iii) and providing further dissolved Na-Fmoc protected amino acids or Fmoc protected peptide and reacting with the deprotected product of step iii) to form a further peptide bond in the sequence of the desired peptide. Suitably, the further dissolved Na-Fmoc protected amino acid ion exchanges with the amine on the solid support. Suitably, the further dissolved protected amino acid ion exchanges with the amine on the solid support. Suitably, the peptide is synthesized by contacting the dissolved Fmoc-protected amino acid solution with the solid particulate support for peptide synthesis in a solid phase process comprising passing a mixture comprising solid phase particles and a liquid component comprising an aqueous solution of one or more protected amino acids through a confined, contact zone having an inlet and an outlet and between the inlet and outlet, a permeable barrier which is permeable to liquids and wherein the solid and liquid component interact and a portion of the liquid component(s) or a liquid product of the interaction between the solid and the liquid component(s) passes through the permeable barrier. Suitably, the synthesis process is carried out in a solid phase process contact apparatus comprising a contact vessel having an inlet for receiving a mixture comprising solid phase particles and one or more liquid components and an outlet for removing the solid phase particles from the contact vessel which provides a confined chamber defining a contact zone between the inlet and outlet wherein at least part of the chamber wall comprises a permeable barrier which is permeable to the liquid component and a pump to convey the mixture into the contact chamber. The said further Fmoc-protected amino acids may be the same or different to other amino acids in the peptide. The terms “protected” and “protecting” refer to the known technique of reacting the Na-terminal of the amino acid with a Fmoc protecting group. After the Fmoc-protected amino acid is coupled to the peptide which is being synthesized and washing, the protecting group of the newly coupled amino acid is removed in preparation for coupling of the next protected amino acid in the sequence. The coupling and washing process as known in the art is then repeated for each successive amino acid until the desired peptide or peptidomimetic is obtained. The peptide is accordingly assembled in a stepwise manner known to those skilled in the art. In a fourth aspect, the invention provides a method of synthesising a peptide or a peptidomimetic comprising: i) contacting a Fmoc-protected amino acid with a solubilizing basic compound selected from an amine, a quaternary ammonium compound, a precursor of an amine, a precursor of a quaternary ammonium compound and mixtures thereof in an aqueous solvent to produce a dissolved salt of the Fmoc-protected amino acid and deprotecting the amino acid; ii) providing at least one further dissolved Fmoc- protected amino acid or Fmoc protected peptide in aqueous solution, the dissolved Fmoc-protected further amino acid having been produced according to the first aspect of the invention, and reacting the further dissolved Fmoc- protected amino acid or Fmoc protected peptide with the deprotected amino acid to form a peptide bond; and iii) optionally deprotecting the Na-Fmoc protected amino acid in step iii) and providing further dissolved Fmoc-protected amino acids or Fmoc protected peptide, produced according to the first aspect of the invention, and reacting with the product of step ii) to form a further peptide bond in the sequence of the desired peptide. The invention allows for removal of excess protected amino acids by extraction from the solution, for example, into organic solvent. The method of synthesising a peptide or a peptidomimetic suitably comprises a step of extraction of a component from the reaction mixture, preferably by contacting the reaction mixture with an organic solvent which is immiscible with aqueous solution. The protected group may be removed in a conventional manner including by the addition of a secondary amine base, for example piperidine. Excess reactant, for example excess Na-Fmoc-protected amino-acid and other reagents, by-products, adducts, or other unwanted materials, for example dibenzofulvene