Syntheses and determination of absolute configurations and biological activities of the enantiomers of the longtailed mealybug pheromone

Article abstract of DOI:10.1021/jo400491n

Preparation and assignment of absolute configurations to both enantiomers of the sex pheromone of the longtailed mealybug, an irregular monoterpenoid with extraordinary biological activity, has been completed. Comparison of the biological activities of bo

Full text of DOI:10.1021/jo400491n

Note
pubs.acs.org/joc
Syntheses and Determination of Absolute Configurations and
Biological Activities of the Enantiomers of the Longtailed Mealybug
Pheromone
Remya Ramesh,† Pandrangi Siva Swaroop,† Rajesh G. Gonnade,† Choppari Thirupathi,‡
Rebeccah A. Waterworth,§ Jocelyn G. Millar,§ and D. Srinivasa Reddy*,†
†CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune, 411008, India
‡Daicel Chiral Technologies (India) Pvt. Ltd., Hyderabad 500078, India
§Department of Entomology, University of California, Riverside California 92521, United States
S
* Supporting Information
ABSTRACT: Preparation and assignment of absolute config-
urations to both enantiomers of the sex pheromone of the
longtailed mealybug, an irregular monoterpenoid with
extraordinary biological activity, has been completed. Compar-
ison of the biological activities of both enantiomers and the
racemate in field trials showed that the (S)-(+)-enantiomer was highly attractive to male mealybugs, strongly suggesting that
female longtailed mealybugs produce this enantiomer. The (R)-(−)-enantiomer was benign, being neither attractive nor
inhibitory.
he longtailed mealybug Pseudococcus longispinus (Order
Hemiptera: Family Pseudococcidae), is one of a group of
the racemate remained active for several months under field
conditions.6
T
However, to date it has not been possible to determine the
absolute configuration of the insect-produced compound
because the pheromone, its corresponding alcohol, and several
analogues were not resolved by GC on chiral stationary
phases.4 We report here the syntheses of both enantiomers of
the pheromone, the determination of their absolute config-
urations, and the results of field trials testing their biological
activity.
The most recently developed synthesis of the racemic
pheromone progressed through the key intermediate 2, in
which the adjacent quaternary carbons were generated by a
Claisen rearrangement of a readily accessible allylic alcohol
precursor, followed by ring-closing metathesis of 2 to close the
cyclopentene ring with the endo double bond in the correct
position.5c Although the alcohol function in 2 was separated
from the stereogenic center by two carbon atoms, we reasoned
that the enantiomers still might be separable as diastereomeric
derivatives of the alcohol. Furthermore, at least one of the two
resulting diastereomers must be crystalline so that the relative
and absolute configurations could be unambiguously deter-
mined from an X-ray crystal structure determination. After
careful examination of the literature, Harada’s camphorsultam
phthalic (CSP) acid appeared to be a suitable candidate for the
optically active auxiliary.7 Thus, alcohol 2, available in gram
quantities from the synthesis of the racemate,5c was readily
esterified (DMAP, DCC) with CSP acid to produce the
mixture of diastereomers 3a and 3b. Although the diaster-
small sucking insects that are widely distributed pests of
agricultural crops and ornamental plants. Damage is caused by
direct feeding, by the growth of sooty mold and other fungi on
the honeydew excreted by the insects, and increasingly, through
the transmission of plant pathogens.1 Females of sexually
reproducing mealybug species have been shown to produce
powerful sex pheromones to attract males for mating, and some
of these pheromones have been identified and commercialized
for use in pest management.2 In addition to their practical
value, the structures of mealybug pheromones are intrinsically
interesting because of their irregular terpenoid skeletons
(Figure 1).3
Figure 1. Gross structure of the sex pheromone of the longtailed
mealybug.
