First preparation of single-enantiomer juvenile hormone III acid and (R)-juvenile hormone III-d3

Article abstract of DOI:10.1080/14786411003752045

The (R)- and (S)-enantiomers of juvenile hormone (JH) III acid [(R)-2 and (S)-2] were prepared by the hydrolysis of (R)- and (S)-JH III [(R)-1 and (S)-1], respectively. Each enantiomer of 2 was purified by preparative reversed-phase high performance liquid chromatography in a single operation. (RS)-2 was methylated with CH₃I and K₂CO₃ in MeCN, yielding (RS)-1. (R)-JH III-d₃ [(R)-3], a single-enantiomer internal standard for quantification, was prepared from (R)-2 with CD₃I and K₂CO₃ in MeCN.

Full text of DOI:10.1080/14786411003752045

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First preparation of single-enantiomer
juvenile hormone III acid and (R)-
juvenile hormone III-d 3
Akio Ichikawa a , Makiko Takenaka b & Hiroshi Ono c
a Division of Insect Sciences , National Institute of Agrobiological
Sciences , Tsukuba, Japan
b Food Engineering Division , National Food Research Institute ,
Tsukuba, Japan
c Analytical Science Division , National Food Research Institute ,
Tsukuba, Japan
Published online: 19 Nov 2010.
To cite this article: Akio Ichikawa , Makiko Takenaka & Hiroshi Ono (2010) First preparation of
single-enantiomer juvenile hormone III acid and (R)-juvenile hormone III-d 3 , Natural Product
Research: Formerly Natural Product Letters, 24:19, 1800-1806, DOI: 10.1080/14786411003752045
To link to this article: http://dx.doi.org/10.1080/14786411003752045
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Natural Product Research
Vol. 24, No. 19, 20 November 2010, 1800–1806
First preparation of single-enantiomer juvenile hormone III acid
and (R)-juvenile hormone III-d3
a
Akio Ichikawa , Makiko Takenaka and Hiroshi Ono
b
c
*
aDivision of Insect Sciences, National Institute of Agrobiological Sciences, Tsukuba,
Japan; Food Engineering Division, National Food Research Institute, Tsukuba,
b
c
Japan; Analytical Science Division, National Food Research Institute,
Tsukuba, Japan
(Received 15 September 2009; final version received 10 November 2009)
The (R)- and (S)-enantiomers of juvenile hormone (JH) III acid [(R)-2 and
(S)-2] were prepared by the hydrolysis of (R)- and (S)-JH III [(R)-1 and
(S)-1], respectively. Each enantiomer of 2 was purified by preparative
reversed-phase high performance liquid chromatography in a single
operation. (RS)-2 was methylated with CH3I and K2CO3 in MeCN,
yielding (RS)-1. (R)-JH III-d3 [(R)-3], a single-enantiomer internal standard
for quantification, was prepared from (R)-2 with CD3I and K2CO3 in
MeCN.
Keywords: enantiomer; juvenile hormone; JH; JH III acid; JH III-d3
1. Introduction
Juvenile hormone (JH) III [(R)-1] was first identified from organ cultures of corpora
allata of the tobacco hornworm moth, Manduca sexta (Judy et al., 1973) (Figure 1).
The titres of JHs in haemolymph control insect development and reproduction.
In the final step of JH biosynthesis, the JH acids (Ismail, Satyanarayana, Bradfield,
Dahm, & Bhaskaran, 1998) are converted to their active forms by JH acid
methyltransferase (JHAMT) in Lepidopterans (Goodman & Granger, 2005; Shinoda
& Itoyama, 2003). Thus, JHAMT has become an important target for the
development of novel insect growth regulators. Currently, we are attempting to
supply single-enantiomer JH III acid (2) for the kinetic study of the JHAMT-
catalysed methylation. For this purpose, enantiopure (R)-2 and (S)-2 were prepared
by the hydrolysis of (R)-1 and (S)-1, respectively. The methylation of (RS)-2 to
produce (RS)-1 was accomplished with CH3I and K2CO3 in MeCN (Sekiguchi et al.,
2008). Finally, (R)-JH III-d3 [(R)-3] was also prepared from (R)-2 using CD3I for
liquid chromatography–mass spectrometry (LC–MS) quantification. JHs are fragile
biomolecules; therefore, the purification of JHs and their derivatives is a practical
problem in insect endocrinology. Here, we report the practical chemical conversion
and efficient purification of JH III (1) and JH III acid (2).
*Corresponding author. Email: ichikawa@affrc.go.jp
ISSN 1478–6419 print/ISSN 1029–2349 online
ß 2010 Taylor & Francis
DOI: 10.1080/14786411003752045
http://www.informaworld.com

