Supporting Information Novel Hexosaminidase-Targeting ...
Supporting Information Bioconjugate Chemistry (Article)
Supporting Information Novel Hexosaminidase-Targeting Fluorescence Probe for Visualizing Human Colorectal Cancer Hiroyuki Matsuzaki, Mako Kamiya, Ryu J. Iwatate, Daisuke Asanuma, Toshiaki Watanabe, Yasuteru Urano
Table of contents p. 1-3: Experimental Procedures General procedure and materials Synthesis of HMRef-βGlcNAc siRNA knockdown of HEXA and HEXB in colorectal cancer cell lines p. 4: Figure S1. Fluorescence spectra of HMRef-βGlcNAc at various pH values. p. 5: Figure S2. Fluorescence intensity change of HMRef-βGlcNAc in the presence of mouse serum. p. 5: Figure S3. HPLC chromatogram of HMRef-βGlcNAc after reaction with hexosaminidase. p. 6: Figure S4. Fluorescence spectra of live-cell imaging with HMRef-βGlcNAc or HMRef. p. 7: Figure S5. Fluorescence imaging of cell lines with HEXA- or HEXB-siRNA. p. 8: Figure S6. ROC curve of HMRef-βGlcNAc in human colorectal cancer specimens. p. 8: Table S1. Photochemical properties of HMRef-βGlcNAc in the presence of 4% (w/v) bovine serum albumin. p. 8: Table S2. Fluorescence intensity change in surgical specimens of human colorectal cancer
Experimental Procedures General procedure and materials NMR spectra were obtained on Bruker NMR AVANCE III 400 spectrometers [1H (400 MHz), 13C (101 MHz)] in deuteriomethanol (CD 3 OD) solution, with CD 2 HOD as an internal standard. High-resolution ESI-mass spectra were obtained on a micrOTOF II (Bruker). HPLC purification was carried out with a JASCO PU-2087 Plus pump equipped with an Inertsil-ODS-3 column (φ10 x 250 mm (semi preparative) and φ20 x 250 mm (preparative)) (GL Science Co., Ltd.) and a UVIDEC-100-V detector (JASCO). The solvents used for HPLC were obtained from Wako Co., Ltd. Silica gel column chromatography was performed on silica gel 60N (spherical, neutral, 63-210 µm; Kanto Chemical Co., Ltd.). TLCs were performed on silica gel plates F 254 (0.25 mm (analytical) and 1.0 mm (preparative); Merck, AKG).
1
Supporting Information Bioconjugate Chemistry (Article) OAc O 1) AcO AcO AcHN Cl NaI, Ag2O MeCN, r.t. 8h
OH
N H
O
O
CF3
HO HO
2) NaOMe MeOH, r.t. 2h 53%
O
OH O AcHN
O
O
N H
CF3
Scheme S1. Synthetic scheme of HMRef-βGlcNAc.
Synthesis of HMRef-βGlcNAc HMRef was synthesized according to the literature23. To a solution of HMRef (12.5 mg, 31.3 μmol, 1.0 eq) in 8 ml of dry MeCN were added 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D-glucopyranosyl chloride (136.0 mg, 372 μmol, 11.9 eq), Ag 2 O (118.8 mg, 513 μmol, 16.4 eq), NaI (36.8 mg, 246 μmol, 7.8 eq) and a sufficient amount of anhydrous Na2SO4 to remove contaminating water. The reaction mixture was stirred at r.t. for 8 h, filtered, and evaporated. The residue was dissolved in 10 ml of MeOH, and 250 μl of NaOMe (28 % solution in MeOH) was added. The mixture was stirred at r.t. for 2 h, neutralized with Amberlite IR120H, filtered, and evaporated. The residue was purified by HPLC (a linear gradient formed from 100 mM TEAA buffer and CH 3 CN 99 %, H 2 O 1 %: isocratic at 90/10 for 5 min, linear gradient to 10/90 in 15 min, then isocratic for 15 min) to afford 9.9 mg of orange powder. Yield: 52.5% in 2 steps. 