Bruchins - Mitogenic 3-(hydroxypropanoyl) esters of long chain diols from weevils of the bruchidae

Article abstract of DOI:10.1016/S0040-4020(00)00650-5

Mono- and bis 3-(hydroxypropanoyl) esters of long chain, unsaturated diols have been isolated and identified from two genera of the family Bruchidae, and have been shown to be responsible for the mitogenic activity observed on pea pods resulting from oviposition by the pea weevil, Bruchus pisorum. The mitogenic compounds have been characterized and synthesized.

Full text of DOI:10.1016/S0040-4020(00)00650-5

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 7633±7641
BruchinsÐMitogenic 3-(Hydroxypropanoyl) Esters of Long
Chain Diols from Weevils of the Bruchidae
b
c
d
James E. Oliver,a, Robert P. Doss, R. Thomas Williamson, John R. Carney and
*
E. David DeVilbissa
aUSDA, Agricultural Research Service, Insect Chemical Ecology Laboratory, Building 007, BARC-W, Beltsville, MD 20705, USA
bHorticultural Crops Research Unit, USDA-ARS, Corvallis, OR 97330, USA
cCollege of Pharmacy, Oregon State University, Corvallis, OR 97331, USA
dKOSAN Biosciences, Hayward, CA 94545, USA
Received 15 June 2000; accepted 22 July 2000
AbstractÐMono- and bis 3-(hydroxypropanoyl) esters of long chain, unsaturated diols have been isolated and identi®ed from two
genera of the family Bruchidae, and have been shown to be responsible for the mitogenic activity observed on pea pods resulting
from oviposition by the pea weevil, Bruchus pisorum. The mitogenic compounds have been characterized and synthesized. Published by
Elsevier Science Ltd.
Pods of the Np mutant of pea (Pisum sativum L.) respond to
oviposition by the pea weevil (Bruchus pisorum L.) with
callus formation at the point of egg attachment.1±3 The
response can also be stimulated by a whole body extract
of adult weevils, and although activity can be detected
from extracts of either sex, egg-bearing adult females
provide extracts of highest activity. In a recent communica-
tion2 we reported that members of an unprecedented class of
natural products are responsible for mitogenesis. We here
provide a more complete account of the identi®cation and
syntheses of these novel and remarkably active (measurable
callus formation is produced by applications as low as
0.5 fmol) compounds, for which we have proposed the
name bruchins.
with phenacyl bromide and re-chromatographed on the C18
column. The esteri®ed fatty acids were moved to a different
chromatographic window, whereas the chromatographic
characteristics of the active material(s) were unchanged.
One of two active fractions thus obtained was then subjected
to preparative thin layer chromatography on silver nitrate-
impregnated silica gel. A band with Rf 0.4 (95:5 CHCl3/
MeOH) provided about 0.4 mg of a white solid 1 that was
highly active in the pea pod bioassay. The major component
of this fraction had limited volatility, but could be success-
fully analyzed by gas chromatography-mass spectrometry
(GC±MS) using a relatively short (15 m) capillary column.
Its electron ionization mass spectrum (EI-MS) did not
provide a reliable molecular ion, but in many respects
resembled the spectrum of oleyl alcohol. Two noteworthy
differences were prominent ions with m/z 73 and 91 in the
spectrum of the unknown. Chemical ionization mass spec-
trometry (CI-MS) with ammonia as reagent gas provided an
ammonium ion adduct with m/z 430, indicating a molecular
weight of 412. CI-MS with deuteroammonia established the
presence of two exchangeable hydrogens. Consistent with
the latter was the formation of a bis-TMS derivative (Mw
556) upon silylation with BSTFA. Although the mass spec-
trum of the TMS derivative had some features in common
with those of certain glycerol ethers, the spectrum of the
unknown could not be reconciled with either a glycerol
ether or ester.
Compound Identi®cation
After ®nding that the response elicited by extracts of pea
weevils was also elicited by extracts of the more easily
reared cowpea weevil (Callosobruchus maculatus L.), we
began a bioassay-guided fractionation of whole body
extracts of the latter. Two complications quickly became
apparent: the active material(s) tended to co-chromatograph
with common fatty acids, and the extreme sensitivity of the
bioassay could be misleading in that very high activity in the
bioassay did not necessarily ensure high purity of a fraction.
In our ®rst successful separation, chromatography on
Florisil followed by low-pressure liquid chromatography
on an ODS column gave an active fraction that was reacted
Treatment of the unknown (1) with NaOH in MeOH gave a
new compound 2 (Scheme 1), Mw 340, that also contained
two exchangeable hydrogens, but whose mass spectrum
lacked the ions with m/z 73 and 91, and also lacked ions
likely to be indicative of rings or chain branches. The
Keywords: biologically active compounds; insects; plants.
* Corresponding author; e-mail: oliverj@ba.ars.usda.gov
0040±4020/00/$ - see front matter. Published by Elsevier Science Ltd.
PII: S0040-4020(00)00650-5

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J. E. Oliver et al. / Tetrahedron 56 (2000) 7633±7641
Scheme 1.
hydrolysis product 2 also formed a bis-TMS derivative
whose mass spectrum contained no ions characteristic of
TMS ethers of secondary or tertiary alcohols, suggesting
an a,v,-diol. Exhaustive hydrogenation/hydrogenolysis of
underivatized 2 with LiAlH4/Pt/Al2O3 at 3008C4 provided
n-docosane, n-heneicosane and n-eicosane, indicating that 2
was a docosene-1,22-diol. This was con®rmed by catalytic
hydrogenation (Pd on C, 1 atm) to produce the known5
docosane-1,22-diol. Ozonolysis provided fragments
corresponding to C9 and C13 hydroxyaldehydes, implying
9-docosene-1,22-diol with double bond geometry yet to be
determined.
the m/z 73 and 91 ions in its EI-MS, synthesized a small
sample of 1 using the Fleming7 (vide infra) oxidative
desilylation technique for introducing an OH into the
hydroxypropanoyl ester.2 Synthetic 1 matched the isolated
compound in all respects including activity in the pea pod
bioassay.2
At this time we began another bioassay-directed fractiona-
tion on a somewhat larger scale. After derivatization of
acids to facilitate chromatography2 and multiple column
chromatographies, three major compounds, 3, 4 and 5,
were obtained, all highly active in the pea pod assay.