and dibenzofulvene adducts may be removed from the reaction mixture. Suitably, such materials are removed by contacting an organic solvent, preferably a hydrophobic organic solvent, for example 2-methyltetrahydrofuran, which is immiscible with the aqueous phase. Suitably, the organic solvent and the aqueous phase are immiscible and the unwanted materials are extracted at the interface of the immiscible phases. The organic solvent containing the extracted by-products, excess reactants or other unwanted materials may be processed to deplete the level of those materials or, preferably to remove them substantially, allowing the organic solvent to be recycled and reused in the peptide synthesis method, thereby reducing the level of organic solvent employed in the process. Suitably, the solubilizing basic compound has a lower pKa than the pKa value of the amine pendant on the solid support upon which the peptide is synthesized. As the growing peptide chain is assembled in an aqueous phase, the higher pKa value of the amine pendant on the solid support compared to the solubilizing basic compound, described below, allow for ion-exchange, effectively immobilizing the Na-Fmoc-protected amino acid on the solid support. Immobilization by ion-exchange may be confirmed experimentally by UV analysis. The peptide synthesis step may be carried out on a solid phase by chemical means or by enzymatic means using enzymes in solution or immobilised enzymes. Depending upon the circumstances it also allows for a combination of both chemical and enzymatic processes for peptide bond formation on the same peptide assembly. The peptide synthesis step may be carried out in solution phase, solid phase, by enzymatic synthesis or a combination of these methods for different amino acids as desired. In particular, the Na-Fmoc protected amino acid may be reacted to form a peptide bond. Solid phase synthesis advantageously involves bonding a Na-Fmoc protected amino acid to a support and thereafter successively forming peptide bonds with desired amino acids to synthesise the peptide to a solid phase, with the Na-Fmoc protected amino acid being deprotected, for example, using a suitable base such as piperidine, and then coupled with a further amino acid. Solid phase synthesis permits washing and the like between successive steps, advantageously simplifying the synthesis process as compared to solution phase synthesis. The method of synthesizing a peptide may comprise a step in which an amino acid is reacted or “capped” to preclude further reaction at that site. The term “solubilizing compound” refers to a compound which, upon contact with the protected amino acid in aqueous solution, preferably water, provides a solution which is preferably clear. Without wishing to be bound by any theory, it is believed that the solubilizing compound may form a salt or another form of interaction, association or conjugation, for example hydrogen-bonding with the protected amino acid which may provide a hydrophilic cage type structure to facilitate or improve solubilization of the protected amino acid in aqueous solution. The invention encompasses variants irrespective of whether or not solubilization is achieved by this hypothetical method of action. In other words, the invention encompasses variants whether or not they work in the same way as this hypothesis. The amino acids are Na-Fmoc-protected amino acids. Suitably, the Fmoc-protected amino acid is activated by contacting in aqueous solution with an activating agent, suitably selected from 1-hydroxybenzotriazole (HOBt), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI), (4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride) (DMTMM), optionally with an activating agent such as HOBt. Suitably, for activation, equimolar amounts of EDCI and HOBt are dissolved in water prior and left for 15 to 30 minutes prior to addition to the solid support. Whilst ethyl cyanohydroxyiminoacetate, for example available under the trade name Oxyma™, may be employed as an activating agent, the risk of evolution of hydrogen cyanides requires extremely careful use. DMTMM releases N-methyl morpholine (NMM) upon activation which may be employed as the solubilizing basic compound. The invention provides in a further aspect a