WO2016020708A1

WO2016020708A1 - Improved polymer synthesis

Info

Publication number: WO2016020708A1
Application number: PCT/GB2015/052310
Authority: WO
Inventors: Andrew Guy Livingston; Jeong Kim; Irina Boyanova VALTCHEVA; Piers Robert James Gaffney; Marc SCHAEPERTOENS
Original assignee: Imperial Innovations Limited
Priority date: 2014-08-08
Filing date: 2015-08-10
Publication date: 2016-02-11
Also published as: GB201414120D0

Abstract

A process for preparing defined monomer sequence polymers is provided comprising successive coupling and deprotection steps, in which the deprotection step is performed simultaneously with at least one diafiltration separation step, thereby markedly reducing the number of unit operations per chain extension cycle. Also provided is a use of an organic solvent resistant nanofiltration membrane in the polymer preparation processes of the invention.

Description

IMPROVED POLYMER SYNTHESIS

INTRODUCTION

[0001] The present invention relates to a process for the preparation of defined monomer sequence polymers, as well as to the use of organic solvent resistant nanofiltration membranes in such processes.

BACKGROUND OF THE INVENTION

[0002] Defined monomer sequence polymers comprise naturally-occurring or non-naturally- occurring polymers, in which the sequence of monomers constituting the polymer is exactly defined.

[0003] Biological examples of defined monomer sequence polymers are oligonucleotides (termed "oligos") and peptides. These are heteropolymers that consist of a series of non- identical monomer units: nucleotides for oligos, and amino acids for peptides. The sequence of the monomers defines the biological function, structure and targets of species. Oligos are being considered as the next generation of therapeutics, as treatment is effected at the protein expression level. After the discovery of RNA interference (RNAi) by small interfering RNAs (siRNA, RNA oligomers ca. 18-25 monomers long), leading to the award of the 2006 Nobel Prize in Physiology or Medicine to Fire and Mello, there has been a renaissance in the search for oligo-based drugs, and the market is growing rapidly. Currently, there are 3 FDA approved drugs in the market, and over 100 oligo-based potential drug candidates are going through clinical trials [1]. The current state-of-the-art synthesis platform is solid-phase oligonucleotide synthesis (SPOS) which is efficient and versatile for small-scale syntheses. However, there is currently no viable synthesis platform that can manufacture such drugs on a batch scale greater than 10 kg, and there is a pressing need to meet the forecast demand, which is expected to grow to several tonnes per annum.

[0004] Non-naturally-occurring polymers can also be prepared by iterative synthesis when a defined monomer sequence is required. Technically, these materials can be prepared by solid phase synthesis or advanced polymerisation techniques, as described in [2] and [3]. Defined monomer sequence polymers of this type can be synthesized by attaching a small building block, or monomer, to the growing product in an iterative manner. Each chain extension cycle is typically composed of two sets of reactions, each followed by a purification step. The first set constitutes chain extension, where the next monomer in the sequence is chemically attached to the growing chain. The monomers must be protected at the reactive site to which subsequent monomers will be attached to prevent uncontrolled chain extension. In oligonucleotide synthesis a dimethoxytri phenyl methyl (Dmtr) ether is almost universally used for temporary protection of the 5'-hydroxyl. In peptide synthesis, either fluorenylmethyl oxycarbonyl (Fmoc), or less frequently te/f-butyl oxycarbonyl (Boc) carbamates, fulfill the same role of protecting the peptide /V-terminus. In the case of the monomer sequence defined polymer non-naturally-occurring polymer poly(ethylene glycol) (PEG), a dimethoxytriphenylmethyl (Dmtr) ether protecting group can also be employed [4,5]. The second reaction then takes off the temporary protecting group (i.e. deprotection of the 5'-hydroxyl for oligos, the /V-terminal amino group for peptides, and the chain terminal hydroxyl for PEG), so that the next round of the chain extension cycle can proceed.

[0005] Between one chain extension and the next, thorough purification must be carried out to remove excess reagents and byproducts; additional purification after chain extension, but before deprotection, also allows for the option to recover excess building block if chemically viable. Apart from inhibiting later reactions, such species can participate in chain extension, generating more complex contaminants that rapidly become much harder to separate from the desired naturally-occurring or non-naturally-occurring polymer. When defined monomer sequence polymers are prepared by classical solution phase organic synthesis, purification (which is predominantly chromatographic) is cumbersome due to the chemical similarities between products and byproducts, and the yield is reduced due to handling and irreversible adsorption. Thus solid-phase synthesis, including solid phase oligo synthesis (SPOS) and solid phase peptide synthesis (SPPS), are attractive alternative platforms for small-scale syntheses.

