Supporting Information (34 pages)

Transcription

1 Supporting Information (34 pages) Benzoboroxoles as Efficient Glycopyranoside-Binding Agents in Physiological Conditions: Structure and Selectivity of Complex Formation Marie Bérubé, Meenakshi Dowlut, Dennis G. Hall* Department of Chemistry, University of Alberta, Edmonton, Alberta, T6G 2G2, Canada Table of Contents S2 S2 S4 S4 S12 S14 S15 S20 S22 S24 S34 1. General experimental conditions 2. Qualitative ARS displacement assay 3. Methodology for K a measurements by 1 H NMR spectroscopy 4. Methodology for K a measurements by the three-component ARS quantitative assay 5. Electrospray mass spectrometry analysis of boronic acid/sugar complexes 6. Synthesis of methyl 3-deoxy-β-D-galactopyranoside 7. 1 H NMR studies showing the dynamic binding of boronic acids with glycopyranosides 8. Measurements of pk a 9. 1 H NMR spectra of new compounds synthesized 10. Atom coordinates from molecular modeling calculations (xyz files) 11. References S - 1

2 1. General experimental conditions Me was distilled over CaH 2. Alizarin red S was purchased from Aldrich and used as received. Me-α-D-fucopyranoside was purchased from IRIS Biotech GmbH (Marktredwitz, Germany). Methyl 6-deoxy-α-D-glucopyranoside 1, methyl 2-acetamido- 2-deoxy-α-D-galactopyranoside 2 and methyl-4-deoxy-α-d-glucopyranoside 3 were synthesized as previously reported. Synthesis of phenylmethylsilanediol (3) was reported elsewhere. 4 Analytical thin layer chromatography (TLC) were conducted on silica gel 60- F 254. Plates were visualized with a cerium ammonium molybdate (CAM) stain followed by heating. The water used for K a measurements and ESMS studies was distilled and further purified with a filtration system. Quartz cuvettes were used for the quantitative ARS assay. All data were plotted on Excel. 1 H and 13 C NMR experiments were recorded on 300, 400 or 500 MHz spectrometers. Chemical shifts were reported in δ (ppm) units using 13 C and residual 1 H signals from deuterated solvents. A TOF mass spectrometer provided the high resolution electrospray ionization mass spectra. 2. Qualitative ARS displacement assay The methodology used for screening the arylboronic acids shown in Figure 3 was described in the supporting information of our preliminary report. 1 In the case of boronic acid 1n, it was dissolved in a minimum of DMSO (a few drops) prior to add the ARS solution. S - 2

3 New assays (ph 7.4): A1 B1 A2 C1 B2 C2 Figure 1S. Vials of ARS (10-4 M in 0.1 M phosphate buffer) and phenylboronic acid (A, 0.01 M), boronic acid 1m (B, 0.01 M) or boronic acid 1n (C, 0.01 M) containing: 1, no sugar; 2, Me-α-D-Glc (0.50 M). Note: A slightly more orange solution is observed for vial B2 compared to all of the other solutions. A B C D Figure 2S. Vials of ARS (10-4 M in 0.1 M phosphate buffer) and boronic acid 2 (0.01 M) containing: A, no sugar; B, Me-α-D-Gal (0.25 M); C, Me-α-D-Fuc (0.25 M); D, Me-αD-Glc (0.25 M). S-3

