S1

Supporting Information

Inhibiting Gas Hydrate Formation Using Small Molecule Ice Recrystallization Inhibitors

Devin Tonelli,a Chantelle J. Capicciotti,a Malay Doshi,a and Robert N. Ben,*a

a Department of Chemistry, D’Iorio Hall 110 Marie Curie, University of Ottawa Ottawa, ON, Canada K1N 6N5

Table of ContentsThermal Hysteresis (TH) Assay ................................................................................................S1

Ice Recrystallization Inhibition (IRI) Assay..............................................................................S1

Clathrate Hydrate Inhibition DSC Measurements.....................................................................S2

General Experimental Conditions .............................................................................................S2

Synthesis of n-octyl-β-D-glucopyranoside (1) and n-octyl-β-D-galactopyranoside (2).............S3

Synthesis of N-Alkyl-D-gluconoamides (3-5) ...........................................................................S4

Synthesis of Azasugar derivatives (6-8)....................................................................................S5

Spectral Data ...........................................................................................................................S14

References ...............................................................................................................................S31

Thermal Hysteresis (TH) AssayNanoliter osmometry was performed using a Clifton nanoliter osmometer (Clifton Technical Physics,Hartford, NY), as described by Chakrabartty and Hew.1 All of the measurements were performed in doubly distilled water. Ice crystal morphology was observed through a Leitz compound microscope equipped with an Olympus 20× (infinity-corrected) objective, a Leitz Periplan 32X photo eyepiece, and a Hitachi KPM2U CCD camera connected to a Toshiba MV13K1 TV/VCR system. Still images were captured directly using a Nikon CoolPix digital camera.

Ice Recrystallization Inhibition (IRI) AssaySample analysis for IRI activity was performed using the “splat cooling” method as previously described.2 In this method, the analyte was dissolved in phosphate buffered saline (PBS) solution and a10 μL droplet of this solution was dropped from a micropipette through a two meter high plastic tube (10cm in diameter) onto a block of polished aluminum precooled to approximately -80 °C. The droplet froze instantly on the polished aluminum block and was approximately 1 cm in diameter and 20 μm thick. This wafer was then carefully removed from the surface of the block and transferred to a cryostage held at -6.4 °C for annealing. After a period of 30 min, the wafer was photographed between crossed polarizing filters using a digital camera (Nikon CoolPix 5000) fitted to the microscope. A total of three images were taken from each wafer.

Electronic Supplementary Material (ESI) for RSC Advances.This journal is © The Royal Society of Chemistry 2015

S2

During flash freezing, ice crystals spontaneously nucleated from the supercooled solution. These initial crystals were relatively homogeneous in size and quite small. During the annealing cycle, recrystallization occurred, resulting in a dramatic increase in ice crystal size. A quantitative measure of the difference in recrystallization inhibition of two compounds X and Y is the difference in the dynamics of the ice crystal size distribution. Image analysis of the ice wafers was performed using a novel domain recognition software (DRS) program.3 This processing employed the Microsoft Windows Graphical User Interface to allow a user to visually demarcate and store the vertices of ice domains in a digital micrograph. The data was then used to calculate the domain areas. All data was plotted and analyzed using Microsoft Excel. The mean grain (or ice crystal) size (MGS) of the sample was compared to the MGS of the control PBS solution for that same day of testing. IRI activity is reported as the percentage of the MGS (% MGS) relative to the PBS control, and the % MGS for each sample was plotted along with its standard error of the mean. Large percentages represent a large MGS, which is indicative of poor IRI activity.

Clathrate Hydrate Inhibition DSC MeasurementsThrough differential scanning calorimetery (Setaram Inc, m-DSC VII) methane hydrate nucleation was observed. Samples were prepared by injecting 1 µL of test solution into approximately 1.8 mg of silica gel isolated in a 1 mm diameter borosilicate capillary tube. 12 capillaries containing identical samples were then loaded into a DSC cell and pressurized to 100 Barr under methane. Starting at 20 °C the DSC was cooled to -12 °C at -0.0085 °C/sec. The cell was then kept at -12 °C for 20 hours before being heated to 20 °C 0.0085 °C/sec. This trial is repeated three times sequentially, resulting in 36 trials for each test solution. Test solutions are prepared from 5 mM stock solutions and are tested at all desired concentrations.

General Experimental ConditionsAll anhydrous reactions were performed in flame-dried glassware under a positive pressure of dry argon. Air or moisture-sensitive reagents and anhydrous solvents were transferred with oven-dried syringes or cannulae. All flash chromatography was performed with E. Merck silica gel 60 (230-400 mesh). All solution phase reactions were monitored using analytical thin layer chromatography (TLC) with 0.2 mm pre-coated silica gel aluminum plates 60 F254 (E. Merck). Components were visualized by illumination with a short-wavelength (254 nm) ultra-violet light and/or staining (ceric ammonium molybdate, ninhydrin stain, potassium permanganate, or phosphomolybdate stain solution).

All solvents used for anhydrous reactions were distilled. Tetrahydrofuran (THF) and diethyl ether were distilled from sodium/benzophenone under nitrogen. Dichloromethane and acetonitrile were distilled from calcium hydride. N,N-dimethylformamide (DMF) was stored over activated 4Å molecular sieves under argon.

1H (300, 400 or 500 MHz) and 13C NMR (76 or 100 or 125 MHz) spectra were recorded at ambient temperature on a Bruker Avance 300, Bruker Avance 400, Bruker Avance 500, or Varian Inova 500 spectrometer. Deuterated chloroform (CDCl3), methanol (CD3OD), DMSO (DMSO-d6) or water (D2O) were used as NMR solvents, unless otherwise stated. Chemical shifts are reported in ppm downfield from trimethylsilane (TMS) or the solvent residual peak as an internal standard. Splitting patterns are designated as follows: s, singlet; d, doublet; t, triplet; q, quartet; quint, quintet; m, multiplet and br, broad. Low resolution mass spectrometry (LRMS)

S3

was performed on a Micromass Quatro-LC Electrospray spectrometer with a pump rate of 20 μL/min using electrospray ionization (ESI).

Synthesis of n-octyl-β-D-glucopyranoside (1) and n-octyl-β-D-galactopyranoside (2)