These were all slightly slower moving on silica gel than 1,
which was, at best, a minor component; they were also later-
eluting from the GC, again requiring relatively short
columns. The diagnostic m/z 73 and 91 ions were evident
in the mass spectra of each. All formed bis-TMS ethers,
which, although also of limited volatility, performed satis-
factorily for GC±MS analysis. Chemical ionization mass
spectra of these bis-TMS ethers indicated molecular weights
of 628, 656, and 654, respectively. EI-Mass spectra of the
TMS ethers all contained ions with m/z 103, 145, 147, and
163; although the ®rst three of these ions are not uncommon
in spectra of TMS-ethers, especially of polyhydroxylated
compounds, collectively the four ions have proved useful
for selected ion monitoring-based identi®cations of
bruchin±TMS-derivatives.
These facts indicated that the smaller fragment (not
observed) produced by base hydrolysis had itself contained
an active hydrogen; possible candidates for 1 would thus be
monoesters of lactic or 3-(hydroxypropanoic) acids.
Available mass spectra of lactate esters did not display
prominent m/z 73 or 91 ions (and neither did the spectra
of either the mono- or diesters from an acid-catalyzed
reaction of nonane-1,9-diol and lactic acid). Our mass
spectra library contained no spectra of esters of 3-(hydroxy-
propanoic) acid.
Esters of 3-(hydroxypropanoic) acid are relatively
uncommon, and as natural products appear to be virtually
unknown. The free acid is a hygroscopic liquid that is
usually obtained only in a hydrated form.6 We initially
prepared a sample of n-decyl 3-(hydroxypropanoate) as a
model compound, and after establishing the presence of
Saponi®cation of 3 gave the same C22 monounsaturated
diol 2 encountered earlier, indicating that 3 was the

J. E. Oliver et al. / Tetrahedron 56 (2000) 7633±7641
7635
Scheme 2.
bis-3-(hydroxypropanoyl) ester of 2. Compounds 4 and
5 also proved to be bis-3-(hydroxypropanoyl) esters;
saponi®cation provided mono- and diunsaturated C24-
diols 6 and 7, respectively, whose double bond locations
were again determined by ozonolysis. We suspected
these compounds to be lipid-derived, and that double
bond geometries would be Z. Note that all the three
diols have the conventional (Z)-9 double bond, i.e.
analogous to oleic acid, and that 6 is simply a two
carbon homolog of 2. The diunsaturated diol 7 also
contains a (Z)-9 double bond (if numbered from the
longer end of the chain), but contrary to our expectation,
was not a `skipped' diene analogous to many polyunsatu-
rated fatty acids, but instead had the two double bonds
separated by six methylenes.
a,v-Diol Syntheses
Construction of C22 and C24 mono- and diunsaturated diols
were achieved using conventional acetylene alkylation
reactions and semihydrogenations with Lindlar9 catalyst
(Scheme 2). 9-Decyn-1-ol 8 was alkylated with the tetra-
hydropyranyl (THP) ether of 12-bromododecan-1-ol 9,10
and the product was subjected to semihydrogenation to
give the monoprotected (Z)-9-docosen-1,22-diol 11.
Removal of the THP ether gave diol 2, indistinguishable
(GC and GC±MS of the diol and of its TMS±ether) from
the saponi®cation product of 1.
Diol 6 was prepared by alkylating 15-hexadecyn-1-ol11 12
with the THP ether of 8-bromooctanol 13 to give the mono
THP 14, followed by semihydrogenation and deprotection
(Scheme 2). Two acetylenic units were used in the synthesis
of 7: the THP ether of 9-decyn-1-ol 15 was deprotonated
with butyllithium and alkylated with excess 1,6-dibromo-
hexane 16. Monoadduct 17 was obtained in 94% yield after
¯ash chromatography; the diadduct accounted for no more
than 3% of the product. The THP ether of 7-octyn-1-ol 18
was similarly deprotonated and allowed to react with 17 to
afford, after deprotection, diyne 19. Semihydrogenation and
deprotection provided (Z,Z)-7,15-tetracosadiene-1,24-diol 7.
Adequate quantities of 3, 4 and 5 were isolated to permit
NMR analyses. NMR spectra supported the assignments
discussed, and con®rmed the (Z)-con®guration of all double
bonds. This was achieved by a recently described pro-
cedure:8 an experiment was designed, which isolates the
individual ole®nic protons that are coupled to the attached
13C atoms and are also coupled to the vicinal ole®nic proton
as well as to the two vicinal methylene protons. In order to
simplify this pattern and to facilitate spectral interpretation,
the vicinal methylene protons were simultaneously
decoupled during acquisition. Ole®nic homonuclear
coupling constants of ,11 Hz for the natural bruchins
indicated that the con®gurations of these double bonds
were indeed (Z).8
3-Hydroxypropanoyl Esters
Because monoester 1 was the ®rst compound identi®ed,

7636
J. E. Oliver et al. / Tetrahedron 56 (2000) 7633±7641
Scheme 3.
mono THP 11 was esteri®ed with acid chloride 20 (from the
known12 methyl 3-(phenyldimethylsilyl) propanoate
(Scheme 3) to give 21. Removal of the THP protecting
group followed by oxidative desilylation (KF, NaHCO3,
H2O2)7,13 gave compound 1 accompanied by diol 2.
Esteri®cation of diol 2 with 20 (2 equiv.) gave diester 22;
the same oxidative desilylation procedure afforded bis
3-(hydroxypropanoyl) ester 3. Yields, however, were dis-
appointing, and 3 was accompanied by the alcohols 1 and
2 resulting from ester hydrolysis. A recent review14
indicates that although a number of methods have been
developed for oxidative desilylation, very few are likely to
be compatible with both double bonds and esters. We
accordingly turned our attention to alternative methods for
constructing 3-(hydroxypropanoyl) esters.
protecting group is incompatible with ole®nic compounds.
