A stochastic, cantilever approach to the evaluation of solution phase thermodynamic quantities

Snyder et al. 10.1073/pnas.0606604104.

Supporting Information

Files in this Data Supplement:

SI Text
SI Figure 6
SI Figure 7




Fig. 6. Calorimetry data and fit. A solution of b-methyl lactose was titrated into a solution of galectin 3 at 25°C. The raw data of heat vs. volume of injectant is integrated to give the above set of points (black points). A nonlinear least squares fit of these points (red line) gives the association constant.





Fig. 7. Force-frequency histogram at 10-3 M soluble lactose.





SI Text

Isothermal Titration Microcalorimetry

Calorimetric measurements were made with a VP-ITC (Microcal) with cell volume 1.4346 ml at 25.0°C. Titration of galectin-3 (0.198 mM) was carried out at pH 8.50 in 10 mM Tris, 1 mM EDTA, 100 mM b-mercaptoethanol. A 2.14 mM solution of b-methyl lactoside in identical buffer was added by 15.0 ml injections, 3.0 s in duration separated by 5 min intervals. The stir rate was set to 310 rpm and reference power was 25 mcal sec-1. Data were integrated to provide a titration curve and, by use of a nonlinear least-squares fit assuming one binding site, the binding constant K and the enthalpy of binding were extracted.

Synthesis of Maleimido-Disulfide

Undecen-11-yl-tetra(ethylene glycol)-methoxymethyl acetal. To a stirred solution of Undecen-11-yl-tetra(ethylene glycol) (2.35 g, 6.8 mmol) in anhydrous dichloromethane (60 ml) were added chloromethyl methyl ether (1.63 ml, 3.0 equiv.) and diisopropylethylamine (2.3 ml, 2 equiv). The reaction was left to stir at room temperature for 16 hours and concentrated at reduced pressure. The residue was resuspended in water (100 ml) and extracted twice with dichloromethane (100 ml). The combined extracts were washed brine (125 ml), dried over MgSO4, and concentrated at reduced pressure. The residual oil (2.65 g, > 95%) was used without further purification. 1H NMR (300 MHz, CDCl3) d 5.80 (m, 1H), 4.93 (m 2H), 4.66 (s, 2H), 3.77 - 3.54 (m, 16H), 3.42 (t, J = 7.0 Hz, 2H), 3.37 (s, 3H), 2.04 (m, 2H), 1.57 (m, 2H), 1.28 (bs, 12H); 13C NMR (75 MHz, CDCl3) d 139.1, 114.0, 96.5, 71.4, 70.5, 70.4, 70.0, 66.7, 55.1, 33.7, 29.5, 29.4, 29.3, 29.0, 28.8, 26.0; IR (cm-1) 2926, 1640, 1463, 1350, 1213, 1112, 1043, 918; HRMS (CI) calculated for C21H42O6 (M+) 390.289, found 345.263 (M+ - MOM).

S-acetyl 1-mercaptoundecan-11-yl-tetra(ethylene glycol)-methoxymethyl acetal. A solution of Undecen-11-yl-tetra(ethylene glycol)-methoxymethyl acetal (2.65 g, 6.8 mmol) in anhydrous tetrahydrofuran (20 ml) was transferred to a quartz flask and sparged with argon gas for 20 minutes with stirring. Azobisisobutyrylnitrile (~50 mg) and thiolacetic acid (1.9 ml) were added and the solution was irradiated in a Rayonettephotochemical reactor for 6 hours and concentrated. The thioacetate was purified via column chromatography (SiO2, gradient of dichloromethane to 1.5% methanol in dichloromethane) to give a yellow oil (2.10 g, 66%). 1H NMR (300 MHz, CDCl3) 4.66 (s, 2H), 3.70 - 3.58 (m, 16H), 3.44 (t, J = 6.8 Hz, 2H), 3.37 (s, 3H), 2.86 (t, J = 7.3 Hz, 2H), 2.32 (s, 3H), 1.57 (m, 4H), 1.27 (bs, 14H); 13C NMR (75 MHz, CDCl3) d 195.8, 96.4, 71.4, 70.5, 70.4, 70.0, 66.7, 55.0, 33.7, 30.5, 29.5, 29.3, 29.0, 28.8, 28.7, 26.0; IR (cm-1) 2926, 2855, 1693, 112, 1042; HRMS (CI) calculated for C23H46O7S (M+) 466.296, found 421.262 (M+ - MOM).