1H NMR (400 MHz, CD 3 OD): δ 7.43-7.36 (m, 2H), 7.27 (t, 1H, J = 7.3 Hz), 6.86 (t, 1H, J = 2.4 Hz), 6.84-6.78 (m, 2H), 6.72-6.68 (m, 2H), 6.51 (d, 1H, J = 2.4 Hz), 6.45 (dd, 1H, J = 8.6, 2.4 Hz), 5.25 (s, 2H), 5.08 (dd, 1H, J = 8.4, 0.8 Hz), 3.91-3.80 (m, 4H), 3.75-3.70 (m, 1H), 3.59-3.54 (m, 1H), 3.49-3.44 (m, 2H), 3.42-3.40 (m, 1H), 1.98 (d, 3H, J = 2.2 Hz); 13C NMR (101 MHz, CD 3 OD): δ 173.9, 159.6, 152.9, 152.7, 152.7, 150.3, 146.1, 146.1, 140.2, 140.2, 130.9, 129.4, 129.3, 126.9 (q, J = 279.8 Hz), 124.7, 124.7, 121.9, 120.3, 120.2, 114.8, 113.5, 113.4, 110.8, 104.7, 104.6, 100.7, 100.7, 99.3, 85.4, 85.3, 78.3, 75.8, 72.7, 71.8, 62.5, 57.3, 57.3, 45.6 (q, J = 33.5 Hz), 23.0; HRMS (ESI+): calcd for [M+Na]+ 625.17682; found 625.17585 (-0.97mmu). HPLC analysis was performed using eluent A (100 mM TEAA buffer) and eluent B (CH 3 CN). A/B: isocratic 90/10 for 5 min, linear gradient to 10/90 in 15 min, then isocratic for 15 min. Absorbance at 498 nm was monitored. Purity: > 98%.
Absorbance
1500000 1000000 500000 0 0
5
10
15
20
25
Time (min)
2
30
Supporting Information Bioconjugate Chemistry (Article)
siRNA knockdown of HEXA and HEXB in colorectal cancer cell lines To evaluate the specificity of HMRef-βGlcNAc for hexosaminidase, we knocked down the HEXA and HEXB genes in four human colorectal cell lines by siRNA transfection. The cells were seeded on 8-well µ-Slides (ibidi) and cultured overnight, then the medium was replaced with antibiotic-free medium, and the cells were transfected with HEXA-targeting siRNA (sc-60783, Santa Cruz Biotechnology, USA), HEXB-targeting siRNA (sc-60786, Santa Cruz Biotechnology, USA) or control siRNA (BannoNegacon; RNAi Inc.) using Lipofectamine RNAiMAX transfection reagent (Invitrogen, USA) according to the manufacturer’s protocol. Two days after transfection, the culture medium was replaced with phenol red-free and serum-free medium containing HMRef-βGlcNAc probe (10 µM), and live cell fluorescence microscopy was performed as described above.
3
Supporting Information Bioconjugate Chemistry (Article)
Supporting Figures
(b)
0.035
12.0 10.0 8.0 7.4 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0
0.030
Absorbance
0.025 0.020 0.015 0.010 0.005 0.000 400
(c)
450 500 550 Wavelength [nm]
1000 800 600 400 200
12.0 10.0 8.0 7.4 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0
0 450 500 550 600 650 700 750 800 Wavelength [nm]
600
0.04
1200 Abs (481.0 nm)
1000
Flu (519.2 nm)
0.03
800 0.02
600 400
Flu (519.2 nm)
Abs (481.0 nm)
1200
Fluorescence intensity (A.U.)
(a)
0.01 200 0
0 2
4
6
8
10
12
pH
Supporting Figure 1. (a) Absorption and (b) emission spectra and (c) titration curves of HMRef-βGlcNAc at various pH values in 0.2 M sodium phosphate buffer containing 0.1% DMSO as a cosolvent. Excitation wavelength for fluorescence spectra was 482 nm. The slit widths of excitation and emission were 2.5 nm. The photomultiplier voltage was 700 V.
4
Supporting Information Bioconjugate Chemistry (Article)
Supporting Figure 2. Time course of fluorescence intensity change accompanying enzymatic reaction of HMRef-βGlcNAc with hexosaminidase in the presence of mouse serum. Hexosaminidase (2 U) was added at 100 sec to a 1 μM solution of HMRef-βGlcNAc in phosphate buffer saline, pH 7.4, containing 5% (v/v) mouse serum at
Absorbance at 498 nm
room temperature.
