Responsive Photonic Crystal Carbohydrate Hydrogel Sensor. Materials for Selective and Sensitive Lectin Protein Detection

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1 Supporting Information Responsive Photonic Crystal Carbohydrate Hydrogel Sensor Materials for Selective and Sensitive Lectin Protein Detection Zhongyu Cai, Aniruddha Sasmal, Xinyu Liu*, Sanford A. Asher* Department of Chemistry, University of Pittsburgh, Pittsburgh, PA USA *Corresponding author. Table of Contents 1. Experimental Section Synthesis of 2-Allylethoxyl-β-D-lactose Synthesis of 2-Allylethoxyl α-d-mannopyranoside H NMR and 13 C NMR Characterization of Carbohydrate Monomers Photographs of 2-D PCs and Debye Diffraction Ring Calculations of limit of detection (LoD), dynamic range and linear range of the lectin protein sensors Calculations for the 2-D PC PAAm-AAc-Lactose sensor Calculations for the 2-D PC PAAm-AAc-Mannose sensor Calculations for the 2-D PC PAAm-AAc-α-Galactose sensor References..18 1

2 1. Experimental Section 1.1 Synthesis of 2-Allylethoxyl-β-D-lactose 2-Allylethoxyl-β-D-lactose was synthesized from commercially available D-lactose in 3 steps. The steps are described below (Schemes S1-S3). Scheme S1. Synthesis of 2,3,4,6-tetra-O-benzoyl-β-D-galactopyranosyl-(1 4)-2,3,6-tri-O-benzoyl-α-Dglucopyranosyl bromide. 2,3,4,6-tetra-O-benzoyl-β-D-galactopyranosyl-(1 4)-2,3,6-tri-O-benzoyl-α-Dglucopyranosyl bromide (S2): To a 0 C solution of D-lactose (3.0 g, 8.8 mmol) in dry pyridine (20 ml) was added benzoyl chloride (12.2 ml, mol). The solution was then slowly warmed to room temperature and stirred for 14 h. The mixture was then diluted with 60 ml CH 2 Cl 2 and washed with an ice-cold saturated NaHCO 3 solution, and room temperature water, and a saturated NaCl solution. Then the solution was dried with Na 2 SO 4, filtered and concentrated in vacuum. The crude residue was directly used without further purification. The per-o-benzoyl-d-lactose was dissolved in CH 2 Cl 2 (30 ml) and the solution was cooled to -10 C and stirred, while 33 wt% hydrobromic acid in glacial acetic acid was slowly added. The mixture was then warmed to room temperature and stirred for 8 h. Then the mixture was diluted with 30 ml CH 2 Cl 2, and poured into cold water (10 C). The organic layer was washed with saturated NaHCO 3 solution, with water, and with a saturated NaCl solution. The solution was dried with Na 2 SO 4, filtered, and concentrated in vacuum to obtain the product S2 as a white solid (9.3 g, 93% yield). 2

3 2-Allylethoxyl Scheme S2. Synthesis of 2-Allylethoxyl 2,3,4,6-tetra-O-benzoyl-β-D-galactopyranosyl-(1 4)-2,3,6-tri-Obenzoyl-β-D-glucopyranoside. 2,3,4,6-tetra-O-benzoyl-β-D-galactopyranosyl-(1 4)-2,3,6-tri-Obenzoyl-β-D-glucopyranoside (S3): To a stirred solution of Hg(CN) 2 (0.51 g, 2.0 mmol) and HgBr 2 (0.72 g, 0.2 mmol) in dry MeCN (10 ml) was added 2-allyloxyethanol (0.81 g, 7.95 mmol). To this solution, a solution of per-o-benzoyl-d-lactosyl bromide S2 (4.5 g, 3.98 mmol) dissolved in MeCN (10 ml) was added by dropwise addition to a stirred solution that was maintained at room temperature for an additional 16 h. The solvent was partially evaporated, and re-dissolved in 60 ml CH 2 Cl 2. The organic layer was successively washed with water, with a saturated NaHCO 3 solution, with a saturated NaCl solution, and then dried over MgSO 4. After the solvent was evaporated the residue was purified by flash chromatography (silica gel, Hexane- EtOAc) to obtain S3 as a white solid (3.6 g, 78% yield). Scheme S3. Synthesis of 2-Allylethoxyl-β-D-lactose. 2-Allylethoxyl-β-D-lactose (S4): Compound S3 (3 g, 2.60 mmol) was dissolved in MeOH (15 ml) and then NaOMe (56 mg, 1.04 mmol) was added to this solution. The solution was stirred at 40 C for 16 h and then cooled to room temperature. The mixture was neutralized 3