1-mercaptoundecan-11-yl-tetra(ethylene glycol)-methoxymethyl acetal. A solution of S-acetyl 1-mercaptoundecan-11-yl-tetra(ethylene glycol)-methoxymethyl acetal (2.08 g, 4.4 mmol) in methanol (20 ml) and triethylamine (10 ml) was sparged with argon gas for 30 minutes. A 25% (w/w) solution of sodium methoxide in methanol (2.04 ml, 2 equiv) was added and the reaction was stirred at room temperature for 6 hours. Subsequent quenching with acidic resin, filtration and concentration at reduced pressure provided the thiol as an odorous oil (1.86 g, >95%), which was used without further purification. 1H NMR (300 MHz, CDCl3) d 4.66 (s, 2H), 3.71 - 3.56 (m, 16H), 3.44 (t, J = 6.3 Hz, 2H) 3.37 (s, 3H), 2.52 (m, 2H), 1.58 (m, 5H), 1.27 (m, 14H); 13C NMR (75 MHz, CDCl3) d 96.4, 71.4, 70.5, 70.0, 66.7, 55.06, 33.9, 33.7, 29.5, 29.4, 29.0, 28.3, 26.0, 24.5; IR (cm-1) 2926, 2855, 1112, 1042, 918.

(Pyridin-2-yl)disulfanyl-11-undecyl-tetra(ethylene glycol)methoxymethyl acetal. Aldrithiol (1.67 g, 7.6 mmol) and 1-mercaptoundecan-11-yl-tetra(ethylene glycol)-methoxymethyl acetal (1.67, 3.9 mmol) were dissolved in methanol (30 ml) and triethylamine (1.2 ml) was added. The reaction was stirred at room temperature for 12 hours and concentrated. The residual yellow oil was chromatographed (SiO2, gradient of dichloromethane to 3% methanol in dichloromethane) to provide the pyridyl disulfide as a yellow oil (710 mg, 33%). 1H NMR (300 MHz, CDCl3) d 8.46 (d, J = 4.4 Hz, 1H), 7.74 (d, J = 7.1 Hz, 1H), 7.64 (dd, J = 7.0 Hz, J = 1.0 Hz, 1H), 7.07 (dd, J = 4.2 Hz, J = 1Hz, 1H), 4.66 (s, 2H), 3.72 - 3.56 (m, 16 H), 3.44 (t, J = 6.9 Hz, 2H), 3.37 (s, 3H), 2.79 (t, J = 7.4 Hz), 1.69 (m, 2H), 1.57 (m, 2H), 1.25 (bs, 14 H); 13C NMR (75 MHz, CDCl3) d 183.1, 149.7, 136.9, 120.3, 120.0, 96.5, 71.5, 70.6, 70.5, 70.0, 66.7, 55.1, 53.4, 39.1, 29.6, 29.5, 29.2, 28.5, 26.1; IR (cm-1) 2925, 2854, 1112, 1042; MS (MALDI) calculated for C26H47O6S2 (M+) 533.284, found 556.1 [(M+Na)+], (FAB+) found 534.3 ([(M+H)+].

Synthesis of Methoxymethyl Acetal protected Acid Disulfide. To a solution of mercaptoundecan-11-ylhexa(ethylene glycol)acetic acid (1) ( 187 mg, 0.35 mmol) in tetrahydrofuran were added (pyridin-2-yl)disulfanyl-11-undecyl-penta(ethylene glycol)methoxymethyl acetal (227 mg, 1.2 equiv) and p-dimethylaminopyridine (52 mg, 1.2 equiv). The reaction was allowed to stir at room temperature for 8 hours and was concentrated at reduced pressure. The residual oil was chromatographed (SiO2, 9:1:0.50 dichloromethane/ methanol/ acetic acid). Fractions containing the desired disulfide were concentrated carefully to approximately 5 ml, then diluted with toluene (20 ml) and this process was repeated twice to remove residual acetic acid. The desired disulfide was recovered as a clear oil (25 mg, 7.5%). 1H NMR (300 MHz, CDCl3) d 4.65 (s, 2H), 3.93 (s, 2H), 3.71 - 3.57 (m, 40 H), 3.43 (2 t, 4H), 3.36 (s, 3H), 2.67 (t, J = 7 Hz, 4H), 1.66 (m, 4H), 1.57 (m, 4H), 1.27 (bs, 28H); MS (ESI+) calculated for C46H92O15S2 (M+) 948.59, found 972.5 [(M+Na)+] and 993.6 [(M+2Na)+], (ESI-) found 947.6([(M-H)-].