[0006] In solid phase synthesis, the growing chain of monomers is tethered to a solid support of resin or glass beads. During the synthesis cycle, excess reagents are simply washed off the solid by flushing solvent through the support, and no oligomer is lost from the synthesis. Liquid phase peptides synthesis (LPPS) is often used for scale-up, although SPPS is also common at Kg scales. Liquid phase oligonucleotide synthesis (LPOS) is, however, much rarer, possibly due to the generally greater chemical sensitivity of these compounds. Reported syntheses of monodisperse and heterobifunctional poly(ethylene glycol) utilized precipitation and chromatography for purification [4,5]. Reported liquid-phase approaches to oligos have employed precipitation [6], extraction [7], and Organic Solvent Nanofiltration (OSN) technology [8,9] for purification. Recently Livingston et al. [8,9] proposed a scalable LPOS platform using organic solvent nanofiltration (OSN) technology for purification, named LPOS-OSN. Three growing oligonucleotide chains were chemically attached to a soluble mono-disperse star polymer. This increased the overall size of the supported oligo relative to the excess monomer, which is the largest contaminant that has to be separated before the chain extension cycle can be repeated. This facilitated the separation using membranes, which are selective for the difference in sizes, and helped with analytical monitoring of reactions.

[0007] OSN is an established membrane technology that can separate solutes in organic media at ambient temperature. It is well known as an energy-efficient separation technology, and is being considered as a key technology to enhance the sustainability of chemical processes. As the name suggests, a characteristic feature of OSN membranes is that they withstand organic solvents, the medium for most chemical reactions, during separation.

[0008] In a typical membrane process, the solution that is retained by the membrane is called the retentate, and the solution that penetrates the membrane is called the permeate. The compound(s) of interest may be in the retentate, the permeate, or both.

[0009] Many OSN applications, including the iterative synthesis platform, require complete separation of a mixture containing at least two or more solutes dissolved in an organic solvent (or a mixture of organic solvents). When performing purifications using membranes, the constant volume diafiltration (CVD) mode is usually employed, where the retentate volume is held constant by matching the permeate volume outflow with pure solvent input.

[0010] In a typical CVD process, a feed tank is first charged with a solution containing at least two solutes. The solution is then circulated around the system under pressure which forces the solution through the membrane to initiate separation. If at least one of the solutes permeates through the membrane faster than the others, then molecular separation is achieved at ambient temperature.

[001 1] The slower the rate of permeation of a solute, the higher its rejection by the membrane. Similarly, the faster the permeation, the lower the rejection of species i (R,, mathematically defined in Equation 1).

Ri = (1 - C_Pi/C_Ri) x 100% (Eq.1 )

[0012] where C_Pi and C_Ri represent the concentrations of solute i in the permeate and retentate, respectively. Consequently, the larger the difference in solute permeation rates (or rejections) between two solutes, the easier the separation becomes. In practice, to obtain a reasonable yield during CVD it is essential that one solute is as near as possible completely rejected by the membrane (i.e. R approaches 100%). Similarly, the rejection (R) of the solutes that permeate through the membrane should be below 80%, or else the separation will require excessive volumes of solvent to achieve acceptable purity. [0013] CVD processes can be characterized using a time-like dimensionless parameter called a diavolume, defined in Equation 2.

Diavolume = ^Fpt (Eq.2)

^system

[0014] where F_p (L.min ¹) represents the permeate flow rate, t (min) the time, V_system (L) represents the system volume. A typical CVD process requires between 10 - 25 diavolumes to purify a product of interest to a high enough purity.

[0015] Oligonucleotides that show therapeutic potentials are typically 18-25 base-pairs long (n = 18-25). Hence, to synthesize any desired oligo, a total of n-1 chain extension cycles will be required. Assuming only 4 unit operations per chain extension (2 reactions and 2 separations), at least 76 unit operations are required to synthesize a 20-mer oligo. Such a large number of steps necessitate high yields at each step to ensure reasonable overall yield. For example, a step-wise yield of 98% only results in 22% overall yield after 76 unit operations. Therefore, it is extremely important to reduce the number of steps as much as possible with high yield and purity at each step. In addition, the batch operation time must be reasonable at large scale.

[0016] The current LPOS-OSN platform [9] adopted the aforementioned 4-step per cycle protocol: 2 sets of reactions and 2 membrane diafiltration steps. In addition to the 4 main unit operations, the LPOS-OSN technique can require up to 9 unit operations per chain extension cycle when multiple solvent exchange steps, as well as a precipitation step [9], are required (see Figure 1). Hence, in order to make the platform more competitive, there is a need for simplifying the overall process and reduce the number of unit operations.

[0017] The present invention was devised with the foregoing in mind.

SUM MARY OF THE INVENTION

[0018] According to a first aspect of the present invention there is provided a process for preparing a defined monomer sequence polymer by iterative addition of at least one subsequent monomer to a starting material, the starting material being either an initial monomer or a polymer whose chain of monomers is to be extended, the process comprising the steps of:

a) reacting the starting material with an excess of a subsequent monomer in a first organic solvent, wherein one of the reactive terminals of the subsequent monomer is protected with a protecting group, b) isolating the product of step a) by a process of diafiltration using a membrane that is stable in the first organic solvent and which provides a rejection for the product of step a) which is greater than the rejection for the residual subsequent monomer and other small reaction debris, c) removing the protecting group present on the product of step a) using at least one deprotecting reagent in a second organic solvent, and d) isolating the product of step c) by a process of diafiltration using a membrane that is stable in the second organic solvent and which provides a rejection for the product of step c) which is greater than the rejection for the removed protecting group and/or one or more degradation products thereof, and deprotecting reagent, wherein deprotection step c) and diafiltration step d) are performed simultaneously.

