SUPRAMOLECULAR GEL SUPPORTED ON OPEN-CELL POLYMER FOAM

Abstract

The present invention relates to a polymer foam, said polymer foam comprising pores forming an open-cell polymer foam, said polymer foam comprising a supramolecular gel inside pores, and said polymer foam comprising at least one enzyme. The present invention relates to a supramolecular gel; its preparation and its applications, notably in chemical synthesis and kinetic resolution, in particular of organic compounds. The present invention also relates to flow chemistry.

Claims

1. A polymer foam, said polymer foam comprising pores forming an open-cell polymer foam, said polymer foam comprising a supramolecular gel inside the pores, and said polymer foam comprising at least one enzyme.

2. The polymer foam according to claim 1, wherein said supramolecular gel comprises nanofibers anchored on the surface of the pores of said polymer foam.

3. The polymer foam according to claim 1, wherein said supramolecular gel is a peptide-based hydrogel.

4. The polymer foam according to claim 1, wherein said supramolecular gel is an enzyme-assisted self-assembly gel.

5. The polymer foam according to claim 1, wherein said supramolecular gel has at least one catalytic activity.

6. The polymer foam according to claim 1, wherein said at least one enzyme is selected from the group consisting of esterase, phosphatase, alkaline phosphatase, β-lactamase, matrix metalloproteinase, matrix metalloproteinase-9 (MMP-9), chymotrypsin, thrombin, galactosidase, lipase, microbial transglutaminase (MTGase), thermolysin, glucose oxidase, peroxidase, tyrosinase, and any combination thereof.

7. The polymer foam according to claim 5, wherein said at least one catalytic activity is an esterase-like activity, or catalyzes a reaction selected from the group consisting of Aldolization, Mannich Reaction, Michael addition, hydrolysis of glycosidic bonds, Diels-Alder reaction in the presence of Cu(II) ions, oxidation of benzyl alcohol in the presence of Pd(II) ions, triazole formation by cycloaddition in the presence of Cu(I) ions, peroxidase activity in the presence of Fe(II) ions and a heme nucleus, CO.sub.2 to carbonate conversion in the presence of Zn(II) ions and any combination thereof.

8. The polymer foam according to claim 1, wherein said polymer foam comprises a layer of polymer material forming a substrate and a layer of said supramolecular gel on said substrate, and wherein said at least one enzyme is adsorbed in said supramolecular gel.

9. The polymer foam according to claim 8, wherein said supramolecular gel comprises a polyelectrolyte multilayer comprising a combination of a polycationic compound and a polyanionic compound which forming bilayers, said polyelectrolyte multilayer being in between the substrate and the supramolecular gel.

10. A method for preparing a polymer foam according to claim 1, said method comprising: (a) providing a polymer foam comprising pores forming an open-cell polymer foam, (b) providing an enzyme inside said pores of said polymer foam, and (c) providing a molecule forming a supramolecular gel inside pores of said polymer foam.

11. The method of claim 10, wherein step (b) comprises coating said pores with a polyelectrolyte multilayer comprising a combination of a polycationic compound and a polyanionic compound which form bilayers prior to providing said enzyme and then coating said polyelectrolyte multilayer with said enzyme.

12. The method of claim 10, wherein the molecule forming said supramolecular gel is a peptide and step (c) further comprises growing the supramolecular gel inside the pores by enzymatic activity of said enzyme.

13. A flow reactor comprising a polymer foam according to claim 1.

14. A method for chemical synthesis comprising putting one or more chemical reactants in contact with a polymer foam according to claim 1, converting said one or more chemical reactants into one or more chemical products by a catalytic reaction performed by said polymer foam.

15. A method for kinetic resolution of chemical components comprising putting a mixture of chemical components in contact with a polymer foam according to claim 1, separating said mixture upon contact with said polymer foam and providing separated compounds from said mixture.

Description

[0098] FIG. 1 represents a monitoring of para-nitrophenol production from PNP hydrolysis by HPLC as illustrated in example 11. Absorbance (405 nm) evolution over residence time is measured for three runs: one day (black line), five days (red line) and twelve months (green line) after the preparation of the supported supramolecular hydrogel (black).

