1

SI Appendix for:

Molecular matchmaking between the popular ‘weight loss’ herb

Hoodia gordonii and GPR119, a potential drug target for metabolic

disorder

Shuyong Zhang1,2, ‡, Yuyong Ma3, ‡, Jing Li2, Junjun Ma3, Biao Yu3,*, Xin Xie1,2,*

1Shanghai Key Laboratory of Signaling and Disease Research, Laboratory of

Receptor-based Bio-medicine, School of Life Sciences and Technology, Tongji

University, Shanghai 200092, China 2CAS Key Laboratory of Receptor Research, National Center for Drug Screening,

Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai

201203, China 3State Key Laboratory of Bio-organic & Natural Products Chemistry, Shanghai

Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingling Road,

Shanghai 200032, China

‡These authors contribute equally.

*Address correspondence to: Dr. Xin Xie, 189 Guo Shou Jing Road, Shanghai

201203, China; Fax: 0086-21-50800721; E-mail: xxie@simm.ac.cn. Or Dr. Biao Yu,

345 Lingling Road, Shanghai 200032, China; Fax: 0086-21-64166128; E-mail:

byu@mail.sioc.ac.cn.

2

SI Appendix Table S1. Selectivity of Gordonoside F on GPCRs

Receptor Calcium mobilization assay [EC50 (M)] Known agonists Gordonoside F

GPR119 AR231453 2.8e-7 4.1e-6 PSN632408 4.7e-6

GPR40 Linoleic Acid 1.0e-5 NR GPR41 Sodium Propionate 9.6e-6 NR GPR120 Linolenic Acid 1.6e-5 NR

CB1 CP55940 3.6e-8 NR CB2 CP55940 1.7e-9 NR

DRD1 Dopamine 9.5e-8 NR DRD2 Dopamine 6.2e-7 NR DP2 PGD2 5.2e-7 NR EP2 PGE2 1.3e-6 NR EP4 PGE2 8.0e-8 NR S1P1 S1P 3.1e-9 NR

AGTR1 Angiotensin II 3.7e-8 NR GLP-1R GLP1 4.5e-9 NR GCGR Glucagon 5.5e-8 NR α1A AR Phenylephrine 1.5e-7 NR α1B AR Phenylephrine 6.0e-9 NR α1D AR Phenylephrine 4.9e-8 NR

ADORA2A NECA 6.5e-10 NR β1 AR Isoproterenol 9.0e-10 NR β2 AR Isoproterenol 1.4e-10 NR DOR DPDPE 3.3e-9 NR KOR U50488 3.4e-9 NR MOR DAGO 7.1e-8 NR CCR5 RANTES 3.1e-9 NR CCR7 CCL19 1.3e-8 NR

CXCR2 IL-8 5.7e-9 NR CXCR4 SDF-1 6.4e-9 NR CXCR6 CXCL16 3.7e-9 NR

NR = No response at concentrations up to 100 µM

3

SI Appendix Figure S1. Product insert of Hoodia extract.

Product Specification

HOODIAPURE

Ref�:

EA149462

Thi s speci f i cat i on sheet cancel s and r epl aces al l pr evi ous January 14, 2014

Irradiation detection : Not irradiated (PPSL <700)

Mi crobi ol ogi cal qual i t y :Total plate count :Yeasts and molds :Enterobacteria :Coliforms :E. coli :Salmonella :

*Cont r ol Pl an, Anal ysi s per f or med once a year .

< 10,000 cfu/g< 300 cfu/g< 1,000 mpn/g*Negative / g*Negative / g*Negative / 25 g*

Packagi ng :Cardboard box with polyethylene bags : 25 kg net

Anal yt i cal qual i t y :Particle size :Moisture :Dry extract :Identification :Arsenic content :Cadmium content :Lead content :Mercury content :

=

100% through 40 mesh< 8%> 92%Conform< 3 ppm*< 1 ppm*< 2 ppm*< 1 ppm*

Speci f i cat i ons :

Aspect :

Descr i pt i on :

=

Color :

=

Botanical name :

Powder

Best Bef ore 24 months under the previously mentioned conditions and in its original packaging.

Plant part used : Aerial part Extract ratio : 3-5/1

Green to greenish-brown

Recommended st orage condi t i ons :

Ambient temperature, protected from light, moisture and oxygen.

=

Hoodi a gor doni i

Composi t i on :

Hoodia gordonii

Flavor :

=

=

Characteristic

Sensory qual i t y :

Naturex SA

250, rue Pierre Bayle - BP 81218 - 84911 Avignon Cedex 9 - France - SA au capital de 11.592.808,50� - RCS Avignon B 384 093 563

T��l : +33 (0)4 90 23 96 89 - Fax : +33 (0)4 90 23 73 40 - E-mail : naturex@naturex.com - Site : www.naturex.com

1/1

4

SI Appendix Figure S2. HPLC analysis of Hoodia extract

A. Hoodia extract (CH2Cl2 extract; the peaks at 4.523 min and 7.847 min are

characterized to be P57 and Gordonoside F, respectively, by MS analysis and by

comparison to the authentic compounds.)

B. P57 (synthetic)

C. Gordonoside F (synthetic)

HPLC conditions: column: Agilent Poroshell 120; mobile phase: H2O/CH3CN

(40 : 60 to 0 : 100 within 8 min); flow rate: 0.8 mL/min then 1.0 mL/min after 8

min; column temperature: 25 °C; detection wavelength: 210 nm.