4 A B C D E Figure 3S. Vials of ARS (10-4 M in 0.1 M phosphate buffer) and boronic acid 1m (0.01 M) containing: A, no sugar; B, Me-α-D-Gal (0.25 M); C, Me-β-D-Gal (0.25 M); D, Meα-D-Fuc (0.25 M); E, Me-3-deoxy-β-D-Gal (0.25 M). Note: Vial E, containing Me-3-deoxy-β-D-Gal is less orange-pink than vials B, C and D. 3. Methodology for Ka measurements by 1H NMR spectroscopy The methodology used for Ka measurements reported in Table 1 was described in the supporting information of our preliminary report.1 4. Methodology for Ka measurements by the three-component ARS quantitative assay (Table 2) The equations used for the determination of Ka by the three-component ARS assay were described in the supporting information of our preliminary report.1 Ka of boronic acid 1m and ARS (KARS) Following the procedure of Wang and co-workers.5 a mm ARS solution was prepared in 0.1 M phosphate buffer at ph 7.4. The solution needs to be sonicated for 2-3 h to obtain complete dissolution of the ARS in the phosphate buffer. A solution of 1m (15 mm) in the ARS solution was prepared in a volumetric flask and adjusted to ph 7.4. By mixing this boronic acid solution with the ARS solution together in the UV cuvette, a range of boronic acid concentrations (1.2-4 mm) was obtained. The UV absorbance of S-4

5 each solution was taken at 450 nm and plotted to determine the K ARS. At least two experiments were carried out to determine an average value of K ARS. (Average value of K ARS of 1m used: 1180 M -1.) K ARS of 1m 4,5 4,3 4,1 3,9 y = 0,0021x + 2,5256 R 2 = 0,9987 3,7 1/!A 3,5 3,3 3,1 K A RS: 1203 M -1 2,9 2,7 2, /[S] K a of boronic acid 1m and α-d-glucose (entry 1) A solution of 1m (3.1 mm) in ARS solution (0.144 mm in 0.1 M phosphate buffer) was prepared in a volumetric flask and adjusted to ph 7.4. Then, a portion of this solution was used to make a 2.0 M α-d-glucose solution at ph 7.4. By mixing the two solutions together in the UV cuvette, a range of sugar concentrations ( M) was obtained. The UV absorbance of each solution was taken at 454 nm and plotted to determine the K a. A plot of [S]/P versus Q is constructed in where Q = [RI]/[I] = (A RI A)/ (A A I ), where R is the receptor (1m), A is measured absorbance, A RI is absorbance of the receptor-indicator complex, and A I is absorbance of free indicator (ARS). P = [R] 1/(QK ARS ) [I 0 ]/(Q+1), where [I 0 ] is total indicator concentration (ARS). The Ka is given by [S]/P = K ARS /K a Q + 1, where [S] is sugar concentration. S - 5

6 Ka of 1m with!-d-glucose y = 38,998x + 34,361 R 2 = 0,9944 [S]/P K a = 31 M ,5 1 1,5 2 2,5 3 Q K a of boronic acid 1m and methyl-αd-glucopyranoside (entry 2) A solution of 1m (3.1 mm) in ARS solution (0.144 mm in 0.1 M phosphate buffer) was prepared in a volumetric flask and adjusted to ph 7.4. Then, a portion of this solution was used to make a 2.0 M methyl-α-d-glucopyranoside solution at ph 7.4. By mixing the two solutions together in the UV cuvette, a range of sugar concentrations ( M) was obtained. The UV absorbance of each solution was taken at 454 nm and plotted to determine the K a. Ka of 1m with methyl-! -D-glucopyranoside y = 53,748x + 389,11 R 2 = 0, [S]/[P] 410 Ka = 22 M ,6 0,7 0,8 0,9 1 1,1 1,2 1,3 Q S - 6

7 K a of boronic acid 1m and methyl-β-d-glucopyranoside (entry 3) A solution of 1m (3.1 mm) in ARS solution (0.144 mm in 0.1 M phosphate buffer) was prepared in a volumetric flask and adjusted to ph 7.4. Then, this solution was used to make a 2.0 M methyl-β-d-glucopyranoside solution at ph 7.4. By mixing the two solutions together in the UV cuvette, a range of sugar concentrations ( M) was obtained. The UV absorbance of each solution was taken at 454 nm and plotted to determine the K a. 600 K a of 1m with methyl-! -D-glucopyranoside 550 y = 121,77x + 405,48 R 2 = 0,92 S/[P] Ka = 9.7 M ,6 0,7 0,8 0,9 1 1,1 1,2 Q K a of boronic acid 1m and methyl-α-d-galactopyranoside (entry 4) A solution of 1m (3.1 mm) in ARS solution (0.144 mm in 0.1 M phosphate buffer) was prepared in a volumetric flask and adjusted to ph 7.4. Then, a portion of this solution was used to make a 1.8 M methyl-α-d-galactopyranoside solution at ph 7.4. By mixing the two solutions together in the UV cuvette, a range of sugar concentrations ( M) was obtained. The UV absorbance of each solution was taken at 453 nm and plotted to determine the K a. S - 7

