Defined monomer sequence polymers

Abstract

Processes of preparing defined monomer sequence polymers are disclosed, in which a backbone portion of the polymer is first prepared by performing one or more sequential monomeric coupling reactions with intervening membrane diafiltration purification/isolation steps, followed by a step of decorating the backbone portion with one or more side chains at predetermined positions along its length. The process represents an improvement on prior art techniques, which impose limitations on the size of the side chains that may be present. Defined monomer sequence polymers that are obtainable by the processes are also disclosed.

Claims

1. A process for the preparation of a first compound being a defined monomer sequence polymer, in which at least two of the monomeric units are distinct from each other; the process comprising the steps of: i) synthesising a backbone portion of the first compound by performing one or more sequential monomeric coupling reactions in a first organic solvent, at least one of the monomeric units used in the sequential monomeric coupling reactions comprising a reactive side chain precursor group, such that the backbone portion comprises one or more reactive side chain precursor groups located at one or more predetermined positions along its length; ii) between each coupling reaction, separating a product of said one or more sequential coupling reactions from at least one second compound, which is a reaction by-product of the synthesis of the product and/or an excess of a reagent used for the synthesis of the product, and iii)attaching one or more side chains to the one or more reactive side chain precursor groups located along the length of the backbone portion; wherein during step (ii) the product of said one or more sequential coupling reactions and at least one second compound are dissolved in a second organic solvent and are separated by a process of diafiltration using a membrane that is stable in the organic solvent and which provides a rejection for the product which is greater than the rejection for the second compound.

2. The process of claim 1, wherein step (i) comprises synthesising a backbone portion comprising a first reactive side chain precursor group and a second reactive side chain precursor group, and step (iii) comprises attaching a first side chain to the first reactive side chain precursor group and a second side chain to the second reactive side chain precursor group.

3. The process of claim 2, wherein the first reactive side chain precursor group and the second reactive side chain precursor group are different, and the first side chain and the second side chain are different.

4. The process of claim 3, wherein a first monomeric unit used in the one or more sequential monomeric coupling reactions comprises the first reactive side chain precursor group and a second monomeric unit used in the one or more sequential monomeric coupling reactions comprises the second reactive side chain precursor group.

5. The process of claim 3, wherein the first reactive side chain precursor group is configured to react exclusively with the first side chain, and the second reactive side chain precursor group is configured to react exclusively with the second side chain.

6. The process of claim 1, wherein each side chain independently comprises a group selected from targeting molecules, active pharmaceutical ingredients, imaging agents, sugars, amino acids, peptides, nucleobases, aptamers, oligonucleotides, and monodisperse synthetic polymers.

7. The process of claim 1, wherein all of the monomeric units used in the one or more sequential monomeric coupling reactions of step (i) have identical backbone moieties.

8. The process of claim 1, wherein the backbone portion of the first compound is homopolymeric and is selected from poly(ethylene glycol) (PEG), poly(propylene glycol) (PPG), poly(butylene glycol), poly(ethylene oxide), poly(propylene oxide), poly(butylene oxide), poly(dimethylsiloxane) (PDMS), polybutadiene, polyisoprene, polystyrene, nylons and polyesters, poly(ethylene imines) (PEI), poly(propylene imines), poly(L-lysine) (PLL), poly(amidoamines) (PAA), poly(methyl methacrylate) (PMMA), poly(vinyl benzoic acid), poly(hydroxystyrene), N-substituted glycines, and poly(lactide-co-glycolide) (PLGA).

9. The process of claim 1, wherein the backbone portion of the first compound is a poly(ethylene glycol) homopolymer.

10. The process of claim 1, wherein not all of the monomeric units used in the one or more sequential monomeric coupling reactions of step (i) have identical backbone moieties.

11. The process of claim 10, wherein the backbone portion of the first compound is a copolymer formed from two or more of ethylene glycol, propylene glycol, butylene glycol, dimethylsiloxane, butadiene, isoprene, styrene, amides and esters, ethylene imines, propylene imines, L-lysine, amidoamines, methyl methacrylate, vinyl benzoic acid, hydroxystyrene, N-substituted glycines, lactide-co-glycolide, and polymers thereof.

