Deuterated analogues of 4,8-dimethyldecanal, the aggregation pheromone of Tribolium castaneum: Synthesis and pheromonal activity

Article abstract of DOI:10.1002/jlcr.881

To elucidate the deuterium isotope effect (DIE) in pheromonal activity and to investigate the biosynthetic pathway of 4,8-dimethyldecanal (4,8-DMD; 1), the aggregation pheromone of the red flour beetle (Tribolium castaneum), deuterated analogues of 4,8-DMDs (2, 3, 4, and 5), were synthesized and their pheromonal activities were tested using a two-hole pitfall olfactometer. Although no apparent DIE was observed in their pheromonal activities, 4,8-DMD-1-d ₁ (2) was less attractive than other analogues, which suggested that the bond distance between the formyl group of 1 and its receptor was critical in pheromone recognition by T. castaneum. Copyright 2004 John Wiley & Sons, Ltd.

Full text of DOI:10.1002/jlcr.881

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2004; 47: 921–934.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.881
Research Article
Deuterated analogues of 4,8-dimethyldecanal, the
aggregation pheromone of Tribolium castaneum:
synthesis and pheromonal activity
Junheon Kim, Shigeru Matsuyama and Takahisa Suzuki*
Division of Life Sciences and Bioengineering, Graduate School of Life and
Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan
Summary
To elucidate the deuterium isotope effect (DIE) in pheromonal activity and to
investigate the biosynthetic pathway of 4,8-dimethyldecanal (4,8-DMD; 1), the
aggregation pheromone of the red flour beetle (Tribolium castaneum), deuterated
analogues of 4,8-DMDs (2, 3, 4, and 5), were synthesized and their pheromonal
activities were tested using a two-hole pitfall olfactometer. Although no apparent DIE
was observed in their pheromonal activities, 4,8-DMD-1-d1 (2) was less attractive than
other analogues, which suggested that the bond distance between the formyl group of
1 and its receptor was critical in pheromone recognition by T. castaneum.
Copyright # 2004 John Wiley & Sons, Ltd.
Key Words: Tribolium castaneum; aggregation pheromone; 4,8-dimethyldecanal;
deuterated 4,8-dimethyldecanals; deuterium isotope effect
Introduction
Deuterated compounds have been utilized for investigation of binding
interactions or structure–odour relationships between molecules of biological
interest and their receptors, the so-called deuterium isotope effect (DIE), and
also used as mass-labeled precursors for biosynthetic studies of insect
pheromones.1–3 Some of the deuterated semiochemicals affected insect
behavior, and those phenomena were interpreted by the spatial and/or
binding affinity between molecules and their receptor surfaces due to the short
1,4,5
(
bond length of C–D (1.1071 A) versus that of C–H (1.1122 A).
(
4,8-Dimethyldecanal (4,8-DMD, 1; Figure 1.) is the male-produced
aggregation pheromone of the red flour beetle, Tribolium castaneum
*Correspondence to: T. Suzuki, Division of Life Sciences and Bioengineering, Graduate School of Life and
Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan. E-mail: tksuzuki@
agbi.tsukuba.ac.jp
Copyright # 2004 John Wiley & Sons, Ltd.
Received 14 June 2004
Revised 21 July 2004
Accepted 23 July 2004

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J. KIM ET AL.
Figure 1. The aggregation pheromone (1) of T. castaneum and its deuterated
analogues (2, 3, 4, and 5)
(Coleoptera; Tenebrionidae), identified by one of the authors.6 T. castaneum is
known as a serious stored-product pest distributed throughout the world. The
natural pheromone is a (4R,8R)-optical isomer, and the racemic 4,8-DMD
shows one hundredth of the attraction of the natural pheromone.7 It is
thought that 4,8-DMD has a four-group-binding site to its receptor or a
pheromone-binding protein, that is, –CHO, 4-CH3, 8-CH3 and the terminal
CH3 group in the molecule, and these groups appear to be responsible for
pheromone recognition by the insect. Therefore, deuterated analogues of 4,8-
DMDs (2, 3, 4, and 5; Figure 1) were synthesized to elucidate their DIEs. Our
final objective is to elucidate the biosynthetic pathway of the pheromone,
which is unclear to date. Deuterium labeled putative precursors and
deuterated 4,8-DMDs are expected to be useful for this purpose.
Here we report the detailed synthesis of the deuterated 4,8-DMDs and their
DIEs in the pheromonal activities of T. castaneum.
Results and discussion
Synthesis
Two synthetic routes (A, B) were employed as described below. The racemic
4,8-DMDs (1a and 1b), prepared by different procedures, are expected to show
equal levels of activity.
Synthesis of 1a, 2, and 3 (Route A). Deuterium at the C-1 position of 2 and
CD3 at the C-4 position of 3 were introduced using LiAlD4 and CD3I,
respectively, as outlined in Scheme 1. The key reaction is alkylation of a
malonate followed by decarboxylation (malonic ester synthesis). Malonate 6,
prepared with CD3I and diethyl malonate, was alkylated with 7 followed by
decarboxylation to give the deuterated ester 9, which was converted into 10 by
LiAlH4 reduction. Hydrogenation and subsequent bromination yielded the
saturated bromide 12. This compound was submitted to malonic ester
synthesis again. The desired aldehyde 3 was obtained by PCC oxidation of 15,
prepared as described above via ester 14. Synthesis of aldehyde 2 was
accomplished by the same procedure using ester 14a, prepared in the same
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DEUTERATED 4,8-DMDS
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Scheme 1. Synthesis of deuterated 4,8-DMDs (2, 3; Route A)
manner as for 14, using diethyl methylmalonate instead of 6 and LiAlD4, while
the unlabeled aldehyde 1a was synthesized using 14a and LiAlH4.
