ARGININE-BASED ANTIBACTERIAL POLYMERS WITH UCST PROPERTY

Abstract

The present invention concerns a polymer comprising repetitive units having the following formula (I) wherein R.sub.1 is H or Me; L is a linker; R.sub.2 is the side chain of an α-amino acid being other than arginine; m is 0 or an integer comprised from 1 to 10; n is an integer comprised from 1 to 10; and X— is a counterion.

##STR00001##

Claims

1. A polymer comprising repetitive units having the following formula (I): ##STR00018## wherein: R.sub.1 is H or Me; L is a linker; R.sub.2 is the side chain of an α-amino acid being other than arginine; m is 0 or an integer comprised from 1 to 10; n is an integer comprised from 1 to 10; and X.sup.− is a counterion.

2. The polymer of claim 1, wherein L is a linker having the following formula (II):
-A.sub.1-A.sub.2-A.sub.3-  (II) wherein: A.sub.1 is a (C.sub.1-C.sub.6)alkylene group; A.sub.2 is a group obtainable by alkyne-azide cycloaddition reaction; and A.sub.3 is a (C.sub.1-C.sub.10)alkylene group, optionally interrupted by one or several oxygen atoms.

3. The polymer of claim 2, wherein A.sub.2 is a triazole group.

4. The polymer of claim 1, wherein L has the following formula: ##STR00019##

5. The polymer of claim 1, wherein m=0.

6. The polymer of claim 5, further comprising repetitive units having the following formula (IV): ##STR00020## wherein: p is an integer comprised from 1 to 10.

7. The polymer of claim 1, having the following formula (V): ##STR00021## wherein: R.sub.1, L, R.sub.2, X.sup.−, m, and n are as defined in claim 1; R.sub.3 represents an aliphatic or aromatic chain, possibly substituted by one or several functional groups and/or optionally interrupted by one or several oxygen atoms; R.sub.4 represents a group —S—C(═S)—Z, Z being the group controlling the reactivity of the C═S bond; and q is comprised from 2 to 100.

8. The polymer of claim 6, having the following formula (VI): ##STR00022## wherein: R.sub.3 represents an aliphatic or aromatic chain, optionally substituted by one or several functional groups and/or optionally interrupted by one or several oxygen atoms; R.sub.4 represents a group —S—C(═S)—Z, Z being the group controlling the reactivity of the C═S bond; q is comprised from 2 to 100; and r is comprised from 2 to 100.

9. The polymer of claim 1, having the following formula (VII): ##STR00023## wherein: L, R.sub.2, X.sup.−, m, and n are as defined in claim 1, and q is comprised from 2 to 100.

10. The polymer of claim 1, having the following formula (VIII): ##STR00024## wherein: X.sup.− and n are as defined in claim 1; and q is comprised from 2 to 100.

11. A method for the preparation of a polymer of claim 1, comprising a step of RAFT polymerization of a monomer having the following formula (IX): ##STR00025## wherein R.sub.1, L, R.sub.2, X.sup.−, m, and n are as defined in claim 1.

12. The method of claim 11, wherein m is 0, and comprising a step of RAFT polymerization of monomers having the following formulae (X) and (XI): ##STR00026## wherein is an integer comprised from 1 to 10.

13. A monomer having one of the following formulae (IX) or (X): ##STR00027## wherein R.sub.1, L, R.sub.2, X.sup.−, m, and n are as defined in claim 1.

14. A method for delivering a drug to a subject in need thereof, comprising encapsulating the drug with the polymer of claim 1, and delivering the drug to the subject.

15. A method for treating a bacterial infection in a patient in need thereof, comprising administering to the patient a pharmaceutically acceptable amount of the polymer of claim 1.

Description

FIGURES

[0098] FIG. 1. UCST behavior of P(MA-R5).sub.10 monitored by turbidimetry on heating (open symbols) and cooling (filled symbols): (a) transmittance versus temperature curves of aqueous solutions at 1 (squares), 2 (triangles) and 5 (circles) mg mL.sup.−1; inset photos of solutions at 20 and 85° C. (b) evolution of the UCST, determined at 5 mg mL.sup.−1, as a function of the degree of polymerization.

