Process For Preparation Of Bioorganic Nylon Polymers And Their Use As Antibacterial Material

Abstract

The present invention relates to new biopolymer, i.e. bioorganic nylons incorporating peptidic blocks obtained by a process of polymerization of amino peptidic blocks. The process of the invention comprises the steps of mixing amino peptidic blocks with or without a diaminoalcane and reacting the mixture according to polycondensation with a diacyl chloride in homogeneous or heterogeneous media.

Claims

1. A process for preparation of bioorganic nylon type polymers comprising the steps of i) preparing a basic solution containing at least one type of peptidic amino-building block selected from the group consisting of the following A, B, C, D and E blocks: wherein AA is an amino-acid residue, PEPTIDE is a peptide residue corresponding to a chain of amino acid residues bound by peptidic bonds; is the N.sub.terminus of AA or PEPTIDE; is the amino derivatized C.sub.terminus of AA PEPTIDE; is the free amino group of the side chain of AA or of an amino-acid residue of PEPTIDE; is the C.sub.terminus of AA or PEPTIDE; is the protected N terminus of the AA or PEPTIDE; and ii) Initiating polymerization by contacting the solution of i) with a solution containing a diacyl G of the following formula (G): X(CO)—Y—(CO)X wherein: X is a halogen; Y is (CH.sub.2).sub.q, C.sub.6H.sub.4, C.sub.6H.sub.4—W—C.sub.6H.sub.4, C.sub.6F.sub.4, C.sub.6F.sub.4—W—C.sub.6F.sub.4, or C.sub.10H.sub.6; W is a bond, SiO.sub.2[(CH.sub.2).sub.rCH.sub.3].sub.2, CH.sub.2, O or S, with r comprised between 1 and 4; and q is comprised between 1 and 12.

2. The process of claim 1, wherein solution of i) further contains a diamino building block F selected from the group consisting of diamino-alcane building block of general formula (F): H.sub.2N—Z—NH.sub.2, wherein: Z is (CH.sub.2).sub.p, C.sub.6H.sub.4, C.sub.6H.sub.4—W′—C.sub.6H.sub.4, C.sub.6F.sub.4, C.sub.6F.sub.4—W′—C.sub.6F.sub.4, or C.sub.10H.sub.6; W′ is a bond, CH.sub.2, O, S, or SiO.sub.2[(CH.sub.2).sub.sCH.sub.3].sub.2 with s comprised between 1 and 4; and p is comprised between 1 and 14.

3. The process of claim 1, wherein the polymerization is achieved in homogeneous or heterogeneous phase.

4. The process of claim 1, wherein the polymerization is achieved in homogeneous phase and the buildings blocks A to E are dissolved in a solvent which is also a solvent of the diacyl compound G.

5. The process of claim 1, wherein the polymerization is achieved in heterogeneous phase and polymerization is initiated through interfacial polymerization.

6. The process of claim 1, wherein the polymer is recovered and washed.

7. The process of claim 1, wherein the basic solution of i) has a pH above 7.

8. The process of claim 1, wherein the diacyl compound (G) is diacyl chloride

9. The process of claim 1, wherein the molar concentration of peptidic-diamino building blocks [A+B+C+D+E] varies from 1 to 100% and the molar concentration of diaminoalcane building block [F] is comprised between 0 and 99%, the sum of both molar quantities being equal to 100% and corresponding to the molar quantity of diacyl compound [G] expressed as 100%.

10. The process of claim 1, wherein the protected N-terminus of the AA or of the PEPTIDE is protected by a protecting group selected from the group consisting of Fmoc, Boc, Cbz, Dde, trityl, NVoc, Alloc and Troc.

11. The process of claim 1, wherein the amino peptidic block comprises, within its peptidic chain or at any of its N terminus or C-terminus, a lysine or an amino acid residue having a side chain that contains the following structure: —(CH.sub.2).sub.m—NH.sub.2 wherein m is comprised between 0 and 10.