Synthesis of Methoxymethyl Acetal protected Maleimido Disulfide. The acid disulfide (25 mg, 26 mmol) was dissolved in dichloromethane (1 ml) and N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide (7 mg, 1.4 equiv) and 2-aminoethyl maleimide trifluoroacetic acid salt (9 mg, 1.4 equiv) were added. The reaction was stirred at room temperature overnight and chromatographed (SiO2, 9:1 dichloromethane/ methanol) to provide the desired maleimido disulfide (12 mg, 43%). 1H NMR (300 MHz, CDCl3) d 7.39 (bt, 1H), 6.71 (s, 2H), 4.66 (s, 2H), 3.93 (s, 2H), 3.72 - 3.57 (m, 40H), 3.50 - 3.40 (m, 4H), 3.36 (s, 3H), 2.68 (t, J = 7.2 Hz, 4H), 1.65 (m, 4H), 1.57 (m, 4H), 1.27 (bs, 28H); 13C NMR (75 MHz, CDCl3) d 170.8, 165.2, 134.2, 96.5, 71.5, 70.9, 70.5, 70.3, 70.0, 66.8, 55.2, 39.1, 37.8, 37.6, 29.5, 29.2, 28.5, 26.1; MS (MALDI) calculated for C52H98O16S2 (M+) 1070.64, found 1094.0 [(M+Na)+].

Synthesis of maleimido disulfide. The methoxymethyl acetal protected maleimido disulfide (12 mg, 11mmol) was dissolved in methanol (1 ml) and concentrated hydrochloric acid (2 ml) was added. The reaction was allowed to stir at room temperature for 3 hours and was concentrated at reduced pressure to provide the desired maleimido disulfide (11 mg, >95%). 1H NMR (300 MHz, CDCl3) d 6.70 (s, 2H), 3.94 (s, 2H), 3.71 - 3.41 (m, 48 H), 2.68 (t, J = 7.1 Hz, 4H), 1.70 - 1.52 (m, 8H), 1.27 (bs, 28H); IR (cm-1) 3391, 2923, 2853, 1711, 1455, 1260, 1101, 799; MS (MALDI) calculated for C50H94O15S2 (M+) 1026.61, found 1049.8 [(M+Na)+].

Synthesis of mercaptopentyl glycoside

Synthesis of peracetylated mercaptopentanyl mactoside. Peracetylated pentenyl lactoside (860 mg, 1.2 mmol) was dissolved in tetrahydrofuran (10 ml) and the solution was sparged with argon gas for 20 minutes. Azobisisobutyrylnitrile (~50 mg) and thiolacetic acid (220 ml) were added and the solution was irradiated in a Rayonette™ Photochemical reactor for 6 hours and concentrated. The thioacetate was purified via column chromatography (SiO2,1:1 hexane/ ethyl acetate) to provide the peracetylated S-acetyl mercaptopentanyl lactose-heptaacetate as a colorless oil (450 mg, 46%). 1H NMR (300 MHz, CDCl3) d 5.22 - 5.07 (m, 2H), 4.96 (dd, J = 3 Hz, J = 10.2 Hz, 1H), 4.87 (dd, J = 1.5 Hz, J = 7.8 Hz, 1H), 4.48 (m, 3H), 4.11 (m, 3H), 3.87 (m, 3H), 3.61 (dd, J = 3.2 Hz, J = 8.4 Hz, 1H), 3.46 (dt, J = 5.9 Hz, J = 9.4 Hz, 1H), 2.85 (t, J = 7.1 Hz, 2H), 2.32 (s, 3H), 2.15 (s, 3H), 2.12 (s, 3H), 2.08 (s, 3H), 2.06 (s, 3H), 2.05 (s, 9H), 1.96 (s, 3H), 1.57 (m, 3H), 1.39 (m, 2H), 1.26, (t, J = 6.9 Hz, 2H); 13C NMR (75 MHz, CDCl3) d 195.6, 170.2, 169.9, 169.8, 16936, 169.4, 168.9, 100.8, 100.3, 72.7, 72.4, 71.5, 70.8, 70.5, 69.5, 68.9, 66.5, 61.9, 60.7, 60.1, 30.4, 29.0, 28.7, 24.8, 20.8, 20.6, 20.4, 20.3, 14.0; IR (cm-1) 2940, 2256, 1750, 1686, 1431, 1370, 1213, 1076, 913, 733, 648; HRMS 780.25 (calc) 781.21 (found MH+).

