Stereospecificity of the (E)- and (Z)-11 myristoyl CoA desaturases in the biosynthesis of spodoptera littoralis sex pheromone

Full text of DOI:10.1021/ja972577v

J. Am. Chem. Soc. 1997, 119, 11335-11336
11335
Scheme 1a
Stereospecificity of the (E)- and (Z)-11 Myristoyl
CoA Desaturases in the Biosynthesis of Spodoptera
littoralis Sex Pheromone
Isabel Navarro, Imma Font, Gemma Fabria`s,* and
Francisco Camps
Department of Biological Organic Chemistry
CID-CSIC, Jordi Girona 18-26, 08034-Barcelona, Spain
ReceiVed July 29, 1997
Unsaturated fatty acids are biosynthesized in nature from
saturated fatty acids by the action of specific desaturases, among
which (Z)-9 stearoyl CoA desaturase is the most ubiquitous
one.1-3 The biosynthetic pathways of lepidopteran sex phero-
mone blends involve the action of rather unusual desaturases,
such as (Z)-11,4 (Z)-10,5 (Z)-13,6 and (Z)-147 acyl CoA
desaturases. Additionally, certain moth pheromone gland
desaturases give rise to (E) fatty acids, also involved in the
biosynthesis of the pheromone blend,4 thus making these tissues
excellent models to study these unique enzymes. The main body
of literature concerning the biosynthetic pathways of (E) fatty
acids deal with bacteria. In these prokaryotic cells, (E)
unsaturated fatty acids are biosynthesized either by anaerobic
pathways8,9 or by isomerization of the corresponding (Z)
compounds.10 In moth pheromone glands, however, (E) fatty
acids are biosynthesized by direct desaturation.11 Our ongoing
interest in desaturase enzymes involved in insect sex pheromone
biosynthesis has led us to study the stereospecificity of the (Z)-
and (E)-11 desaturases of myristic acid,13 implied in the
biosynthesis of Spodoptera littoralis sex pheromone.14
To carry out this research, we performed experiments with
two different sets of probes: erythro- and threo-(11,12,13,13,
14,14,14-2H7)myristic acids (erythro- and threo-1) and (12R)-
and (12S)-(2,2,3,3,12-2H5)myristic acids ((12R)- and (12S)-1).
The first two probes, erythro- and threo-1, would reveal the
relative stereochemistry of hydrogens removed at C11 and C12,
whereas enantiomerically pure (12R)- and (12S)-1 would allow
for assessing the stereospecificity of hydrogen removal at C12.
Both erythro- and threo-1 were synthesized from alkyne 2 by
way of well-established reactions (Scheme 1). Deuteration of
double bonds was carried out using the Wilkinson catalyst to
ensure stereospecificity and to prevent deuterium scrambling.15,16
a Reagents: a, MeLi/THF, then ICD2CD3/HMPA (99%); b, H2/Pd-
BaSO4/quinoline (96%); c, Na/NH3 (86%); d, HCl/MeOH ((Z)-isomer,
83%; (E)-isomer, 74%); e, D2/RhCl(PPh3)3/C6H6 (erythro-isomer, 94%;
threo-isomer, 80%); f, CrO3/H2SO4/H2O (erythro-isomer, 96%; threo-
isomer, 80%);
On the other hand, (12R)- and (12S)-1 were prepared from
alkyne 6 as shown in Scheme 2. Deuterium atoms at R and â
positions of ester 8 were introduced by reaction of acetylenic
ester 7 with magnesium in MeOD,17 and the stereogenic center
at C12 was generated by reaction of aldehyde 10 with the system
Et2Zn/Ti(O-i-Pr)4/1,2-N,N′-bis(trifluoromethylsulfonylamino)-
cyclohexane.18 In this reaction, the use of the (1R,2R)-
sulfonamide afforded the hydroxyester (12S)-11, whereas the
(1S,2S)-sulfonamide furnished (12R)-11.19 Reduction of tosyl-
derivatives of (R)- and (S)-11 with lithium aluminium deuteride
followed by Jones oxidation gave the expected probes (12R)-
and (12S)-1, respectively.16 Probes were topically administered
to the pheromone gland as dimethyl sulfoxide solutions (0.1
µL, 2.5 mg/mL).14 A total dose of 2 µg was given in eight
subsequent 1-h incubations with 0.25 µg. Fatty acid methyl
esters were obtained by base methanolysis of pheromone gland
lipidic extracts,14 and analyses were performed by GC-MS as
previously reported14 using a polar SGE BP-20 capillary column
(30 m × 0.20 mm) programmed from 60 °C to 150 °C at 2
°C/min and then to 260 °C at 7 °C/min after an initial delay of
2 min. The selected ion monitoring mode was used, and ions
selected were the corresponding molecular ions for all isoto-
pomers.