The pheromone of the longtailed mealybug was identified
after collection of headspace odors produced by thousands of
live unmated females over many weeks. Isolation by liquid and
preparative gas chromatography produced a few micrograms of
the pure material, sufficient for identification of the basic
structure as 2-(1,5,5-trimethyl-2-cyclopent-2-en-yl)ethyl acetate
1.4 Several syntheses of the racemate have been developed,
using different strategies.4,5 The synthetic pheromone proved
to be extremely biologically active; lures loaded with 25 μg of
Received: March 10, 2013
© XXXX American Chemical Society
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Note
eomers were not separable on achiral stationary phases, they
were resolved to baseline on an enantioselective column,
eluting with n-hexane:EtOH:MeOH (90:10:2). NMR data of
compounds 3a and 3b were very similar (See Table S1 in the
Supporting Information).
After recrystallization from petroleum ether, the relative and
absolute configurations of 3b were determined through X-ray
crystal structure analysis, revealing that the chiral center in the
synthetic intermediate had the (R)-configuration (Scheme 1).
Scheme 1
Figure 2. Male longtailed mealybugs caught in traps baited with each
of the enantiomers of the pheromone, and the racemate. No
mealybugs were caught in solvent treated controls.
only slightly more attractive than solvent-treated controls. This
very slight attraction may have been due to the contamination
of this enantiomer with 1.7% of the bioactive (S)-enantiomer.
Furthermore, as seen with many other mealybug species,2 the
racemate was as attractive as the pure enantiomer, indicating
that the (R)-(−)-enantiomer is not inhibitory. Thus, the
cheaper and more readily synthesized racemic pheromone
should be entirely adequate for practical applications in pest
management.
EXPERIMENTAL SECTION
■
General Methods. All reactions were carried out in oven-dried
glassware under argon or nitrogen unless otherwise specified, with
magnetic stirring. Air sensitive reagents and solutions were transferred
via syringe or cannula and were introduced to the apparatus via rubber
septa. All reagents, starting materials, and solvents were obtained from
commercial suppliers and used without further purification. Reactions
were monitored by thin layer chromatography (TLC) with 0.25 mm
precoated silica gel plates (60 F254). Visualization was accomplished
with either UV light, iodine vapors, or by immersion in ethanolic
solutions of phosphomolybdic acid, para-anisaldehyde, or KMnO4
followed by heating with a heat gun for ∼15 s. Flash column
chromatography was performed on silica gel (100−200 or 230−400
mesh size). High resolution mass spectra (HRMS, ESI) were recorded
with an ORBITRAP mass analyzer (Q Exactive). Mass spectra were
measured with electrospray ionization with an MSQ LCMS mass
spectrometer. Infrared (IR) spectra were recorded on a FT-IR
spectrometer as thin films. Optical rotations were recorded on a
polarimeter at 589 nm.
CSP Esters (3a and 3b). A solution of 3,4,4-trimethyl-3-vinyl-
hept-6-en-1-ol 2 (50 mg, 0.27 mmol) and N-(2-carboxybenzoyl)-
(−)-10,2-camphorsultam (131 mg, 0.36 mmol, prepared according to
literature procedures7a,b,8) in CH2Cl2 (5 mL) was treated with
dimethylaminopyridine (DMAP, 44 mg, 0.36 mmol) followed by
dicyclohexylcarbodiimide (DCC, 61 mg, 0.30 mmol) at 0 °C. After
stirring at rt for 24 h, the reaction mixture was filtered, and the filtrate
was concentrated under reduced pressure. The residue was purified by
flash chromatography yielding the mixture of diastereomers (105 mg,
74%). Rf = 0.2 (20% EtOAc:hexanes). The mixture was resolved by
chiral preparative HPLC on a Daicel CHIRALPAK AD-H column
(amylose tris(3,5-dimethylphenyl carbamate) coated on 5 μm silica
gel; 30 mm id × 250 mm length), eluting with n-hexane/ethanol/
methanol (90/10/2, v/v/v) at 40 mL/min and monitoring with a UV
detector at 225 nm. The diastereomeric mixture was dissolved in the
mobile phase (10 mg/mL), and 3 mL aliquots were injected.