[0019] According to a second aspect of the present invention, there is provided a use of a solvent resistant nanofiltration membrane in a process defined herein.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

[0020] The term "monomer" or "monomeric unit" is used herein to refer to a polymer building block which has a defined and unique molecular structure and which can be reacted to form a part of a polymer.

[0021] The term "defined monomer sequence polymer" is used herein to refer to a polymer, comprising at least two monomers, in which at least two of the monomers are distinct from each other and in which the monomers are present in the same order in the polymer chain for all molecules of the polymer. The defined monomer sequence polymer may be a naturally- occurring polymer or a non-naturally-occurring polymer.

[0022] The term "synthesis support" is used herein to relate to a chemical entity that allows the first compound to stay in solution during the reaction and diafiltration step, and optionally to provide an increased molecular bulk to enhance membrane separation. The synthesis support may be a branch point molecule, or a polymer, dendrimer, dendron, hyperbranched polymer, organic/inorganic nanoparticle, fullerenes or 2-D material such as graphene or boron nitride. [0023] The term "branch point molecule" is used herein to refer to a polyfunctional organic molecular "hub", having at least 2 reactive moieties, and the ability to covalently bind to a terminal of an initial monomer.

Processes of the invention

[0024] As discussed hereinbefore, the present invention provides a process for preparing a defined monomer sequence polymer by iterative addition of at least one subsequent monomer to a starting material, the starting material being either an initial monomer or a polymer whose chain of monomers is to be extended, the process comprising the steps of:

[0025] In comparison to the LPOS-OSN platform of the prior art, the process of the invention provides a considerably simplified protocol for preparing defined monomer sequence polymers by reducing the number of unit operations per chain extension cycle. For illustrative purposes, Fig. 1 shows an LPOS-OSN protocol comprising a total of 9 unit operations per chain extension cycle. In contrast to this, the process of the invention, which combines deprotection step c) and at least diafiltration step d) into a single operation, is able to reduce the complexity of the overall process to as few as 3 unit operations in total per chain extension cycle. By carrying out the deprotection reaction (step c)) and simultaneously removing the protecting group and deprotection reagent using diafiltration, the reaction can be driven to completion by the process of diafiltration (therefore no cation trap is required), and the overall process can be significantly simplified. Fig. 2 provides a schematic representation of a process of the invention in which the number of unit operations per cycle has been reduced to a total of 3.

[0026] It will be understood that the term "simultaneously" means "at the same time", in the sense that the reaction of step c) is conducted in the presence of the membrane of step d).

[0027] In an embodiment, steps c) and d) are performed in a reactor. In its most primitive sense, the reactor is in the form of an enclosure for containing a reaction medium and a diafiltration membrane for separating one or more contents of the reaction medium into a retentate and a permeate. The reactor may include dedicated means (e.g. conduits) for introducing one or more reagents (e.g. the feed stream, and reagents used to effect deprotection) into the enclosure, and dedicated means for removing the permeate. Where it is desired that steps c) and d) are performed under increased pressure, the reactor may be substantially sealed.

[0028] It will be understood that the term "other small reaction debris" relates to one or more reagents or by-products present in the reaction medium following step a). Examples of such debris will be readily apparent to the skilled person.

[0029] In another embodiment, during steps c) and d), the residence time of the removed protecting group and/or degradation product(s) thereof within the diafiltration retentate is shorter than the residence time of the deprotecting reagents within the diafiltration retentate. The process of the invention is ideally conducted with a deprotecting group that has been selected for its suitability with a given diafiltration membrane. Once a deprotecting reagent (e.g. acid) has been added to the feed stream, the deprotection reaction of step c) proceeds according to Equation 3 shown below:

Growing polymer (Protected) -> Growing polymer (Deprotected) + Protecting Group and/or degradation product(s) thereof (Eq.3)

where " ->" represents the presence of a thermodynamic equilibrium. It is therefore preferred that the residence time of the protecting group and/or degradation product(s) thereof within the retentate (as being governed by its molecular weight and the molecular weight cut-off of the membrane) is as low as possible with respect to that of the deprotecting reagents. Fig. 3 provides a schematic illustration of combined steps c) and d) of the present process. [0030] In another embodiment, during steps c) and d), the deprotecting reagent is added during the first 1 - 10 diavolumes, at typical concentrations between 0.1 - 10 vol%. Suitably, during steps c) and d), the deprotecting reagent is added during the first 1 - 7 diavolumes, and/or at typical concentrations between 0.1 - 5 vol%. More suitably, during steps c) and d), the deprotecting reagent is added during the first 1 - 5 diavolumes, and/or at typical concentrations between 0.1 - 3 vol%. Most suitably, during steps c) and d), the deprotecting reagent is added during the first 1 - 5 diavolumes, at typical concentrations between 0.1 - 1 vol%. Diafiltration may then proceed with pure solvent for a further 10 - 15 diavolumes to remove both the protecting group and/or degradation product(s) thereof and the deprotecting reagent from the retentate, leaving a retentate consisting substantially of the growing defined monomer sequence polymer, which is ready for the next round of chain extension. Suitably, the deprotecting reagent exposure to the product needs to be minimized during steps c) and d), and both the deprotecting reagent and the protecting group and/or degradation product(s) thereof must be removed (suitably >99% removal) at the end of the diafiltration.