(1) Fox, B. G.; Shanklin, J.; Somerville, C.; Munck, E. Proc. Natl. Acad.
Sci. U.S.A. 1993, 90, 2486-2490.
(2) Stukey, J. E.; McDonough, V. M.; Martin, C. E. J. Biol. Chem. 1990,
265, 20144-20149.
(3) Enoch, H. G.; Catala, A.; Strittmatter, P. J. Biol. Chem. 1976, 251,
5095-5103.
(4) Roelofs, W. L.; Bjostad, L. Bioorg. Chem. 1984, 12, 279-298.
(5) Foster, S. P.; Roelofs, W. L. Arch. Insect Biochem. Physiol. 1988, 8,
1-9.
(6) Arsequell, G.; Fabrias, G.; Camps, F. Arch. Insect Biochem. Physiol.
1990, 14, 47-56.
(7) Zhao, C.; Lofstedt, C.; Wang, X. Arch. Insect Biochem. Physiol. 1990,
15, 57-65.
(8) Keweloh, H.; Heipieper, H. J. Lipids 1996, 31, 129-137.
(9) Sommerfeld, M. Prog. Lipid Res. 1983, 22, 221-233.
(10) Loffeld, B.; Keweloh, H. Lipids 1996, 31, 811-815.
(11) Isomerization processes have been ruled out in Argyrotaenia
Velutinana (Bjostad, L. B.; Roelofs, W. L. J. Biol. Chem. 1990, 256, 7936-
7940) and S. littoralis.12 The possibility that (E)-11-tetradecenoic acid is
formed by dehydration of hydroxyacid intermediates in S. littoralis has also
been discarded (Navarro, I.; Fabrias, G.; Camps, F. Lipids, 1997, 32, 407-
412).
(16) The purity of probes and location of deuterium label in all substrates
were determined by elemental analysis, MS, and 1H and 13C NMR analyses.
(17) Hutchins, R. O.; Suchismita; Zipkin, R. E.; Taffer, I. M.; Sivakumar,
R.; Monaghan, A.; Elisseou, E. M. Tetrahedron Lett. 1989, 30, 55-56.
(18) Takahashi, H.; Kawakita, S.; Kobayashi, S. Tetrahedron 1992, 48,
5691-5700.
(19) Enantiomeric purity was >95% in both enantiomers, as determined
by 19F NMR analysis of the corresponding Mosher esters. The absolute
configuration of hydroxyesters 11 is inferred from the known mechanism
of the addition reaction of Et2Zn to aldehydes in the presence of titanium
complexes of chiral disulfonamides.18
(12) Topical application of (13,13,14,14,14-2H5)-(Z)-11-tetradecenoic acid
to S. littoralis pheromone glands did not show any labeled methyl (E)-11-
tetradecenoate in base methanolyzed pheromone gland lipidic extracts
analyzed by gas chromatography coupled to mass spectrometry under
selected ion monitoring mode.