8 Ka of 1m with methyl-!-d-galactopyranoside y = 41,277x + 99,377 R 2 = 0,9927 [S]/P K a : 29 M ,5 0,7 0,9 1,1 1,3 1,5 1,7 1,9 2,1 2,3 2,5 Q K a of boronic acid 1m and methyl-β-d-galactopyranoside (entry 5) A solution of 1m (3.1 mm) in ARS solution (0.144 mm in 0.1 M phosphate buffer) was prepared in a volumetric flask and adjusted to ph 7.4. Then, a portion of this solution was used to make a 1.8 M methyl-β-d-galactopyranoside solution at ph 7.4. By mixing the two solutions together in the UV cuvette, a range of sugar concentrations ( M) was obtained. The UV absorbance of each solution was taken at 454 nm and plotted to determine the K a. Ka of 1m with methyl-!-d-galactopyranoside y = 50,44x + 88,937 R 2 = 0,9906 [S]/P K a = 23 M ,5 0,7 0,9 1,1 1,3 1,5 1,7 1,9 2,1 Q S - 8

9 K a of boronic acid 1m and methyl-αd-mannopyranoside (entry 6) A solution of 1m (3.1 mm) in ARS solution (0.144 mm in 0.1 M phosphate buffer) was prepared in a volumetric flask and adjusted to ph 7.4. Then, a portion of this solution was used to make a 1.2 M methyl-α-d-mannopyranoside solution at ph 7.4. By mixing the two solutions together in the UV cuvette, a range of sugar concentrations ( M) was obtained. The UV absorbance of each solution was taken at 454 nm and plotted to determine the K a. Ka of 1m with methyl-!-d-mannopyranoside y = 48,44x + 81,582 R 2 = 0, [S]/P 160 K a = 24 M ,5 1 1,5 2 2,5 3 3,5 Q K a of boronic acid 1m and methyl-α-d-fucopyranoside (entry 7) A solution of 1m (3.1 mm) in ARS solution (0.144 mm in 0.1 M phosphate buffer) was prepared in a volumetric flask and adjusted to ph 7.4. Then, a portion of this solution was used to make a 1.0 M methyl-αd-fucopyranoside solution at ph 7.4. By mixing the two solutions together in the UV cuvette, a range of sugar concentrations ( M) was obtained. The UV absorbance of each solution was taken at 454 nm and plotted to determine the K a. S - 9

10 Ka of 1m with methyl-!-d-fucopyranoside y = 46,639x + 115,13 R 2 = 0, [S]/P K a : 25 M ,9 1,1 1,3 1,5 1,7 1,9 2,1 Q K a of boronic acid 2 and ARS (K ARS ) Following the procedure of Wang and co-workers. 5 a mm ARS solution was prepared in 0.1 M phosphate buffer at ph 7.4. The solution needs to be sonicated for 2-3 h to obtain complete dissolution of the ARS in the phosphate buffer. A solution of 2 (15 mm) in the ARS solution was prepared in a volumetric flask and adjusted to ph 7.4. By mixing this boronic acid solution with the ARS solution together in the UV cuvette, a range of boronic acid concentrations ( mm) was obtained. The UV absorbance of each solution was taken at 453 nm and plotted to determine the K ARS. K ARS of 2 5 4,5 y = 0,0008x + 2,1642 R 2 = 0, /!A 3,5 3 K A RS: 2705 M -1 2, /[S] S - 10