5

SI Appendix Figure S3. Dose effect of Gordonoside F on food intake in WT and

GPR119 KO mice

(A and B) Age-matched WT (A) or GPR119 KO (B) male mice were fasted overnight,

Gordonoside F (100, 200, 400 mg/kg), or vehicle (0.5% methyl-cellulose) was

administered orally. Cumulative food intake upon refeeding was recorded at 2, 4, 8,

and 24 hours. (C) Comparison of the appetite-suppressing effect of 400 mg/kg

Gordonoside F in WT and GPR119 KO mice. Data are Means ± SEM (n=8). *p<0.05,

**p<0.01, ***p<0.001 versus vehicle control. #p<0.05 versus WT.

6

SI Appendix Figure S4. Dose effect of Hoodia extract on food intake in WT and

GPR119 KO mice

(A and B) Age-matched WT (A) or GPR119 KO (B) male mice were fasted overnight,

Hoodia extract (500, 1000, 2000 mg/kg), or vehicle (0.5% methyl-cellulose) was

administered orally. Cumulative food intake upon refeeding was recorded at 2, 4, 8,

and 24 hours. (C and D) Comparison of the appetite-suppressing effect of 1000 mg/kg

(C) and 2000 mg/kg (D) Hoodia extract in WT and GPR119 KO mice. Data are

Means ± SEM (n=8). *p<0.05, **p<0.01, ***p<0.001 versus vehicle control. #p<0.05

versus WT.

7

Materials and Methods

Cell culture and transfection

For transient transfection, about 1×106 HEK293 cells or CHO cells were mixed

with 2 to 4 µg plasmids in 200 µL transfection buffer, and electroporation was carried

out. To generate cell lines stably expressing GPCRs in combination with Gα16 or

cAMP responsive element-driven luciferase reporter (CRE-luc), the transfected cells

were seeded onto 10 cm dishes and selected with and proper antibiotics (400 µg/mL

G418 and/or 20 µg/mL blasticidin). Single colonies formed in the selection medium

were picked, expanded, and tested for the expression of transfected genes.

Calcium mobilization assay

Cells expressing GPR119 (or other GPCRs) and Gα16 were seeded onto 96-well

plates at a density of 3×104 cells/well and cultured overnight. Cells were then

incubated with 2 µM Fluo-4 AM in Hank’s buffered saline solution (HBSS)

supplemented with 250 µM sulfinpyrazone at 37 °C for 45 min. After a thorough

washing, 50 µL of HBSS was added. After incubation at room temperature for 10 min,

25 µL of agonist were dispensed into the well using a FlexStation III microplate

reader and intracellular calcium change was recorded.

cAMP assay

Cells were harvested and re-suspended in PBS containing 500 µM IBMX at a

density of 8x105 cells/mL. Cells were then plated onto 384-well assay plates at 4000

cells/5 µL/well. Another 5 µL buffer containing compounds at various concentrations

were added to the cells and the incubation lasted for 1 h at room temperature.

Intracellular cAMP measurement was carried with a Cisbio HTRF Dynamic 2 cAMP

kit and EnVision multiplate reader according to the manufacturer’s instructions.

Western blotting

Cells were serum starved for 2 h and then treated with compounds for the

indicated duration at 37 °C. Cells were lysed, sonicated, and boiled at 95 °C for 5 min

in sample buffer. Aliquots of protein were fractionated by SDS-PAGE on 10%

polyacrylamide gels and transferred to PVDF membranes. The membranes were

8

incubated first with blocking buffer (TBS with 0.05% Tween 20 and 5% nonfat milk)

for 1 h at room temperature and then incubated overnight at 4 °C in buffer containing

antibodies against GAPDH, ERK, or p-ERK. The membranes were washed thrice and

incubated with secondary antibody for 1 h. After washing, immunostaining was

visualized using Amersham ECL Plus western blotting detection reagents (GE

Healthcare).

Flow cytometry

HEK293 cells expressing HA-tagged GPR119 (HEK293/HA-GPR119) were

harvested and resuspended at 5×105/mL in PBS supplemented with 1% BSA (FACS

buffer). Cells were incubated at 37 °C for the indicated time periods in the presence or

absence of 10 µM Gordonoside 1. Cells were then washed thrice in ice-cold FACS

buffer and fixed with 4% paraformaldehyde. After washing, cells were incubated at

4 °C for 1h with FITC-conjugated anti-HA antibody. Cells were then washed,

resuspended in ice-cold FACS buffer and the surface staining was analyzed with a

Guava EasyCyte™ flow cytometers (Millipore).

Immunofluorescence staining

Sections (10 µm) of pancreas were blocked with 10% goat serum (GS) and 0.1%

Triton-X-100 in PBS for 30 min and then incubated with a combination of a rabbit

anti-insulin and a mouse anti-glucagon antibodies in PBS containing 0.1%

Triton-X-100 and 1% GS at 4 °C overnight. After washing with PBS, the sections

were incubated with a cocktail of secondary antibodies conjugated to Alexa fluor 488

or Alexa fluor 555 for 2 h at room temperature. Nuclei were counterstained with

hoechst 33342. Fluorescent images were obtained with an Olympus IX51 inverted

fluorescent microscope.

9

Chemical Syntheses of Gordonoside F

A) General Remarks for the Synthesis

B) Experimental Procedures

C) 1H and 13C NMR Spectra of the Key Compounds

A) General Remarks for the Syntheses

All solvents were purchased from commercial sources and were used as received

unless otherwise stated. Crushed 4Å molecular sieves were activated through

flame-drying immediately prior to use. Optical rotations were measured at room

temperature with a Perkin–Elmer 241 MC polarimeter. 1H and 13C NMR spectra were

recorded on a Bruker Avance 400 or 500 in CDCl3. TMS was used as the internal

standard and all J values are given in hertz. High-resolution mass spectra were

recorded with APEXIII 7.0 TESLA FTMS (ESI) or IonSpec 4.7 Tesla FTMS

(MALDI). Flash column chromatography was performed on silica gel H (10-40 µ).

Analytical thin layer chromatography (TLC) was performed on glass plates pre-coated

with a 0.25 mm thickness of silica gel. The TLC plates were visualized with UV light

and/or by staining with sulfuric acid/ethanol (10%, v/v).