12. The process of claim 1, wherein during synthesis of the first compound, the product is covalently attached to a synthesis support by an initial monomeric unit.

13. The process of claim 12, wherein the synthesis support is a branch point molecule having two or more reactive moieties capable of covalently binding to the initial monomeric unit.

14. The process of claim 1, wherein the one or more reactive side chain precursor groups each comprise a functional group.

15. The process of claim 14, wherein the functional group is selected from NH.sub.2, CC, SH, CO.sub.2H, N.sub.3 and CHCH.sub.2.

16. The process of claim 1, wherein the one or more sequential monomeric coupling reactions each comprise the steps of: a) reacting a starting material with an excess of an additional monomeric unit, the additional monomeric unit having one of its reactive terminal protected by a protecting group, and b) removing the protecting group so as to expose the reactive terminal such that it is ready for reaction with a subsequent additional monomeric unit, wherein the starting material is either an initial monomeric unit having at least one of its reactive terminals protected, or the polymeric product of the one or more sequential monomeric coupling reactions.

17. The process of claim 16, wherein the step (ii) is performed after step a) and again after step b).

Description

EXAMPLES

(1) Examples of the invention will now be described, for the purpose of illustration only, with reference to the accompanying drawings in which:

(2) FIG. 1 shows the synthesis routes for various pentagol-based monomer building blocks used for fabrication of PEG defined monomer sequence backbone portions with distinct reactive side chain precursor groups.

(3) FIG. 2 shows the full synthesis routes for the pentagol-based building blocks BnOBB, N.sub.3BB and PmbSBB.

(4) FIG. 3 shows .sup.1H NMR spectra for the building blocks identified in FIG. 2.

(5) FIG. 4 shows mass spectra for the building blocks identified in FIG. 2.

(6) FIG. 5 shows the preparation of sequence-defined PEGs for use as backbone portion with multiple (4) reactive side chain precursor groups attached to a synthesis support, and subsequent cleavage from the synthesis support.

(7) FIG. 6 shows the attachment of distinct side-chains (R.sup.A,B,C,D) to defined monomer sequence PEG backbone portions comprising multiple distinct reactive side chain precursor groups.

(8) FIG. 7 shows the preparation of sequence-defined PEG backbone portions with identical reactive side chain precursor groups suitable for click chemistry. Synthesis of the backbone portions on a synthesis support is followed by cleavage from the support.

(9) FIG. 8 shows the attachment by click chemistry of various side-chains to defined monomer sequence PEG backbone portions containing identical reactive side chain precursor groups.

(10) FIG. 9 shows the preparation of a sequence-defined PEG backbone portion containing multiple distinct reactive side chain precursor groups for click chemistry. Synthesis of the backbone portion on a synthesis support is followed by cleavage from the support.

(11) FIG. 10 shows the attachment by click chemistry of multiple distinct side-chains to PEG backbone portions containing multiple distinct reactive side chain precursor groups.

(12) FIG. 11 shows the synthesis of a high-molecular-weight brush PEG homostar by click reaction between Hub.sup.3-Octagol-(N.sub.3BB).sub.5-OThp and DmtrO-EG.sub.60-Alkyne.

(13) FIG. 12 shows (a) Click reaction between DmtrO-EG.sub.12-Alkyne and PEG2k-N.sub.3. (b) .sup.1H NMR and (c) .sup.13C NMR spectra of the target product DmtrO-EG.sub.12-PEG2k.

(14) FIG. 13 shows (a) Click reaction between DmtrO-EG.sub.60-Alkyne and N.sub.3BB. (b) .sup.1H NMR and (c) .sup.13C NMR spectra of the target product (DmtrO-EG.sub.60-N.sub.3BB).

(15) FIGS. 14 and 15 show various side chains suitable for use in the invention.

(16) FIG. 16 shows the synthesis of Hub.sup.3-Octagol-(N.sub.3BB).sub.5-OThp involving 5 chain-extension and deprotection cycles.