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1801
Figure 1. Chemical conversions of JH III (1) and related compounds.
2. Results and discussion
First, the cis–trans isomers, (RS)-1 and (RS)-4, were separated by preparative HPLC
using a pre-packed silica gel glass column (100 mm ꢀ 22 mm i.d.; hexane/
EtOAc ¼ 9 : 1; 2 mL minꢁ1). The third operation gave geometrically pure (RS)-1.
Then, the enantioseparation of (RS)-1 was performed by normal-phase enantiose-
lective HPLC (Chiralpak IA, 250 mm ꢀ 10 mm i.d.; hexane/EtOH ¼ 98 : 2;
1.5 mL minꢁ1) (Ichikawa, Ono, Furuta, Shiotsuki, & Shinoda, 2007).

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A. Ichikawa et al.
Table 1. NMR data for JH III acid (2) and 2Z-isomer (5).
13C-NMR
Number (126 MHz)
1H-NMR
(800 MHz)
13C-NMR
(126 MHz)
1H-NMR
(500 MHz)
1
2
3
30
4
5
6
7
70
8
170.32
114.81
162.65
19.12
40.98
25.82
123.37
135.44
16.01
–
5.69 (1H, br s)
–
2.17 (3H, br d, J ¼ 1 Hz)
2.2 (2H, m)
2.2 (2H, m)
5.14 (1H, m)
–
170.47
115.54
163.11
25.64
33.49
26.62
123.91
135.10
15.88
–
5.69 (1H, br s)
–
1.93 (3H, br d, J ¼ 1 Hz)
2.65 (2H, br t, J ¼ 8 Hz)
2.18 (2H, br q, J ¼ 8 Hz)
5.20 (1H, br t, J ¼ 7 Hz)
–
1.63 (3H, br s)
2.16 (1H, m)
2.10 (1H, m)
1.63 (3H, br d, J ¼ 1 Hz)
36.33
2.2 (1H, m)
2.10 (1H, m)
1.6 (2H, m)
36.26
9
10
11
110
12
27.35
64.17
58.40
18.74
24.85
27.32
64.20
58.48
18.72
24.86
1.6 (2H, m)
2.71 (1H, t, J ¼ 6 Hz)
2.70 (1H, t, J ¼ 6 Hz)
–
–
1.26 (3H, s)
1.30 (3H, s)
1.26 (3H, s)
1.30 (3H, s)
Note: The solvent used is CDCl3.
When (RS)-1 was subjected to hydrolysis (Goodman & Adams, 1984), the
procedure was frequently accompanied by epoxide ring opening. To prevent
this, the acidification procedure was modified as follows: (1) The pH was set at
3 (ꢂ pKa ꢁ 2); (2) 0.1 mol Lꢁ1 hydrochloric acid was used instead of 1 mol Lꢁ1; and (3)
the pH was monitored with a pH indicator. In addition, trace hydrochloric acid
remaining after solvent extraction was removed with brine. With these modifications,
(RS)-2 was routinely obtained as a single major product. Previously, acid 2 had been
purified using a LiChrosorb Diol column with CHCl3/EtOH (99 : 1). Considering the
toxicity of CHCl3, the crude (RS)-2 obtained was purified by preparative
octadecylsilane (ODS) HPLC with MeCN/H2O in a single operation. The yield of
(RS)-2 was 88% in hydrolysis. Subsequently, (R)-1, (S)-1 and (RS)-4 were subjected to
hydrolysis, yielding (R)-2, (S)-2 and (RS)-5, respectively. Table 1 shows the NMR
signals of 2 and 5 assigned on the basis of the 2-D NMR spectra (i.e. DQFCOSY,
NOESY, HSQC and HMBC). JH acids have also been prepared by enzymatic
hydrolysis. It was reported that the incubation of (RS)-1 with crude haemolymph of
the adult Colorado potato beetle, Leptinotarsa decemlineata, resulted in the prefer-
ential formation of (S)-2 (up to 55.4% ee) (de Kort, Peter, & Koopmanschap, 1983).
Next, (RS)-2 was methylated with CH3I and K2CO3 in MeCN, to produce
(RS)-1. This methylation would be useful for the stereochemical analysis of natural
2. Previously, diazomethane had been used for the methylation of 2 (de Kort et al.,