[0099] FIG. 2 represents a graphic illustrating that supported esterase-like hydrogel encapsulating AP allows to catalyze two reactions (phosphate and ester hydrolyses) on the same substrate Fmoc-pY-OMe, leading to Fmoc-Y—OH. HPLC monitoring has been done according to the residence time (example 13).

[0100] FIG. 3 represents a TEM image: piece of supramolecular hydrogel generated from melamine foam in presence of gold nanoparticles (diameter: 15 nm). The catalytically-active nanoojects (black dots) are encapsulated within the supramolecular hydrogel (example 14).

EXAMPLES

[0101] All chemicals used in this work are gathered in the following table. They were all used as received, without further purifications.

TABLE-US-00001 Name, acronym MW (abbreviation) (g.mol.sup.−1) Supplier CAS number Bovine serum albumin  66 000   Sigma-Aldrich    9048-46-8 (BSA) Alkaline Phosphatase from 170 000   Sigma-Aldrich    9001-78-9 bovine intestinal mucosa (AP) Poly(ethylene imine) (PEI) 750 000   Alfa Aesar    9002-98-6 Poly(styrene sulfonate)  70 000   Sigma-Aldrich   25704-18-1 (PSS) Deuterated Water (D.sub.2O)      20.03 Sigma-Aldrich    7789-20-0 Dimethylformamide (DMF)      73.09 Acros Organics      68-12-2 Dichloromethane (DCM)      84.93 Acros Organics      75-09-2 Trifluoroacetic acid (TFA)     114.02 Alfa Aesar      76-05-1 Deuterated DMSO      84.17 SDS    2206-27-1 (DMSO-d.sub.6) N-Ethyldiisopropylamine     129.25 Alfa Aesar    7087-68-5 (DIEA) Fmoc-L-phenylalanine     387.43 Iris biotech   35661-40-6 (Fmoc-F-OH) Fmoc-L-Tyrosine Phosphate     483.41 Bachem  147762-53-6 (Fmoc-Y(PO.sub.3H.sub.2)-OH Fmoc-L-Glycine     297.31 Iris biotech   29022-11-5 (Fmoc-G-OH) Fmoc-trityl-L-Histidine     619.71 Iris biotech  109425-51-6 (Fmoc-H(Trt)-OH Triisopropylsilane (TIPS)     158.36 Sigma-Aldrich    6485-79-6 Resin 2-chlorotrityl chloride — Sigma-Aldrich   42074-68-0 (2-CTC) 1-Hydroxybenzotriazole     135.12 Sigma-Aldrich  123333-53-9 hydrate (HOBt) N,N,N′,N′-Tetramethyl-o-     379.24 Alfa Aesar   94790-37-1 (IH-benzotriazol-1- yl)uranium hexafluoro- phosphate (HBTU) Diethyl ether      74.12 Acros Organics      60-29-7 Polymer Foam (melamine) / FoamPartner / BASOTCT V3012 white L-Phenylalanine methyl ester     215.68 Sigma-Aldrich    7524-50-7 hydrochloride 4-Nitrophenyl acetate (PNA)     181.15 Sigma-Aldrich     830-03-5 Fmoc-L-glutamic acid     425.47 Sigma-Aldrich   71989-18-9 5-tert-butyl ester (Fmoc-L-Glu(OtBu)-OH) Fmoc-D-glutamic acid     425.47 Sigma-Aldrich  104091-08-9 5-tert-butyl ester (Fmoc-D-Glu(OtBu)-OH) Fmoc-L-aspartic acid     411.25 Sigma-Aldrich   71989-14-5 4-tert-butyl ester (Fmoc-L-Asp(OtBu)-OH) Fmoc-L-Lysine t-butyl ester     460.98 Iris Biotech  940941-43-5 hydrochloride GMBH (Fmoc-L-Lys-OtBu) Fmoc-D-Lysine t-butyl ester     460.98 Iris Biotech 2250436-42-9 hydrochloride GMBH (Fmoc-D-Lys-OtBu) 4-Hydroxybenzoïc acid     180.20 Sigma-Aldrich    4191-73-5 isopropyl ester (Isopropyl 4-hydroxybenzoate) Boc-L-Tyrosine methyl ester     295.33 Sigma-Aldrich    4326-36-7 (Boc-L-Tyr-OMe) Fmoc-L-tyrosine tert-butyl     237.29 Sigma-Aldrich   16874-12-7 ester (Fmoc-L-Tyr-OtBu) D-tyrosine tert-butyl ester     237.29 Alfa Aesar   87553-74-0 (H.sub.2N-D-Tyr-OtBu) Fmoc Chloride     258.70 Sigma-Aldrich   28920-43-6