(17) FIG. 17 shows (a) .sup.1H NMR and (b) .sup.13C NMR for the PEG homostar (Hub.sup.3-Octagol-(N.sub.3BB).sub.5-OThp) illustrated in FIG. 16.

(18) FIG. 18 shows the MALDI-TOF-MS spectrum for the PEG homostar (Hub.sup.3-Octagol-(N.sub.3BB).sub.5-OThp) illustrated in FIG. 16.

(19) FIG. 19 shows the permeance (top) and rejection (bottom) of BnOBB (10) in methanol using three different OSN membranes; PEEK membrane was cast as reported in Journal of Membrane Science 493(2015) 524-538, while bench cast PBI18-DBX-JM2005 and PBI19-DBX-JM2005 membranes were cast as described above.

(20) FIG. 20 shows the rejection of N.sub.3BB (15) in methanol (top) and the rejection of PmbSBB (18) in methanol (bottom) using three different OSN membranes; PEEK membrane was cast as reported in Journal of Membrane Science 493(2015) 524-538, while bench cast PBI18-DBX-JM2005 and PBI19-DBX-JM2005 membranes were cast as described above.

(21) FIG. 21 shows the rejection of N.sub.3BB (15) and Hub.sup.3-Octagol in methanol using PBI18-DBX-JM2005 membrane.

(22) FIG. 22 (top) shows the rejection of Hub.sup.3-Octagol and DmtrO-EG.sub.60-Alkyne in methanol using PBI18-DBX-JM2005 membrane; and (bottom) the rejection of Hub.sup.3-Octagol and DmtrO-EG.sub.60-N.sub.3BB in methanol using PBI18-DBX-JM2005 membrane.

(23) Abbreviations

(24) The following abbreviations are used throughout the figures and Examples: Bn=benzyl; Dmtr=4,4-dimethoxytrityl; Tbdms=tert-butyldimethylsilyl; Ms=Methanesulfonic; DMF=dimethyl formamide; THF=tetrahydrofuran; DCA=dichloroacetic acid; NMI=1-methylimidazole; Tbdps=tert-butyldiphenylsilyl; TEA=triethylamine DCM=dichloromethane; DHP=dihydropyran BB=building block HMTETA=1,1,4,7,10,10-hexamethyltriethylenetetramine TBAF=tetra-n-butylammonium fluoride; OSN=organic solvent nanofiltration; PMDETA=N,N,N,N,N-pentamethyldiethylenetriamine; EDC=1-ethyl-3-(3-dimethylaminopropyl)carbodiimide; NHS=N-hydroxysuccinimide; PBI=polybenzimidazole; PEEK=poly(ether ether ketone)

Example 1

(25) FIG. 1 shows four pentagol-containing (Eg.sub.5) protected monomeric building blocks (tetragol=(ethylene glycol).sub.4) with different reactive side chain precursor groups (R.sup.2). The monomeric building blocks (BBs) have the general structure TsOEg4OCH(CH.sub.2R.sup.2)CH.sub.2OPG [R.sup.2=N.sub.3 (10a), CC-Tbdps (10b), SPmb (10c) and OCH.sub.2C(O.sub.3C.sub.6H.sub.9) (10d)], which are obtained according to the synthetic route described in FIG. 1. Different protecting groups [PG=-Dmtr, -Mip, -Cyc, -Mthp, -Thp and SO.sub.3.sup.] are used for simple protection and effective deprotection, with a range of stability/controllable rate of acid-labile deprotection during each chain-extension cycle.

(26) Three hetero-functionalized tetragol derivatives with different reactive side-groups, namely TsO-EG.sub.4(R)OThp (R=OBn, N.sub.3 and SPmb), were synthesised as building blocks according to the procedure shown in FIG. 2. Notably, the coupling reaction between compound 2 and compound 5 generates some side products. Thus, it is preferable to completely dry the starting materials using acetonitrile before adding NaH catalyst to initiate the reaction. The hydrogenolysis of compound 8 to produce compound 11 was found to work best in acetonitrile and ethyl acetate. Here, the acid-labile Thp group was selected as a protecting group due to its acid-sensitivity and its small size, which means that it can be both effectively deprotected after each chain-extension cycle and, also easily removed by OSN diafiltration. The Tosyl group has high reactivity with OH group using NaH as a catalyst, making it suitable for chain extension of PEGs. The three different side-groups including BnO, N.sub.3 and PmbS on these building blocks can be readily converted into highly reactive groups after deprotection procedure, such as OH, NH.sub.2 and SH, respectively.