O

AcO

OAc

AcOR1 O

6

O

AcO

OAc

AcOR1 OAc

11:Glc: R1=OAc, R2=H12:Gal: R1=H, R2=OAc

HO

6BF3OEt2

CH2Cl2, 4Å MS, 0°C

O

HO

OH

HOR1 O

6

NaOMe/MeOH

13:Glc: R1=OAc, R2=H14:Gal: R1=H, R2=OAc

1:Glc: R1=OH, R2=H2:Gal: R1=H, R2=OH

R2R2 R2

n-Octyl-2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside (13)To a mixture of 1,2,3,4,6-Penta-O-acetyl-β-D-glucopyranose (11, 275 mg, 0.70 mmol), 1-octanol (215 μL, 1.36 mmol) and 4 Å MS in anhydrous CH2Cl2 (6 mL) stirring at 0 °C under Ar, was slowly added boron trifluoride diethyl etherate (160 μL, 1.27 mmol). The reaction mixture was stirred overnight, then diluted with CH2Cl2 and quenched with sodium bicarbonate. The solution was filtered through Celite®, then extracted with CH2Cl2. The organic layer was washed with sodium bicarbonate, water, saturated brine, then dried over MgSO4 and concentrated. Flash column chromatography (7:3 hexanes/EtOAc) afforded 13 as a white powder (103 mg, 32%). 1H NMR (400 MHz, CDCl3): δ 5.20 (t, J = 9.5 Hz, 1H), 5.09 (t, J = 9.7 Hz, 1H), 4.98 (dd, J = 9.6, 8.0 Hz, 1H), 4.49 (d, J = 8.0 Hz, 1H), 4.26 (dd, J = 12.3, 4.7 Hz, 1H), 4.13 (dd, J = 12.3, 2.5 Hz, 1H), 3.87 (dt, J = 9.6, 6.4 Hz, 1H), 3.67 (ddd, J = 10.0, 4.7, 2.5 Hz, 1H), 3.47 (dt, J = 9.6, 6.8 Hz, 1H), 2.09 (s, 3H), 2.04 (s, 3H), 2.02 (s, 3H), 2.01 (s, 3H), 1.64-1.48 (m, 2H), 1.34-1.21 (m, 10H), 0.88 (t, J = 6.9 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ 170.7, 170.3, 169.4, 169.3, 100.8, 72.9, 71.7, 71.4, 70.3, 68.5, 62.0, 31.8, 29.4, 29.3, 29.2, 25.8, 22.6, 20.7, 20.6, 20.6, 20.6, 14.1. LRMS (ESI): m/z calcd. for C22H40NO10 [M+NH4]+ 478.5, found, 478.4.

n-Octyl-β-D-glucopyranoside (1)Compound 13 (103 mg, 0.22 mmol) was dissolved in a solution of sodium methoxide in methanol (5 mL) and stirred for one hour at room temperature. The solution was then neutralized with Amberlite® IR-120 (H+) ion-exchange resin, filtered and concentrated. The product was purified by column chromatography (9:1 CH2Cl2/MeOH) to afford 1 as a white powder (64 mg, 98%). 1H NMR (400 MHz, D2O): δ 4.44 (d, J = 8.0 Hz, 1H), 3.95-3.87 (m, 2H), 3.75-3.62 (m, 2H), 3.50-3.33 (m, 3H), 3.24 (dd, J = 9.2, 8.0 Hz, 1H), 1.62 (quint, J = 7.1 Hz, 2H), 1.38-1.24 (m, 10H), 0.86 (t, J = 7.0 Hz, 3H). 13C NMR (100 MHz, D2O): δ 102.1, 75.8, 75.7, 73.0, 70.6, 69.5, 60.7, 31.1, 28.7, 28.5, 28.4, 25.1, 22.0, 13.4. LRMS (ESI): m/z calcd. for C14H28NaO6 [M+Na]+ 315.4, found, 315.3.

n-Octyl-2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside (14)To a mixture of 1,2,3,4,6-Penta-O-acetyl-β-D-galactopyranose (12, 500 mg, 1.28 mmol), 1-octanol (280μL, 1.79 mmol) and 4 Å MS in anhydrous CH2Cl2 (10 mL) stirring at 0 °C under Ar, was slowly added boron trifluoride diethyl etherate (210 μL, 1.66 mmol). The reaction mixture was stirred overnight, then diluted with CH2Cl2 and quenched with sodium bicarbonate. The solution was filtered through Celite®,then extracted with CH2Cl2. The organic layer was washed with sodium bicarbonate, water, saturated brine, then dried over MgSO4 and concentrated. Flash

S4

column chromatography (7:3 hexanes/EtOAc) afforded 14 as a white powder (228 mg, 39%). 1H NMR (400 MHz, CDCl3): δ 5.38 (dd, J = 3.4, 0.9 Hz, 1H), 5.20 (dd, J = 10.5, 7.9 Hz, 1H), 5.01 (dd, J = 10.5, 3.4 Hz, 1H), 4.45 (d, J = 8.0 Hz, 1H), 4.23-4.08 (m, 2H), 3.93-3.84 (m, 2H), 3.47 (dt, J = 9.6, 6.9 Hz, 1H), 2.15 (s, 3H), 2.05 (s, 3H), 2.05 (s, 3H), 1.98 (s, 3H), 1.62-1.53 (m, 2H), 1.34-1.22 (m, 10H), 0.88 (t, J = 6.9 Hz, 3H). 13C NMR (100 MHz, CDCl3): δ 170.4, 170.3, 170.2, 169.4, 101.4, 71.0, 70.6, 70.3, 68.9, 67.1, 61.3, 31.8, 29.4, 29.3, 29.2, 25.8, 22.6, 20.7, 20.7, 20.7, 20.6, 14.1. LRMS (ESI): m/z calcd. for C22H36KO10 [M+K]+ 499.6,found, 499.4.

n-Octyl-β-D-galactopyranoside (2)Compound 14 (183 mg, 0.40 mmol) was dissolved in a solution of sodium methoxide in methanol (5mL) and stirred for one hour at room temperature. The solution was then neutralized with Amberlite®IR-120 (H+) ion-exchange resin, filtered and concentrated. The product was purified by column chromatography (9:1 CH2Cl2/MeOH) to afford 2 as a white powder (109 mg, 94%). 1H NMR (400 MHz, D2O): δ 4.38 (d, J = 7.9 Hz, 1H), 3.95-3.89 (m, 2H), 3.81-3.72 (m, 2H), 3.70-3.61 (m, 3H), 3.49 (dd, J = 9.9, 7.9 Hz, 1H), 1.62 (quint, J = 7.0 Hz, 2H), 1.39-1.24 (m, 10H), 0.86 (t, J = 7.0 Hz, 3H). 13C NMR (100 MHz, D2O): δ 102.7, 75.0, 72.7, 70.7, 70.6, 68.5, 60.8, 31.0, 28.7, 28.4, 28.3, 25.0, 21.9, 13.3. LRMS (ESI): m/z calcd. for C14H28NaO6 [M+Na]+ 315.4, found, 315.3.

Compounds 1 and 2 were synthesized according to a known literature procedure and characterization was consistent with that previously reported. 3

Synthesis of N-Alkyl-D-gluconoamides (3-5)

O

HO

OH

HOHO

O

NH2

n

MeOH, reflux

HN

O

OH

OH

OH

OHn OH

3, n=64, n=45, n=1

N-Octyl-D-gluconamide (3)To a solution of D-gluconic acid-δ-lactone (1.4 g, 7.86 mmol) in MeOH (30 mL) was added n-octylamine (1.3 mL, 7.86 mmol). The mixture was refluxed for 1 hour then cooled in an ice bath. The precipitate was filtered off and washed with cold MeOH to afford 3 as a white powder. 1H NMR (500 MHz, DMSO-d6): δ 7.59, (t, J = 6.0 Hz, 1H), 5.34 (d, J = 5.1 Hz, 1H), 4.53 (t, J = 4.8 Hz, 1H), 4.47 (d, J = 5.1 Hz, 1H), 4.39 (d, J = 7.2 Hz, 1H), 4.33 (d, J = 5.8 Hz, 1H), 3.97 (dd, J = 4.9, 3.8 Hz, 1H), 3.89 (ddd, J = 7.2, 3.7, 2.2 Hz, 1H), 3.57 (m, 1H), 3.46 (m, 2H), 3.37 (m, 1H), 3.06 (m, 2H), 1.40 (quint, J = 6.6 Hz, 2H), 1.31-1.18 (m, 10H), 0.86 (t, J = 6.7 Hz, 3H). 13C NMR (125 MHz, DMSO-d6): δ 172.2, 73.6, 72.4, 71.5, 70.1, 63.4, 38.3, 31.3, 29.2, 28.8, 28.7, 26.4, 22.1, 14.0. LRMS (ESI): m/z calcd. for C14H30NO6 [M+H]+ 308.2, found 308.3.

