METHODS AND PRODUCTS

Abstract

A method of producing a biofilm-like microorganism-polymer complex, the method comprising the step of exposing, in an aqueous medium, cells of a microorganism to a polymer comprising groups as defined by formula (la) or formula (lb), wherein: Y comprises an imine; and R.sup.2 comprises a CI-30 group. A biofilm-like microorganism-polymer complex comprising cells of a microorganism and a polymer comprising groups as defined by formula (la) and/or formula (lb). A method for producing a chemical and/or biological product using the microorganism-polymer complex, and the chemical and/or biological products obtained therefrom. A method of dispersing the microorganism-polymer complex. Polymers of formula (la) and of formula (lb).

Claims

1-24. (canceled)

25. A microorganism-polymer complex comprising cells of a microorganism and a polymer comprising groups as defined by formula (Ia) and/or formula (Ib): ##STR00159## ##STR00160## wherein: Y comprises an imine; and R.sup.2 comprises a C1-30 group.

26. The microorganism-polymer complex of claim 25, wherein the complex is obtainable by a method comprising exposing, in an aqueous medium, cells of the microorganism to the polymer.

27. The microorganism-polymer complex of claim 25, wherein the imine is part of a group selected from the list consisting of hydrazone, oxime, acyl hydrazone, acyl thiohydrazone, semicarbazone, semithiocarbazone, hydrazone carboximidamide, carbazate hydrazone, thiocarbazate hydrazone, dithiocarbazate hydrazone, carbazone, thiocarbazone, azacarbazone, 2-hydrazoneyl pyridine, 2-hydrazoneyl pyrimidine and 2-hydrazoneyl triazine.

28. The microorganism-polymer complex of claim 27, wherein the imine is part of an acyl hydrazone.

29. The microorganism-polymer complex of claim 25, wherein Y further comprises one or more groups selected from the list consisting of: amide (such as a secondary or tertiary amide), ester, amine (such as a secondary or tertiary amine), ether, dialkyl peroxide, thioether, disulfide, sulfoxide, sulfone, sulfonamide, sulfonate ester, thioketone, thioester, phosphine, phosphonate ester, phosphate ester, boronic ester, borinic ester, borane, ketone, carbamate, carbonate, carboxylic acid anhydride, urea, ketal, acetal, orthoester, orthocarbonate, imide, diimide, hydrazine, hydroxylamine, 1,2,3-triazole, alkyl, alkenyl, alkynyl, aryl and heterocyclyl.

30. The microorganism-polymer complex of claim 29, wherein the list consists of amine, ether, aryl, heteroaryl and alkyl.

31. The microorganism-polymer complex of claim 25, wherein R.sup.2 comprises a C3-30 group.

32. The microorganism-polymer complex of claim 31, wherein R.sup.2 comprises a C5-30 group.

33. The microorganism-polymer complex of claim 32, wherein R.sup.2 comprises a C5-30 aryl or heteroaryl group.

34. The microorganism-polymer complex of claim 25, wherein R.sup.2 is selected from the list consisting of: ##STR00161## ##STR00162## ##STR00163## ##STR00164## ##STR00165## ##STR00166## ##STR00167## ##STR00168## ##STR00169## with the proviso that if the group of the polymer is of formula (Ia′): ##STR00170## then R.sup.2 can be selected from the list consisting of: ##STR00171## ##STR00172## ##STR00173## ##STR00174## ##STR00175## .

35. The microorganism-polymer complex of claim 25, wherein the polymer and/or the cells are in a dispersed state in the aqueous medium.

36. The microorganism-polymer complex of claim 25, wherein the clogD and/or clogP of the R.sup.2 group is -2.0 or higher.

37. The microorganism-polymer complex of claim 25, wherein the microorganism is a bacteria or a fungi.

38. The microorganism-polymer complex of claim 37, wherein the microorganism is a bacteria.

39. A product comprising a microorganism-polymer complex according to claim 25, wherein the product is: a) a composition for oral administration; b) a composition for topical application; or c) a coated article comprising an article coated with the microorganism-polymer complex.

40. The product of claim 39, wherein option c) applies and wherein the article is: a) made of a plastic, a glass, a metal, or a natural material; or b) is a live object.

41. The product of claim 40, wherein the article is a seed.

42. A method for producing a chemical and/or biological product, the method comprising exposing a substrate to a microorganism-polymer complex as defined by claim 25.

43. The method of claim 42, wherein the chemical and/or biological product is a medicament, an intermediate of a medicament, an organic molecule, a protein binding fragment, an antibody, a fine chemical and/or a bulk chemical.

44. A method of dispersing a microorganism-polymer complex, wherein the microorganism-polymer complex is as defined by claim 25, and wherein the method comprises the step of cleaving the polymer.

45. The method of claim 44, wherein the polymer is cleaved by hydrolysis.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0236] The invention will be further illustrated in a non-limiting manner by the accompanying drawings, in which:

[0237] FIG. 1 shows imine-containing functional groups that may be used as part of the linker group Y;

[0238] FIG. 2 shows the results of laser diffraction analysis comparing the size distribution (by %volume) of complexes induced by polymer pAH-2AFP and biofilms formed without the polymer after 48 h of incubation;

[0239] FIG. 3 shows the absorbance of cultures incubated for 24 hours (bottom graph) or 48 hours (top graph) with polymer pAH or a polymer according to the invention, following staining with crystal violet;

[0240] FIG. 4 shows the green fluorescent protein (GFP) fluorescence of cultures over 48 hours’ incubation with or without polymer pAH-2AFP;

[0241] FIG. 5 shows the green fluorescent protein (GFP) fluorescence of cultures over 48 hours’ incubation with or without a polymer containing a carbazate hydrazone linker;

[0242] FIG. 6 shows the normalised GFP expression at 48 hours for cultures with or without polymer pAH-2AFP;

[0243] FIG. 7 shows, for cultures with polymer pAH or polymers according to the invention, the fold change in GFP fluorescence, compared to a control culture without polymer;

[0244] FIG. 8 shows, for cultures with polymers containing carbazate hydrazone linkers, the change in GFP fluorescence, compared to a control culture without polymer;

[0245] FIG. 9 shows, for cultures with or without polymer pAH-2AFP, the effect of pH change on crystal violet absorbance;

[0246] FIG. 10 shows the percentage conversion of 5-fluoroindole to 5-flurotryptophan in cultures with polymer pAH or polymers according to the invention after 48 hours, correlated with the clogD for the R.sup.2 group of that polymer; and

[0247] FIG. 11 shows the kinetic trace for the hydrolysis of 4-nitrophenyl dodecanoate to 4-nitrophenol in cultures with or without polymer pAH-Bn or polymer pAH-In.

