POLY(ETHYLENE GLYCOL) HAVING C1 TO C3-ALKYLOXYMETHYL SIDE CHAINS, BIOCONJUGATES THEREOF, PROCESS FOR ITS PREPARATION AND ITS USE

Abstract

Polyether polymers represented by the following formula [I]:

(CH.sub.2CHRO).sub.m(I)

wherein 10 to 90% of the residues R are hydrogen and 10% to 90% of the residues R are independently from each other selected from the group made of, methoxymethyl, ethoxymethyl, n-propoxymethyl and iso-propoxymethyl; 1 to 100% of the residues R are methoxymethyl; up to 50% of the residues R may be selected from the group made of ethoxymethyl, n-propoxymethyl and iso-propoxymethyl; and m is in the range of from 10 to 1000 and the dispersity is 1.15 or less. A process for their preparation, conjugates thereof and the use thereof.

Claims

1. Polyether polymers represented by the following formula [I]:
(CH.sub.2CHRO).sub.m[I] wherein 10 to 90% of the residues R are hydrogen and 10% to 90% of the residues R are independently from each other selected from the group consisting of, methoxymethyl, ethoxymethyl, n-propoxymethyl and iso-propoxymethyl; at least 1% of the residues R are methoxymethyl; up to 50% of the residues R may be selected from the group consisting of ethoxymethyl, n-propoxymethyl and iso-propoxymethyl; and m is in the range of from 10 to 1000, wherein the dispersity is 1.15 or less measured by size exclusion chromatography in DMF by calibration with PEG standards.

2. The polyether polymer according to claim 1, wherein the polyether is a poly(glycidyl methyl ether-co-ethylene oxide) copolymer.

3. The polyether polymer according to claim 1, wherein 30 to 70% of the residues R are hydrogen and the remaining residues R are methoxymethyl.

4. The polyether polymer according to claim 1, wherein the polymer is a random copolymer or a statistical copolymer.

5. The polyether polymers according to claim 1 represented by the following formula [II]:
X(CH.sub.2CHRO).sub.mCH.sub.2CHRY[II] wherein X is selected from the group consisting of hydrogen, alkyl, hydroxy, sulfanyl, C1-C10-alkoxy, C1-C10-thioalkoxy, amino, amino-C1-C10-alkoxy, dibenzylamino-C1-C10-alkoxy, amide, N-heterocyclic carbenes and N-heterocyclic olefins; Y is selected from the group consisting of hydrogen, hydroxy, alkoxy, CH.sub.2C(?O)R wherein R is as defined above, CHO, alkylcarbonyloxy, alkyoxycarbonyloxy, amine, C1-C10-alkylamine, carboxamido, azide, halogen, sulfanyl, thioalkoxy, N-succinimidyl carbonate and sulfonate; X and/or Y may also be selected from the group consisting of low molecular weight drugs, nanocarriers, liposomal structures, peptides, polypeptides, proteins, glycoproteins, polynucleotide, polysaccharides, lipid structures, liposomes, surfaces and interfaces, which are bonded directly by a covalent bond or by a spacer to the polymer, m is in the range of from 9 to 999, wherein the dispersity is 1.15 or less measured by size exclusion chromatography in DMF by calibration with PEG standards.

6. The polyether polymer according to claim 5, wherein X is alkoxy and Y is hydroxy.

7. The polyether polymer according to claim 5, wherein X and Y are each hydroxy.

8. The polyether polymers according to claim 1 represented by the following formula [II]:
X(CH.sub.2CHRO).sub.mCH.sub.2CHRY[II] wherein X is selected from the group consisting of hydrogen or X, wherein X is selected from the group consisting of ##STR00026## wherein R?H or alkyl, alkenyl, aryl, alkoxymethyl, alkenyloxymethyl, aryloxymethyl, aminomethyl and thioalkoxymethyl and p=1, 2, 3, 4 or 5, and k=5-500; ##STR00027## wherein R?H or alkyl, alkenyl, aryl, alkoxymethyl, alkenyloxymethyl, aryloxymethyl, aminomethyl and thioalkoxymethyl, and p=1, 2, 3, 4 or 5 and k=5-500; and ##STR00028## wherein R?H or alkyl, alkenyl, aryl, alkoxymethyl, alkenyloxymethyl, aryloxymethyl, aminomethyl and thioalkoxymethyl q=3, 4 or 5 and k=5-500; and ##STR00029## wherein k=5-500; and wherein Y is ZW meaning composed of substituent Z and W linked via a covalent bond, wherein Z is ORO and W is RCHCH.sub.2(OCHRCH2).sub.mV; wherein V is X or X; wherein R is selected from the group consisting of ##STR00030## wherein R?H or alkyl, alkenyl, aryl, alkoxymethyl, alkenyloxymethyl, aryloxymethyl, aminomethyl and thioalkoxymethyl, and s=0-20 t=0-20; and further R is selected from ##STR00031## and wherein m is in the range of from 9 to 999, wherein the dispersity is 1.15 or less measured by size exclusion chromatography in DMF by calibration with PEG standards.