[0102] Transmission Electronic Microscopy (TEM)

[0103] The TEM images were performed with sample prepared in liquid (diluted solutions (1 mg/mL of Fmoc-GFFpYGHpY and Fmoc-GFFYGHY) or gels (10 mg/mL of Fmoc-GFFpYGHpY or Fmoc-GFFYGHY (prepared as described in section 2 just above))). All the samples were freshly prepared before TEM measurements. To make the observations 20 μL of the sample is dropped off on a shelf. Then, the sample is observed by a TEM Tecnai G2 machine in negative staining. To make the observations, 5 μL of the different gels are deposited onto a freshly glow discharged carbon-covered grid (400 mesh). The gel is left for 2 minutes and the grid is negatively stained with 5 microliters uranyl acetate (2% in water) for another minute and finally dried using a filter paper. The grids were observed at 200 kV with a Tecnai G2 (FEI) microscope. Images were acquired with a camera Eagle 2k FEI) ss CD camera.

[0104] Analytic High-Performance Liquid Chromatography (HPLC)

[0105] Analytic High-Performance Liquid chromatography (HPLC) was carried out with a 1100 Series from Agilent technologies.

[0106] For characterization of the different peptides and the catalytic assays: the column is a Supelcosil ABZ+Plus with the following dimensions 15 cm×4.6 mm, 3 μm. The eluent used for all analyses was acetonitrile/deionized water in different ratios depending on the experiment. Ratio 80/10 in isocratic conditions, at 1 mL/min for the first catalytic assays but a ratio 50/50 in isocratic conditions, at 1 mL/min for the peptide characterization.

[0107] For the racemic discrimination assays: the column is a chiral column is a Cosmosil 3B with the following dimensions 4.6 mm I.D.×250 mm. The eluent used for all analyses was n-hexane/isopropanol in ratio 90/10 in isocratic conditions, at 1 mL/min except for the racemic solution of methyl-3-(4-methoxyphenyl) oxirane-2-carboxylate where the ratio was 97.5/2.5 in isocratic conditions at 1 mL/min. Chromatograms were recorded by the software OpenLab Agilent 1100.

[0108] All samples were observed in solution in diluted conditions (ten times under the gelation condition (1 mg/mL of peptides)).

Example 1—Synthesis and Characterization of Fmoc-GFFYGHY, Fmoc-GFFpYGHY, Fmoc-GFFYGHpY and Fmoc-GFFpYGHpY

[0109] All peptides were prepared using solid support chemistry. The “Fmoc strategy” was used based on 2-CTC resin. The following synthetic pathway is given in the scheme below:

