Interpenetrating Polymer Network Capturing FRET-Sensitive Polymers Available for an Enzyme Assay

Abstract

Fluorogenic glycomonomers have been used for biological evaluations, and water-soluble and Förster resonance energy transfer (FRET)-sensitive glycopolymers have also been reported. A FRET-sensitive polymer was conveniently prepared from a fluorogenic donor monomer and a fluorogenic acceptor monomer by means of simple radical polymerization in high yield. Continuous fluorospectroscopic monitoring of the polymer in the presence of an enzyme was performed, and the results showed the possible application of the FRET-sensitive glycopolymer for practical use. In addition to the use of aqueous solution phase, the water-soluble and FRET-sensitive glycopolymer was completely captured into an interpenetrating polymer network (IPN) by means of radical polymerization with a combination of acrylamide and bis-acrylamide as used for the cross-linking reagent system. The IPN including the FRET-sensitive glycopolymer was allowed to react with amylases in an aqueous buffer solution at 37 °C, and the enzymatic reaction was continuously and conveniently monitored by means of fluorometric spectroscopy.

Figure 1

Schematic diagram of the polymerization. (a) Synthetic assembly of a FRET-sensitive polymer by means of radical polymerization of a fluorogenic glycomonomer, a dansyl monomer, and acrylamide (AAm), and fluorometric monitoring of the polymer in the enzymatic reaction. (b) Synthetic assembly of an IPN using the FRET-sensitive polymer by means of the radical polymerization with AAm and bis-acrylamide (bis-AAm).

Scheme 1. Reagents and conditions: (i) 4-Penten-1-ol (5 equiv), BF₃·OEt₂ (10 equiv), N₂, 0 °C → rt, 2 h, DCM, (ii) NaOMe, N₂, rt, 2 h, MeOH, (iii) 2-Naphthaldehyde di-i-propyl Acetal (1.5 equiv), CSA (0.8 equiv), Reduced Pressure, 55 °C, 2 h, DMF, then, Ac₂O, rt, 3 d, pyr, (iv) BH₃·NMe₃ (7 equiv), AlCl₃ (7 equiv), MS 4A, Ar, 0 °C → rt, 4 h, THF, (v) NaOMe, N₂, rt, 2 h, MeOH, (vi) Cysteamine Hydrochloride (10 equiv), UV Light, Ar, rt, 2 h, MeOH, (vii) Acryloyl Chloride (1.2 equiv), Et₃N (3 equiv), Ar, 0 °C → rt, 1 h, MeOH, then, Ac₂O, rt, 3 d, Pyr, (viii) NaOMe, N₂, rt, 2 h, MeOH

Scheme 2. Reagents and conditions: (i) TEMED, APS, Ar, rt, 24 h, H₂O, then 0.1 M AcOH–Pyr Buffer (pH 5.2)

Figure 2

(a) Time course (0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 24, 26, 28, and 30 min) of the relative fluorescence emission of FRET-sensitive polymer 11a during hydrolysis with human salivary α-amylase in 10 mM HEPES buffer (pH 7.0) at 37 °C. The appropriate concentration of FRET-sensitive polymer 11a (based on the sugar unit, x) was used for the assay. (b) Time course of the relative fluorescence intensity from both the dansyl moiety (red ■) at 515 nm and the naphthyl moiety (blue ⧫) at 333 nm in FRET-sensitive polymer 11a during hydrolysis with α-amylase. (c) Hanes–Woolf plot of hydrolysis of FRET-sensitive polymer 11a by α-amylase (means ± SE, n = 5). (d) Initial rates of hydrolysis by α-amylase as a function of sugar concentration based on the sugar unit in the polymer.

Figure 3

(a) Appearance of xerogel dried from the IPN gel. (b) Appearance of the IPN gel in a cuvette in an aqueous medium. (c) Time course (0, 10, 20, 30, 40, 50, 60, 120, 180, 240, 300, 360, 420, 480, 540, 600, 660, 720, 780, 840, 900, and 960 min) of the relative fluorescence emission of FRET-sensitive polymer 11b alone in 10 mM HEPES buffer (pH 7.0) at 37 °C. (d) Time course (0, 10, 20, 30, 40, 50, 60, 120, 180, 240, 300, 360, 420, 480, 540, 600, 660, 720, 780, 840, 900, and 960 min) of the relative fluorescence emission of FRET-sensitive polymer 11b with α-amylase from B. subtilis (1.5 U/3.00 mL) in 10 mM HEPES buffer (pH 7.0) at 37 °C. IPN with the FRET-sensitive polymer 11b (3.13 mg) was used for the assay.

Figure 4

Time course (0, 10, 20, 30, 40, 50, 60, 120, 180, 240, 300, 360, 420, 480, 540, 600, 660, 720, 780, 840, 900, and 960 min) of the relative fluorescence emission of the FRET-sensitive polymer 11b with porcine pancreatic α-amylase (1.5 U/3.00 mL) in 10 mM HEPES buffer (pH 7.0) at 37 °C. IPN having the FRET-sensitive polymer 11b (2.90 mg) was used for the assay.

Figure 5

Summary of this study. (a) The water-soluble glycopolymer having maltooligosaccharide chains in a cuvette with UV irradiation. (b) Treatment with α-amylase for the FRET-sensitive polymer giving fluorescence emission from a naphthyl moiety of the hydrolyzed maltooligosaccharide at the nonreducing end. (c) The IPN including the FRET-sensitive polymer on the bottom of the cuvette with UV irradiation. (d) Treatment with α-amylase for the IPN including the FRET-sensitive polymer on the bottom of the cuvette, giving fluorescence emission due to a naphthyl moiety of the hydrolyzed maltooligosaccharide at the nonreducing end.