4 with Dowex H + resin, filtered, and evaporated. The product was purified by flash chromatography (silica gel, DCM-MeOH) to obtain S4 as a white solid in 85% yield. 1.2 Synthesis of 2-Allylethoxyl α-d-mannopyranoside Mannose monomer (2-allylethoxyl α-d-mannopyranoside) with the same 2-allylethoxy linker at its reducing end was synthesized from per-acetate mannose in 2 steps. The synthesis protocol is shown below (Schemes 4 and 5). Scheme S4. Synthesis of 2-Allylethoxyl 2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside. 2-Allylethoxyl 2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside (S6): α-d-mannose pentaacetate S5 (3.50 g, 8.97 mmol) was added to a round-bottom flask and dissolved in CH 2 Cl 2 (50 ml) and placed under nitrogen. The stirred solution was cooled to 0 ºC and BF 3 OEt 2 O (1.36 ml, mmol) was added followed by 2-allyloxyethanol (1.37 g, mmol). The reaction mixture was warmed up to room temperature and stirred for 12 hours. Then the solution was quenched with a saturated solution of NaHCO 3 at 0 ºC and diluted with water. The organic layer was separated and the aqueous layer was extracted with 30 ml CH 2 Cl 2 twice. The combined organic layer was dried with MgSO 4, filtered, and evaporated. Purification by column chromatography afforded compound S6 as a clear oil (1.4 g, 36% yield). Scheme S5. Synthesis of 2-Allyloethoxyl α-d-mannopyranoside. 4

5 2-Allylethoxyl α-d-mannopyranoside (S7): NaOMe (25 mg, 0.46 mmol) was added to compound S6 (1 g, 2.31 mmol) dissolved in MeOH (15 ml). The reaction mixture was stirred at 40 C for 16 h and then cooled to room temperature. The solution was neutralized with Dowex H + resin, filtered, and evaporated. The crude residue was purified by column chromatography to obtain S7 as a white solid in 89% yield. The 1 H and 13 C NMR spectra of these two carbohydrate monomers and their analysis are shown in Figures S1-S5. The structure of commercial allyl α-d-galactose and allyl β-d-galactose was also confirmed with 1 H NMR spectra (Figures S6 and S7). 5

6 2. 1 H NMR and 13 C NMR Characterization of Carbohydrate Monomers Figure S1. Chemical structure and 1 H NMR spectroscopy of 2,3,4,6-tetra-O-benzoyl-β-D-galactopyranosyl- (1 4)-2,3,6-tri-O-benzoyl-β-D-glucopyranoside. 6