B) Experimental Procedures

1. Preparation of oleandrose donor S10 from triacetylglucal

OAcO

AcOAcO

OTsOHO

TBSO

OAcOTBSO

OAcOHO

2) TsCl, DCM,pyridine, rt

NaBr, NaHCO3,DMF, 80 oC, 89%

CH3I, Ag2O,DCM, rt, 91% OAcO

MeO

3HF-Et3N, MeCN,60 oC, 90%

OTsOHOHO

TBSCl, imidazole,DMF, rt;

OBr

HOTBSO

Ac2O, DMAP,Et3N, DCM, 98% O

BrAcOTBSO

Bu3SnH, AIBN,toluene, 80 oC

OAcOMeO

OAcI

NIS, HOAc,toluene, reflux

N2H4-H2O, DCM,MeOH, 100%

OO

AcOMeO CF3

NPhICs2CO3, DCM, 80%OH

OAcOMeO

I

ClF3C

NPh

S1 S2

S3 S4 S5

S6 S7 S8

S9 S10

1) NaOMe, DCM,MeOH, rt;

47% (3 steps)

99%

54%

10

1.1.

O

TsOHO

TBSO S2 To a solution of triaceylglucal (16.5 g, 60.7 mmol) in CH2Cl2/MeOH (20 mL/40 mL)

was added NaOMe (330 mg, 6.07 mmol) at rt. After stirring for 4 h, the mixture was

evaporated in vacuo to give a residue. To a solution of the residue in CH2Cl2/pyrdine

(30 mL/30 mL) was added TsCl (17.4 g, 91.0 mmol) at 0 oC. After stirring for 8 h at

rt, the mixture was quenched by adding water. After concentration, the mixture was

diluted with EtOAc and washed with 1 N HCl solution. The water solution was

extracted with EtOAc for three times. The combined organic layer was washed with

saturated NaHCO3 solution and brine, dried over Na2SO4, filtered, and evaporated in

vacuo. The resulting yellow syrup S1S1 (12.10 g) was used without further

purification.

To a solution of the crude S1 (12.10 g) in dry DMF (40.0 mL) were added

t-butyldimethylsilylchloride (6.03 g, 40.0 mmol) and imidazole (5.48 g, 80.6 mmol).

After being stirred for 1 h at rt, the mixture was quenched with methanol (10.0 mL)

and concentrated in vacuo. The residue was diluted with EtOAc and washed with

H2O and saturated brine. The organic layer was dried over anhydrous Na2SO4 and

concentrated. The residue was purified by column chromatography on silica gel

(petroleum/ethyl acetate, 9/1 to 6/1) to provide S2 (11.73g, 47%) as a colorless syrup:

[α]D26 = 47.0 (c 1.5, CHCl3); 1 H NMR (400 MHz, CDCl3) δ 7.79 (d, J = 8.4 Hz, 2 H),

7.34 (d, J = 7.6 Hz, 2 H), 6.16 (d, J = 6.0 Hz, 1 H), 4.64 (dd, J = 6.4, 2.8 Hz, 1 H),

4.41 (dd, J = 11.4, 5.8 Hz, 1 H), 4.22 (dd, J = 11.4, 1.8 Hz, 1 H), 4.14-4.11 (m, 1 H),

4.05-3.98 (m, 1 H), 3.73-3.67 (m, 1 H), 2.48 (d, J = 5.2 Hz, 1 H), 2.44 (s, 3 H), 0.87

(s, 9 H), 0.08 (s, 6 H); 13C NMR (100 MHz, CDCl3) δ 144.9, 142.7, 132.6, 129.8,

127.9, 103.4, 75.5, 69.0, 68.5, 68.1, 25.6, 21.6, 17.9, -4.6, -4.7; MS (ESI) m/z calcd

C19H30O6SSiNa [M+Na]+ 437.1, found 437.2.

1.2.

O

BrHO

TBSO S3 To a solution of S2 (11.53 g, 27.8 mmol) in DMF (180.0 mL) was added NaBr (14.3

g, 139.0 mmol) and NaHCO3 (6.0 g, 71.5 mmol) at rt. After stirring for 10 h at 80 oC,

the mixture was cooled to rt and concentrated in vacuo. The mixture was diluted with

11

EtOAc, washed with water and brine, dried over Na2SO4, filtered, and evaporated in

vacuo. The resulting residue was purified by column chromatography on silica gel

(petroleum/ethyl acetate, 20/1) to provide S3 (7.11g, 89%) as a colorless syrup: [α]D25

= -14.9 (c 1.8, CHCl3); 1H NMR (400 MHz, CDCl3) δ 6.28 (dd, J = 6.0, 1.2 Hz, 1 H),

4.66 (dd, J = 6.2, 2.6 Hz, 1 H), 4.21 (dt, J = 6.4, 1.8 Hz, 1 H), 4.03-3.97 (m, 1 H),

3.81-3.67 (m, 3 H), 2.33 (d, J = 4.4 Hz, 1 H), 0.89 (s, 9 H), 0.09 (s, 6 H); 13C NMR

(100 MHz, CDCl3) δ 143.0, 103.6, 76.4, 71.1, 69.4, 32.1, 25.7, 18.0, -4.4, -4.6.

1.3.

O

BrAcOTBSO S4 To a solution of S3 (7.11g, 22.1 mmol) in CH2Cl2 (270.0 mL) was added Ac2O (3.8

mL, 40.2 mmol) at rt under nitrogen. After stirring for 10 min, Et3N (5.8 mL, 41.5

mmol), DMAP (183 mg, 1.5 mmol) was added to the mixture, and the whole solution

was kept stirring overnight. The solution was evaporated under reduced pressure. The

resulting syrup was dissolved in EtOAc, and washed sequentially by water, saturated

NaHCO3 (twice), and brine. The organic layer was dried over Na2SO4, filtered, and

concentrated in vacuo. The residue was purified by flash column chromatography on

silica gel (petroleum ether/ethyl acetate, 30/1) to provide S4 (7.83 g, 98%) as a

colorless syrup: [α]D26 = -50.8 (c 2.1, CHCl3); 1H NMR (400 MHz, CDCl3) δ 6.36 (dd,

J = 6.4, 0.8 Hz,1 H), 5.09 (t, J = 4.2 Hz, 1 H), 4.78 (ddd, J = 6.0, 4.0, 0.8 Hz, 1 H),

4.28-4.22 (m, 1 H), 4.13-4.09 (m, 1 H), 3.66-3.55 (m, 2 H), 2.09 (s, 3 H), 0.87 (s, 9

H), 0.09 (s, 3 H), 0.08 (s, 3 H); 13C NMR (100 MHz, CDCl3) δ 169.5, 142.7, 102.5,

75.3, 71.3, 64.1, 29.8, 25.6, 20.9, 17.8, -4.8, -5.0; MS (ESI) m/z calcd

C14H25O4BrSiNa [M+Na]+ 389.1, found 389.1.