[0031] In another embodiment, steps c) and d) are performed under increased pressure.

[0032] In one embodiment, diafiltration step b) is performed using a separate membrane to that used in diafiltration step d). In such embodiments, the apparatus used to perform the process of the invention contains two membranes in total, with step b) being performed separately, and prior to simultaneous steps c) and d).

[0033] In another embodiment, steps b), c) and d) are performed simultaneously. In such embodiments, the process suitably makes use of a single membrane for both of diafiltration steps b) and d). The single membrane may be selected for its suitability in permeating the reagents of step a) (i.e. the excess unreacted subsequent monomer), and the reagents and by-products of step c) (i.e. the deprotecting reagent and the cleaved protecting group).

[0034] The protecting group is selected according to the monomers being coupled (and hence the specific functional group needing to be protected). Suitable examples include ether protection derivatives, substituted methyl ether protection derivatives, substituted ethyl ether derivatives, substituted benzyl ether derivatives, silyl ether derivatives, ester protection derivatives, carbonate and carbamate derivatives, and sulfonate and sulfonamide protection derivatives. Suitably, protecting groups should be acid cleavable protections such as, but not limited to, triphenylmethyl ether, 4,4'-dimethoxytriphenylmethyl ether (Dmtr), tetrahydropyran-2- yl (Thp) acetal, 4-methoxytetrahydropyran-4-yl (Mthp) acetal, benzyloxyisopropyl (BnOlp) acetal, phenoxyisopropyl (Pip) acetal and methoxyisopropyl (Mip) acetal protections.

[0035] Suitably, protecting groups should be of small enough size to permeate through OSN membranes with short residence times. More suitably, the protecting groups have a molecular weight of less than or equal to 500Da. Yet more suitably, the protecting groups have a molecular weight of less than or equal to 400Da. Yet more suitably, the protecting groups have a molecular weight of less than or equal to 300Da. Yet more suitably, the protecting groups have a molecular weight of less than or equal to 250 Da.

[0036] In another embodiment, the protecting groups have a molecular weight of less than or equal to 200 Da. Suitably, the protecting groups have a molecular weight of less than or equal to 150 Da. Most suitably, the protecting groups have a molecular weight of less than or equal to 100 Da. Protecting groups described in [10] are suitable for use in the present invention.

[0037] The particular deprotecting reagent used in step c) is dependent on the nature of the protecting group employed. The deprotecting agent could be, but is not limited to, dichloroacetic acid, trifluoroacetic acid, acetic acid, and hydrochloric acid.

[0038] Suitable membranes for use in the invention include polymeric and ceramic membranes, and mixed polymeric/inorganic membranes.

[0039] The membranes used in steps b) and/or d) may be formed from any polymeric or ceramic material. Suitable membranes are formed from, or comprise, a material selected from polymeric materials suitable for fabricating microfiltration, ultrafiltration, nanofiltration or reverse osmosis membranes, including polyethylene, polypropylene, polytetrafluoroethylene, polyvinylidene difluoride, polysulfone, polyethersulfone, polybenzimidazole (PBI), polyetheretherketone, polyacrylonitrile, polyamide, polyimide, polyetherimide, cellulose acetate, polyaniline, polypyrrole and mixtures thereof. The membranes can be made by any technique known to the art, including sintering, stretching, track etching, template leaching, interfacial polymerisation or phase inversion. More preferably, membranes may be crosslinked or treated so as to improve their stability in the working solvents. Membranes described in [11] are preferred for use in the present invention.

[0040] In another embodiment, the membranes used in steps b) and/or d) may also be a composite material comprising a support and a thin selectively permeable layer, and the non- porous, selectively permeable layer thereof is formed from, or comprises, a material selected from modified polysiloxane based elastomers including polydimethylsiloxane (PDMS) based elastomers, ethylene-propylene diene (EPDM) based elastomers, polynorbornene based elastomers, polyoctenamer based elastomers, polyurethane based elastomers, butadiene and nitrile butadiene rubber based elastomers, natural rubber, butyl rubber based elastomers, polychloroprene (Neoprene) based elastomers, epichlorohydrin elastomers, polyacrylate elastomers, polyethylene, polypropylene, polytetrafluoroethylene (PTFE), polyvinylidene difluoride (PVDF) based elastomers, polyetherblock amides (PEBAX), polyurethane elastomers, crosslinked polyether, polyamide, polyester, polyaniline, polypyrrole, and mixtures thereof. [0041] In another embodiment, the membranes used in steps b) and/or d) may be integrally- skinned asymmetric membranes.

[0042] The membrane of the present invention may also be fabricated from an inorganic material such as by way of non-limiting example silicon carbide, silicon oxide, zirconium oxide, titanium oxide, or zeolites, using any technique known to those skilled in the art such as sintering, leaching or sol-gel processing.