11 K a of boronic acid 2 and methyl-α-d-galactopyranoside A solution of 2 (3.1 mm) in ARS solution (0.144 mm in 0.1 M phosphate buffer) was prepared in a volumetric flask and adjusted to ph 7.4. Then, a portion of this solution was used to make a 1.8 M methyl-αd-galactopyranoside solution at ph 7.4. By mixing the two solutions together in the UV cuvette, a range of sugar concentrations ( M) was obtained. The UV absorbance of each solution was taken at 457 nm and plotted to determine the K a. Ka of 2 with methyl-!-d-galactopyranoside y = 101,92x + 53,1 R 2 = 0, [S]/P 100 K a = 27 M ,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1 1,1 Q K a of boronic acid 2 and methyl-α-d-fucopyranoside A solution of 2 (3.1 mm) in ARS solution (0.144 mm in 0.1 M phosphate buffer) was prepared in a volumetric flask and adjusted to ph 7.4. Then, a portion of this solution was used to make a 1.0 M methyl-α-d-fucopyranoside solution at ph 7.4. By mixing the two solutions together in the UV cuvette, a range of sugar concentrations ( M) was obtained. The UV absorbance of each solution was taken at 457 nm and plotted to determine the K a. S - 11

12 Ka of 2 with methyl-!-d-fucopyranoside y = 160,58x + 75,768 R 2 = 0,9969 [S]/P K a = 17 M ,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 Q 5. Electrospray mass spectrometry analysis of boronic acid/sugars complexes Samples preparation: (a) Boronic acid 1m (13 mg, 0.1 mmol) and Me-α-D-Gal (2.1 mg, 0.01 mmol) were weighed in a vial and dissolved in deionized water (0.5 ml) and HPLC grade CH 3 CN (0.5 ml). The sample was analyzed by low resolution electrospray mass spectrometry. (b) Boronic acid 1m (13 mg, 0.1 mmol) and mannitol (1.8 mg, 0.01 mmol) were weighed in a vial and dissolved in deionized water (0.5 ml) and HPLC grade CH 3 CN (0.5 ml). The sample was analyzed by low resolution electrospray mass spectrometry. (c) Boronic acid 1m (13 mg, 0.1 mmol) and lactulose (3.4 mg, 0.01 mmol) were weighed in a vial and dissolved in deionized water (0.5 ml) and HPLC grade CH 3 CN (0.5 ml). The sample was analyzed by low resolution electrospray mass spectrometry. S - 12

13 Electrospray mass spectra of boronic acid/sugar complexes (with 1m) (a) x O B + Me-!D-Gal 1: 1 complex M = m Me-α-D-Gal 1m/Me-α-D- Gal 1:1 complex (M= 309.1) (b) O B + mannitol 1: 1 complex M = :1 complex -2.H 2 O M = x 1m/mannitol 1:1 complex (M= 297.1) 1m mannitol 1m/mannitol 2:1 complex (M= 413.2) S - 13

14 (c) O B + lactulose 1: 1 complex M = :1 complex -2.H 2 O M = x 1m 1m/lactulose 1:1 complex (M= 457.1) 1m/lactulose 2:1 complex (M= 573.2) Figure 4S. Low resolution electrospray mass spectrometry of boronic acid 1m (0.1 mmol) with (a) Me-α-D-Gal (0.01 mmol) (b) mannitol (0.01 mmol) or (c) lactulose (0.01 mmol). 6. Synthesis of methyl 3-deoxy-β-D-galactopyranoside (4) Ph HO HO O OMe ref 6 O O O OAc OMe Ac/H 2 O 50 o C, 3 h 83% HO O OAc OMe MeONa, Me rt, 16 h 98% HO O OMe Methyl 2-O-acetyl-3-deoxy-β-D-galactopyranoside (6): Methyl 2-O-acetyl-4,6-O-benzylidene-3-deoxy-β-D-galactopyranoside (5) 6 (682 mg, 2.21 mmol) was dissolved in Ac (35 ml) and H 2 O (9 ml). The mixture was stirred at 50 C for 3 h. Then, the solvent was evaporated under reduced pressure and the mixture S - 14