1.4.

OAcOTBSO S5 To a solution of S4 (7.39 g, 20.2 mmol) and AIBN (330 mg, 2.0 mmol) in dry toluene

(70.0 mL) at 80 oC, Bu3SnH (8.2 mL, 30.5mmol) was added under an Ar atmosphere.

After stirring at this temperature for 30 min, the mixture was concentrated in vacuo.

The residue was purified by column chromatography on silica gel (petroleum

ether/dichloromethane, 1/1) to provide S5 (5.74 g, 99%) as a colorless syrup: [α]D23 =

12

-46.5 (c 1.1, CHCl3); 1 H NMR (400 MHz, CDCl3) δ 6.29 (d, J = 5.6 Hz,1 H), 4.90

(dd, J = 8.0, 6.4 Hz, 1 H), 4.66 (dd, J = 6.4, 2.8 Hz,1 H), 4.25 (d, J = 5.2 Hz, 1 H),

4.02-4.00 (m, 1 H), 2.08 (s, 3 H), 1.30-1.25 (m, 5 H), 0.93-0.86 (m, 9 H), 0.07 (s, 3

H), 0.06 (s, 3 H); 13C NMR (100 MHz, CDCl3) δ 169.8, 143.5, 103.2, 75.0, 72.6, 66.7,

25.6, 21.0, 17.9, 16.7, -4.6, -4.9; HRMS (ESI) m/z calcd C14H26O4Na [M+Na]+

309.1493, found 309.1498.

1.5.

OAcOHO S6

To a solution of S5 (5.34g, 18.6 mmol) in CH3CN (200 mL) at 70 oC, 3HF•Et3N (6.1

mL, 37.2 mmol) was added under an Ar atmosphere. After stirring at this temperature

for 10 h, the mixture was quenched by adding saturated NaHCO3 and extracted with

EtOAc. The combined organic layer was washed with saturated NaHCO3 solution and

brine, dried over Na2SO4, filtered, and evaporated in vacuo. The residue was purified

by column chromatography on silica gel (petroleum ether/ethyl acetate, 4/1) to

provide S6 (3.23 g, 97%) as a colorless syrup: [α]D23 = 50.3 (c 1.2, CHCl3); 1HNMR

(400 MHz, CDCl3) δ 6.33 (d, J = 6.0 Hz, 1 H), 4.78 (dd, J = 6.4, 2.4 Hz,1 H), 4.75

(dd, J = 9.2, 7.2 Hz, 1 H), 4.25 (brs, 1 H), 4.00-3.92 (m, 1 H), 2.72 (brs, 1 H), 2.12 (s,

3 H), 1.28 (d, J = 6.0 Hz, 3 H); 13C NMR (100 MHz, CDCl3) δ 171.2, 144.3, 102.9,

76.7, 72.4, 67.6, 20.9, 16.8; HRMS (ESI) m/z calcd C8H12O4Na [M+Na]+ 195.0628,

found 195.0636.

1.6.

OAcOMeO S7 To a solution of S6 (3.82 g, 22.2 mmol) in freshly distilled dichloromethane (100.0

mL), 4Å MS (15.0 g) and MeI (3.6 mL, 57.8 mmol) were added at room temperature.

After being stirred for 30 min, Ag2O (16.50 g, 71.2 mmol) was added and the stirring

was continued for another 20 h. The extra MeI was quenched with NaOAc and the

resulting mixture was filtrated through a pad of celite. The filtrate was concentrated in

vacuo to give a residue, which was purified by column chromatography on silica gel

(petroleum ether/ethyl acetate, 10/1) to give S7 (3.77 g, 91%) as a colorless syrup:

[α]D23 = -20.4 (c 1.1, CHCl3); 1HNMR (400 MHz, CDCl3) δ 6.37 (dd, J = 6.4,1.2 Hz,

13

1 H), 5.00 (dd, J = 7.6, 6.0 Hz, 1 H), 4.82 (dd, J = 6.0, 2.8 Hz, 1 H), 4.05-4.01 (m, 1

H), 3.86 (brs,1 H), 3.32 (s, 3 H), 2.08 (s, 3 H), 1.27 (d, J = 6.4 Hz, 3 H); 13C NMR

(100 MHz, CDCl3) δ 169.8, 144.7, 99.3, 73.9, 72.3, 71.4, 55.0, 20.8, 16.2; HRMS

(ESI) m/z calcd C9H14O4Na [M+Na]+ 209.0784, found 209.0787.

1.7.

OAcOMeO

OAcOMeO OAc

I

NIS, HOAc,toluene, reflux

S7 S8 (54%)

OAcOMeO

OAc

I

S11 (45%)

+

To a solution of S7 (3.67 g, 19.8 mmol) in toluene (230 mL) were added HOAc (4.6

mL, 81.0 mmol) and NIS (13.4 g, 59.7 mmol) at 150 oC. After stirring for 15 min at

this temperature, the mixture was cooled to rt and stirred with 10% Na2S2O3 solution

until the mixture turned colorless. The resulting mixture was diluted with EtOAc,

washed with saturated Na2S2O3 solution (twice) and with brine, respectively. The

organic layer was dried over Na2SO4, filtered, and evaporated in vacuo. The residue

was purified by column chromatography on silica gel (petroleum ether/ethyl acetate,

10/1 to 4/1) to give S8 (3.95 g, 54%) as a white solid and S11 (3.30 g, 45%) as a

colorless syrup. S8: 1H NMR (400 MHz, CDCl3) δ 6.12 (d, J = 3.2 Hz, 1 H, α), 5.69

