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.

1. Introduction

Fluorescence resonance energy transfer or Förster resonance energy transfer (FRET) is a significant physical phenomenon that was initially introduced by Förster¹ more than 70 years ago, and the FRET technique was later used as a spectroscopic ruler in biological chemistry by Stryer and Haugland.² FRET techniques have been widely used in chemical, biochemical, biophysical, medicinal, and other fields for the evaluation of many biological events.³ For effective FRET from a fluorogenic donor to a fluorogenic acceptor, an appropriate pair of fluorescence probes affording spectral overlap of the donor emission spectrum with the absorption spectrum of the acceptor is needed.⁴ The distance between the donor fluorescence probe and the acceptor fluorescence probe is obviously important, and the most suitable distance is estimated to be around 10–60 Å for a pair of the donor probe and acceptor probe in order to obtain effective intramolecular FRET. Successful monitoring of an enzymatic reaction on the basis of FRET technique by using oligosaccharide derivatives having fluorogenic probes was reported⁵ and we further expanded the FRET effect for a FRET-based polymer substrate for a continuous assay of α-amylase.⁶ Simple and convenient preparation by means of a typical radical copolymerization of FRET-component monomers in different ratios of the fluorogenic donor and acceptor gave the corresponding water-soluble FRET-sensitive polymers in high yields. Polymers having maltooligosaccharide chains, which are repeating units via the α1 → 4 glycosidic bond of d-glucose residues and are partial structures of amylose and showed remarkable FRET due to the corresponding pair of fluorogenic probes, were successfully used for a continuous assay of human salivary α-amylase in aqueous media. α-Amylase (EC.3.2.1.1) is a well-known enzyme and belongs to endotype enzyme families that selectively cleave α1 → 4 glucosidic linkages in an amylose and maltooligosaccharides.⁷ The formation of an interpenetrating polymer network (IPN) is a unique characteristic phenomenon in polymer chemistry⁸ and the physical property of the IPN⁹ was of great interest in capture of the water-soluble and FRET-sensitive polymer into the polymer network for evaluation of the enzyme activities of α-amylase. In this paper, we describe synthetic assembly of a FRET-sensitive polymer with a maltohexaose unit and the enzymatic behavior for amylase. We also describe the convenient preparation of an IPN consisting of water-soluble FRET-sensitive glycopolymers by means of one-step assembly of acrylamide (AAm) and bis-acrylamide (bis-AAm) using a simple radical polymerization reaction.

A schematic diagram of our synthetic plan for the simple preparation of an IPN consisting of FRET-sensitive glycopolymers by a one-step hybridization is shown in Figure 1. The aim of this study was to capture the water-soluble and FRET-sensitive glycopolymer into the IPN without time-consuming methodology. In addition to the formation of the IPN, continuous monitoring of the enzyme reaction by the fluorometric spectroscopy according to the IPN was also carried out.

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).

2. Experimental Section

2.1. Materials and Methods

Unless otherwise stated, all commercially available solvents and reagents were used without further purification. Fully acetylated maltohexaose 1 was prepared by the method previously reported.¹⁰ A dansylated monomer 10 was prepared by the method previously reported.¹¹ Acrylamide (AAm) was recrystallized from CHCl₃ before use. Reaction under UV illumination was conducted using a UV-LT-10 UV radiation apparatus with a low-pressure mercury-vapor lamp (Ishii Scientific Instruments, Co. Ltd., Tokyo, Japan). Infrared (IR) spectra were obtained using a Shimadzu IR Prestige-21 spectrometer (SHIMADZU CORPORATION, Kyoto, Japan). NMR spectra were recorded at 500 MHz for ¹H and at 125.8 MHz for ¹³C with a Bruker AVANCE 500 spectrometer in CDCl₃, DMSO-d₆ or deuterium oxide (D₂O). Chemical shifts are expressed as parts per million (ppm, δ) and are relative to an internal TMS in CDCl₃ (δ 0.00), a methyl group in DMSO-d₆ (δ 2.50) or HDO in D₂O (δ 4.78) for ¹H and CDCl₃ in CDCl₃ (δ 77.00), a methyl group in DMSO-d₆ (δ 39.50) or CH₃ of MeOD (49.00) in D₂O for ¹³C. Measurement of fluorescence emission spectra and excitation spectra was conducted at 37 °C in a Teflon-stoppered cuvette (12.5 mm in width × 45 mm in height) containing a 3.00 mL sample using a Shimadzu RF-5300PC spectrometer (SHIMADZU CORPORATION, Kyoto, Japan). The excitation wavelength was used at 290 nm and the measured wavelength was from 300 to 600 nm. The slit width of the excitation and emission was 5.0 nm, and the scan speed was medium. Thin layer chromatography (TLC) on a precoated plate of Silica Gel 60F₂₅₄ (layer thickness, 0.25 mm; E. Merck, Darmstadt, Germany) was used for monitoring the reactions. In order to detect the progress of the reaction, TLC sheets were dipped in (a) a solution of 85:10:5 (v/v/v) MeOH–p-anisaldehyde–concd H₂SO₄ and heated for a few minutes (for carbohydrate) or (b) an aq solution of 5 wt % KMnO₄ and heated similarly (for detection of C=C double bonds). Purification by dialysis was performed against distilled water using a dialysis tubing [molecular cutoff (MWCO): 12 k–16 kDa, Viskase Companies, Inc., Illinois, USA].

2.2. Synthesis

2.2.1. n-Pentenyl 2,3,4,6-Tetra-O-acetyl-α-d-glucopyranosyl-(1 → 4)-2,3,6-Tetra-O-acetyl-α-d-glucopyranosyl-2,3,6-tetra-O-acetyl-α-d-glucopyranosyl-2,3,6-tetra-O-acetyl-α-d-glucopyranosyl-2,3,6-tetra-O-acetyl-α-d-glucopyranosyl-1,2,3,6-tetra-O-acetyl-α-d-glucopyranoside (2)