7 Figure S2. Chemical structure and 13 C NMR spectroscopy of 2,3,4,6-tetra-O-benzoyl-β-Dgalactopyranosyl-(1 4)-2,3,6-tri-O-benzoyl-β-D-glucopyranoside. 1 H NMR (600 MHz, CDCl 3 ): δ (m, 11H), (m, 2H), (m, 2H), (m, 4H), (m, 14H), 7.21 (t, 2H, J=7.8 Hz), 7.13 (t, 2H, J= 7.8 Hz), 5.79 (dd, 1H, J= 9 Hz), (m, 2H), (m, 1H), 5.46 (dd, 1H, J = 10.2 Hz, 7.8 Hz), 5.35 (dd, 1H, J = 10.2 Hz, 3 Hz), 5.07 (ddd, 1H, J = 16 Hz, 4.2 Hz, 1.8 Hz), 4.99 (ddd, 1H, J = 10.8 Hz, 4.2 Hz, 1.8 Hz), 4.86 (d, 1H, J = 7.8 Hz), 4.83 (d, 1H, J = 7.8 Hz), 4.58 (dd, 1H, J = 12.0 Hz, 2.4 Hz), 4.48 (dd, 1H, J = 12 Hz, 4.2 Hz), 4.25 (app t, 1H, J =9.6 Hz), (m, 2H), 3.82 (ddd, 1H, J = 9.6 Hz, 4.2 Hz, 1.8 Hz), (m, 2H), 3.71 (dd, 1H, J= 10.2 Hz, 7.2 Hz ), 3.71 (dd, 1H, J= 10.8 Hz, 6.0 Hz), 3.66 (dd, 1H, J= 10.8 Hz, 7.2 Hz), 3.48 (t, 2H, J= 4.2 Hz). 13 C NMR (150 MHz, CDCl 3 ): δ 165.8, 165.5, 165.4, 165.2, 164.8, 134.4, 134.5, (2), 134.2, 134.1(2), 130.0, 129.8, 129.7(2), 129.6(3), 129.5, 129.4, 128.8, 128.6(3), 128.5(3), 128.4, 128.3, 128.2(2), 116.7, 101.3, 100.9, 76.0, 72.9(2), 72.0, 71.7(2), 71.3, 69.8, 69.2(2), 67.5, 62.4,

8 Figure S3. Chemical structure and 1 H NMR spectroscopy of 2-allylethoxyl β-d-lactose. 8

9 Figure S4. Chemical structure and 13 C NMR spectroscopy of 2-allylethoxyl β-d-lactose. 1 H NMR (600 MHz, D 2 O): 5.93 (ddd, 1H, J = 18 Hz, 10.2 Hz, 6 Hz), 5.33 (d, 1H, J = 18 Hz), 5.26 (d, 1H, J = 10.2 Hz), 4.49 (d, 1H, J = 7.8 Hz), 4.43 (d, 1H, J = 7.2 Hz), (m, 3H), 3.96 (app d, 1H, J = 12 Hz), 3.90 (d, 1H, J= 2.4 Hz), (m, 7H), (m, 3H), (m, 2H), 3.33 (app t, 1H, J= 8.4 Hz). 13 C NMR (150 MHz, D 2 O): δ 133.4, 118.6, 102.9, 102.0, 78.3, 75.3, 74.7, 74.2, 72.7, 72.4, 71.7, 70.9, 68.7(2), 68.5, 60.9,

10 Figure S5. Chemical structure and 1 H NMR spectroscopy of 2-allylethoxyl α-d-mannose. 1 H NMR (400 MHz, D 2 O): 5.94 (tdd, 1H, J = 14 Hz, 6 Hz, 6 Hz), 5.32 (dd, 1H, J = 16 Hz, 4 Hz), 5.26 (dd, 1H, J = 10 Hz, 2 Hz), 4.86 (d, 1H, J = 1.6 Hz), 4.07 (ddd, 2H, J = 6 Hz, 1.78 Hz, 1.3 Hz), 3.95 (dd, 1H, J = 3.4 Hz, 1.6 Hz), (m, 3H), 3.76 (dd, 1H, J = 4.4 Hz, 1.72 Hz), (m, 2H), 3.68 (dd, 1H, J = 4 Hz, 3.6 Hz), (m, 2H). 10

11 Figure S6. Chemical structure and 1 H NMR spectroscopy of ally α-d-galactose. 1 H NMR (400 MHz, D 2 O): 5.98 (dddd, 1H, J = 18 Hz, 10 Hz, 6 Hz, 6 Hz), 5.36 (dd, 1H, J = 17.3 Hz, 3.6 Hz), 5.25 (dd, 1H, J = 10 Hz, 3.2 Hz), 4.98 (d, 1H, J = 2.8 Hz), 4.22 (ddd, 1H, J = 12.8 Hz, 5.6 Hz, 1.8 Hz), 4.07 (ddd, 1H, J = 13.2 Hz, 6 Hz, 1.8 Hz), 3.96 (d, 1H, J =3.2 Hz ), 3.94 (dd, 1H, J = 8.2 Hz, 8.2 Hz), 3.85 (dd, 1 H, J = 10.4 Hz, 3.2 Hz), 3.81 (dd, 1 H, J = 10.6 Hz, 4 Hz), 3.72 (d, 2H, J = 6 Hz). 11