Synthesis of 1b, 4, and 5 (Route B). CD3 at the C-8 position of 4 and CD3 at
the C-10 position of 5 were introduced using CD3I and LiAlD4, respectively
(Scheme 2). The main reactions are the Grignard coupling reaction in the
presence of LiCuCl4 and oxidative cleavage of a double bond to an aldehyde.
The Grignard coupling of citronellyl bromide 22 and iodide 21, prepared by
malonic ester synthesis, gave alkene 23, which was submitted to oxidative
cleavage to yield the target aldehyde 4.8–10 The a,b-unsaturated ester 26 was
obtained by the acetoacetic ester synthesis of 22 followed by the Wittig–
Horner reaction. The desired aldehyde 5 was produced by oxidative cleavage
of alkene 30 prepared by selective hydrogenation of 26 with Red-AlTM and
CuBr(I).
Bioassay
The bioassay results of 4,8-DMD and its deuterated analogues on T.
castaneum are summarized in Table 1. All compounds (1a, 2, and 3) prepared
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J. KIM ET AL.
Scheme 2. Synthesis of deuterated 4,8-DMDs (4, 5; Route B)
Table 1. Attraction of 4,8-DMD and its analogues on T. castaneum
Attractiveness (%, mean Æ SD)a
Compounds (Route A)
Compounds (Route B)
Dose (ng/disk) 1a
2
3
1b
4
5
1
10
À1.3 Æ 22.0 À1.7 Æ 18.5 À4.2 Æ 30.2
2.9 Æ 20.1 15.0 Æ 21.5 8.8 Æ 12.3 23.8 Æ 22.8* 15.0 Æ 13.9* 33.3 Æ 14.6**
9.2 Æ 14.4 À7.1 Æ 30.6 À4.2 Æ 12.8
100
1000
38.8 Æ 22.4** 20.0 Æ 21.2** 30.0 Æ 29.3** 43.3 Æ 11.8** 28.3 Æ 20.4** 30.8 Æ 19.2**
46.7 Æ 22.5** 27.9 Æ 13.0* 42.9 Æ 16.9* 30.0 Æ 15.0** 29.2 Æ 22.2** 38.3 Æ 14.9**
a Thirty 4-day-old beetles were used per replicate (8 replicates). * **: Significant at p50.05 and p50.01,
,
respectively, by t-test.
in Route A were active to the extent of more than 100 ng, while all compounds
(1b, 4, and 5) prepared in Route B showed an attraction of more than 10 ng.
Although the racemic 4,8-DMDs (1a and 1b) prepared by different synthetic
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925
routes (Routes A and B) were expected to show equal levels of activity, 1b was
ten times more active than 1a (the lowest active limit: 1a – 100 ng; 1b – 10 ng).
The pheromonal activity of 4,8-DMD was affected by the composition of
optical isomers involved.7 Since compounds 9 and 22 were optically inactive,
the ratio of optical isomers at the C-4 position of 4,8-DMDs in each route
would be same. Therefore, the different activity between the compounds
prepared by Routes A and B would be the result of the different ratio of
optical isomers at the C-8 position of 4,8-DMDs, which was supported by the
fact that the 8:2 mixture of the (4R,8R)- and (4R,8S)-isomers showed
maximum activity, although the natural pheromone was identified as the
(4R,8R)-isomer.7
No apparent DIE was observed in all deuterated 4,8-DMDs, whereas 2
showed relatively less attraction than 1a at doses of 100 and 1000 ng.
Pheromonal activity, in general, decreased or disappeared when the
functional group and/or carbon chain length of a pheromone was converted.
Compound 2 has a chain length equal to the original compound 1a and
maintains the characteristics of the functional group. However the binding
distance to its receptor would change due to the shorter bond length of C–D
versus that of C–H. Therefore, the lower attraction of 2 suggests that the bond
distance between the CHO group of 4,8-DMD and its receptor was critical in
pheromone recognition by T. castaneum.
Experimental
General analytical procedure
GC analyses were performed on a HP 5890 Series II gas chromatograph
equipped with FID and a 007-5MS column [25 m  0.25 mm (I.D)  0.25 mm,
Quardrex]. Mass spectra were obtained either on a HP MSD 5972 Series
coupled with a HP 5890 Series II Plus equipped with a HP-5MS column
[30 m  0.25 mm (I.D)  0.25 mm, J&W Scientific] or a JEOL MS Router MS-
600 coupled with a HP 6890N equipped with a HP-1MS column
[30 m  0.25 mm (I.D)  0.25 mm, J&W Scientific]. NMR spectra were
recorded in CDCl3 with tetramethylsilane (TMS) as an internal standard
1
using a Bruker Avance 500 spectrometer at 500 MHz for H spectra and at
125 MHz for 13C spectra. IR spectra were run on a JASCO IRA-1 (Japan
Spectroscopic Co., Ltd.) spectrometer.
Reagents and solvents
Deuterium-labeled reagents (CD3I and LiAlD4) were purchased from Aldrich.
Tetrahydrofuran (THF) was dried and distilled from sodium benzophenone
ketyl prior to use. Diethyl ether was dried over sodium wire and used without
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purification. Acetone was dried over K2CO3 and distilled prior to use. Column
chromatography was performed using Wakogel1 (C-200, Wako, Japan).