Diastereomers 3a and 3b eluted at 9 and 12 min, respectively. The
purity of the collected fractions was checked on a 4.6 × 250 mm
Chiralpak IA-3 column (3 μm particle size) eluted with n-hexane:
EtOH (95/5), flow 1.0 mL/min, 25 °C, monitoring by UV at 224 nm.
Diastereomer 3a was hydrolyzed with K2CO3 in MeOH to
obtain alcohol (S)-2, which was subsequently transformed to
the target pheromone (S)-(+)-1 by acetylation followed by
ring-closing metathesis as previously described (Scheme 2).5c
As expected, the spectral data of (S)-(+)-1 were in agreement
with those of the racemate. The other diasteromer 3b was
converted to (R)-(−)-1 in similar fashion.
With both enantiomers of the pheromone in hand, the
biological activities of each enantiomer and the racemate were
tested in field trials. The results showed that the (S)-
(+)-enantiomer was highly attractive to male mealybugs
(Figure 2), strongly suggesting that female longtailed mealy-
bugs produce this enantiomer. The (R)-(−)-enantiomer was
Scheme 2
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Note
Diastereomer-I (3a): [α]26 −106.6° (c 0.2, CHCl3); IR γmax (thin
4.02 (m, 2H), 4.93−5.02 (m, 3H), 5.13 (dd, J = 11.0, 1.5 Hz, 1H),
5.77−5.84 (m, 2H).
D
film applied as CHCl3 solution) 2963, 1721, 1686, 1635, 1336, 1300,
1168, 1112, 1083, 912, 776 cm−1; HRMS (ESI) m/z calculated for
C30H42NO5S [M + H]+ 528.2778, found 528.2787; Chiral HPLC
A solution of (−)-(3,4,4-trimethyl-3-vinyl-hept-6-enyl) acetate (22
mg, 0.10 mmol) in dry CH2Cl2 (5 mL) was degassed for 10 min with a
stream of argon and then treated with Grubbs’ second generation
catalyst (9 mg, 10 mol %; Aldrich, cat# 569747) in one portion. After
stirring at 40 °C for 16 h, the mixture was treated with a drop of
DMSO, and stirring was continued for 1 h. Evaporation of the solvent
and purification by column chromatography furnished the (S)-(+)-2-
(1,5,5-trimethylcyclopent-2-en-1-yl)ethyl acetate (S)-1 (14.2 mg,
1
purity = 98.3% (96.6% ee); tR:10.57 min. H and 13C NMR data are
provided in Supporting Information.
Diastereomer-II (3b): [α]24D −71.0° (c 0.2, CHCl3); IR γmax (thin
film applied as CHCl3 solution) 2963, 1720, 1685, 1635, 1333, 1299,
1168, 1137, 1082 cm−1; HRMS (ESI) m/z calculated for C30H42NO5S
[M + H]+ 528.2778, found 528.2787; Chiral HPLC purity = 99.3%
75%): Rf = 0.7 (20% EtOAc: hexanes); [α]25 +27.8° (c 0.16,
1
(98.6% ee); tR:13.63 min. H and 13C NMR data are provided in
D
CH2Cl2). The 1H NMR matched that of the racemate. 1H NMR: (400
MHz, CDCl3) δ 0.89 (s, 3H), 0.94 (s, 3H), 0.95 (s, 3H), 1.52−1.58
(m, merged with CDCl3 moisture, 1H), 1.66−1.74 (m, 1H), 2.04 (s,
3H), 2.13 (t, J = 1.6 Hz, 2H), 4.05−4.23 (m, 2H), 5.55−5.64 (m, 2H).
Diastereomer 3b was converted to (R)-1 in analogous fashion.
Spectral data matched those of the corresponding enantiomers
described above. Yields and optical rotations were as follows. Alcohol
Supporting Information.