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1983). However, because of its instability and toxicity, the storage and transport of
diazomethane is not advisable. Moreover, Kagei et al. (1981) reported the
1,3-dipolar additions of diazomethane for conjugated carbonyl compounds (Kagei
et al., 1981). The cis–trans isomer (RS)-5 was also methylated, giving (RS)-4.
Similarly, (RS)-2 and (R)-2 were methylated with CD3I and K2CO3 in MeCN,
yielding (RS)-3 (Ichikawa et al., 2007) and (R)-3, respectively. Considering the
interactions between JHs and proteins, the single-enantiomer (R)-3 would be
desirable over (RS)-3 as an internal standard for the LC–MS quantification of (R)-1.
Both enantiomers of JH III acid (2) were prepared by hydrolysis and purified by
preparative ODS HPLC. The NMR signals of acid 2 and its cis–trans isomer 5 were
unambiguously assigned. (RS)-2 was methylated with CH3I and K2CO3 in MeCN to
obtain (RS)-JH III [(RS)-1]. Similarly, (R)-JH III-d3 [(R)-3] was prepared from (R)-2
using CD3I in MeCN in the presence of K2CO3. To the best of our knowledge, this is
the first preparation of enantiopure (R)-2, (S)-2 and (R)-3. These procedures will
contribute to the biosynthetic and physiological studies of JHs.
3. Experimental
3.1. General
The NMR spectra were obtained with Bruker Avance 800 and Avance 500
spectrometers (Bruker BioSpin, Rheinstetten, Germany), equipped with cryoprobes.
The 1H-NMR chemical shifts were reported relative to the residual protons of CDCl3
(7.26 ppm). The 13C-NMR spectra were obtained with 1H composite pulse
decoupling. The infrared (IR) spectra were recorded as the chloroform solutions in
a potassium bromide cell with an FT-IR-8200 spectrophotometer (Shimadzu, Kyoto,
Japan). The MS data were obtained with an HP1100 LC/MSD instrument (Agilent,
Palo Alto, CA, USA) attached to a Zorbax SB-C18 column (50 mm ꢀ 2.1 mm i.d.;
Agilent) using electrospray ionisation (ESI). HPLC was performed using the
LC10AT VP systems (Shimadzu) and the following columns: a pre-packed silica gel
column (100 mm ꢀ 15 or 22 mm i.d.; Kusano, Tokyo, Japan), a pre-packed ODS
column (100 mm ꢀ 15 mm i.d.; Kusano), a silica SG80 column (250 mm ꢀ 10 or
4.6 mm i.d.; Shiseido, Tokyo, Japan), a Capcell Pak C18 MG column (150 mm ꢀ 4.6
or 3 mm i.d.; Shiseido), and a Chiralpak IA column (250 mm ꢀ 10 or 4.6 mm i.d.;
Daicel, Tokyo, Japan). Crude (RS)-1 was purchased from Sigma–Aldrich (St. Louis,
MO, USA). All procedures were conducted in semi-darkness.
3.2. Preparation of (RS)-2
A mixture of (RS)-1 (8.0 mg, 30 mmol), methanol (0.3 mL) and aqueous NaOH
solution (1 mol Lꢁ1, 0.3 mL; Wako, Osaka, Japan) was kept at 40ꢃC for 6 h (Goodman
& Adams, 1984). After dilution with 2 mL of ice water, a drop of pH indicator
(0.04%, w/v%, bromocresol purple solution; Wako) was added to the mixture.
While stirring the mixture on ice, iced hydrochloric acid (0.1 mol Lꢁ1, ca 0.26 mL;
Wako) was added dropwise until the colour of the iced solution changed from
bluish-purple to yellow. The solution was extracted four times with dichloromethane
(4 mL ꢀ 4). After each extraction, a few drops of hydrochloric acid were added to
gradually lower the pH. The organic layer was washed three times with brine and dried

