Glucosylated suspensosides, water-soluble pheromone conjugates from the oral secretions of male Anastrepha suspensa

Article abstract of DOI:10.1021/np800096k

A diastereomeric mixture of the glycosylated pheromones (6R)- (1a) and (6S)-β-D-glucopyranosyl 2-(2,6-dimefhyl-6-vinylcyclohex-1-enyl)acetate (1b), which we named respectively suspensoside A and suspensoside B, was isolated from the oral secretions of male Caribbean fruit flies, Anastrepha suspensa. The absolute stereochemical configurations were established using microsample NMR instrumentation, chiral gas chromatography, and chemical synthesis utilizing pure enantiomers of anastrephin, (3aS,4R,7aS)- (4a) or (3aR,4S,7aR)-4,7a-dimethyl-4-vinylhexahydrobenzofuran-2(3H)one (4b), as the aglycon precursor.

Full text of DOI:10.1021/np800096k

1726
J. Nat. Prod. 2008, 71, 1726–1731
Glucosylated Suspensosides, Water-Soluble Pheromone Conjugates from the Oral Secretions of
Male Anastrepha suspensa
Spencer S. Walse,† Fang Lu, and Peter E. A. Teal*
United States Department of Agriculture, Agricultural Research SerVice, Center for Medical, Agricultural, and
Veterinary Entomology 1700 S.W. 23rd DriVe, GainesVille, Florida 32604
ReceiVed February 11, 2008
A diastereomeric mixture of the glycosylated pheromones (6R)- (1a) and (6S)-ꢀ-D-glucopyranosyl 2-(2,6-dimethyl-6-
vinylcyclohex-1-enyl)acetate (1b), which we named respectively suspensoside A and suspensoside B, was isolated from
the oral secretions of male Caribbean fruit flies, Anastrepha suspensa. The absolute stereochemical configurations were
established using microsample NMR instrumentation, chiral gas chromatography, and chemical synthesis utilizing pure
enantiomers of anastrephin, (3aS,4R,7aS)- (4a) or (3aR,4S,7aR)-4,7a-dimethyl-4-vinylhexahydrobenzofuran-2(3H)one
(4b), as the aglycon precursor.
Volatile pheromones produced by male Anastrepha suspensa
(Loew), the Caribbean fruit fly, are key factors in the natural
aggregation strategy of this agricultural pest.1-4 To attract conspecifics,
males deposit aqueous oral secretions (∼33 wt % sugar) at lek mating
sites that emit, over many days, two diastereomeric pairs of the trans-
fused γ-lactone 4,7a-dimethyl-4-vinylhexahydrobenzofuran-2(3H)-
one: (3aS,4R,7aS)-(-)-anastrephin (4a), (3aR,4S,7aR)-(+)-anastrephin
(4b), (3aS,4S,7aS)-(-)-epianastrephin (4c), and (3aR,4R,7aR)-(+)-
epianastrephin (4d).5 Since the γ-lactones (4a-d) have a greater
potential for water to air (or lipid) phase transfer than the other
structures with which they are in aqueous equilibria,6,7 we became
interested in elucidating precursory chemistry, such as hydrolysis,
dehydration, and conjugation reactions, which enable the oral secretions
to carry and release the volatile aggregation pheromones over
prolonged periods. We describe herein the isolation of natural
diastereomeric pheromone glucoconjugates named suspensoside A and
suspensoside B, respectively (6R)- (1a) and (6S)-ꢀ-D-glucopyranosyl
2-(2,6-dimethyl-6-vinylcyclohex-1-enyl)acetate (1b), as well as the
chemical synthesis used to confirm absolute stereochemical configuration.
standard and was used to confirm that the liberated hexose
monosaccharide was glucose. With respect to the aglycon, the acid-
catalyzed hydrolysis of the purified conjugate (1 M HCl at 80 °C,
3 h) resulted in 4a-d, 3a-d, and 2a and 2b; alternatively, the
base-catalyzed hydrolysis described above resulted in only 2a and
2b. These products, which were identified on the basis of
chromatographic and spectrometric agreement with purchased or
synthetic standards (Figures S2 and S9, Supporting Information),
suggested that the glucosylated component was either 1a, 1b, or a
mixture of the diastereomers. It is interesting to note that all of
these pheromone components are related through aqueous equilibria,
and all occur in freshly collected oral secretions of males.