[0043] In a further embodiment, the membranes may comprise a polymer membrane with dispersed organic or inorganic matrices in the form of powdered solids present at amounts up to 20wt% of the polymer membrane. Carbon molecular sieve matrices can be prepared by pyrolysis of any suitable material as described in [12]. Zeolites as described in [13] may also be used as an inorganic matrix. Metal oxides, such as titanium dioxide, zinc oxide and silicon dioxide may be used, for example the materials available from Evonik Industries (Germany) under their Aerosol and AdNano trademarks. Mixed metal oxides such as mixtures of cerium, zirconium, and magnesium oxides may be used. Other partiicles or objects comprised of metal organic frameworks (MOF)s, graphene, graphene oxide, boron nitride, or carbon nanotubes may also be used. Preferred matrices will be particles less than 1.0 micron in diameter, preferably less than 0.1 microns in diameter, and more preferably less than 0.01 microns in diameter.

[0044] Suitably, the membranes used in steps b) and/or d) are solvent resistant nanofiltration membranes. Nanofiltration membranes are understood by those of skill in the art to have pores of size 0.5 - 5 nm, and molecular weight cut-offs in the range of 100 - 3000 Da.

[0045] The term "organic solvent" will be well understood by the skilled person and includes, for example, an organic liquid with molecular weight less than 300 Daltons. It is understood that the term solvent also includes a mixture of solvents.

[0046] Suitable examples of solvents include aromatics, alkanes, ketones, glycols, chlorinated solvents, esters, ethers, amines, nitriles, aldehydes, phenols, amides, carboxylic acids, alcohols, furans, and dipolar aprotic solvents, and mixtures thereof and with water. Specific examples of solvent include toluene, xylene, benzene, styrene, anisole, chlorobenzene, dichlorobenzene, chloroform, dichloromethane, dichloroethane, methyl acetate, ethyl acetate, butyl acetate, methyl ether ketone (MEK), methyl isobutyl ketone (MIBK), acetone, ethylene glycols, ethanol, methanol, propanol, butanol, hexane, cyclohexane, dimethoxyethane, methyl tert-butyl ether (MTBE), diethyl ether, adiponitrile, Ν,Ν-dimethylformamide, dimethylsulfoxide, Ν,Ν-dimethylacetamide, dioxane, nitromethane, nitrobenzene, pyridine, carbon disulfide, tetrahydrofuran (THF), methyl-tetrahydrofuran, N- methyl pyrrolidone, N-ethyl pyrrolidone, acetonitrile, and mixtures thereof and with water. [0047] In one embodiment, the first and second solvents are the same.

[0048] In another embodiment, the starting material (i.e. the initial monomer or the polymer whose chain of monomers is to be extended) is covalently attached to a synthesis support with a reactive terminal located at one end of the starting material. The synthesis support may be a branch point molecule, or a polymer, dendrimer, dendron, hyperbranched polymer, or organic/inorganic nanoparticle, or fullerene of 2-D material such as graphene or boron nitride. Once the desired defined monomer sequence polymer has been synthesised, the synthesis support may be cleaved from the polymer and separated therefrom.

[0049] When used as a synthesis support, suitable polymers include polycondensation matrices or polymerisation matrices containing heteroatom functions. Such heteroatom functions may contain oxygen, nitrogen, or can contain more than one heteroatom, such as acid amide groups. Examples of polymeric synthesis supports include polyalkylene glycols including polyethylene glycol, polycaprolactone, polyethylene glycol esterified with citric acid, copolymers of polyethyleneglycol and succinic acid, of vinylpyrrolidone and acrylic acid or beta-hydroxy-ethylacrylate, or of acrylamide and vinylactetate.

[0050] When used as a synthesis support, suitable dendrimers include poly(amidoamine), also known as PAMAM dendrimers; phosphorous dendrimers; polylysine dendrimers; and polypropylenimine (PPI) dendrimers which can have surface functionalities including -OH, - NH₂ and COOH groups.

[0051] When used as a synthesis support, suitable nanoparticles may be prepared from Si0₂, Ti0₂, or other organic or inorganic materials.

[0052] In another embodiment, the synthesis support is a branch point molecule (i.e. a polyfunctional molecule) having two or more reactive moieties capable of covalently binding to a reactive terminal located at one end of the starting material. Chemistries suitable for covalently binding the starting material to the branch point molecule will be readily apparent to a person of skill in the art, and include amide, ester, ether and silyl ether couplings.

[0053] In another embodiment, the branch point molecule may have any of the structures shown below:

Uses of the invention

[0054] As discussed hereinbefore, the present invention also provides a use of a solvent resistant nanofiltration membrane in a process defined herein.

[0055] It will be understood that features common to both the processes and uses of this invention may be further defined by reference to any of the definitions, embodiments and examples recited in the preceding paragraphs.

EXAMPLES

[0056] The invention will now be exemplified, for the purpose of reference and illustration only, with reference to the accompanying figures, in which:

Fig. 1 is a schematic representation of an LPOS-OSN protocol comprising a total of 9 unit operations per chain extension cycle.