15 was co-evaporated with toluene (3x 40 ml). The crude was purified on SiO 2 in DCM with Me (2 to 10%) to afford the titled compound (404 mg, 1.83 mmol, 83% yield) as a white solid. [αd] (c = 1.03 in CHCl 3 ); IR (CHCl 3 cast film Microscope, cm -1 ) 3424, 2939, 1737, 1374, 1245, 1047; 1 H NMR (400 MHz, CDCl 3 ) δ 1.64 (ddd, J = 3.2, 11.4, 13.5 Hz, 1H), 2.33 (ddd, J = 3.4, 5.3, 13.5 Hz, 1H), 2.05 (s, 3H), 3.51 (s, 3H), 3.60 (dt, J = 1.5, 5.0 Hz, 1H), 3.88 (d, J = 5.0 Hz, 2H), 4.04 (dt, J = 1.4, 3.2 Hz, 1H), 4.39 (d, J = 7.8 Hz, 1H), 5.01 (ddd, J = 5.2, 7.7, 11.4 Hz, 1H); 13 C NMR (100 MHz, CDCl 3 ) δ 170.1, 103.6, 77.1, 67.9, 66.9, 62.8, 56.6, 35.5, 21.2; HRMS (ESI, m/z) calcd for C 9 H 16 O 6 Na , found Methyl 3-deoxy-β-D-galactopyranoside (4): A freshly prepared solution of MeONa (1 M in Me, 0.11 ml, 0.11 mmol) was added to compound 6 (246 mg, 1.12 mmol) dissolved in dry Me (6 ml) under N 2. The reaction mixture was stirred at room temperature overnight. Then, Amberlite IR 120 H+ resin (200 mg) was added to neutralize the solution and the mixture was stirred 30 min. The reaction was filtered and the filtrate was evaporated to dryness to afford the 3-deoxy sugar (196 mg, 1.10 mmol, 98% yield) as a white solid. [αd] (c = 0.54 in H 2 O), [lit. 7 [αd] 67 (c = 0.5 in H 2 O)]; IR (Microscope, cm -1 ) 3274, 2448, 1094, 1052, 1025; 1 H NMR (400 MHz, D 2 O) δ 1.71 (ddd, J = 2.4, 10.0, 11.2 Hz, 1H), 2.18 (ddd, J = 2.4, 4.2, 11.0 Hz, 1H), 3.55 (s, 3H), (m, 4H), 3.96 (t, J = 2.4 Hz, 1H), 4.29 (d, J = 6.8 Hz, 1H); 13 C NMR (125 MHz, CDCl 3 ) δ 106.1, 78.6, 66.0, 65.7, 61.5, 57.1, 37.4; HRMS (ESI, m/z) calcd for C 7 H 14 O 5 Na , found H NMR studies showing the dynamic binding of boronic acids with glycopyranosides Sample preparation with boronic acid 1m The deuterated buffer solution was prepared by evaporating off an aqueous solution of 0.1 M phosphate monobasic solution (at ph 7.4) three times with D 2 O, and again the ph S - 15

16 was adjusted to 7.4 (with 4N Na or saturated sodium phosphate monobasic solution). Due to the hygroscopic nature of D 2 O, a certain amount of water as H 2 O was always present in 1-2%. Boronic acid 1m (0.06 mmol, 8 mg) was dissolved in deuterated phosphate buffer (6 ml) in order to prepare a 0.01 M solution. To 0.7 ml of the boronic acid solution was added 25 equiv of carbohydrates (0.175 mmol) [Me-αD-Gal (37 mg); Me-αD-Fuc (32 mg); Me-4-deoxy-αD-Glc (32 mg); Me-3-deoxy-βD-Gal (32 mg); Me-2- deoxy-αd-galnac (41 mg)]. 1 H NMR spectra was taken on i500 NMR and peak broadening in the aromatic part were compared to the spectra without added carbohydrate (see Figure 6). Sample preparation with boronic acid 2 Boronic acid 2 (0.05 mmol, 10.6 mg) was dissolved in deuterated phosphate buffer as described above (5 ml) in order to prepare a 0.01 M solution. To 0.7 ml of the boronic acid solution was added 25 equiv of sugars (0.175 mmol) [Me-αD-Gal (37 mg); Me-αD- Fuc (31 mg)]. 1 H NMR spectra was taken on i500 NMR and peak broadening in the aromatic part were compared to the spectra without added carbohydrate (see below). S - 16