9. The polyether polymer according to claim 5, wherein end-group fidelity of the polymer in regard to group X and/or in regard to group Y is at least 95%.

10. The conjugate, comprising a polyether polymer of claim 1 and a substrate.

11. The conjugate according to claim 10, wherein the substrate is selected from the groups consisting of low molecular weight drugs, nanocarriers, liposomal structures, peptides, polypeptides, proteins, glycoproteins, polynucleotides, polysaccharides, lipid structures, liposomes, surfaces and interfaces.

12. The process for the preparation of a polyether polymer according to claim 1, by anionic ring-opening copolymerization comprising the steps of: providing an anion An.sup.?, adding at least one monomer of the formula ##STR00032## where in R is as defined in claim 1, allowing the polymerization to proceed at a temperature in the range of ?10 to 90? C. wherein the monomer comprises less than 1 wt % of epichlorohydrin.

13. The process according to claim 12, wherein the step of adding at least one monomer and the step of allowing the polymerization to proceed are repeated at least one time, whereby at least one different monomer is used than the first time.

14. The process according to claim 12, wherein the counter ion to the An.sup.? anion is selected from the group consisting of Na.sup.+, K.sup.+ and Cs.sup.+.

15. A process for the preparation of a conjugate of the polymers with a bioactive compound using the polyether polymers according to claim 1.

16. The process according to claim 15 for the preparation of conjugated lipids for use in vaccinesUse according to claim 15 for the preparation of conjugated lipids for use in vaccines.

17. The process according to claim 16 based on lipid nanoparticles.

18. The process according to claim 17 wherein these nanoparticles are used against COVID-19.

19. The polyether polymer according to claim 8, wherein end-group fidelity of the polymer in regard to group X and/or in regard to group Y is at least 95%.

Description

BRIEF DESCRIPTION OF THE FIGURES:

[0088] FIG. 1: FIG. 1 shows the results of ELISA test of three of the polymers of the present invention and mPEG.

[0089] FIGS. 2 to 4: These figures show the SEC traces of polymers of the present invention synthesized in DMSO and FIG. 4 shows in addition the SEC trace of commercially available mPEG.

[0090] FIG. 5: FIG. 5 shows the .sup.1H NMR spectrum of ?-BzO-P(EG.sub.0.51-co-GME.sub.0.49).

[0091] FIG. 6: FIG. 6 shows the MALDI-TOF spectrum of ?-BzO-P(EG.sub.0.51-co-GME.sub.0.49).

[0092] FIG. 7: FIG. 7 shows cloud point of ?-BzO-P(EG.sub.0.51-co-GME.sub.0.49).

[0093] FIG. 8: FIG. 8 shows the MALDI-TOF spectrum of ?-MeOP(PEG.sub.0.88-b-PGME.sub.0.22), i.e. a block copolymer of the present invention.

[0094] FIG. 9: FIG. 9 shows the decreasing .sup.1H NMR signals of monomers during the preparation of a polymer of the present invention.

[0095] FIG. 10: FIG. 10 shows the result of a study of the relative reactivity of ethylene oxide and glycidyl methyl ether in the anionic ring opening polymerization.

[0096] FIG. 11: FIG. 11 shows the mechanism and side reactions of monomer-activated ring-opening polymerization using triisobutylaluminium leading to low end-group fidelity, wherein a) shows the epoxide activation; b) the formation of ate complex; c) initiation and propagation; d) termination and e) side reactions.

[0097] FIG. 12: FIG. 12 shows SEC traces of EG-GME copolymers synthesized in toluene at different temperatures.

[0098] FIG. 13: FIG. 13 shows MALDI TOF MS of BnOP(EG-co-GME) synthesized in toluene at 25? C.

[0099] FIG. 14: FIG. 14 shows stacked NMR spectra of copolymerization of EO and GME in toluene-d.sub.8 at different times of the copolymerization.

[0100] FIG. 15: FIG. 15 shows composition profile based on obtained reactivity ratios.

[0101] FIG. 16: FIG. 16 shows turbidity measurements of P(EG-co-GME) with different mol % GME at a constant concentration.

[0102] FIG. 17: FIG. 17 shows the synthesis of P(EG-co-GME) with different end groups.

[0103] FIG. 18: FIG. 18 shows SEC traces of EO-GME copolymers with different end groups.

[0104] FIG. 19: FIG. 19 shows SEC traces of ?,?-OHP(EG-co-GME) and two PLLA-b-P(EG-co-GME)-b-PLLA.

[0105] FIG. 20: FIG. 20 shows .sup.1H NMR spectrum of PLLA-b-P(EG-co-GME)-b-PLLA.

[0106] FIG. 21: FIG. 21 shows .sup.1H NMR DOSY spectrum of PLLA-b-P(EG-co-GME)-b-PLLA.

EXAMPLES

Reagents

[0107] Chemicals were purchased from TCI, Acros Organics, Roth, Sigma Aldrich and Honeywell, unless otherwise noted. Ethylene oxide was obtained from Air Liquide. Deuterated solvents were purchased from Deutero GmbH. THF was flashed over basic aluminum oxide before usage. Glycidyl methyl ether was dried over CaH.sub.2 and cryo-transferred before the polymerizations.