Synthesis
Diethyl trideuteriomethylmalonate (6). To a solution of NaH (2.21 g,
60.0 mmol; 65%) in THF (60 ml), diethyl malonate (8.00 g, 50.0 mmol) in
THF (20 ml) was added at 08C under N2. After stirring for 30 min at room
temperature, iodomethane-d3 (3.73 ml, 60.0 mmol) in THF (20 ml) was added
at 08C. The solution was refluxed for 2 h, and then ice-cold water (200 ml) was
added. The mixture was extracted with ether (3 Â 60 ml). The combined
extracts were washed with saturated Na2SO3, H2O, brine, then dried and
concentrated in vacuo. Distillation of the residue gave 8.22 g of 6, containing
ca. 30% diethyl ditrideuteriomethylmalonate as a by-product. This material
was employed in the next step without further purification. bp: 95–968C/
1
21 mmHg. H NMR: 4.20 (4H, q, J=7.1), 3.41 (1H, s), 1.27 (6H, t, J=7.14).
13C NMR: 170.6 (C Â 2), 61.7 (CH2 Â 2), 46.4 (CH), 14.4 (CH3 Â 2). GC-MS:
177 (M+, 8.3), 150 (21.1), 132 (100), 105 (31.1), 77 (74.0), 60 (31.2), 59 (35.2).
IR: 2950 (s), 2220 (w), 1730 (s), 1360 (m), 1230 (s), 1120 (s), 1020 (s).
1-Bromo-4-methyl-3-hexene (7). Compound 7 was prepared in the usual
manner.13 Yield 53.2% (3E:3Z=3:1, calculated on GC). bp: 85–868C/
35 mmHg. H NMR: 5.13 (1H, tq, J=7.1,1.4), 3.34 (2H, t, J=7.4), 2.57
1
(2H, dq, J=7.3, 0.7), 2.01 (2H, q, J=7.4), 1.63 (3H, s), 1.00 (3H, t, J=7.4).
13C NMR: 140.8 (C), 119.8 (CH), 33.4 (CH2), 32.7 (CH2), 32.1 (CH2), 16.6
(CH3), 13.0 (CH3). GC-MS (A): 178 (M+, 10.2), 176 (11.0), 97 (40.3), 83
(17.7), 55 (100). IR: 2950 (s), 1650 (w).
Diethyl (4-methyl-3-hexenyl)(trideuteriomethyl)malonate (8). To a suspen-
sion of NaH (0.98 g, 26.55 mmol) in THF (10 ml), 6 (5.76 g, 22.13 mmol, 70%
pure) in THF (15 ml) was added at 08C under N2, and the solution was stirred
for a further 30 min at room temperature and then a solution of 7 (4.70 g,
26.55 mmol) in THF (15 ml) was added at 08C. After the mixture was refluxed
for 12 h, ice-cold water (70 ml) was added. The mixture was extracted with
ether (3 Â 60 ml). The combined extracts were washed with saturated Na2SO3,
H2O, brine, then dried and concentrated to give 9.52 g of crude 8. This
material was employed in the next step without further purification. GC-MS:
228 (M+-OMe, 2.2), 177 (100), 131 (73.2), 103 (30.0), 81 (12.2).
Ethyl 2-trideuteriomethyl-6-methyl-5-octenoate (9). According to the modified
procedure in the literature, 9 was prepared.14 To a solution of crude 8 (9.52 g,
34.87 mmol) and LiCl (2.96 g, 69.74 mmol) in DMSO (70 ml), H2O (4.5 ml) was
added, and then the mixture was refluxed for 4 h by stirring. After being cooled
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to room temperature, water (200 ml) was added to the mixture. The solution
was extracted with ether (3 Â 60 ml) and the organic phases were washed with
H2O, brine, then dried and concentrated in vacuo. Distillation of the residue
gave 3.45 g of 9 (17.20 mmol, 77.6% based on 7). bp: 109–1118C/15 mmHg. 1H
NMR: 5.08 (1H, tq, J=7.1, 1.3), 4.13 (2H, q, J=7.1), 2.41 (1H, t, J=7.0),
2.03-1.96 (4H, m), 1.70 (1H, dq, J=6.7, 0.9) 1.59 (3H, s), 1.43 (1H, dq, J=6.3,
1.6), 1.26 (3H, t, J=7.1), 0.98 (3H, t, J=7.4). 13C NMR: 177.3 (C), 138.0 (C),
122.5 (CH), 60.5 (CH2), 39.3 (CH), 34.2 (CH2), 32.7 (CH2), 25.6 (CH2), 16.2
(CH3), 14.7 (CH3), 13.1 (CH3). GC-MS: 201 (M+, 1.5), 156 (11.6), 105 (100),
97 (25.4), 96 (29.1), 77 (96.8). IR: 2900 (s), 2210 (m), 2130 (m), 2050 (w), 1730
(s), 1150 (s), 1010 (s), 840 (m).
2-Trideuteriomethyl-6-methyloctanol (11). Ester 9 was converted to the
corresponding alcohol 10 with LiAlH4 according to the usual procedure
(Yield 66.3%). Compound 10 (1.81 g, 11.38 mmol) was submitted for catalytic
hydrogenation to give crude 11, which was purified by column chroma-
1
tography (silica 20 g, hexane:ether=1:1). Yield 67.1%. H NMR: 3.51 (1H,
ddd, J=10.5, 5.8, 2.0), 3.41 (1H, dd, J=10.5, 6.6), 1.59 (1H, m), 1.47 (1H,
br. s), 1.40-1.23 (6H, m), 1.18-1.01 (3H, m), 0.86 (3H, t, J=7.2), 0.85 (3H, dd,
J=6.5, 1.1). 13C NMR: 68.8 (CH2), 37.3 (CH2), 36.0 (CH), 34.8 (CH),
33.8 (CH2), 30.0 (CH2), 24.8 (CH2), 19.6 (CH3), 11.8 (CH3). GC-MS: 143
(M+-H2O, 0.4), 114 (64.4), 88 (20.1), 83 (18.2), 74 (30.5), 72 (63.0), 71
(61.1), 70 (100), 57 (78.6), 43 (72.5). IR: 3300 (br, s), 2900 (s), 2300 (w),
2200 (m), 1020 (s).