(d, J = 9.6 Hz, 1 H, β), 4.66 (t, J = 9.5 Hz, 1 H, α), 4.63 (t, J = 9.2 Hz, 1 H, β), 3.93

(dd, J = 11.2, 3.5 Hz, 1 H, α), 3.85-3.76 (m, 1 H, α/β), 3.60-3.48 (m, 1 H, α/β),

3.43-3.38 (m, 1 H, β), 3.41 (s, 3 H, α/β), 2.06 (s, 3 H, α), 2.03 (s, 3 H, β), 2.01 (s, 3 H,

α/β), 1.09 (d, J = 6.0 Hz, 3 H, β), 1.03 (d, J = 6.0 Hz, 3 H, α); 13CNMR (100 MHz,

CDCl3) δ 169.1 (β), 169.0 (α), 168.4 (β), 168.3 (α), 93.7 (β), 91.7 (α), 84.0 (β), 80.5

(α), 75.0 (α), 74.6 (β), 70.8 (β), 68.1 (α), 59.5 (α), 59.3 (β), 28.9 (β), 26.6 (α), 20.6

(β, α), 20.5 (β, α), 17.0 (α), 16.8 (β); HRMS (MALDI) Calcd forC11H17IO6Na

[M+Na]+394.9962, found 394.9957. S11: [α]D22 = 4.0 (c 0.9, CHCl3); 1H NMR (400

MHz, CDCl3) δ 6.28 (d, J = 1.6 Hz, 1 H), 4.96 (t, J = 9.8 Hz, 1 H), 4.43 (dd, J = 4.0,

1.6 Hz, 1 H), 3.91-3.83 (m, 1 H), 3.27 (s, 3 H), 2.84 (dd, J = 9.0, 4.2 Hz, 1 H), 2.06 (s,

3 H), 2.02 (s, 3 H), 1.13 (d, J = 6.0 Hz, 3 H); 13C NMR (100 MHz, CDCl3) δ 169.5,

168.3, 94.8, 75.5, 73.1, 69.3, 56.4, 29.8, 20.7, 17.3; HRMS (ESI) Calcd for

C11H17IO6Na [M+Na]+ 394.9962, found 394.9953.

1.8.

14

OAcOMeO OH

IS9

To a solution of S8 (3.95 g, 10.6 mmol) in CH2Cl2/MeOH (60 mL/60 mL) was added

N2H4•H2O (2.6 mL) at rt. After stirring for 2.5 h at rt, the mixture was filtered through

a pad of silica gel (petroleum ether/ethyl acetate, 2:1). The filtrate was concentrated to

yield the corresponding hemiacetal S9 (3.50 g, 100%) as a colorless syrup: 1H NMR

(400 MHz, CDCl3) δ 5.25 (t, J = 3.2 Hz, 1 H, α), 5.06 (brs, -OH, β), 4.82 (d, J = 8.8

Hz, 1 H, β), 4.66-4.58 (m, 1 H, α/β), 4.57 (brs, -OH, α), 4.07-4.01 (m, 1 H, α), 3.87

(dd, J = 11.2, 2.8 Hz, 1 H, α), 3.78 (dd, J = 11.2, 9.2 Hz, 1 H, β), 3.68 (dd, J = 11.0,

9.0 Hz, 1 H, α), 3.49-3.38 (m, 2 H, β), 3.42 (s, 3 H, α), 3.41 (s, 3 H, β), 2.03 (s, 3 H,

α/β), 1.12 (d, J = 6.4 Hz, 3 H, β), 1.04 (d, J = 6.4 Hz, 3 H, α); 13CNMR (100 MHz,

CDCl3) δ 170.0, 169.8, 96.9, 93.2, 84.0, 80.1, 75.7, 74.7, 69.9, 85.5, 59.4, 58.9, 33.2,

29.9, 20.78, 20.75, 17.04, 16.98; HRMS (ESI) Calcd for C9H15IO5Na [M+Na]+

352.9856, found 352.9855.

1.9.

S10O

OAcOMeO CF3

NPhI

To a solution of hemiacetal S9 (3.50 g, 10.66 mmol) in CH2Cl2 (70.0 mL) was added

Cs2CO3 (8.9 g, 27.5 mmol) and N-phenyl-2,2,2-trifluoroacetimidoyl chloride (2.4 mL,

21.9 mmol) at rt. After stirring for 2.5 h, the mixture was filtered through a pad of

celite. The filtrate was evaporated in vacuo to give a residue, which was subjected to

chromatography on DavisilTM silica (pH = 7.0, petroleum ether/ethyl acetate, 20:1) to

give S10 (4.92 g, 80%) as a colorless syrup: 1H NMR (400 MHz, CDCl3) δ 7.35 (t, J

= 8.0 Hz, 2 H), 7.16 (t, J = 7.6 Hz, 1 H), 6.9 (d, J = 7.2 Hz, 2 H), 5.96 (brs, 1 H),

4.82 (t, J = 9.2 Hz, 1 H), 4.01 (t, J = 9.0 Hz, 1 H), 3.55 (s, 3 H), 3.59-3.42 (m, 1 H),

2.13 (s, 3 H), 1.25 (d, J = 6.0 Hz, 3 H); 13CNMR (100 MHz, CDCl3) δ 169.3, 143.1,

128.7, 124.4, 119.1, 96.4, 84.0, 74.5, 71.2, 59.4, 28.0, 20.7, 17.0.

2. Synthesis of Gordonoside F (3)

15

S10

OOAcO

MeO CF3

NPhI

Bu3SnH, AIBN,toluene, 80 oC, 92%

2) EDCI, DIPEA, DMAP,DCM, rt, 97%.

CO2H

Hoodigogenin A

OH

O

O

O

O

O

MeO

OO

OMe

OO

MeO

OORO

MeO

OMPO

MeO

OO

OMe

OO

MeO

OOAcO

MeOR

O

MeO

OO

OMe

OO

MeO

OOAcO

MeO

O

O

S13 R = IS14 R = H

S15

TBSOTf, DCM,5Å MS, -72 oC, 97%

KOH, MeOH,toluene, rt, 94%

OMPO

MeO

OO

OMe

OO

MeO

HO

S12+

1) Ag(DPAH)2, CH3CN,H2O, 98%.