(27) The chemical structure and molecular weight of the resulting building blocks including BnOBB, N.sub.3BB and PmbSBB, have been confirmed by NMR spectroscopy and mass spectroscopy in FIGS. 3 and 4. The clearly observed m/z peaks at 614.2, 549.3 and 660.3 are assigned to BnOBB, N.sub.3BB and PmbSBB, respectively.

Example 2

(28) FIGS. 5, 7, and 9 show coupling and deprotection processes used to form suitable defined monomer sequence backbone portions. The monomeric building blocks obtained in FIG. 1 are coupled onto a synthesis support (support-OEg.sub.4OH) in any preselected order, using organic solvent nanofiltration (OSN) technology for purification during each round, to produce monodisperse sequence-defined PEG backbone portions, each bearing up to four possible reactive side chain precursor groups (15 in FIG. 5). PEG backbone portions with identical reactive side chain precursor groups (18b and 19c in FIG. 7), and PEG backbone portions with different reactive side chain precursor groups (23 in FIG. 9) are also prepared. These defined monomer sequence backbone portion PEGs have a plurality of identical or different reactive side chain precursor groups, which can then be post-conjugated with a large range of side chains. In addition, the clickable reactive side chain precursor groups (e.g. SH and CCH) incorporated into these monodisperse sequence-defined PEGs enhance their conjugation efficiency.

Example 3

(29) FIGS. 6, 8, and 10 show the post-modification process for the monodisperse sequence-defined backbone portion PEGs of Example 2. By using different conjugation approaches, diverse and distinct side-chains (see FIGS. 14 and 15) can be precision conjugated to specific reactive side chain precursor groups of the target PEGs to generate site-specific multifunctional PEGs with precision-guided side-chains. A range of potential functional side-chains are illustrated in FIGS. 14 and 15, including targeting molecules, drugs, imaging agents, sugars, amino acids, peptides, nucleobases, aptamers, oligonucleotides, and monodisperse synthetic polymers. In order to achieve effective conjugation to the target PEGs chains, particular side-chains are selectively coupled to reactive side chain precursor groups on the backbone portions, such as NHS-esters, CHCH.sub.2, N.sub.3, NH.sub.2.

(30) FIG. 11 shows the post-modification process for a monodisperse azide-based PEG homostar (prepared according to Example 4 below). To provide efficient post modification, click chemistry was chosen to achieve efficient coupling between the alkyne group terminating the DmtrO-EG.sub.60-Alkyne which is the side chain (SC) and the azide side chain precursor group on the Hub.sup.3-Octagol-(N.sub.3BB).sub.5-OThp. The click reaction between Hub.sup.3-Octagol-(N.sub.3BB).sub.5-OThp and DmtrO-EG.sub.60-Alkyne was carried out in DMF at 60 C. using CuCl as catalyst and HMTETA as chelating agent. After 24 hrs, the target brush PEG homostar with large molecular weight was obtained with a high yield of >95%. At this stage, OSN diafiltration can be used to separate the Brush PEG Homostar from the DmtrO-EG.sub.60-Alkyne.

(31) The feasibility of the click reaction illustrated in FIG. 11 is further exemplified in FIG. 12. The coupling between PEG 2000 with an azido end group (PEG2k-N.sub.3) and DmtrO-EG12-Alkyne was performed to produce DmtrO-EG.sub.12-PEG2k. After the click reaction between alkyne and azide groups, a characteristic triazole unit was formed. The new proton signal (a) and carbon signals (a and b) can been clearly observed in FIG. 12. Compared to other reaction types, click chemistry has several unique advantages including high selectivity, high coupling efficiency and mild reaction condition.