We intended to explore the use of 3-(p-methoxybenzyl-
oxy)propanoate esters because the p-methoxybenzyl
protecting group should be removable under oxidative
conditions;17 p-methoxybenzyl alcohol reacted exothermi-
cally with acrylonitrile to form the desired 1,4 adduct 23
(Scheme 3); 23 in turn reacted readily with methanol and
anhydrous HCl (using conditions employed for methanoly-
sis of 3-(benzyloxy)propanenitrile), but did not give the
expected imidate 24. Instead, two volatile compounds
were formed, and although they have not been exhaustively
characterized, their mass spectra indicate them to be
p-methoxybenzyl chloride 25 and p-methoxybenzyl methyl
ether 26. This approach was not pursued further.
Greene and Wuts17 list a single example of a 2,2,2-trichloro-
ethyl ether as a protecting group for alcohols, although the
use of trichloroethyl esters, carbonates, and carbamates
along with trichloroethoxymethyl ethers has been better
documented.17 Zinc, in acetic acid and other media, has
frequently been used to remove these groups, conditions
we felt likely to be compatible with both esters and double
bonds. 3-(2,2,2-Trichloroethoxy)propanoic acid 28 was
prepared by HBr hydrolysis of the known18 methyl ester
27 (hydrolysis of 27 under alkaline conditions was accom-
panied by considerable retroadditon of trichloroethanol).
Esters of 28 such as 29 (Scheme 4) could be converted to
3-(hydroxypropanoyl) esters with zinc in methanol contain-
ing triethylamine and acetic acid;19 however, yields were
unsatisfactory and the products were accompanied by
byproduct(s) that, although not rigorously characterized,
appeared to provide the analogous esters containing one
less chlorine (and which were unreactive toward the
desired reductive elimination). Precedent for this type of
Acylation of long chain alcohols with propiolactone did not
proceed well, and in any event, the products, themselves
being primary alcohols, might be expected to continue to
react with additional reagent. Limited attempts toward
deprotonation of acetates with subsequent reaction
with formaldehyde were unrewarding (small amounts of
3-(hydroxypropanoyl)esters were detectable, but were
accompanied by larger amounts of unchanged acetate
along with the primary alcohol resulting from deacetyl-
ation), as were reactions of bromoacetates with zinc and
formaldehyde (Reformatsky conditions). Hydrogenolysis
of 3-(benzyloxy)propanoate esters has been described,15,16
the acids having been obtained by addition of benzyl alcohol
to acrylonitrile followed by methanol/HCl conversion to a
methyl imidate and ®nally hydrolysis to the methyl ester or
free acid (analogous to line 4 of Scheme 3). We have used
this route for the construction of several saturated analogs of
3±5, but the hydrogenolysis used to remove the benzyl

J. E. Oliver et al. / Tetrahedron 56 (2000) 7633±7641
7637
Scheme 4.
dechlorination has been reported during the removal of the
trichloroethoxymethyl group with zinc±copper or zinc±
silver couples in methanol.19 Other reductive conditions
including zinc in acetic acid, zinc in buffered THF20 or
chromium perchlorate in DMF21 failed to produce more
satisfactory results.
tion of 1 and 3±5 was from extracts of the cowpea weevil
(Callosobruchus maculatus L.). We next turned our atten-
tion back to the species with which the mitogenic activity
had originally been associated, the pea weevil Bruchus
pisorum L. Active chromatographic fractions of a whole
body extract were assayed by GC±MS using selected ion
monitoring (the m/z 73 and 91 ions for underivatized
samples, and m/z 103, 145, 147 and 163 for trimethyl-
silylated samples). Although we detected several additional,
albeit very minor, components providing spectra character-
istic of 3-(hydroxypropanoyl) esters, the major components
were the same bis-3-(hydroxypropanoyl) esters 3±5
previously characterized. This was somewhat surprising in
view of the fact that although belonging to the same family
(Bruchidae), the two insects occupy different genera. Thus
our results indicate that in two different bruchids, bis
3-(hydroxypropanoyl) esters 3±5 are the principal
mitogens, and that monoesters such as 1, although active
mitogens, are in general less abundant. Neither these esters
(1,3,4,5) nor their diol precursors (2, 6, 7) appear to have
been described previously, and it is noteworthy that the diols
themselves do not promote callus formation. With the
exception of 5 and 7, which are liquids at room temperature
but solidify in a refrigerator, the esters and diols are crystal-
line solids that are stable for at least several months at room
temperature. We are continuing to explore the scope and
generality of this insect±plant interaction, and to examine
the structure±activity relationships of these compounds and
synthetic analogs.
Although 3-hydroxypropanoic acid is inconvenient to work
with, its methyl ester 30 can be conveniently prepared from
3-hydroxypropionitrile22 (Scheme 4). We converted 30 to
the known t-butyldiphenylsilyl ether 31.23 Saponi®cation
with lithium hydroxide in methanol±water was accompa-
nied by some desilylation, but provided satisfactory yields
of acid 32 (the previous preparation featured monosilylation
of propane-1,3-diol and Jones oxidation24). This acid, via its
acid chloride, was readily esteri®ed with diols 6 and 7 to
provide esters 33 and 34, respectively. Tetrabutylammo-
nium ¯uoride in THF smoothly promoted deprotection at
room temperature to give the bis-3-(hydroxypropanoyl)
esters 4 and 5, respectively. Both proved indistinguishable
from the isolated materials, and both stimulated callus
formation at applications of 1 pg or less in the pea pod assay.
In summary, 3-(benzyloxy)propanoic acid is probably the
most practical precursor of 3-(hydroxypropanoyl) esters of
alcohols compatible with catalytic hydrogenation.
However, esters of 3-((t-butyldiphenylsilyl)oxy)propanoic
acid 32 provide viable alternatives in cases where hydro-
genation is not an option.
