The absolute configurations of hydroxy fatty acids from the royal jelly of honeybees (Apis mellifera)

Article abstract of DOI:10.1007/s11745-010-3497-x

9-Hydroxy-2E-decenoic acid (9-HDA) is a precursor of the queen-produced substance, 9-oxo-2E-decenoic acid (9-ODA), which has various important functions and roles for caste maintenance in honeybee colonies (Apis mellifera). 9-HDA in royal jelly is considered to be a metabolite of 9-ODA produced by worker bees, and it is fed back to the queen who then transforms it into 9-ODA. Recently we found that 9-HDA is present in royal jelly as a mixture of optical isomers (R:S, 2:1). The finding leads us to suspect that chiral fatty acids in royal jelly are precursors of semiochemicals. Rather than looking for semiochemicals in the mandibular glands of the queen bee, this study involves the search for precursors of pheromones from large quantities of royal jelly. Seven chiral hydroxy fatty acids, 9,10-dihydroxy-2E-decenoic, 4,10-dihydroxy-2E-decenoic, 4,9-dihydroxy-2E-decenoic, 3-hydroxydecanoic, 3,9-dihydroxydecanoic, 3,11-dihydroxydodecanoic, and 3,10-dihydroxydecanoic acids were isolated. The absolute configurations of these acids were determined using the modified Mosher's method, and it was revealed that, similar to 9-HDA, five acids are present in royal jelly as mixtures of optical isomers. AOCS 2010.

Full text of DOI:10.1007/s11745-010-3497-x

Lipids (2011) 46:263–270
DOI 10.1007/s11745-010-3497-x
ORIGINAL ARTICLE
The Absolute Configurations of Hydroxy Fatty Acids
from the Royal Jelly of Honeybees (Apis mellifera)
•
Tetsuya Kodai Takafumi Nakatani
•
Naoki Noda
Received: 20 June 2010 / Accepted: 22 October 2010 / Published online: 17 November 2010
Ó AOCS 2010
Abstract 9-Hydroxy-2E-decenoic acid (9-HDA) is a
precursor of the queen-produced substance, 9-oxo-2E-
decenoic acid (9-ODA), which has various important
functions and roles for caste maintenance in honeybee
colonies (Apis mellifera). 9-HDA in royal jelly is consid-
ered to be a metabolite of 9-ODA produced by worker
bees, and it is fed back to the queen who then transforms it
into 9-ODA. Recently we found that 9-HDA is present in
royal jelly as a mixture of optical isomers (R:S, 2:1). The
finding leads us to suspect that chiral fatty acids in royal
jelly are precursors of semiochemicals. Rather than looking
for semiochemicals in the mandibular glands of the queen
bee, this study involves the search for precursors of
pheromones from large quantities of royal jelly. Seven
chiral hydroxy fatty acids, 9,10-dihydroxy-2E-decenoic,
4,10-dihydroxy-2E-decenoic, 4,9-dihydroxy-2E-decenoic,
3-hydroxydecanoic, 3,9-dihydroxydecanoic, 3,11-dihydrox-
ydodecanoic, and 3,10-dihydroxydecanoic acids were iso-
lated. The absolute configurations of these acids were
determined using the modified Mosher’s method, and it was
revealed that, similar to 9-HDA, five acids are present in
royal jelly as mixtures of optical isomers.
HMBC Two-dimensional heteronuclear multiple-bond
connectivity
MTPA 2-Methoxy-2-(trifluoromethyl)phenylacetic acid
TMS
Tetramethylsilane
Introduction
The so-called ‘‘queen substance,’’ 9-oxo-2-decenoic acid
(9-ODA), was isolated from the honeybee queen mandib-
ular glands by Butler et al. [1]. This compound is a
well-known semiochemical which has various important
functions, such as queen recognition and inhibition of
ovary development in worker bees for caste maintenance in
honeybee colonies [2].
In 1965, Johnston and co-workers reported that 9-ODA
was rapidly metabolized to 9-hydroxy-2E-decenoic (9-HDA)
or 9-hydroxydecanoic acids [3]. They further postulated a
‘‘pheromone cycle,’’ in which worker bees would receive
9-ODA from the queen and convert it into 9-HDA. The
metabolite would then be transported to the worker man-
dibular glands where it would be passed back to the queen
through royal jelly. The queen would then convert it back
into the active oxo-form, 9-ODA. In fact, significant
amounts of 9-HDA have been found in royal jelly, but
9-ODA has not yet been detected. Furthermore, our pre-
vious study revealed that 9-HDA is present as a mixture of
optical isomers (R:S, 2:1) in royal jelly [4]. This interesting
finding is important with regard to searching for precursors
of unknown semiochemicals in royal jelly.