EXAMPLES

Synthesis of Polymers

Materials and Methods

[0248] Chemicals were purchased from Sigma-AldrichO® , Fisher Scientific®, VWR® or AcrosO® , and were used without further purification. All solvents were reagent grade or above, purchased from Sigma-AldrichO® , Fisher ScientificO® or VWRO® , and were used without further purification.

[0249] Nuclear Magnetic Resonance (NMR) spectra were recorded on either a Bruker Avance III 300 MHz, a Bruker Avance III 400 MHz spectrometer, a Varian Mercury 300 MHz or a Varian Inova 500 MHz spectrometer. Chemical shifts are reported in ppm (δ units) referenced to the following solvent signals: DMSO-d.sub.6 δH 2.50, D.sub.2O δH 4.79 and CDC1.sub.3, δH 7.26. Infrared (IR) spectra were recorded on a Perkin Elmer Spectrum Two FT-IR spectrometer. Ultraviolet-visible (UV-vis) spectra were recorded on a Campsec M550 Double Beam Scanning UV-vis Spectrophotometer or a Cary 50 Spectrophotometer. Size Exclusion Chromatography (SEC), AKA Gel Permeation Chromatography (GPC) spectra were recorded on a Shimadzu Prominence LC-20A fitted with a Thermo Fisher Refractomax 521 Detector or a SPD20A UV-vis Detector. In all cases flow rate was 1 mL min.sup.-1. Reactive polymers were analysed using Dulbecco’s Phosphate Buffered Saline 0.0095 M (PO.sub.4) without Ca and Mg as the eluent and a flow rate of 1 mL min.sup.-1. The instrument was fitted with an Agilent PL aquagel-OH column (300 × 7.5 mm, 8 mm) and run at 35° C. Molecular weights were calculated based on a standard calibration method using polyethyleneglycol standards. Protected polymers were analysed using 0.05 M LiBr in dimethylformamide (DMF) at 60° C. as the eluent. The instrument was fitted with a Polymer Labs PolarGel guard column (50 × 7.5 mm, 5 .Math.m) followed by two PLGel PL1110-6540 columns (300 × 7.5 mm, 5 .Math.m). Alternatively, molecular weights were calculated based on a standard calibration method using polymethylmethacrylate standards. Dialysis was carried out in deionised water at room temperature for a minimum of 48 hours using a Spectra/Por 6 1000 Molecular weight cut-off (MWCO) 38 mm width membrane.

Synthesis of Polymers

[0250] Synthesis of Poly(acryloyl hydrazide) pAHx

##STR00103##

The synthesis of pAHx is described in Crisan D.N. et al., Polym. Chem., 2017, 8, 4576-4584 (where pAHx is described as “Px”) and in the supporting information relating to Angew. Chem. Int. Ed. 2016, 55, 7492-7495; DOI: 10.1002/anie0.201601441; https://onlinelibrary.wiley.com/action/downloadSupplement?doi=10.1002%2Fanie0.201601441 &file=anie201601441-sup-0001-misc.sub.-information. pdf, accessed 2 Sep. 2019 (where pAHx is described as “P1”).

[0251] It will be understood that the “x” of pAHx signifies the approximate number of monomer units of the polymer. The end groups of the pAH polymer have been shown as methyl groups for simplicity only.

Calculation of DP Using .SUP.1.H-NMR

[0252] The degree of polymerisation (DP) in pAHx was calculated from the .sup.1H-NMR spectra by comparing the integration of the methyl substituents in the 2-(2-methylpropionic acid) end-group (0.95 and 1.01 ppm, 6 H) to the integration from the aliphatic region in the polymer backbone (1.59-2.08 ppm) (as shown in Figure S2 and Table S1 of the supporting information relating to Angew. Chem. Int. Ed. 2016, 55, 7492-7495; DOI: 10.1002/anie0.201601441; https://onlinelibrary.wiley.com/action/downloadSupplement?doi=10. 1002%2Fanie0.201601441&file=anie201601441-sup-0001-misc_information.pdf, accessed 2 Sep. 2019).

[0253] DP may alternatively be calculated by dividing the polymer mass by the monomer mass, where polymer mass can be determined from the decrease in integration of the vinylic proton peaks of the monomer (6.2 and 5.6 ppm, 3 H) as observed by .sup.1H NMR.

Synthesis of Poly(acetylene)s

Synthesis of Acetylene Monomers

[0254] O-propargyl-N-(tert-butoxycarbonyl)amino carbamate PBocAC

##STR00104##

[0255] Propargyl Alcohol (259 mg, 4.67 mmol) was dissolved in EtOAc (10 ml). Carbonyldiimidazole (665 mg, 5.13 mmol) was dissolved in EtOAc (10 ml). The two solutions were combined and the mixture was stirred at 60° C. for 2 hours. tert-Butyl carbazate (925 mg, 7.00 mmol) was dissolved into EtOAc (10 ml). The resulting solution was added to the previous crude, and the mixture was allowed to react for a further 2 hours. The organic layer was concentrated under reduced pressure, then suspended again in EtOAc (20 ml). The organic layer was washed with deionized H.sub.2O (9 × 20 ml) and 0.1 M HC1 (3 × 20 ml). The solvent was then removed on a rotary evaporator and to give the target compound as white powder (500 mg, 47% yield). .sup.1H NMR (400 MHz, CDC13) /ppm; 6.52 (s, 1H, H-N), 6.30 (s, 1H, H-N), 4.79 (d, 2H, J= 2.5 Hz, H—C═), 2.52 (t, 1H, J= 2.5 Hz, H—CΞ), 1.50 (s, 9H, H-C). .sup.13C NMR (400 MHz, CDC13) /ppm; 155.69 (OC(O)N), 81.87 (CC═C), 75.15 (C═CH), 53.32 (C2CO), 27.93 (CCH3). FTIR vmax /cm.sup.1; 3279 (s, sh, H—C═), 3024 (w, sh, H-N), 2979 (w, sh, H-C), 2136 (w, sh, C═C), 1752 (m, sh, C═O), 1730 (s, sh, C═O), 1692 (s, sh, N-H). UV-Vis /nm (CDC1.sub.3); λ.sub.max = 280.

[0256] 1H-Imidazole-1-carbohydrazide

##STR00105##

tert-butyl carbazate (595 mg, 4.5 mmol) was dissolved in water (15 ml) at room temperature and cooled to 0-5° C. Then, carbonyldiimidazole (1459 mg, 9.0 mmol) was added and the reaction mixture stirred for 1 hr at 0-5° C. and then allowed to warm up to room temperature. The precipitated product was then filtered and washed with cold water 3 to 4 times and dried to obtain a white powder (871 mg, 85% yield). .sup.1H NMR (400 MHz, DMSO-d6) / ppm: δ 10.43 (s, 1H, H-N), 9.27 (s, 1H, H-N), 8.27 (d, J= 1.2 Hz, 1H, H-C), 7.69 (t, J= 1.5 Hz, 1H, H-C), 7.03 (m, 1H, H-C), 1.44 (s, 9H, H-C). .sup.13C NMR (101 MHz, DMSO-d6) / ppm: δ 155.84 (OC(O)N), 136.52 (NCH═HCN), 130.48(NCH═HCN), 116.95 (NCH═N), 80.43 (OCC2), 28.46 (CCH3).