(32) In a further demonstration, the azido building block (N.sub.3BB) was reacted with DmtrO-EG.sub.60-Alkyne to generate the larger monomeric building block (DmtrO-EG.sub.60-N.sub.3BB) under similar reaction conditions. After column purification, its chemical structure was confirmed using NMR spectroscopy (FIG. 13).

Example 4

(33) FIG. 16 shows the coupling and deprotection processes used to form PEG homostar with N.sub.3-based reactive side chain precursor groups. The monomeric N.sub.3-building blocks obtained in FIG. 2 were coupled onto a synthesis support (Hub3-Octagol-OH) in any preselected order using column chromatography technology for purification during each round. After 5 coupling and deprotection cyles, the monodisperse PEG homostar (Hub.sup.3-Octagol-(N.sub.3BB).sub.5-OThp) (ca. 65 mg) was obtained with a total yield of 26.4%, which contains 15 identical reactive azide groups (15 in FIG. 16). Thus, the azide-based PEG homostar is then ready for subsequent modification with a large range of side chains using highly efficient click chemistry, to further produce, for example, larger monodisperse PEG brush molecules.

(34) The chemical structure of the N.sub.3-based PEG homostar (Hub.sup.3-Octagol-(N.sub.3BB).sub.5-OThp) prepared as illustrated in FIG. 16 was confirmed by .sup.1H NMR and .sup.13C NMR spectroscopy (FIG. 17), and MALDI-TOF mass spectroscopy. Furthermore, the molecular weight was determined using MALDI-TOF-MS (FIG. 18), and its m/z peak at 6085.1 was clearly observed, which is consistent with its calculated molecular weight value.

Example 5

(35) Example 5 demonstrates the feasibility of the diafiltration-based synthetic process of the invention. A laboratory scale cross-flow nanofiltration unit was used with 4 cross flow cells in series. Membrane discs of active area 14 cm.sup.2 were used. An 80 mL feed tank was charged with a feed solution consisting of 0.04-0.07 g of BnOBB (10), or N.sub.3BB (15), or Pmbs-BB (18) in MeOH, or 0.01-0.04 g Hub.sup.3-Octagol in MeOH (see FIG. 2). The feed solution was re-circulated at a flow rate of 150 L h.sup.1 using a Micropump (GD series, Michael Smith Engineers Ltd., UK). A pressure of 20 bar in the cells was generated using a backpressure regulator which was located down-stream of a pressure gauge. During operation, permeate samples were collected from individual sampling ports for each cross-flow cell and the retentate sample was taken from the recycle line. Solvent flux was calculated as in Equation 1.

(36) $\begin{array}{cc}{N}_v=\frac{V}{\mathrm{At}}& \left(1\right)\end{array}$
where V=volume of a liquid sample collected from the permeate stream from a specific cross-flow cell, t=time over which the liquid sample is collected, A=membrane area.

(37) Membrane rejection R.sub.i, was calculated as in Equation 2.

(38) $\begin{array}{cc}{R}_i=\left(1-\frac{C_{\mathrm{Pi}}}{C_{\mathrm{Ri}}}\right)100\%& \left(2\right)\end{array}$

(39) where C.sub.P,i=concentration of species i in the permeate (permeate being the liquid which has passed through the membrane), and C.sub.R,i=concentration of species i in the retentate (retentate being the liquid which has not passed through the membrane).

(40) The solute concentrations were measured using an Agilent HPLC machine. A reverse phase column (C4-300, 250 mm4.6 mm, ACE Hichrom) was used and the mobile phases were MeOH and DI water buffered with 5 mM ammonium acetate. The HPLC pump flow rate was set at 1 ml min.sup.1 and the column temperature was set at 30 C.

(41) Integrally skinned asymmetric PBI membranes were prepared by phase inversion as reported in Journal of Membrane Science 457 (2014) 62-72 using 18 to 19 wt % PBI dope solutions. Bench cast membranes were cast with the knife set at 250 m and the casting machine set at a speed of 3.5 cm s.sup.1 (Elcometer, UK). Continuous cast membranes were cast with the knife set at 200 m and a speed of 3 cm s.sup.1(SepraTek, Korea). The PBI membranes were cross-linked using a,a-dibromo-p-xylene in MeCN at 80 C. for 24 hours, followed by reaction with a polyetheramine conditioning agent (Jeffamine 2005). Finally, the membrane surfaces were rinsed with IPA and the membranes were immersed in a solution of PEG400-IPA 1:1, stirring continuously for at least 4 hours, before drying.