12. The process of claim 1, wherein the PEPTIDE corresponds to the homo- or hetero-oligomer according to the following motif: ##STR00019## wherein: n is comprised between 2 and 25; R is the side chain of an amino-acid selected from the group consisting of natural aminoacids side chains, non-natural aminoacids side chains, and modified aminoacids side chains; R′ is H or a C3-C6 alkyl linked to R thus forming a R-R′ ring; and d and e being such that d+e=0 to 10.

13. The process of claim 1, wherein the amino-building block is labelled with a probe.

14. A bioorganic nylon type polymer obtainable by a process according to claim 1.

15. A bioorganic nylon type polymer comprising or consisting of at least one group of repeated unit, said group being selected from: a) group a, wherein group a is selected from the group consisting of repeated units of formula A1 and A2 ##STR00020## b) group b, wherein group b is selected from the group consisting of repeated units of formula B1 to B8: ##STR00021## ##STR00022## c) group c, wherein group c is selected from the group consisting of repeated units of formula C1 and C2: ##STR00023## d) group d, wherein group d is selected from the group consisting of repeated units of formula D1 and D2: ##STR00024## e) group e, wherein group e is selected from the group consisting of repeated units of formula E1 and E2: ##STR00025## wherein each AA is an amino-acid residue; PEPTIDE is a peptide residue corresponding to a chain of amino acid residues bound by peptidic bonds; Y is (CH.sub.2).sub.q, C.sub.6H.sub.4, C.sub.6H.sub.4—W—C.sub.6H.sub.4, C.sub.6F.sub.4, C.sub.6F.sub.4—W—C.sub.6F.sub.4, or C.sub.10H.sub.6; Z is (CH.sub.2).sub.p, C.sub.6H.sub.4, C.sub.6H.sub.4—W′—C.sub.6H.sub.4, C.sub.6F.sub.4, C.sub.6F.sub.4—W′—C.sub.6F.sub.4, or C.sub.10H.sub.6; PG(Nter) denotes the protected N.sub.terminus of the AA or PEPTIDE; and (Cter) denotes the C.sub.terminus of AA PEPTIDE.

16. The bioorganic nylon type polymer of claim 15, consisting of at least one group repeated units selected from group a, group c, group d, and/or group e.

17. The bioorganic nylon type polymer of claim 16, wherein it is of the following formula (IA), (IC), (ID) or (IE): ##STR00026## wherein each AA is an amino-acid residue; PEPTIDE is a peptide residue corresponding to a chain of amino acid residues bound by peptidic bonds; Y is (CH.sub.2).sub.q, C.sub.6H.sub.4, C.sub.6H.sub.4—W—C.sub.6H.sub.4, C.sub.6F.sub.4, C.sub.6F.sub.4—W—C.sub.6F.sub.4, or C.sub.10H.sub.6; Z is (CH.sub.2).sub.p, C.sub.6H.sub.4, C.sub.6H.sub.4—W′—C.sub.6H.sub.4, C.sub.6F.sub.4, C.sub.6F.sub.4—W′—C.sub.6F.sub.4, or C.sub.10H.sub.6; PG(Nter) denotes the protected N.sub.terminus of the AA or PEPTIDE; (Cter) denotes the C.sub.terminus of AA or PEPTIDE; m and m′ are independently selected from positive real numbers, for instance integers selected from 0 and 2000, m and m′ may not be simultaneously 0, and t is a positive integer from 1 to 2000.

18. An antibacterial material comprising the bioorganic nylon type polymer of claim 14.

19. A fabric comprising a bioorganic nylon type polymer of claim 14.

20. An antibacterial material comprising the bioorganic nylon type polymer of claim 17.

Description

[0173] FIG. 1 depicts the general procedure for the preparation of bioorganic nylon type polymer according to the present invention.

[0174] FIG. 2 depicts the general procedure of synthesis route for the preparation of amino peptide building blocks 2 and 4 of Table 1.

[0175] FIG. 3 depicts the general procedure of synthesis route for the preparation of amino peptide building blocks 3 and 5 of Table 1.

EXAMPLES

Example 1: Preparation of a Nylons N1 with Amino Building Block Compound 1 DanLysNHCH.SUB.2.CH.SUB.2.NH.SUB.2..