EXAMPLES

[0099] Materials

[0100] 2,2′-Azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (VA-044, TCI, 98%), 2-chlorotrityl chloride (CTC) resin (100-200 mesh, 1.6 mmol g.sup.−1, Iris Biotech), Cuprisorb (Seachem), Na-Fmoc-N,-(2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl)-L-arginine (Fmoc-Arg(Pbf)-OH, Fluorochem, 95%) and pyridine (Fluka, 99.5%) were purchased. Acetic acid (AcOH, 100%) and hydrochloric acid (HCl, 37%) were purchased from Roth. Acetonitrile (ACN, 99.7% HPLC grade), n-butanol (99%), N,N-diisopropylethylamine (DIPEA, 99%), N,N,N′,N″,N″-pentamethyl-diethylenetriamine (PMDETA, 98%), O-(benzotriazol-1-yl)-N,N,N′,N′-tetramethyl uronium hexafluorophosphate (HBTU, 98%), propargyl methacrylate ester (97%), trifluoroacetic acid (TFA, 99%) and 2,2,2-trifluoroethanol (TFE, 99%) were purchased from Alfa Aesar. Anhydrous dichloromethane (DCM, 99.9%), anhydrous N,N-dimethylformamide (DMF, 99.8%), and ninhydrin (95%) were purchased from Acros Organics. α-Cyano-4-hydroxy-cinnamic acid (CHCA), deuterium oxide (D.sub.2O, 99.96%), formic acid (HCOOH, 98%), isopropanol (99.7%), piperidine (89%), phenol (99%), potassium cyanide (KCN, 98%), sodium hydroxide (NaOH, 98%) and triisopropylsilane (TIPS, 98%), were purchased from Sigma Aldrich. 4-Cyano-4-(phenylcarbonothioylthio)pentanoic acid (CPABD, 97%) and 1-hydroxybenzotriazole hydrate (HOBt, 99%, water content >20 wt.-%) were purchased from ABCR. Absolute ethanol (EtOH, 99.8), DMF (99.8%), magnesium sulfate (MgSO.sub.4, 98.7%), methanol (MeOH, 99.8%), and sodium chloride (NaCl, 99%) were purchased from VWR Chemicals. Acetone (99.8%), %), DCM (99.8%) was purchased from the Dasit Group (Carlo Erba reagents). All compounds if not mentioned are employed as received. Copper(I) bromide (CuBr, 98%) was purchased from Sigma Aldrich, washed with AcOH, subsequently with absolute EtOH, to be filtered and stored under inert atmosphere prior to use. 6-Azidohexanoic acid was synthesized as previously reported (D. Chan-Seng, J.-F. Lutz, ACS Macro Lett., 2014, 3, 291). The Kaiser test was conducted as described in the literature (E. Kaiser, R. L. Colescott, C. D. Bossinger, P. I. Cook, Anal. Biochem., 1970, 34, 595). All the syntheses on solid support were performed in solid phase extraction (SPE) tubes (60 mL polypropylene SPE tubes with polyethylene frits, 20 μm porosity purchased from SUPELCO®).

[0101] Characterization Techniques

[0102] Nuclear magnetic resonance (NMR) spectra were recorded on a 400 MHz Bruker Avance spectrometer equipped with Ultrashield magnets at 25° C. or a 400 MHz Bruker Avance III HD at 65° C. For the determination of the monomer conversion, the kinetic points of the polymerization mixtures were determined at 65° C. by 1-D NOESY experiments (M. Findeisen and S. Berger, in 50 and more essential NMR experiments: A detailed guide, Wiley, Weinheim, 2013) with 256 scans and a recycle delay (d1) of 2.4 s. The water suppression was achieved with pre-saturation occurring during the relaxation delay and the mixing time 100 ms.

[0103] Fourier transform infrared (FTIR) spectra were recorded on a Bruker Vertex 70 spectrometer using the attenuated total reflectance (ATR) technique.