Acetate 1 (25.5 g, 13.9 mmol) was treated with NaOMe (0.75 g, 13.9 mmol) in MeOH at rt for 2 h. To the mixture was added acetic acid until pH 7, and the mixture was filtered through cotton. The filtrate was concentrated in vacuo, and the residual light-yellowish foam was used for the next step without further purification. To a suspension of NaOAc (6.85 g, 83.5 mmol) in Ac₂O (95.9 mL) was portionwise added the form at 110 °C and the reaction mixture was stirred at 120–130 °C for 1.5 h. TLC indicated complete conversion of the alcohol, and the mixture was poured into ice-cold water. After a while, CHCl₃ was added to the mixture, and the organic solution was partitioned. The organic layer was successively washed with ice-cold water, satd aq NaHCO₃, and brine, dried over anhyd MgSO₄, filtered, and evaporated in vacuo to yield the corresponding light-yellow foam, which was used for the next step without purification. The whole foam and 4-penten-1-ol (7.13 mL, 69.5 mmol) were dissolved in dichloromethane (DCM) (128 mL) at rt. Boron trifluoride-diethyl ether complex (BF₃·OEt₂) (17.6 mL, 0.139 mol) was dropwise added to the solution at 0 °C, and the solution was stirred for 2 h at rt under a N₂ atmosphere. The reaction mixture was diluted with CHCl₃, and the organic solution was successively washed with ice-cold water, satd aq NaHCO₃, and brine and dried over anhyd MgSO₄. The suspension was filtered on a pad of Celite, and the filtrate was concentrated in vacuo. The residual brownish syrup was subjected to a column of silica gel with 1:2 (v/v) n-hexane–EtOAc as the eluent to give light-yellowish foam 2 (9.48 g) in 36.7% yield: R_f 0.36 [1:3 (v/v) n-hexane–EtOAc]; [α]_D³¹ + 114.9° (c 1.00, CHCl₃); IR (KBr) 2960 (ν_C–H), 1751 (ν_C=O, ester), 1371 (ν_C–H), 1238 (ν_C–O, ester), 1035 (ν_C–O, ether) cm^–1; ¹H NMR (CDCl₃, 500 MHz): δ 5.85–5.74 (m, 1H, −CH=CH₂), 5.44–5.23 (m, 11H, H-1^II, H-1^III, H-1^IV, H-1^V, H-1^VI, H-3, H-3^II, H-3^III, H-3^IV, H-3^V, H-3^VI), 5.07 (t, 1H, J = 9.9 Hz, H-4^VI), 5.00 [d, 1H, J_trans = 19.6 Hz, –CH=CH₂(trans)], 4.96 [d, 1H, J_cis = 10.4 Hz, –CH=CH₂(cis)], 4.87–4.84 (m, 1H, H-2^VI), 4.81 (t, 1H, J = 7.8 Hz, H-2), 4.74–4.71 (m, 4H, H-2^II, H-2^III, H-2^IV, H-2^V), 4.51–4.46 (m, 6H, H-1, H-6a, H-6a^II, H-6a^III, H-6a^IV, H-6a^V), 4.38–4.15 (m, 6H, H-6b, H-6b^II, H-6b^III, H-6b^IV, H-6b^V, H-6b^VI), 4.06–3.84 (m, 13H, H-4, H-4^II, H-4^III, H-4^IV, H-4^V, H-5, H-5^II, H-5^III, H-5^IV, H-5^V, H-5^VI, H-6^VI, –OCH_aCH₂), 3.73–3.68 (m, 1H, H-5), 3.51–3.43 (m, 1H, –OCH_bCH₂), 2.20, 2.20, 2.19, 2.19, 2.15, 2.10, 2.10, 2.06, 2.03, 2.03, 2.02, 2.01, 2.01, 2.01, 2.00, 2.00, 1.99, 1.98, 1.98 (each s, 57H, 19 OAc), 2.08 (m, 2H, –CH₂CH=CH₂), 1.76–1.63 (m, 2H, –CH₂CH₂CH=CH₂); ¹³C NMR (CDCl₃, 125.8 MHz): δ 170.82, 170.77, 170.74, 170.63, 170.54, 170.46, 170.27, 169.84, 169.73, 169.63, 169.56, 137.93, 115.17, 100.36, 95.84, 95.74, 75.54, 74.01, 73.63, 73.46, 73.36, 72.40, 72.14, 71.85, 71.76, 70.60, 70.53, 70.15, 69.47, 69.32, 69.05, 68.56, 68.06, 63.12, 63.06, 62.65, 62.58, 62.54, 62.46, 62.28, 61.48, 29.95, 28.69, 20.96, 20.91, 20.77, 20.66; MALDI-TOF-MS calcd for [M + Na]⁺, 1879.580; found, m/z 1879.537, calcd for [M + K]⁺, 1895.554; found, m/z 1895.536.

Anal. Calcd for C₇₉H₁₀₈O₅₀: C, 51.077; H, 5.860; N, 0.000%. Found: C, 50.81; H, 5.73; N, 0.00%.

2.2.2. n-Pentenyl α-d-Glucopyranosyl-(1 → 4)-α-d-glucopyranosyl-α-d-glucopyranosyl-α-d-glucopyranosyl-α-d-glucopyranosyl-α-d-glucopyranoside (3)

Acetyl glycoside 2 (16.9 g, 9.09 mmol) was dissolved in MeOH (169 mL) at rt, and NaOMe (0.932 g, 17.3 mmol) was added to the methanolic solution at rt. The reaction mixture was allowed to react at rt for 2 h under a N₂ atmosphere, and acetic acid was added to the reaction mixture until a neutral condition was reached. The solution was passed through cotton, and the filtrate was concentrated to afford a light-yellowish foam (9.65 g) in quantitative yield; R_f 0.07 [65:25:4 (v/v/v) CHCl₃–MeOH–H₂O]; [α]_D³⁰ + 130.4° (c 1.00, MeOH); IR (KBr) 3399 (ν_O–H), 2926 (ν_C–H), 1637 (ν_C=C), 1363 (ν_C–H), 1026 (ν_C–O, ether) cm^–1; ¹H NMR (D₂O, 500 MHz): δ 5.87–5.79 (m, 1H, −CH=CH₂), 5.32–5.30 (m, 5H, H-1^II, H-1^III, H-1^IV, H-1^V, H-1^VI), 5.02 [d, 1H, J_trans = 17.2 Hz, –CH=CH₂(trans)], 4.94 [d, 1H, J_cis = 10.2 Hz, –CH=CH₂(cis)], 4.38 (d, 1H, J = 8.0 Hz, H-1), 3.89–3.49 (m, 36H, H-2^II, H-2^III, H-2^IV, H-2^V, H-2^VI, H-3, H-3^II, H-3^III, H-3^IV, H-3^V, H-3^VI, H-4, H-4^II, H-4^III, H-4^IV, H-4^V, H-5, H-5^II, H-5^III, H-5^IV, H-5^V, H-5^VI, H-6a, H-6b, H-6a^II, H-6b^II, H-6a^III, H-6b^III, H-6a^IV, H-6b^IV, H-6a^V, H-6b^V, H-6a^VI, H-6b^VI, –OCH_aCH₂), 3.33 (t, 1H, J = 9.6 Hz, H-4^VI), 3.21 (d, 1H, J = 8.0 Hz, J = 9.4 Hz, H-2), 2.08–2.04 (m, 2H, –CH₂CH=CH₂), 1.69–1.62 (m, 2H, –CH₂CH₂CH=CH₂); ¹³C NMR (D₂O, 125.8 MHz): δ 138.92, 114.95, 102.21, 99.91, 99.79, 99.77, 99.59, 77.07, 77.01, 76.99, 76.91, 76.36, 74.64, 73.48, 73.45, 73.16, 73.02, 72.85, 71.88, 71.69, 71.61, 71.32, 70.07, 69.46, 60.81, 60.62, 60.60, 60.54, 29.87, 29.48, 28.14; MALDI-TOF-MS calcd for [M + Na]⁺, 1081.379; found, m/z 1081.421.

Anal. Calcd for C₄₁H₇₀O₃₁·2H₂O: C, 44.971; H, 6.812; N, 0.000%. Found: C, 44.91; H, 6.81; N, 0.00%.