N-Hexyl-D-gluconamide (4)To a solution of D-gluconic acid-δ-lactone (1.4 g, 7.86 mmol) in MeOH (30 mL) was added n-hexylamine (1.03 mL, 7.86 mmol). The mixture was refluxed for 1 hour then cooled in an ice

S5

bath. The precipitate was filtered off and washed with cold MeOH to afford 4 as a white powder (945 g, 43%). 1H NMR (300 MHz, DMSO-d6): δ 7.60 (t, J = 5.9 Hz, 1H), 5.35 (d, J = 5.0 Hz, 1H), 4.55-4.53 (m, 1H), 4.48-4.46 (m, 1H), 4.39 (d, J = 7.2 Hz, 1H), 4.34 (t, J = 5.4 Hz, 1H), 3.96 (t, J = 4.2 Hz, 1H), 3.90-3.88 (m, 1H), 3.59-3.55 (m, 1H), 3.46-3.43 (m, 2H), 3.37 (t, J = 0.4 Hz, 1H), 3.12-3.02 (m, 2H), 1.42-1.38 (m, 2H), 1.25 (d, J = 10.3 Hz, 6H), 0.86 (t, J = 6.7 Hz, 3H). 13C NMR (76 MHz, DMSO-d6): δ 172.2, 73.7, 72.4, 71.5, 70.1, 63.4, 38.3, 31.1, 29.2, 26.1, 22.1, 14.0. LRMS (ESI): m/z calcd. for C12H26NO6 [M+H]+ 280.3; found 280.2.

N-Propyl-D-gluconamide (5)To a solution of D-gluconic acid-δ-lactone (500 mg, 2.81 mmol) in MeOH (15 mL) was added n-propylamine (230 μL, 2.81 mmol). The mixture was refluxed for 1 hour then cooled in an ice bath. The precipitate was filtered off and washed with cold MeOH to afford 5 as a white powder (158 mg, 24%). 1H NMR (300 MHz, DMSO-d6): 7.61 (t, J = 5.9 Hz, 1H), 5.36 (d, J = 5.1 Hz, 1H), 4.54 (d, J = 5.0 Hz, 1H), 4.47 (d, J = 5.5 Hz, 1H), 4.40 (d, J = 7.2 Hz, 1H), 4.33 (t, J = 5.7 Hz, 1H), 3.97 (t, J = 4.4 Hz, 1H), 3.91-3.87 (m, 1H), 3.59-3.54 (m, 1H), 3.49-3.43 (m, 2H), 3.40-3.37 (m, 1H), 3.04 (qd, J = 6.5, 3.1 Hz, 2H), 1.48-1.36 (m, 2H), 0.82 (t, J = 7.4 Hz, 3H). 13C NMR (76 MHz, DMSO-d6): 172.3, 73.7, 72.4, 71.5, 70.1, 63.4, 40.0, 22.4, 11.4 LRMS (ESI): m/z calcd. for C9H20NO6 [M+H]+ 238.3; found 238.2.

Compounds 3-5 were synthesized according to a known literature procedure and characterization was consistent with that previously reported. 3

Synthesis of Azasugar derivatives (6-8)

O-Allyl-2,3,4,6-tetra-O-benzyl-D-glucose (15)

O

HO

OH

HOHO

OH1) Allyl alcohol, BF3OEt22) NaH, BnBr, DMF

O

BnO

OBn

BnOBnO

O

15D-glucose (10g, 55.5mmol) was dissolved in allyl alcohol (120mL) and boron trifluoride diethyl etherate (0.5mL) was added. The mixture was refluxed for 3h and concentrated in vacuo. to remove the excess allyl alcohol. The resulting syrup (14g, 63.57mmol) was dissolved in DMF (100mL) and 60% NaH (12.21g, 305.30mmol) was added in portions at 0°C. The mixture was then stirred for 2h to ensure complete deprotonation. Benzyl bromide (45.3mL, 381.42mmol) was then added and the reaction was left to stir overnight. The reaction was diluted with water and ethyl acetate and the two phases were separated. The organic phase was washed twice with water and once with brine. The organic phase was dried with MgSO4 and concentrated in vacuo. and purified by flash chromatography (9:1 petroleum ether/EtOAc) to give 15 a yellow syrup (10g, 72%) over two steps. Characterization was consistent with that previously reported.4 1H NMR (300 MHz, CDCl3): δ 7.40-7.24 (m, 18H), 7.17-7.12 (m, 2H), 5.87-5.75 (m, 1H), 5.18 (dd, 1H, J = 17.2, 1.5 Hz), 5.12 (dd, 1H, J = 10.4, 1.5 Hz), 4.91 (d, 1H, J = 1.7 Hz), 4.86 (d, 1H, J = 10.8 Hz), 4.71 (s, 2H), 4.64 (d, 1H, J = 12.0 Hz), 4.60 (s, 2H), 4.52 (d, 1H, J = 12.0 Hz), 4.48 (d, 1H, J = 10.8 Hz), 4.13 (dd, 1H, J = 13.0, 5.0 Hz), 4.01-3.88 (m, 3H), 3.80-3.67 (m, 4H).13C NMR (100 MHz, CDCl3): δ 138.8, 138.7, 134.1, 138.6, 138.6, 128.5, 128.4, 128.3, 128.2, 128.0,

S6

127.9, 127.8, 127.6, 117.4, 97.4, 80.5, 75.4, 75.2, 75.0, 73.6, 72.8, 72.4, 72.1, 69.6, 68.1. LRMS (ESI): m/z calcd. for C9H19NaO6 [M+Na]+ 603.7, found 603.6.

2,3,4,6-tetra-O-benzyl-D-glucose (16)

PdCl2, H2OO

BnO

OBn

BnOBnO

OMeOH

O

BnO

OBn

BnOBnO

OH

15 16

Compound 15 (10g, 17.22mmol) was dissolved in a mixture of MeOH (150mL) and H2O (0.5mL) and PdCl2 (610.7mg, 3.44mmol) was added to mixture and the reaction was allowed to stir till starting material was consumed by TLC. The mixture was then concentrated and purified by flash chromatography (3:1 hexanes/EtOAc) to give 16 (6.2g, 67%) as a yellow oil. Characterization was consistent with that previously reported.5 1H NMR (300 MHz, CDCl3) δ 7.52-7.13 (m, 20H), 4.94 (t, J = 9.4 Hz, 1H), 4.89-4.65 (m, 4H), 4.64-4.41 (m, 3H), 4.10-3.91 (m, 1H), 3.76- 3.46 (m, 5H), 3.45-3.03 (m, 1H), 1.62 (s, 1H). 13C NMR (100 MHz, CDCl3): δ 138.6, 138.4, 138.2, 138.1, 137.8, 137.8, 137.7, 137.6, 128.4, 128.3, 128.3, 128.0, 127.9, 127.8, 127.8, 127.7, 127.7, 127.6, 127.6, 127.6, 127.5, 97.4, 91.1, 84.5, 83.0, 81.6, 79.9, 77.7, 77.6, 75.6, 75.5, 74.9, 74.9, 74.6, 74.5, 73.4, 73.4, 73.1, 70.1, 68.8, 68.5. LRMS ESI-MS m/z calcd for C34H40NO6 [M + NH4]+: 558.70; C34H40KO6 [M + K]+: 579.8. Found 558.5, 579.4.