[0257] N-propargyl-N′-(tert-butoxycarbonyl)amino urea PBocAU

##STR00106##

1H-Imidazole-1-carbohydrazide (566 mg, 2.5 mmol) and propargyl amine (248 mg, 4.5 mmol) were dissolved separately in EtoAc (15 mL) at room temperature. The two solutions were combined, and the mixture was stirred at room temperature overnight. The reaction was quenched with 0.5 M HC1 (20 mL). The organic layer was separated and washed with 0.5 M HC1 (2 × 20 mL) with deionized H.sub.2O (6 × 20 mL) and brine (1 × 20 mL), and dried over sodium sulphate. The solvent was then removed by rotary evaporated to give an off-white colour powder at (241 mg, 45% yield). .sup.1H NMR (400 MHz, DMSO-d6) / ppm: 8 8.53 (s, 1H, H-N), 7.77 (s, 1H, H-N), 6.65 (s, 1H, H-N), 3.78 (dd, J= 5.8, 2.5 Hz, 2H, H2—CC═), 3.03 (t, J= 2.5 Hz, 1H, H—C≡), 1.40 (s, 9H, H-C). .sup.13C NMR (101 MHz, DMSO-d6) / ppm: δ 156.38 (NC(O)N), 82.79 (CC═C), 79.45 (CCO), 72.86 (C═CH), 29.17 (HNCH2C═), 28.55 (CCH3).

[0258] 1-(Chloromethyl)-4-ethynylbenzene

##STR00107##

1-(hydroxymethyl)-4-ethynylbenzene (3.00 g, 22.7 mmol) dissolved in CHC1.sub.3 (105 mL) and cooled to 0° C., methanesulfonyl chloride (1.2 mol equiv.) and Et3N (1.2 mol equiv.) were dissolved in the reaction mixture, and then heated to reflux for 45 h. Reaction was quenched with sat. NaHCO3, the organic layer was further washed with sat. NaHCO.sub.3, dried with Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The crude was purified by column chromatography (20:1 hexane/ EtOAc) to afford a pale yellow oil as the product (2.50 g, 73% yield). 1H-NMR (400 MHz, CDC13) 8(ppm): 7.50 (d, J= 8 Hz, 2H), 7.36 (d, J= 8 Hz, 2H), 4.58 (s, 2H), 3.12 (s, 1H).

[0259] N-(ethynylbenzyl)oxy)phthalimide EBPHT

##STR00108##

N-Hydroxyphthalimide (2.17 g, 13.3 mmol) and Na.sub.2CO.sub.3 (1 mol equiv.) were dissolved in a mixture DMF, MeCN and water (43.5 mL, 0.457: 0.1875: 0.457 ratio). 1-(Chloromethyl)-4-ethynylbenzene (1 mol equiv.) was added to reaction mixture forming a deep red suspension and reacted for 4 h at room temperature. The mixture was filtered off and the precipitate was washed with water (100 mL × 3) and ice-cold methanol (100 mL × 3) to afford a white powdered solid as the product (3.58 g, 97% yield). .sup.1H-NMR (400 MHz, DMSO-d6) 8(ppm): 7.86 (s, 4H), 7.57 - 7.46 (m, 4H), 5.19 (s, 2H), 4.25 (s, 1H)

Oligoethylene Glycol Containing Monomers

[0260] 1-(Bromomethyl)-4-ethynylbenzene

##STR00109##

1-(Hydroxymethyl)-4-ethynylbenzene (4.85 g, 37.83 mmol) dissolved in THF (162 mL) and cooled to 0° C. in an ice bath for 20 minutes. Phosphorous tribromide (0.5 equiv.) was added dropwise to the solution and allowed to stir for an additional 15 minutes and then brought gradually to room temperature. The reaction was stirred at room temperature for 6 h, after which the solvent was removed in vacuo and the crude oil was purified by column chromatography (10 % EtOAc/ hexane) to afford an orange oil as the product (5.61 g, 76% yield). .sup.1H-NMR (400 MHz, CDC13) 8(ppm): 7.49 (d, J= 8.4, 2H), 7.37 (d, J = 8.4, 2H), 4.50 (s, 2H), 3.15 - 3.11 (s, 1H).

[0261] a-Ethynylbenzyl) tri(ethylene glycol)

##STR00110##

A solution of tri(ethylene glycol) (6.65 g, 43.85 mmol) in anhydrous THF (10 mL) was added to a suspension of NaH (438.4 mg, 10.96 mmol) in THF (40 mL)and heated to reflux. Then, 1-(bromomethyl)-4-ethynylbenzene (2.14 g, 10.96 mmol) was added dropwise to this suspension and refluxed for a further 3 h. The reaction was then cooled to room temperature, quenched with methanol (10 mL) and 5% HC1 (50 mL) and the solvent removed under vacuum. The product was then extracted with CHC1.sub.3 (3 × 25 mL) and 5% HC1 (50 mL), dried with Na.sub.2SO.sub.4, filtered and concentrated in vacuo. The crude was purified by column chromatography (9:1 hexane/ EtOAc) to afford a colourless oil as the product (2.09 g, 72% yield). .sup.1H-NMR (400 MHz, DMSO-d6) 8(ppm): 7.49 - 7.40 (d, J = 8 Hz, 2H), 7.40 - 7.30 (d, J= 8 Hz, 2H), 4.57 (t, J= 5.5 Hz, 1H, OH), 4.52 (s, 2H), 4.17 (s, 1H), 3.60 - 3.39 (m, 12H).

[0262] a-Ethynylbenzyl) di(ethylene glycol)

##STR00111##

The same synthetic procedure and ratios were used as those described for a-(4-ethynylbenzyl) tri(ethylene glycol), but di(ethylene glycol) was used instead of tri(ethylene glycol). The crude was purified by column chromatography (9:1 hexane/ EtOAc) to afford a colourless oil as the product (1.90, 84% yield). .sup.1H-NMR (300 MHz, DMSO-d6) 8(ppm): 7.46 (d, J= 8.3 Hz, 2H), 7.35 (d, J= 8.49 Hz, 2H), 4.59 (t, J= 4.5 Hz, 1H, OH), 4.51 (s, 4H), 3.57 (s, 4H), 3.15-3.13 (m, 4H).