2.2.3. n-Pentenyl 2,3-Di-O-acetyl-4,6-O-(naphthylmethylidene)-α-d-glucopyranosyl-(1 → 4)-2,3,6-Tetra-O-acetyl-α-d-glucopyranosyl-2,3,6-tetra-O-acetyl-α-d-glucopyranosyl-2,3,6-tetra-O-acetyl-α-d-glucopyranosyl-2,3,6-tetra-O-acetyl-α-d-glucopyranosyl-1,2,3,6-tetra-O-acetyl-α-d-glucopyranoside (4)

To a solution of polyol 3 (9.63 g, 9.09 mmol) in DMF (96.3 mL) was added 2-naphthaldehyde di-i-propyl acetal¹² (1.5 equiv molar for 3) and camphor sulfonic acid (CSA) (1.69 g, 7.28 mmol) at rt, and the reaction mixture was stirred at 55 °C under reduced pressure for 2 h. In order to neutralize the reaction mixture, Et₃N was added to the mixture at 0 °C and the mixture was concentrated in vacuo. The residue was treated with Ac₂O (144.5 mL, 1.53 mol) in Pyr (144.5 mL) at rt for 3 d. The reaction mixture was evaporated in vacuo, and the residue was poured into ice-cold water. After a while, the mixture was extracted with CHCl₃, successively washed with 1 M aq H₂SO₄, ice-cold water, satd aq NaHCO₃, and brine, dried over anhyd MgSO_4, filtered on a pad of Celite, and concentrated in vacuo. Chromatographic purification of the brownish syrup on a column of silica gel with 1:2 (v/v) n-hexane–EtOAc as the eluent yielded a light-yellowish foam 4 (6.12 g, 35.3% yield): R_f 0.29 [1:2 (v/v) n-hexane–EtOAc]; [α]_D³⁰ + 103.7° (c 1.00, CHCl₃); IR (KBr) 2960 (ν_C–H), 1751 (ν_C=O, ester), 1371 (ν_C–H), 1236 (ν_C–O, ester), 1033 (ν_C–O, ether) cm^–1; ¹H NMR (CDCl₃, 500 MHz): δ 7.90–7.82 (m, 4H, naphthyl), 7.54–7.47 (m, 3H, naphthyl), 5.82–5.74 (m, 1H, −CH₂CH=CH₂), 5.63 (s, 1H, naphthyl–CHO₂-), 5.49 (t, 1H, J = 9.9 Hz, H-3^VI), 5.42–5.38 (m, 5H, H-1^VI, H-3^II, H-3^III, H-3^IV, H-3^V), 5.29–5.22 (m, 5H, H-1^II, H-1^III, H-1^IV, H-1^VI, H-3), 5.01 [d, 1H, J_trans = 18.1 Hz, –CH=CH₂(trans)], 4.96 [d, 1H, J_cis = 10.5 Hz, –CH=CH₂(cis)], 4.90–4.88 (m, 1H, H-2^VI), 4.80 (t, 1H, J = 7.3 Hz, H-2), 4.77–4.72 (m, 4H, H-2^II, H-2^III, H-2^IV, H-2^V), 4.61–4.47 (m, 6H, H-1, H-6a^II, H-6a^III, H-6a^IV, H-6a^V, H-6a^VI), 4.39–4.20 (m, 6H, H-6a, H-6b^II, H-6b^III, H-6b^IV, H-6b^V, H-6b^VI), 4.05–3.84 (m, 11H, H-4, H-4^II, H-4^III, H-4^IV, H-4^V, H-5^II, H-5^III, H-5^IV, H-5^V, H-5^VI, –OCH_aCH₂), 3.78 (t, 1H, J = 10.3 Hz, H-6b), 3.70–3.66 (m, 2H, H-4^VI, H-5), 3.51–3.43 (m, 1H, –OCH_bCH₂), 2.21, 2.20, 2.20, 2.19, 2.17, 2.16, 2.14, 2.13, 2.09, 2.08, 2.04, 2.02, 2.02, 2.01, 2.01, 1.98, 1.98 (each s, 51H, 17 OAc), 2.08 (m, 2H, –CH₂CH=CH₂), 1.70–1.61 (m, 2H, –CH₂CH₂CH=CH₂); ¹³C NMR (CDCl₃, 125.8 MHz): δ 171.05, 170.89, 170.85, 170.54, 170.52, 170.48, 170.33, 170.29, 169.75, 169.64, 169.56, 137.94, 134.15, 133.82, 132.92, 128.53, 128.21, 127.78, 126.56, 126.24, 125.92, 123.89, 115.18, 102.06, 100.35, 96.49, 95.83, 95.80, 95.72, 79.06, 75.56, 73.98, 73.54, 73.27, 73.04, 72.43, 72.20, 72.16, 72.02, 71.87, 71.80, 70.99, 70.63, 70.56, 69.31, 69.12, 69.04, 68.73, 68.53, 63.87, 63.10, 62.61, 62.45, 62.25, 62.13, 29.96, 28.69, 21.02, 20.98, 20.90, 20.86, 20.76, 20.66; MALDI-TOF-MS calcd for [M + Na]⁺, 1933.606; found, m/z 1933.580, calcd for [M + K]⁺, 1949.580; found, m/z 1949.564.

Anal. Calcd for C₈₆H₁₁₀O₄₈·2H₂O: C, 53.030; H, 5.899; N, 0.000%. Found: C, 52.91; H, 5.57; N, 0.00%.

2.2.4. n-Pentenyl 2,3-Di-O-acetyl-6-O-naphthylmethyl-α-d-glucopyranosyl-(1 → 4)-2,3,6-Tetra-O-acetyl-α-d-glucopyranosyl-2,3,6-tetra-O-acetyl-α-d-glucopyranosyl-2,3,6-tetra-O-acetyl-α-d-glucopyranosyl-2,3,6-tetra-O-acetyl-α-d-glucopyranosyl-1,2,3,6-tetra-O-acetyl-α-d-glucopyranoside (5)

A suspension of cyclic acetal 4 (6.00 g, 3.14 mmol) and molecular sieves 4A (6.0 g) in THF (60 mL) was stirred at rt under an Ar atmosphere for 30 min. To the suspension was added borane–trimethylamine complex (Me₃N·BH₃) (1.60 g, 21.9 mmol) at rt under an Ar atmosphere, and the stirring was continued for 30 min at rt. AlCl₃ (2.93 g, 22.0 mmol) was added to the mixture at 0 °C under an Ar atmosphere, and the mixture was allowed to react at rt for 4 h under an Ar atmosphere. Since TLC showed complete consumption of 4, the suspension was filtered. The filtrate was diluted with CHCl₃, and the organic solution was successively washed with 1 M aq HCl, ice-cold water, satd aq NaHCO₃, and brine, dried over anhyd MgSO_4, filtered on a pad of Celite, and concentrated in vacuo to give white foam, which was chromatographed on silica gel with 1:3 (v/v) n-hexane–EtOAc as the eluent to afford a white foam 5 (3.69 g) in 61.4% yield: R_f 0.13 [1:2 (v/v) n-hexane–EtOAc]; [α]_D³³ + 113.0° (c 1.00, CHCl₃); IR (KBr) 3502 (ν_O–H), 2951 (ν_C–H), 1751 (ν_C=O, ester), 1371 (ν_C–H), 1240 (ν_C–O, ester), 1033 (ν_C–O, ether) cm^–1; ¹H NMR (CDCl₃, 500 MHz): δ 7.85–7.78 (m, 4H, naphthyl), 7.50–7.45 (m, 3H, naphthyl), 5.83–5.74 (m, 1H, −CH=CH₂), 5.42–5.36 (m, 5H, H-1^VI, H-3^III, H-3^IV, H-3^V, H-3^VI), 5.29–5.23 (m, 5H, H-1^II, H-1^III, H-1^IV, H-1^V, H-3^II),5.20 (t, 1H, J = 9.5 Hz, H-3), 5.01 [d, 1H, J_trans = 17.2 Hz, –CH=CH₂(trans)], 4.97 [d, 1H, J_cis = 10.3 Hz, –CH=CH₂(cis)], 4.84–4.70 (m, 8H, H-2, H-2^II, H-2^III, H-2^IV, H-2^V, H-2^VI, naphthyl–CH₂O−), 4.51 (d, 1H, J = 8.2 Hz, H-1), 4.49–4.46 (m, 5H, H-6a^II, H-6a^III, H-6a^IV, H-6a^V, H-6a^VI), 4.37 (dd, 1H, J = 3.8 Hz, J = 12.4 Hz, H-6b^V), 4.32–4.26 (m, 2H, H-6b^III, H-6b^IV), 4.19 (dd, 1H, J = 3.0 Hz, J = 12.4 Hz, H-6b^II), 4.15 (dd, 1H, J = 3.2 Hz, J = 12.4 Hz, H-6b^VI), 4.06–3.67 (m, 15H, H-4, H-4^II, H-4^III, H-4^IV, H-4^V, H-4^VI, H-5, H-5^II, H-5^III, H-5^IV, H-5^V, H-5^VI, H-6a, H-6b, –OCH_aCH₂), 3.51–3.47 (m, 1H, –OCH_bCH₂), 2.90–2.89 (m, 1H, –OH), 2.20, 2.20, 2.19, 2.16, 2,09, 2.06, 2.04, 2.02, 2.01, 2.01, 1.99, 1.99, 1.97, 1.98, 1.98, 1.97, 1.90 (each s, 51H, 17 OAc), 2.08 (m, 2H, –CH₂CH=CH₂), 1.71–1.62 (m, 2H, –CH₂CH₂CH=CH₂); ¹³C NMR (CDCl₃, 125.8 MHz): δ 171.30, 170.82, 170.75, 170.69, 170.63, 170.47, 170.40, 170.36, 170.17, 169.65, 169.63, 169.51, 169.45, 137.82, 135.05, 133.23, 133.05, 128.34, 127.89, 127.70, 126.19, 125.99, 125.62, 115.07, 100.24, 95.82, 95.72, 95.60, 75.44, 73.93, 73.89, 73.50, 73.34, 72.96, 72.35, 72.31, 72.04, 72.02, 71.97, 71.66, 70.50, 70.42, 70.01, 69.80, 69.20, 69.10, 68.97, 68.92, 68.80, 63.00, 62.53, 62.50, 62.49, 62.44, 62.35, 62.32, 29.84, 28.58, 20.90, 20.87, 20.80, 20.65, 20.55;