Bruchins
Experimental
As stated in the introduction, the isolation and characteriza-
Melting points are uncorrected. NMR spectra were obtained

7638
J. E. Oliver et al. / Tetrahedron 56 (2000) 7633±7641
with a Bruker DRX 600 spectrometer (Billerica, MA) on
deuterochloroform solutions at 600 MHz for 1H, and
150 MHz for 13C. Mass spectra were obtained from a
Finnigan-MAT Incos-50 GC±MS equipped with a 15 m
DB-5 fused silica column. Electron ionization spectra
were collected at 70 eV and a source block temperature of
1508. Chemical ionization spectra were collected using
ammonia as reagent gas (reagent gas pressure 0.5 Torr.) at
a source temperature of 608. Elemental analyses were
performed by Galbraith Laboratories, Knoxville, TN.
Mention of a proprietary product does not imply endorse-
ment by the US Department of Agriculture.
ether 11. A solution of 9-decyn-1-ol 8 (3.85 g, 25 mmol)
in dry THF (50 mL) was cooled to 2708 and treated drop-
wise with 22 mL of 2.5 M butyllithium in hexanes. A thick
precipitate separated. Hexamethylphosphoric triamide
(HMPA, 10 mL) was added, and the mixture was mechani-
cally stirred and warmed to room temperature, then again
cooled to 2708. Sodium iodide (ca. 200 mg) was added
followed by the THP ether of 12-bromo-1-dodecanol25
9
(8.4 g, 24 mmol). The mixture was allowed to warm to
room temperature and stir overnight, then was partitioned
between water and ether±hexane (1:1). The crude product
(10.8 g) was ¯ash chromatographed (20±25% EtOAc in
hexanes) to give 5.30 g of the mono THP ether of 9-doco-
syne-1,22-diol 10. This material (CI-MS m/z 440, M118)
was used without further characterization. A portion was
semihydrogenated as described above, and the resulting
11 (CI-MS m/z 442, M118) was used without further
puri®cation.
Microreactions of unknowns
Saponi®cation. Typically, a solution containing 10±30 mg
of the unknown ester was evaporated to dryness in a conical
vial, and the residue was treated with 10 mL MeOH plus
5 mL of 25% NaOH in 1:1 MeOH/H2O. The vial was tightly
capped and heated 30 min at 708, then was cooled and the
contents neutralized with 10 mL 2N HCl and concentrated
with a stream of N2. The white residue was extracted with
several 10 mL portions of EtOAc which were passed
through a micro column of silica gel (contained in a
capillary drawn from a Pasteur pipette) into a clean conical
vial. The volume was adjusted for GC or subsequent
transformations.
(Z)-9-Docosene-1,22-diol 2. A solution of 11 (3.0 g) in
methanol (50 mL) containing toluenesulfonic acid mono-
hydrate (0.15 g) was stirred at room temperature for 5 h.
The solvent was concentrated and the residue was parti-
tioned between ether and aq. Na2CO3. The ether solution
was dried and concentrated, and the residue was crystallized
from isooctane and 1±2% EtOAc to give 1.38 g of diol 2,
mp 56±578. 1H NMR: 5.25 (2H, m), 3.64 (4H, t, Jˆ7.4 Hz),
2.01 (2H, m), 1.56 (2H, q, Jˆ6.6 Hz), 1.39±1.22 (12H,
ovlp). 13C NMR: 129.9, 129.8, 63.1, 63.1, 32.8, 32.8, 29.7,
29.6, 29.6, 29.5, 29.5, 29.4, 29.4, 29.2, 29.2, 27.2, 25.7,
25.7. CI-MS m/z 358, M118; EI-MS, m/z (%) 123 (10),
110 (16), 109 (21), 97 (19), 96 (46), 95 (41), 94 (12), 83
(30), 82 (62), 81 (55), 80 (22), 79 (14), 69 (45), 68 (36), 67
(58), 56 (12), 55 (100), 54 (28), 43 (32), 42 (16), 41 (66).
Further chilling of the mother liquor afforded a second crop
(0.19 g). Anal. Calcd for C22H44O2: C, 77.58; H, 13.02.
Found: C, 77.34; H, 13.33.
Trimethylsilylation. A solution containing ca. 10 mg of the
compound(s) to be derivatized was concentrated with a
stream of nitrogen, and the residue was treated with
10 mL of bis(trimethylsilyl)tri¯uoroacetamide (BSTFA)
that had been amended with 1% chlorotrimethylsilane.
The vial was tightly capped and heated at 608 for 15±
20 min, then a stream of N2 was used to evaporate excess
reagent and an appropriate volume of hexane was added for
GC or GC±MS.Ozonolysis. A solution of ca. 10 mg of an
ole®n in 5 mL CH2Cl2 was cooled with dry ice±acetone and
treated with 20 mL of a blue solution of freshly generated
ozone trapped in CH2Cl2 at 2788. The vial was removed
from the cooling bath for several seconds, then again cooled
and dimethylsul®de (2±3 mL) was added. After warming to
room temperature and concentrating with a stream of N2, an
appropriate volume of solvent was added for GC or GC±
MS. In addition to the expected hydroxyaldehydes and
dialdehyde from unsaturated diols 2, 6 and 7, aldehyde±
acids were in some cases also observed, apparently from
oxidation of the primary alcohols under the conditions.
(Z)-9-Tetracosene-1,24-diol 6. A solution of 15-hexa-
decyn-1-ol11 (12, 4.76 g, 20 mmol) in THF (40 mL) was
stirred mechanically and cooled to 2758 under nitrogen
and treated dropwise with 16 mL 2.5 M butyllithium.
After warming to about 08 then again cooling with dry
ice±acetone, a solution of the THP ether of 8-bromooctanol
(13, 5.37 g, 18 mmol) in THF (10 mL) and HMPA (5 mL)
was added. The mixture was allowed to warm to room
temperature and stir overnight, then was partitioned
between hexane and water to give 9.21 g of an amber oil.