[0263] a-Ethynylbenzyl ethylene glycol)

##STR00112##

The same synthetic procedure and ratios were used as those described for a-(4-ethynylbenzyl) tri(ethylene glycol), but ethylene glycol was used instead of tri(ethylene glycol). The crude was purified by column chromatography (9:1 hexane/ EtOAc) to afford a colourless oil as the product (84% yield). .sup.1H-NMR (300 MHz, CDC13) 8(ppm): 7.48 (d, J= 7.99, 2H), 7.30 (d, J= 8.55, 2H), 4.56 (s, 2H), 3.87 - 3.70 (m, 2H), 3.66 - 3.55 (m, 2H), 3.07 (s, 1H), 2.01 (m, 1H, OH).

[0264] a-Ethynylbenzyl) co-tosyl tri(ethylene glycol)

##STR00113##

a-(4-Ethynylbenzyl) tri(ethylene glycol) (1 g, 3.78 mmol) and NaOH (3.5 mol equiv.) were dissolved in a dry THF and distilled water mixture (5.5 mL, 1:1 ratio) and cooled to 0° C. A separate THF solution (3.5 mL) ofp-toluenesulfonyl chloride (874.3 mg, 4.54 mmol) was then added dropwise to the stirred suspension over the course of 30 mins and was stirred at 0° C. for a further 2 h, before allowing it to warm to room temperature and reacted for a further 20 h. The reaction was then cooled to 0° C., poured to 5% HC1 (50 mL) on ice and extracted with CHC1.sub.3 (3 × 25 mL), dried with Na.sub.2SO.sub.4, filtered and concentrated in vacuo to afford a colourless oil as the product. This product was then used without further purification in subsequent syntheses (1.84 g, 100% yield; 90% purity). .sup.1H- NMR (300 MHz, CDC13) 8(ppm): 7.85 - 7.72 (m, 2H), 7.49 - 7.43 (m, 2H), 7.36 - 7.27 (m, 3H), 4.55 (s, 2H), 4.18 - 4.11 (m, 2H), 3.74 - 3.60 (m, 3H), 3.06 (s, 1H), 2.43 (s, 3H).

[0265] a-Ethynylbenzyl) co-tosyl di(ethylene glycol)

##STR00114##

The same synthetic procedure and ratios were used as those described for a-(4-ethynylbenzyl) ω-tosyl tri(ethylene glycol)to give a colourless oil (1.68 g, 99% yield; 94% purity). .sup.1H-NMR (300 MHz, CDC13) 8(ppm): δ 7.84 - 7.74 (m, 2H), 7.51 - 7.41 (m, 2H), 7.35 - 7.28 (m, 4H), 4.53 (s, 2H), 4.21- 4.13 (m, 2H), 3.80 - 3.52 (m, 6H), 3.07 (s, 1H), 2.43 (s, 3H).

[0266] a-Ethynylbenzyl) co-phthalimide tri(ethylene glycol) EBTEGPHT

##STR00115##

a-(4-Ethynylbenzyl) ω-tosyl tri(ethylene glycol) (1.0 g, 2.39 mmol) and N-hydroxyphthalimide (1.2 mol equiv.) were suspended in dry MeCN (9 mL). Et3N (1.2 mol equiv.) was added to afford a deep red solution. The reaction was heated to reflux and left for 17 h. Solvent was then removed under vacuum and the resultant crude oil was redissolved in CHC1.sub.3 (50 mL) and washed with sat. NaHCO.sub.3 (4 × 25 mL). The organic phase was dried with Na.sub.2SO.sub.4, filtered and concentrated in vacuo to afford a crude yellow oil. The product was then purified by flash chromatography (1:1 hexane/EtOAc) to afford a yellow oil as the product (730.6 mg, 75% yield). .sup.1H NMR (400 MHz, DMSO-d6) 8(ppm): 7.86 (s, 4H), 7.49 - 7.40 (m, 2H), 7.39 -7.28 (m, 2H), 4.49 (s, 2H), 4.32 - 4.23 (m, 2H), 4.16 (s, 1H), 3.78 - 3.68 (m, 2H), 3.60 - 3.41 (m, 8H).

[0267] a-Ethynylbenzyl) co-phthalimide di(ethylene glycol) EBDEGPHT

##STR00116##

The same synthetic procedure and ratios were used as those described for a-(4-Ethynylbenzyl) ω-phthalimide tri(ethylene glycol). The product purified by flash chromatography (1:1 hexane/EtOAc) to afford a yellow oil as the product (1.05 g, 63% yield). .sup.1H-NMR (300 MHz, 20 CDC13) 8(ppm): 7.85 - 7.77 (m, 2H), 7.77 - 7.69 (m, 2H), 7.47 - 7.41 (m, 2H), 7.25 - 7.21 (m, 2H), 4.48 (s, 2H), 4.44 - 4.35 (m, 2H), 3.93 - 3.85 (m, 2H), 3.73 - 3.64 (m, 2H), 3.61 - 3.50 (m, 2H), 3.06 (s, 1H).

Polymerisation of Acetylene Monomers

[0268] All polymerisation were performed in a similar manner to Masuda et al. (Polymerization of Substituted Acetylenes, in Handbook of Metathesis: Catalyst Development. (2008)). One of the four following rhodium catalysts were employed, [Rh(NBD)nPh—BPh.sub.3], [(Rh(NBD)C1).sub.2], [(Rh(COD)C1).sub.2], and [Rh(COD).sub.2].sup.⊕ BF.sub.6.sup.⊖. [Rh(nbd)B(Ph).sub.4] was synthesised as in Schrock, R. R.; Osborn, J. J. Inorg. Chem. 1970, 9, 2339.

[0269] In a typical procedure, protected monomer (PBocAC, PBocAU, EBPHT, EBTEGPHT, EBDEGPHT) (1.43 mmol) was dissolved in a suitable solvent (6 mL) at 30° C. When fully dissolved rhodium catalyst (0.0143 mmol, 0.01 mol equiv. per monomer, target x = 100) was added, either in solution or solid. The mixture was stirred at 30° C. for 24 hours. The reaction mixture was precipitated over excess n-hexane. The resulting precipitate was then dried on a rotary evaporated to produce a pale-yellow powder. Other experimental conditions for the synthesis of poly(acetylene)s are described in Masuda, T. et al. Polymerization of Substituted Acetylenes, in Handbook of Metathesis: Catalyst Development. (2008).