Results and Discussion
An initial structural analysis with HPLC-ESIMS of crude oral
secretions from A. suspensa males revealed a component with
prominent buffer-adduct ions of m/z 401.4 [M + formate]- and
374.4 [M + NH4]+ in negative- and positive-ionization modes,
respectively (Figure 1). These results supported a molecular formula
of C18H28O7, which was later confirmed with HRESIMS. Based
on column and mobile phase considerations, the retention time of
this component, 20.3 ( 0.1 min, was diagnostic of nonpolar
character. Yet, MSn experiments showed the loss of m/z 162, which
typifies the cleavage of a hexose monosaccharide structural unit.
Degradation studies aided the characterization of both the glycon
and aglycon of 1a and 1b. In the case of the glycon, base-catalyzed
hydrolysis (1 M NaOH at 45 °C, 3 h) of the purified component
yielded a hexose monosaccharide that coeluted with 13C6 D-glucose
during HPLC-ESIMS analysis ( Figure S1, Supporting Information).
Acidic methanolysis of the purified component (1 M HCl/MeOH,
80 °C, 18 h) followed by treatment with Tri-Sil (Pierce, Rockford,
IL) in pyridine (80 °C, 0.5 h) yielded per-O-trimethylsilyated
derivatives of the glycon. Subsequent analysis with GC-MS, as
described in Merkle and Poppe,8 agreed with that of an authentic
Microsample NMR studies were used to confirm that the
glucosylated pheromone component naturally exists as a mixture
of 1a and 1b. Using a novel a 1 mm triple-resonance high-
temperature superconducting probe developed by Brey et al.,9 we
1
performed H NMR, COSY, HMQC, and HMBC experiments on
∼40 µg of isolated material in 10 µL of d4-methanol (Table 1 and
Figure 2). One salient 1H NMR spectroscopic feature was the
doubling of peaks diagnostic of a diastereomeric pair that was not
resolved during chromatographic purification (Figure 3). The C-7
(δC 34.8) AB spin system clearly illustrates this relationship and
* To whom correspondence should be addressed. Tel: (352) 374-5730.
Fax: (352) 374-5707. E-mail: pteal@gainesville.usda.ufl.edu.
† Current address: United States Department of Agriculture, Agricultural
Research Service, San Joaquin Valley Agricultural Sciences Center, Parlier,
CA 93648.
10.1021/np800096k CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 09/18/2008

Glucosylated Suspensosides from Anastrepha suspensa
Journal of Natural Products, 2008, Vol. 71, No. 10 1727
three vinylic protons (δH 4.91, 5.04, 5.71), as well as the two furthest
upfield methylene proton systems (δH 1.46-1.53, 1.54-1.65),
correlated with the aliphatic quaternary carbon at C-6 (δC 41.9).
Another set of methylene protons (δH 1.98-2.12) showed analogous
correlation to the two vinylic carbons at C-1 (δC 127.0) and C-2
(δC 134.5), which have no attached protons, but not to the aliphatic
quaternary carbon at C-6 (δC 41.9). The methylene protons of the
C-7 AB spin system (δH 3.01-3.24) were particularly useful in
deciphering the core cyclohexene acetic acid structure of the
aglycon, as they coupled to the four quaternary carbons (C-1, C-2,
C-6, C-8) only, and not to the carbons at C-3, C-4, and C-5, which
have methylene protons, respectively, at δH 1.98-2.12, 1.54-1.65,
1
and 1.46-1.53. H-1H COSY was used to probe the three-bond
vicinal (C-3/C-4, C-4/C-5, C-1′/C-2′, C-2′/C-3′, C-3′/C-4′, C-4′/
C-5′, C-5′/C-6′), four-bond (C-3/C-5, C-2′/C-4′, C-3′/C-5′), vinylic
(C-11/C-12), and geminal (C-3, C-4, C-5, C-7, C-6′) proton
interactions at the specified carbons. In combination with COSY
results, HMQC correlations were used to assign the glucose carbons
(C-1′ through C-6′).
The relative stereochemical configurations of the aglycons and
glucoconjugates were confirmed with chemical synthesis (Scheme
1). Briefly, the γ-hydroxy carboxylates (3a or 3b) were quantita-
tively obtained from enantiomerically pure (>97% ee) 4a or 4b
starting material via base-catalyzed hydrolysis (2-propanol: H2O,
1:1 v/v, KOH). Methyl esterification, to form 5a or 5b, was then
accomplished using methanolic CH2N2. Tertiary alcohol dehydration
(E1), using triphenylphosphine and iodine,10 resulted in the
formation of 6a or 6b (∼65% yield) and the regeneration of 4a or
4b (∼25% yield), which were both purified using semipreparative
normal-phase HPLC. Hydrolysis of 6a or 6b under mild basic
conditions (K2CO3 aqueous in methanol, 15 °C) gave 2a or 2b. A
stereoselective ꢀ-O-glucosylation of 6a or 6b with 2,3,4,6-tetra-
acyl-R-D-glucopyranosyl bromide was achieved by refluxing them
in ACN containing mercuric cyanide.11 Another hydrolysis under
mild basic conditions allowed for the removal of glycon acetyl
groups and the formation of 1a or 1b.