2,3,4,6-tetra-O-benzyl-1,5-dideoxy-1,5-imino-D-glucitiol (17)

O

BnO

OBn

BnOBnO

OHLiAlH4

THFOH

BnO

OBn

BnOBnO

OH

16 17

Compound 16 (1g, 1.85mmol) was dissolved in THF (15mL) and LiAlH4 (247mg, 6.5mmol) was added in small portions at 0°C.The reaction mixture was stirred overnight, allowing it to warm to rt. The excess LiAlH4 was quenched via Fieser quench. The mixture was diluted with EtOAc and washed with sat. aq. NH4Cl (3x). The organic phase was dried with MgSO4 and concentrated in vacuo. The crude was not purified and carried onto the next step.

2,3,4,6-tetra-O-benzyl-1,5-dideoxy-1,5-imino-D-glucitiol (18)

OH

BnO

OBn

BnOBnO

OH

1)DMSO, (COCl)2,CH2Cl2, -75°C, 2 h;2) Et3N, -75 to 0°C, 2 h

3) NaBH3CN, NH4HCO2, MeOH, 0°C to rt, 20 h

NH

BnO

OBn

BnOBnO

17 18

A solution of oxalylchloride (0.64mL) in DCM (7.4mL) was cooled to -78°C. After dropwise addition of a solution of DMSO (0.66mL) in DCM (4.6mL) over 10 minutes, the reaction mixture was stirred for 40 minutes while being kept below -70 °C. Next, a dry solution of the glucitol intermediate 17 (1g, 1.84mmol) in DCM (3.7mL) was added dropwise to the reaction

S7

mixture over a 15 minute period, while keeping the reaction mixture below -70°C. After stirring the reaction mixture for 2 hours below -65°C, Et3N (3mL) was added dropwise over a 10 minute period, while keeping the reaction mixture below -65 °C. After addition, the reaction mixture was allowed to warm to -5 °C over 2 hours. The Swern reaction mixture was concentrated at a moderate temperature (~30 °C) with simultaneous co-evaporation of toluene (3x). The residue was dissolved in MeOH (37mL) and NH4HCO2 (2.3g, 36.8mmol) was added. The mixture was cooled to 0 °C and stirred until all NH4HCO2 had dissolved. Activated 3Å molsieves (10 g/mmol) were added and reaction mixture was stirred for 20 minutes, after which NaBH3CN (463mg, 7.36mmol) was added. The reaction mixture was kept at 0 °C for one hour after which the cooling source was removed and the reaction was stirred for an additional 20 hours. After removal of the mol. sieves by filtering over Celite®, the filtrate was concentrated, dissolved in EtOAc and washed with sat. aq. NaHCO3. The aqueous phase was back-extracted with EtOAc (3x) and the combined organic layers were dried with MgSO4 and concentrated in vacuo. and purified by flash chromatography (8:2 petroleum ether/EtOAc) giving 18 (703mg, 73%) as a light yellow crystalline solid over three steps. 1H NMR (300 MHz, CDCl3): δ 7.35-7.14 (m, 20H), 4.97 (d, J = 12.9 Hz, 1H), 4.87-4.82 (m, 2H), 4.68 (d, J = 11.7 Hz, 1H), 4.64 (d, J = 11.7 Hz, 1H), 4.48 (d, J = 11.0 Hz, 1H), 4.45 (d, J = 11.8 Hz, 1H), 4.40 (d, J = 11.8 Hz, 1H), 3.65 (dd, J = 9.0 Hz, 2.6 Hz, 1H), 3.57-3.45 (m, 3H), 3.34 (dd, J = 8.8 Hz, 1H), 3.22 (dd, J = 12.2 Hz, 4.9 Hz, 1H), 2.71 (ddd, J = 9.8, 5.9 Hz, 2.6 Hz, 1H), 2.48 (dd, J = 12.2 Hz, 10.3 Hz, 1H), 1.89 (br s, 1H, NH). 13C NMR (100 MHz, CDCl3): δ 138.8, 138.4, 138.3, 137.8, 128.2, 128.2, 127.8, 127.7, 127.7, 127.6, 127.5, 127.4, 87.2, 80.5, 80.0, 75.5, 75.0, 73.2, 72.6, 70.1, 59.6, 48.0. LRMS ESI-MS m/z calcd for C37H34NNaO4 [M + Na]+: 546.7. Found 546.5.

D-gluco-1-Deoxynojirimycin (6)

NH

BnO

OBn

BnOBnO Pd/C, H2, HCl (1M)

EtOHNH

HO

OH

HOHO

18 6

A solution of 18 (150mg, 0.286 mmol) in EtOH (10 mL) was acidified to pH ~2 with 1M aq HCl. Pd/C (10 wt%, 72 mg) was added and the mixture was exposed to 4 bar of hydrogen for 20 hours. The reaction mixture was filtered over Celite® and the filter cake was rinsed successively with MeOH (4×20 mL) and H2O (2×20 mL). The combined filtrate was concentrated and co-evaporated with MeOH (3×50 mL). The residue was purified by flash column chromatography with aluminum oxide (isocratic 16:3.7:0.3, n-propanol:H2O:NH4OH) to provide 6 (46.7 mg, 98%) as a colorless oil. 1H NMR (300 MHz, D2O) δ 3.77 (dd, J = 12.7, 3.2 Hz, 1H), 3.70 (dd, J = 12.8, 5.3 Hz, 1H), 3.60 (ddd, J = 10.5, 5.2, 3.1 Hz, 1H), 3.44-3.39 (m,1H), 3.36-3.30 (m,2H), 3.04-2.99 (m, 1H), 2.82-2.76 (m,1H). 13C NMR (100 MHz, D2O) δ 76.1, 67.8, 67.0, 60.0, 57.7, 45.9. LRMS ESI-MS m/z calcd for C6H14NO4 m/z [M+H]+ 164.2, found 164.1.

S8

N-methyl-D-gluco-1-deoxynojirimycin (8)

NH

BnO

OBn

BnOBnO

1) HCOH, NaCNBH3,ACN/AcOH (30:1)

2) BCl3, DCM, 0CN

HO

OH

HOHO

18 8

Formaldehyde (43.05mg, 1.43 mmol) and NaCNBH3 (54.05 mg, 0.8601 mmol) were successively added to a solution of 18 (150 mg, 0.2867 mmol) in CH3CN/AcOH (3mL, 30:1, v/v). The reaction mixture was stirred for 20 hours after which sat. aq. NaHCO3 (10 mL) was added and the resulting mixture was extracted with Et2O (3×10 mL). The combined organic phases were dried (Na2SO4) and concentrated to provide the crude N-methylated intermediate. The crude intermediate was co-evaporated with dichloroethane and dissolved in CH2Cl2 (3.55 mL). The solution was cooled to 0 °C and BCl3 (3.86 mL, 1M in CH2Cl2) was added. After stirring for 20 hours at 0 °C MeOH (5 mL) was carefully added. The mixture was concentrated and co-evaporated with toluene. The residue was purified by flash silica gel chromatography (0-20% MeOH in CHCl3 + 1% NH4OH) to produce 8 (16.1 mg, 10.7%) over two steps as a colorless oil. 1H NMR (300 MHz, D2O) δ 3.97-3.82 (m, 2H), 3.71-3.59 (m, 1H), 3.50 (m, 1H), 3.42-3.32 (m, 2H), 2.95-2.82 (m, 2H), 2.80-2.75 (s, 3H). 13C NMR (100 MHz, D2O) δ 72.6, 69.8, 66.1, 64.3, 59.0, 57.1, 40.8. LRMS ESI-MS m/z calcd for C7H15NNaO4 m/z [M + Na]+ 200.2 , found 200.5.