This material was deprotected by stirring in methanol
(200 mL containing 0.5 g toluenesulfonic acid hydrate) for
1 h; after removal of most of the methanol, the residue was
partitioned between EtOAc and dilute HCl. After rinsing
with water and aq. NaHCO3, the solution was dried and
concentrated, and the residue (7.6 g) was ¯ash chromato-
graphed (20, 40 and 60% EtOAc in hexanes). A major
component was unreacted 15-hexadecyn-1-ol 12 (3.03 g);
also obtained was the desired 9-tetracosyne-1,24-diol
(2.83 g, 43%), mp 738 (isooctane), 1H NMR (CDCl3,
600 MHz) 3.36 (4H, q, 6.6 Hz), 2.10 (4H, t, 6.6 H), 1.42±
1.20 (32 H, ovlp). 13C NMR 85.0, 65.8, 37.6, 34.2, 34.2,
34.1, 34.1, 34.1, 34.0, 33.7, 34.1, 33.6, 33.6, 33.2, 33.2,
23.1. CI-MS m/z 384, M118). Two grams of this material
was semihydrogenated and the ole®n was recrystallized
Semihydrogenations. Reductions of alkynes to (Z)-alkenes
were conducted with lead-poisoned, 5% palladium on
calcium carbonate (Lindlar9 catalyst, Strem Chemicals,
Inc., 50±75 mg per g of alkyne) at one atmosphere and
room temperature, using freshly distilled cyclohexene as
solvent or as a cosolvent with absolute ethanol or ethyl
acetate. The use of an ole®nic solvent eliminates or greatly
reduces possible overreduction, and the reactions, some-
times dif®cult to monitor with precision, could be allowed
to proceed several hours, or even overnight in at least two
cases, to ensure completion.
9-Docosyne-1,22-diol, 22-tetrahydropyranyl ether 10
and (Z)-9-docosene-1,22-diol, 22-tetrahydropyranyl

J. E. Oliver et al. / Tetrahedron 56 (2000) 7633±7641
7639
1
from isooctane to give 6 (1.65 g), mp 62±638. H NMR:
5.32 (2H, m), 3.36 (4H, t, Jˆ6.6 Hz), 1.98 (4H, q,
Jˆ6.4 Hz), 1.39 (m), 1.32±1.2 (ovlp). 13C NMR: 129.6,
129.6, 60.7, 32.5, 29.1, 29.1, 29.0, 29.0, 29.0, 28.9, 28.9,
28.9, 28.9, 28.8, 28.6, 28.5, 26.6, 26.5, 25.5. CI-MS m/z 386
(M118); EI-MS m/z (%): 125 (13), 123 (11), 111 (21), 110
(16), 109 (29), 97 (26), 95 (37), 94 (11), 83 (37), 82 (45), 81
(46), 80 (18), 71 (13), 70 (12), 69 (100), 68 (32), 67 (50), 57
(29), 56 (14), 55 (99), 54 (23), 43 (32), 42 (12), 41 (47).
Anal. Calcd for C24H48O2: C, 78.19; H, 13.12. Found: C,
77.80; H, 13.42.
of 11 (424 mg, 1 mmol) in benzene (5 mL) and pyridine
(0.25 mL) was treated with 3-(phenyldimethylsilyl)pro-
panoyl chloride 20 (prepared from 0.33 g of the correspond-
ing acid13 and excess oxalyl chloride). The mixture was
stirred overnight, then partitioned between hexanes and
water to give 0.68 g of a clear oil. Flash chromatography
(5 then 10% EtOAc in hexanes) gave 0.30 g of 22 as an oil,
CI-MS m/z 632 (M1NH4).
(Z)-9-Docosene-1,22-diol mono 3-(hydroxypropanoyl)
ester 1. A. By oxidative desilylation: A solution of 22
(73 mg) in MeOH (2.5 mL) containing about 10 mg TsOH
was stirred for 1.5 h at room temperature, then was concen-
trated and the residue partitioned between aq. NaHCO3 and
ether±hexane (1:1) to give 60 mg crude product. To 53 mg
of this material was added dichloromethane (400 mL) and
¯uoroboric acid etherate (125 mL). After 5 h at room
temperature the product was partitioned between ether and
aq. NaHCO3; the organic portion was treated with THF
(400 mL), MeOH (400 mL), NaHCO3 (44 mg), KF
(44 mg), and 30% H2O2 (200 mL). After 45 min the mixture
was partitioned between dichloromethane and water, and
the organic products (9 mg) were ¯ash chromatographed
with 25±50% EtOAc in hexanes. About 2.8 mg of 1 was
obtained that was indistinguishable from the natural product
and that was very active in the pea pod assay.
(Z,Z)-7,15-Tetracosadiene-1,24-diol 7. A solution of the
THP ether of 9-decyn-1-ol (15, 2.35 g, 10 mmol) in dry
THF was cooled to 2758 under nitrogen, and butyllithium
(4.4 mL, 2.5 M) was added by syringe. The solution was
allowed to warm to about 108, then was again cooled to
2758 and HMPA (2 mL) and NaI (about 50 mg) were
added, followed by 1,6-dibromohexane (16, 5 mL,
32 mmol). The solution was allowed to warm to room
temperature and stir overnight, then was partitioned
between water and hexanes. Evaporation of solvent left
9.17 g of a liquid that was added to a silica gel column
(35 g). Elution with hexane gave 4.44 g of unreacted 16;
subsequent elution with 10% ethyl acetate in hexanes
provided 4.93 g of an oil that was ¯ash chromatographed
on 100 g silica gel, eluting with 2.5±10% ethyl acetate in
hexanes to provide 3.79 g (94%) of the tetrahydropyranyl
ether of 16-bromohexadec-9-yn-1-ol 17 (CI-MS m/z 418
and 420, NH14 adducts). A solution of the THP ether of
7-octyn-1-ol (18, 1.05 g, 5 mmol) in dry THF (20 mL)
was cooled to 2758 under nitrogen, and 2.4 mL of 2.5 M
butyllithium was added. The cooling bath was removed for a
few minutes, then reapplied. HMPA (1 mL), NaI (50 mg),
and 17 (2.0 g, 5 mmol) were added. The solution was
allowed to warm to room temperature and stirred overnight,
then was partitioned between water and hexanes. The crude
product, which also contained both reactants 17 and 18, was
¯ash chromatographed (5 then 10% ethyl acetate in
hexanes) to afford 1.27 g (48%) of the bis-tetrahydropyranyl
ether of 7,15-tetracosadiyne. Nearly all of this material was
semihydrogenated to give 1.23 g of crude diene that was
added to methanol (25 mL) and treated with toluenesulfonic
acid (25 mg). The initially insoluble diene gradually
dissolved as deprotection occurred; after stirring overnight
most of the methanol was removed and the residue was
partitioned between methyl t-butyl ether and aqueous
sodium carbonate. A colorless oil (0.84 g) was thus obtained
that was ¯ash chromatographed with 40, then 50% ethyl
acetate in hexanes to provide 0.53 g of 7 as a clear viscous
B. By F-catalyzed desilylation: A solution of 32 (0.90 g) in
benzene (5 mL) was treated with 0. 5 mL oxalyl chloride
and 1 mL DMF. After 1.5 h at room temperature the solvent
and excess oxalyl choride were stripped followed by another
5 mL portion of benzene. The residue was again dissolved
in 5 mL benzene, and a solution of 10 (1.06 g, 2.5 mmol) in
benzene (5 mL) and pyridine (0.6 mL) was added dropwise.