##STR00004## ##STR00005## [0110] Step a: loading of the resin 2-chlorotrityl chloride (2-CTC, called as “resin” or “r” in the rest of this procedure). Addition of 3 eq/r of Fmoc-Tyr(OR)—OH+6 eq/r of DIEA in 3 mL of DMF for 300 mg of resin. The solution in contact with the resin is stirred at RT for 2 h. Then, the solution is removed and a solution of MeOH is added at RT for 1 h. [0111] Step b: Fmoc group deprotection: 3 mL of a 20% of piperidine in DMF solution is added and stirred at RT for 15 min. [0112] Step c: Coupling step: 3eq/r of Fmoc-amino acid+3 eq/r of HOBt+3 eq/r of HBTU+6 eq/r of DIEA are added in 3 mL of DMF and let in contact with the resin at RT for 30 min. [0113] Step d: Cleavage of the resin and lateral chains deprotection: addition of 3 mL of a solution containing 95% TFA+2.5% H.sub.2O+2.5% triisopropylsilane it's stirred at RT for 2 h. Then the solution is filtered. The solvent is then removed. Finally the product is precipitated by using a small amount of cold ether. [0114] Between each step a, b, c and d, a rinsing stage is executed by using 5 times 3 mL of DMF and then a Kaiser test is made to confirm the achievement of the coupling or deprotection steps.

[0115] When the amino acid Fmoc-Tyrosine-OH was required in the final sequence, the protection of the phenol group was ensured by a tBu group. When a tyrosine-phosphate is required, the Fmoc-Tyr(OPO3H2)-OH is directly introduced during the synthesis.

Example 2—Preparation of Peptide Solution and Gel Formation

[0116] All gels were prepared in PBS buffer (pH 7.4). The PBS buffer is prepared the day of the gel preparation.

[0117] PBS Buffer (pH 7.4): one tablet of commercially available PBS (P4417 from Sigma Aldrich) was dissolved in 200 mL of ultrapure water (Milli-Q Plus system, Millipore, Billerica, Mass.) leading to 0.001 M phosphate buffer, 0.0027 M potassium chloride and 0.137 M sodium chloride. If necessary, the pH of this buffer was adjusted to 7.4 value by addition of few drops of HCl (0.01 M) or NaOH (0.01 M) solution. The pH value was monitored using a pH meter.

[0118] General preparation of peptide solution: an adequate amount of peptide was dissolved in PBS to get the suitable concentration (usually 1 and 10 mg/mL). This solution was vortexed 2-5 minutes and sonicated in an ultrasound bath during 1 minute. The resulting peptide solution was thus used for all kinds of analyses described in this ESI. Fmoc-GFFYGHY gel formation were obtained by dissolving Fmoc-GFFYGHY (10 mg/mL) in PBS buffer. This solution was vortexed during 2 minutes and then dipped into an ultrasound bath during 1 minute. The solution was heated at around 100° C. to solubilize the peptide. When the solution cooled down the gel formed. Fmoc-GFFpYGHpY gel formation were obtained by dissolving Fmoc-GFFpYGHpY (10 mg/mL) in PBS buffer. This solution was vortexed 2 minutes and then dipped into an ultrasound bath during 1 minute. 1 mg/mL of commercial AP (P7640 from Sigma Aldrich) was added (ratio AP/peptide (1/10). The resulting mixture was vortexed 10 seconds. The resulting gel was obtained after 5 minutes

[0119] Upside-down vial tests with peptides for the gelation assays. Gels were obtained from Fmoc-GFFYGHY by heating and cooling and from Fmoc-GFFpYGHpY using AP, as described above. No gels were obtained from mono-phosphorylated peptides Fmoc-GFFYGHpY or Fmoc-GFFpYGHY using heating/cooling, AP or by decreasing the pH.

Example 3—Multilayer Film Preparation and Localized Gel Formation at the Liquid-Solid Interface

[0120] All polyelectrolytes (1 mg/mL), proteins (AP 1 mg/mL and BSA 1 mg/mL), and peptides or amino acid (1 mg/mL) were prepared in PBS buffer freshly prepared as described in example 2. Different solid substrates were used depending on the characterization technique investigated: gold coated quartz crystal for QCM-D monitoring, ZnSe crystal for ATR-FTIR experiments, Glass slide and Si Wafer for Cryo-SEM analyses, glass slide for fluorescence emission assays (using a multiplate reader (FLX-Xenius®, SAFAS, Monaco)) and melamine foam (from Foam Partner industry) for activity assays in the continuous flow reactor experiments. The growth of the supramolecular gel from the chosen substrate was done as following: after the deposition of a PEI (1 mg/mL) precursor layer on the chosen surface by dipping (for 10 minutes in the PEI solution), the multilayer film was built up by alternately exposing the surface to PSS (1 mg/mL in PBS buffer) and PEI (1 mg/mL in PBS buffer) solutions for 10 minutes with an intermediate rinsing step with PBS buffer during 5 minutes. AP (1 mg/mL) or BSA (1 mg/mL), all prepared in PBS, were put in contact with the substrate during 20 minutes followed by 5 minutes of rinsing step with PBS buffer. Finally, the peptide Fmoc-GFFpYGHpY solution (1 mg/mL in PBS buffer) was let in contact with the modified surface overnight. The volume of each solution brought in contact with the substrate was 1 mL except for the QCM-D experiment where it was 700 μL. All steps were done at RT.