17 (a) boronic acid 2 alone B() 2 HOOC NO 2 (b) with methyl-α-d-galactopyranoside (K a 27 M -1 ) HO O OMe (c) with methyl-α-d-fucopyranoside (K a 17 M -1 ) HO Me O OMe Figure 5S. 1 H NMR spectra of boronic acid 2 in phosphate buffer D 2 O (0.01 M) at ph 7.4, alone (a) or with 0.25 M of different hexopyranosides (b-c). S - 17

18 Sample preparation with boronic acid 1n Boronic acid 1n (0.05 mmol, 7.4 mg) was dissolved in a minimum of DMSO and deuterated phosphate buffer, as described above (5 ml), was added in order to prepare a 0.01 M solution. To 0.7 ml of the boronic acid solution was added 25 equiv of Me-α-D- Glc (0.175 mmol, 34 mg). 1 H NMR spectra was taken on i500 NMR and peaks broadening in the aromatic part were compared to the spectra without added carbohydrate (Figure 6S). (a) boronic acid 1n alone O B (b) with methyl-α-d-glucopyranoside HO HO O OMe Figure 6S. 1 H NMR spectra of boronic acid 1n in phosphate buffer D 2 O (0.01 M) at ph 7.4, alone (a) or with 0.25 M of Me-α-D-Glc (b). S - 18

19 Sample preparation with phenylboronic acid Phenylboronic acid (0.05 mmol, 6.1 mg) was dissolved in a minimum of DMSO and deuterated phosphate buffer, as described above (5 ml), was added in order to prepare a 0.01 M solution. To 0.7 ml of the boronic acid solution was added 25 equiv of Me-α-D- Glu (0.175 mmol, 34 mg). 1 H NMR spectra was taken on i500 NMR and peak broadening in the aromatic part were compared to the spectra without added carbohydrate (Figure 7S). (a) phenylboronic acid alone HO B (b) with methyl-α-d-glucopyranoside HO HO O OMe Figure 7S. 1 H NMR spectra of phenylboronic acid in phosphate buffer D 2 O (0.01 M) at ph 7.4, alone (a) or with 0.25 M of Me-α-D-Glc (b). S - 19

20 8. Measurements of pk a pk a of benzoboroxole 1m (7.2) The measurement of pk a of benzoboroxole 1m was reported in our preliminary report. 1 pk a of benzoboroxole 1n (8.4) by 11 B NMR titration Phosphate buffer solution: In a volumetric flask (50 ml), 690 mg of NaH 2 PO 4 were suspended in 5 ml of D 2 O. The flask was filled to 50 ml with H 2 O. Boronic acid solution: In a volumetric flask (25 ml), 59 mg of boronic acid 1n was dissolved in a minimum of DMSO. The flask was filled to 25 ml with the phosphate buffer solution (resulting solution: 16 mm of 1n in 0.1 M phosphate buffer; 90/10 H 2 O/D 2 O). Solution for 11 B NMR: 1 ml of the boronic acid solution was placed in a vial. This solution was adjusted to the desired ph with an aqueous Na solution and the 11 B NMR of this solution was recorded on a 400 MHz spectrometer and plotted (Figure 8S). pka of 1n B NMR ph Figure 8S. 11 B NMR chemical shifts of 1n with increasing ph (10% D 2 O in H 2 O, 16 mm in 0.10 M phosphate buffer). Reference to Et 2 O-BF 3 in deuterated chloroform as zero. (pk a of 1n: 8.4) S - 20