Keywords Absolute configuration Á Apis mellifera Á
Honeybees Á Hydroxy fatty acid Á Modified Mosher’s
method Á Royal jelly
Abbreviations
1
COSY Two-dimensional H-1H shift correlation
T. Kodai Á T. Nakatani Á N. Noda (&)
The present study was undertaken to examine the con-
stituents of royal jelly in the hope of discovering precursors
of unknown queen substances. We conducted a more
Faculty of Pharmaceutical Sciences, Setsunan University,
45-1, Nagaotoge-cho, Hirakata, Osaka 573-0101, Japan
e-mail: noda@pharm.setsunan.ac.jp
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Lipids (2011) 46:263–270
detailed survey of the components, particularly the fatty
acid fraction, and isolated seven chiral fatty acids in the
pure state. The absolute configurations of the chiral acids
were determined using the modified Mosher’s method
[5–7]. Similar to 9-HDA found in royal jelly, five acids are
present as mixtures of optical isomers.
to give four fractions, fr. 1 (30.0 g), fr. 2 (1.8 g), fr. 3
(0.9 g), and fr. 4 (0.5 g). Fr. 2 was chromatographed on
Sephadex LH-20 (methanol) and Cosmosil 75C18-OPN
(methanol/water, 1:1 vol/vol ? methanol) columns to give
three fractions, fr. 5 (115 mg), fr. 6 (314 mg), and fr. 7
(340 mg). Fr. 5 was subjected to preparative HPLC on a
reversed-phase column (Inertsil ODS-3) using acetonitrile/
water/trifluoroacetic acid, 30:70:0.25 by vol to give com-
pound 1 (7.6 mg). Fr. 3 was chromatographed on Sephadex
LH-20 (methanol) and Cosmosil 75C18-OPN (methanol/
water, 3:7 ? 1:1 by vol ? methanol) columns to give
four fractions, fr. 8–11. Fr. 8 (31.2 mg) was subjected to
preparative HPLC on a reversed-phase column (Mightysil
RP-18 GP) using a mobile phase (methanol/water/tri-
fluoroacetic acid, 25:75:0.5 by vol) to give compounds 2
(11.7 mg) and 3 (1.3 mg). Fr. 9 (499 mg) was separated by
an Inertsil ODS-3 column using a mobile phase (methanol/
water/trifluoroacetic acid, 25:75:0.5 by vol) to give com-
pounds 5 (5.4 mg) and 7 (118 mg). Fr. 10 was subjected to
HPLC on an Inertsil ODS-3 column using a solvent
(methanol/water/trifluoroacetic acid, 37:63:0.5 by vol) to
give compounds 4 (3.4 mg) and 6 (4.2 mg). Compound 7
was identified as 3R,10-dihydroxydecanoic acid by com-
parison of the NMR and FABMS spectroscopic data with
those of an authentic sample [4].
Experimental Procedure
Materials
Lyophilized royal jelly powder was supplied by Yamada
Apiculture Center Inc. (Okayama, Japan). A voucher
specimen was deposited in the Faculty of Pharmaceutical
Sciences, Setsunan University.
Instrumentation
Optical rotations were measured at 25 °C with a JASCO
DIP-140 polarimeter (JASCO, Tokyo, Japan). 1H- and 13C-
NMR spectra were recorded on JEOL JMN GX400 or ECA
600SN spectrometers (JEOL, Tokyo, Japan), using tetra-
methylsilane (TMS) as an internal reference. All samples
were measured at a probe temperature of 35 °C. The two-
dimensional heteronuclear multiple bond connectivity
1: High resolution negative ion FABMS m/z: 201.1120
[M-H]- (calcd. for C10H17O4: 201.1127). [a]D -5.5° at
0.8 g/100 mL in methanol. 2: High resolution negative ion
FABMS m/z: 201.1118 [M-H]- (calcd. for C10H17O4:
201.1127). 3: High resolution negative ion FABMS m/z:
201.1117 [M-H]- (calcd. for C10H17O4: 201.1127). [a]D
-3.1° at 0.2 g/100 mL in methanol. 4: High resolution
negative ion FABMS m/z: 187.1339 [M-H]- (calcd. for
C10H19O3: 187.1334). [a]D ?1.1° at 0.3 g/100 mL in
methanol. 5: High resolution negative ion FABMS m/z:
203.1291 [M-H]- (calcd. for C10H19O4: 203.1283). [a]D
?3.3° at 0.5 g/100 mL in methanol. 6: High resolution
negative ion FABMS m/z: 231.1588 [M-H]- (calcd. for
C12H23O4: 231.1596). [a]D ?3.4° at 0.4 g/100 mL in
methanol. 1H- and 13C-NMR spectroscopic data of 1–6 are
shown in Tables 1 and 2.
(HMBC) spectrum was recorded at 600 MHz with 64
2,3
scans (
J
= 8 Hz). Fast atom bombardment mass
CH
spectrometry (FAB-MS) including high-resolution mass
was recorded on a JEOL JMS-700T spectrometer (JEOL).