1-Bromo-2-trideuteriomethyl-6-methyloctane (12). To a solution of triphenyl-
phosphine (2.20 g, 8.40 mmol) in CH2Cl2 (20 ml), bromine (0.43 ml, 8.40 mmol)
was added dropwise at 08C and the mixture was stirred for 30 min with
cooling. To this solution, 11 (1.23 g, 7.64 mmol) and triethylamine (1.60 ml,
11.46 mmol) in CH2Cl2 (10 ml) was added. After stirring for 2 h at 08C, the
mixture was filtered and the solvent was evaporated in vacuo. The residue was
extracted with petroleum ether (100 ml) and filtered off. The filter cake was
rinsed with petroleum ether (2 Â 50 ml). The combined filtrates were
concentrated. After this procedure was done twice, the residue was
chromatographed over silica (20 g, hexane) to give 1.30 g of 12 (5.80 mmol,
76.0%). 1H NMR: 3.40 (1H, ddd, J=9.8, 4.9, 1.9), 3.32 (1H, ddd, J=9.8, 6.2,
1.1), 1.77 (1H, m), 1.46-1.04 (9H, m), 0.86 (3H, t, J=7.2), 0.85 (3H, dd, J=6.1,
0.8). 13C NMR: 42.0 (CH2), 37.0 (CH2), 35.5 (CH2), 35.4 (CH), 34.7 (CH),
29.9 (CH2), 24.7 (CH2), 19.6 (CH3), 11.8 (CH3). GC-MS: 196 (M+-Et, 1.6),
194 (M+-Et, 1.6), 168 (60.4), 166 (62.4), 114 (36.1), 72 (55.3), 57 (100.0), 56
(45.6), 55 (36.1). IR: 2910 (s), 2200 (m), 1040 (m).
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J. KIM ET AL.
Ethyl 4-trideuteriomethyl-8-methyldecanoate (14). Crude malonate 13
(1.82 g), prepared by a method similar to that used for 8 with DMF, was
decarboxylated as described previously to give 0.71 g of 14 (53.0% yield in 2
1
steps). H NMR: 4.12 (2H, q, J=7.1), 2.38-2.26 (2H, m), 1.70-1.63 (1H, m),
1.48-1.38 (2H, m), 1.37-1.20 (6H, m), 1.26 (3H, t, J=7.1), 1.16-1.02 (3H, m),
0.85 (3H, t, J=7.3), 0.84 (3H, dd, J=6.1, 0.7). 13C NMR: 174.6 (C), 60.6
(CH2), 37.3 (CH2), 37.2 (CH2), 34.8 (CH), 32.6 (CH), 32.6 (CH2), 32.3 (CH2),
29.9 (CH2), 24.8 (CH2), 19.6 (CH3), 14.6 (CH3), 11.8 (CH3). GC-MS: 231
(M+, 0.5), 174 (11.0), 171 (10.0), 143 (4.3), 101 (100), 73 (26.2). IR: 2900 (s),
2090 (m), 1730 (s), 1350 (m), 1240 (m), 1150 (s).
4-Trideuteriomethyl-8-methyldecanal (3). Alcohol 15, obtained from the
corresponding ester 14 by LiAlH4 reduction, was submitted for PCC
oxidation. To a suspension of PCC (0.72 g, 3.33 mmol) in CH2Cl2 (10 ml), 15
(0.42 g, 2.22 mmol) in CH2Cl2 (10 ml) was added. After the mixture was stirred
for 1 h at room temperature, the supernatant was decanted, and the black gum
was washed with dry ether (3 Â 50 ml). The combined organic layers were
passed through a short pad of florisil column and concentrated. The residue
was chromatographed over silica (10 g, hexane:ether=95:5) and distilled to
yield 0.10 g of 3 (24.8%). Compound 3 was 91.5% pure and it was used for
bioassay without further purification. 1H NMR: 9.78 (1H, t, J=1.9), 2.45-2.40
(2H, m), 1.70-1.62 (1H, m), 1.48-1.40 (2H, m), 1.37-1.20 (6H, m), 1.17-1.02
(3H, m), 0.86 (3H, t, J=7.1), 0.84 (3H, dd, J=6.5, 0.8). 13C NMR: 203.5
(CH), 42.1 (CH2), 37.3 (CH2), 37.2 (CH2), 34.8 (CH), 32.5 (CH), 29.8 (CH2),
29.3 (CH2), 24.8 (CH2), 19.6 (CH3), 11.7 (CH3). GC-MS: 187 (M+, 0.4), 143
(46.5), 140 (24.9), 114 (53.5), 98 (50.2), 84 (76.5), 70 (100), 57 (88.6). IR: 2920
(s), 2710 (m), 2190 (m), 1710 (s).
4,8-Dimethyldecanol-1,1-d2 (16). Ethyl 4,8-dimethyldecanoate 14a (0.78 g,
3.42 mmol), prepared by the same procedure as used for compound 14
(starting from diethyl methylmalonate), was reduced with LiAlD4 to give
0.42 g of 16 (2.28 mmol, 65.3%), which was purified by column chromato-
1
graphy (silica 10 g, hexane:ether = 1:1). H NMR: 1.63-1.48 (2H, m), 1.44-
1.22 (8H, m), 1.40 (1H, br. s), 1.20-1.03 (4H, m), 0.87 (3H, dd, J=6.4, 0.6),
0.86 (3H, t, J=7.1), 0.84 (3H, d, J=6.1). 13C NMR: 37.7 (CH2), 37.3 (CH2),
34.8 (CH), 33.3 (CH2), 33.0 (CH), 30.5 (CH2), 30.0 (CH2), 24.9 (CH2), 20.0
(CH3), 19.6 (CH3), 11.8 (CH3). GC-MS: 170 (M+-H2O, 0.6), 140 (15.3), 111
(17.0), 99 (11.9), 85 (30.4), 71 (100), 70 (70.0), 57 (62.1). IR:3300 (br, s), 2170,
2070 (m), 1115 (m), 960 (m).