[0270] poly(O-propargyl-N-(tert-butoxycarbonyl)amino carbamate) p(PBocAC)

##STR00117##

Following the polymerization conditions described above, PBocAC (4.67 mmol) and [Rh(nbd)B(Ph).sub.4] (0.01 mol equiv. per monomer0.01 mol equiv. per monomer) were reacted in THF (15.7 ml) to give 610 mg (61% yield) of the title compound. .sup.1H NMR (400 MHz, DMSO-d6) /ppm; 8.87 (s, 1H, H-H), 8.61 (s, 1H, H-N), 6.35 (s, 1H, H—C═), 4.66 (s, 2H, H-CC2), 1.38 (s, 9H, H-CC). .sup.13C NMR (400 MHz, DMSO-d6) /ppm; 155.77, 155.63 & 155.52 (OC(O)N), 79.40, 77.56 (C═CH), 52.09 (C2CO), 28.05 (CCH3). FTIR vmax /cm.sup.-1; 3290 (m, H-N), 2979 (w, sh, H-C), 1707 (s, sh, C=O). UV-Vis /nm (DMSO); λ.sub.max = 288 nm. GPC; MwGPC = 28340 (±10673); MnGPC = 20115 (±7324); ÐGPC = 1.41 (±0.095).

[0271] poly(N-propargy1-N′-(tert-butoxycarbony1)amino urea) p(PBocAU)

##STR00118##

Following the polymerization conditions described above, PBocAU (1.43 mmol) was dissolved in chloroform (6 mL) at 30° C. When fully dissolved [Rh(nbd)B(Ph).sub.4] (0.01 mol equiv. per monomer) was added. 249 mg (96% yield) of the title compound were isolated. .sup.1H NMR (400 MHz, DMSO-d6) / ppm: δ 8.46 (s, 1H, H-N), 7.69 (s, 1H, H-N), 6.55 (s, 1H, H—N) 60.11 (s, 1H, H—C═), 3.78 (s, 2H, H—CCNH) 10.39 (d, J = 5.9 Hz, 9H).

[0272] polyr[N-((4-ethynylbenzyl)oxy)phthalimide] p(EBPHT)

##STR00119##

Following the polymerization conditions described above, EBPHT (1.07 mmol) was dissolved in DMF (6 mL) and Et3N (0.01 mol equiv.) at 30° C. When fully dissolved [Rh(nbd)B(Ph).sub.4] (0.01 mol equiv. per monomer) was added. The title polymer was precipitated into methanol, filtered, diluted in water then dialysed for four days. .sup.1H NMR (400 MHz, DMSO-d6) 8(ppm): 7.53 (br m, 4H), 7.29 - 6.88 (br s, 2H), 6.62 (br s, 2H), 5.70 (s, 1H), 5.27 - 4.41 (m, 2H).

[0273] polyra-(α-(4-Ethynylbenzy1) co-phthalimide di(ethylene glycol)] p(EBDEGPHT)

##STR00120##

[0274] Following the polymerization conditions described above, EBDEGPHT (1.07 mmol) was dissolved in DMF (6 mL) and Et3N (0.01 mol equiv.) at 30° C. When fully dissolved [Rh(nbd)B(Ph).sub.4] (0.01 mol equiv. per monomer) was added. The title polymer was isolated by diluting in water, dialysed for four days, and freeze-drying. .sup.1H NMR (400 MHz, CDC13) δ(ppm): 8 7.59 - 7.48 (m, 4H), 6.72 (s, 2H), 6.44 (s, 2H), 5.63 (s, 1H), 4.60 (s, 1H), 4.21 - 4.05 (m, 2H), 3.63 (s, 2H), 3.39 (s, 2H), 3.21 (s, 2H).

Deprotection of Acetylene-Based Polymers

[0275] Reactive polymers may then be produced by deprotection. Certain protecting can be removed under acidic conditions, e.g. the Boc groups of p(PBocAC) and p(PBocAU) to make p(PAC) and p(PAU) respectively. Other protecting groups must be deprotected using basic conditions, e.g. the phthalic acid protecting groups of p(EBPHT) and p(EBDEGPHT) to make (p(EBHA) and p(EBOEGHA). Other experimental conditions for the deprotection of amines, for example as carbamates or phthalimides, can be found in Peter G. M. Wuts Theodora W. Greene, Protection for the Amino Group in Protective Groups in Organic Synthesis (2007).

[0276] poly(O-propargyl-N-amino carbamate) p(PAC)

##STR00121##

p(PBocAC) (1 g, 60.7 mmol) was dissolved in 10 ml TFA. The mixture was stirred for 2 hours at room temperature. Excess TFA was blown off with a steady stream of Argon. 10 ml of deionised water was added to the reaction vessel, and the solution was neutralised with sodium bicarbonate. The polymer was then dialysed against 100 mM acetic acid and freeze dried to yield a pale-yellow powder (465.3 mg, 46.53% yield) that was stored under vacuum. FTIR vmax /cm.sup.-.sup.1; 3292 (m, br, H-N), 2950 (w, br, H-C), 1690 (s, br, C═O), 1616 (w, br, C═O). UV/Vis /nm (HC1); λ.sub.max = 275. Fluorescence /nm (HC1); λexc = 355, 385, 413; λem = 491. LD (Thin film) /dOD @ nm; 4.9 ×10.sup.-4 @ 510. DSC / °C; 139, 205.

[0277] poly(N-propargyl-N′-amino urea) p(PAU)

##STR00122##

p(PBocAU) (249 mg, 2.6 mmol) was dissolved in a minimum amount of TFA, and the mixture stirred for 20 hr at room temperature. The TFA was then removed by a steady stream of Argon until the high viscosity of the reaction mixture is seen. Thereafter, the solution was neutralised with sodium bicarbonate. The polymer was then dialysed again 100 mmol acetic for 4-5 days and freeze-dried to give a pale-yellow powder (149 mg, 60% yield). .sup.1H NMR (400 MHz, DMSO-d6) / ppm: 8 9.68 (s, 2H, H2-N), 9.13 (s, 1H, H-N), 7.42 (s, 1H, H-N), 6.14 (s, 1H, H—C═), 3.88 (s, 1H, H-CCNH).

Synthesis of Polymers of Formula (I)

[0278] To a solution of reactive polymer (e.g. pAH, p(PAC), p(PAU), p(EBHA), p(EBOEGHA)) in solvent (200 .Math.l, 100 mM reactive polymer solution in reactive functional group (e.g. hydrazide) moieties as determined by .sup.1H NMR; by default, for example, for pAHx, x may be 50) was added an aldehyde in a water-miscible solvent (200 .Math.l, 100 mM). This mixture was shaken at 60° C. for 3 to 24 h. Polymers were used without further purification.