21 pk a of boronic acid 2 (7.0) by 11 B NMR titration Phosphate buffer solution: In a volumetric flask (50 ml), 690 mg of NaH 2 PO 4 were placed in 5 ml of D 2 O. The flask was filled to 50 ml with H 2 O. Boronic acid solution: In a volumetric flask (25 ml), 84 mg of boronic acid 2 was dissolved in the phosphate buffer solution and Na was added until a clear solution was obtained (resulting solution: 16 mm of 2 in 0.1 M phosphate buffer; 90/10 H 2 O/D 2 O, ph 5.0). Solution for 11 B NMR: 1 ml of the boronic acid solution was placed in a vial. This solution was adjusted to the desired ph with an aqueous Na solution and the 11 B NMR of this solution was recorded on a 400 MHz spectrometer and plotted (Figure 9S). 11 B NMR pk a of ph Figure 9S. 11 B NMR chemical shifts of 2 with increasing ph (10% D 2 O in H 2 O, 16 mm in 0.10 M phosphate buffer). Reference to Et 2 O-BF 3 in deuterated chloroform as zero. (pk a of 2: 7.0) S - 21

22 9. 1 H NMR spectra of new compounds synthesized Methyl 2-O-acetyl-3-deoxy-β-D-galactopyranoside (6): HO O OMe OAc 6 S - 22

23 Methyl 3-deoxy-β-D-galactopyranoside (4): HO 4 O OMe S - 23

24 10. Atom coordinates from molecular modeling calculations (xyz files) Figure 9 Structure I H C C C C C C B H H C H H O H O O C C H H H H H H S - 24

25 Structure II B C C C C C C H H H H H O O O H C C H H H H H H S - 25

26 Figure 10 A. galactopyranoside 3,4-complex, alpha-aryl O C C H H C C O O H C H O H C O O H C H B H H H H H C C C C C C C H H H H O H H S - 26

27 B. galactopyranoside 3,4-complex, beta-aryl O C C H H C C O O H C H O H C O O H C H B H H H H H C C C C C C C H H H H O H H S - 27

28 C. galactopyranoside 4,6-complex, alpha-aryl O C C H H C C O O H C H H C O O H C H H H H H B O H C C C C C C H C H H H H H O S - 28

29 D. galactopyranoside 4,6-complex, beta-aryl O C C H H C C O O H C H H C O O H C H H H H H C C C C C C H H C H H B O H H O H S - 29

30 E. glucopyranoside 3,4-complex, alpha-aryl O C C H H C C O H C H O H C O O H C H B O H H H H C C C C C C C H H H H O H H H S - 30

31 F. glucopyranoside 3,4-complex, beta-aryl O C C H H C C O H C H O H C O O H C H O H H H H C C C C C C H H H H H B C H H O S - 31

32 G. glucopyranoside 4,6-complex, alpha-aryl O C C H H C C O H C H H C O O H C H H H H C C C C C C H H H O H H O H C H H O B S - 32

33 H. glucopyranoside 4,6-complex, beta-aryl O C C H H C C O H C H H C O O H C H H H H C C C C C C H H H O H H O H B C H H O S - 33

34 11. References (1) Dowlut, M.; Hall, D.G. J. Am. Chem. Soc. 2006, 128, (2) Abdallah, Z.; Doisneau, G.; Beau, J.M. Angew. Chem. Int. Ed. 2003, 42, (3) (a) Sinha, S.K.; Brew, K. Carbohydr. Res. 1980, 81, (b) Reist, E.J.; Spencer, R.R.; Calkins, D.F.; Baker, B.R.; Goodman, L. J. Org. Chem. 1965, 30, (c) Bock, K.; Duus, J. J. Carb. Chem. 1994, 13, (4) Anderson, T.F.; Statham, M.A.J.; Carroll, M.A. Tetrahedron Lett. 2006, 47, (5) Springsteen, G.; Wang, B. Tetrahedron 2002, 58, (6) Kihlberg, J.; Frejd, T.; Jansson, K.; Magnusson, G. Carbohydr. Res. 1986, 152, (7) Lin, T.H.; Kovac, P.; Glaudemans, C.P.J. Carbohydr. Res. 1989, 188, S - 34