4,8-Dimethyldecanal-1-d1 (2). Alcohol 16 (0.42 g, 2.22 mmol) was oxidized
with PCC (0.72 g, 3.33 mmol) by stirring for 2 h, yielding 0.18 g of 2 (42.4%).
Purification by column chromatography (silica 10 g, hexane:ether = 95:5) and
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bulb-to-bulb distillation gave 98.8% purity of 2 and it was used for bioassay.
1H NMR: 9.78 (trace, 0.01H, t, J=1.9), 2.45-2.40 (2H, m), 1.70-1.62 (1H, m),
1.48-1.40 (2H, m), 1.37-1.20 (6H, m), 1.17-1.02 (3H, m), 0.88 (3H, d, J=6.1),
0.86 (3H, t, J=7.2), 0.84 (3H, dd, J=6.5, 0.8). 13C NMR: 41.9 (CH2), 37.4
(CH2), 37.2 (CH2), 34.8 (CH), 32.8 (CH), 29.8 (CH2), 29.3 (CH2), 24.8 (CH2),
19.7 (CH3), 19.6 (CH3), 11.8 (CH3). GC-MS: 185 (M+, 0.4), 140 (26.6), 138
(19.3), 111 (40.5), 96 (43.3), 83 (48.7), 70 (100), 57 (69.2). IR: 2910 (s), 2010 (s),
1700 (s).
4,8-Dimethyldecanal (1a). This compound was synthesized according to
Scheme 1 from nondeuterated diethyl methylmalonate as the starting material.
Yield 0.12 g (24.4% based on 4,8-dimethyldecanol). Further purification gave
1
95.3% purity of 1a and it was used for bioassay. H NMR: 9.78 (1H, t,
J=1.9), 2.45-2.40 (2H, m), 1.70-1.62 (1H, m), 1.48-1.40 (2H, m), 1.37-1.20
(6H, m), 1.17-1.02 (3H, m), 0.88 (3H, d, J=6.1), 0.86 (3H, t, J=7.1), 0.84 (3H,
dd, J=6.5, 0.8). 13C NMR: 203.5 (CH), 42.1 (CH2), 37.4 (CH2), 37.2 (CH2),
34.8 (CH), 32.7 (CH), 29.9 (CH2), 29.3 (CH2), 24.7 (CH2), 19.7 (CH3), 19.6
(CH3), 11.7 (CH3). GC-MS: 184 (M+, 0.2), 140 (38.5), 137 (18.0), 111 (40.7),
95 (50.7), 85 (59.5), 81 (48.9), 70 (100), 69 (72.4), 57 (96.8). IR: 2920 (s), 2700
(m), 1710 (s).
Diethyl (ethyl)(trideuteriomethyl)malonate (17). This compound was pre-
pared in the same manner as that used for 6 from NaH (1.77 g, 48.00 mmol),
diethyl ethylmalonate (7.52 g, 40.00 mmol), and iodomethane-d3 (3.00 ml,
48.00 mmol) as the starting materials. Yield 85.7%. bp: 120–1218C/45 mmHg.
1H NMR: 4.18 (4H, q, J=7.0), 1.91 (2H, q, J=7.5), 1.25 (6H, t, J=7.5). 0.87
(3H, t, J=7.5). 13C NMR: 172.8 (C Â 2), 61.4 (CH2 Â 2), 54.2 (C), 28.8 (CH2),
14.4 (CH3 Â 2), 9.0 (CH3). GC-MS: 205 (M+, 0.2), 177 (100), 160 (45.8), 132
(66.1), 118 (57.1), 90 (91.8), 76 (44.0). IR: 2990 (s), 2230 (m), 1720 (s), 1230 (s),
1130 (s), 1100 (s), 1015 (s).
2-Trideuteriomethylbutan-1-ol (19). Decarboxylation of 17 (7.03 g), as de-
scribed previously, gave ester 18 (2.33 g, 51.1%), followed by reduction with
LiAlH4 to afford the corresponding alcohol 19 (1.13 g, 70.9%). bp: 748C/
1
76 mmHg. H NMR: 3.51 (1H, dd, J=10.5, 5.9), 3.41 (1H, dd, J=10.5, 6.5),
1.82 (1H, br. s), 1.52 (1H, m), 1.49-1.42 (1H, m), 1.14 (1H, dquin, J=13.5,
7.4), 0.91 (3H, t, J=7.4). 13C NMR: 68.3 (CH2), 37.5 (CH), 26.1 (CH2), 11.7
(CH3). GC-MS: 91 (M+, 0.2), 73 (49.4), 60 (100), 59 (90.0). IR: 3300 (s), 2900
(s), 2200 (m), 2050 (m), 1240 (m), 1040 (s).
1-Iodo-2-trideuteriomethylbutane (21). To
a solution of NaI (3.50 g,
23.34 mmol) in dry acetone (40 ml), tosylate 20 (2.86 g), derived from 19,
was added. The solution was stirred and refluxed for 5 h. After cooling to
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room temperature, water (100 ml) was added. The solution was extracted with
pentane (3 Â 70 ml). The organic phases were washed with saturated Na2SO3,
brine and dried. After the solvent was evaporated, the residue was distilled to
1
afford 1.61 g of 21 (8.01 mmol, 64.5% in 2 steps). bp: 628C/70 mmHg. H
NMR: 3.23 (1H, dd, J=9.6, 4.8), 3.17 (1H, dd, J=9.6, 5.8), 1.44-1.35 (2H, m),
1.29-1.24 (1H, m), 0.89 (3H, t, J=7.3). 13C NMR: 36.6 (CH), 29.5 (CH2), 17.8
(CH3), 11.7 (CH3). GC-MS: 201 (M+, 12.4), 127 (16.3), 74 (100). IR: 2930 (s),
2220 (m), 2050 (m), 1270 (m), 1250 (m), 1180 (s), 1050 (s).