MALDI-TOF-MS calcd for [M + Na]⁺, 1935.622; found, 1935.522, calcd for [M + K]⁺, 1951.595; found, 1951.495.

Anal. Calcd for C₈₆H₁₁₂O₄₈·H₂O: C, 53.469; H, 5.943; N, 0.000%. Found: C, 53.62; H, 5.87; N, 0.00%.

2.2.5. n-Pentenyl 6-O-Naphthylmethyl-α-d-glucopyranosyl-(1 → 4)-α-d-Glucopyranosyl-α-d-glucopyranosyl-α-d-glucopyranosyl-α-d-glucopyranosyl-α-d-glucopyranoside (6)

NaOMe (0.153 g, 2.83 mmol) was added to a solution of acetate 5 (3.20 g, 1.67 mmol) in MeOH (32 mL) at rt under a N₂ atmosphere, and the reaction mixture was stirred for 2 h under a N₂ atmosphere. The mixture was neutralized by using IRA 120B (H⁺), and the suspension was passed through a cotton filter. The filtrate was evaporated in vacuo to give the corresponding polyol 6 (2.42 g) in quantitative yield: R_f 0.10 [65:25:4 (v/v/v) CHCl₃–MeOH–H₂O]; [α]_D³⁰ + 107.8° (c 1.00, MeOH); IR (KBr) 3381 (ν_O–H), 2927 (ν_C–H), 1026 (ν_C–O, ether) cm^–1; ¹H NMR (CD₃OD, 500 MHz): δ 7.89–7.85 (m, 4H, naphthyl), 7.53–7.46 (m, 3H, naphthyl), 5.90–5.81 (m, 1H, −CH₂CH=CH₂), 5.21–5.17 (m, 5H, H-1^II, H-1^III, H-1^IV, H-1^V, H-1^VI), 5.04 [d, 1H, J_trans = 17.2 Hz, –CH=CH₂(trans)], 4.95 [d, 1H, J_cis = 10.1 Hz, –CH=CH₂(cis)], 4.77 (m, 2H, naphthyl–CH₂O−), 4.28 (d, 1H, J = 7.8 Hz, H-1), 3.92–3.48 (m, 35H, H-2^II, H-2^III, H-2^IV, H-2^V, H-2^VI, H-3, H-3^II, H-3^III, H-3^IV, H-3^V, H-3^VI, H-4, H-4^II, H-4^III, H-4^IV, H-4^V, H-5^II, H-5^III, H-5^IV, H-5^V, H-5^VI, H-6a, H-6b, H-6a^II, H-6b^II, H-6a^III, H-6b^III, H-6a^IV, H-6b^IV, H-6a^V, H-6b^V, H-6a^VI, H-6b^VI, –OCH₂CH₂), 3.38–3.32 (m, 2H, H-4^VI, H-5), 3.25 (dd, 1H, J = 8.0 Hz, J = 9.1 Hz, H-2), 2.18–2.14 (m, 2H, –CH₂CH=CH₂), 1.76–1.69 (m, 2H, –CH₂CH₂CH=CH₂); ¹³C NMR (CD₃OD, 125.8 MHz): δ 139.48, 137.09, 134.76, 134.46, 129.06, 128.94, 128.66, 127.44, 127.10, 126.89, 126.86, 115.20, 104.27, 102.75, 102.66, 102.62, 102.56, 81.47, 81.36, 81.26, 81.23, 81.13, 77.79, 76.50, 75.15, 74.91, 74.84, 74.67, 74.57, 74.16, 73.84, 73.80, 73.74, 73.71, 73.32, 73.29, 71.65, 71.04, 70.25, 62.31, 62.21, 62.16, 49.85, 31.21, 30.07; MALDI-TOF calcd for [M + Na]⁺, 1221.442; found, 1221.458, calcd for [M + K]⁺, 1237.416; found, 1237.446.

Anal. Calcd for C₅₂H₇₈O₃₁·1.5H₂O: C, 50.935; H, 6.658; N, 0.000%. Found: C, 50.97; H, 6.61; N, 0.00%.

2.2.6. 5-(2-Aminoethylthio)-pentyl 6-O-Naphthylmethyl-α-d-glucopyranosyl-(1 → 4)-α-d-Glucopyranosyl-α-d-glucopyranosyl-α-d-glucopyranosyl-α-d-glucopyranosyl-α-d-glucopyranoside Hydrochloride Salt (7)