The absolute stereochemical configurations of the natural phero-
mone glucoconjugates were verified by analyzing the derivatives
of the base-catalyzed hydrolysates, using GC-FID with a ꢀ-Dex
120 chiral analytical column capable of enantiomeric resolution.
Specifically, GC-amenable methyl esters of the natural aglycon were
prepared with methanolic diazomethane for retention comparisons
against synthetic 6a and 6b. Figure 4 illustrates that synthetic 6a
(tR 20.64 ( 0.01 min) and 6b (tR 20.82 ( 0.01 min) resolve
chromatographically and that the methylated natural algycon was
nearly racemic (48 ( 8% 6a; 52 ( 9% 6b; mean ( SE, n ) 10).
For glucose determination, retention comparisons were made
between the alditol trifluoroacetate derivatives12 of purchased
D-glucose (tR 25.50 ( 0.1 min) or L-glucose (tR 24.53 ( 0.1 min)
standards and similarly derivatized glycon, which coeluted only
with the former.
Figure 1. HPLC-(-)ESIMS of 1a or 1b over m/z 80-800 (A),
MS2 spectrum of m/z 401 [M + formate]- showing loss of formate
(B), and MS3 spectrum of the m/z (401f355) ions (C) that results
from the carboxylate of the aglycon. HPLC-(+)ESIMS of 1a or
1b over m/z 80-800 (D), MS2 spectrum of m/z 374 ([M + NH4]+)
showing loss of a hexose monosaccahride (E), and MS3 spectrum
of the m/z (374f195) ions (F) that results from an acylium ion,
consistent with the (+)ESIMSn spectra of 3a-d and 4a-d (shown
in the Supporting Information).
showed geminal coupling values (2J ≈ 17.7 Hz) consistent with
those expected for methylene protons of this type (δH 3.01-3.24).
The proton signals (δH 5.46-5.48) at the anomeric C-1′ position
(δC 94.2) of the glycon also exhibited conspicuous peak doubling,
as well as coupling (3J1′-2′ ) 8.15 Hz) associated with a diaxial
arrangement of vicinal protons and a ꢀ-glucosidic orientation.
The pheromone aglycon is most likely conjugated enzymatically
within the crop, or related digestive tissue,13,14 as glucosidases and
glucotransferases often function in an anabolic capacity15,16 and
abiotic O-glucosyl esterification of 2a or 2b was not observed under
simulated physiologic conditions of 0.01 M NaHCO3 buffer at crop
pH (5.5) at 35 °C over a week. In other controlled experiments
under these conditions, only 8 ( 3% of 1a and 1b was converted
to 2a or 2b. The communicative function(s) of the glucoconjugates
for A. suspensa is likely tied to the integrity of the O-glucosyl ester
linkage. Enzymic, or abiotic, hydrolysis of the glucoconjugates in
the oral secretions would prolong the emission of 4a-d from lek
sites. This is curious from an evolutionary perspective, because
many fruits control the release of their volatile attractants from
sugar-based matrixes in this manner.17 Alternatively, pheromone
activity of the glucoconjugates, or the enzymatically liberated
aglycon, may be related to the tasting of oral secretion scent
1
Multiple-bond H-13C connectivity, assessed using HMBC, was
observed between the anomeric proton at C-1′ and the most upfield
carbon resonance, C-8 (δC 171.2). This result, in combination with
base-catalyzed hydrolysis and mass spectrometry, was indicative
of an O-glucosyl ester linkage to the aglycon. Three other quaternary
1
carbons, not amenable to single-bond H-13C HMQC correlation
experiments, were also detected with HMBC. The more deshielded
methyl protons (δH 23.7) showed connectivity to an aliphatic carbon,
C-6 (δC 41.9), and the more shielded methyl protons (δH 19.5)
showed connectivity to a vinylic carbon, C-2 (δC 134.5); both
groups of methyl protons also exhibited connectivity, albeit
relatively weaker, to another vinylic carbon at C-1 (δC 127.0). The

1728 Journal of Natural Products, 2008, Vol. 71, No. 10
Walse et al.