Measurements

[0108] .sup.1H and .sup.13C NMR spectra were recorded on a Bruker Avance III HD 300 spectrometer with 300 MHz and referenced internally to residual proton signals of the deuterated solvent. In case of .sup.1H DOSY NMR, spectra were recorded on a Bruker Avance III HD 400 spectrometer with 400 MHz and referenced internally to residual proton signals of the deuterated solvent.

[0109] M.sub.w, M.sub.n and dispersities (M.sub.w/M.sub.n=PDI) of all samples were determined from the corresponding size exclusion chromatograms (refractive index (RI) detector, DMF, calibration with PEG standards).

[0110] Size-exclusion chromatography (SEC) were performed with dimethylformamide (DMF with 1 g/L LiBr) as the mobile phase (flow rate 1 mL/min) on poly(2-hydroxyethylmethacrylat) (PHEMA) 300/100/40 columns at 50? C. Polymer concentrations were 1 mg/mL. Calibration was carried out using poly(ethylene glycol) standards (from Polymer Standard Service, Mainz, Germany).

[0111] Differential scanning calorimetry (DSC) measurements were carried out in the temperature range of ?100 to 100? C. with a heating rate of 10 K/min at a PerkinElmer DSC 8500. The thermal history of the samples was excluded via two cooling and two heating cycles. For each sample, the glass transition and the melting temperatures were obtained from the second heating curve.

[0112] MALDI-ToF MS measurements were carried out at a Bruker autoflex max MALDI-TOF/TOF. The potassium salt of trifluoroacetic acid and trans-2-[3-(4-tert-Butylphenyl)-2-methyl-2-propenylidene]malononitrile (DCTB) were utilized as ionization salt and matrix, respectively.

Example 1: Synthesis of Glycidyl methyl Ether (GME)

[0113] ##STR00022## [0114] a) Allyl methyl ether (10.0 g, 139 mmol) was dissolved in 274 mL dichloromethane (DCM) and m-chloroperoxybenzoic acid (m-CPBA, 70%, 37.6 g, 153 mmol (based on m-CPBA)) was added to the solution. After stirring overnight, the solution was filtrated and slowly concentrated under reduced pressure. Crude glycidyl methyl ether was separated from solid impurities via cryo-transfer. Slow evaporation of residual dichloromethane in vacuo gave pure glycidyl methyl ether as a colorless liquid; Yield 41%. [0115] b) 1-Chloro-3-methoxy-propan-2-ol (3.00 g, 2.56 mL, 24.1 mmol) and anhydrous sodium sulfate (1.02 g, 7.23 mmol) were added to a flask equipped with a magnetic stirrer and cooled with a water bath. Finely grounded sodium hydroxide (1.25 g, 31.3 mmol) was added under stirring. After complete reaction (TLC control) the crude product was cryo-transferred in vacuo from the reaction flask and dried over CaH2 under cooling. After an additional cryo-transfer, GME was obtained as a colorless liquid with a yield of 93%.

Example 2: General Procedure for the Preparation of Random Copolymers Synthesis of ?-BzO-P(EG.SUB.0.51.-co-GME.SUB.0.49.)

[0116]
Ph-CH.sub.2OCH.sub.2CH.sub.2O[CH.sub.2CH.sub.2O].sub.0.51[CH.sub.2CH(CH.sub.2OCH.sub.3)O].sub.0.49H

[0117] Cesium hydroxide monohydrate (28.0 mg, 118 ?mol) was dissolved in a THF-water mixture and transferred into a reaction flask, equipped with a Teflon stopcock and a septum. 2-Benzyloxy-ethanol (20.0 mg, 18.7 ?L, 131 ?mol) was dissolved in benzene and added to the reaction flask. After slow evaporation of the solvent, the resulting solid was dried at 60? C. under high vacuum overnight. The residue was dissolved in DMSO (5 mL) and the solution was cooled to ?78? C. GME (367 mg, 374 ?L, 4.16 mmol) was added via syringe and EO (183 mg, 189 ?L, 4.16 mmol) was cryo-transferred from a graduated ampule into the reaction flask. The solution was stirred for 48 h at room temperature under vacuum. Afterwards, the solution was poured into excess chloroform and the organic phase was extracted against water (3 times) and brine, dried over MgSO.sub.4 and filtrated. P(EG.sub.0.51-co-GME.sub.0.49) was obtained in quantitative yield as a viscous liquid after evaporation of the solvent and drying under high vacuo. The data of the product can be found in Tables 2 and 3, entry e.