Compounds 6, 8, and 18 were synthesized according to a known literature procedure and characterization was consistent with that previously reported. 6

1-thio-phenyl-2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside (19)

O

AcO

OAc

AcO OAcO

AcO

OAc

AcO SPhPhSH, BF3OEt2

DCM

OAc OAc

19

1,2,3,4,6-Penta-O-acetyl-β-D-galactopyranose (10.0 g, 25.6 mmol) was dissolved in DCM under argon. The flask was cooled to 0 ºC and thiophenol (7.35 mL, 72 mmol) was added. boron trifluoride diethyl etherate (9.01 mL, 72 mmol) was then added drop wise, the ice bath was removed, and stirring was continued for an additional 12 hours where upon the reaction was diluted with DCM. The reaction mixture was washed three times with 2 M sodium hydroxide, followed three times with water. The organic phase was dried with MgSO4 and concentrated in vacuo. and purified through flash chromatography (4: 1 hexanes: ethyl acetate to 1: 1 hexanes: ethyl acetate) to provide 19 (11.3 g, 98%) as a white solid. Characterization was consistent with that previously reported.7 1H NMR (400 MHz, CDCl3) δ 7.54-7.49 (2H, m), 7.34-7.29 (3H, m), 5.42 (1H, dd, J = 3.3, 0.9 Hz), 5.24 (1H, dd, J = 10.0, 10.0 Hz), 5.05 (1H, dd, J = 9.9, 3.3 Hz), 4.72 (1H, d, J = 10.0 Hz), 4.19 (1H, dd, J = 11.3, 7.0 Hz), 4.12 (1H, dd, J = 6.7, 4.7 Hz), 3.94 (1H, ddd, J = 7.0, 6.2, 0.9 Hz), 2.12 (3H, s), 2.10 (3H, s), 2.04 (3H, s), 1.97 (3H, s); 13C NMR (100 MHz, CDCl3) δ 170.3, 170.1, 170.0, 169.4, 132.5, 132.4, 128.8, 128.1, 86.6, 74.4, 72.0,

S9

67.2, 67.2, 61.6, 20.8, 20.6, 20.6, 20.5. LRMS (ESI): m/z calcd for C20H24NaO9S [M + Na]+: 463.5. Found 463.2.

1-thio-phenyl-β-D-galactopyranoside (20)

O

AcO

OAc

AcO SPhNaOMe

MeOH

O

HO

OH

HO SPh

OAc OH

19 20

Compound 19 (11.3 g, 25.6 mmol) was dissolved in 1mL of 0.1 M sodium methoxide in methanol and the reaction was stirred at room temperature overnight. Amberlite®IR-120 (H+) ion-exchange resin was added and stirred till a pH of 7 was achieved. The beads were removed by filtration and solvent was removed under reduced pressure to provide 6.98 g of the crude deprotected sugar as a white solid. The crude product was not purified at this stage and carried forward to the next reaction.

1-thio-phenyl-2,3,4,6-tetra-O-benzyl-β-D-galactopyranoside (21)

O

HO

OH

HO SPh

NaH, BnBr

DMFO

BnO

OBn

BnO SPh

OH OBn

20 21

Compound 20 (6.98 g, 25.7 mmol) was dissolved in anhydrous DMF (30 mL) and cooled to 0 ºC. This mixture was cannulated into a suspension of sodium hydride (3.70 g, 154 mmol) in anhydrous DMF (40 mL) under argon at 0ºC. Following the completion of addition, the reaction was stirred for 20 minutes before benzyl bromide (18.2 mL, 154 mmol) was added dropwise. The ice bath was removed and the reaction was stirred overnight. The reaction was diluted with water and ethyl acetate and the two phases were separated. The organic phase was washed twice with water and one with brine. The organic phase was dried and concentrated in vacuo. and the crude product was re-dissolved in minimum boiling methanol and was allowed to recrystallize. The crystals were collected, washed with ice-cold methanol, and dried in vacuo to provide 21 (12.6 g, 78%) of the as a white solid. Characterization was consistent with that previously reported.8 1H NMR (400 MHz, CDCl3) δ 8.00-7.00 (25H, m), 4.89 (1H, d, J = 11.5 Hz), 4.71 (1H, d, J = 10.2 Hz), 4.67 (1H, d, J = 11.9 Hz), 4.66 (1H, d, J = 9.4 Hz), 4.63 (1H, d, J = 11.8 Hz), 4.57 (1H, dd, J = 9.7, 0.9 Hz), 4.53 (1H, d, J = 11.6 Hz), 4.40 (1H, d, J = 11.7 Hz), 4.34 (1H, d, J = 11.6 Hz), 3.91 (1H, d, J = 2.5 Hz), 3.86 (1H, dd, J = 9.4, 9.4 Hz), 3.60-3.50 (4H, m); 13C NMR (100 MHz, CDCl3) δ 138.7, 138.3, 138.2, 137.8, 134.1, 131.5, 128.8, 128.4, 128.3, 128.2, 127.9, 127.8, 127.8, 127.7, 127.7, 127.5, 127.4, 127.1, 87.7, 84.2, 77.3, 77.2, 75.6, 74.4, 73.6, 73.4, 72.7, 68.7. LRMS (ESI): m/z calcd for C40H44NO5S [M + NH4]+: 650.8; C40H40NaO5S [M + Na]+: 655.8. Found 650.5, 655.5.

S10

2,3,4,6-tetra-O-benzyl-D-galactose (22)

O

BnO

OBn

BnO SPh

NBS

Acetone/H2O (9:1)O

BnO

OBn

BnO OH

OBn OBn

21 22

Compound 21 (3.35g, 5.30 mmol) was dissolved in a mixture of acetone (36 mL) and water (4mL) with stirring. N-bromosuccinamide was added (2.2g, 12.2 mmol) and the reaction was stirred for 10 minutes. Solid sodium bicarbonate was added (5g), and the reaction was concentrated. The solution was diluted with water and ethyl acetate and the organic layer was separated. The organic phase was further extracted with saturated sodium bicarbonate, water and brine. The organic phase was dried with MgSO4 and concentrated in vacuo. The reaction was purified by flash chromatography (7:3 to 6:4 hexanes: ethyl acetate) to provide 22 (2.82g, 80%) of an amorphous white solid. Characterization was consistent with that previously reported.9 1H NMR (400 MHz, CDCl3) δ 7.34-7.12 (40H, m), 5.20 (1H, d, J = 3.5 Hz), 4.89-4.83 (2H, m), 4.76-4.60 (7H, m), 4.58-4.48 (3H, m), 4.43-4.28 (4H, m), 4.13-4.07 (2H, m), 3.99-3.93 (2H, m), 3.90-3.84 (3H, m), 3.81-3.77 (1H, m), 3.74-3.66 (1H, m), 3.56-3.33 (6H, m); 13C NMR (100 MHz, CDCl3) δ 138.6, 138.4, 138.2, 137.7, 91.7, 78.6, 76.5, 74.7, 74.5, 73.33, 73.27, 72.8, 69.3, 69.0; 138.6, 138.4, 138.3, 137.6, 128.3, 128.3, 128.3, 128.2, 128.2, 128.1, 128.1, 128.0, 127.9, 127.9, 127.8, 127.7, 127.6, 127.6, 127.5, 127.5, 127.4, 127.4, 97.7, 82.1, 80.6, 75.0, 74.4, 73.5, 73.4, 73.4, 69.3, 68.8. LRMS ESI-MS m/z calcd for C34H40NO6 [M + NH4]+: 558.7; C34H36KO6 [M + K]+: 579.7. Found 558.5, 579.4.