TABLE-US-00001 Details of specific examples are shown in the table below Designation of Polymer Aldehyde Solvent % functionalisation Polymer structure pAH-2AFP [00123] 100 mM AcOH in D.sub.2O (pH 2.9) 61 [00124] pAH-IMI [00125] 100 mM AcOH in D.sub.2O (pH 2.9) 75 [00126] pAH-IVA [00127] 95 vol.% DMSO-d.sub.6 5 vol.% 100 mM AcOH 80 [00128] pAH-Bn [00129] 95 vol.% DMSO-d.sub.6 5 vol.% 100 mM AcOH 65 [00130] pAH-In [00131] 95 vol.% DMSO-d.sub.6 5 vol.% 100 mM AcOH 53 [00132] pAH - Pyr [00133] 95 vol.% DMSO-d.sub.6 5 vol.% 100 mM AcOH 63 [00134] pAH - Naph [00135] 95 vol.% DMSO-d.sub.6 5 vol.% 100 mM AcOH 62 [00136] pAH - Anthr [00137] 95 vol.% DMSO-d.sub.6 5 vol.% 100 mM AcOH 54 [00138] pAH - Pyrene [00139] 95 vol.% DMSO-d.sub.6 5 vol.% 100 mM AcOH 58% [00140] p(PAC)-2AFP [00141] 50 vol.% DMSO-d.sub.6 50 vol.% 0.1 M HC1, pH 3 74 [00142] p(PAC)-IMI [00143] 50 vol.% DMSO-d.sub.6 50 vol.% 0.1 M HC1, pH 3 72 [00144] p(PAC)-IVA [00145] 50 vol.% DMSO-d.sub.6 50 vol.% 0.1 M HC1, pH 3 78 [00146] p(PAC)-Bn [00147] 50 vol.% DMSO-d.sub.6 50 vol.% 0.1 M HC1, pH 3 61 [00148] p(PAC)-In [00149] 50 vol.% DMSO-d.sub.6 50 vol.% 0.1 M HC1, pH 3 64 [00150]

[0279] It will be appreciated that similar yields of imine-containing compounds may be achieved using the variety template polymers described above, which have for example different backbones and different linker groups. Therefore, a wide variety of polymers of formula (I) can be synthesised using the disclosure of the present application.

[0280] It has been recognised that when the loading of aldehyde is increased, in this example specifically from 1 equivalent to 2 equivalents of aldehyde, the % functionalisation of the resulting polymer increases.

General Procedure for Production of Microorganism-Polymer Complex

[0281] The microorganism (e.g. Escherichia ( E.) coli) was grown overnight in LB broth at 30 degrees and stirred with an orbital shaker with a throw of 19 mm at 150 rpm. The resulting culture was re-inoculated with fresh LB (1% re-inoculation) and grown in the same conditions for around 3 h until the culture reached an OD of 0.2. The cells were washed in water and isolated by centrifuge. The isolated cells were suspended in sufficient KH.sub.2PO.sub.4/K.sub.2HPO.sub.4 buffer (0.1 M, pH 7.0) to give an OD of 0.2. The polymer was added to the resulting solution. Then M63 media (equal volume to that of the KH.sub.2PO.sub.4/K.sub.2HPO.sub.4) was added. The final polymer concentration was determined to be 0.053 mg/mL. The culture was incubated at 30 degrees and mixed with an orbital shaker with a throw of 19 mm at 150 rpm for 24 h or 48 h.

Investigations Relating to Microorganism-Polymer Complexes

Size Distribution of Complexes

[0282] PHL644 E. coli were grown overnight in LB media. The next morning, the cells were washed with water and re-suspended in KH.sub.2PO.sub.4/K.sub.2HPO.sub.4 (0.1 M in deionised water) to an OD of 1.0. Half of the cells were then mixed with polymer pAH-2AFP (final concentration 0.5 mg/ml). The two samples of cells (i.e. with and without pAH-2AFP) were mixed for 48 h before the size distribution was determined.

[0283] FIG. 2 shows that after 48 hours the sample of E. coli that was not mixed with the polymer (bottom graph) retained a large proportion of planktonic cells. There were some small clusters of natural biofilm, having a peak size of about 20 .Math.m. There were few clusters greater in size than 200 .Math.m, but no recorded clusters greater in size than 300 .Math.m.

[0284] The E. coli that was mixed with pAH-2AFP (top graph) had a lower proportion of planktonic cells and had a higher proportion of clusters of high diameter, up to 1000 .Math.m in size.

[0285] This indicates that the polymer formed complexes with the cells of the microorganism.

Crystal Violet Staining

[0286] The interaction of polymers according to the invention and E. coli was also studied by Crystal Violet staining. Crystal Violet (CV) is employed to stain the extracellular material in biofilms (in particular the negatively charged components of bacterial cells envelopes and extracellular matrices). This technique allows the amount of biomass in a culture to be correlated with microorganism-polymer complex formation.

[0287] Cultures of weak biofilm-forming E. coli strain MC4100 with pAH or polymers of the invention were washed with deionised water and the supernatant was discarded. The washed residue was stained with 1% crystal violet solution for 1 h. The stained residue was washed three times with deionised water, discarding the supernatant from each wash, to remove excess crystal violet. The stained and washed residue was dissolved in a controlled volume of 33% acetic acid and the absorbance at 550 nm was measured using a ClarioStar Plus BMG plate reader. In a control experiment, the bacteria was not mixed with any polymer but was exposed to Crystal violet. The amount of complex formation by MC4100 E. coli, in the presence of pAH or polymers of the invention was determined using CV staining at 24 and 48 hours. These results were correlated with the clogD value calculated for the R.sup.2 group of the polymer. FIG. 3 shows the results of this analysis.

[0288] It was observed that each of the cultures formed complexes akin to biofilms in the presence of the polymers of the invention.

[0289] Therefore, it is possible to produce biofilm-like microorganism-polymer complexes in cultures with the polymers of the present invention.

[0290] Furthermore, cultures including certain polymers exhibited significantly more cluster formation than the control culture at 24 hours and/or 48 hours (MC4100 24 h, MC4100 48 h). These polymers were pAH-AFP, pAH-IMI, pAH-Bn, pAH-In, pAH-Naph, pAH-Anthr and pAH-Pyrene.

[0291] Surprisingly, where a polymer selected from pAH- AFP, pAH-Bn, pAH-In, pAH-Naph, pAH-Anthr and pAH-Pyrene was used, the cultures of MC4100 reached levels of absorbance approaching, equalling or even improving upon control cultures of the stronger biofilm-forming strain PHL644 at 24 hours and/or 48 hours.

[0292] Surprisingly, where a polymer selected from pAH-Naph, pAH-Anthr and pAH-Pyrene was used, the cultures of MC4100 reached levels of absorbance significantly improving upon control cultures of the stronger biofilm-forming strain PHL644 at 24 hours.

[0293] Crystal violet staining of the complex may not be the only metric to determine the most useful polymers, as the polymers producing lower crystal violet staining may produce stronger complexes with microorganisms and/or be more beneficial for other strains and/or species of microorganism.

[0294] Thus, as shown by FIG. 3, it is possible to produce biofilm-like clusters, microorganism-polymer complexes, in cultures with the polymers of the present invention.