12 Figure S7. Chemical structure and 1 H NMR spectroscopy of ally β-d-galactose. 1 H NMR (400 MHz, D 2 O): 5.97 (dddd, 1H, J = 17.6 Hz, 11 Hz, 5.6 Hz, 5.6 Hz), 5.37 (dd, 1H, J = 17.2 Hz, 4 Hz), 5.27 (dd, 1H, J = 10.4 Hz, 3.2 Hz), 4.43 (d, 1H, J = 7.8 Hz), 4.38 (ddd, 1H, J = 12.4 Hz, 5.8 Hz, 1.8 Hz), 4.21 (ddd, 1H, J = 12.8 Hz, 6.4 Hz, 2.1 Hz), 3.90 (dd, 1H, J =3.6 Hz, 0.8 Hz), 3.77 (dd, 1H, J = 10.8 Hz, 7.8 Hz), 3.66 (dd, 1 H, J = 7.8 Hz, 1.2 Hz), 3.62 (dd, 1 H, J = 10.0 Hz, 7.4 Hz). 12

$3. Photographs of 2-D PCs and Debye Diffraction Ring Figure S8.$ $650 nm diameter; (b) Debye ring diffraction of 2-D PC array on$

13 3. Photographs of 2-D PCs and Debye Diffraction Ring Figure S8. (a) 2-D photonic crystals (PCs) made of PS colloidal spheres of 650 nm diameter; (b) Debye ring diffraction of 2-D PC array on the surface of a 2-D PC PAAm-AAc-Lactose-40 hydrogel sensor. 13

14 4. Calculations of limit of detection (LoD), dynamic range and linear range of the lectin protein sensors In this study, the limit of detection (LoD), dynamic range and linear range of the lectin protein sensors are determined with the following procedures: (1) Plot the curve of lectin concentration vs 2-D PC sensor particle spacing changes; (2) Linear fit (y l =a + bx) and nonlinear fit (y e =A*exp(-x/t) + y 0 ) the curve; (3) Limit of detection (LoD) is defined as 3 standard deviations, that is LoD=(a-3*( a)-a)/b; Limit of quantitation (LoQ) is defined as 10 standard deviations; (4) Dynamic range is defined as LoQ to 3 standard deviations from saturation, solve x from y 0 +3 y 0 =A*-exp(-x/t)+y 0 by using Matlab ; (5) Linear range, if defined as from LoD to where 5% deviate from linearity, solve x from (y e - y l )/y l =0.05 by using Matlab. 4.1 Calculations for the 2-D PC PAAm-AAc-Lactose sensor As shown in Figure S9, we did both linear fit and nonlinear fit for the response of the 2-D PC PAAm-AAc-Lactose-120 sensor to 2 ml ricin solutions. The results are shown as follows: A= ± 3.48, t= ± , y0= ± 2.74 (all points, R 2 =0.992) a = ± 3.13, b= ± (fitting from 0 to 0.05, R 2 =0.964) LoD=0.009 mg/ml= M=75 nm; Dynamic range is from to mg/ml, which equals that from M to M; Linear range is from to mg/ml, which equals that from M to M. 14

15 Figure S9. Ricin concentration dependence of 2-D PC PAAm-AAc-Lactose-120 hydrogel particle spacing changes. There are many other more sensitive methods for ricin detection. Interested readers are referred to a more detailed review on methods for ricin detection Calculations for the 2-D PC PAAm-AAc-Mannose sensor Figure S10. Con A concentration dependence of 2-D PC PAAm-AAc-Mannose-160 hydrogel particle spacing changes. 15