peptide or a peptidomimetic obtainable and preferably obtained by a method according to the invention. Advantageously, the method according to the invention enables peptides to be produced with the Fmoc-protected amino acid being at a higher concentration in the aqueous solvent than typically achievable in organic solvents. Accordingly, the invention affords benefits as regards yield and efficiency in addition to environmental and health and safety benefits. Suitably, the solubilizing basic compound has a lower pKa than the pKa value of the amine pendant on the solid support upon which the peptide is synthesized. The amine or quaternary ammonium compound should be basic. Preferably the amine which is capable of forming an ammonium analogue of the amine and preferably comprises a tertiary amine. The amine may be aliphatic, aromatic, cyclic or polymeric. The amine may comprise a linear or branched amine comprising one or more amine groups, preferably including at least one tertiary amine; a heterocyclic compound including a 5-membered or 6-membered heterocyclic ring, including morpholine derivatives, piperazine derivatives, a bismorpholine, and a bispiperazine; a 5-membered or 6-membered aromatic ring. The amine may include other heteroatoms, for example oxygen and sulphur, and / or be optionally substituted with functional groups, for example hydroxyl, thiol and further amine groups. Examples of suitable amines include N-methylmorpholine (NMM), N-hydroxyethyl morpholine, 4,4'-(oxydi-2,1-ethanediyl)bismorpholine, N-ethylmorpholine (NEM), 4-(3-hydroxypropyl)morpholine, 4-(3-hydroxypropyl)morpholine (HEM) N,N-dimethylaminoethanol (DMAE), triethanolamine, N,N’-dihydroxyethyl piperazine, In a preferred embodiment, the solubilizing compound is selected from an amine compound which comprises a compound comprising a 6-membered heterocyclic ring of formula (II) which may be substituted at any of the carbon atoms in the heterocyclic ring: .......................- / Y (II) wherein: X is independently selected from -NZ’- and -N+Z’Z”-; Y is independently selected from -NZ’-, -N+Z’Z”-, O and S; Z’ Z” are independently selected from Ci to C22, (CH2)2-12N R1R2, (CH2)2.12ORi, (CH2)2.12SRi and a divalent ether group linking two compounds of formula (II); Rt and R2 are as defined above or an immobilizing solid of formula -C(O)CH2[solid] wherein [solid] represents a water compatible solid support. Preferably, Z’ and Z” are selected from (CH2)2.12N R1R2, (CH2)2.12ORi and (CH2)2.i2SRi. Examples of preferred compounds of formula (II) are N-methyl morpholine, N-ethyl morpholine, N-hydroxyethyl morpholine, triethanolamine and compounds of the following formulae and their amine or ammonium analogues: wherein the solid circle is a solid for immobilizing the amine Morpholine derivatives having a substituent bonded to the nitrogen atom in the morpholine ring are preferred, preferably morpholines having an optionally substituted 15 hydrocarbyl group bonded to the N atom of the morphine, especially where the hydrocarbyl group has 1 to 4 carbon atoms. The optionally-substituted hydrocarbyl group may be substituted with any suitable group including hydroxy and amine. The carbon atoms of the morpholine ring may be substituted. The morpholine derivate may be linked to a solid support. Examples of especially preferred morpholine derivatives are N-methylmorpholine and N-ethylmorpholine and substituted analogues thereof. Precursors of the morpholine derivatives, for example N-methyldiethanolamine (used to manufacture NMM) and triethanolamine (used to manufacture HEM) may be employed. The solid may be any suitable known solid support employed in synthetic chemistry for use in an aqueous environment and is suitably hydrophilic. Examples of suitable solids particles or supports are described in GB2473814, WO2011 / 032703, WO2011 / 032704, WO2011 / 032704, WO2011 / 032705, WO2012 / 143508, WO2013 / 041250 and WO2016 / 139322. Preferred solid supports include self-assembled microparticles available under the trade name SpheriSomes® and cross-linked poly-e-lysine supports which may be particulate or non-particulate available under the trade name SpheriTide®, both from SpheriTech Ltd. Suitably, the solid phase particles comprise a cross-linked poly-c-lysine