Table 1. NMR Spectroscopic Data (600 MHz, CD3OD) of Natural 1a and 1b
position
δc, mult
δH (J in Hz)
HMBC (H f C#)
COSY
1
2
3
4
5
6
7
127.0, qC
134.5, qC
32.1, CH2
18.5, CH2
37.5, CH2
41.9, qC
3, 7, 9, 10, 11
3, 4, 7, 9
4, 5, 9
1.98-2.12, m
1.54-1.65, m
1.46-1.54, m
3, 4, 5
3, 4, 5
3, 4, 5
3, 5
3, 4, 10, 11
4, 5, 7, 10, 11, 12
34.8, CH2
3.07, d(17.7)a; 3.01, d(17.8)b
3.18, d(17.7)a; 3.24, d(17.8)b
7
7
8
9
10
11
12
171.2, qC
19.5, CH3
23.7, CH3
146.4, CH
112.0, CH2
1,7
3
5, 11
5, 10
1.66, s
1.12, s
5.71, dd(3Jcis ) 10.6, 3Jtrans ) 17.5)
4.91, dd (2Jjem ) 1.6, 3Jtrans ) 17.5)
5.04, dd (2Jjem ) 1.6, 3Jcis ) 10.6)
5.46, d (8.15)a; 5.48, d (8.15)b
3.32, d (8.15)
12
11
11
2′
1′, 3′
4′, 5′
3′, 5′
3′, 4′
5′
1′
2′
3′
4′
5′
6′
94.2, CH
72.8, CH
77.5, CH
70.2, CH
77.2, CH
61.4, CH2
2′
3.34-3.38, m
2′, 5′
3.34-3.38, m
3′, 5′, 6′
3′, 4′, 6′
5′
3.34-3.38, m
3.68, dd
3.81, dd
5′
a,b Proton signals of the 6R (1a) and 6S (1b) diastereomers, respectively.
injector, a deactivated fused silica column that served as a retention
gap (l 10 m, i.d. 0.25 mm, df 0.25 µm), and a Supelco ꢀ-Dex 120
analytical column (l 30 m, i.d. 0.25 mm, df 0.25 µm). The oven program
was as follows: isothermal at 80 °C for 5 min, heated at 7 °C/min to
150 °C, then isothermal for 35 min. Analyte tR (min): 6a 20.64 ( 0.01,
6b 20.82 ( 0.01, L-glucose 24.53 ( 0.1, D-glucose 25.50 ( 0.1, 4b
46.77 ( 0.02, 4a 47.49 ( 0.02, 4d 48.24 ( 0.02, and 4c 48.76 (
0.02.
Gas Chromatography-Mass Spectrometry. A Varian 3400 gas
chromatograph and a Finnigan MAT Magnum ion trap mass spectrom-
eter (GC-ITMS) were operated with 70 eV electron impact (EI)
ionization or isobutane chemical ionization (CI). Full-scan spectra (m/z
40 to 400) were acquired at 0.85 s per scan. Cool on-column injection
(1 µL) was at 40 °C with He carrier gas (1.4 mL/min). Transfer-line
and manifold temperatures were 240 and 220 °C, respectively. The
oven program was as follows: isothermal at 40 °C for 5 min, heated at
11 °C/min to 200 °C, isothermal for 10 min, heated at 25 °C/min to
250 °C, then isothermal for 15 min. The three-column system described
above was used, except that the analytical column was a J&W DB-1
(l 30 m, i.d. 0.25 mm, df 0.25 µm). Analyte tR (min): 6a and 6b 15.27
( 0.01, 4a and 4b 17.15 ( 0.01, 3a-d 17.22 ( 0.01, 4c and 4d 17.31
( 0.01, and 2,3,4,6-tetra-acyl-R-D-glucopyranosyl bromide 21.28 (
0.01.
1
Figure 2. Selected multiple-bond H-13C correlation for 1a and
1b as revealed by HMBC.
markings by conspecifics, which is a common behavior within
Tephritidae.3 To substantiate the latter, future investigations will
explore the physiological effects of feeding 1a and 1b to adult male
and female A. suspensa.