Example 4—Esterase-Like Activity Assays

[0121] Esters Samples preparation: Different concentrations of esters were used: 0.275 mM, 0.55 mM, 1.35 mM, 2.2 mM, 2.76 mM, 2.76 mM, 8.2 mM, 10 mM, 22 mM, 28 mM and 41 mM. Esters were solubilized in ultrapure water. 20 μL of the solution is diluted in 300 μL of isopropanol and then 50 μL of the new solution is added in 450 μL of the eluent ratio of n-hexane and isopropanol.

[0122] Solutions were prepared in the eluent ratio of ACN/water and n-hexane/isopropanol. All solutions were filtrated with a PTFE 0.2 μm filter before each injection.

Example 4.1. With Para-Nitrophenyl Acetate (PNA)

[0123] Microplate reader UV spectroscopy (FLX-Xenius®, SAFAS, Monaco) using SP2000V7 software was the main device entailed in esterase-like activity measurement toward PNA. The activity of the Fmoc-GFFYGHY gel (formed by the dephosphorylation of Fmoc-GFFpYGHpY in presence of AP) was measured on three kinds of samples: first one in solution with a self-standing gel; second by generating the gel on a surface on a glass slide coated beforehand with the following multilayer PEI/(PSS/PEI)2/AP (protocol described in example 3) and finally by coating on a polymer open-cell foam with the multilayer PEI/(PSS/PEI)2/AP. Concentration (1 mM) and volume (1 mL) ensure a large excess of p-NPA to monitor the esterase like activity through the absorbance measurements at 405 nm (corresponding to the para-nitrophenol maximum absorption).

Example 4.2. With One of the Three Classes of Non-Activated Esters

[0124] All experiments were followed by HPLC. Esters (1 mM) were dissolved in a freshly prepared PBS buffer at pH 7.4. Then, 504 of this ester solution were dropped off on the catalytic gel (see figure below). The gel is formed in the bulk in a 4 mL glass vial, from a PBS buffer solution thanks to the dephosphorylation of Fmoc-GFFpYGHpY (10 mg/mL) in the presence of AP (1 mg/mL). 200 μL of this catalytically-active supramolecular gel (CASH) is formed and 50 μL of different ester solutions (1 mM) are brought in contact with it. The solution of ester diffuses within the CASH and the monitoring of the ester hydrolysis is monitored as following: (i) the CASH containing the ester solution is vortexed and mixed with 1 mL of deionized water, (ii) 10 μL of this solution is mixed with 1 mL of deionized water and (iii) and 54 is analyzed by HPLC.

Example 5—Esterase-Like Activity and Kinetic Resolution of the Supported CASH in a Continuous Flow Reactor

[0125] Enzymatically active multilayer film deposition on foam surface: all polyelectrolytes (1 mg/mL), enzyme (AP 1 mg/mL), and Fmoc-GFFpYGHpY (1 mg/mL) were freshly prepared in PBS buffer as described in example 3 above. After the first deposition of a PEI (1 mg/mL) precursor layer on the foam surface by dipping, the multilayer film was built up by alternately exposing the foam in PSS (1 mg/mL in PBS buffer) and PEI (1 mg/mL in PBS buffer) solutions for 10 minutes with an intermediate rinsing step with PBS buffer during 5 minutes. AP (1 mg/mL) solution was prepared in PBS, put in contact during 20 minutes followed by 5 minutes of rinsing step with PBS buffer. Finally, the Fmoc-GFFpYGHpY solution (1 mg/mL in PBS buffer) was brought in contact with the modified foam installed already within the reactor (column) through a continuous flow (in a closed circuit) of 0.5 mL/min during 12 h.