[0104] Matrix-assisted laser desorption/ionization-Time of flight (MALDI-ToF) mass spectra were acquired on a TOF spectrometer (Autoflex Speed LRF, Bruker Daltonics, Bremen, Germany) equipped with a nitrogen laser (λ=337 nm). An external multi-point calibration was carried out before each measurement using the singly charged peaks of a standard peptide mixture (0.4 μM in water with 1% HCOOH). Scan cumulation and data processing were performed with FlexAnalysis 3.0 software. Matrix solutions were freshly prepared from a saturated CHCA solution in H.sub.2O/ACN/HCOOH (50/50, 1%). A 1/1 sample solution/matrix was prepared and 1 μL deposited on the stainless-steel plate.

[0105] Visible spectroscopy was conducted on a CARY 5000 spectrophotometer (Agilent Technologies) equipped with a thermostatic Peltier multicell holder (−10 to 100° C.).

[0106] Preparation of Monomers

[0107] Synthesis of Pentaarginine-g-Methacrylate (Ma-R5)

##STR00016##

[0108] Loading of the resin. CTC resin (1.5 g, 2.40 mmol, 1 eq.) was weighed in a SPE tube and washed six times with anhydrous DCM (15 mL). Fmoc-Arg(Pbf)-OH (3.1 g, 4.80 mmol, 2 eq.) was added to the resin. The tube was degassed by performing three vacuum/argon cycles. Under argon, 15 mL of anhydrous DCM was added to the tube, followed by 1.7 mL of DIPEA (9.60 mmol, 4 eq.). The tube was agitated using an orbital stirrer for 2 h (500 rpm) at room temperature. The solution was filtered and the resin was rinsed six times with 15 mL of DMF after 2 min stirring. 30 mL of DCM/MeOH/DIPEA (80/15/5) was added to the tube and stirred for 10 min at room temperature (twice). The resin was then rinsed six times with DMF (15 mL). The deprotection of the Fmoc group was performed by adding 20 mL of a 25 v % piperidine solution in DMF to the tube that was agitated for 6 min. After filtration, this step was repeated with an agitation of 40 min. The resin was washed six times with DMF (15 mL), six times with DCM (15 mL), three times with MeOH (15 mL) and finally six times with DCM (15 mL). The resin was dried under vacuum for 36 h. The loading density of the resin was determined by gravimetry as 1.17 mmol Pbf-protected arginine per gram of resin (73%).

[0109] Addition of protected arginine residues. Fmoc-Arg(Pbf)-OH (3.4 g, 5.26 mmol, 3 eq.), HOBt (0.7 g, 5.26 mmol, 3 eq.), HBTU (2.0 g, 5.26 mmol, 3 eq.) were added to the resin followed by three vacuum/argon cycles. DIPEA (1.8 mL, 10.5 mmol, 6 eq.) and anhydrous DMF (15 mL) were added to the tube under argon. The reaction was agitated for 4 h at room temperature with an orbital stirrer (500 rpm). The resin was washed six times with DMF (15 mL). The Kaiser test was performed: i) if blue, the coupling reaction was repeated or ii) if yellow, the Fmoc deprotection reaction was performed as previously using a solution of piperidine in DMF.

[0110] Insertion of 6-azidohexanoic acid. To the vessel, 6-azidohexanoic acid (1.7 g, 5.26 mmol, 3 eq.), HOBt (0.7 g, 5.26 mmol, 3 eq.), HBTU (2.0 g, 5.26 mmol, 3 eq.) were added. Three vacuum/argon cycles were performed. 30 mL anhydrous DMF and DIPEA (1.8 mL, 10.5 mmol, 6 eq.) were added to the tube that was agitated at room temperature with an orbital shaker (500 rpm) for 2 h. The solution was filtered and the resin was rinsed six times with DMF (15 mL), six times with MeOH (15 mL), and six times with DCM (15 mL). FTIR spectroscopy was performed to confirm the presence of the azide (2100 cm.sup.−1).