PPh3AuOTf, DCM,0 oC, 98% (β/α = 1/1)

OH

O

O

O

O

O

MeO

OO

OMe

OO

MeO

OOAcO

MeO

S16β R = Ac3 R = H

S16α

+

2.1.

OMPO

MeO

OO

OMe

OO

MeO

OOAcO

MeOI

S13 To a solution of the trisaccharide acceptor S12S2 (1.90 g, 3.4 mmol) and donor S10

(3.04 g, 5.3 mmol) in CH2Cl2 (10.0 mL) was added 5Å MS at rt. After stirring at rt for

30 min, the mixture was cooled to -72 oC. TBSOTf (77 µL, 0.34 mmol) was then

added to the mixture. After stirring for 0.5 h at this temperature, Et3N was added to

quench the reaction. Filtration and evaporation in vacuo gave a residue, which was

purified by column chromatography on silica gel (petroleum

ether/dichloromethane/ethyl acetate, 5/1/2) to afford S13 (2.86 g, 97%) as a white

solid: [α]D25 = 36.2 (c 0.9, CHCl3);1 H NMR (400 MHz, CDCl3) δ 6.91 (d, J = 8.8 Hz,

2 H), 6.75 (d, J = 8.8 Hz, 2 H), 5.26 (d, J = 8.8 Hz, 1 H), 4.74 (d, J = 8.8 Hz, 1 H),

4.68 (t, J = 9.4 Hz, 1 H), 4.57 (d, J = 8.8 Hz, 1 H), 3.96-3.76 (m, 7 H), 3.70 (s, 3 H),

3.44 (s, 3 H), 3.41 (s, 6 H), 3.37 (s, 3 H), 3.27 (d, J = 7.6 Hz, 1 H), 3.22-3.17 (m, 2 H),

2.25 (d, J = 12.4 Hz, 1 H), 2.13-2.06 (m, 2 H), 2.06 (s, 3 H), 1.78 (t, J = 10.8 Hz, 1 H),

1.66-1.51 (m, 2 H), 1.37 (d, J = 5.6 Hz, 3 H), 1.23-1.15 (m, 9 H); 13C NMR (100 MHz,

CDCl3) δ 169.4, 154.5, 151.2, 117.4, 114.2, 103.9, 99.4, 96.6, 84.3, 83.4, 82.3, 81.8,

77.2, 76.8, 76.5, 76.2, 74.7, 69.9, 68.7, 68.2, 58.9, 57.8, 57.7, 55.4, 35.1, 35.0, 34.6,

16

31.2, 20.7, 19.2, 18.1, 18.0, 17.2; HRMS (MALDI) m/z calcd C37H57O15INa [M+Na]+

891.2634, found 891.2653.

2.2.

OMPO

MeO

OO

OMe

OO

MeO

OOAcO

MeO

S14 To a solution of S13 (2.86 g, 3.3 mmol) and AIBN (55 mg, 0.34 mmol) in dry toluene

(50 mL) at 80 oC, Bu3SnH (1.1 mL, 4.1 mmol) was added under an Ar atmosphere.

After stirring at this temperature for 30 min, the mixture was concentrated in vacuo.

The residue was purified by column chromatography on silica gel (petroleum

ether/dichloromethane/ethyl acetate, 6/2/3) to provide S14 (2.25 g, 92%) as a white

solid: [α]D25= -7.9 (c 0.4, CHCl3);1 H NMR (400 MHz, CDCl3) δ 6.96 (d, J = 8.8 Hz,

2 H), 6.80 (d, J = 8.8 Hz, 2 H), 5.31 (dd, J = 9.6, 1.6 Hz, 1 H), 4.80-4.75 (m, 2 H),

4.65 (t, J = 9.2 Hz, 1 H), 4.51 (dd, J = 9.6, 1.6 Hz, 1 H), 4.05-3.98 (m, 1 H), 3.92-3.78

(m, 5 H), 3.76 (s, 3 H), 3.45 (s, 6 H), 3.44 (s, 3 H), 3.40-3.28 (m, 6 H), 3.26-3.20 (m,

2 H), 2.35-2.28 (m, 2 H), 2.16-2.20 (m, 2 H), 2.095 (s, 3 H), 1.87-1.78 (m, 1 H),

1.70-1.59 (m, 3 H), 1.27 (d, J = 6.0 Hz, 3 H), 1.23 (d, J = 6.4 Hz, 6 H), 1.19 (d, J =

6.4 Hz, 3 H); 13C NMR (100 MHz, CDCl3) δ 170.0, 154.6, 151.2, 117.4, 114.2, 101.2,

99.6, 99.5, 96.6, 82.5, 82.3, 81.9, 77.6, 76.9, 76.8, 76.5, 75.1, 69.8, 68.7, 68.3, 68.1,

58.2, 57.9, 57.7, 56.3, 55.4, 36.0, 35.5, 35.1, 34.6, 20.9, 18.1, 17.5; HRMS (MALDI)

m/z calcd C37H58O15Na [M+Na]+ 765.3668, found 765.3679.

2.3.