[0126] Ester solution: according to the desired ester concentration, the desired amount of ester was dissolved in 20 mL of deionized water before flow catalysis.

[0127] Protocol for flow catalysis: the solution of substrate (ester) was introduced in the reactor at 1.5 mL/min flow in a closed circuit. In open circuit (continuous flow conditions) the flow was adapted (decreased) to the desired residence time. For all substrates (except in case of PNA) the conversion in the corresponding carboxylic acid and the enantiomeric excess were followed by HPLC using an adequate column.

[0128] Washing step of the flow reactor (column): a solution of 10 mL of deionized water was passed through the flow reactor at 0.5 mL/min flow in an open circuit in the case of PNA and at 1.5 mL/min for all others esters. The resulting solution was checked by HPLC or by UV (in the case of PNA) to be sure that all residual ester or acid have been removed from the reactor. This absence of gelator in the resulting solution supports that the gel is not delaminated.

[0129] Quantitative production and isolation of chemically pure and enantiopure Fmoc-L-Glu(OH)—OH: when the conversion of Fmoc-L-Glu(OtBu)—OH in Fmoc-L-Glu(OH)—OH reached 80% after 30 min of time residence (monitored by HPLC/before the hydrolysis of Fmoc-D-Glu(OtBu)—OH in Fmoc-D-Glu(OH)—OH), the reaction is stopped: all the reaction medium is removed from the reactor and this latter is washed with deionized water (see paragraph just above). Then, the reaction medium and the washing solution are gathering and basified up to pH 10 using NaHCO.sub.3 (0.5 M) solution. This aqueous phase is extracted three times with dichloromethane (equivalent volume as the aqueous phase). The organic phase is dried on magnesium sulfate and the solvent is removed under reduced pressure. The aqueous phase is neutralized using 0.1 HCl and then freeze-dried. The Fmoc-L-Glu(OH)—OH is isolated as a white solid from the aqueous phase and the enantio-enriched mixture of Fmoc-L-Glu(OtBu)—OH and Fmoc-D-Glu(OtBu)—OH is isolated as a colourless oil from the organic phase.

Example 6—Repeatability of the Catalytic Process

[0130] The flow reactor has been prepared as described in Example 5. A solution of the model ester substrate, i.e. para-nitrophenylacetate PNA, (1 mM in 20 mL of deionized water) is injected through the catalytic flow reactor using the flow rate value: 1.5 mL/min. The conversion of PNA in the product para-nitrophenol is monitored over time by HPLC analysis. Once the conversion is completed, the flow reactor is washed with deionized water until no traces of residual para-nitrophenol is detected by HPLC analysis (washing step). This allows to get the kinetic profile of run 1. Then, a freshly prepared new solution of PNA (1 mM in 20 mL of deionized water) is injected through the catalytic reactor at 1.5 mL/min of flow rate. The HPLC monitoring of the PNA conversion over time allows to get the run 2 kinetic profile. Then, a third, fourth and fifth catalytic cycle were successively realized using the same catalytic hydrogel supported foam, corresponding to run 3, 4 and 5 respectively. Comparison between the kinetic profiles of run 1, 2, 3, 4 and 5 shows an excellent repeatability of the catalytic process.

Example 7—Robustness of the Catalytic Process Overtime

[0131] Five successive runs were performed as described in example 8, transforming PNA in para-nitrophenol. Then, the tubular column containing the catalytic hydrogel supported on the polymer foam is stored at 4° C. One month later, this column is adapted again for continuous flow reaction in conditions described in example 8. A solution of PNA (1 mM in 20 mL of deionized water) is injected through the catalytic flow reactor using the flow rate value: 1.5 mL/min. The conversion of PNA in the product para-nitrophenol is monitored over time by HPLC analysis (run 6). The graph showing the evolution to the para-nitrophenol production over time of run 6 can be overlapped to those corresponding to run 1, 2, 3, 4 and 5.