Example 3: Synthesis of mPEG-b-PGME

[0118]
MeO[CH.sub.2CH.sub.2O].sub.0.78[CH.sub.2CH(CH.sub.2OCH.sub.3)O].sub.0.22H

[0119] Potassium tert-butoxide (13.0 mg, 118 ?mol) was dissolved in a tetrahydrofuran (THF)water mixture and transferred into a reaction flask, equipped with a Teflon stopcock and a septum. Methoxypoly(ethylene glycol) (mPEG, M.sub.n 2 kg/mol; 250 mg, 125 ?mol) was dissolved in benzene and added to the reaction flask. After slow evaporation, the residue was dried at 80? C. under high vacuum overnight. The residue was dissolved in dimethyl sulfoxide (DMSO, 5 mL) and the solution was cooled to ?78? C. Glycidyl methyl ether (253 mg, 258 ?L, 2.88 mmol) was cryo-transferred into the reaction flask. The solution was heated to 55? C. and stirred for 24 h under vacuum. Afterwards, the solution was poured into excess chloroform and the organic phase was extracted against water (3 times) and brine, dried over MgSO.sub.4 and filtrated. mPEG-b-PGME 4 k was obtained as a colorless solid after precipitation in ice-cold diethyl ether; yield quantitative. Table 2 and 3, entry I shows the data for this polymer.

Example 4: Synthesis of ?-BzO-?-N-succinimidyl carbonate-P(EG-co-GME)

[0120] ##STR00023##

R?H, CH.sub.2OCH.sub.3 N,N-disuccinimidyl carbonate (3.5 mg, 13.8 ?mol) is added to a stirred solution of ?-BzO-P(EG-co-GME) (50.0 mg, 4.6 ?mol) from Example 2 in dry CH.sub.3CN (1 ml) at room temperature for 18 h. The reaction mixture was dissolved in dichloromethane extracted with a saturated NaHCO.sub.3, water and brine, dried over MgSO.sub.4 and filtered. The solvent was removed under reduced pressure and the polymer dried under vacuum. The product was obtained as a white solid; yield quantitative.

Example 5: Conjugation of ?-BzO-?-N-succinimidyl carbonate-P(EG-co-GME) to Bovine Serum Albumin (BSA)

[0121] ##STR00024##

R?H, CH.sub.2OCH.sub.3 Bovine serum albumin (5 mg, 0.07 ?mol) and ?-BzO-?-N-succinimidyl carbonate-P(EG-co-GME) (16.9 mg, 2.6 ?mol) from Example 4 were stirred in phosphate-buffered saline (PBS buffer) for 1 h. The unreacted polymer was removed by dialysis in deionized water with a 25 kDa membrane filter. The conjugate was then dried by lyophilization and obtained in quantitative yield. In the preparation of this conjugate, the outer lysing group (30-35) are targeted for the conjugation.

Example 6: Competitive Enzyme-Linked Immunosorbent Assay (ELISA)

[0122] The interaction of the copolymer samples entry c, d and e were evaluated by a competitive PEG ELISA kit using a murine monoclonal, horseradish peroxidase conjugated anti PEG antibody (HRP anti-PEG) (Life diagnostics, West Chester, PA, USA). Samples of concentrations ranging from 0 to 4600 ?g ml-1 were prepared in dilution buffer. Additionally, mPEG with a molecular weight of 5000 g mol-1 was utilized as internal standard for comparison. 50 ?l of each prepared sample was dispensed to a PEG pre-coated 96-well plate and 50 ?l of HRP anti-PEG was added to each well. The solutions were incubated for 1 h at 25? C. with a micro-plate shaker and then washed six times with 400 ?l of wash buffer per each well. After removal of residual droplets, 100 ?l of 3,3,5,5-Tetramethylbiphenyl-4,4-diamine was added to each well and the solutions were mixed on a micro-plate shaker for 20 min. The reaction was stopped by addition of 100 ?l of stop solution and the absorbance at 450 nm was read within 5 minutes.

[0123] Analysis of ELISA Data: The determined absorbance values were normalized to visualize the percent of maximal binding. The sample concentrations were transformed to a function of log.sub.10. The sigmoidal fits were calculated using the following equation with a representing the upper, b the lower limit, c the inflection point and d the hill slope.

[00001]$y=a+\left(b-a\right)/1+10^{\left(c-x\right)*d}$

Example 7: Kinetic of the Synthesis of ?-BzO-P(EG-co-GME)

[0124] The synthesis of ?-BzO-P(EG-co-GME) was repeated in fully deuterated DMSO for an online in situ .sup.1H NMR kinetic measurement. 95 mg glycidyl methyl ether (95 ?l, 1.1 ?mol) and 32 mg ethylene oxide (33 ?l, 7.1 ?mol), i.e. 60% glycidyl methyl ether and 40% ethylene oxide where solved in fully deuterated DMSO. The consumption of ethylene oxide and glycidyl methyl ether over time was measured by .sup.1H-NMR spectroscopy. As measure for the ethylene oxide concentration, the signal of the protons of ethylene oxide at about 2.6 ppm was used. For glycidyl methyl ether the signals of the protons at the oxirane ring with absorptions at about 2.75 and 3.1 ppm were used. FIG. 9 shows the decrease of the selected .sup.1H NMR signals of the two monomers over time. FIG. 10 shows plot of monomer consumption M.sub.x,t/M.sub.x,t=0 versus total conversion. As can be seen, ethylene oxide and glycidyl methyl ether are consumed at exactly the same rate. This data shows that the copolymers of the present invention are almost ideal random copolymers.