Citronellyl bromide (22). Racemic citronellol (15.60 g, 100.00 mmol) was
brominated in the same way as 12 to give 18.23 g of 22 (83.24 mmol, 83.2%).
bp: 103–1058C/12 mmHg. 1H NMR: 5.09 (1H, tq, J=7.1, 1.4), 3.48-3.37 (2H,
m), 2.05-1.84 (3H, m), 1.71-1.57 (2H, m), 1.68 (3H, d, J=1.3), 1.61 (3H, s),
1.38-1.32 (1H, m), 1.21-1.14 (1H, m), 0.90 (3H, d, J=6.5). 13C NMR: 131.9
(C), 125.0 (CH), 40.4 (CH2), 37.0 (CH2), 32.4 (CH2), 31.8 (CH), 26.1 (CH3),
25.8 (CH2), 19.3 (CH3), 18.1 (CH3). GC-MS: 220 (M+, 8.4), 218 (M+, 8.0), 83
(56.2), 69 (100). IR: 2910 (s), 1250 (w), 1100 (w), 870 (w), 810 (w).
2,6-Dimethyl-10-trideuteriomethyl-2-dodecene (23). To a solution of citronel-
lylmagnesium bromide prepared from Mg 0.77 g (0.032 g atom) and 22 (7.02 g ,
32.04 mmol) in THF (40 ml), a solution of 21 (1.61 g, 8.01 mmol), N-methyl
pyrrolidinone (12.30 ml, 128.16 mmol) and Li2CuCl4 (4.0 ml, 0.40 mmol;
0.1 M) in THF (20 ml) was added at 08C under N2. The mixture was stirred
for 1.5 h at 08C and 1 h at room temperature. The excess Grignard reagent was
destroyed by dropwise addition of 2N HCl (50 ml). The precipitate formed was
removed by filtration through defatted cotton and the filtrate was extracted
with hexane (3 Â 60 ml). The organic phase was washed successively with 2N
HCl, saturated Na2SO3, H2O, and brine, then dried, and concentrated in
vacuo. The residue was distilled to give 0.46 g of 23 (2.16 mmol, 27.0% based
on 21). bp: 102–1048C/4 mmHg. 1H NMR: 5.10 (1H, tq, J=7.1, 1.4), 2.00-1.92
(2H, m), 1.68 (3H, d, J=1.1), 1.60 (3H, s), 1.42-1.35 (1H, m), 1.34-1.17 (7H,
m), 1.18-1.02 (4H, m), 0.86 (3H, d, J=6.6), 0.85 (3H, t, J=7.3). 13C NMR:
131.3 (C), 125.5 (CH), 37.7 (CH2), 37.6 (CH2), 37.3 (CH2), 34.6 (CH), 32.8
(CH), 29.8 (CH2), 26.0 (CH3), 25.7 (CH2), 24.8 (CH2), 20.0 (CH3), 18.0 (CH3),
11.8 (CH3). GC-MS: 213 (M+, 20.4), 111 (14.7), 73 (26.6), 69 (100). IR: 2920
(s), 2200 (m), 2050 (m), 1440 (s), 1370 (s), 1050 (m).
2,3-Epoxy-2,6-dimethyl-10-trideuteriomethyldodecane (24). Compound 24
was prepared according to the procedure of Suzuki.10 Yield 84.9%. GC-MS:
229 (M+, 0.4), 86 (31.0), 85 (37.5), 73 (46.5), 59 (100). IR: 2930 (s), 2200 (m),
2030 (m), 1240 (w), 1110 (m), 1040 (w).
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DEUTERATED 4,8-DMDS
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8-Trideuteriomethyl-4-methyldecanal (4). Epoxide 24 was cleaved with HIO4
to give the expected aldehyde 4. Yield 31.3%. Purification of a small portion of
4 by column chromatograph yielded 95.2% purity of 4 and it was used for
bioassay. 1H NMR: 9.78 (1H, t, J=1.8), 2.45-2.40 (2H, m), 1.70-1.62 (1H, m),
1.46-1.41 (2H, m), 1.36-1.20 (6H, m), 1.14-1.04 (3H, m), 0.88 (3H, d, J=6.3),
0.85 (3H, t, J=7.3). 13C NMR: 203.1 (CH), 41.7 (CH2), 37.0 (CH2), 36.8
(CH2), 34.2 (CH), 32.9 (CH), 29.4 (CH2), 28.9 (CH2), 24.4 (CH2), 19.4 (CH3),
11.4 (CH3). GC-MS: 187 (M+, 0.5), 143 (61.0), 140 (28.1), 114 (54.0), 95
(55.9), 85 (68.7), 73 (100), 60 (77.6), 57 (90.0). IR: 2910 (s), 2700 (s), 2190 (m),
2050 (m), 1730 (s).
Citronellylacetone (25). A solution of t-BuOK (10.95 g, 97.80 mmol) in t-
BuOH (70 ml) was heated and refluxed to dissolve t-BuOK and then cooled to
508C. After ethyl acetoacetate (11.65 g, 89.65 mmol) was added, the mixture
was stirred for 30 min, and 22 (17.84 g, 81.50 mmol) was added to the solution.