Example 8— Stability of the Supported Supramolecular Hydrogel

[0132] Five successive runs (run 1, 2, 3, 4 and 5) and one additional run one month later (run 6) were realized as described in examples 9 and 10. HPLC monitoring was carried out during all these six runs to detect any leaching of the peptide hydrogelator Fmoc-GFFYGHY during both the catalytic flow processes and the washing steps. In any cases, no traces of the hydrogel-constituting peptide was measured proving no delamination of the peptide self-assembled structure. This observation is in full agreement with the preservation of the catalytic activity over the several runs, highlighting the good stability the supported supramolecular hydrogel in the flow catalytic process.

Example 9—Determination of the Proportion of Peptides Involved in the Catalytic Process

[0133] The number of peptide Fmoc-GFFYGHY that has been reacted with PNA is estimated from FIG. 3a and is equivalent to 141 μmoles: this is the amount of PNA converted when 22, 28 and 41 mM of PNA is used. We postulate that one PNA has reacted with one histidine. To determine the ratio of peptides Fmoc-GFFYGHY that have reacted with PNA against peptides present in the CASH within the investigated tubular column (15 cm length and 4 mm diameter, see 4b in the manuscript), the supported gel was entirely dissolved using a flow of acetone. After the removal of this organic solvent under reduced pressure, the residue was first analyzed by 1H NMR and HPLC to confirm the solely presence of Fmoc-GFFYGHY, and then weight. A mass of 1.66 mg (1.49 mmoles) was isolated as a white solid. Thus, the ratio r of peptide involved in the catalytic process against the whole number of peptide Fmoc-GFFYGHY engaged in the CASH is: r=1490/141≈111.

Example 10—Experimental Determination of Km, Vmax and Kcat Using Methyl Ester 9 and the Michaelis-Menten Equation

[0134] To determine the characteristic values of our catalytic system, two hypotheses were made. As our system of catalysis is a gel supported on porous polymer foam contained in a reactor in a continuous flow system, the first hypothesis made is that the catalysis can be related to a packed bed reactor under continuous flow. The second is that the gel can be considered as a synzyme, which acts as a “Michaelian” enzyme during its steady state. Due to the catalyst and its confinement inside a reactor, a pre-steady state is observed. To determine the Km, Vmax and kcat, only the steady state is taken in account. The Michaelis-Menten equation describes the kinetic curve of V.sub.0-[S]:

[0135] To determine Km and Vmax values, the Lineweaver-Burk graphical method was choosen. In order to plot the 1/V0 in function of 1/[S].sub.0, the V.sub.0 values of the different concentration of substrate, were graphically determined by the slope present at the beginning of the steady state of the catalysis (after the inflection point) in the graph of the evolution of the product concentration as a function of time.

[0136] We obtained:

[0137] 1/Vmax=0.0481 s/μmol=>Vmax=20.79 μmol/s

[0138] Km: Km/Vmax=1.35 s=>Km=1.35×20.79=28 mM

[0139] Then, it is possible to determine the kcat by using the following equation:

[00001]$k_{\mathrm{cat}}=\frac{V_{\max }}{\left[\mathrm{enzyme}\right]}$

[0140] The hypothesis is that [enzyme]=[“catalytic site” of the gel] (μmol/L). Then:

[00002]$k_{\mathrm{cat}}=\frac{V_{\max }}{\left[\mathrm{enzyme}\right]}=\frac{V_{\max }}{\left[\mathrm{catalytic}\mathrm{site}\mathrm{of}\mathrm{the}\mathrm{hydrogel}\right]}=\frac{20.79}{0.007}=3\times {10}^3{s}^{-1}$

[0141] The concentration of catalytic site called [Enzyme] has been determined as following: the number of peptides involved in the catalytic process and present in our flow reactor (length 15 cm; diameter 4 mm) is 141 μmol (see Section 17 page 25). Considering the volume in the flow reactor, it represents a concentration [enzyme] 0.007 moles.Math.L−1.