2,3,4,6-tetra-O-benzyl-D-galactono-δ-lactone (23)

O

BnO

OBn

BnO OH

OBn TPAP, NMOO

BnO

OBn

BnO

OBn

OACN

22 23

To a solution of 5 gram of compound 22 in 50mL acetonitrile was added tetrapropylammonium perruthenate (176mg, 0.05eq) and N-Methylmorpholine-N-oxide (1.7g, 1.5eq). This solution was stirred till TLC indicated complete consumption of the starting material. The crude mixture was filtered over Celite® and concentrated under vacuo. The resulting crude mixture was diluted with ethyl acetate and washed 2x with a saturated solution of sodium thiosulfate. Afterwards, it was washed with brine, dried over magnesium sulfate and concentrated in vacuo. The product was used for further reactions without further purification.

S11

2,3,4,6-tetra-O-benzyl-D-galactonamide (24)

O

BnO

OBn

BnO

OBn

O

NH3MeOH

OH

BnO

OBn

BnO

OBn

O

NH2

23 24

Compound 23 (3g, 5.57 mmol) was dissolved in 40mL solution of methanol saturated with ammonia. The resulting mixture was stirred at room temperature for 1.5 hour under argon atmosphere. The reaction mixture was then concentrated in vacuo. Crystallization of the resulting white solid from ethyl acetate and petroleum ether afforded 24 (2.56 g, 85%) as a white powder. 1H NMR (300 MHz, CDCl3): δ 7.50-7.00 (m, 20H), 5.62 (bs, 1H), 4.80-4.40 (m, 8H), 3.63 (2x dd, 2H, J = 10.5, 4.7, 2.3 Hz), 2.85 (d, 1H, J = 2.9 Hz). 13C NMR (100 MHz, CDCl3): δ 128.6, 128.4, 128.2, 128.2, 128.0, 128.0, 127.8, 127.7, 127.4, 104.5, 79.8, 79.4, 75.0, 73.7, 73.3, 73.3, 71.3, 69.2. LRMS ESI-MS m/z calcd for C34H37NO6 [M + H]+: 556.3. Found 557.3.

2,3,4,6-tetra-O-benzyl-D-galactono-δ-lactam (25)

OH

BnO

OBn

BnO

OBn

O

NH2

1) Ac2O, DMSO

2) NaCNBH3, HCO2HACN

NH

BnO

OBn

BnO

OBn

O

24 25

Compound 24 (2.56 grams, 4.61mmol) was dissolved in 19mL of dimethyl sulfoxide and 11mL of acetic anhydride. The mixture was stirred overnight. After, 60mL of water was added and the mixture is stirred for 15 more minutes during which a yellow oil precipitated. The water layer was removed and the residue was extracted with water 3x. The residue was dissolved in dichloromethane and extracted with brine 2x. The organic fractions were combined and dried over MgSO4 and concentrated in vacuo. The product (2.12 g, 83%) was used for further reactions without further purification. This mixture was dissolved in 60mL of acetonitrile and 15mL of formic acid. To this mixture, sodium cyanoborohydride (1.2 g, 3.4 eq) was added and the reaction was refluxed for two hours. The mixture was then cooled in ice and the reaction was quenched by adding aq. HCl-solution (0.1M). After stirring for 15 minutes, the mixture was poured into a mixture of ethyl acetate/ saturated aqueous NaHCO3 solution (1:1, 200mL). The water layer was separated and extracted with ethyl acetate; the combined organic fractions were then washed with brine and dried over MgSO4 and concentrated in vacuo. Flash chromatography 2:1 petroleum ether/ ethyl acetate) gave 25 (1.55 g, 63%) as a yellow syrup. 1H NMR (CDCl3, 300MHz): δ 7.44-7.20 (m, 2OH), 5.85 (bs, 1H), 5.21 (d, 1H, J = 11.3 Hz), 4.91 (d, 1H, J = 11.5 Hz), 4.81 (d, 1H, J = 11.3 Hz), 4.79 (d, 1H, J = 12.0 Hz), 4.69 (d, 1H, J = 11.9 Hz), 4.57 (d, 1H, J = 11.5 Hz), 4.49 (d, 1H, J = 11.6 Hz), 4.43 (d, 1H, J = 11.8 Hz), 4.34 (d, 1H, J = 9.1 Hz), 3.97 (bs, 1H), 3.83 (dd, 1H, J = 9.2 Hz, 1.4 Hz), 3.61-3.52 (m, 2H) 3.43 (dd, 1H, J = 8.0, 3.1 Hz). 13C NMR (100 MHz, CDCl3): δ 171.1, 138.4, 138.2, 138.0, 137.5, 128.6, 128.5, 128.4, 128.4, 128.3,

S12

128.1, 128.1, 128.0, 127.9, 127.8, 127.7, 127.6, 80.7, 77.5, 77.5, 77.2, 76.9, 75.4, 74.2, 73.6, 73.1 53.6. LRMS ESI-MS m/z calcd for C34H36NO5 [M + H]+: 538.7. Found 538.2.

2,3,4,6-tetra-O-benzyl-1,5-dideoxy-1,5-imino-D-galactiol (26)