Fig. 2 is a schematic representation of a process of the invention, in which step c) and at least step d) are performed simultaneously.

Fig. 3 is a schematic representation of simultaneous steps c) and d) of a process of the invention; D.R represents the deprotection reagent and P is the protecting group debris.

Fig. 4 is a schematic representation of a membrane diafiltration rig used for performing the process of the invention. Fig. 5 shows stacked HPLC chromatograms taken throughout the diafiltration process exemplified in Example 1.

Fig. 6 shows stacked HPLC chromatograms for the deprotecting reagent peak taken throughout the diafiltration process exemplified in Example 1.

Fig. 7 shows stacked HPLC chromatograms taken throughout the diafiltration process exemplified in Example 2.

Fig. 8 shows stacked HPLC chromatograms for the deprotecting reagent peak taken throughout the diafiltration process exemplified in Example 2.

Fig. 9 shows the deprotection of dinucleotides, wherein the dimer is protected with Dmtr and Mip protecting groups.

Fig. 10 shows the synthesis of monodisperse PEGs where the building blocks are protected with different acid labile protecting groups.

Fig. 11 shows the synthesis of monodisperse PEGs where the building blocks are protected with Thp protecting groups.

Example 1

[0057] Having regard to Fig. 9, deprotection of a dinucleotide (an oligo dimer) attached to a soluble polyethyleneglycol benzylic hub (1), where the dimer is protected with 4,4'- dimethoxytriphenylmethyl ether (Dmtr) [14]. The deprotection reaction to give 2a proceeds in the membrane diafiltration rig (see Fig. 4). Polybenzimidazole membranes crosslinked with dibromoxylene [10] are used.

[0058] Having regard to Fig. 5, upon addition of 1 vol% deprotecting reagent, e.g. dichloroacetic acid (DCA), the deprotection proceeds. The deprotecting reagent is added to the system during the first 5 diavolumes. DCA permeates through the membrane quicker than the Dmtr protecting group debris [Dmtr-OMe (3), MW 334 Da, rejection R, ca. 80%], since the residence time of the protecting group is longer than the residence time of the deprotection reagent. Hence, once addition of DCA stops after the 5^th diavolume, the deprotection reaction begins to reverse, and some of the Dmtr protecting group re-attaches to the growing oligos.

[0059] Fig. 6 shows HPLC chromatograms for the deprotecting reagent peaks. By the 20^th diavolume, it can be seen that the deprotecting reagent peak has almost disappeared. As the deprotecting reagent passes into the permeate, the deprotection reaction begins to reverse. Example 2

[0060] Deprotection of a dinucleotide attached to a soluble polyethyleglycol benzylic hub (6a) where the dimer is protected with methoxyisopropyl acetal (Mip). This material was prepared similarly to Dmtr-dinucleotide 1 [9] from uridine loaded hub 3 and the appropriate Mip-protected phosphoramidite (5a). The deprotection reaction proceeds in the single-stage membrane diafiltration rig (see Fig. 4). Polybenzimidazole membranes crosslinked with dibromoxylene [10] are used.

[0061] Having regard to Fig. 7, upon addition of 1 vol% deprotecting reagent, e.g. dichloroacetic acid (DCA), the deprotection proceeds. The deprotecting reagent is added to the system during the first 5 diavolumes. DCA permeates through the membrane at a similar rate to the Mip protecting group debris (acetone, MW 58 Da, and methanol), since the residence time of the protecting group is similar to the residence time of the deprotection reagent. Hence, once addition of DCA stops after the 5^th diavolume, the deprotection reaction does not reverse and the reaction instead reaches completion. Since the rate of permeation for Mip protecting group debris is within the same order of magnitude as that of DCA, the reaction proceeds to completion without re-attaching to the growing oligos. Both the protecting group and the deprotecting reagent are removed from the rig, leaving the growing oligos pure and ready for the next round of chain extension. Apart from combining the deprotection reaction and separation, employing the Mip protecting group reduced the number of required unit operations from 9 to 3 as solvent exchange steps were no longer necessary.

[0062] Fig. 8 shows HPLC chromatograms for the deprotecting reagent peaks. By the 16^th diavolume, it can be seen that the deprotecting reagent peak has almost disappeared.

[0063] The yield of chain-extended dinucleotide in a single-stage rig (Fig. 4) is good at around 75%, but diaflitration in protic solvents leads to a low level of deprotection of cytosine. The same series of reactions were conducted using both acetyl and benzoyl protected cytosine phorphoramidites (5a and b respectively) to give Mip-protected dinucleotide 6 (Fig. 9). This time, a two-stage rig was used to maximise yield, and diafiltration conducted in an aprotic solvent to suppress cytosine deacylation. A solution of 1 % DCA in DMF-MeCN 1 :19 was circulated through the rig under pressure for 27 minutes, ensuring that both stages became sufficiently acidified to effect Mip deprotection, before quenching the acid with pyridine. After 12 system volumes of solvent had permeated, highly purified dinucleotides 2a and 2b were obtained in ca. 95% yield. Example 3

[0064] Synthesis of monodisperse PEGs where the building block is protected with different acid labile protecting groups, ranging from methoxyisopropyl acetal (Mip) to 4,4'- dimethoxytriphenylmethyl ether (Dmtr) is illustrated in Fig. 10. The deprotection reaction proceeds in the membrane diafiltration rig (see Fig. 4). Polybenzimidazole membranes crosslinked with dibromoxylene [10] are used for diafiltration in acetonitrile.