[0176] Compound 1 was prepared by Fmoc SPPS using trityl chloride resin which was first reacted with ethylene diamine.

[0177] The introduction of a ethyle diamine of formula H.sub.2N—(CH.sub.2).sub.2—NH.sub.2 on 2-chlorotrityl is described in numerous paper and, as an example in ‘A New Way to Silicone Based Peptide Polymers’, Said Jebors, Jeremie Ciccione, Soultan Al-Halifa, Benjamin Nottelet, Christine Enjalbal, Céline M'Kadmi, Muriel Amblard, Ahmad Mehdi, Jean Martinez and Gilles Subra. Angew.Chem.Int.Ed., Volume 54, Issue 12, pages 3778-3782, Mar. 16, 2015.

[0178] In this case, the introduction of diamine on chloride resin was achieved as follows: To a pre-swollen suspension of 2-chlorotrityl chloride resin (1.44 mmol Cl/g, 5 g) in CH.sub.2Cl.sub.2, was added a solution of ethylene diamine (1.44 ml, 3 equiv.), DIEA (8.8 ml, 7 equiv.) in DMF(50 ml). The reaction mixture was agitated at room temperature overnight, then washed with DMF(3×), DCM (3×), MeOH(1×) and DCM (1×).

[0179] After coupling of FmocLys(Boc)OH on the primary amine group, Compound 1 was obtained according to the following scheme and used without further purification.

##STR00018##

[0180] Fluorescent nylons N1 were prepared first by solubilizing adipoyl dichloride in DCM (0.4 M). Then a thin layer of 0.5 M NaOH solution was deposited with a pipette over the organic phase. Finally, a mixture of 1 and 1,6 diamino hexane (10/100, 1/100 and 0.1/100; total concentration=0.4 M) in 0.5 M NaOH was slowly deposited over the organic phase.

[0181] Polymerization happens immediately at the interface. The resulting polymer is pulled out manually with a stick from the beaker. White and resistant wire of over one meter long can be obtained with variable thickness, depending on its length.

[0182] After washing in various solvents to eliminate unreacted species and drying under vacuum, the wire appears to be harsher and resistant but not so much elastic even though it remains flexible. The yield was nearly quantitative (>99%) implying that the peptide was probably fully consumed as diamine building block in the polymer.

Example 2: Preparation of Various Nylon

[0183] Similarly and using the described experimental procedure as in example 1, four other peptide sequences (2-5 in Table 1) were synthesized either on Rink amide-PS (type C), or on 2-Chloro-Chloro-Trityl-PS resin first functionalized with Fmoc ethylene diamine (type L).

[0184] After cleavage from the resin, the peptides were purified by preparative RP-HPLC. Compounds 2 and 3 shared the same Gly-Phe-Arg peptide sequence, incorporated in the polymer backbone in a linear way (type L) or as pendant chains (type C) through a N-terminus lysine. Peptides 4 and 5 are cationic sequences derived from the short antibacterial amphipathic Palm-Arg-Arg-NH2 peptide designed in our laboratory. Interestingly, derivatives of such peptide were grafted on a glass surface or incorporated in new bioorganic polymers including peptide-silicone. The resulting hybrid materials kept antibacterial properties against E. Coli, consolidating the fact that the mode of action of this antibacterial peptide involves the destabilization of the bacterial membrane. Peptide 5 leads to a nylon containing the antibacterial peptide sequence in a comb configuration, while peptide 4 lead to a linear nylon containing the antibacterial peptide sequence. The same procedure used for N1 was applied to the synthesis of other peptide nylons. Peptides 2-5 were used in different x/y ratios with 1,6-diamino hexane yielding twelve different nylons (Table 1). In addition, a fluorescent nylon N6, incorporating the dansyl derivative 1 (1%) and the peptide 4 (9%) was prepared.

[0185] All nylons, including N0 not containing any peptide sequence, were characterized by ATG and DSC to determine glass transition temperature, fusion and solidification temperature. The profiles were comparable with conventional 1-6 nylons indicating that introduction of 1-10% peptide related to conventional 1.6-diamino, does not really affect the mechanical properties.