[0125] Further, in-situ .sup.1H NMR kinetics were conducted to investigate the influence of the solvent change from DMSO to toluene on the copolymerization kinetics as can be seen in FIG. 14. In addition, based on the decrease of the monomer signals during copolymerization, copolymerization parameters of r.sub.EO=0.61 and r.sub.GME=1.65 were obtained, showing a slighty preferred incorporation of GME repeating units over EO at the beginning of the copolymerization due to the higher reactivity of GME over EO leading to a statistical copolymer as can be seen in FIG. 15.

Example 8: Synthesis of mP(EG.SUB.0.66.-co-GME.SUB.0.34.) (1) according to FIG. 17

[0126] mP(EG.sub.0.66-co-GME.sub.0.34) (1): Diethylene glycol monomethyl ether (50.8 mg, 420 ?mol) was dissolved in benzene (5 mL) and transferred via syringe into a flame-dried flask under stationary vacuum. Potassium-tert-butoxide (42.7 mg, 380 ?mol) was dissolved in stabilizer-free THF (3 mL) and small quantities of Millipore water and transferred into the flask. High vacuum was applied to the flask and the solvents were removed under high vacuum. The resulting initiator salt was dried under high vacuum at 60? C. overnight and finally dissolved in dry DMSO (5 mL) under stationary vacuum. After freezing the resulting solution at ?90? C., GME (1.03 g, 11.6 mmol) were added via syringe to the flask. EO (1.04 g, 1.00 mL, 23.6 mmol) was added to the flask via cryo-transfer before the cooling bath was removed and the reaction mixture was allowed to warm up to room temperature. The reaction was allowed to stir for 72 h at room temperature. Subsequently, a mixture of 2M HCl (500 ?L) and MeOH (500 ?L) was added to terminate the polymerization.

[0127] The reaction mixture was added to chloroform (20 mL), washed three times with Millipore water (10 mL) and once with brine. The organic phase was dried over MgSO4 before the solvent was removed under vacuum. The resulting polymer was dialyzed against MeOH (MWCO of 2 kDa) for 24 h and freeze-dried under high vacuum over night to give 1.82 g (86%) of the polymer (1) of FIG. 17 as a viscous pale-yellow liquid. The data of the product can be found in Tables 2 and 3, entry n.

[0128] Further, ?-BzO-P(EG-co-GME) with varying amounts of the monomers were prepared according to the same procedure with a polymerization temperature of 55? C. and benzyloxy ethanol as initiator, respectively, instead of diethylene glycol monomethyl ether (entries c and d of Tables 2 and 3).

Example 9: Synthesis of mP(EG.SUB.0.66.-co-GME.SUB.0.34.)-Ms (2) according to FIG. 17

[0129] mP(EG.sub.0.66-co-GME.sub.0.34)-Ms (2): Polymer (1) (600 mg, 120 ?mol) was dissolved in benzene (5 mL) and added to a flame-dried flask under argon-flow. Benzene was slowly removed under high vacuum. The polymer was heated to 60? C. and dried under high vacuum overnight. Under argon-flow, the dry polymer was dissolved in dry DCM (10 mL) and triethyl amine (97.1 mg, 960 ?mol) and methanesulfonyl chloride (110 mg, 960 ?mol) were added under ice-cooling. The mixture was allowed to stir for 72 h at room temperature under argon. Afterwards, the mixture was washed three times with Millipore water and once with brine. The organic phase was dried with MgSO4 and the solvent was removed under high vacuum to give 427 mg (70%) of the mesylated polymer (2) as a viscous dark yellow liquid. The data of the product can be found in Tables 2 and 3, entry o.

Example 10: Synthesis of mP(EG.SUB.0.66.-co-GME.SUB.0.34.)-N.SUB.3 .(3) according to FIG. 17

[0130] Polymer (2) (400 mg, 80.0 ?g) was dissolved in benzene (5 mL) and added to a flame-dried flask under argon-flow. Benzene was removed under high vacuum. The polymer was further heated to 60? C. and dried under high vacuum overnight. Subsequently, the dry polymer was dissolved in dry DMF (8 mL) and sodium azide (41.6 mg, 640 ?mol) was added as a suspension in dry DMF (2 mL) under argon-flow. The reaction mixture was stirred at 65? C. for 72 h. The solvent was removed under high vacuum and the residue was suspended in DCM (10 mL) and treated in an ultrasonic bath for 20 min. The remaining residue was filtered off and the filtrate was washed three times with Millipore water and once with brine. The organic phase was dried over MgSO.sub.4 and the solvent was removed to give 284 mg (71%) of polymer (3) as a viscous dark yellow liquid. The data of the product can be found in Tables 2 and 3, entry p.