polymers, preferably cross-linked with di carboxylic acids. Preferably, the solid phase particle comprises a particulate support comprising a cross-linked poly-e-lysine polymer comprising poly-e-lysine and a cross linker linked by amide bonds wherein the cross linker comprises two or more carboxylic acid groups and an aliphatic chain linking the two or more groups adapted to react with an alpha carbon amine of poly-e-lysine. In a preferred embodiment, the solid phase particles comprise a self-assembled microparticle comprising an acid having two or more acid groups and an organic base in which the molar ratio of acid groups to basic groups in the acid and base is from 0.6 to 1.4:1, as described in WO2016 / 139322. More preferably, the solid phase particle comprises self-assembled microparticles comprising an acid having two or more acid groups and an organic base wherein the said acid comprises a compound selected from i) a compound of general formula HOOC-(CH2)n-COOH wherein n is at least 5 and not more than 40; and ii) a C7 to C13 bis carboxylic fatty acid in combination with a further acid selected from EDTA, nitrolotriacetic acid and a monocarboxylic acid; wherein the molar ratio of acid groups in the acid to basic groups in the base is from 0.6 to 1.4:1, preferably 0.8 to 1.2:1, and the microparticle comprises a multi-lamellar structure. The microparticle may be any micron scale particle size, preferably 0.1 to 100 microns, especially 0.5 to 50 microns, particularly 1 to 20 microns, for example 1 to 5 microns. The acid and organic base in the microparticle align upon contact to suitably do not react to form a particle having a multi-lamellar structure and are not covalently bonded to each other. WO2016 / 139322 further describes a process for preparing microparticles involving the self-assembly of a bis-carboxy fatty acid microparticle in water followed by crosslinking with a suitable amine. Poly-epsilon lysine is especially preferred to provide a solid phase particle suitable for use in the present invention. The selection of fatty acid determines the diameter of the resulting particle. Suberic acid (C8) provides ~0.5pm particles, azelaic acid (C9) gives ~1pm, sebacic acid (C10) produces particles of ~2pm diameter, brassylic acid (C13) gives ~3pm particles. The 2 or 3pm SpheriSome® particles afford a desirable combination of flow, reaction kinetics, handling and utility in the process of this invention. Several different peptides have been manually assembled including Leu-enkephalin, insulin B-chain, and glucagon with superior products being obtained in each case with SpheriSomes®. Suitably, the loading of the poly-epsilon lysine may be up to 2.5 mmol / g. For micron scale particles, such as SpheriSomes, having a particle size of 1 to 10 microns, a permeable barrier having a mesh size of 0.2 to 1 micron, for example 0.2, 0.5 and 0.65 microns may be suitable. In another preferred embodiment, the solid phase particles are as described in WO2012 / 143508. Suitably, the solid phase particles comprise cross-linked poly-e-lysine polymer comprising poly-e-lysine and a cross linker linked by amide bonds wherein the cross linker comprises at least two functional groups capable of reacting with an alpha carbon amine of poly-e-lysine. Preferably the cross-linker comprises two or more carboxylic acid groups and an aliphatic chain linking the two or more groups. Preferred solid supports include self-assembled microparticles available under the trade name SpheriSomes® and cross-linked poly-e-lysine supports which may be particulate or non-particulate available under the trade name SpheriTide®, both from SpheriTech Ltd. These solid supports are readily pumpable in aqueous solution at 5000 cm3 / minute in apparatus according to the invention, for example having a permeable barrier of 1cm diameter x 50cm long tubular filters at 5000 cm3 / minute without any observable damage to the particles. SpheriTide® is a solid support and, when solvated, is noticeably more mechanically stable than traditional polymer supports used for this purpose such as cross-linked polystyrene. SpheriTide® can be made in a range of particle sizes down to sub-micron if needed. The inherent mechanical stability of this support allows for it to be pumped