NH

BnO

OBn

BnO

OBn

O

LiAlH4

THFNH

BnO

OBn

BnO

OBn

25 26

Compound 25 (200 mg, 0.4 mmol), was dissolved in 10 mL THF. Then, lithium aluminiumhydride (55mg, 3.6eq) was added and the reaction mixture was stirred for 3 hours at 70°C under argon atmosphere. The reaction mixture was then poured into a stirred mixture of 50 mL ice water and 50 mL diethyl ether. After stirring for 10 minutes 75 mL sodium hydroxide solution (0.5 M) was added and the mixture was stirred for another 10 minutes. The water layer was then removed and extracted with 25 mL of diethyl ether (2x); the combined organic fractions were washed with brine and water. Flash chromatography (2:1 petroleum ether / ethyl acetate) gave 26 (198 mg, 98%) as a light yellow oil.1H NMR (CDCl3, 300MHz): δ 7.40-7.25 (m, 20H), 4.90 (d, 1H, J = 11.4 Hz), 4.78 (d, 1H, J = 12.0 Hz), 4.73 (d, 1H, J = 9.7 Hz), 4.64 (d, 1H, J = 11.6 Hz), 4.55 (d, 1H, J = 11.5 Hz), 4.47 (d, 1H, J = 11.8 Hz), 4.46 (d, 1H, J = 11.8 Hz), 4.00-3.95 (m, 1H), 3.90 (ddd, 1H, J = 9.8, 9.8, 5.3 Hz), 3.53 (dd, 1H, J = 8.9, 6.7 Hz), 3.47 (dd, 1H, J = 9.2, 2.6 Hz), 3.34-3.41 (m, 1H), 3.31 (dd, 1H, J = 13.3, 4.6 Hz), 2.86 (t, 1H, J = 7 Hz), 2.52 (dd, 1H, J = 10.4, 2.4 Hz). 13C NMR (100 MHz, CDCl3): δ 138.6, 138.6, 138.5, 137.8, 128.4, 128.4, 128.3, 128.2, 128.2, 128.0, 127.8, 127.6, 127.8, 127.6, 127.5, 127.5, 84.0, 76.2, 74.4, 74.4, 74.1, 73.5, 73.2, 72.8, 69.0, 58.2,47.8. LRMS ESI-MS m/z calcd for C34H37NNaO4 [M + Na]+: 546.7. Found 546.5.

S13

D-galacto-1-deoxynojirimycin (7)

NH

BnO

OBn

BnO

OBnBCl3DCM

NH

HO

OH

HO

OH

26 7

Boron trichloride (2.5 mL, 1M in CH2Cl2) was added to a cooled (0 °C) solution of compound 26 (200 mg, 0.38 mmol) in CH2Cl2 (4 mL). The reaction mixture was stirred for 20 hours at 0 °C after which MeOH (0.5 mL) was carefully added. The reaction mixture was concentrated and co-evaporated with toluene. Flash column purification of the residue over aluminumoxide (1:2, MeOH/EtOAc to MeOH to 1:3 H2O/MeOH) provided 7 (180 mg, 96%) as a colorless oil. 1H NMR (300 MHz, D2O): δ 3.87 (d, 1H, J = 2.6 Hz), 3.62 (dt, 1H, J = 10.6, 5.2 Hz), 3.43-3.55 (m, 2H), 3.30 (dd, 1H, J = 9.9, 3.3 Hz), 3.00 (dd, 1H, J = 12.9, 5.6), 2.68 (t, 1H, J = 6.8 Hz), 2.29 (dd, 1H, J = 10.9, 1.5 Hz). 13C NMR (100 MHz, D2O): δ 74.8, 69.0, 67.8, 61.1, 58.9, 48.8, 48.7. LRMS ESI-MS m/z calcd for C6H14NO4 [M + H]+: 164.2, C6H14NNaO4 [M + Na]+: 186.1. Found 164.2, 186.1.

Characterizations of 7 and 23-26 were consistent with that previously reported. 10