[0111] End-capping with methacrylate group. CuBr (126 mg, 0.877 mmol, 0.5 eq.) was added to the tube followed by three vacuum/argon cycles. Propargyl methacrylate ester (661 μL, 5.26 mmol, 3 eq.), PMDETA (366 μL, 1.87 mmol, 1.00 eq.) were degassed, dissolved in 20 mL anhydrous DCM and transferred to the previously degassed tube. The tube was agitated with an orbital shaker (500 rpm) for 16 h. The solution was filtered and the resin was rinsed multiple times (at least six times for each solvent, 20 mL) with DMF, MeOH and DCM until the color blue/green was not observed. Completion was monitored by FTIR spectroscopy (disappearance of the azide band at 2100 cm.sup.−1).

[0112] Cleavage from the resin. The resin was dried under vacuum overnight prior to be mixed in a 100 mL round bottom flask with 9.7 mL of a TFA/TIPS/H.sub.2O (95/2.5/2.5) mixture. The solution was stirred at room temperature for 2 h and then the filtrate was collected. These steps were repeated twice and the filtrate was concentrated by rotary evaporation at room temperature. After dilution in 40 mL MilliQ water and 10 mL DCM, the product was stirred with Cuprisorb overnight. After filtration and rinsing the Cuprisorb beads with 50 mL water, the filtrate was extracted with 50 mL DCM three times. The aqueous phase was slightly acidified (1 mL 0.3 N HCl is added) and precipitated in cold acetone (250 mL). The white solid was redissolved in 5 mL 0.3 N HCl solution and precipitated again in cold acetone (250 mL). The precipitation was repeated once. After filtration the white solid was dissolved in MilliQ water prior to freeze-drying. The compounds were kept in the dark and stored at −20° C.

[0113] 676 mg (36.3%). .sup.1H NMR (400 MHz, D.sub.2O) δ 8.12 (s, 1H), 6.17 (s, 1H), 5.76 (s, 1H), 5.35 (s, 2H), 4.47 (t, J=6.8 Hz, 2H), 4.39 (dd, J=14.6, 5.8 Hz, 4H), 4.32-4.24 (m, 1H), 3.35-3.18 (m, 10H), 2.32 (t, J=7.2 Hz, 2H), 2.06-1.55 (m, 29H), 1.39-1.20 (m, d, 2H). .sup.13C NMR (126 MHz, D.sub.2O) δ 176.9, 175.3, 174.0, 173.5, 169.1, 156.7, 142.7, 135.5, 127.3 125.4, 57.7, 53.3, 53.2, 52.6, 51.8, 50.3, 40.5, 35.0, 29.0, 28.0, 27.7, 25.0, 24.4, 17.3. MALDI-ToF MS (m/z) [M+H].sup.+ calculated for C.sub.43H.sub.80N.sub.23O.sub.9, 1062.651; found 1062.705.

[0114] Preparation of Polymers

[0115] Here is described the first fully charged polyelectrolyte based on oligopeptide side chains with a reversible UCST behavior in pure water without addition of any specific counterions. This polymer is a cationic comb homopolymer with oligoarginine pendent chains synthesized by RAFT polymerization of pentaarginine-g-methacrylate (MA-R5). The preparation of methacrylate-based monomers bearing a peptide sequence requires the development of synthesis strategies to minimize the Michael side reaction affecting strongly the purity of the macromonomers synthesized. Here an original strategy is proposed by combining solid-phase peptide synthesis and copper-assisted alkyne-azide cycloaddition. MA-R5 was prepared by solid-phase peptide synthesis through the iterative addition of Fmoc-Arg(Pbf)-OH onto a 2-chlorotrityl chloride resin using HBTU as coupling agent and HOBt as racemization inhibitor. The on-resin protected pentaarginine was amidated with 6-azidohexanoic acid followed by a copper-assisted alkyne-azide cycloaddition with propargyl methacrylate. The macromonomer was cleaved from the resin using a solution of trifluoroacetic acid (TFA) and isolated by precipitation in cold acetone in 22% yield with a purity of higher than 90%. The structure of the macromonomer was confirmed by .sup.1H and .sup.13C NMR spectroscopies, and mass spectrometry. RAFT polymerization of MA-R5 targeting different degree of polymerization (DP.sub.n,th) was performed in water/methanol mixture (1:1 v/v) at pH 3 using 4-(cyanopentanoic acid)-4-dithiobenzoate (CPABD) as chain transfer agent and an azo initiator (2,2′-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride, VA-044) for 24 h at 60° C. as depicted in Scheme 1 using conditions gathered in Table 51.