Curli Expression

[0295] Curli is a type of amyloid fibre produced by certain strains of enterobacteria. They are extracellular fibres located on bacteria such as E.coli and Salmonella. Curli are primarily known to be the adhesin by which cells attach to biotic and abiotic surfaces. There is evidence that Curli proteins are involved in cell-cell attachment. Thus, Curli expression may be indicative of the formation of a microorganism-polymer complex.

[0296] MC4100 E. coli was genetically modified to express GFP (green fluorescent protein) as a function of Curli expression. A plasmid containing a gene reporter for the csgBAC operon (pJLC-A) was used, as described by James Thomas Leech “Development of an Escherichia coli Biofilm Platform for use in Biocatalysis”, PhD thesis, University of Birmingham (UK), 2017, https://etheses.bham.ac.uk/id/eprint/8055/1/Leech18PhD.pdf accessed 11 Oct. 2019 (especially pages 70-76).

[0297] The fluorescence of the genetically modified E. coli was measured over 48 hours in the presence and absence of polymer pAH-2AFP using a BMG Clariostar plus (excitation wavelength 488 nm; emission wavelength 510 nm). This allowed for measurement of Curli expression, and therefore monitoring of the microorganism-polymer complex formation by E. coli in the presence or absence of polymer. The results of this analysis are shown in FIG. 4.

[0298] The results show that for an initial period of about 24 hours the fluorescence of the E. coli was low in the bacteria with or without pAH-2AFP. Without being bound by theory, this may be because the MC4100 strain of E. coli used in this experiment naturally produces Curli slowly, or because it takes time for the cells to respond to the environmental stimuli that initiate biofilm formation.

[0299] After about 24 hours the fluorescence of both cultures began to increase until it reached a plateau at about 35 to 40 hours. The culture including the polymer reached a maximum fluorescence of about 150% of the maximum fluorescence of the culture without the polymer.

[0300] Therefore, culturing microorganisms with a polymer as defined by formula (I) can produce higher levels of biofilm-like microorganism-polymer complex, than without the polymer.

[0301] The fluorescence of the culture without the polymer decreased up to the 48 hour point. However, whilst some decrease in the fluorescence of the culture with the polymer was observed, this decrease was less severe than in the sample without the polymer.

[0302] This suggests that the complex in the culture with the polymer is more stable than the biofilm in the sample without the polymer.

[0303] The same experiment was performed with the carbazate hydrazone version of pAH-2AFP (shown below) according to a similar procedure to that reported above.

##STR00151##

FIG. 5 shows the fluorescence by GFP over time.

[0304] The results show that for an initial period of about 24 hours the fluorescence of the E. coli was similar for the bacteria with and without the polymer. After about 24 hours the fluorescence of both cultures began to increase further until it reached a plateau at about 30 to 36 hours. The culture including the polymer reached a maximum fluorescence of about double the maximum fluorescence of the culture without the polymer, indicating significantly more formation of biofilm-like structures in the presence of the polymer.

[0305] Therefore, culturing microorganisms with a polymer as defined by formula (I) that contain a carbazate hydrazone group as the linker can produce higher levels of biofilm-like microorganism-polymer complex than without the polymer.

[0306] The normalised GFP expression of cultures of MC4100 and PHL644 strains of E. coli was determined after 48 hours with or without pAH-2AFP.

[0307] As shown by FIG. 6, each of these E. coli strains had significantly higher normalised GFP expression when pAH-2AFP was present. MC4100, a poor biofilm forming strain of E. coli, was able to produce a strong microorganism-polymer complex with pAH-2AFP. PHL644, already a strong biofilm forming strain of E. coli, was able to produce an even stronger microorganism-polymer complex with pAH-2AFP.

[0308] This shows that the polymers of the present invention enable weak biofilm-forming microorganisms to be used in applications that have been developed for, or are more suited towards, (stronger) biofilms/microorganism-polymer complexes.

[0309] It also shows that strong biofilm forming microorganisms can produce stronger biofilm-like complexes when they are cultured with a polymer, according to the present invention.

[0310] The GFP fluorescence of cultures with polymer pAH or a polymer of the invention over 48 hours was determined with PHL644 and MC4100 strains of E. coli. The fold change in fluorescence of the culture with each polymer was determined and is shown in FIG. 7. As indicated by FIG. 7, the “fold change” of a polymer refers to the GFP fluorescence of that polymer in the culture at 48 hours divided by that of the control sample. The unmarked bars signify control experiments with either DMSO or AcOH.

[0311] The results show that, in most cases, the polymers of the invention significantly increased the fluorescence of the culture. Every polymer of the invention increased the fluorescence of the MC4100 culture. Thus, it is currently proposed that biofilm-like characteristics of a microorganism are enhanced by polymers of the present invention.

[0312] It can be seen that polymers pAH-2AFP and pAH-In provided the best results with E. coli strain PHL644. These polymers each include a heteroaromatic ring in the R.sup.2 group. It can be seen that polymers pAH-2AFP, pAH-Bn and pAH-In provide the best results with E. coli strain MC4100.

[0313] The same experiment was performed with polymers that contained units of poly(acetylene) backbone and carbazate hydrazone linkers:

##STR00152##

Where the R groups were:

##STR00153##

##STR00154##

##STR00155##

##STR00156##

##STR00157##

[0314] FIG. 8 shows the maximum GFP expression following incubation of these polymers with MC4100, as described above, correlated against the clogP for the monomer unit of the polymer. A dashed line shows the baseline emission for a comparative culture of MC4100.

[0315] It can be seen that there is generally a decrease in the expression as clogP increases, indicating that formation of the complex is lower when polymers with monomer units having a higher clogP were used. Therefore, it may be beneficial to use polymers with monomer groups having a low clogP to encourage matrix formation with these bacteria.

[0316] The polymers including, as R.sup.2 groups, imidazolyl, 2-aminopyridyl, phenyl and s-butyl groups provided more matrix formation than the comparative culture.

Reversibility of Microorganism-polymer Complex Formation with Polymers

[0317] Investigations were performed into the reversibility of microorganism-polymer complex formation using E. coli strain PHL644 with polymer pAH-2AFP.

[0318] A sample of the microorganism was exposed to pAH-2AFP under the normal conditions detailed above. A control sample of the microorganism was subjected to the same conditions but without pAH-2AFP. After incubation for 22 hours, the amount of biofilm/complex formed in the sample with pAH-2AFP and the control sample was determined by crystal violet staining and absorbance measurement at 550 nm.

[0319] The results of this analysis are shown by the bars labelled “22 h Biofilm (no pH change)” in FIG. 9. There was an increase in absorbance for the sample with pAH-2AFP compared to the control culture. This relative increase in absorbance suggests that the biofilm-like microorganism-polymer complex had formed.