The mixture was stirred and refluxed for 24 h. The reacted solution was cooled,
filtered and concentrated. The residue was poured into a stirred 2N NaOH
(300 ml) solution and heated for 3 h at 50–558C. After cooling to room
temperature, the alkaline solution was acidified with acetic acid (40 ml). The
mixture was extracted with hexane (3 Â 100 ml). The organic phases were
washed with saturated NaHCO3, dried and concentrated in vacuo. The residue
was distilled to give 10.63 g of 25 (54.23 mmol, 66.5%). bp: 108–1118C/
4 mmHg. 1H NMR: 5.09 (1H, t, J=7.1), 2.40 (2H, t, J=7.6), 2.13 (3H, s), 1.96
(2H, m), 1.68 (3H, s), 1.60 (3H, s), 1.55 (2H, m), 1.41 (1H, m), 1.29 (2H, m),
1.13 (2H, m), 0.87 (3H, d, J=6.6). 13C NMR: 209.7 (C), 131.5 (C), 125.2 (CH),
44.5 (CH2), 37.3 (CH2), 36.8 (CH2), 32.6 (CH), 30.2 (CH3), 26.1 (CH3), 25.9
(CH2), 21.8 (CH2), 19.9 (CH3), 18.0 (CH3). GC-MS: 196 (M+, 4.8), 138 (6.4),
69 (78.8), 43 (100). IR: 2910 (s), 1710 (s), 1150 (m).
Ethyl 3,7,11-trimethyl-2,10-dodecadienoate (26). Compound 26 was prepared
1
by the usual Wittig–Horner reaction procedure. Yield 79.9%. H NMR: 5.66
(1H, q-like, J=1.1), 5.09 (1H, t-like, J=7.1), 4.14 (2H, q, J=7.1), 2.15 (3H, d,
J=1.2), 2.11 (2H, t-like, J=7.7), 1.95 (2H, m), 1.68 (3H, d, J=0.9), 1.60 (3H,
s), 1.48 (2H, m), 1.40 (1H, m), 1.33-1.25 (2H, m), 1.26 (3H, t, J=7.1), 1.12
(2H, m), 0.87 (3H, d, J=6.6). 13C NMR: 167.3 (C), 160.7 (C), 131.5 (C), 125.3
(CH), 115.8 (CH), 59.8 (CH2), 41.6 (CH2), 37.4 (CH2), 36.8 (CH2), 32.6 (CH),
26.1 (CH3), 25.9 (CH2), 25.2 (CH2), 19.9 (CH3), 19.1 (CH3), 18.0 (CH3), 14.7
(CH3). GC-MS: 266 (M+, 10.4), 221 (18.2), 128 (50.4), 109 (98.0), 69 (100). IR:
2910 (s), 1705 (s), 1640 (m), 1210 (s), 1130 (s).
Ethyl 3,7,11-trimethyl-1-dodecenoate (27). Selective hydrogenation of 26 was
carried out using a modified procedure described in the literature.11,12 To a
suspension of CuBr (I) (12.13 g, 84.87 mmol) in dry THF (50 ml), Red-AlTM
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(19.78 ml, 65.28 mmol; 65%) was added dropwise at À108C under N2 and the
solution was stirred for a further 30 min. A solution of 26 (2.17 g, 8.16 mmol)
in dry THF (15 ml) was added to the mixture and stirred for 1 h at À108C and
for 2 h at room temperature. After the reacted solution was cooled to 08C, ice-
cold water was added carefully. The black gum which formed was washed with
ether (4 Â 50 ml). The organic phase was filtered on a short pad of SiO2,
washed with 2N HCl, saturated NaHCO3, brine, then dried and concentrated
in vacuo. The residue was chromatographed over silica (20 g, hexa-
1
ne:ether=9:1) to give 1.57 g of 27 (5.86 mmol, 71.8%). H NMR: 5.10 (1H,
t-like, J=7.1), 4.13 (2H, q, J=7.1), 2.28 (1H, ddd, J=14.0, 6.0, 2.0), 2.09 (1H,
ddd, J=14.0, 8.0, 2.0), 2.03-1.90 (3H, m), 1.68 (3H, s), 1.60 (3H, s), 1.39 (1H,
m), 1.36-1.02 (8H, m), 1.26 (3H, t, J=7.1), 0.95 (3H, d, J=6.7), 0.86 (3H, d,
J=6.5). 13C NMR: 173.8 (C), 131.4 (C), 125.3 (CH), 60.4 (CH2), 42.3 (CH2),
37.5 (CH2), 37.5 (CH2), 37.44 (CH2), 32.8 (CH), 30.8 (CH), 26.1 (CH3), 26.0
(CH2), 24.7 (CH2), 20.1 (CH3), 19.9 (CH3), 18.0 (CH3), 14.7 (CH3). GC-MS:
268 (M+, 2.8), 222 (7.7), 180 (5.2), 125 (27.5), 69 (100). IR: 2910 (s), 1735 (s),
1170 (s), 1010 (m), 900 (m).
3,7,11-Trimethyl-10-dodecen-1-ol-1,1-d2 (28). Reduction of 27 (1.57 g,
5.86 mmol) with LiAlD4 (0.49 g, 11.72 mmol) in the usual way gave 1.09 g of
28 (4.78 mmol, 81.6%). This was purified by column chromatography (silica
1
15 g, hexane:ether = 7:3). H NMR: 5.10 (1H, t-like, J=7.1), 3.67 (trace;
0.04H, m), 1.95 (2H, m), 1.68 (3H, s), 1.64-1.54 (2H, m), 1.60 (3H, s), 1.44 (1H,
br. s), 1.42-1.00 (10H, m), 0.89 (3H, d, J=6.6), 0.86 (3H, d, J=6.6). 13C NMR:
131.4 (C), 125.5 (CH), 40.2 (CH2), 37.8 (CH2), 37.6 (CH2), 37.5 (CH2), 32.8
(CH), 29.9 (CH), 26.1 (CH3), 26.0 (CH2), 24.7 (CH2), 20.1 (CH3), 20.0 (CH3),
18.0 (CH3). GC-MS (B): 228 (M+, 11.7), 210 (5.6), 125 (53.3), 109 (26.4), 83
(46.3), 69 (100). IR: 3350 (br, s), 2160 (m), 2080 (m), 950 (m).