A solution of olefin 6 (1.98 g, 1.64 mmol) and 2-aminoethanethiol hydrochloride (1.86 g, 16.4 mmol) in MeOH (3.96 mL) and H₂O (3.96 mL) was UV-irradiated at rt for 2 h under an Ar atmosphere. When TLC showed complete conversion of 6, the reaction mixture was directly applied to a column of Sephadex LH-20 with MeOH as the eluent. The fractions giving a positive ninhydrin test were stored and concentrated in vacuo to provide the corresponding amine salt 7 (2.12 g) in 98.1% yield: R_f 0.52 [3:3:1 (v/v/v) CHCl₃–MeOH–H₂O]; IR (KBr) 3396 (ν_O–H), 2928 (ν_C–H), 1647 (ν_N–H), 1026 (ν_C–O, ether) cm^–1; ¹H NMR (D₂O, 500 MHz): δ 7.79–7.70 (m, 4H, naphthyl), 7.43–7.38 (m, 3H, naphthyl), 5.24–5.11 (m, 5H, H-1^II, H-1^III, H-1^IV, H-1^V, H-1^VI), 4.65–4.60 (m, 2H, naphthyl–CH₂O−), 4.15 (d, 1H, J = 8.0 Hz, H-1), 3.92–3.39 (m, 35H, H-2^II, H-2^III, H-2^IV, H-2^V, H-2^VI, H-3, H-3^II, H-3^III, H-3^IV, H-3^V, H-3^VI, H-4, H-4^II, H-4^III, H-4^IV, H-4^V, H-5^II, H-5^III, H-5^IV, H-5^V, H-5^VI, H-6a, H-6b, H-6a^II, H-6b^II, H-6a^III, H-6b^III, H-6a^IV, H-6b^IV, H-6a^V, H-6b^V, H-6a^VI, H-6b^VI, –OCH₂CH₂), 3.31–3.24 (m, 2H, H-4^VI, H-5), 3.16–3.13 (m, 1H, H-2), 3.10 (t, 2H, J = 6.7 Hz, –SCH₂CH₂N–), 2.74 (t, 2H, J = 6.7 Hz, –SCH₂CH₂N–), 2.47 (t, 2H, J = 7.4 Hz, −CH₂CH₂SCH₂−), 1.51–1.41 (m, 4H, –OCH₂CH₂CH₂CH₂CH₂S–), 1.40–1.25 (m, 2H, –CH₂CH₂CH₂S–); ¹³C NMR (D₂O, 125.8 MHz): δ 135.30, 133.10, 132.94, 128.39, 128.01, 127.82, 126.64, 126.26, 126.50, 126.26, 102.21, 100.37, 100.34, 100.01, 100.00, 99.66, 78.14, 78.06, 77.85, 77.74, 77.48, 76.25, 74.50, 73.51, 73.43, 73.32, 73.15, 72.93, 71.89, 71.83, 71.80, 71.71, 71.57, 71.49, 71.41, 71.25, 71.10, 70.48, 69.90, 69.27, 60.58, 60.48, 60.41, 38.48, 30.71, 28.47, 28.27, 24.45; MALDI-TOF-MS calcd for [M + H]⁺, 1276.490; found, 1276.476, calcd for [M + Na]⁺, 1298.472; found, 1298.480, calcd for [M + K]⁺, 1314.446; found, 1314.467.

2.2.7. 5-(2-Acrylamidoethylthio)-pentyl 2,3-Di-O-acetyl-6-O-naphthylmethyl-α-d-glucopyranosyl-(1 → 4)-2,3,6-Tetra-O-acetyl-α-d-glucopyranosyl-2,3,6-tetra-O-acetyl-α-d-glucopyranosyl-2,3,6-tetra-O-acetyl-α-d-glucopyranosyl-2,3,6-tetra-O-acetyl-α-d-glucopyranosyl-1,2,3,6-tetra-O-acetyl-α-d-glucopyranoside (8)

To a solution of amine 7 (2.00 g, 1.52 mmol) and Et₃N (0.635 mL, 4.56 mmol) in MeOH (20 mL) was dropwise added acryloyl chloride (147 μL, 1.82 mmol) with stirring at 0 °C under an Ar atmosphere, and the reaction mixture was further stirred at rt under an Ar atmosphere for 1 h. The reactant was concentrated in vacuo, followed by acetylation in Ac₂O (30 mL) and Pyr (30 mL) at rt for 3 d. After concentration of the reaction mixture in vacuo, the resulting mixture was poured into ice-cold water. After a while, CHCl₃ was added to the mixture, followed by extraction and partitioning of each layer, and the organic solution was successively washed with 1 M aq H₂SO₄, ice-cold water, satd aq NaHCO₃, and brine, dried over anhyd MgSO_4, filtered, and concentrated at diminished pressure. Chromatographic purification of the residue using a silica gel column with 1:4 (v/v) CHCl₃–EtOAc as the eluent gave the corresponding acrylamide acetate 8 (2.26 g) in 71.3% yield: R_f 0.32 [1:4 (v/v) CHCl₃–EtOAc]; [α]_D³² + 120.0° (c 1.00, CHCl₃); IR (KBr) 2947 (ν_C–H), 1747 (ν_C=O, ester), 1676 (ν_C=O, amide I), 1630 (ν_C=C), 1521 (δ_N–H, amide II), 1371 (ν_C–H), 1240 (ν_C–O, ester), 1035 (ν_C–O, ether) cm^–1; ¹H NMR (CDCl₃, 500 MHz): δ 7.85–7.75 (m, 4H, naphthyl), 7.49–7.43 (m, 3H, naphthyl), 6.31–6.27 [m, 1H, CH=CH₂ (trans)], 6.12 (dd, 1H, J_trans = 16.8 Hz, J_cis = 10.2 Hz, −CH=CH₂), 6.12 (m, 1H, –NHCO−), 5.66–5.64 [m, 1H, –CH=CH₂ (cis)], 5.45 (d, 1H, J = 3.8 Hz, H-1^IV), 5.43–5.19 (m, 11H, H-1^II, H-1^III, H-1^IV, H-1^V, H-3, H-3^II, H-3^III, H-3^IV, H-3^V, H-3^VI, H-4^VI), 4.87 (dd, 1H, J = 3.8 Hz, 10.5 Hz, H-2^VI), 4.79 (t, 1H, J = 8.4 Hz, H-2), 4.76–4.46 (m, 6H, H-2^II, H-2^III, H-2^IV, H-2^V, naphthyl–CH₂O−), 4.51 (d, 1H, J = 7.4 Hz, H-1), 4.49–4.46 (m, 5H, H-6a, H-6a^II, H-6a^III, H-6a^IV, H-6a^V), 4.38 (dd, 1H, J = 3.2 Hz, J = 12.5 Hz, H-6b^V), 4.31–4.26 (m, 2H, H-6b^III, H-6b^IV), 4.21–4.19 (m, 1H, H-6b^II), 4.16–4.14 (m, 1H, H-6b), 4.04–3.82 (m, 11H, H-4, H-4^II, H-4^III, H-4^IV, H-4^V, H-5^II, H-5^III, H-5^IV, H-5^V, H-5^VI, –OCHaCH₂CH₂CH₂CH₂S–), 3.73–3.68 (m, 1H, H-5), 3.55–3.44 (m, 5H, H-6a^VI, H-6b^VI, –OCHbCH₂CH₂CH₂CH₂S–, –SCH₂CH₂N–), 2.69 (t, 2H, J = 6.2 Hz, –SCH₂CH₂N–), 2.52 (t, 2H, J = 7.3 Hz, −CH₂CH₂SCH₂CH₂N–), 2.20, 2.20, 2.20, 2.17, 2.05, 2.02, 2.02, 2.02, 2.01, 2.01, 2.01, 1.99, 1.99, 1.98, 1.98, 1.98, 1.98, 1.81 (each s, 54H, 18 OAc), 1.78–1.54 (m, 4H, –OCH₂CH₂CH₂CH₂CH₂S–), 1.50–1.36 (m, 2H, –OCH₂CH₂CH₂CH₂CH₂S–); ¹³C NMR (CDCl₃, 125.8 MHz): δ 170.68, 170.57, 170.41, 170.35, 170.30, 170.10, 169.87, 169.69, 169.58, 169.53, 169.47, 169.38, 165.43, 134.80, 133.13, 133.00, 130.71, 128.21, 127.86, 127.60, 126.80, 126.53, 126.09, 125.93, 125.86, 100.17, 95.66, 95.66, 95.55, 95.54, 95.53, 75.33, 73.80, 73.72, 73.39, 73.22, 73.12, 73.12, 72.24, 72.01, 71.99, 71.77, 71.64, 71.62, 71.61, 71.58, 70.44, 70.37, 70.01, 69.68, 69.58, 69.51, 68.91, 68.86, 68.44, 67.40, 62.99, 62.91, 62.44, 62.37, 62.27, 62.24, 38.39, 31.37, 31.59, 29.12, 28.89, 25.02, 20.90, 20.83, 20.82, 20.80, 20.78, 20.64, 20.58, 20.56, 20.50, 20.48; MALDI-TOF-MS calcd [M + Na]⁺, 2108.673; found, 2108.555, calcd for [M + K]⁺, 2124.647; found, 2124.545.