0
HMRef HMRef
28.4 min
HMRef-bGlcNAc HMRef-βGlcNAc
24.9 min
HMRef-bGlcNAc HMRef-βGlcNAc ++Hexosaminidase Hexosaminidase
28.5 min
5
30
10 15 20 25 Retention time (min)
35
Supporting Figure 3. HPLC chromatogram of HMRef-βGlcNAc after reaction with hexosaminidase. Chromatograms of unreacted dye and HMRef are also shown. Analytical HPLC was performed on an Inertsil ODS-3 (4.6 x 250 mm) column (GL Sciences Inc.) using an HPLC system composed of a pump (PU-2080, JASCO) and a detector (MD-2010, JASCO). HPLC elution was done with a linear gradient formed from 100 mM TEAA buffer and CH 3 CN 99%, H 2 O 1% (isocratic at 90/10 for 5 min, linear gradient to 10/90 in 15 min, then isocratic for 15 min). Detection wavelength was 498 nm.
5
Supporting Information Bioconjugate Chemistry (Article)
(a)
(b)
Supporting Figure 4. Spectral scanning imaging in vitro or in cells. (a) Emission spectra of HMRef-βGlcNAc (blue line) or HMRef (red line) solution in vitro in 0.2 M sodium phosphate buffer (pH 5). (b) Emission spectra of fluorescence signal in cells loaded with HMRef-βGlcNAc (blue line) or HMRef (red line). Cells were incubated at 37 °C for 30 minutes after the application of 10 µM HMRef-βGlcNAc or HMRef. Spectral scanning imaging (lambda scan) was conducted with a detection window of 10 nm.
6
Supporting Information Bioconjugate Chemistry (Article)
Supporting Figure 5. (a) Fluorescence confocal imaging of human colon cancer cell lines transfected with HEXAor HEXB-targeting siRNA or control siRNA and loaded with 10 μM HMRef-βGlcNAc, at 30 minutes after probe application. Scale bar represents 100 μm. (b) Quantified fluorescence intensities of images in (a), normalized to the controls. Data are shown as mean + SD (n=10). *: p
Supporting Information Novel Hexosaminidase-Targeting Fluorescence Probe for Visualizing Human Colorectal Cancer Hiroyuki Matsuzaki, Mako Kamiya, Ryu J. Iwatate, Daisuke Asanuma, Toshiaki Watanabe, Yasuteru Urano
Table of contents p. 1-3: Experimental Procedures General procedure and materials Synthesis of HMRef-βGlcNAc siRNA knockdown of HEXA and HEXB in colorectal cancer cell lines p. 4: Figure S1. Fluorescence spectra of HMRef-βGlcNAc at various pH values. p. 5: Figure S2. Fluorescence intensity change of HMRef-βGlcNAc in the presence of mouse serum. p. 5: Figure S3. HPLC chromatogram of HMRef-βGlcNAc after reaction with hexosaminidase. p. 6: Figure S4. Fluorescence spectra of live-cell imaging with HMRef-βGlcNAc or HMRef. p. 7: Figure S5. Fluorescence imaging of cell lines with HEXA- or HEXB-siRNA. p. 8: Figure S6. ROC curve of HMRef-βGlcNAc in human colorectal cancer specimens. p. 8: Table S1. Photochemical properties of HMRef-βGlcNAc in the presence of 4% (w/v) bovine serum albumin. p. 8: Table S2. Fluorescence intensity change in surgical specimens of human colorectal cancer
Experimental Procedures General procedure and materials NMR spectra were obtained on Bruker NMR AVANCE III 400 spectrometers [1H (400 MHz), 13C (101 MHz)] in deuteriomethanol (CD 3 OD) solution, with CD 2 HOD as an internal standard. High-resolution ESI-mass spectra were obtained on a micrOTOF II (Bruker). HPLC purification was carried out with a JASCO PU-2087 Plus pump equipped with an Inertsil-ODS-3 column (φ10 x 250 mm (semi preparative) and φ20 x 250 mm (preparative)) (GL Science Co., Ltd.) and a UVIDEC-100-V detector (JASCO). The solvents used for HPLC were obtained from Wako Co., Ltd. Silica gel column chromatography was performed on silica gel 60N (spherical, neutral, 63-210 µm; Kanto Chemical Co., Ltd.). TLCs were performed on silica gel plates F 254 (0.25 mm (analytical) and 1.0 mm (preparative); Merck, AKG).