at the very high flow rates required for TFF without damage to the particles. Suitably, the solid phase particles for solid phase peptide synthesis and solid phase oligonucleotide synthesis, the solid particle is suitably highly solvated soft particles to improve diffusion of reagents throughout the particle. Use of smaller particles also improves diffusion of reagents. In a preferred embodiment, the solid phase particles are small, preferably at least 0.1. Suitably the particles are not more than 250 microns, preferably not more than 150 microns, especially not more than 100 microns. In one embodiment, the particles may be 1 to 50 microns, for example 1 to 20 microns and are highly solvated. Advantageously, the present invention enables such particles to be readily processed and filtered whereas, small highly solvated particles would typically either not be capable of filtration using the conventional flat bed techniques or be prone to blockage and other process complications. Typically, by way of example, solid phase peptide synthesis is carried out on particles in the size range 75-150pm as dry particle size which swell when solvated in an appropriate solvent. Traditionally cross linked polystyrene is used for solid phase peptide synthesis and 1g of 75-150pm diameter particles will swell in N,N-dimethylformamide to 10cm3 so the diameter swollen particle size range will be ~120-250pm. Reagents used in solid phase peptide synthesis reach the active site by diffusion throughout the polymer matrix so the rate of reaction is limited by diffusion. The volume of a 100pm diameter bead is approximately 1 / 16th the volume of a 250pm bead so the rate of diffusion through the bead will be proportionately less. Similarly, the volume of a 50pm bead is 125 times less than a 250pm bead. At another extreme the volume of a 3pm bead will -580,000 times smaller than a 250pm bead. The Fmoc- protected amino acid and the solubilizing compound are suitably contacted at a molar ratio of 5:1 to 1:5, preferably: 3:1 to 1 to 3, especially 1.5:1 to 1:1.5, for example 1:1. The molar ratio of the Fmoc- protected amino acid and the solubilizing compound is suitably selected so as to provide an aqueous solution which is substantially clear to visual inspection. Suitably, the aqueous solution has a concentration of the Na-Fmoc-protected amino acid of greater than 0.4 mol / dm3, preferably greater than 0.8 mol / dm3 and especially at least 1 mol / dm3. In known solid phase synthesis processes in DMF, the concentration of Na-Fmoc-protected amino acid in DMF is typically about 0.4 mol / dm3. The present invention accordingly enables a higher concentration of Fmoc-protected amino acid to be employed, may provide improved process efficiency by forming a salt of the protected amino acid with the terminal amine on the solid support prior to activation in-situ while providing a desirable alternative to and reducing or avoiding the disadvantages of organic solvents. The invention is described by the following non-limiting examples. All quantities and amounts are by weight or weight percent unless otherwise specified. Example 1 A selection of Fmoc-protected amino acids as shown in Table 1, were each slurried in water. To each slurry, one mole equivalent and one molar equivalent of N-methylmorpholine (NMM) was added with stirring. In each mixture, a clear or substantially clear solution resulted, indicating that the Fmoc protected amino acid had been solubilized by the addition of NMM. The pH of each solution was measured and is recorded in Table 1. Table 1 - Fmoc-amino acids dissolution Fmoc Derivative Initial pH on dissolution Fmoc-Ala-OH.H2O 7.29 Fmoc-Arg(HCI)-OH 1.24 Fmoc-Arg(Pbf)-OH 7.50 Fmoc-Asn-OH 8.00 Fmoc-Asp(OtBu)-OH 7.10 Fmoc-Cys(Acm)-OH 7.19 Fmoc-GIn-OH 7.98 Fmoc-Glu(OtBu)-OH 7.40 Fmoc-Gly-OH 7.25 Fmoc-His(Boc)-OH 7.45 Fmoc-lle-OH 7.20 Fmoc-Leu-OH 7.30 Fmoc-Lys(Boc)-OH 7.25 Fmoc-Met-OH 9.45* Fmoc-Phe-OH 8.70* Fmoc-Pro-OH 7.36 Fmoc-Ser-OH 7.44 Fmoc-Ser(tBu)-OH 7.47 Fmoc-Thr-OH 7.41 Fmoc-Thr(tBu)-OH 7.31 Fmoc-Trp-OH 7.60 Fmoc-Tyr(tBu)-OH 9.16* Fmoc-Val-OH 7.28 * pH adjusted down to pH7 with MES buffer The pH of each solution was less than pH 8, indicating that Fmoc would not be removed, 5 this typically requiring a pH of 8 or higher. The stability