16 We did both linear fit and nonlinear fit for the curve of the response of the 2-D PC PAAm-AAc-Mannose-160 sensor to 2 ml Con A solutions (Figure S10). The results are shown as follows: A= ± 3.72, t=0.093 ± 0.005, y0= ± 2.74 (all points, R 2 =0.997) a = 2.39 ± 2.57, b= ± (fitting from 0 to 0.05, R 2 =0.993) LoD=0.004 mg/ml= M=38 nm Dynamic range is from to mg/ml, which equals that from M to M; Linear range is from to 0.044, which equals that from M to M. We summarized some methods for the detection of Con A as shown in Table S1. In general, comparing to these methods, our method is much simpler and less expensive. Table S1. Comparison of different methods for the detection of Con A Methods Linear range/nm Detection limit / nm References Colorimetry (silver) (gold) this study Electrochemistry Surface plasmon resonance Fluorimetry Calculations for the 2-D PC PAAm-AAc-α-Galactose sensor 16

17 Figure S11. Jacalin concentration dependence of 2-D PC PAAm-AAc-α-Galactose-160 hydrogel particle spacing changes. As shown in Figure S11, we did both linear fit and nonlinear fit for the curve of the response of our 2-D PC PAAm-AAc-α-galactose-160 sensor to 2 ml jacalin solutions. The results are shown as follows: A=110.11± 4.54, t=0.166 ± 0.018, y0= ± 3.99 (all points, R 2 =0.986) a = ± 4.13, b= ± (from 0 to 0.05, R 2 =0.902) LoD=0.015 mg/ml= M Dynamic range is from to mg/ml, which equals that from M to M; Linear range is from to mg/ml, which equals that from M to M. 17

18 5. References 1. Bozza, W. P.; Tolleson, W. H.; Rivera Rosado, L. A.; Zhang, B., Ricin detection: Tracking active toxin. Biotechnol. Adv. 2015, 33, Schofield, C. L.; Haines, A. H.; Field, R. A.; Russell, D. A., Silver and Gold Glyconanoparticles for Colorimetric Bioassays. Langmuir 2006, 22, Sugawara, K.; Kadoya, T.; Kuramitz, H., Electrochemical sensing of concanavalin A using a non-ionic surfactant with a maltose moiety. Anal. Chim. Acta 2014, 814, Liu, B.; Zhang, B.; Chen, G.; Yang, H.; Tang, D., Proximity Ligation Assay with Three-Way Junction-Induced Rolling Circle Amplification for Ultrasensitive Electronic Monitoring of Concanavalin A. Anal. Chem. 2014, 86, Babu, P.; Sinha, S.; Surolia, A., Sugar Quantum Dot Conjugates for a Selective and Sensitive Detection of Lectins. Bioconjugate Chem. 2007, 18, Huang, C.-F.; Yao, G.-H.; Liang, R.-P.; Qiu, J.-D., Graphene oxide and dextran capped gold nanoparticles based surface plasmon resonance sensor for sensitive detection of concanavalin A. Biosens. Bioelectron. 2013, 50, Chen, Q.; Wei, W.; Lin, J.-M., Homogeneous detection of concanavalin A using pyreneconjugated maltose assembled graphene based on fluorescence resonance energy transfer. Biosens. Bioelectron. 2011, 26, Pu, K.-Y.; Shi, J.; Wang, L.; Cai, L.; Wang, G.; Liu, B., Mannose-Substituted Conjugated Polyelectrolyte and Oligomer as an Intelligent Energy Transfer Pair for Label-Free Visual Detection of Concanavalin A. Macromolecules 2010, 43, Liu, H.; Bai, Y.; Qin, J.; Chen, Z.; Feng, F., A novel fluorescent concanavalin A detection platform using an anti-concanavalin A aptamer and graphene oxide. Anal. Methods 2017, 9, Lim, K. R.; Ahn, K.-S.; Lee, W.-Y., Detection of concanavalin A based on attenuated fluorescence resonance energy transfer between quantum dots and mannose-stabilized gold nanoparticles. Anal. Methods 2013, 5,

Supporting Information: Regioselective esterification of vicinal diols on monosaccharide derivatives via

Supporting Information: Regioselective esterification of vicinal diols on monosaccharide derivatives via Mitsunobu reactions. Guijun Wang,*Jean Rene Ella-Menye, Michael St. Martin, Hao Yang, Kristopher