1
Supporting Information Bioconjugate Chemistry (Article) OAc O 1) AcO AcO AcHN Cl NaI, Ag2O MeCN, r.t. 8h
OH
N H
O
O
CF3
HO HO
2) NaOMe MeOH, r.t. 2h 53%
O
OH O AcHN
O
O
N H
CF3
Scheme S1. Synthetic scheme of HMRef-βGlcNAc.
Synthesis of HMRef-βGlcNAc HMRef was synthesized according to the literature23. To a solution of HMRef (12.5 mg, 31.3 μmol, 1.0 eq) in 8 ml of dry MeCN were added 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-α-D-glucopyranosyl chloride (136.0 mg, 372 μmol, 11.9 eq), Ag 2 O (118.8 mg, 513 μmol, 16.4 eq), NaI (36.8 mg, 246 μmol, 7.8 eq) and a sufficient amount of anhydrous Na2SO4 to remove contaminating water. The reaction mixture was stirred at r.t. for 8 h, filtered, and evaporated. The residue was dissolved in 10 ml of MeOH, and 250 μl of NaOMe (28 % solution in MeOH) was added. The mixture was stirred at r.t. for 2 h, neutralized with Amberlite IR120H, filtered, and evaporated. The residue was purified by HPLC (a linear gradient formed from 100 mM TEAA buffer and CH 3 CN 99 %, H 2 O 1 %: isocratic at 90/10 for 5 min, linear gradient to 10/90 in 15 min, then isocratic for 15 min) to afford 9.9 mg of orange powder. Yield: 52.5% in 2 steps. 1H NMR (400 MHz, CD 3 OD): δ 7.43-7.36 (m, 2H), 7.27 (t, 1H, J = 7.3 Hz), 6.86 (t, 1H, J = 2.4 Hz), 6.84-6.78 (m, 2H), 6.72-6.68 (m, 2H), 6.51 (d, 1H, J = 2.4 Hz), 6.45 (dd, 1H, J = 8.6, 2.4 Hz), 5.25 (s, 2H), 5.08 (dd, 1H, J = 8.4, 0.8 Hz), 3.91-3.80 (m, 4H), 3.75-3.70 (m, 1H), 3.59-3.54 (m, 1H), 3.49-3.44 (m, 2H), 3.42-3.40 (m, 1H), 1.98 (d, 3H, J = 2.2 Hz); 13C NMR (101 MHz, CD 3 OD): δ 173.9, 159.6, 152.9, 152.7, 152.7, 150.3, 146.1, 146.1, 140.2, 140.2, 130.9, 129.4, 129.3, 126.9 (q, J = 279.8 Hz), 124.7, 124.7, 121.9, 120.3, 120.2, 114.8, 113.5, 113.4, 110.8, 104.7, 104.6, 100.7, 100.7, 99.3, 85.4, 85.3, 78.3, 75.8, 72.7, 71.8, 62.5, 57.3, 57.3, 45.6 (q, J = 33.5 Hz), 23.0; HRMS (ESI+): calcd for [M+Na]+ 625.17682; found 625.17585 (-0.97mmu). HPLC analysis was performed using eluent A (100 mM TEAA buffer) and eluent B (CH 3 CN). A/B: isocratic 90/10 for 5 min, linear gradient to 10/90 in 15 min, then isocratic for 15 min. Absorbance at 498 nm was monitored. Purity: > 98%.
Absorbance
1500000 1000000 500000 0 0
5
10
15
20
25
Time (min)
2
30
Supporting Information Bioconjugate Chemistry (Article)
siRNA knockdown of HEXA and HEXB in colorectal cancer cell lines To evaluate the specificity of HMRef-βGlcNAc for hexosaminidase, we knocked down the HEXA and HEXB genes in four human colorectal cell lines by siRNA transfection. The cells were seeded on 8-well µ-Slides (ibidi) and cultured overnight, then the medium was replaced with antibiotic-free medium, and the cells were transfected with HEXA-targeting siRNA (sc-60783, Santa Cruz Biotechnology, USA), HEXB-targeting siRNA (sc-60786, Santa Cruz Biotechnology, USA) or control siRNA (BannoNegacon; RNAi Inc.) using Lipofectamine RNAiMAX transfection reagent (Invitrogen, USA) according to the manufacturer’s protocol. Two days after transfection, the culture medium was replaced with phenol red-free and serum-free medium containing HMRef-βGlcNAc probe (10 µM), and live cell fluorescence microscopy was performed as described above.