Example 11: mP(EG.SUB.0.66.-co-GME.SUB.0.34.)-NH.SUB.2 .(4) according to FIG. 17

[0131] Polymer (3) (180 mg, 40.0 ?g) was dissolved in EtOH (10 mL). Catalytic amounts of Pd/C 10 wt % (13.8 mg, 10.0 ?mol of Pd) were added to the solution and a balloon filled with an excess of H2-gas was connected to the flask. Vacuum was applied to the flask until the solvent started to evaporate and the flask was subsequently flushed with argon. This process was repeated two more times. Afterwards, vacuum was applied until the solvent started to evaporate and the flask was flushed with H2-gas. The last step was repeated two more times. The reaction was allowed to stir under 1 atm of H2-gas for 48 h. After 24 h the H2 balloon was refilled to guarantee a saturation of the atmosphere with H2. After 48 h the balloon was removed, the catalyst was filtered off over zeolite and the solvent was removed under high vacuum to give 142 mg (79%) of the amino-terminated polymer (4). The data of the product can be found in Tables 2 and 3, entry q.

Example 12: Synthesis of ?,?-OHP(EG-co-GME)

[0132] 2,2-((Propane-2,2-diylbis(4,1-phenylene))bis(oxy))bis(ethan-1-ol) (BHEPP) (123 mg, 389 ?mol) was dissolved in benzene (5 mL) and transferred via syringe into a flame-dried flask under argon. Potassium-tert-butoxide (43.6 mg, 389 ?mol) was dissolved in stabilizer-free THF (3 mL) and small quantities of Millipore water and transferred into the flask. High vacuum was applied to the flask and the solvents were removed under high vacuum. The resulting initiator salt was dried under high vacuum at 70? C. overnight and finally dissolved in dry DMSO (5 mL) under stationary vacuum. After freezing the resulting solution at ?78? C., GME (873 mg, 890 ?L, 9.91 mmol) were added via syringe to the flask. EO (1.46 g, 1.50 mL, 33.1 mmol) was added to the flask via cryo-transfer before the cooling bath was removed and the reaction mixture was allowed to warm up to room temperature. The reaction was allowed to stir for 22 h at 55? C. Dowex? (30 mg) and water (4 mL) were added to the solution after venting the flask. After dialysis against water and lyophilization, the polymer was obtained as a viscous liquid (1.36 g, 57%). The data of the product can be found in Tables 2 and 3, entry r.

Example 13: Synthesis of PLLA.SUB.69.-b-P(EG-co-GME)-b-PLLA.SUB.70

[0133] In a glove box, dry ?,?-OHP(EG-co-GME) (400 mg, 66.7 ?mol) and L-Lactide (577 mg, 4.00 mmol) were dissolved in dry DCM (3 mL). DBU (6.08 mg, 40.0 ?mol) dissolved in DCM (70.0 ?L) was added and the resulting solution was stirred for 30 min. After addition of benzoic acid (24.4 mg, 200 ?mol) in DCM (3 mL), the crude polymer was precipitated in cold diethyl ether. The last step was repeated two times. The block copolymer was obtained after lyophilization from benzene as a colorless solid (79%).

Example 14: Synthesis of ?-BzO-P(EG.SUB.0.50.-co-GME.SUB.0.50.) in toluene

[0134] Other copolymerization conditions, but temperature, and solvent were held constant compared to Example 8 to merely investigate the influence of temperature and solvent on the copolymerization. In the investigated temperature range (25-50? C.), the GME-EO copolymerization in toluene results in well-defined copolymers with low dispersities <1.10 and monomodal narrow molecular weight distributions as can be seen in FIG. 12.

[0135] The MALDI-TOF MS of ?-BzO-P(EG.sub.0.50-co-GME.sub.0.50) (toluene, 25? C.) shows a clear overlay of theoretical and experimental M.sub.n values (2 kg mol.sup.?1) and reveals an exceptional high end-group fidelity (>99%), known from the copolymerization in DMSO as can be seen in FIG. 13.

[0136] The data of the product can be found in Tables 2 and 3, entry s to u.

Discussion of the Results

[0137] Tables 2, 3, 4 and 5 and FIGS. 1 to 21 show the properties of the polymers prepared. They are discussed in the following.

TABLE-US-00002 TABLE 2 Composition of polymers prepared Entry (Example) Sample DP.sub.EO NMR DP.sub.GE NMR mol %.sub.EO NMR mol %.sub.GME NMR c ?-BzO P(EG.sub.0.84-co-GME.sub.0.16) 112 21 84 16 (9) d ?-BzO P(EG.sub.0.76-co-GME.sub.0.24) 85 26 76 24 (9) e ?-BzO P(EG.sub.0.51-co-GME.sub.0.49) 35 33 51 49 (9) l ?-MeOPEG.sub.0.88-b-PGME.sub.0.22 50 14 78 22 (6) n ?-MeOP(EG.sub.0.66-co-GME.sub.0.34) 56 29 66 34 (8) o ?-MeO-?-MsP(EG.sub.0.66-co-GME.sub.0.34) 56 29 66 34 (9) p ?-MeO-?-N.sub.3P(EG.sub.0.66-co-GME.sub.0.34) 56 29 66 34 (10) q ?-MeO-?-NH.sub.2 P(EG.sub.0.66-co-GME.sub.0.34) 56 29 66 34 (11) r ?,?-OHP(EG-co-GME) 92 28 77 23 (12) s ?-BzOP(EG.sub.0.50-co-GME.sub.0.50) 15 15 50 50 (14) (toluene, 25? C.) t ?-BzOP(EG.sub.0.50-co-GME.sub.0.50) 15 15 50 50 (14) (toluene, 35? C.) u ?-BzOP(EG.sub.0.50-co-GME.sub.0.50) 15 15 50 50 (14) (toluene, 50? C.) GE = Glycidyl ether; DP = degree of polymerization