(13) These enzymes afford a ratio of (E)- and (Z)-11-tetradecenoic acid
of 2:1 in the insect pheromone gland.
(14) Martinez, T.; Fabrias, G.; Camps, F. J. Biol. Chem. 1990, 265,
1381-1387.
(15) Tulloch, A. P. Chem. Phys. Lipids 1979, 24, 391-406.
S0002-7863(97)02577-8 CCC: $14.00 © 1997 American Chemical Society

11336 J. Am. Chem. Soc., Vol. 119, No. 46, 1997
Communications to the Editor
Scheme 2a
probe, which indicated the number of deuterium atoms present
in the unsaturated compounds and thus the number of deuterium
atoms that had been lost from each substrate to form each
isomer. The data obtained with erythro- and threo-1 clearly
showed that formation of (E)-11-tetradecenoic acid occurs by
elimination of C11-H and C12-H of opposite stereochemistry,
whereas (Z)-11-tetradecenoic acid is formed by removal of
C11-H and C12-H of the same stereochemistry. In these
experiments, erythro-1 gave rise to two isotopomers, d5 and
d7, of (Z)-11-tetradecenoic acid, arising from loss of either two
deuterium or two hydrogen atoms from C11 and C12, respec-
tively. The same result was obtained in the formation of (E)-
11-tetradecenoic acid from threo-1. However, in both cases,
the relative abundances of the d5 isotopomers were lower than
those of the d7, probably due to the existence of kinetic isotope
effects.20 On the other hand, experiments with the enantio-
merically pure substrates (12R)- and (12S)-1 showed that in
formation of the (E)-isomer the pro-(S) C12-H is specifically
removed, whereas specific cleavage of the pro-(R) C12-H takes
place to form the (Z)-isomer. In summary, the overall results
demonstrate that formation of (E)-11-tetradecenoic acid takes
place by stereospecific removal of the pro-(R) C11-H and the
pro-(S) C12-H and desaturation to the (Z)-isomer occurs by
stereospecific cleavage of pro-(R) C11-H and pro-(R) C12-H.
Therefore, the results presented in this paper show opposite
stereospecificities in removal of C12-H by both the (E)- and
(Z)-11 myristoyl CoA desaturases. The results found for the
(Z) enzyme are in agreement with those reported previously on
the related (Z)-11 palmitoyl CoA desaturase21 and (Z)-9
stearoyl CoA desaturase,3,22,23 which specifically remove the pro-
(R) hydrogen atoms at C11 and C12, and suggest a common
steric course for all (Z) desaturase enzymes. On the other hand,
the results reported in this Communication represent the first
determination of the stereospecificity of a (E) fatty acyl
desaturase in nature. Additional stereochemical studies on other
(E) desaturase enzymes will establish if the steric course found
here for the (E)-11 myristoyl CoA desaturase is common to all
(E) desaturase enzymes. Biological desaturation of fatty acids
to the corresponding (Z)-isomers is a syn-elimination reaction.24
Assuming that desaturation to the (E)-isomers is also a syn-
elimination process and that both (Z)- and (E)-desaturases are
structurally related enzymes, the geometry of the resulting
double bond would result from the different conformation
adopted by the acyl substrate at the enzyme active site, which
might be influenced by different amino acid sequence alignments
in the enzyme active center of both (Z)- and (E)-enzymes.25
Further work along these lines is in progress in our laboratories.
a Reagents: a, MeLi/THF, then ClCOOMe/THF (88%); b, Mg/
MeOD (73%), see ref. 17; c, HCl/MeOH (83%); d, (CO)2Cl2/DMSO/
Et3N (75%); e, Et2Zn/Ti(O-i-Pr)4/1,2-N,N′-bis(trifluoromethylsulfonyl-
amino)cyclohexane/toluene ((12S)-11, 66%; (12R)-11, 89%), see ref
18; f, TsCl/pyridine ((12R)-12, 72%; (12S)-12, 70%); g, LiAlD4/Et2O,
then CrO3/H2SO4/H2O ((12R)-1, 38%; (12S)-1, 43%).
