si appendix for...1 si appendix for: molecular matchmaking between the popular ‘weight loss’...
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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: [email protected]. Or Dr. Biao Yu,
345 Lingling Road, Shanghai 200032, China; Fax: 0086-21-64166128; E-mail:
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 : [email protected] - 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