O

MeO

OO

OMe

OO

MeO

OOAcO

MeO

O

OS15

To a solution of S14 (2.25 g, 3.0 mmol) in CH3CN/H2O (25mL/25mL) was added

Ag(DPAH)2 (3.07 g, 6.7 mmol) at rt. After being stirred in darkness for 3 h, the

reaction was quenched with saturated NaHCO3. The aqueous phase was extracted

with ethyl acetate. The combined organic phase was washed with brine, dried over

Na2SO4, filtered, and evaporated in vacuo. The residue was purified by column

chromatograph on silica gel (petroleum ether/dichloromethane/ethyl acetate, 2/1/1) to

provide the corresponding hemiacetal (1.88 g, 98%). The hemiacetal (1.78 g, 2.8

17

mmol) was dissolved in anhydrous CH2Cl2 (20.0 mL) at rt, to which

2-(cyclopropylethynyl)- benzoic acid (629 mg, 3.4 mmol), DMAP (340 mg, 2.4

mmol), EDCI (646 mg, 5.2 mmol), and DIPEA (0.85 mL) were added. After being

stirred overnight at rt, the mixture was diluted with CH2Cl2, washed with 1N HCl,

saturated aqueous NaHCO3, and brine, respectively. The organic phase was dried

over anhydrous Na2SO4 and concentrated in vacuo. The crude product was purified

by column chromatography on silica gel (petroleum ether/dichloromethane/ethyl

acetate, 6/1/2.5) to provide S15 (2.17 g, 97%) as a white solid: [α]D25 = 11.2 (c 1.6,

CHCl3); 1 H NMR (400 MHz, CDCl3) δ 7.92 (d, J = 7.6 Hz, 1 H), 7.46 (d, J = 7.6 Hz,

1 H), 7.40 (t, J = 7.4 Hz, 1 H), 7.28 (d, J = 7.6 Hz, 1 H), 6.23 (dd, J = 9.4, 1.8 Hz, 1

H), 4.82-4.75 (m, 2 H), 4.85 (t, J = 9.4 Hz, 1 H), 4.52 (d, J = 8.8 Hz, 1 H), 4.16-4.06

(m, 1 H), 3.92-3.76 (m, 5 H), 3.48 (s, 3 H), 3.46 (s, 3 H), 3.45 (s, 3 H), 3.41-3.33 (m,

3 H), 3.33 (s, 3 H), 3.26-3.22 (m, 2 H), 2.38-2.30 (m, 2 H), 2.18-2.09 (m, 2 H), 2.09

(s, 3 H), 1.87-1.78 (m, 1 H), 1.70-1.59 (m, 3 H), 1.5-1.48 (m, 1 H), 1.28 (d, J = 6.4

Hz, 3 H), 1.23 (d, J = 6.0 Hz, 6 H), 1.19 (d, J = 6.8 Hz, 3 H), 0.91-0.85 (m, 4 H); 13CNMR (100 MHz, CDCl3) δ 170.1, 164.3, 134.1, 131.6, 131.1, 130.4, 126.8, 124.8,

101.3, 99.6, 99.4, 91.8, 82.6, 82.3, 81.4, 77.6, 77.2, 76.93, 76.86, 76.1, 75.2, 74.4,

69.9, 68.3, 68.2, 58.3, 57.9, 57.5, 56.3, 36.0, 35.6, 35.1, 33.3, 29.5, 20.9, 18.1, 18.0,

17.6, 8.7, 0.5; HRMS (ESI) Calcd forC42H60O15Na [M+Na]+ 827.3824, found

827.3821.

2.4.

OH

O

O

O

O

O

MeO

OO

OMe

OO

MeO

OOAcO

MeO

S16βOH

O

O

O

O

O

MeO

OO

OMe

OO

MeO

OOAcO

MeO

S16α

To a solution of donor S15 (80 mg, 0.10 mmol) in dry CH2Cl2 (5.0 mL) were added

Hoodigogenin AS3 (60 mg, 0.14 mmol) and 5Å MS (200 mg). After being stirred for

30 min at rt, to the mixture was added PPh3AuOTf (0.2 mL, 0.05M in in CH2Cl2) at 0 oC. The stirring was continued for 1 h and the mixture was then filtered through

Celite and concentrated in vacuo. The residue was purified by column

chromatography on silica gel (petroleum ether /ethyl acetate, 2/1) to provide S16β

(52 mg, 50%) and S16α (50 mg, 48%) as white solids. S16β: [α]D28 = 6.4 (c 1.3,

CHCl3); 1 H NMR (400 MHz, CDCl3) δ 6.92 (qd, J = 7.0, 1.4 Hz, 1 H), 5.41 (brs, 1

18

H), 4.84 (d, J = 9.6 Hz, 1 H), 4.76 (d, J = 9.2 Hz, 1 H), 4.74 (d, J = 8.8 Hz, 1 H),

4.68-4.61 (m, 2 H), 4.51 (dd, J = 9.8, 1.4 Hz, 1 H), 4.25 (brs, 1 H), 3.90-3.76 (m, 6 H),

3.58-3.47 (m, 1 H), 3.45 (s, 3 H), 3.441 (s, 3 H), 3.437 (s, 3 H), 3.39-3.33 (m, 2 H),

3.33 (s, 3 H), 3.25-3.12 (m, 4 H), 2.41-2.30 (m, 3 H), 2.20 (s, 3 H), 1.26-1.18 (m, 12

H), 1.06 (s, 3 H), 0.98 (s, 3 H); 13C NMR (100 MHz, CDCl3) δ 217.0, 170.1, 167.6,

139.0, 137.7, 128.7, 121.9, 101.3, 99.7, 99.6, 95.8, 85.6, 82.6, 82.44, 82.42, 77.7, 77.2,

76.7, 75.8, 75.2, 70.0, 68.5, 68.3, 68.2, 58.3, 57.94, 57.92, 57.1, 56.4, 53.7, 43.0, 38.6,

37.2, 37.0, 36.1, 35.65, 35.58, 35.5, 35.2, 34.3, 33.1, 29.6, 29.4, 27.3, 26.0, 24.3, 21.0,

19.2, 18.2, 18.1, 17.6, 14.4, 12.1, 9.8; HRMS (MALDI) m/z calcd C56H88O18Na

[M+Na]+ 1071.5863, found 1071.5876. S16α: [α]D28 = 44.1 (c 1.3, CHCl3); 1 H NMR

(400 MHz, CDCl3) δ 6.93 (q, J = 6.8 Hz, 1 H), 5.37 (brs, 1 H), 4.88 (d, J = 4.0 Hz, 1

H), 4.79 (d, J = 8.8 Hz, 1 H), 4.76 (d, J = 8.8 Hz, 1 H), 4.67-4.62 (m, 2 H), 4.51 (d, J

= 8.4 Hz, 1 H), 4.28-4.17 (m, 2 H), 3.91-3.71 (m, 5 H), 3.45 (s, 3 H), 3.43 (s, 3 H),