1804
A. Ichikawa et al.
over anhydrous Na2SO4. After filtration, the Na2SO4 was washed with dichlor-
omethane four times. The combined solution was evaporated under reduced pressure,
yielding 7.1 mg of crude (RS)-2. The crude product was purified by preparative HPLC
in a single operation using a pre-packed ODS column (100 mm ꢀ 15 mm i.d.), which
was eluted with MeCN/H2O 65 : 35 at a flow rate of 1 mL minꢁ1 and with detection at
250 nm. The fraction containing (RS)-2 was extracted three times with dichloro-
methane. The organic layer was washed twice with brine, dried over anhydrous
Na2SO4 and evaporated under reduced pressure, giving an 88% yield of (RS)-2
(6.7 mg, 27 mmol). A trace amount of (RS)-1 was recovered. Acid (RS)-2 was kept at
ꢁ20ꢃC as an MeCN solution under argon.
3.2.1. (2E,6E)-(RS)-10,11-Epoxy-3,7,11-trimethyldodeca-2,6-dienoic acid
[(RS)-2]
1
For H-NMR (800 MHz, CDCl3) and 13C-NMR (126 MHz, CDCl3), see Table 1;
LC–MS [ESI, negative ion mode, (MeCN, 0.1% w/v NH4OAc; Sigma–Aldrich)/
H2O ¼ 7 : 3]: 251 ([M ꢁ H]ꢁ, 100%).
3.3. Preparation of (R)-2
(R)-1 (5.4 mg, 20 mmol) was converted to (R)-2 (4.0 mg, 16 mmol) by incubation
for 7 h with 1 mol Lꢁ1 aqueous NaOH solution (0.3 mL) and methanol (0.3 mL) in a
78% yield (see Section 3.2 for details). The (R)-2 was enantiopure (499% ee)
(Section 3.9).
3.3.1. (2E,6E)-(R)-10,11-Epoxy-3,7,11-trimethyldodeca-2,6-dienoic acid [(R)-2]
LC–MS [ESI, negative ion mode, (MeCN, 0.1% w/v NH4OAc)/H2O ¼ 7 : 3]: 251
([M ꢁ H]ꢁ, 100%).
3.4. Preparation of (S)-2
(S)-1 (4.8 mg, 18 mmol, 499% ee) was converted to (S)-2 (3.0 mg, 12 mmol) by
incubation for 6.3 h with 1 mol Lꢁ1 aqueous NaOH solution (0.3 mL) and methanol
(0.3 mL) in a 66% yield (see Section 3.2 for details).
3.4.1. (2E,6E)-(S)-10,11-Epoxy-3,7,11-trimethyldodeca-2,6-dienoic acid [(S)-2]
LC–MS [ESI, negative ion mode, (MeCN, 0.1% w/v NH4OAc)/H2O ¼ 7 : 3]: 251
([M ꢁ H]ꢁ, 100%).
3.5. Preparation of (RS)-5
(RS)-4 (2.4 mg, 9 mmol) was converted to (RS)-5 (1.1 mg, 4 mmol) by incubation
for 4 h with 1 mol Lꢁ1 aqueous NaOH solution (0.4 mL) and methanol (0.4 mL) in
48% yield (see Section 3.2 for details).