Table 1. Relative Stereochemistry of Hydrogens Removed at C11
and C12 in ∆11 Desaturations of Myristic Acid
isotopomer (mol %)a
tracer
productb
d5
d6
d7
erythro-1
E
Z
E
Z
6.1 ( 3.1
29.0 ( 1.9
29.0 ( 3.0
0.5 ( 0.5
85.9 ( 16.3
3.4 ( 3.0
5.8 ( 6.0
96.2 ( 3.3
8.0 ( 13.8
67.6 ( 1.9
65.2 ( 3.4
3.3 ( 2.3
threo-1
a Percentages of isotopomers correspond to the average ( standard
deviation of three independent experiments performed with groups of
four glands. Percentages were corrected for the abundances of M•+
+
1 and M•+ - 1 ions in the substrate probes. b Products refer to E, (E)-
11-tetradecenoic acid and Z, (Z)-11-tetradecenoic acid, analyzed as
methyl esters.
Acknowledgment. We thank Comisio´n Asesora de Investigacio´n
Cient´ıfica y Te´cnica (Grant AGF 95-0185), Comissionat per a Uni-
versitats i Recerca from the Generalitat de Catalunya (Grant GRQ 93-
8016) and SEDQ S. A. for financial support, and Germa´n La´zaro for
insect rearing.
Table 2. Absolute Stereochemistry of Hydrogen Removed at C12
in ∆11 Desaturations of Myristic Acid
Supporting Information Available: Experimental procedures, 1H
and 13C NMR, and IR spectroscopic data for the products (9 pages).
See any current masthead page for ordering and Internet access
instructions.
isotopomer (mol %)a
tracer
productb
d4
d5
(12R)-1
E
Z
E
Z
9.5 ( 7.0
91.7 ( 6.2
91.7 ( 4.0
12.0 ( 5.2
90.5 ( 7.0
8.2 ( 6.2
8.3 ( 4.0
88.0 ( 5.2
(12S)-1
JA972577V
(20) Buist, P. H.; Behrouzian, B. J. Am. Chem. Soc. 1996, 118, 6295-
6296.
a Percentages of isotopomers correspond to the average ( standard
deviation of three independent experiments performed with groups of
(21) Boland, W.; Frossl, C.; Schottler, M.; Toth, M. J. Chem. Soc., Chem.
Commun. 1993, 1155-1157.
four glands. Percentages were corrected for the abundances of M•+
+
1 and M•+ - 1 ions in the substrate probes. b Products refer to E, (E)-
11-tetradecenoic acid and Z, (Z)-11-tetradecenoic acid, analyzed as
methyl esters.
(22) Buist, P. H.; Marecak, D. M. J. Am. Chem. Soc. 1992, 114, 5073-
5080.
(23) Schroepfer, G. J.; Bloch, K. J. Biol. Chem. 1965, 240, 54-63.
(24) Cook, H. In Biochemistry of lipids and membranes; Vance, D. E.,
Vance, J. E., Eds.; The Benjamin Cummings Publishing Co. Ltd.: Menlo
Park, CA, 1985; pp 191-203.
The results obtained in the deuterium-labeling experiments
are summarized in Tables 1 and 2. In these experiments, the
stereochemistry of hydrogens removed at both C11 and C12
was deduced from the mass of the most abundant isotopomer
of both (Z)- and (E)-11-tetradecenoic acid resulting from each
(25) Chain-length recognition by fatty acid desaturases from plants is
influenced by the amino acid residues of the enzyme portion that binds to
the substrate beyond the point of double bond insertion (Cahoon, E. B.;
Lindqvist, Y.; Schneider, G.; Shanklin, J. Proc. Natl. Acad. Sci. U.S.A. 1997,
94, 4872-4877).