Anal. Calcd for C₉₃H₁₂₃O₅₀NS: C, 53.521; H, 5.940; N, 0.671%. Found: C, 53.28; H, 5.91; N, 0.60%.

2.2.8. 5-(2-Acrylamidoethylthio)-pentyl 6-O-Naphthylmethyl-α-d-glucopyranosyl-(1 → 4)-α-d-Glucopyranosyl-α-d-glucopyranosyl-α-d-glucopyranosyl-α-d-glucopyranosyl-α-d-glucopyranoside (9)

Sodium methoxide (85.2 mg, 1.58 mmol) was added to a solution of acetate 8 (1.83 g, 0.876 mmol) in MeOH (18.3 mL) at rt under a N₂ atmosphere, and the reaction mixture was further stirred at rt for 2 h. To the mixture was added IRA 120B (H⁺) for neutralization, and the suspension was filtered on a pad of cotton, followed by concentration to give the desired monomer 9 (1.17 g) in quantitative yield: R_f 0.09 [65:25:4 (v/v/v) CHCl₃–MeOH–H₂O]; [α]_D²⁹ + 90.2° (c 1.00, MeOH); IR (KBr) 3404 (ν_O–H), 2926 (ν_C–H), 1658 (ν_C=O, amide I), 1624 (ν_C=C), 1550 (δ_N–H, amide II), 1028 (ν_C–O, ether) cm^–1; ¹H NMR (CD₃OD, 500 MHz): δ 7.89–7.85 (m, 4H, naphthyl), 7.53–7.47 (m, 3H, naphthyl), 6.29–6.21 (m, 2H, –CH=CH₂), 5.67 (dd, 1H, J = 4.0 Hz, J = 8.1 Hz, −CH=CH₂), 5.20–5.16 (m, 5H, H-1^II, H-1^III, H-1^IV, H-1^V, H-1^VI), 4.77 (m, 2H, naphthyl–CH₂O−), 4.29 (d, 1H, J = 7.8 Hz, H-1), 3.92–3.77 (m, 20H, H-3^II, H-3^III, H-3^IV, H-3^V, H-5^II, H-5^III, H-5^IV, H-5^V, H-6a, H-6a^II, H-6b^II, H-6a^III, H-6b^III, H-6a^IV, H-6b^IV, H-6a^V, H-6b^V, H-6a^VI, H-6b^VI, –OCHaCH₂CH₂−), 3.72 (dd, 1H, J = 6.1 Hz, J = 10.7 Hz, H-6b), 3.67–3.62 (m, 2H, H-3, H-3^VI), 3.57–3.47 (m, 13H, H-2, H-2^II, H-2^III, H-2^IV, H-2^V, H-2^VI, H-4, H-4^II, H-4^III, H-4^IV, H-4^V, H-5^VI, –OCHbCH₂CH₂−), 3.43 (t, 2H, J = 6.9 Hz, –SCH₂CH₂N–), 3.39–3.31 (m, 2H, H-4^VI, H-5), 3.24 (dd, 1H, J = 7.8 Hz, J = 9.3 Hz, H-2), 2.66 (t, 2H, J = 7.2 Hz, –SCH₂CH₂N–), 2.56 (t, 2H, J = 7.3 Hz, −CH₂CH₂SCH₂−), 1.67–1.59 (m, 4H, –OCH₂CH₂CH₂CH₂CH₂S–), 1.52–1.47 (m, 2H, –CH₂CH₂CH₂S–); ¹³C NMR (CD₃OD, 125.8 MHz): δ 168.15, 137.12, 134.79, 134.48, 131.99, 129.08, 128.96, 128.68, 127.46, 127.12, 126.91, 126.88, 126.84, 104.27, 102.77, 102.71, 102.68, 102.59, 81.51, 81.41, 81.34, 81.32, 81.17, 77.84, 76.54, 75.17, 74.93, 74.86, 74.70, 74.59, 74.20, 73.88, 73.84, 73.77, 73.73, 73.35, 71.68, 71.07, 70.70, 62.33, 62.25, 62.18, 62.17, 40.39, 32.60, 31.94, 30.52, 30.30, 26.27; MALDI-TOF-MS calcd for [M + Na]⁺, 1352.482; found, 1352.493, calcd for [M + K]⁺, 1368.456; found, 1368.472.

Anal. Calcd for C₅₇H₈₇O₃₂NS·2H₂O: C, 51.461; H, 6.592; N, 1.053%. Found: C, 50.36; H, 6.61; N, 0.93%.

2.2.9. Synthesis of a FRET-Sensitive Polymer (11a)

A solution of a fluorogenic carbohydrate monomer 9 (75.0 mg, 56.3 μmol) and an acrylamide AAm (40.0 mg, 563 μmol) in deionized water (1.00 mL) was deaerated under reduced pressure for 30 min and then Ar gas was added. To the solution was added a solution of dansyl monomer 10(11) (2.0 mg, 5.6 μmol) in MeOH (30 μL) and the solution was deaerated again under reduced pressure for 10 min, and then Ar gas replacement was conducted. N,N,N′,N′-tetramethylethylenediamine (TEMED) (1.7 μL, 11 μmol) and ammonium persulfate (APS) (1.3 mg, 5.6 μmol) were added under an Ar atmosphere at rt. The mixture was stirred at rt for 24 h and diluted with 0.1 M aq Pyr—AcOH buffer (pH 5.2). The viscous solution was dialyzed against distilled water, followed by lyophilization to provide the corresponding white powdery glycopolymer 11a (104 mg) in 88.7% yield.

2.2.10. Synthesis of a FRET-Sensitive Polymer (11b)

Monomer 9 (24.95 mg, 18.8 μmol), AAm (13.75 mg, 193 μmol), and a solution of dansyl monomer 10(11) (0.653 mg, 1.88 μmol) in MeOH (30.6 μL) were used in deionized water (330 μL) for the polymerization reaction under an Ar atmosphere. TEMED (0.564 μL, 3.76 μmol) and a solution of APS (0429 mg, 1.88 μmol) in deionized water (44 μL) were used, and the same workup procedure as that described for 11a was applied to provide the corresponding white powdery glycopolymer 11b (31 mg) in 79% yield.

2.2.11. Synthetic assembly of the IPN Involving a Water-Soluble Glycopolymer

A solution of the FRET-sensitive polymer 11b (7.39 mg) and a 30w/v % AAm/bis-AAm mixed solution (251.2 μL) in deionized water (100 μL) was deaerated under reduced pressure for 15 min, and then Ar gas was added. TEMED (0.444 μL, 2.96 μmol) and a solution of APS (0.344 mg, 1.28 μmol) in deionized water (20 μL) were added to the solution under an Ar atmosphere at rt. The mixture was deaerated again under reduced pressure for 5 min and then Ar gas was added. The gel-like mixture was stored at rt on and diluted with 0.1 M aq Pyr—AcOH buffer (pH 5.2). The gel was directly dialyzed against distilled water, followed by lyophilization to provide the corresponding colorless xerogel.

2.2.12. Size Exclusion Chromatography Method

The weight-average molecular weight () and number-average molecular weight () were estimated by the following conditions. Size exclusion chromatography (SEC) was performed using a CLASS-VP system (Shimadzu Corp., Tokyo, Japan) equipped with a Shodex Asahipak GF510HQ column (0.46 mm × 250 mm, Shoko, Co., ltd., Tokyo, Japan) at 40 °C. The column was equilibrated with 3:2 (v/v) DMF–H₂O solution and run at a flow rate of 0.300 mL/min. For detection of the sample by SEC, a differential refractometer (Waters Corp., MA, USA) was used. Calibration curves were obtained using pullulan standards (5.9, 9.6, 21.1, 47.1, 117, 200, and 344 kDa; Shodex P-82).