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3.5.1. (2Z,6E)-(RS)-10,11-Epoxy-3,7,11-trimethyldodeca-2,6-dienoic acid
[(RS)-5]
1
For H-NMR (500 MHz, CDCl3) and 13C-NMR (126 MHz, CDCl3), see Table 1;
LC–MS [ESI, negative ion mode, (MeCN, 0.1% w/v NH4OAc)/H2O ¼ 7 : 3]: 251
([M ꢁ H]ꢁ, 100%); IR (KBr, CHCl3): 2964 cmꢁ1, 2928, 1693, 1647, 1456, 1379, 1117
and 866.
3.6. Methylation of (RS)-2
A mixture of (RS)-2 (ca 1 mg, ca 4 mmol), K2CO3 (151 mg, 1.09 mmol), CH3I
(0.5 mL, 8 mmol, use with caution) and MeCN (1 mL) was stirred at room
temperature for 3 h. The mixture was subjected to chromatography using a short
column of silica gel (Wakogel C-200, CH2Cl2). The eluent was evaporated under
reduced pressure, and the mixture obtained was chromatographed on Wakogel
C-200 eluting successively with CH2Cl2 and hexane : EtOAc (7 : 3). The
hexane : EtOAc solution was evaporated under reduced pressure. The crude product
was purified by preparative HPLC (column, pre-packed ODS; eluent, MeCN : H2O;
flow rate, 1 mL minꢁ1; detection, 240 nm). This procedure yielded 0.9 mg of (RS)-1
(ca 3 mmol). The structure of (RS)-1 was confirmed by 1H-NMR (800 MHz, CDCl3),
13C-NMR (201 MHz, CDCl3) and LC–MS (m/z 289 [M þ Na]þ).
3.7. Methylation of (RS)-5
(RS)-5 (1.0 mg, ca 4 mmol) was converted to (RS)-4 (0.7 mg, ca 3 mmol) by stirring for
2.5 h with K2CO3 (149 mg, 1.08 mmol), CH3I (0.5 mL, 8 mmol) and MeCN (1 mL)
1
(see Section 3.6 for details). The structure of (RS)-4 was confirmed by H-NMR
(500 MHz, CDCl3) and 13C-NMR (126 MHz, CDCl3) (see Ichikawa et al. (2007) for
spectral data of (RS)-4).
3.8. Preparation of (RS)-3
(RS)-2 (ca 1 mg, ca 4 mmol) was converted to (RS)-3 (0.6 mg, ca 2 mmol) by stirring
for 4 h with K2CO3 (242 mg, 1.75 mmol), CD3I (0.5 mL, 8 mmol; 99.5þ at.% D;
Isotec, OH, USA; use with caution) and MeCN (0.9 mL). The reaction time was 4 h
1
(see Section 3.6 for details). The structure of (RS)-3 was confirmed by H-NMR
(800 MHz, CDCl3), 13C-NMR (201 MHz, CDCl3) and LC–MS (m/z 292 ([M þ Na]þ)
(see Ichikawa et al. (2007) for spectral data of (RS)-3).
3.9. Preparation of (R)-3
(R)-2 (0.4 mg, ca 2 mmol) was converted to (R)-3 (0.3 mg, ca 1 mmol; 499%
ee, enantioselective HPLC) by stirring for 3.5 h with K2CO3 (250 mg, 1.81 mmol),
CD3I (0.5 mL, 8 mmol) and MeCN (0.75 mL) (see Section 3.6 for details).

1806
A. Ichikawa et al.
3.9.1. [2H3]Methyl (2E,6E)-(R)-10,11-epoxy-3,7,11-trimethyldodeca-2, 6-
dienoate [(R)-3]
1H-NMR (500 MHz, CDCl3): see Ichikawa et al. (2007) for the spectral data of
(RS )-3; 13C-NMR (126 MHz, CDCl3): see Ichikawa et al. (2007); LC–MS [ESI,
positive ion mode, (MeCN, 0.1% w/v NH4OAc)/H2O ¼ 85 : 15]: m/z 328
([M þ NH4 þ MeCN]þ, 40), 292 ([M þ Na]þ, 10), 270 ([M þ H]þ, 47), 235
([M ꢁ OCD3]þ, 100).
Acknowledgements
The authors are grateful to Ms I. Maeda (NFRI) for the NMR measurements. A.I. is grateful
to Dr T. Shinoda (NIAS), Dr T. Shiotsuki (NIAS) and Dr K. Furuta (NIAS) for valuable
discussions, LC–MS measurements and the use of Chiralpak columns.
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