[0142] This example supports the enzyme-like activity of the gel.

Example 11— Enzymatic Activity of the Supported Supramolecular Hydrogel

[0143] The flow reactor has been prepared as described in Example 5. A solution of the phosphorylated model substrate, i.e. para-nitrophenylphosphate PNP, (8.2 mM in 20 mL of deionized water) is injected through the catalytic flow reactor using the flow rate value: 1.5 mL/min. The conversion of PNP in the product para-nitrophenol was monitored over time by HPLC analysis in order to evaluate the enzymatic activity of the alkaline phosphatase entrapped during the hydrogel formation. After 20 minutes, a complete conversion of PNP was observed. Then the flow reactor was extensively washed with deionized water and a second run was carried out using PNP solution in the same condition as described just above in the first run. The kinetic profile of this second run is similar to the one obtained for the first run, showing that no leaching of the enzyme occurs during these two runs without any loss of its activity.

[0144] The supported supramolecular hydrogel (on melamine) within the tubular column was stored at 4° C. after each run. Example 11 was repeated 30 times over 12 months. Kinetic profiles showing the conversion of PNP in para-nitrophenol of all runs overlap (FIG. 1). These results highlight the high stability of the enzymatic activity.

Example 12— Supramolecular Hydrogel Coating of Both the Tubular Column and all Pipes Used in the Flow Reactor (without the Foam Supported Hydrogel)

[0145] The whole interior surface of the tubular column and all pipes used in the flow reactor have been coated by the catalytic supramolecular hydrogel according to the protocol described in example 5. No open cell polymer foam has been used. Then a solution of the model ester substrate, i.e. para-nitrophenylacetate PNA, (1 mM in 20 mL of deionized water) is injected through the catalytic flow reactor using the flow rate value: 1.5 mL/min. The conversion of PNA in the product para-nitrophenol is monitored over time by HPLC analysis. Quasi no conversion of the substrate PNA is measured over time.

Example 13— Dual Enzymatic Activities Coming from Enzyme-Immobilized Hydrogel and Catalytic Peptide Self-Assembly

[0146] The flow reactor has been prepared as described in Example 5 (using a melamine foam). The alkaline phosphatase (AP) used to trigger the hydrogel formation thus is encapsulated within the catalytic material. This means that two catalytic activities are present: an esterase-like activity coming from the peptide self-assembly and a phosphatase activity (enzymatic activity) coming from AP. A solution of N-Fmoc tyrosine phosphate methyl ester amino acid, Fmoc-pY-OMe, (5 mM is deionized water) is injected through the catalytic flow reactor using the flow rate value: 1.5 mL/min. The hydrolysis of both the phosphate group and the methyl ester group, leading to the product Fmoc-Y—OH, is monitored by HPLC (FIG. 2). This work shows the possibility to mix different kinds of catalyst within the same reactor for tandem or domino transformations.

Example 14: Encapsulation of Gold Nanoparticles (Θ=15 nm) within the Supported Supramolecular Hydrogel

[0147] Supramolecular hydrogel was prepared according to example 5 using a solution of the phosphorylated tripeptide Fmoc-FFpY instead of Fmoc-GFFpYGHpY. The solution of the tripeptide Fmoc-FFpY was prepared by mixing a 10 mg/mL solution of Fmoc-FFpY with a 1OD gold nanoparticles solution in PBS purchased from Sigma-Aldrich (ref.: 777089) (1:1 v/v). Gold nanoparticles can be used as catalyst for various kind of chemical transformations. The resulting supported supramolecular hydrogel on melamine foam was red colored because of the presence of nanoparticles. After flowing PBS buffer through in a column (150*4.6 mm) for 16 h at a rate of 1 ml/min, the foam remained equally red TEM images of the gold nanoparticles-contained hydrogel shows the presence of nanoparticles within the gel at the micrometer scale (FIG. 3).