2.3. Biological Evaluation

2.3.1. Continuous Assay of α-Amylase with FRET-Sensitive Polymer 11a by Intramolecular Fluorescence Quenching

Measurement of fluorescence emission spectra carried out at 37 °C, in a Teflon-stoppered cuvette (12.5 mm wide × 45 mm high) containing a 3.0 mL sample. The slit width of the excitation and the emission was 5.0 nm, and the scan speed was high. The sensitivity was high, and the interval of sampling was 1.0 nm. Emission spectra of FRET-sensitive polymer 11a by excitation at 290 nm were uncorrected and were recorded with a Shimadzu RF-5300PC fluorescence spectrophotometer (SHIMADZU CORPORATION, Kyoto, Japan). An enzymatic reaction is carried out at 37 ± 0.1 °C as follows: appropriate amounts of FRET-sensitive polymer 11a in 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, 50 mM NaCl, 10 mM CaCl₂, and 0.01% NaN₃, pH 7.0 were added in a Teflon-stoppered cuvette, and an appropriate amount of human salivary α-amylase was added and quickly mixed by inversion. The initial spectrum as 0 min was taken immediately after the enzyme was added. The quantification of FRET-sensitive polymer 11a by measurements of naphthyl emission and dansyl emission was estimated by subtracting the naphthyl emission (333 nm) or the dansyl emission (515 nm) from the original fluorescence intensity, respectively.

2.3.2. Test of Exudation of the Water-Soluble FRET-Sensitive Polymer 11b from the IPN into an Aqueous Solvent

Dried IPN xerogel including the water-soluble FRET-sensitive polymer 11b (3.13 mg) was added to a Teflon-stoppered cuvette. Ten mM HEPES containing 50 mM NaCl, 10 mM KCl, and 0.02% NaN₃, pH 7.0 (3.000 mL) was added to the cuvette, and the cuvette was quickly mixed by inversion. The cuvette was placed on a fluorospectrometric meter and left for 30 min at 37 °C. The initial spectrum as 0 min was then taken.

2.3.3. Hydrolysis of the IPN Including the FRET-Sensitive Polymer 11b with α-Amylase from Bacillus subtilis

Dried IPN xerogel including the water-soluble FRET-sensitive polymer 11b (3.13 mg) was added to a Teflon-stoppered cuvette. Ten mM HEPES containing 50 mM NaCl, 10 mM KCl, and 0.02% NaN₃, pH 7.0 (2.997 mL) was added to the cuvette, and the cuvette was quickly mixed by inversion. The cuvette was placed on a fluorospectrometric meter and left for 30 min at 37 °C. To the cuvette was added 2.65 μL (1.5 U) of a stock solution of α-amylase from Bacillus subtilis (0.567 U/μL), and the cuvette was quickly mixed 5 times by inversion. The initial spectrum as 0 min was obtained immediately after the enzyme had been added.

2.3.4. Hydrolysis of the IPN including the FRET-Sensitive Polymer 11b with α-Amylase from a Porcine Pancreas

Dried IPN xerogel including the water-soluble FRET-sensitive polymer 11b (2.90 mg) was added to a Teflon-stoppered cuvette. Ten mM HEPES containing 50 mM NaCl, 10 mM KCl, and 0.02% NaN₃, pH 7.0 (2.997 mL) was added to the cuvette, and the cuvette was quickly mixed by inversion. The cuvette was placed on a fluorospectrometric meter and left for 30 min at 37 °C. To the cuvette was added 2.86 μL (1.5 U) of a stock solution of α-amylase from a porcine pancreas (0.525 U/μL), and the cuvette was quickly mixed 5 times by inversion. The initial spectrum as 0 min was obtained immediately after the enzyme had been added.

3. Results and Discussion

A water-soluble FRET-sensitive polymer was initially prepared from a naphthyl methylated maltohexaosyl monomer 9, a dansylated monomer 10(11), and AAm. A fluorogenic carbohydrate monomer 9 was synthesized from fully acetylated maltohexaoside 1(10) as shown in Scheme 1. Thus, a known acetate 1(10) was glycosylated with an n-pentenyl alcohol in the presence of a Lewis acid to give the corresponding β-maltohexaoside 2 in 36.7% yield, which underwent quantitative transesterification¹³ to afford the polyol 3. The polyol 3 was treated with a known 2-naphthaldehyde di-i-propyl acetal¹² in the presence of CSA followed by complete acetylation to afford the cyclic acetalized glycoside 4 with 2-naphthylmetyliden at the 4 and 6 positions on the nonreducing glucose residue¹⁴ in 35.3% yield in 2 steps. The reductive ring opening reaction of 4 gave the corresponding mono alcohol 5 with a naphthylmethyl group at the nonreducing terminal glucose in 61.4% yield. Further chemical modification was performed to the alkenyl glycoside 5 by quantitative transesterification to yield polyol 6, which was allowed to react with 2-aminoethanthiol in a radical manner to give the amine hydrochloride 7 in 98.1% yield. Acryloylation of 7 provided acrylamide 8 after temporal acetylation in 71.3% yield. Acetyl protections in 8 were removed by the Zemplén manner¹³ to afford the desired water-soluble fluorogenic glycomonomer 9 in quantitative yield.

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.

On the other hand, a known dansylated monomer 10 was prepared by the previously reported method.¹¹ In brief, ethylene diamine as a starting material was directly dansylated for one of the amino groups and the remaining amino group was condensed with acrylic chloride to give the desired dansyl monomer 10.

Since the preparation of fluorogenic monomers was done, our attention was turned toward the polymerization of both monomers with AAm. Thus, the carbohydrate monomer 9 and 0.1 mol equiv of dansyl monomer 10 were polymerized with 10 M equivalents of AAm in the presence of APS and TEMED as a radical initiator system in water to yield a FRET-sensitive glycopolymers 11a and 11b as white powder after lyophilization as shown in Scheme 2. Structural characterization was performed by a combination of ¹H NMR spectroscopy and infrared spectroscopy (IR), and the polymer composition in glycopolymer 11a was estimated by the results of ¹H NMR spectroscopy. The weight-average molecular weight () and number-average molecular weight () of 11a were determined to be : 208 kDa and : 158 kDa by means of SEC, and the polydispersity (/ ) was calculated to be 1.31. In the case of 11b, SEC measurement could not be conducted because of the higher molecular weight than the exclusion limit of the column used in this study.