After stirring at room temperature for 1.25 h, water was
added and the mixture was partitioned between water and
ether±hexane. The organic extracts were rinsed with dilute
HCl and aq. NaHCO3, dried, and concentrated to yield
2.12 g of a clear liquid. Flash chromatography (5 and 10%
EtOAc in hexanes) provided 1.01 g of the desired ester
(56%) as a clear glass. Most of this sample was semihydro-
genated as described above. To the product was added THF
(6 mL) and 1 M tetrabutylammonium ¯uoride in THF
(2.7 mL), and the solution was allowed to remain at room
temperature 1.5 h. It was then partitioned between 1N HCl
and ether to give 1.01 g of a product that was treated directly
with methanol (40 mL) and toluenesulfonic acid hydrate
(95 mg). After 2.5 h at room temperature, most of the
methanol was evaporated and the residue was partitioned
between ethyl acetate and water. The organic phase was
rinsed with aq. NaHCO3, then dried and concentrated to
give 0.86 g of an oil that solidi®ed upon standing.
Recrystallization from hexane gave 0.31 g of 1, mp 47±
1
oil. H NMR: 5.39 (4H, m), 3.64 (4H, t, Jˆ5.8 Hz), 2.01
(8H, m), 1.56 (4H, q, Jˆ5.8 Hz), 1.45±1.25 (26 H, ovlp).
13C NMR: 130.4, 130.3, 130.1, 63.5, 63.4, 33.1, 30.1, 30.1,
30.1, 30.1, 29.9, 29.8, 29.6, 29.5, 29.5, 27.6. 27.5, 26.1,
26.0. CI-MS m/z 384 (Mˆ18); EI-MS, m/z (%), 135 (17),
123 (11), 121 (23), 110 (14), 109 (26), 108 (12), 107 (14), 97
(22), 96 (48), 95 (62), 94 (28), 93 (24), 83 (27), 82 (54), 81
(73), 80 (41), 79 (31), 69 (43), 68 (39), 67 (99), 57 (15), 56
(11), 55 (100), 54 (35), 53 (11), 43 (25), 41 (75). Anal. Calcd
for C24H46O2: C, 78.62; H, 12.65. Found: C, 78.67; H, 12.86.
1
488 that was identical to the sample described above. H
NMR: d 5.34 (2H, m, ole®nic), 4.11 (2H, t, Jˆ4.4 Hz,
CH2CO2R), 3.86 (2H, t, Jˆ3.9 Hz, ±O2CCH2CH2OH),
3.63 (2H, t, Jˆ4.6 Hz, alkyl-CH2OH), 2.70 (2H, t Jˆ
3.8 Hz, ±O2CCH2CH2OH), 2.09 (4H, m, allylic), 1.78 (br.
s., OH). EIMS, m/z (%): 412 [M]1 (ca. 0.4), 124 (14), 123
(16), 121 (14), 111 (11), 109 (28), 97 (25), 96 (59), 95 (56),
94 (21), 93 (12), 91 (58), 83 (34), 82 (74), 81 (69), 80 (36),
79 (18), 73 (83), 71 (11), 69 (51), 68 (36), 67 (67), 57 (19),
56 (14), 55 (100), 54 (31), 45 (13), 43 (42), 42 (14), 41 (58).
(Z)-9-Docosene-1,22-diol,3-(phenyldimethylsilyl)pro-
panoate ester, 22-tetrahydropyranyl ether 21. A solution

7640
J. E. Oliver et al. / Tetrahedron 56 (2000) 7633±7641
Anal. Calcd for C25H48O4: C, 72.76; H, 11.72. Found: C,
72.49; H, 12.04. 1-bis(Trimethylsilyl) ether, EIMS m/z
(%): 556 [M]1 (ca. 0.2), 541 (ca. 0.2), 235 (10), 219 (22),
163 (31), 149 (10), 147 (100), 109 (13), 105 (28), 103 (79),
97 (12), 96 (14), 95 (20), 91 (12), 83 (17), 82 (14), 81 (23),
75 (58), 73 (57), 69 (24), 67 (23), 55 (41), 43 (15), 41 (16).
above procedure applied to diol 7 (173 mg, 0.47 mmol)
gave 159 mg of 5 (66% overall) as a clear glass, H NMR:
1
5.39 (m), 4.10 (t, Jˆ5.8 Hz), 3.86 (t, Jˆ5.8 Hz), 2.01 (m),
1.64 (q, Jˆ5.8 Hz), 1.45±1.25 (ovlp). 13C NMR 173.0,
130.0, 129.8, 129.7, 129.5, 64.8, 58.2, 36.7, 29.6, 29.6,
29.5, 29.3, 29.1, 29.1, 28.7, 28.5, 28.5, 28.4, 27.1 ovlp,
27.0, 25.8, 25.7 CI-MS m/z 528, (M118), EI-MS, m/z
(%), 149 (11), 136 (10), 135 (18), 123 (11), 122 (13), 121
(25), 110 (11), 109 (26), 108 (16), 107 (16), 97 (16), 96 (26),
95 (50), 94 (27), 93 (22), 91 (36), 83 (19), 82 (28), 81 (63),
80 (38), 79 (28), 73 (100), 69 (33), 68 (23), 67 (73), 55 (82),
54 (28), 45 (15), 443 (32), 41 (39). Anal. Calcd for
C24H46O2: C, 70.55; H, 10.66. Found: C, 70.20; H, 10.87.