TABLE-US-00003 TABLE 3 Characterization data of polymers prepared Entry/ M.sub.n, NMR M.sub.n, MALDI M.sub.n, SEC End-group (Example) Sample [kg/mol] [kg/mol] [kg/mol] PDI.sub.SEC fidelity.sub.MALDI c (9) ?-BzO P(EG.sub.0.84-co-GME.sub.0.16) 6.9 4.9 3.7 1.05 >99% d (9) ?-BzO P(EG.sub.0.76-co-GME.sub.0.24) 6.0 5.2 3.8 1.06 >99% e (9) ?-BzO P(EG.sub.0.51-co-GME.sub.0.49) 4.2 3.8 2.4 1.09 >99% l (6) ?-MeOPEG.sub.0.88-b-PGME.sub.0.22 3.6 3.7 2.8 1.05 >99% n (8) ?-MeOP(EG.sub.0.66-co-GME.sub.0.34) 5.0 5.0 3.9 1.08 >99% o (9) ?-MeO-?-MsP(EG.sub.0.66-co-GME.sub.0.34) 5.0 5.1 3.9 1.09 >99% p (10) ?-MeO-?-N.sub.3P(EG.sub.0.66-co-GME.sub.0.34) 5.0 N/A 3.9 1.07 N/A q (11) ?-MeO-?-NH.sub.2 P(EG.sub.0.66-co-GME.sub.0.34) 5.0 N/A 4.0 1.09 N/A r (12) ?,?-OHP(EG-co-GME) 6.0 6.1 4.3 1.09 >99% s (14) ?-BzOP(EG.sub.0.50-co-GME.sub.0.50) 2.1 2.1 1.6 1.06 >99% (toluene, 25? C.) t (14) ?-BzOP(EG.sub.0.50-co-GME.sub.0.50) 2.2 2.2 1.8 1.07 >99% (toluene, 35? C.) u (14) ?-BzOP(EG.sub.0.50-co-GME.sub.0.50) 2.2 2.2 1.9 1.07 >99% (toluene, 50? C.) M.sub.n = molecular weight (number average molar mass), PDI = dispersity.

TABLE-US-00004 TABLE 4 Thermal properties Cloud [GME] M.sub.n.sup.b Point Sample (mol % ).sup.a (kg/mol) PDI.sup.c (? C.) ?-BzOP(EG.sub.0.41-co-GME.sub.0.59) 59 4.8 1.08 81 ?-BzOP(EG.sub.0.28-co-GME.sub.0.72) 72 4.7 1.06 73 ?-BzOP(EG.sub.0.19-co-GME.sub.0.81) 81 4.8 1.10 70 ?-BzOP(EG.sub.0.14-co-GME.sub.0.86) 86 4.8 1.07 66 .sup.acalculated via .sup.1H NMR spectroscopy .sup.bdetermined by MALDI-ToF mass spectrometry .sup.cdetermined by SEC (DMF, calibration with PEG standards)

[0138] Table 4 shows the thermal properties of the respective compounds. The polymers were prepared according to Example 8 with varying amounts of the monomers and with benzyloxy ethanol as initiator, respectively, instead of diethylene glycol monomethyl ether. The cloud point of the random copolymers of EO and GME was investigated via turbidimetry measurements of copolymers containing high amounts (? 59 mol %) of GME. Cloud point was defined at a transmittance of 50%. Benzyloxy-initiated copolymers synthesized via anionic ring-opening polymerization (AROP) in DMSO were used for the measurements.

[0139] The cloud point of the copolymers decreases with increasing amount of GME in the polymer backbone. Despite the decrease in the cloud point with increasing mol % GME, solubility in water under physiologically conditions is ensured for all copolymers.

Immunogenicity

[0140] As can be seen from Table 3, entries c to e and I show copolymers of the present invention which consisting of ethylene oxide repeating units and glycidyl methyl ether repeating units with about the same molecular weight, i.e. with an M.sub.n of about 4 to 5 kg/mol. The amount of glycidyl methyl ether repeating units is increasing from c to e from 16% to 49% (see Table 2, entries c to e and l). FIG. 1 shows the ELISA test results of polymers c to e and of commercially available mPEG. It shows the function of the normalized absorption at a wavelength of 450 nm versus the log10 function of the polymer concentration in nanograms per ml, therefore illustrating the anti-PEG antibody interaction with the investigated polymer concentrations. The ELISA data shows that a strong influence of an increasing concentration of alkyl side chains can be observed (it is important to note that the x-axis has a logarithmic scale). With increasing amount of GME incorporated into the polyether structure, significantly higher polymer concentrations are necessary to observe interactions between the copolymer and the APA. Remarkably, at 49 mol % of GME, absolutely no interaction between APA and the copolymer is present. This means that the respective copolymer cannot be detected by APAs, i.e. it is not immunogenic.