S14

Spectral Data

O

HO

OH

HOHO

O

61

S15

O

HO

OHHO

HO O

62

S16

HN

O

OH

OH

OH

OH

OH6

3

S17

HN

O

OH

OH

OH

OH

OH4

4

S18

HN

O

OH

OH

OH

OH

OH

5

S19

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

1.591

3.595

3.602

3.648

3.653

3.657

3.666

3.686

3.736

3.750

4.010

4.029

4.047

4.464

4.468

4.486

4.493

4.581

4.618

4.623

4.642

4.658

4.682

4.778

4.802

4.825

4.837

4.846

4.857

5.001

5.023

5.212

5.214

5.233

5.235

5.340

5.343

7.133

7.136

7.147

7.151

7.265

7.276

7.282

7.290

7.301

7.308

7.311

7.317

7.324

7.326

7.332

7.336

7.342

7.358

7.362

7.373

1.23

2.09

4.41

2.61

1.52

3.15

2.20

4.63

5.53

8.63

2.61

2.00

1.86

1.47

4.50

42.94

220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

68.199

68.400

70.210

73.234

73.469

74.853

74.926

75.109

75.768

76.793

77.047

77.300

77.665

77.864

79.828

82.121

82.281

84.698

95.688

102.719

117.266

118.290

127.710

127.784

127.924

127.937

127.983

128.128

128.381

128.414

128.436

137.915

138.200

138.852

O

BnO

OBn

BnOBnO

O

15

S20

1.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5 ppm

3.132

3.406

3.545

3.571

3.592

3.614

3.621

3.630

3.642

3.681

3.699

3.956

3.973

3.990

4.028

4.045

4.464

4.474

4.489

4.496

4.573

4.597

4.679

4.700

4.724

4.751

4.765

4.772

4.784

4.808

4.829

4.846

4.911

4.934

4.956

5.222

7.138

7.143

7.261

7.271

7.297

7.304

7.309

7.318

7.332

0.80

0.25

6.24

1.24

1.23

2.79

1.55

3.35

3.00

1.99

1.00

3.26

28.22

220 200 180 160 140 120 100 80 60 40 20 0 ppm

68.543

70.272

73.304

73.494

74.771

75.023

75.688

75.744

77.662

77.805

79.943

81.742

83.110

84.561

91.366

127.703

127.714

127.877

127.945

127.989

128.101

128.370

128.379

128.408

128.519

137.809

137.832

138.152

138.303

138.486

138.645

O

BnO

OBn

BnOBnO

OH

16

S21

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

2.459

2.493

2.500

2.534

2.704

2.713

2.716

2.725

2.736

3.211

3.226

3.251

3.268

3.313

3.342

3.374

3.476

3.495

3.506

3.548

3.643

3.652

3.673

3.682

4.433

4.447

4.464

4.487

4.500

4.676

4.809

4.835

4.845

4.871

4.952

4.989

7.172

7.178

7.188

7.195

7.204

7.241

7.248

7.253

7.266

7.269

7.274

7.280

7.284

7.290

7.305

7.307

7.316

7.322

7.325

7.339

7.348

1.14

0.99

1.00

0.99

1.02

3.11

1.07

3.16

2.18

2.07

1.04

2.06

17.99

220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

48.106

59.726

70.249

72.778

73.383

75.174

75.658

80.060

80.614

87.308

127.509

127.653

127.747

127.769

127.849

127.932

128.002

128.345

128.365

128.392

137.955

138.376

138.477

138.889

NH

BnO

OBn

BnOBnO

18

S22

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

2.767

2.790

2.815

3.002

3.009

3.013

3.019

3.023

3.030

3.034

3.040

3.307

3.315

3.317

3.333

3.342

3.352

3.397

3.418

3.436

3.578

3.589

3.597

3.602

3.607

3.612

3.620

3.630

3.680

3.691

3.706

3.716

3.752

3.758

3.777

3.784

1.01

0.99

2.08

0.97

1.00

1.02

1.00

220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

45.829

57.684

59.836

66.952

67.777

76.151

NH

HO

OH

HOHO

6

S23

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

2.785

2.943

3.330

3.339

3.345

3.370

3.386

3.401

3.467

3.501

3.533

3.596

3.613

3.627

3.634

3.644

3.651

3.664

3.682

3.828

3.838

3.873

3.882

3.907

3.912

3.951

3.00

1.99

2.05

1.01

1.14

1.10

0.99

220 200 180 160 140 120 100 80 60 40 20 0 ppm

40.82

57.10

59.03

64.27

66.08

69.76

72.64

N

HO

OH

HOHO

8

S24

1.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5 ppm1.955

2.024

2.077

2.102

3.902

3.904

3.919

3.935

3.937

4.074

4.090

4.103

4.118

4.150

4.168

4.178

4.196

4.684

4.709

5.013

5.022

5.038

5.047

5.197

5.222

5.394

5.396

5.402

5.404

7.288

7.296

7.305

7.483

7.489

7.498

7.507

3.00

3.05

2.97

3.02

1.08

2.21

1.07

1.08

1.07

1.07

3.15

2.11

210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

20.60

20.66

20.69

20.86

61.63

67.22

67.26

72.01

74.43

76.71

77.03

77.35

86.64

128.18

128.91

132.47

132.57

169.46

170.08

170.21

170.40

O

AcO

OAc

AcO SPh

OAc

19

S25

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

1.589

2.212

3.455

3.710

3.732

3.744

3.774

4.050

4.083

4.103

4.506

4.544

4.571

4.609

4.710

4.749

4.762

4.794

4.829

4.876

4.904

4.938

5.075

5.113

7.289

7.296

7.500

7.521

7.700

7.710

0.56

4.12

1.97

2.04

6.20

1.00

25.66

220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

68.73

72.70

73.56

74.42

75.63

77.26

84.16

87.69

127.00

127.43

127.54

127.66

127.71

127.80

127.90

128.16

128.75

131.47

134.12

137.84

138.23

138.29

138.74

O

BnO

OBn

BnO SPh

OBn

21

S26

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

3.476

3.482

3.495

3.528

3.540

3.546

3.559

3.601

3.757

3.772

3.776

3.791

3.905

3.911

3.925

3.931

3.967

4.028

4.036

4.048

4.055

4.166

4.398

4.492

4.575

4.703

4.726

4.737

4.745

4.809

4.822

4.845

4.923

4.938

4.946

5.283

5.290

7.262

7.269

7.271

7.293

7.304

7.319

7.333

7.337

7.350

7.353

7.363

7.367

7.384

1.53

1.77

0.89

0.41

1.63

1.20

1.20

1.15

1.63

1.67

1.76

6.59

1.94

1.00

33.47

160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 ppm

14.257

21.104

60.475

68.958

69.145

69.474

73.007

73.463

73.493

73.564

73.655

74.592

74.698

74.817

75.125

76.602

76.821

77.139

77.456

78.783

80.763

82.232

91.898

97.845

127.557

127.608

127.676

127.787

127.807

127.874

128.009

128.052

128.212

128.274

128.294

128.349

128.421

128.445

128.477

137.776

137.870

138.324

138.460

138.534

O

BnO

OBn

BnO OH

OBn

22

S27

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

1.254

1.587

2.488

3.500

3.513

3.519

3.532

3.576

3.589

3.595

3.608

3.885

3.887

3.901

3.904

4.141

4.154

4.158

4.171

4.175

4.189

4.193

4.327

4.350

4.391

4.414

4.435

4.460

4.484

4.548

4.572

4.600

4.620

4.623

4.692

4.713

5.541

5.548

6.609

6.616

7.167

7.180

7.183

7.262

7.278

7.283

7.308

7.322

7.334

1.74

0.68

1.00

0.99

1.00

3.00

1.53

2.64

3.07

1.03

0.97

0.97

1.81

16.71

220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

69.176

71.338

73.232

73.261

73.687

75.022

76.605

77.029

77.161

77.453

79.349

79.746

127.393

127.721

127.836

128.043

128.180

128.210

128.352

128.389

128.597

136.715

137.725

137.865

137.935

174.756

OH

BnO

OBn

BnO

OBn

O

NH2

24

S28

1.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5 ppm

3.489

3.499

3.507

3.576

3.605

3.852

3.856

3.875

3.879

4.032

4.384

4.407

4.438

4.468

4.530

4.591

4.620

4.702

4.732

4.825

4.848

4.876

4.933

4.962

5.257

5.285

6.303

7.258

7.273

7.277

7.298

7.314

7.319

7.328

7.338

7.346

7.370

7.459

7.476

0.91

1.87

1.06

1.00

3.39

1.09

1.13

1.91

0.89

0.92

0.91

6.95

10.00

1.93

220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

53.550

70.360

73.040

73.537

74.046

75.357

80.555

127.542

127.850

127.979

128.242

128.334

128.403

128.500

170.951

NH

BnO

OBn

BnO

OBn

O

25

S29

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

2.480

2.515

2.523

2.557

2.835

2.858

2.881

3.271

3.288

3.313

3.331

3.353

3.408

3.440

3.448

3.506

3.527

3.534

3.557

3.867

3.886

3.900

3.916

3.932

3.950

4.368

4.407

4.437

4.476

4.530

4.568

4.625

4.664

4.713

4.745

4.906

4.944

7.242

7.280

7.301

7.312

7.318

7.346

0.93

1.10

0.50

0.86

0.77

1.03

1.07

1.03

0.96

2.20

1.49

3.78

1.00

19.63

220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

58.142

72.751

73.180

73.440

74.058

74.410

127.454

127.755

127.955

128.146

128.231

128.329

128.352

128.416

137.771

138.517

138.550

138.610

NH

BnO

OBn

BnO

OBn

26

S30

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 ppm

2.250

2.292

2.329

2.660

2.682

2.704

2.989

3.006

3.032

3.048

3.320

3.331

3.354

3.364

3.475

3.515

3.597

3.612

3.631

3.647

3.648

3.664

3.865

3.874

0.93

0.96

0.95

1.10

1.96

1.07

1.00

220 210 200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 10 0 ppm

48.709

58.841

61.123

67.783

68.978

74.785

NH

HO

OH

HO

OH

7

S31

References

1. A. Chakrabartty and C. L. Hew, Eur. J. Biochem., 1991, 202, 1057-1063.2. C. A. Knight, J. Hallett and A. L. DeVries, Cryobiology., 1988, 25, 55-60.3. C. J. Capicciotti, M. Leclère, F. A. Perras, D. L. Bryce, H. Paulin, J. Harden, Y. Liu and

R. N. Ben, Chem. Sci., 2012, 3, 1408-1416.4. R. Rodebaugh and B. Fraser-Reid, Tetrahedron, 1996, 52, 7663-7678.5. B. Dasari, S. Jogula, R. Borhade, S. Balasubramanian, G. Chandrasekar, S. S. Kitambi

and P. Arya, Org. Lett., 2013, 15, 432-435.6. T. Wennekes, A. J. Meijer, A. K. Groen, R. G. Boot, J. E. Groener, M. van Eijk, R.

Ottenhoff, N. Bijl, K. Ghauharali, H. Song, T. J. O’Shea, H. Liu, N. Yew, D. Copeland, R. J. van den Berg, G. A. van der Marel, H. S. Overkleeft and J. M. Aerts, J. Med. Chem., 2009, 53, 689-698.

7. A. K. Balcerzak, S. S. Ferreira, J. F. Trant and R. N. Ben, Bioorg. Med. Chem. Lett., 2012, 22, 1719-1721.

8. S. R. Vidadala, S. A. Thadke, S. Hotha and S. Kashyap, J. Carbohydr. Chem., 2012, 31, 241-251.

9. J. N. Gorantla, D. Kovval and R. S. Lankalapalli, Tetrahedron Lett., 2013, 54, 3230-3232.

10. H. S. Overkleeft, J. van Wiltenburg and U. K. Pandit, Tetrahedron, 1994, 50, 4215-4224.