5-Bis(trimethylsilyl ether) EI-MS, m/z (%), 163 (30), 147
(59), 146 (14), 145 (91), 135 (11), 121 (13), 109 (11), 105
(38), 104 (11), 103 (100), 95 (23), 94 (13), 93 (13), 91 (14),
83 (11), 82 (10), 81 (29), 80 (24), 79 (18), 75 (37), 73 (42),
69 (16), 67 (34), 55 (37), 43 (11), 41 (16).
(Z)-9-Docosen-1,22-diol bis (3-hydroxypropanoate) ester
3. Diol 2 (0.5 mmol) was esteri®ed with 32 (1 mmol) as
described above. The crude diester (1H NMR: 5.34 (2H,
m), 4.11 (4H, Jˆ5.8 Hz), 3.87 (4H, q, Jˆ5.8 Hz), 2.57
(4H, t, Jˆ5.8 Hz), 2.43 (2H, t, Jˆ5.8 Hz), 2.01 (4H,
Jˆ5.8 Hz), 1.63 (4H, quin, Jˆ7 Hz), 1.39±1.22 (28 H,
ovlp); 13C NMR 173.0, 173.0, 130.0, 129.8, 70.6, 70.6,
64.9, 64.9, 58.3, 36.7, 36.7, 29.7, 29.7, 29.6, 29.5, 29.5,
29.5, 29.4, 29.3, 29.2, 29.2, 28.6, 28.5, 27.2, 27.1 ovlp,
25.9. CI-MS M/Z 978, M118) was passed through a short
column of silica gel with 5% EtOAc in hexanes, then was
treated with 1.1 mmol tetrabutylammonium ¯uoride in THF
(4 mL). After 2 h at room temperature, the desilylated
product was partitioned between EtOAc and dilute HCl,
and the organic phase was rinsed with aq. NaHCO3, dried,
and concentrated. Flash chromatography of the residue (50
then 60% EtOAc in hexanes) gave 114 mg of 3 (47%) that
crystallized on standing. Recrystallization from hexanes
B. By reductive elimination of the bis-3-(2,2,2-trichloro-
ethoxy)propanoate ester of (Z,Z)-7,15-tetracosadiene-
1,24-diol 29: Diol 7 (366 mg, 1 mmol) was esteri®ed with
2.5 mmol of 3-(2,2,2-trichloroethoxy)propanoyl chloride
generated from 28 and oxalyl chloride. The crude product
(0.75 g, 97%, single spot TLC, Rf 0.55 15% EtOAc in
hexanes) was stirred in MeOH (18 mL) containing HOAc
(2 mL) and Et3N (1.4 mL) at 358 and zinc dust (2.7 g) was
added in portions over about 20 min. Within 2 h the starting
diester had been consumed, and the composition underwent
no further change with time. The mixture was cooled,
diluted with ether (50 mL) and ®ltered through Celite,
then was washed twice with water, with aq. NaHCO3, and
®nally with sat. NaCl. After drying and removal of solvent,
a clear syrup (0.41 g) remained that was ¯ash chromato-
graphed (25±50% EtOAc in hexanes). One product moved
just behind the solvent front (132 mg) and may have
consisted of dechlorinated diester (CI-MS m/z 624, 626,
628 ca. 100, 80, 12%, respectively, M118 ions consistent
with two chlorines), but was not investigated further. A later
eluting fraction (133 mg) was followed by 111 mg 5 that
was rechromatographed with 30±50% methyl t-butyl ether
in benzene to give 96 mg (19% from 7) of bis 3-(hydroxy-
propyl)ester 5, identical to the sample described above.
1
gave 107 mg, mp 478. H NMR: 5.35 (2H, m), 4.11 (4H, t,
Jˆ6.62), 3.86 (4H, q, Jˆ6.6 Hz), 2.57 (4H, t, Jˆ5.52), 2.43
(2H, t, Jˆ5.8 Hz), 2.01 (4H, q, Jˆ5.8 Hz), 1.63 (4H, quin,
Jˆ7.0 Hz), 1.39±1.22 (28 H, ovlp). 13C NMR 173.0, 173.0,
130.0, 129.8, 64.9, 64.9, 58.3, 58.3, 36.7, 36.7, 29.7, 29.7,
29.6, 29.6, 29.5, 29.5, 29.4, 29.3, 29.2, 29.2, 29.2 ovlp. CI-
MS m/z 502 (M118); EI-MS, m/z (%), 121 (11), 110 (13),
109 (17), 97 (16), 96 (28), 95 (32), 94 (14), 91 (57), 83 (19),
82 (34), 81 (40), 80 (23), 79 (12), 73 (100), 69 (28), 68 (22),
67 (37), 57 (11), 55 (74), 54 (21), 45 (13), 43 (35), 41 (31).
Anal. Calcd for C28H52O6: C, 69.38; H, 10.81. Found: C,
69.18; H, 11.21. 3-Bis(trimethylsilyl ether) EI-MS, m/z
(%), 628 (M1, ca. 1%), 467 (ca. 3%), 235 (14), 163 (34),
147 (71), 146 (16), 145 (100), 105 (41), 104 (12), 103 (92),
95 (11), 81 (14), 75 (19), 73 (33), 69 (14), 67 (15), 55 (29),
43 (11), 41 (13).