3.42 (s, 3 H), 3.39-3.29 (m, 6 H), 3.23 (dd, J = 9.6, 2.4 Hz, 1 H), 3.16-3.10 (m, 1 H),

2.38-2.26 (m, 4 H), 2.20 (s, 3 H), 1.26-1.18 (m, 12 H), 1.06 (s, 3 H), 0.98 (s, 3 H); 13C

NMR (100 MHz, CDCl3) δ 216.9, 170.1, 167.6, 139.4, 137.7, 128.7, 121.5, 101.3,

99.6, 99.4, 93.8, 85.7, 82.6, 82.3, 81.5, 77.7, 77.2, 76.9, 75.9, 75.5, 75.2, 69.9, 68.4,

68.2, 62.8, 58.3, 57.8, 57.4, 57.1, 56.4, 53.7, 42.9, 39.8, 37.1, 37.0, 36.1, 35.64, 35.56,

35.0, 34.3, 33.0, 32.8, 29.6, 27.4, 27.2, 26.0, 24.3, 21.0, 19.3, 18.14, 18.11, 17.62,

17.60, 14.4, 12.1, 9.8; HRMS (MALDI) m/z calcd C56H88O18Na [M+Na]+ 1071.5863,

found 1071.5869.

2.5.

OH

O

O

O

O

O

MeO

OO

OMe

OO

MeO

OOHO

MeO

3 (Gordonoside F) To a solution of S16β (50 mg, 0.048 mmol) in toluene (8.0 mL) was added KOH (0.8

mL, 11 mg/mL in MeOH). The mixture was stirred for 40 min at rt, and was then

neutralized with 1 M HCl solution to pH = 6~7. The mixture was diluted with EtOAc.

The organic phase was washed with H2O and brine, dried over anhydrous Na2SO4,

and then concentrated in vacuo. The residue was purified by column chromatography

on silica gel (petroleum ether /ethyl acetate, 1/1) to provide 3 (45 mg, 94%) as a white

19

solid: [α]D27 = 6.1 (c 2.1, CHCl3); 1 H NMR (400 MHz, CDCl3) δ 6.93 (q, J = 6.8 Hz,

1 H), 5.41 (brs, 1 H), 4.84 (d, J = 8.4 Hz, 1 H), 4.76 (d, J = 7.6 Hz, 1 H), 4.74 (d, J =

8.0 Hz, 1 H), 4.64 (dd, J = 12.0, 4.0 Hz, 1 H), 4.50 (d, J = 8.4 Hz, 1 H), 4.26 (brs, 1

H), 3.92-3.78 (m, 6 H), 3.58-3.48 (m, 1 H), 3.45 (s, 6 H), 3.44 (s, 6 H), 3.39 (s, 3 H),

3.32-3.10 (m, 7 H), 2.59 (brs, 1 H), 2.40-2.26 (m, 3 H), 2.20 (s, 3 H), 2.14-2.09 (m, 1

H), 1.33-1.12 (m, 12 H), 1.06 (s, 3 H), 0.98 (s, 3 H); 13C NMR (100 MHz, CDCl3) δ

217.0, 167.6, 138.9, 137.7, 128.7, 121.9, 101.4, 99.7, 99.6, 95.8, 85.6, 82.5, 82.42,

82.38, 80.5, 77.2, 76.9, 75.8, 75.3, 71.5, 68.5, 68.3, 68.2, 58.1, 58.0, 57.9, 57.1, 56.2,

53.7, 43.0, 38.6, 37.2, 37.0, 35.6, 35.5, 35.4, 35.3, 35.2, 34.3, 33.1, 29.6, 29.4, 27.3,

26.0, 24.3, 19.2, 18.2, 18.14, 18.11, 17.9, 14.4, 12.1, 9.8; 1 H NMR (400 MHz,

CD3OD) δ 6.97 (q, J = 7.0 Hz, 1 H), 5.46 (brs, 1 H), 4.82-4.79 (m, 2 H), 4.69 (dd, J =

12.0, 4.0 Hz, 1 H), 4.62 (d, J = 9.5 Hz, 1 H), 3.93-3.77 (m, 6 H), 3.57-3.47 (m, 1 H),

3.46-3.41 (m, 12 H), 3.35-3.18 (m, 5 H), 3.12-3.06 (m, 1 H), 3.00 (t, J = 9.0 Hz, 1 H),

1.30 (d, J = 6.4 Hz, 3 H), 1.24 (d, J = 6.4 Hz, 3 H), 1.22 (d, J = 6.0 Hz, 3 H), 1.20 (d,

J = 6.0 Hz, 3 H), 1.08 (s, 3 H), 1.04 (s, 3 H); 13C NMR (100 MHz, CD3OD) δ 217.4,

169.0, 140.4, 139.2, 129.8, 123.0, 102.8, 101.23, 101.18, 97.2, 87.0, 83.8, 81.6, 78.9,

78.54, 78.50, 78.47, 77.5, 76.9, 73.2, 69.9, 69.84, 69.79, 58.9, 58.6, 58.51, 58.49, 57.4,

55.0, 44.5, 39.7, 38.30, 38.26, 37.5, 37.4, 36.7, 36.4, 36.3, 34.9, 32.4, 30.6, 28.3, 27.1,

24.9, 19.9, 18.6, 18.5, 18.4, 14.5, 12.3, 10.6; HRMS (MALDI) m/z calcd C54H86O17Na

[M+Na]+ 1029.5757, found 1029.5758.

Reference

S1. Schou, C.; Pedersen, E. B.; Nielsen, C. Acta Chem. Scand. 1993, 47, 889-895.

S2. Yuyong Ma, PhD Theses, Shanghai Institute of Organic Chemsitry, Chinese

Academy of Sciences, 2013.

S3. Zhang, J.; Shi, H.; Ma, Y.; Yu, B. Chem. Commun. 2012, 48, 8679-8681.

20

C) 1H and 13C NMR Spectra of the Key Compounds

Compound S10

21

Compound S15

22

Compound 3