TABLE-US-00001 TABLE 1 Peptide Nylons Peptide Peptide Type [a] Peptide sequence nylon x/y ratio[b] Std nylon 6.6 prepared with diaminohexane N0 0/100 1 DanLysNHCH.sub.2CH.sub.2NH.sub.2 N1-10% 1/10 ″ N1-1% 1/100 ″ N1-0.1% 1/1000 2 L HGlyPheArgNHCH.sub.2CH.sub.2NH.sub.2 N2-10% 1/10 L ″ N2-1% 1/100 L ″ N2-0.1% 1/1000 3 C HLysGlyPheArgNH.sub.2 N3-10% 1/10 C ″ N3-1% 1/100 C ″ N3-0.1% 1/1000 4 L HArgArgNHCH.sub.2CH.sub.2NH.sub.2 N4-10% 1/10 L ″ N4-1% 1/100 L ″ N4-0.1% 1/1000 5 C HLysAhxArgArgNH.sub.2 N5-10% 1/10 C ″ N5-1% 1/100 C ″ N5-0.1% 1/1000 4 and 1 M HArgArgNHCH.sub.2CH.sub.2NH.sub.2 N6-10% 9/1/100[c] DanLysNHCH.sub.2CH.sub.2NH.sub.2 [a] see FIG. 1: L means linear peptide-nylon, C means comb-like peptide-nylon, M means multifunctional peptide nylon [b]x/y molar ratio of peptide/diamino hexane [c]x/y/z molar ratio of peptide 4/diamino hexane/compound 1

Example 3: Procedure for the Synthesis of Peptide-Nylon N2-1%.

[0186] Firstly, a 0.4 M solution of adipoyl chloride (625 μL) in dichloromethane (10.7 ml) was introduced in a 20 mL beaker.

[0187] Then a solution of 0.4M of a mixture of 1.6 diamino hexane (449 mg) and peptide sequence 3 (221 mg) in 10.7 mL of 0.5 M NaOH is gently deposited with a pipette on the top of the organic solution of adipoyl chloride.

[0188] Polymerization happens immediately at the interface. The resulting polymer is pulled out manually with a stick from the beaker and washed with 10 mL of methanol (3×), 10 mL of water (3×), 10 mL of acetone (3×) and dried.

Example 4: Procedure for the Synthesis of Peptide-Nylon N2-100%

[0189] Firstly, a 0.4 M solution of adipoyl chloride (312.5 μL) in dichloromethane (5.3 ml) was introduced in a 20 mL beaker.

[0190] Then a solution of 0.4M of a peptide sequence 2 (1.4 g) in 5.3 mL of 0.5 M NaOH is gently deposited with a pipette on the top of the organic solution of adipoyl chloride.

[0191] Polymerization happens immediately at the interface. The resulting polymer is pulled out manually with a stick from the beaker and washed with 10 mL of methanol (3×), 10 mL of water (3×), 10 mL of acetone (3×) and dried.

Example 5 Procedure for the Synthesis of Peptide-Nylon N2-100%

[0192] To a 0.1 M solution of peptide sequence 2 (0.35 g) in DMF was added DIEA (6 éq., 684 μL) and adipoyl chloride (1 éq., 78.1 μL). The reaction mixture was allowed to stir until precipitation of the polymer. The resulting polymer is filtered and washed with 10 mL of methanol (3×), 10 mL of water (3×), 10 mL of acetone (3×) and dried.

Example 6 Procedure for the Synthesis of Peptide-Nylon N3-1%

[0193] Firstly, a 0.4 M solution of adipoyl chloride (625 μL) in dichloromethane (6.15 ml) was introduced in a 20 mL beaker.

[0194] Then a solution of 0.4M of a mixture of 1.6 diamino hexane (449 mg) and peptide sequence 3 (221 mg) in 6.15 mL of 0.5 M NaOH is gently deposited with a pipette on the top of the organic solution of adipoyl chloride.

[0195] Polymerization happens immediately at the interface. The resulting polymer is pulled out manually with a stick from the beaker and washed with 10 mL of methanol (3×), 10 mL of water (3×), 10 mL of acetone (3×) and dried.