X-ray Crystal Structure Details. Single crystals of compound 3b
were obtained from petroleum ether. X-ray intensity data were
collected on a Bruker SMART APEX II CCD diffractometer with
graphite-monochromatized (Mo Kα = 0.71073 Å) radiation at low
temperature, 150(2) K. The X-ray generator was operated at 50 kV
and 30 mA. Diffraction data were collected with a ω scan width of 0.5°
and at different settings of φ and 2θ. The sample-to-detector distance
was fixed at 5.00 cm. The X-ray data acquisition was monitored by the
APEX2 program suite.9 All the data were corrected for Lorentz-
polarization and absorption effects using SAINT and SADABS
programs integrated in the APEX2 program package.9 The structures
were solved by the direct method and refined by full matrix least-
squares, on the basis of F2, using SHELX-97.10 Molecular diagrams
were generated using XSHELL program integrated in SHELXTL
package.11 All the H-atoms were placed in geometrically idealized
position (C−H = 0.95 Å for phenyl H-atoms, C−H = 0.99 Å for
methylene H-atoms, C−H = 1.00 Å for methine H-atoms, and C−H =
0.98 Å for methyl H-atoms) and constrained to ride on their parent
atoms [Uiso(H) = 1.2Ueq(C) for the phenyl, methylene, and methine
group, and Uiso(H) = 1.5Ueq(C) for the methyl group]. Crystallo-
graphic data for 3b (C30H41NO5S): M = 527.70, Crystal dimensions
0.40 × 0.22 × 0.02 mm3, monoclinic, space group P21, a = 9.8209(13),
b = 11.2394(16), c = 13.1369(18) Å, β = 107.284(10)°, V = 1384.6(3)
Å3, Z = 2, ρcalcd = 1.266 gcm−3, μ(Mo Kα) = 0.157 mm−1, F(000) =
568, 2θmax = 50.00°, T = 150(2) K, 8872 reflections collected, 4482
unique, 3055 observed (I > 2σ(I)) reflections, 340 refined parameters,
R value 0.0524, wR2 = 0.0902, (all data R = 0.0954, wR2 = 0.1053), S =
0.996, minimum and maximum transmission 0.940 and 0.997;
maximum and minimum residual electron densities +0.26 and −0.23
e Å−3. The absolute configuration was established by anomalous
dispersion effect (Flack parameter of 0.07(11)) in X-ray diffraction
measurements, caused by the presence of the sulfur atom in the
molecule.
2b: Yield 94%; [α]25 −2.0° (c 0.15, CHCl3). Acetate (R)-2: yield
D
94%; [α]25 +3.3° (c 0.15, CHCl3). Pheromone (R)-1: yield 83%;
D
[α]25 −24.0° (c 0.13, CH2Cl2).
D
Field Trial of the Pheromone Enantiomers and the
Racemate. A field bioassay of the pheromone was conducted at a
nursery in Bonsall, California, USA, in a 0.49 ha plot of Ruscus
hypoglossum L. (plot coordinates: 33°17′18.36′′ N, 117°16′55.61′′ W
elev 113 m) that was known to be infested with P. longispinus. The plot
was divided into seven hoop houses (63 m long × 7 m wide), six of
which were used in this study. Each house (block) was covered in
plastic with open ends. Airflow between houses was not restricted
because the plastic cover began 1 m above the plant canopy. Four delta
sticky traps were spaced every 12.5 m along a transect within each
house, suspended directly above the ruscus canopy. Each trap
contained an 11 mm gray rubber septum impregnated with a hexane
solution of one of four treatments: solvent control, 5 μg of (S)-
(+)-enantiomer, 5 μg of (R)-(−)-enantiomer, 10 μg of the racemate.
Treatments were assigned randomly along each transect. Traps were
replaced, and treatments were repositioned once after 6 d. Traps
remained in place for another 11 d. Trap count data were analyzed by
analysis of variance after √(x + 0.5) transformation of the data to
meet the assumptions of normality and equal variances. Differences
among means were tested using Tukey’s honestly significant
differences (HSD) test. There was no significant interaction between
the two sampling periods (date) and the treatments (F = 3.23, df = 2,
30, and P = 0.054). Thus, data for each date were combined for the
final analysis. There was both a significant effect of date (F = 10.19, df
= 2, 32, P = 0.0032) and treatment (F = 130.04, df = 2, 32, P <
0.0001). Controls were not included in the analysis because their zero
values and lack of variance violate the assumptions of ANOVA.