[0065] Three-arm, triply benzylic hub 7 was used as a synthesis support for preparing monodisperse PEG because of its high rejection by crosslinked polybenzimidazole membranes. Building blocks 8 were prepared from tetragol (n = 4) and could be extended to octagol derivatives (n = 8), using methods similar to those reported [4,5]. The hub was loaded with the first PEG building block 8 under standard Williamson etherification conditions (the same procedure a as for later chain extension), using an excess of 8 to drive the reaction to completion. The excess building block 8 and other reagents were then removed by diafiltration (procedure b). Observing that toluene sulfonate (Ts) esters, and especially Dmtr- ethers, both increased rejection of building block 8 to levels that required too much solvent to allow practical purification of protected PEG homostar 9 by diafiltration, a selection of new leaving groups (LG) and protecting groups (PG) were tested. Combinations including PG = Mip, Pip and BnOlp, and LG = methyl sulfonate (Ms) and Br, all provided building blocks 8 with acceptable rejections for synthesis of PEGs with recovery of excess building block 8 by diafiltration.

[0066] Once the great majority of building block 8 had been separated from triply protected PEG homostar 9 by diafiltration (procedure b), dichloroacetic acid (DCA) was added to the system and diafiltration continued (procedures c and d combined). The deprotection of Mip and BnOlp proceeded to completion during the permeation of 5 diavolumes of 1% DCA, but the removal of Pip required more concentrated acid. DCA was then removed over the permeation of a further 10 diavolumes of solvent. This second diafiltration also allowed complete removal of final traces of building block which, after concomitant deprotection to 11 , was more easily separated from highly rejected tri-hydroxy PEG homostar 10, than initial building block 8 from protected homostar 9. Once the retentate had been concentrated under reduced pressure, PEG homostar 10 was then ready for chain extension. Procedures a, chain extension, b, diafiltration with building block recovery, c, homostar deprotection, and d, homostar purification by diafiltration whilst simultaneously driving the deprotection (c) to completion, were repeated to give chain extended PEG homostar homologue 12. This cycle may be repeated several times to obtain longer monodisperse PEGs on a benzylic hub synthesis support. [0067] Building blocks may also be protected with the readily available tetrahydropyranyl (Thp) acetal protecting group (Fig. 11). Excess tetragol (13) was reacted with dihydropyran (DHP) under acid catalysed conditions, and the resultant Thp-tetragol (14) activated as its toluene sulfonate (15). This building block may also be prepared more efficiently by reversing the above procedure, via 16, but the first route is also valuable because intermediate 14 can be used to load the hub rapidly and cleanly.

[0068] To increase the rejection of homostars, and therefore to correspondingly increase the yields of each chain extension cycle, a hub with an even larger rigid diameter was prepared. Hub 17 may be obtained in three steps and good yield from the commercially available corresponding tris-carboxylic acid: the carboxylic acid groups were esterified with methanol in THF, plus sulfuric acid catalyst; the crystalline tris-methyl ester was an excellent substrate for reduction with lithium triethylborohydride; the resultant triol was readily converted to reactive tribromide 17 by treatment with thionyl bromide and the crude material crystallised from toluene.

[0069] Tribromide 17 reacted rapidly and completely with a small excess of Thp-tetragol (14) (Fig. 11). After chromatographic purification, homostar 18 was found to have a rejection of over 95% by a PEEK membrane in EtOH-THF 1 :9. Addition of 1 % toluene sulfonic acid (TsOH) to the rig caused rapid unblocking of the Thp-acetal to give homostar 19, plus protecting group debris (presumed 20), which was removed by diafiltration, along with TsOH, using 12 diavolumes of solvent.

[0070] After chain extension of tetragol homostar 19 with tetragol building block 15 under our standard Williamson etherification conditions [4,5], the resultant tris-Thp octagol homostar 21 was purified by diafiltration. Thp-tetragol tosylate (15) was found to have a somewhat higher rejection than tetragol tosylate (16), but it was still low enough so that most of the unreacted excess building block 15 could be recovered by diafiltration, without significant loss of chain extended homostar 21 , before unblocking Thp from 21.

[0071] While specific embodiments of the invention have been described herein for the purpose of reference and illustration, various modifications will be apparent to a person skilled in the art without departing from the scope of the invention as defined by the appended claims.

[0072] The work leading to this invention has received funding from the [European Community's] Seventh Framework Programme ([FP7/2007-2013] under grant agreement n° 238291. REFERENCES

[I] Sanghvi, Y.S., Curr. Protoc. Nucleic Acid Chem., 2011 , 4.1.1 - 4.1.22.

[2] Lutz J-F et al., "Sequence-Controlled Polymers", Science 9 August 2013, Vol 341 , page 628

[3] Hartmann L and Borner HG, "Precision Polymers: Monodisperse, Monomer-Sequence- Defined Segments to Target Future Demands of Polymers in Medicine" Advanced Materials. 2009, Vol 21 , pp3425-3431.