of the dissolved Fmoc-amino acids was monitored by HPLC. The solutions were all tested and shown to be stable in solution overnight at room temperature. HPLC traces for Fmoc-Val-OH and Fmoc-Pro-OH initially and after overnight storage are shown 10 in Figures 1 and 2, indicating no material change and hence the solutions are stable. The solutions shown in Table 1 were all made at a concentration in excess of 0.4 mol / dm3 and up to 1 mol / dm3, significantly higher concentrations than typically employed in known organic solvent synthesis. The higher concentration aqueous solutions permit 15 more efficient coupling reactions than hitherto envisaged with organic phase peptide synthesis processes. Example 2 Similarly to Example 1, a selection of Na-Fmoc-protected amino acids were each slurried in water. To each slurry, one mole equivalent and one molar equivalent of triethanolamine (TEOA) was added with stirring. In each mixture, a clear or substantially clear solution resulted, indicating that the Na-Fmoc-protected amino acid had been solubilized by the addition of TEOA. It was noted that Fmoc-Phe-OH was not solubilized using TEOA but provides a clear solution using NMM. Example 3 Ion-exchange with SpheriTide Aq-M H-Pro-SpheriTide Aq-M (0.7g, 0.91mmol / g free amine) was suspended in water (10cm3). Fmoc-Leu-OH (0.353g, 1mmol) was dissolved in water (2.5cm3) containing NMM (0.111g, 1.1 mmol) and added to the H-Pro-SpheriTide Aq-M suspension. The mixture was left on a roller to mix for 1h. The resultant Fmoc-Leu-OH. H-Pro-SpheriTide Aq-M was washed with water (5x 10cm3) to remove any excess Fmoc-Leu-OH not immobilized by ion-exchange. The Fmoc-Leu-OH.H-Pro-SpheriTide Aq-M was suspended in aqueous HOI in EtOH (40cm3, 0.5mol / dm3 HOI in 1:1v / v) to displace Fmoc-Leu-OH leaving the H-Pro-SpheriTide Aq-M as a hydrochloride salt, then diluted to 50cm3. A sample of this solution was diluted 100-fold and analysed by reversed phase HPLC to compare the peak area (5927.84) with a standard sample. A standard sample of amino acid solution was prepared by diluting a 1mg / cm3 solution of Fmoc-Leu-OH (1.0cm3) to 20.0cm3. This was analysed by reversed phase HPLC. The area by HPLC of the Fmoc-Leu-OH (6469.84). By comparison of peak area, it was confirmed that 0.64mmol of Fmoc-Leu-OH was immobilized by ion-exchange. Example 4 Synthesis of Leu-enkephalin Fmoc-Leu-OH (1.4g, 4mmol) was dissolved in N,N-dimethylformamide (DMF) (10cm3) and added to 4-hydroxymethylbenzoic amide(HMBA)-SpheriTide® Aq (0.84g, 1mmol). 4-Dimethylaminopyridine (DMAP) (49mg, 0.4mmol) was added followed by diisopropylcarbodiimide (DIC) (0.8cm3, 5mmol) and the coupling left overnight to produce Fmoc-Leu-HMBA-SpheriTide® Aq. The Fmoc-Leu-HMBA-SpheriTide® Aq was washed with DMF (5x10cm3) then treated with morpholine (30cm3) for 90 minutes to remove the Fmoc group. The H-Leu-HMBA-SpheriTide® Aq was washed with aqueous NMM (5x10cm3, 5%v / v) and water (5x10cm3) then washed and dried with MeCN. Fmoc-Phe-OH (1.55g, 4mmol) was suspended in water (4cm3) and NMM (0.808g, 8mmol) added. The mixture was swirled to dissolve producing a clear solution with a concentration of approximately 0.8mol / dm3. This solution was added to the H-Leu-HMBA-SpheriTide® Aq. EDCI (1.534g, 8mmol) and HOBt (0.616g, 4mmol) were dissolved in water (4cm3) and added to the mixture. The coupling was monitored to completion by Kaiser test. The Fmoc group was removed with morpholine as described above and further Fmoc-amino acids coupled as described to produce the peptide resin, H-Tyr(Bu‘)-Gly-Gly-Phe-Leu-HMBA-SpheriTide® Aq. This peptide resin was treated with trifluoracetic acid (20cm3) for 1 h, filtered, then washed with water before washing and drying in MeCN. A sample of the peptide resin produced above (200mg) was treated with aqueous sodium hydroxide (2cm3, 0.25mol / dm3) for 1h before adding acetic acid (0.5cm3). This produced Leu-enkephalin (yield 86.4%) with a purity of 90.45% by HPLC (Figure 3). Example 5 Synthesis of Bivalirudin This 20 amino acid residue peptide was assembled on H-Leu-HMBA-SpheriTide® Aq-M (5.4g, 5mmol). Fmoc-Tyr(Bu‘)-OH (4.6g, 10mmol) was suspended in water (15cm3) and TEOA (3g, 20mmol) added. The mixture was swirled to dissolve producing a clear solution with a concentration of approximately 0.6mol / dm3. This solution was added to the H-Leu-HMBA-SpheriTide® Aq-M (5mmol) to ion-exchange with the amine on the solid support thereby forming an immobilized salt with the terminal amine. EDCI (2.4g, 12.5mmol) and HOBt (2.1g, 13.8mmol) were dissolved in water (7.5cm3) and added to the mixture. The coupling was monitored to completion by Kaiser test and shown to be complete in less than 4h. The peptide-solid support was washed with aqueous TEOA (10%w / v, 1 bed volume, 5x), water (3x1 bed volume) and MeCN (2x1 bed volume). 