[0116] Polymerization of MA-R5 (P(MA-R5))

##STR00017##

[0117] All the polymerizations were conducted with a constant amount of MA-R5 (106 mg, 100.0 μmol) and solvent (5 mL), while the concentration of initiator (VA-044) and RAFT agent (CPABD) was adjusted to the targeted degree of polymerization (DP.sub.target).

[0118] MA-R5 was placed in a 10 mL Schlenk tube and dissolved in 2.5 mL HCl aqueous solution (pH=2.3). From solutions of CPABD (40 mmol L.sup.−1) and VA-044 (16 mmol L.sup.−1) prepared in MeOH, a specific volume was withdrawn and added to the monomer solution according to DP.sub.target (Table S1). MeOH was added to reach an equivolumetric ratio of water and MeOH. Five freeze-pump-thaw cycles were performed and the reaction mixture was stirred at 60° C. for 24 h. An aliquot was taken for the determination of the conversion while the reaction mixture was still hot. The conversion was determined by .sup.1H NMR spectroscopy at 65° C. in D.sub.2O. The conversion was obtained by estimation of the monomer C═C signal (6.17 ppm or 5.76 ppm) as compared to the proton signal from the triazole ring (8.12 ppm). After polymerization, the reaction mixture was cooled down, exposed to air and let to precipitate at 5° C. overnight. The supernatant was removed, while the white sticky precipitate was dissolved in 1 mL HCl aqueous solution (pH=2.3), heated for 1 min at 60° C. until complete dissolution, and let to precipitate at 5° C. overnight. This operation was repeated once. Subsequently the supernatant was removed and the white solid dissolved in 1 mL HCl aqueous solution (pH=2.3) to be dried by rotary evaporation without heat. The purified polymer P(MA-R5) was characterized by 1H NMR spectroscopy at 65° C. in D.sub.2O, for which the DP.sub.exp was evaluated from the chain-end aromatic proton assigned at 7.83 ppm and the polymer backbone from protons assigned at 1.20-1.09 ppm.

TABLE-US-00001 TABLE S1 RAFT polymerization conditions for P(MA-R5) H.sub.2O CPABD VA-044 MeOH (pH = 2.3) polymers MA-R5:CPABD:VA-044.sup.[a] DP.sub.target V.sup.[b]/μL V.sup.[b]/μL V.sup.[b]/μL V.sup.[b]/mL P(MA-R5).sub.3 100:10:4 10 250 250 2000 2.5 P(MA-R5).sub.10 100:5:0.8 20 125 50 2325 2.5 P(MA-R5).sub.29 100:2:0.32 50 50 20 2430 2.5 P(MA-R5).sub.50 100:1:0.16 100 25 10 2465 2.5 .sup.[a]molar ratio of macromonomer MA-R5, RAFT agent (CPABD) and initiator (VA-044), .sup.[b]volumes used for 100.0 μmol MA-R5 using 40 mmol L.sup.−1 CPABD and 16 mmol L.sup.−1 VA-044 solutions.

[0119] The polymer obtained (P(MA-R5)) was isolated by precipitation from the polymerization medium on cooling. The degree of polymerization of each polymer (Table 1) was determined by .sup.1H NMR spectroscopy, either via the conversion of the polymerization (DP.sub.n,th) assuming the “living” character of RAFT polymerization or by analyzing the integrals of the terminal group (7.83 ppm, aromatic proton of RAFT agent) and the repeat units (1.09-1.20 ppm, methyl of the polymethacrylate backbone) of the purified polymer (DP.sub.n,exp). Due to the high molecular weight of the macromonomer (1061.6 g mol.sup.−1), the determination of DP.sub.n,exp was not possible when targeting high DP.sub.n,th. Poly(methacrylate-g-pentaarginine)s P(MA-R5)s were obtained with a DP.sub.n of 3, 10, 29 and 50.