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

Given the success of the preparation of fluorogenic glycopolymers, the biological activity of the polymer 11a was evaluated by means of enzymatic digestion with continuous monitoring by photoluminescence. Thus, the enzymatic reaction was conducted in the presence of α-amylase in 10 mM HEPES buffer (pH 7.0) at 37 °C, and the time course of the enzymatic hydrolysis of the oligosaccharide was monitored by fluorescence measurement. When the enzyme was added to the substrate solution, the emission spectrum as the 0 min spectrum was taken immediately. Subsequently, the emission spectra excited at 290 nm were taken at appropriate intervals and the results of the continuous measurement are shown in Figure 2a. A decrease in dansyl emission at around 510 nm by diminishing the energy transfer from the naphthyl moieties was clearly observed, and an increase in naphthyl emission at around 330 nm by eliminating the quenching by dansyl moieties was also observed. In addition to the spectral transformation, since an isoemissive point at around 355 nm was clearly observed in these emission spectra, enzymatic scission by α-amylase for the oligosaccharide part of the FRET-sensitive polymer proceeded efficiently. The fluorescence intensities of the FRET-sensitive polymer 11a determined by measurements of both naphthyl emission and dansyl emission at each time point were plotted at the corresponding naphthyl emission (333 nm; blue color) and dansyl emission (515 nm; red color) from the value at 0 min until 30 min, respectively [Figure 2b]. The naphthyl emission at 333 nm gradually increased up to 25 min, after which the reaction tapered off until it had almost stopped at 30 min. On the other hand, decreases in dansyl emission at 515 nm as the reaction progressed were also monitored. Kinetic analyses of the enzymatic reaction for the polymer 11a were performed on the basis of both the Hanes–Woolf (HB)¹⁵ plot and a nonlinear regression fitting¹⁶ and the results are shown in Figure 2c,d. The HB plot of 11a showed linear relation between [S]/V and [S], and the Michaelis constant as represented by K_m and the maximum rate as represented by V_max were estimated. In addition to the HB plot, the nonlinear regression fitting was also used in order to evaluate the differences of K_m and V_max values estimated by the HB plot. The Michaelis constant K_m of α-amylase was reported using a maltotriaoside derivative with 2.2 mM¹⁷ and a maltotetraoside derivative with 0.887 mM.¹⁸ The FRET-sensitive polymer 11a showed K_m = 6.6 μM on the basis of analyses by using the HB plot and nonlinear regression. The K_m value of the polymeric substrate was smaller than the values of monomeric maltooligosaccharides. The results suggested that greater enzymatic activity was obtained when a substrate was used as a polymer-type substrate. Because of the close proximity of adjacent saccharide units in the polymeric substrate, it was considered that the enzymatic reaction could proceed efficiently. In addition to the K_m values, the V_max value was also estimated by means of both analyses, and it was 0.40 nmol/min.

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.

Since the preparation of a water-soluble FRET-sensitive polymer having maltohexaoside and the biological response for enzymatic reaction were accomplished, our next area of interest was incorporation of the water-soluble FRET-sensitive polymer into an IPN. A schematic image of the IPN is shown in Figure 1b. The water-soluble FRET-sensitive polymer 11b was allowed to react with AAm and bis-AAm in the presence of radical initiators to yield the desired IPN having water-soluble glycopolymer 11b as colorless hydrogel, which was purified by means of dialysis against distilled water in order to remove soluble polymers in water and unreacted monomers. The IPN gel was freeze-dried to yield the desired xerogel, and the xerogel that is standing under natural light is shown in Figure 3a. Although the xerogel was colorless under natural light, the xerogel illuminated a dark blue color under a handy-type UV light at 254 nm, while the xerogel excited at 365 nm by using a handy-type UV light emitted a strong bright blue color. UV illumination with a longer wavelength could directly excite the dansyl moieties in the FRET-sensitive polymer 11b immobilized within the IPN. Similar behavior was also observed in an aqueous medium in a cuvette as shown in Figure 3b. The hydrogel of the IPN was insoluble in water in the cuvette. UV irradiation with short and long wavelengths excited the naphthyl moieties in the FRET-sensitive polymer 11b, giving corresponding resonance energy at around 330 nm, and the energy was used to excite the dansyl moieties in the polymer 11b to afford fluorescence emission at around 510 nm. Since the basic physical properties of the IPN were confirmed, our next interest was in exudation of the water-soluble FRET-sensitive polymer 11b from the IPN into an aqueous solvent. The FRET-sensitive polymer 11b was left alone in 10 mM HEPES buffer (pH 7.0) at 37 °C, and the time course of the relative fluorescence emission measured by using a fluorescence spectroscopic meter is shown in Figure 3c. The results strongly suggest that there was no exudation of the polymer 11b from the IPN into the buffer solution. Given the success of the complete capture of the water-soluble polymer into the IPN, the next chemical manipulation was enzymatic digestion of the maltooligosaccharides in the FRET-sensitive polymer 11b on the basis of fluorometric measurement. Continuous monitoring of hydrolysis of the maltooligosaccharide part in the FRET-sensitive polymer 11b by α-amylase from B. subtilis(19) was examined. The fluorometric profiles of the time course of the enzymatic reaction were measured in the presence of α-amylase in 10 mM HEPES buffer (pH 7.0) at 37 °C. The emission spectrum was taken immediately after addition of the enzyme, and this was regarded as the 0 min spectrum. Subsequently, the emission spectra were taken at appropriate intervals, and the results of the continuous measurement are shown in Figure 3d. As expected, enzymatic digestion caused an increase in naphthyl emission at around 330 nm in an ambient solvent by diminishing the energy transfer to the dansyl chromophore left as a part of glycopolymer in the IPN.

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.

Although a successful continuous assay of α-amylase from B. subtilis by means of an IPN having the water-soluble FRET-sensitive polymer was accomplished, further measurement using the IPN for another α-amylase was conducted in order to evaluate the applicability of the IPN. Thus, porcine pancreatic α-amylase²⁰ was used and the results of the continuous assay of α-amylase by means of an IPN having the water-soluble FRET-sensitive polymer 11b are shown in Figure 4. The assay system also effectively worked in this case and similar fluorometric profiles were obtained. Since the quantity of the IPN was smaller than that for α-amylase from B. subtilis, intensities of the naphthyl moieties were lower. It was found that this approach enabled efficient monitoring of the enzymatic reaction and can be used for other amylases.

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.

4. Conclusions

A water-soluble glycopolymer having maltooligosaccharide chains as shown in Figure 5a was reacted with α-amylase, that allowed to monitor continuously the enzymatic reaction [Figure 5b]. In addition, the FRET-sensitive glycopolymer was completely captured in an IPN by means of radical polymerization with acrylamide and bis-acrylamide as shown in Figure 5c. Figure 5a shows that the water-soluble glycopolymer having maltooligosaccharide chains in an aqueous solvent emits from the acceptor fluorophore when an appropriate excitation light is used for excitation of the donor fluorophore. Successful monitoring the enzymatic reaction was accomplished on the basis of fluorescence measurement, that canceled the FRET in the glycopolymer as shown in Figure 5b. On the other hand, a solution including the FRET-sensitive polymer on the bottom of the cuvette does not show any fluorescence at all due to complete immobilization of the FRET polymer in the IPN as shown in Figure 5c. A solution including the FRET-sensitive polymer on the bottom of the cuvette was allowed to react with amylases as shown in Figure 5d. The α-amylase hydrolyzes a maltooligosaccharide chain into the aqueous solution, and the disconnected portion of the maltooligosaccharide shows an emission due to the donor fluorophore at around 340 nm. The FRET from the donor fluorophore with maltooligosaccharide to the acceptor fluorophore is gradually canceled with digestion of the maltooligosaccharide by amylase. The whole enzymatic reaction can be monitored by using a fluorometric spectrometer. The IPN including the FRET-sensitive polymer will be used for continuous monitoring of amylase activities in biomedical applications, and the results will be published elsewhere in the near future.

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.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.biomac.4c00622.

• Spectral data of the desired compound, IR, ¹H and ¹³C NMR, and SEC profile (PDF)

Author Contributions

Conceptualization: K.M. and K.M., methodology: K.M. and K.M., validation: T.M. and K.H., formal analysis: T.K., investigation: H.A. and T.K., data curation: T.M., writing—original draft preparation: K.M., writing—review and editing: K.M., supervision: K.M., project administration: K.M., funding acquisition: K.M. All authors have read and agreed to the published version of the manuscript.

The authors declare no competing financial interest.