5 The Fmoc group was removed with morpholine saturated with ferric chloride (~1% w / v) over 1 h, then washed with MeCN (2x1 bed volume) and water (8x1 bed volume). The remaining Fmoc amino acids were coupled as described above to produce the peptide resin, H-d-Phe-Pro-Arg(Pbf)-Pro-Gly-Gly-Gly-Gly-Asn-Gly-Asp(OBut)-Phe-10 Glu^BuyGlu^Buyile-Pro-Glu^BuyGlu^BuyTy^BuyLeu-HMBA-SpheriTide® Aq- M. A sample of this peptide resin (200mg) was treated with trifluoracetic acid (5cm3) containing anisole (5%w / v) and phenol (5%w / v) for 1 h, filtered, then washed with water before washing and drying in MeCN. 15 The deprotected peptide resin produced above was treated with aqueous sodium hydroxide (10cm3, 0.1mol / dm3) for 1h before adding acetic acid (0.5cm3). This produced Bivalirudin (yield 71% from resin weight) with a purity of 84% by HPLC (Figure 4).

Claims

1. An aqueous solution of a Fmoc-protected amino acid comprising an Fmoc protected amino acid, a solubilizing basic compound selected from an amine, a quaternary ammonium compound, a precursor of an amine, a precursor of a quaternary ammonium compound and mixtures thereof, and water.

2. An aqueous solution of a Fmoc protected amino acid according to claim 1 wherein the solubilizing compound is selected from one or more of a linear or branched amine; a 5-membered or 6-membered heterocyclic amine ring.

3. An aqueous solution of a Fmoc protected amino acid according to claim 1 or claim 2 wherein the solubilizing compound is selected from a tertiary amine.

4. An aqueous solution of a Fmoc protected amino acid according to claim 1 or claim 2 wherein the solubilizing compound is selected from a 5-membered or 6membered heterocyclic amine ring;5. An aqueous solution of a Fmoc protected amino acid according to claim 4 wherein the heterocyclic amine ring is selected from a substituted morpholine, morpholine derivatives, piperazine derivatives, a bismorpholine, and a bispiperazine.

6. An aqueous solution of a Fmoc protected amino acid according to claim 1 or claim 2 wherein the solubilizing basic compound is selected from N-methylmorpholine (NMM), N-hydroxyethyl morpholine (HEM), 4,4'-(oxydi-2,1-ethanediyl)bismorpholine, N-ethylmorpholine (NEM), 4-(3-hydroxypropyl)morpholine, N,N-dimethylaminoethanol (DMAE), triethanolamine N,N’-dimethyl piperazine,7. An aqueous solution of a Fmoc protected amino acid according to claim 1 wherein the solubilizing compound is selected from an amine and a quaternary ammonium compound which comprises a compound comprising a 6-membered heterocyclic ring of formula (II) which may be substituted at any of the carbon atoms in the heterocyclic ring:--------------------...-- / Y (II)wherein:X is independently selected from -NZ’- and -N+Z’Z”-;Y is independently selected from -NZ’-, -N+Z’Z”-, O and S;Z’ Z” are independently selected from Ci to C22, (CH2)2-12N R1R2, (CH2)2. 12OR1, (CH2)2.12SRi and a divalent ether group linking two compounds of formula (II);Rt and R2 are independently selected from H, Ci to C22-8. An aqueous solution of a protected amino acid according to claim 11 wherein the compounds of formula (II) is selected from N-methyl morpholine, N-ethyl morpholine, N-hydroxyethyl morpholine, triethanolamine and compounds of the following formulae and their amine or ammonium analogues:wherein the solid circle is a solid for immobilizing the amine;R4 / A iX \ / ^r' / \ / ”wherein to R4 are independently selected from H, Ci to C22-9. An aqueous solution of a Fmoc protected amino acid according to any one of claims 1 to 8 wherein the concentration of the Fmoc-protected amino acid is preferably greater than 0.4 mol / dm3, preferably greater than 0.8 mol / dm3 and especially at least 1 mol / dm3.