HPLC-Mass Spectrometry. A Thermo Separation Products Spectra
System P4000 pump, a ThermoFinnigan UV6000LP LDC photodiode
array detector (PDA), a YMC-Pack ODS-AQ analytical column (l 250
mm, i.d. 4.6 mm, S 5 µm), and a Finnigan LCQ DecaXP Max mass
spectrometer (HPLC-MS) were used. Mass spectra were obtained using
electrospray ionization ((ESI) with a 5 kV spray voltage and a 275
°C capillary temperature. Sheath and sweep gas flow rates (arb) were
40 and 20, respectively. The mobile phase (1 mL/min) was split after
the PDA; ∼10% was directed to the MS and the remainder collected.
Eluant composition was (a) 0.1% formic acid in ACN, (b) 10 mM
ammonium formate, and (c) 10 mM ammonium formate in 90% ACN.
The elution program was isocratic (4a:72b:24c) for 13.5 min, to 4:0:
96 over 4.5 min, isocratic (4:0:96) for 17 min. Analyte tR (min): 3c
and 3d 10.3 ( 1.5, 3a and 3b 10.8 ( 1.6, 2a and 2b 12.4 ( 1.7, 1a
and 1b 20.1 ( 0.4, 4c and 4d 21.8 ( 0.4, and 4a and 4b 21.9 ( 0.4.
Fractions containing synthetic or natural pheromone components
were pooled, and the pH was adjusted to 7 with NaHCO3 prior to being
concentrated to dryness. The residue was dissolved in H2O, diluted to
1 mL, transferred to 1 mL Supelco DSC-18 1 mL solid-phase extraction
cartridges, and then flushed into 2 mL tubes. Salts were removed by
rinsing the cartridges with H2O (3 × 1 mL), and the analytes were
eluted with three 1 mL rinses of 50% ACN into 2 mL tubes. The
purified sample was then concentrated to dryness before being dissolved
in d4-methanol for NMR.
Experimental Section
General Experimental Procedures. Enantiomerically pure (>97%
ee) standards (>99%) of 4a-d (lot # 882608-1:5) were purchased from
Nitto Denko Co. (Osaka, Japan), and their spectroscopic and spectro-
metric properties are consistent with previous characterizations.18-26
The external standard for HPLC-MS, (+)-sclareolide, was purchased
from Sigma (St. Louis, MO). HRESIMS analysis was performed on a
Bruker APEX II 4.7 T Fourier transform ion cyclone resonance mass
spectrometer (Bruker Daltonics, Billerica, MA). Optical rotations were
obtained using a Perkin-Elmer 241 polarimeter with a 0.5 mL cell.
UV-vis spectra were acquired using a NanoDrop ND-1000 spectro-
photometer. NMR data were collected at 27 °C using a Bruker Avance
II 600 MHz spectrometer (Bruker 600 Ultrashield magnet) and a novel
1 mm triple-resonance superconducting probe with Z-gradients.9
Chemical shift values were referenced to the residual CD3OD solvent
signal at δC 49.2, δH 3.31. Acquisition parameters for all NMR
experiments are available with their respective spectra in Figures
S10-S15, Supporting Information.
Gas Chromatography-Flame Ionization Detection. An Agilent
6890 N gas chromatograph equipped with a flame ionization detector
(FID) was used for analyses of chirality. Holox (Charlotte, NC) high-
purity helium was used as the carrier gas (1.4 mL/min). Cool on-column
injection (1 µL) was at 83 °C and the detector maintained at 250 °C.
GlasSeal connectors (Supelco Inc.) fused three silica columns in series:
a deactivated silica column (l 8 cm, i.d. 0.53 mm) inserted into the
Insects and Natural Product Isolation. A. suspensa males were
cultured as previously described.5 After squeezing their abdomens with

Glucosylated Suspensosides from Anastrepha suspensa
Journal of Natural Products, 2008, Vol. 71, No. 10 1729
Scheme 1
mL each) yielded methyl esters (5a-d and 6a and 6b) amenable to
GC within the organic phase. In pooled samples of freshly isolated
male OS, the diastereomeric distribution of 4a and 4b to 4c and 4d
and 3a and 3b to 3c and 3d was consistently ∼2.5:1 and is in close
agreement with other studies.21,22 The lactone (4a-d) and acid
(3a-d) pheromone forms, which were collectively present at ∼35
( 5 ng/µL, accounted for 28 ( 7% and 61 ( 6% (grand mean (
SE, n ) 18)27 of the pheromone analytes, respectively. By
comparison, the “free” aglycon (2a and 2b) and the glucoconjugates
(1a and 1b) represented the remaining 11 ( 4% and were found at
Figure 3. Within the 1H NMR spectra of the natural pheromone
glucoconjugates, we observed a diasteromeric relationship
between 1a and 1b that was indicative of approximately racemic
distribution of the aglycons 2a and 2b. (A) The C-7 AB spin
system clearly illustrates this relationship and shows geminal
coupling values of 17.7 and 17.8 Hz for 1a and 1b, respectively.