Dispersity

[0141] As can be seen from Table 3, entries c to e and l, all polymers of the present invention have a very low dispersity, i.e. 1.10 or lower. For visualization, the SEC traces of these polymers are shown in FIGS. 2 to 4 and 12. FIG. 4 shows in addition the SEC trace of mPEG. It can clearly be seen that the present invention provides polymers with a very narrow molecular weight distribution.

Preparation and Purity of the Polymers of the Present Invention

[0142] FIG. 5 shows the .sup.1H NMR spectrum of ?-BzO P(EG.sub.0.51-co-GME.sub.0.49), i.e., the polymer of Example 2 (entry e of Tables 2 and 3). The product is as obtained from Example 2, without further purification. As can be seen, the present method does not only provide polymers of narrow molecular weight distribution, but also very pure polymers without the need for laborious purification steps. This is an important feature for the use in pharmaceutical applications.

[0143] The synthesis of bifunctional initiated ?,?-OHP(EG-co-GME) copolymers enables the synthesis of ABA triblock copolymers. In this case, P(EG-co-GME) bearing two hydroxyl-termini (Example 12) was used as an macroinitiator for ring-opening polymerization of L-lactide, enabling the synthesis of PLLA-b-P(EG-co-GME)-b-PLLA, wherein PLLA is

##STR00025##

The polymerization is catalyzed by the organic base DBU (1,8-Diazabicyclo [5.4.0]undec-7-ene).

[0144] FIG. 19 shows an overlay of the SEC traces of the macroinitiator ?,?-OHP(EG-co-GME) (polymer of Example 12) and two PLLA-b-P(EG-co-GME)-b-PLLA (PLLA.sub.46-b-P(EG-co-GME)-b-PLLA.sub.47 is polymer of Example 13) with different degrees of polymerization (k=46, 47 and 69, 70), confirming successful chain extension from the macroinitiator.

[0145] FIG. 20 shows the 1H NMR sprectrum of Example 13, confirming the successful polymerization of L-lactide.

[0146] FIG. 21 shows the .sup.1H DOSY NMR confirming that the blocks within the copolymer are linked via a covalent bond.

End-Group Fidelity

[0147] As can be seen from Table 3, the end-group fidelity for all polymers of the present invention is nearly 100%. FIG. 6 shows the MALDI TOF mass spectrum of ?-BzO P(EG.sub.0.51-co-GME.sub.0.49), i.e. the polymer of Example 2 (entry e of Tables 2 and 3) with potassium and sodium cations. The only peaks visible here are the peaks of the product molecule. There are no peaks form macromolecules with a different end group. This shows that end-group fidelity of the polymers of the present invention is very high. In addition, the spectrum confirms the narrow molecular weight distribution.

Solubility

[0148] FIG. 7 shows the cloud point measurement of ?-BzO-P(EG.sub.0.51-co-GME.sub.0.49), i.e. the polymer of Example 2 (entry e of Tables 2 and 3). The cloud point determines the temperature at which immiscibility in water/in aqueous solution is observed upon heating. As an example, for the copolymer with 49% GME, the cloud point can be observed at 95? C., showing excellent water solubility of the P(EG-co-GME) copolymers. The homopolymer PEG shows a cloud point of ca. 100? C. All other copolymers of the present invention with 26-49% GME are in between PEG and this copolymer, showing cloud points of 96-100? C. For copolymers of 25 mol % GME incorporation and lower no cloud point was detected in water at temperatures ranging from 0 to 99? C. Thus, GME copolymerization hardly affects water solubility, which is a feature of the similar structure of polyethylene glycol and methyl ethyl ether. This is an important feature of the polymers of the present invention as it shows that they can substitute PEG in aqueous systems without causing problems with phase separation or even precipitation.

Comparison of Dispersity of End Group Fidelity of the Polymers of the Present Invention With That of mPEG

[0149] FIG. 8 shows an overlay of MALDI TOF mass spectra of commercially available mPEG and a block copolymer of the present invention (polymer of Example 3 (entry I of Tables 2 and 3). As can be seen, a clear shift between the macroinitiator mPEG and entry I is observed, indicating a successful block copolymer synthesis. Absence of macroinitiator in the MALDI-ToF MS of entry I further proves quantitative block copolymer formation. This is further proven by a distance of the signals of 44 g mol.sup.?1.

Crystallinity

[0150] Table 5 shows thermal properties of some of the polymers of Tables 2 and 3:

TABLE-US-00005 T.sub.g T.sub.m ?H.sub.PEG Sample [? C.] [? C.] [J/g] X.sub.c, PEG a ?56 46 88.63 0.45 b ?56 45 76.79 0.39 c ?59 37 56.26 0.29 l ?59 45 80.16 0.41 e ?62 N/A N/A 0.00 T.sub.g = Glass transition temperature; T.sub.m = Melting point; ?H.sub.PEG = Enthalpy of fusion; X.sub.c = Crystallinity (all measurements by DSC)I

[0151] As can be seen, crystallinity of the inventive polymers is low and can be completely interrupted by the incorporation of GME monomer in the polymer structure. This prevents for example accumulation of the polymers in the kidney or in the liver.