(42) FIGS. 19-20 show the permeance and rejection of three different OSN membranes, using BnOBB (10), N.sub.3BB (15), PmbSBB (18) in MeOH. PEEK membrane was cast as reported in Journal of Membrane Science 493 (2015) 524-538 using a 12 wt % polymer dope solution and drying the membrane from acetone, while bench cast PBI18-DBX-JM2005 and PBI19-DBX-JM2005 membranes were cast as described above.

(43) As is clear from FIGS. 19-20, PBI membranes have higher permeance than PEEK membranes. Furthermore, PBI19-DBX-JM2005 has higher rejection than PBI18-DBX-JM2005 for all building blocks tested.

(44) Continuously cast PBI18-DBX-JM2005 membranes were also tested for separation of Hub.sup.3-Octagol from N.sub.3BB, as shown in FIG. 21 (having regard to the reaction described in FIG. 16). As shown in FIG. 21, the rejection of Hub.sup.3-Octagol is significantly higher than the rejection of N.sub.3BB, showing that the main product (i.e. the growing homostar) can be easily purified from the building block via diafiltration. Moreover, the sequential attachment of building block units to Hub.sup.3-octagol, for example to result in (Hub.sup.3-Octagol-(N.sub.3BB).sub.5-OThp) from example 4, will necessarily increase the product size and therefore product rejection, thereby improving at the same time the membrane selectivity and process yield.

(45) The preparation of the desired Brush PEG Homostar polymer on Hub.sup.3 could be performed in at least two ways:

(46) Method 1 following the present invention, the N.sub.3BB can be reacted with Hub.sup.3-octagol to give Hub.sup.3-octagol-EG.sub.4(N.sub.3), and this can be repeatedwith interspersed diafiltration to separate the growing polymer from the excess unreacted building blockto obtain the desired Hub.sup.3-Octagol-(N.sub.3BB)5OThp homostar with the desired monomer sequence including reactive side chain precursor groups; next, the reactive side chain precursor groups can be reacted with DmtrO-EG.sub.60-Alkyne side chains; and then the resulting Brush-PEG-Homostar may be separated from residual EG.sub.60 by diafiltration; or

(47) Method 2: following the state-of-the-art, Hub.sup.3-octagol could be reacted directly with DmtrO-EG.sub.60-N.sub.3BB (i.e. building blocks that have already been modified with the side chain), until the desired polymer length is obtained.

(48) The two scenarios are characterized by different separation challenges:

(49) Insofar as Method 1 is concerned, FIG. 21 illustrates that the Hub.sup.3-octagol and N.sub.3BB have substantially different rejections using PBI18-DBX-JM2005, meaning that the growing homostar can be easily purified from the building block via diafiltration. Moreover, FIG. 22 (top) shows that the Hub.sup.3-octagol and the DmtrO-EG.sub.60-Alkyne side chain have substantially different rejections using PBI18-DBX-JM2005 (Ri>90% and Ri<60% respectively). This means that the finished homostar (decorated with reactive side chain precursor groups)which will incidentally be more massive than the Hub.sup.3-octagol starting materialcan be reacted with the DmtrO-EG.sub.60-Alkyne side chains, and the excess unreacted the DmtrO-EG.sub.60-Alkyne side chains can then be separated from the Brush PEG homostar using diafiltration.

(50) Insofar as Method 2 is concerned, FIG. 22 (bottom) shows that the Hub.sup.3-octagol and the DmtrO-EG.sub.60-N.sub.3BB (i.e. building blocks that have already been modified with the side chain) have highly similar rejections using PBI18-DBX-JM2005 (Ri>90%). As a consequence, the preparation of Brush PEG homostars by step-wise coupling of DmtrO-EG.sub.60-N.sub.3BB to Hub.sup.3-octagol with interspersed diafiltration purification steps is notably more difficult.

(51) 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.

(52) 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.