(B) The protons at the anomeric position (C-1′) of the glycon
also exhibited conspicuous peak doubling, as well as 8.2 Hz
coupling associated with a diaxial arrangement of vicinal protons
and a ꢀ-glucosidic linkage.
the fingertips until regurgitation, oral secretion from 11-14-day-old
adults was harvested with a glass capillary (1 mm i.d.) that penetrated
a vial under slight negative pressure at 4 °C. Collections were made 2
( 0.5 h prior to sunset, pooled until ∼0.6 mL was accumulated, and
stored at -70 °C.
Oral secretion (0.5 mL) was thawed and diluted to 1 mL. These
samples were extracted with hexane (1 mL) containing 0.8 nL of
tetradecane internal standard and analyzed for 4a-d by GC-MS.
Alternatively, these samples were transferred to the solid-phase
extraction cartridges as described above. The cartridges were flushed
with water (3 × 1 mL), and then the analytes were eluted, with 3
× 1 mL rinses of 0.05% formic acid in 50% ACN, into 3 mL tubes.
Eluants were concentrated to 0.5 mL and split. To one 0.25 mL
subsample was added 120 µL of ACN containing 360 µg of external
standard prior to HPLC-MS analysis. The remaining subsample was
concentrated to dryness and dissolved in a methanolic solution of
diazomethane. Extraction with hexane and subsequently H2O (0.5
Figure 4. The natural aglycons were liberated from their conjuga-
tion to glucose via base-catalyzed hydrolysis. GC-FID analysis of
the methyl-esterified aglycons were compared to synthetic standards
and indicated an approximately racemic distribution (48 ( 8% 6a:
52 ( 9% 6b, n ) 10).

1730 Journal of Natural Products, 2008, Vol. 71, No. 10
Walse et al.
∼2.1 ( 0.5 and ∼2.2 ( 0.5 ng/µL, respectively. In light of these
amounts, an alternative method of isolation was developed to
facilitate NMR studies on the natural glucosides; 1 ft × 1 ft × 1 ft
plexiglass rearing cages were wiped with paper towels saturated
with methanol/water (1:1). The towels were collected and rinsed
with methanol in a Buchner funnel. The filtrate was evaporated to
dryness, dissolved in 1 mL, and purified as described above.
Determination of D-Glucose. Corresponding alditol trifluoroac-
etates were prepared for GC-FID with the method of Linqvist and
Jansson12 by dissolving 0.1-1 mg of purchased D- or L-glucose sugar
standards, or the dried base-catalyzed hydrolysate of the glucoside,
in ∼0.3 mL of 1 M NH4OH containing NaBH4 (1 mg/mL) for 30
min at 22 °C. The sample was then repeatedly quenched with
methanol containing 10% acetic acid and evaporated to dryness.
This process was repeated until neutralization. Acetonitrile (∼250
µL), containing 10% trifluoroacetic acid, and ∼100 µL trifluoroacetic
anhydride were added to the dry sample prior to heat treatment (60
°C for 15 min). After cooling to 25 °C, the samples were dried
with a gentle stream of N2, dissolved in ∼0.3 mL of dichlo-
romethane, and analyzed. The resulting glycon derivative coeluted
with that of D-glucose (tR 25.50 ( 0.1 min), but not with that of
L-glucose (tR 24.53 ( 0.1 min).
177 (14), 135 (7), 135 (4); 5a and 5b, m/z (% rel int) 209 [MH]+
(62), 195 (18), 177 (12), 135 (9); 6a and 6b, m/z (% rel int) 209
[MH]+ (75), 177 (7), 153 (11), 135 (7).
2-(2-Hydroxy-2,-6-dimethyl-6-vinylcyclohexanyl)acetic acid (3a-d).