Instead, confidence intervals were constructed, showing that the low
trap counts for the (R)-(−)-enantiomer were significantly different
than zero, i.e., that the (−)-enantiomer was slightly more attractive
than controls.12
(+)-3,4,4-Trimethyl-3-vinyl-hept-6-en-1-ol (S)-2. K2CO3 (282
mg, 2.04 mmol) was added to a solution of 3a (90 mg, 0.170 mmol) in
methanol at rt. After stirring for 2 h, the reaction mixture was
concentrated under reduced pressure and purified by column
chromatography to give (+)-3,4,4-trimethyl-3-vinyl-hept-6-en-1-ol
(S)-2 (28 mg, 90%): Rf = 0.4 (20% EtOAc:hexanes); [α]25 +1.7°
D
1
(c 0.38, CHCl3). The H NMR spectrum was identical to that of the
racemate. 1H NMR: (400 MHz, CDCl3) δ 0.82 (s, 6H) (gem-
dimethyl), 0.98 (s, 3H) (quaternary methyl), 1.64−1.71 (m, 1H)
(-CH2-CH2−OH), 1.78−1.85 (m, 1H) (-CH2-CH2−OH), 2.02 (d, J =
7.3 Hz, 2H) (allylic CH2), 3.60 (t, J = 7.3 Hz, 2H) (CH2-CH2−OH),
4.93−5.03 (m, 3H) (terminal olefin), 5.12 (dd, J = 10.8, 1.5 Hz, 1H)
(terminal olefin), 5.76−5.95 (m, 2H) (internal olefin).
ASSOCIATED CONTENT
■
S
* Supporting Information
Copies of chiral HPLC traces demonstrating enantiomeric
purities of chiral intermediates; NMR data comparison of 3a
and 3b; copies of NMR spectra and CIF file for the X-ray
crystal structure of compound 3b. This material is available free
of charge via the Internet at http://pubs.acs.org.
(+)-2-(1,5,5-Trimethylcyclopent-2-en-1-yl)ethyl acetate (S)-
1. A solution of (+)-(S)-3,4,4-trimethyl-3-vinyl-hept-6-en-1-ol (S)-2
(24 mg, 0.13 mmol) and Et3N (73 μL, 0.52 mmol) in dry CH2Cl2 (3
mL) was treated with acetic anhydride (26 μL, 0.26 mmol) and a
catalytic amount of DMAP (2.5 mg) at rt. After stirring for 2 h, the
reaction mixture was concentrated under reduced pressure and directly
purified by column chromatography to give (−)-(3,4,4-trimethyl-3-
AUTHOR INFORMATION
■
Corresponding Author
vinyl-hept-6-enyl) acetate (28 mg, 95%): Rf = 0.75 (20%
*E-mail: ds.reddy@ncl.res.in.
1
EtOAc:hexanes); [α]23 −4.6° (c 0.1, CHCl3). The H NMR was
D
identical to that of the racemate. 1H NMR: (500 MHz, CDCl3) δ 0.82
(s, 6H), 0.97 (s, 3H), 1.70−1.83 (m, 2H), 2.01−2.04 (m, 5H), 3.95−
Notes
The authors declare no competing financial interest.
C
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Note
ACKNOWLEDGMENTS
■
D.S.R. thanks CSIR, New Delhi, for the support through
NaPAHA program (XII Five Year Plan, CSC0130). We thank
Suresh Kurhade, Advinus Therapeutics, Ltd., for his help at the
initial stage of the project, Mr. Vijay Bhaskar and Mr. Lakshmi
Narayana of Daicel Chiral Technologies for their assistance
with HPLC separation, and Juan Paz and Ken Taniguchi
(Mellano and Company, Bonsall, California, USA) for
providing access to field sites. We thank Mr. Kashinath
(CSIR-NCL, Pune) for his help in NMR analysis, and R.R.
thanks CSIR, New Delhi, for the award of research fellowship.
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