(Z)-9-Tetracosene-1,24-diol bis (3-hydroxypropanoate)
ester 4. Diol 6 (0.6 mmol) was esteri®ed with 32 as
described above, and the crude product was worked up
and ¯ash chromatographed (5 then 10% EtOAc in hexanes)
to give 421 mg (79%) of the bis t-butyldiphenylsiloxypro-
panoate ester as a clear glass. Desilylation as described
above gave 0.45 g crude product that was crystallized
from hexane to give 0.16 g (66%) of 4, mp 53±548. CI-
MS m/z 530 (M118); EI-MS, m/z (%), 123 (10), 110 (13),
109 (18), 97 (17), 96 (27), 95 (32), 94 (15), 93 (11), 93 (11),
91 (60), 83 (22), 82 (34), 81 (43), 80 (25), 79 (11), 73 (100),
69 (32), 68 (20), 67 (42), 57 (12), 55 (69), 54 (19), 45 (12),
43 (34), 42 (10), 41 (29). Anal. Calcd for C30H56O6: C,
70.27; H, 11.01. Found: C, 69.52; H, 11.21. 4-Bis(tri-
methylsilyl ether) EI-MS, m/z (%), 163 (38), 147 (75),
146 (16), 145 (100), 105 (35), 104 (10), 103 (91), 96 (12),
95 (17), 83 (13), 82 (13), 81 (22), 75 (59), 73 (38), 69 (22),
55 (45), 45 (11), 43 (14), 41 (19).
3-[(2,2,2-trichloroethoxy)propanoic acid 28. A mixture of
methyl 3-[(2,2,2-trichloroethoxy)propanoate18 (11.78 g,
50 mmol) and 48% HBr (50 mL) plus tetrabutylammonium
bromide (1 g) was stirred and heated at 908 2 h, then was
cooled and partitioned between ice and EtOAc. The EtOAc
solution was rinsed with water, diluted with one-half its
volume of hexane, and again rinsed with water, then with
sat. NaCl, and ®nally dried, concentrated, and distilled to
1
give 9.7 g (88%) of 28, bp 104±106, 0.05 Torr. H NMR:
4.13 (2H, s), 4.08 (2H, t, Jˆ6.2 Hz), 2.74 (2H, t, Jˆ6.2 Hz),
13C NMR 177.5, 97.4, 84.0, 68.5, 35.3. Anal. Calcd for
C5H7Cl3O3: C, 27.11; H, 3.19. Found: C, 27.21; H, 3.29.
3-[[t-Butyldiphenylsilyl]oxy]propanoic acid 32. A solu-
tion of methyl 3-[[t-butyldiphenylsilyl]oxy]propanoate23
31 (15.8 g, 46 mmol) in methanol (100 mL) was treated
dropwise at room temperature with 1N LiOH (60 mL).
After stirring 4 hr at room temperature, the solution was
diluted with ice water and extracted three times with
(Z,Z)-7,15-Tetracosadiene-1,24-diol bis 3-hydroxypro-
panoate ester 5. A. By F-catalyzed desilylation: The

J. E. Oliver et al. / Tetrahedron 56 (2000) 7633±7641
7641
4. Bierl-Leonhardt, B. A.; DeVilbiss, E. D. Anal. Chem. 1981, 53,
936.
small volumes of ether±hexanes (1:1). It was then neutra-
lized with 35 mL 2N KHSO4 and extracted thoroughly with
ether±hexanes (1:1). The organic extracts were rinsed with
water, then dried and concentrated to provide 8.47 g of 32 as
a white solid. The neutral portion, which consisted of a
mixture of unsaponi®ed 31 and t-butyldiphenylsilanol,
was concentrated (5.16 g) and dissolved in MeOH. The
resulting solution was treated with 25 mL 1N LiOH as
described above, and after stirring at room temperature
and partitioning, afforded an additional 2.81 g of 32. The
two portions were combined and recrystallized from
5. Slezak, F. B.; Stallings, J. P.; Wagner, D. H.; Wotiz, J. H. J. Org.
Chem. 1961, 26, 3137.
6. Read, R. R. Organic Synthesis Collective Volume 1; 1964;
p 321.
7. Fleming, I. Pure Appl. Chem. 1988, 60, 71.
8. Williamson, R. T.; Carney, J. R.; Gerwick, W. H. J. Nat. Prod.
2000 (in press).
9. Lindlar, H. Helv. Chim. Acta 1952, 35, 446.
10. Kulkarni, S. M.; Mamdapur, V. R.; Chandha, M. S.; Indian
J. Chem., Sect. B. 1983, 22, 683.
1
hexanes to give 10.55 g (70%) of pure 32, mp 1098. H
11. Brown, C. A.; Yamashita, A. J. Chem. Soc., Chem. Commun.
1976, 959.
NMR: 7.52±7.49 (2H, m), 7.67 (2H, d, Jˆ7.3 Hz), 7.45±
7.37 (3H, m, ovlp), 3.95 (2H, t, Jˆ5.8 Hz), 2.61 (2H, t,
Jˆ5.8 Hz), 1.05 (9H,s). 13C NMR 135.5, 135.5, 133.2,
129.7, 129.7, 59.5, 37.3, 26.6, 19.0. CI-MS m/z 346
(M118). EI-MS m/z (%), 271 (36), 200 (17), 199 (100),
197 (16), 193 (19), 181 (13), 139 (23), 105 (11), 91 (10),
78 (11), 77 (30), 57 (15), 45 (19), 41 (19).
12. Takeshita, K.; Seki, Y.; Kawamoto, K.; Murai, S.; Sonoda, N.
J. Org. Chem. 1987, 52, 4864.
13. Polniaszek, R. P.; Dillard, L. W. J. Org. Chem. 1992, 57, 4103.
14. Jones, G. R.; Landais, Y. Tetrahedron 1996, 52, 7599.
15. Gutman, A. L.; Meyer, E.; Boltanski, A. Synth. Commun.
1988, 18, 1311.
16. Malmberg, M.; Rehnberg, N. J. Carbohydr. Chem. 1996, 15,
459.
Acknowledgements
17. Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic
Synthesis, 2nd ed.; Wiley: New York, 1991; p 53.
18. Mamedov, S.; Ismiev, I.; Khydyrov, D. N. J. Gen. Chem.
USSR (Eng. Transl.) 1964, 34, 1072.
We are grateful to Dr Denys Voaden for the preparation and/
or distillation of several starting materials and to Mr
William R. Lusby for valuable consultation.
19. Jacobson, R. M.; Clader, J. W. Synth. Commun. 1979, 9, 57.
20. Just, G.; Grozinger, K. Synthesis 1976, 457.
21. Kochi, J. M.; Singleton, D. M. J. Am. Chem. Soc. 1968, 90,
1582.
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