Enantiomerically pure (>97% ee) 4a-d (40 mg, 0.20 mmol) were
hydrolyzed quantitatively to their corresponding γ-hydroxy car-
boxylates, 3a-d, by the dropwise addition of 10 mM NaOH (2 mL)
to 2 mL of 2-propanol that was rapidly stirred at 35 °C for 8 h. The
reaction mixture was then concentrated in vacuo to 1 mL, adjusted
to pH 6 with 0.1 M HCl, and flushed through Supelco DSC-18 1
mL solid-phase extraction cartridges. The cartridges were then rinsed
with water (3 × 1 mL) to remove salt. Compounds 3a-d were eluted
with three 1 mL portions of 0.05% formic acid in 50% ACN, which
were collected, combined, and concentrated to dryness. The
spectroscopic properties agreed well with previous reports.18,19
25
25
25
Colorless; 3a [R]D +27.3, 3b [R]D -25.6, 3c [R]D +42.1, 3d
25
[R]D -39.9 (c 1.0, ethanol); UV (MeOH) λmax (log ε) 220 (3.15)
nm; 1H NMR (600 MHz, methanol-d4) 3a and 3b δ 1.05 (3H, s,
H3-10), 1.09 (3H, s, H3-9), 1.29-1.87 (6H, m, H2-3,4,5), 1.99 (1H,
3
3
2
dd, J ) 3.8, 6.9 Hz, H-1), 2.35 (1H, dd, J ) 3.8 Hz, J ) 15.9
3
2
Hz, HA-7), 2.45 (1H, dd, J ) 6.9 Hz, J ) 16.0 Hz, HB-7), 5.02
2
3
(1H, dd, Jgem ) 1.9 Hz, Jtrans ) 18.6 Hz, HCdCHHtrans-12), 5.07
(1H, dd, 2Jgem ) 1.8 Hz, 3Jcis ) 10.8 Hz, HCdCHHcis-12), 6.04 (1H,
ꢀ-D-Glucopyranosyl 2-(2,6-dimethyl-6-vinylcyclohex-1-enyl)ac-
etate (1a and 1b). 2,3,4,6-Tetra-acyl-R-D-glucopyranosyl bromide
(103 mg, 0.25 mmol), 6a or 6b (20 mg, 0.1 mmol), and HgIICN (57
mg, 0.25 mmol) were refluxed in anhydrous ACN/ether (1:1)11 to
form 7a and 7b; O-glucosylation was monitored by disappearance
of the brominated glycon via GC-MS. Upon completion (∼8 h),
the reaction mixture was diluted with ether, washed with brine, dried
with MgSO4, and concentrated to a residue. A 5% methanolic
solution of sodium methoxide at 22 °C removed the glycon acetyl
groups in ∼3 h. After neutralization with 10% acetic acid, the
mixture, which contained 1a or b in ∼35% overall yield, was
concentrated to dryness. Synthetic glucoside was purified by HPLC
in the same manner as the natural isolate. Colorless; 1a [R]D25 -35,
3
3
dd, Jcis ) 10.7 Hz, Jtrans ) 17.7 Hz, HCdCHH-11); 3c and 3d δ
0.95 (3H, s, H3-10), 1.18 (3H, s, H3-9), 1.25-1.85 (6H, m, H2-
3
3
3,4,5), 1.89 (1H, dd, J ) 4.8, 7.1 Hz, H-1), 2.14 (1H, dd, J ) 4.7
2
3
2
Hz, J ) 16.0 Hz, HA-7), 2.28 (1H, dd, J ) 7.0 Hz, J ) 16.1 Hz,
2
3
HB-7), 4.91 (1H, dd, Jgem ) 1.8 Hz, Jtrans ) 18.0 Hz, HCdCH-
2
3
Htrans-12), 4.99 (1H, dd, Jgem ) 1.7 Hz, Jcis ) 10.7 Hz, HCdCH-
Hcis-12), 5.77 (1H, dd, 3Jcis ) 10.6 Hz, 3Jtrans ) 17.8 Hz, HCdCHH-
11); HMBC (600 MHz, methanol-d4) 3a and 3b δ 20.0 (CH2, C-4),
21.5 (CH3, C-9), 29.5 (CH3, C-10), 34.0 (CH2, C-7), 37.5 (CH2,
C-5), 39.5 (CH2, C-6), 42.5 (C, C-3), 53.5 (CH2, C-1), 75.1 (C,
C-2), 110.5 (HCdCHH, C-12), 151.2 (HCdCHH, C-11), 175.5
(CdO, C-8); 3c and 3d δ 19.5 (CH3, C-10), 20.5 (CH2, C-4), 23.2
(CH3, C-9), 34.5 (CH2, C-7), 38.2 (CH2, C-5), 38.0 (CH2, C-6), 42.4
(C, C-3), 51.5 (CH2, C-1), 73.5 (C, C-2), 112.5 (HCdCHH, C-12),
153.2 (HCdCHH, C-11), 179.1(CdO, C-8); HRESIMS m/z 213.2415
(calcd for [M + H]+ C12H20O3, 213.1491).