[0320] Two fresh samples of the microorganism were exposed to pAH-2AFP under the normal conditions detailed above. After incubation for 16 hours the samples were acidified from the usual pH of about 7.0 to 5.8 by adding KH.sub.2PO.sub.4 (https://www.unl.edu/cahoonlab/phosphate%20buffer.pdf, accessed 11 Oct. 2019). The samples were incubated for a further 6 h. Two control samples of the microorganism were subjected to the same conditions but without pAH-2AFP. The amount of biofilm/complex formed in one sample with pAH-2AFP and one control sample was determined by crystal violet staining and absorbance measurement at 550 nm.

[0321] As shown by the bars labelled “22 h Biofilm after pH change” in FIG. 9, the absorbance of the sample with pAH-2AFP at this stage was similar to that of the control sample, suggesting that the microorganism-polymer complex had been at least partially, or fully, dispersed by the change in pH. This also suggests that the amount of bacteria in the microorganism-polymer complex form may be able to be decreased by controlling (for example decreasing) the pH of the culture, where the culture includes a polymer according to the present invention. Where the amount of bacteria in the microorganism-polymer complex form has an effect on, for example, the rate of a reaction that is mediated by the microorganism-polymer complex, the rate of reaction may be decreased by controlling (for example decreasing) the pH of the culture.

[0322] The acidified samples that were not stained, i.e. one control sample and one sample with pAH-2AFP, were treated with K.sub.2HPO.sub.4 to restore their pH to 7.0, and the resulting samples were incubated for another 6 h before and biofilm/complex formation was determined by crystal violet staining and absorbance measurement at 550 nm.

[0323] As shown by the “28 h Biofilm (reverse pH change)” bars in FIG. 9, an increase in absorbance was observed in each sample, suggesting an increase in the amount of microorganism-polymer complex/biofilm. However, it is notable that the absorbance of the culture containing pAH-2AFP increased by about three times, whereas the absorbance of the control culture did not increase by even two times.

[0324] This indicates that the culture containing pAH-2AFP was able to reform a microorganism-polymer complex, whereas the control culture remained in a predominantly planktonic form.

[0325] Thus, the amount of bacteria in the microorganism-polymer complex may be able to be controlled (i.e. decreased and/or increased) by adjusting (i.e. decreasing and/or increasing) the pH of the culture, where the culture includes a polymer according to the present invention. Where the amount of bacteria in the microorganism-polymer complex form has an effect on, for example, the rate of a reaction that is mediated by the microorganism-polymer complex, the rate of reaction may be controlled by the pH of the culture.

[0326] Without being bound by theory, it is postulated that the decrease in pH after the initial 22 hours of incubation hydrolysed the imine bonds of the polymer. Thus, it is postulated that the R.sup.2 groups of the polymer were detached from the polymer backbone. This may mean that the R.sup.2 groups would no longer hold the cells in a complex, and the complex was dispersed. The increase in absorbance after the pH was returned to its initial value suggests that the cells were held in a complex once more. This may be because the polymer backbone had reattached to the R.sup.2 groups.

Use of Complexes to Perform Microorganism-Mediated Reactions

Transformation of 5-fluoroindole Into 5-fluorotryptophan

[0327] The influence of polymers according to the present disclosure on the microorganism-mediated transformation of 5-fluoroindole into 5-fluorotryptophan mediated was studied. This biotransformation has been described previously using a range of spin coated E. coli K-12 biofilms matured in M63 medium for 7 days. As an example, MC4100 spin coated biofilms were able to convert 10% of 5-fluoroindole to 5-fluorotryptophan after 24 h, whereas the higher biofilm forming PHL644 was able to convert 50% in 24 h (FIG. 5 of Perni et al., AMB Express, 2013, 3:66).

##STR00158##

[0328] The microorganism-polymer complex was prepared for pAH and polymers according to the invention with E. coli strain MC4100, as described above, and left for 48 h to stabilise. The microorganism-polymer complexes were isolated by centrifuge and washed with KH.sub.2PO.sub.4/K.sub.2HPO.sub.4 buffer (0.1 M). The supernatants were removed and 1 ml of reaction buffer (0.1 M KH.sub.2PO.sub.4/K.sub.2HPO.sub.4, 7 mM serine, 1 mM PLP and 1.5 mM fluoroindole) was added to each microorganism-polymer complex. Each sample of complex in the reaction buffer was left to react for 24 h.

[0329] The conversion of fluoroindole to fluorotryptophan was determined according to the following equation:

[00001]$\%Conversion=\frac{Yieldoffluorotryptophan\left(\%\right)}{Depletionoffluoriindole\left(\%\right)}\times 100$

[0330] The conversion for the sample containing each polymer is shown in FIG. 10.

[0331] FIG. 10 shows that the microorganism-polymer complexes of the present invention can be used to perform chemical transformations.

[0332] It was surprising that cultures including complexes with polymers according to the invention each provided conversion exceeding that reported in the literature.

[0333] It was also surprising that cultures including complexes with polymers pAH-Bn, pAH-In, pAH-Naph or pAH-Anthr provided exceptional conversions, similar to that seen when using the strong biofilm-forming strain PHL644.

[0334] This shows that the performance of weaker biofilm-forming microorganisms can be improved by culturing with a polymer as defined by formula (I).

[0335] As shown by the trend line, as clogD of the R.sup.2 group increases, the conversion generally increases. Thus, there may be a correlation between conversion and the clogD of the monomer unit.

Hydrolysis of 4-nitrophenyl Dodecanoate

[0336] E. Coli Can Naturally Produce Esterase/lipase Enzymes, Which Hydrolyse Esters.

[0337] Samples of microorganism-polymer complexes were prepared as described above and left incubating at 30 degrees, 150 rpm for 48 h. 4-nitrophenyl dodecanoate (as a solution in isopropyl alcohol, to make a final concentration of 0.08 mM of 4-nitrophenyl dodecanoate and 50% isopropyl alcohol by volume) was added. Esterase activity was monitored by monitoring absorbance of the hydrolysis product, 4-nitrophenol, at 410 nm over 240 h.

[0338] The significant amount of isopropyl alcohol used in this experiment was considered to be challenging to the microorganism. This was used to dissolve the 4-nitrophenyl dodecanoate as this chemical is poorly soluble in water.

[0339] FIG. 11 shows the absorbance over time in the presence of the complex of E. coli strain MC4100 with either pAH-Bn or pAH-In. As shown in FIG. 1, the challenging conditions were handled surprisingly well by each complex.

[0340] The initial rates of increase in absorbance for the cultures including the complexes were higher than the bacteria alone.

[0341] Furthermore, the final yields of hydrolysis product indicated by the absorbance were significantly higher for the cultures including the complexes than for the bacteria alone.

[0342] Thus, it can be seen that cultures including complexes of a polymer according to formula (I) (i.e. including a complex of the polymer with the microorganism) can be used to perform chemical transformations.

[0343] Furthermore, cultures including complexes according to the invention can be less susceptible to challenging conditions than similar cultures without the polymer.