10. An aqueous solution of a Fmoc protected amino acid according to any one of claims 1 to 9 wherein the protected amino acid and the solubilizing compound are present at a molar ratio of 5:1 to 1:5, preferably: 3:1 to 1:3, especially 1.5:1 to 1:1.5 and 1:

111. A method of producing an aqueous solution as defined in any one of claims 1 to 10 of a Na-Fmoc-protected amino acid comprising contacting a Na-Fmoc-protected amino acid with a solubilizing basic compound selected from an amine, a quaternary ammonium compound, a precursor of an amine, a precursor of a quaternary ammonium compound and mixtures thereof in an aqueous solvent to produce a dissolved salt of the Fmoc-protected amino acid.

12. A method of synthesising a peptide or a peptidomimetic comprising:i) providing a Fmoc-protected amino acid dissolved in an aqueous according to any one of claims 1 to 10,;ii) optionally adding the dissolved protected amino acid to a solid support containing a terminal amine in order to ion-exchange with the solid support to form a salt of the Fmoc-protected amino acid with the terminal amine on the solid support and removing the Fmoc-protection group from the protected amino acid;iii) providing at least one further dissolved Fmoc-protected amino acid or Fmoc protected peptide and reacting the further dissolved protected amino acid or Fmoc protected peptide with the de-protected amino acid to form a peptide bond; andiv) optionally deprotecting the Na-Fmoc protected amino acid in step iii) and providing further dissolved Fmoc protected amino acids and reacting with the deprotected product of step iii) to form a further peptide bond in the sequence of the desired peptide.

13. A method of synthesising a peptide or a peptidomimetic comprising:i) contacting a Fmoc protected amino acid with a solubilizing basic compound selected from an amine, a quaternary ammonium compound, a precursor of an amine, a precursor of a quaternary ammonium compound and mixtures thereof in an aqueous solvent to produce a dissolved salt of the Fmoc protected amino acid and deprotecting the amino acid;ii) providing at least one further dissolved Fmoc protected amino acid in aqueous solution, the dissolved protected further amino acid having been produced according to any one of claims 1 to 10, and reacting the further dissolved Fmoc protected amino acid or Fmoc protected peptide with the deprotected amino acid to form a peptide bond; andiii) optionally deprotecting the Fmoc protected amino acid in step iii) and providing further dissolved Fmoc protected amino acids or Fmoc protected peptide, produced according to any one of claims 1 to 10, and reacting with the product of step ii) to form a further peptide bond in the sequence of the desired peptide.

14. A method of synthesizing a peptide or a peptidomimetic comprising contacting a dissolved a Fmoc protected amino acid solution with a solid particulate support for peptide synthesis in a solid phase process comprising passing a mixture comprising the solid phase particles and a liquid component comprising an aqueous solution of one or more Fmoc protected amino acids through a confined, contact zone having an inlet and an outlet and between the inlet and outlet, a permeable barrier which is permeable to liquids and wherein the solid and liquid component interact and a portion of the liquid component(s) or a liquid product of the interaction between the solid and the liquid component(s) passes through the permeable barrier.

15. A peptide or a peptidomimetic obtainable by a method according to any one of claims 1 to 14.

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