3
Supporting Information Bioconjugate Chemistry (Article)
Supporting Figures
(b)
0.035
12.0 10.0 8.0 7.4 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0
0.030
Absorbance
0.025 0.020 0.015 0.010 0.005 0.000 400
(c)
450 500 550 Wavelength [nm]
1000 800 600 400 200
12.0 10.0 8.0 7.4 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0
0 450 500 550 600 650 700 750 800 Wavelength [nm]
600
0.04
1200 Abs (481.0 nm)
1000
Flu (519.2 nm)
0.03
800 0.02
600 400
Flu (519.2 nm)
Abs (481.0 nm)
1200
Fluorescence intensity (A.U.)
(a)
0.01 200 0
0 2
4
6
8
10
12
pH
Supporting Figure 1. (a) Absorption and (b) emission spectra and (c) titration curves of HMRef-βGlcNAc at various pH values in 0.2 M sodium phosphate buffer containing 0.1% DMSO as a cosolvent. Excitation wavelength for fluorescence spectra was 482 nm. The slit widths of excitation and emission were 2.5 nm. The photomultiplier voltage was 700 V.
4
Supporting Information Bioconjugate Chemistry (Article)
Supporting Figure 2. Time course of fluorescence intensity change accompanying enzymatic reaction of HMRef-βGlcNAc with hexosaminidase in the presence of mouse serum. Hexosaminidase (2 U) was added at 100 sec to a 1 μM solution of HMRef-βGlcNAc in phosphate buffer saline, pH 7.4, containing 5% (v/v) mouse serum at
Absorbance at 498 nm
room temperature.
0
HMRef HMRef
28.4 min
HMRef-bGlcNAc HMRef-βGlcNAc
24.9 min
HMRef-bGlcNAc HMRef-βGlcNAc ++Hexosaminidase Hexosaminidase
28.5 min
5
30
10 15 20 25 Retention time (min)
35
Supporting Figure 3. HPLC chromatogram of HMRef-βGlcNAc after reaction with hexosaminidase. Chromatograms of unreacted dye and HMRef are also shown. Analytical HPLC was performed on an Inertsil ODS-3 (4.6 x 250 mm) column (GL Sciences Inc.) using an HPLC system composed of a pump (PU-2080, JASCO) and a detector (MD-2010, JASCO). HPLC elution was done with a linear gradient formed from 100 mM TEAA buffer and CH 3 CN 99%, H 2 O 1% (isocratic at 90/10 for 5 min, linear gradient to 10/90 in 15 min, then isocratic for 15 min). Detection wavelength was 498 nm.
5
Supporting Information Bioconjugate Chemistry (Article)
(a)
(b)
Supporting Figure 4. Spectral scanning imaging in vitro or in cells. (a) Emission spectra of HMRef-βGlcNAc (blue line) or HMRef (red line) solution in vitro in 0.2 M sodium phosphate buffer (pH 5). (b) Emission spectra of fluorescence signal in cells loaded with HMRef-βGlcNAc (blue line) or HMRef (red line). Cells were incubated at 37 °C for 30 minutes after the application of 10 µM HMRef-βGlcNAc or HMRef. Spectral scanning imaging (lambda scan) was conducted with a detection window of 10 nm.
6
Supporting Information Bioconjugate Chemistry (Article)
Supporting Figure 5. (a) Fluorescence confocal imaging of human colon cancer cell lines transfected with HEXAor HEXB-targeting siRNA or control siRNA and loaded with 10 μM HMRef-βGlcNAc, at 30 minutes after probe application. Scale bar represents 100 μm. (b) Quantified fluorescence intensities of images in (a), normalized to the controls. Data are shown as mean + SD (n=10). *: p