2,6,10-Trimethyl-2-dodecene-12,12,12-d3 (30). Tosylate 29 (2.35 g), derived
from 28, was reduced with LiAlD4 (1.00 g, 23.90 mmol) in the usual way. After
the workup, the residue was chromatographed over silica (10 g, Hexane)
yielding 0.63 g of 30 (2.96 mmol, 61.9% in 2 steps). 1H NMR: 5.10 (1H, t-like,
J=7.1), 2.03-1.90 (2H, m), 1.68 (3H, d, J=1.1), 1.60 (3H, s), 1.41 (1H, m),
1.35-1.20 (7H, m), 1.18-1.00 (4H, m), 0.86 (3H, d, J=6.6), 0.84 (3H, d, J=6.1).
13C NMR: 131.3 (C), 125.5 (CH), 37.7 (CH2), 37.5 (CH2), 37.3 (CH2), 34.8
(CH), 32.8 (CH), 29.6 (CH2), 26.1 (CH3), 26.0 (CH2), 24.8 (CH2), 20.0 (CH3),
19.6 (CH3), 18.0 (CH3). GC-MS: 213 (M+, 47.6), 111 (35.6), 69 (100). IR: 2980
(s), 2200 (w), 1450 (m).
2,3-Epoxy-2,6,10-trimethyldodecane-12,12,12-d3 (31). Compound 31 was pre-
1
pared by the same procedure described for 24. Yield 88.5%. H NMR: 2.70
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DEUTERATED 4,8-DMDS
933
(1H, t, J=6.3), 1.62-1.02 (14H, m), 1.31 (3H, s), 1.27 (3H, s), 0.88 (3H, d,
J=6.6), 0.84 (3H, d, J=6.3). 13C NMR: 65.2 (CH), 58.6 (C), 37.6 (CH2), 37.3
(CH2), 34.8 (CH), 33.9 (CH2), 33.1 (CH), 29.6 (CH2), 26.8 (CH2), 25.3 (CH3),
24.8 (CH2), 20.0 (CH3), 19.6 (CH3), 19.0 (CH3). GC-MS: 229 (M+, 5.7), 83
(72.5), 73 (87.1), 59 (100). IR: 2950 (s), 2200 (m), 2020 (w), 1100 (m), 720 (m).
4,8-Dimethyldecanal-10,10,10-d3 (5). Cleavage of 31 with HIO4, according to
the procedure used for 4, gave the desired aldehyde 5. Yield 65.3%. A small
portion was purified by column chromatography yielding 97.7% purity of 5
and it was used for bioassay. 1H NMR: 9.78 (1H, t, J=1.9), 2.45-2.40 (2H, m),
1.70-1.62 (1H, m), 1.48-1.40 (2H, m), 1.37-1.20 (6H, m), 1.17-1.02 (3H, m),
0.88 (3H, d, J=6.1), 0.84 (3H, d, J=6.5). 13C NMR: 203.4 (CH), 42.1 (CH2),
37.4 (CH2), 37.2 (CH2), 34.7 (CH), 32.7 (CH), 29.7 (CH2), 29.3 (CH2), 24.7
(CH2), 19.8 (CH3), 19.6 (CH3). GC-MS: 187 (M+, 0.7), 143 (64.0), 137 (26.2),
111 (61.1), 95 (67.0), 85 (65.0), 81 (68.9), 73 (100), 60 (56.8). IR: 2910 (s), 2700
(s), 2200 (m), 2050 (m), 1720 (s).
4,8-Dimethyldecanal (1b). 4,8-Dimethyldecanal (1b) was prepared from 27 in
the same way as described for 5 (95.2% pure), using LiAlH4 instead of
LiAlD4, and it was used for bioassay. The spectra were identical to those of 1a.
Behavioral tests
Insects
A laboratory colony of T. castaneum was used in all experiments. The beetles
were reared on a mixture of whole wheat flour and 5% brewer’s yeast at
27 Æ 18C, ca. 70% RH on a 16 h:8 h l/d cycle. New adult progenies were used
for bioassay 4 days after emergence.
Bioassay
Pheromonal activity of the synthetic compounds was estimated with a two-
hole pitfall olfactometer, which consisted of a four-compartment plastic Petri
dish (f 9 cm; Vermak, Canada) and a surface-roughened walking arena with
two holes (f 6 mm) located directly opposite from each other, 1 cm from the
edge. A filter paper disk (f 21 mm; Kiriyama, Japan) treated with the
compounds in hexane (20 ml) and a control disk treated with hexane (20 ml)
alone were placed in opposite chambers of the Petri dish after evaporation of
the solvent, and then the walking arena was loaded on. Thirty adult mixed-sex
beetles were released on the walking arena in each replicate. After the disk was
covered, the whole device was covered with a black box. The number of beetles
in each chamber were recorded after 15 min, and attraction (%) was calculated
from an equation [100 Â (TÀC)/30], where T and C are the numbers of beetles
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J. KIM ET AL.
in the treated and control chambers, respectively. Each test was repeated eight
times with new beetles. Dose-response studies of the compounds were assessed
at doses ranging from 1 to 1000 ng. The data were statistically analyzed using a
t-test.
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