TABLE-US-00002 TABLE 1 Characteristics of the comb polymers synthesized. Sample DP.sub.target.sup.[a] Conversion.sup.[b] [%] DP.sub.n,th.sup.[c] DP.sub.n,exp.sup.[d] N.sub.R.sup.[e] P(MA-R5).sub.3 10 25 3 3 15 P(MA-R5).sub.10 20 52 10 7 50 P(MA-R5).sub.29 50 57 29 n.d. 145 P(MA-R5).sub.50 100 50 50 n.d. 250 .sup.[a]Targeted degree of polymerization for 100% monomer conversion. .sup.[b]Determined by .sup.1H NMR spectroscopy. .sup.[c]Degree of polymerization obtained from monomer conversion. .sup.[d]Degree of polymerization determined by analysis of the terminal group on the .sup.1H NMR spectrum. .sup.[e]Number of arginine residues per polymer chain calculated from DP.sub.n,th. n.d. stands for not determined.

[0120] Turbidimetry Experiments by Visible Spectroscopy

[0121] The samples were introduced in a closed quartz cuvette and let 10 min to stabilize at the desired temperature. The experiment started with a cooling step from 95 to 10° C. at a rate of 1° C. min.sup.−1, followed by an equilibrating step for 10 min, before to heat the sample back to the final temperature at the same rate. The absorbance was measured for each degree at 600 nm after zeroing the absorbance at high temperature.

[0122] The UCST behavior of the different polymers was investigated in water by turbidimetry at a wavelength of 600 nm. Polymer solutions were prepared in pure water at different concentrations (1, 2 and 5 mg mL.sup.−1) and underwent a heating and cooling process between 10 and 85° C. at 1° C. min.sup.−1. FIG. 1a depicts turbidity measurements of P(MA-R5).sub.10 showing sharp UCST-type transitions with narrow hysteresis during heating and cooling cycles. The cloud point (T.sub.CP) and clearing point (T.sub.CL) were determined at the inflection point (Z. Osváth, B. Iván, Macromol. Chem. Phys. 2017, 218, 1600470) on the cooling and heating curves, respectively. T.sub.CP increased from 31 to 51° C. (from 32 to 52° C. for T.sub.CL) with the polymer concentration (Table 2). Interestingly at 2 mg mL.sup.−1, P(MA-R5).sub.10 gave a phase transition temperature at 36° C. on heating, near the body temperature. Though the monomer MA-R5 had no UCST, all the other polymers, i.e. P(MA-RA).sub.3, P(MA-R5).sub.29 and P(MA-R5).sub.50, showed a UCST with a less sharp transition. Even with a degree of polymerization of 3, a phase transition was observed between 23 and 17° C. thanks to the presence of fifteen arginine residues per polymer chains. The same dependency of UCST on polymer concentration was obtained in the range of 10 to 85° C. with a loss at 1 mg mL.sup.−1. The phase transition was found to be dependent on the molecular weight of the polymers. T.sub.CP increased from 22° C. up to a plateau around 87° C. for P(MA-R5).sub.29 and P(MA-R5).sub.50 (FIG. 1b). This evolution of T.sub.CP could be attributed to the influence of the end groups for the shortest polymer chains that became insignificant above a certain degree of polymerization. We can underline that although arginine residues were protonated, poly(methacrylate-g-pentaarginine)s possessed an UCST in pure water even for a high number of arginine residues (at least up to 250 cationic charges per chain).

TABLE-US-00003 TABLE 2 Thermoresponsive properties of P(MA-R5)s. Sample Concentration [mg mL.sup.−1] T.sub.CP.sup.[a] [° C.] T.sub.CL.sup.[b] [° C.] MA-R5 2 none none P(MA-R5).sub.3 5 22 23 2 17 18 1 none none P(MA-R5).sub.10 5 51 52 2 33 36 1 31 32 P(MA-R5).sub.29 5 86 86 2 60 66 1 none none P(MA-R5).sub.50 5 87 89 2 54 55 1 none none Cloud (T.sub.CP) and clearing (T.sub.CL) temperatures at the inflexion point upon .sup.[a] cooling and .sup.[b] heating, determined by turbidimetry in water (600 nm, 1° C. min.sup.−1).