Synthesis of mercaptopentanyl lactoside. Lactoside (233 mg, 0.52 mmol) was dissolved in methanol (2 ml) and sparged gently for 10 minutes. Sodium methoxide (25% in methanol) was added and the reaction was left to stir for 8 hours at room temperature. The solution was neutralized with the addition of acidic cation exchange resin, filtered and concentrated under reduced pressure. The residual oil was chromatographed (C-18 silica, 10% methanol in water) to provide the desired mercaptopentanyl lactoside (61 mg, 46%). 1H NMR (300 MHz, D2O) d 4.36 (m, 2H), 3.84 (m, 3H), 3.73 - 3.41 (m, 11H), 3.21 (t, J = 8.0 Hz, 2H), 2.68 (t, J = 6.8 Hz), 1.57 (m, 4H), 1.38 (m, 2H); 13C NMR (75 MHz, D2O) d103.1, 102.2, 78.6, 75.5, 74.9, 74.6, 73.0, 72.7, 71.1, 70.6, 68.7, 61.2, 60.3, 38.3, 28.5, 28.3, 24.2; MS (ESI) 444.17 (calc.), 467.3 (found M+Na+).

The radius of curvature of the tips used here was 50 nm and indentation depths were on the order of angstroms. Thus, the spherical cap - with radius of 50 nm, height of 0.3 nm - has a surface area of 94 nm2, and the approximate number of ligands displayed in this area can be calculated from structural data for SAMs. X-ray diffraction studies show that alkanethiolate adsorbed on gold have an area per chain of 0.2 nm2.(2) Using a ratio of 9:1 blank disulfide to maleimido-disulfide, we calculate a density of one ligand molecule per 4 nm2, which corresponds to 24 ligand molecules in the 94 nm2 spherical cap. We next determine the number of receptors covered by this spherical cap. In the original manuscript for the surface immobilization of histidine-tagged proteins, the authors report amounts of adsorbed protein consistent with a monolayer on the functionalized quartz slide.(3) The crystal structure of human galectin 3 (4) shows a roughly cylindrical protein with a diameter of approximately 3.1 nm; these dimensions translate into 1 molecule per 8 nm2. Hence, there is a nearly two fold excess of ligand to receptor per unit area, and the device should observe binding to a majority of these receptors, barring any geometric barriers to binding. Thus, for a probed surface area of 94 nm2, we expect to observe on the order of 13 binding events per contact.

1. Roberts C, Chen CS, Mrksich M, Martichonok V, Ingber DE, Whitesides GM (1998) J Am Chem Soc 120:6548-6555.

2. Strong L, Whitesides GM (1988) Langmuir 4:546-558.

3. Schmid EL, Keller TA, Dienes Z, Vogel H (1997) Anal Chem 69:1979-1985.

4. Seetharaman J, Kanigsberg A, Slaaby R, Leffler H, Barondes SH, Rini JM (1998) J Biol Chem 273:13047-13052.

This Article

  1. Published online before print February 16, 2007, doi: 10.1073/pnas.0606604104 PNAS February 20, 2007 vol. 104 no. 8 2579-2584
  1. AbstractFree
  2. Figures Only
  3. Full Text
  4. Full Text (PDF)
  5. » Supporting Information
About Our New Site Design >>
Link: Advanced Search

This Week's Issue

  1. July 22, 2008, 105 (29)
  1. Current Issue
  1. Alert me to new issues of PNAS

Most