[4] Szekely G, Schaepertoens M., Gaffney PRJ and Livingston, AG, "Iterative Synthesis of Monodisperse PEG Homostars and Linear Heterobifunctional PEG", Polym. Chem., 2014, 5 (3), 694 - 697

[5] Szekely G, Schaepertoens M., Gaffney PRJ and Livingston, AG, "Beyond PEG2000: Synthesis and Functionalisation of Monodisperse PEGylated Homostars and Clickable Bivalent Polyethyleneglycols", DOI: 10.1002/chem.201402186

[6] Bonora, G.M., Scremin, C.L, Colonna, F.P., Garbesi, A., Nucleic Acids Res., 1990, 18, 3155.

[7] de Koning, M.C., Ghisaidoobe, A.B.T., Duynstee, H.I., Ten Kortenaar, P.B.W., Filippov, D.V., van der Marel, G.A., Org. Process Res. Dev., 2006, 10, 1238-1245.

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[9] Gaffney, P.R.J, Kim, J.F, Valtcheva, I.B., Williams, G.D, Anson, M.S., Buswell, A.M. and Andrew G. Livingston, A.G. "Liquid-Phase Synthesis of 2'-Methyl-RNA on a Homostar Support through Organic-Solvent Nanofiltration", Chemistry - A European Journal, 2015, vol. 21 , DOI: 0.1002/chem.201501001.

[10] T.W. Greene, P.G.M. Wuts, Protective groups in organic synthesis, John Wiley & Sons, Inc, 1999.

[I I] PCT/GB201 1/051361

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Claims

1. A process for preparing a defined monomer sequence polymer by iterative addition of at least one subsequent monomer to a starting material, the starting material being either an initial monomer or a polymer whose chain of monomers is to be extended, the process comprising the steps of: a) reacting the starting material with an excess of a subsequent monomer in a first organic solvent, wherein one of the reactive terminals of the subsequent monomer is protected with a protecting group, b) isolating the product of step a) by a process of diafiltration using a membrane that is stable in the first organic solvent and which provides a rejection for the product of step a) which is greater than the rejection for the residual subsequent monomer and other small reaction debris, c) removing the protecting group present on the product of step a) using at least one deprotecting reagent in a second organic solvent, and d) isolating the product of step c) by a process of diafiltration using a membrane that is stable in the second organic solvent and which provides a rejection for the product of step c) which is greater than the rejection for the removed protecting group and/or one or more degradation products thereof, and deprotecting reagent, wherein deprotection step c) and diafiltration step d) are performed simultaneously.

2. The process of claim 1 , wherein during steps c) and d), the residence time of the

protecting group and/or one or more degradation products thereof within the diafiltration retentate is shorter than or equal to the residence time of the deprotecting reagents within the diafiltration retentate.

3. The process of claim 1 or 2, wherein during steps c) and d), the molecular weight of the protecting group and/or one or more degradation products thereof is less than or equal to the molecular weight of the deprotecting reagent.

4. The process of claim 1 , 2 or 3, wherein the molecular weight of the protecting group and/or one or more degradation products thereof is less than or equal to 250 Da.

5. The process of any preceding claim, wherein the molecular weight of the protecting group and/or one or more degradation products thereof is less than or equal to 150 Da.

6. The process of any preceding claim, wherein deprotection step c) and diafiltration step d) are performed in a reactor, the reactor comprising an enclosure for containing a reaction medium, and a diafiltration membrane for separating one or more contents of the reaction medium into a retentate and a permeate.

7. The process of any preceding claim, wherein a single membrane is used for both of diafiltration steps b) and d), such that steps b), c) and d) are all performed

simultaneously.

8. The process of any preceding claim, wherein the membranes used in steps b) and/or d) are selected from polymeric, ceramic, and mixed polymeric/inorganic membranes.

9. The process of any preceding claim, wherein the membranes used in steps b) and/or d) are formed from, or comprise, a material selected from polymeric materials suitable for fabricating microfiltration, ultrafiltration, nanofiltration or reverse osmosis membranes, including polyethylene, polypropylene, polytetrafluoroethylene, polyvinylidene difluoride, polysulfone, polyethersulfone, polybenzimidazole (PBI), polyetheretherketone, polyacrylonitrile, polyamide, polyimide, polyetherimide, cellulose acetate, polyaniline, polypyrrole and mixtures thereof.

10. The process of any preceding claim, wherein the membranes used in steps b) and/or d) have porosity in the nanofiltration range.

11. The process of any preceding claim, wherein the membranes used in steps b) and/or d) are cross-linked.

12. The process of any preceding claim, wherein the first and second solvents are the same.

13. The process of any preceding claim, wherein deprotection step c) and diafiltration step d) are performed under increased pressure.

14. The process of any preceding claim, wherein the defined monomer sequence polymer is a naturally-occurring polymer or a non-naturally-occurring polymer.

15. A use of a solvent resistant nanofiltration membrane in the process of any of claims 1 to 14.