25
1b [R]D 37 (c 1.0, CHCl3); UV (MeOH) λmax (log ε) 229 (4.05)
1
nm; H and 13C NMR, see Table 1; HRESIMS m/z 357.2837 (calcd
for [M + H]+ C18H29O7, 357.1914).
2-(2,6-Dimethyl-6-vinylcyclohex-1-enyl)acetic acid (2a and 2b).
Methyl ester 5a or 5b was formed in quantitative yield when 40
mg (0.19 mmol) of 3a or 3b, respectively, was dissolved in a ∼1
mL of methanolic solution of diazomethane. The solution was diluted
with pentane prior to an extraction with H2O (0.5 mL each). The
organic phase, which contained 5a or 5b, was then dried (MgSO4),
filtered, and concentrated to ∼200 µL prior to being dissolved in
0.5 mL of CH2Cl2. As reported previously,10 this solution was then
added dropwise to a solution of CH2Cl2 (2 mL) containing
triphenylphosphine (58 mg, 0.22 mmol) and iodine (56 mg, 0.22
mmol) that has been stirring for ∼10 min. Tertiary alcohol
dehydration, which was monitored by GC-MS until completion (∼2
h), resulted in the formation of 24 mg of 6a or 6b (12 mmol; 65%
yield)28 and 9 mg of 4a or 4b byproduct (5 mg; 25% yield). The
reaction mixture was concentrated to an oily residue and flash
chromatographed on silica gel (230-400 mesh) with hexane/ethyl
acetate (8:2) to remove polar reaction components. Utilizing an
equivalent mobile phase composition at 3 mL/min flow rate, HPLC
with an refractive index detector and an Econosil 10 µm column
(Alltech 6233) was used for final purification of 6a or 6b (tR 6.1 (
0.3 min) and 4a or 4b (tR 10.8 ( 0.4 min). Hydrolysis of 6a or 6b
under mild basic conditions (K2CO3(aq) in methanol, 15 °C) yielded
Acknowledgment. This work was supported by the United States
Department of Agriculture. Partial support was from the DOE-funded
(DE-FG09-93ER-20097) Center for Plant and Microbial Complex
Carbohydrates, which provided complimentary data on glucose
characterization, and the NSF through the External User Program
of the National High Magnetic Field Laboratory. NMR studies,
facilitated by an invaluable collaboration with Arthur S. Edison,
were done at the Advanced Magnetic Resonance Imaging and
Spectroscopy (AMRIS) facility in the McKnight Brain Institute of
the University of Florida. HRESIMS was performed in the mass
spectroscopy laboratory at the University of Florida. We would like
to extend our gratitude to J. Johnson and J. R. Rocca for guidance
on spectrometric and spectroscopic measurements, respectively.
Supporting Information Available: Spectroscopic data are available
free of charge via the Internet at http://pubs.acs.org.
References and Notes
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25
2a or 2b with >95% efficiency. Colorless; 2a [R]D25 -45, 2b [R]D
55 (c 1.0, CHCl3); UV (MeOH) λmax (log ε) 232 (3.05) nm; 1H NMR
(600 MHz, methanol-d4) δ 1.21 (3H, s, H3-10), 1.44-1.51 (2H,
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2
(2H, m, H2-3), 3.08 (1H, d, J ) 17.7 Hz, HA-7), 3.10 (1H, d, J )
2
3
17.7 Hz, HB-7), 4.95 (1H, dd, Jgem ) 1.6 Hz, Jtrans ) 17.5 Hz,
HCdCHHtrans-12), 5.10 (1H, dd, Jgem ) 1.6 Hz, Jcis ) 10.6 Hz,
2
3
3
3
HCdCHHcis-12), 5.65 (1H, dd, Jcis ) 10.6 Hz, Jtrans ) 17.5 Hz,
HCdCHH-11); HMBC (600 MHz, methanol-d4) δ 18.7 (CH2, C-4),
19.1 (CH3, C-9), 23.5 (CH3, C-10), 32.4 (CH2, C-3), 35.8 (CH2,
C-7), 36.9 (CH2, C-5), 42.5 (C, C-6), 113.0 (HCdCHH, C-12), 127.5
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C-8); HRESIMS m/z 195.2232 (calcd for [M + H]+ C12H19O2,
195.1385); GC-CIMS of 4a and 4b, m/z (% rel int) 195 [MH]+ (79),

Glucosylated Suspensosides from Anastrepha suspensa
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NP800096K