(Accelerating voltage, 5 kV; matrix, glycerin or trietha-
nolamine; collision gas, He). Column chromatography was
carried out on Diaion HP-20 (Mitsubishi Chemical Co.,
Tokyo, Japan), Sephadex LH-20 (Amersham Pharmacia
Biotech AB, Uppsala, Sweden), silica gel (Kiesergel 60,
Merck), and Cosmosil 75C18-OPN (Nacalai Tesque, Inc.,
Tokyo, Japan). Preparative HPLC was conducted over
Mightysil RP-18 GP (5 lm, 4.6 9 250 mm, Kanto
Chemical Co. Inc. Tokyo, Japan) and Inertsil ODS-3
(4 lm, 4.6 9 100 mm, GL Sciences Inc., Tokyo, Japan)
columns with a JASCO 980-PU (JASCO, Tokyo, Japan).
The elution profile was monitored by a refractive index
detector, RI504R (GL Sciences Inc., Tokyo, Japan).
Preparation of 2-Methoxy-2-
(trifluoromethyl)phenylacetyl (MTPA) Ester
Derivatives
Isolation of Compounds, 1–7
Lyophilized royal jelly powder (1 kg) was percolated with
chloroform to remove a major component, 10-hydroxy-2E-
decenoic acid. The residue was extracted with chloroform/
acetone (1:1, vol/vol), and the solvent was removed in
vacuo to give an extract (36.1 g). This was chromato-
graphed on silica gel and eluted successively with chloro-
form/methanol/water (7:2:0.2 ? 7:3:0.5 ? 6:4:1 by vol)
Compounds 1 through 6 (ca. 1 mg) were each treated with
diazomethane in diethyl ether. (-)- and (?)-MTPA Chlo-
rides (Tokyo Kasei Kogyo Co. Ltd., 20 mg each) were
separately added to a solution of the above product in
carbon tetrachloride (0.3 mL) and pyridine (1.0 mL), and
the mixture was stirred at room temperature for 12 h. After
removal of the solvent under a nitrogen stream, the reaction
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Lipids (2011) 46:263–270
265
Table 2 13C-NMR chemical shifts of 1–3 and 5–6 (CD3OD)
1
2
3
5
6
C-1
171.4
123.7
149.5
33.0
73.1
67.3
–
170.0
120.9
152.9
71.6
170.6
121.7
152.2
71.6
68.6
23.5
–
174.1
43.3
69.4
173.8
43.3
69.3
C-2
C-3
C-4
C-9
68.6
23.5
–
C-10
C-11
C-12
63.0
–
68.5
23.5
–
–
–
–
d in ppm from TMS
mixture was subjected to silica gel column chromatography
(chloroform) to give a MTPA derivative.
Bis[(-)-MTPA] Ester of Methyl Ester of 1 (1a) 1H-NMR
(CDCl3, 600 MHz) d: 1.18–1.17 (8H, m, CH2 9 4), 2.12
(0.5H, dt, J = 7.2, 7.2 Hz, H2-4 of S-form), 2.16 (1.5H, dt,
J = 7.2, 7.2 Hz, H2-4 of R-form), 3.42 (2.25H, m, OCH3
of R-form), 3.44 (2.25H, m, OCH3 of R-form), 3.48
(0.75H, m, OCH3 of S-form), 3.49 (0.75H, m, OCH3 of
S-form), 3.73 (3H, s, CO2CH3), 4.27 (0.75H, dd, J = 4.8,
12.0 Hz, Ha-10 of R-form), 4.30 (0.25H, dd, J =
6.0, 12.0 Hz, Ha-10 of S-form), 4.54 (0.75H, dd, J = 3.0,
12.0 Hz, Hb-10 of R-form), 4.62 (0.25H, dd, J = 3.0,
12.0 Hz, Hb-10 of S-form), 5.28–5.32 (1H, m, H-9), 5.79
(0.25H, d, J = 15.6 Hz, H-2 of S-form), 5.80 (0.75H, d,
J = 15.6 Hz, H-2 of R-form), 6.92 (0.25H, dt, J = 15.6,
7.2 Hz, H-3 of S-form), 6.93 (0.75H, dt, J = 15.6, 7.2 Hz,
H-3 of R-form), 7.33–7.40 (6H, m, H-ph), 7.43–7.50 (4H,
m, H-ph).
Bis[(-)-MTPA] Ester of Methyl Ester of 2 (2a) 1H-NMR
(CDCl3, 400 MHz) d: 1.22–1.72 (10H, m, CH2 9 5),
3.53–3.55 (6H, m, OCH3 9 2), 3.74 (1.5H, s, CO2CH3 of
R-form), 3.76 (1.5H, s, CO2CH3 of S-form), 4.24–4.34
(2H, m, H2-10), 5.57–5.60 (1H, m, H-4), 5.86 (0.5H, d,
J = 15.6 Hz, H-2 of R-form), 5.98 (0.5H, d, J = 15.6 Hz,
H-2 of S-form), 6.78 (0.5H, dd, J = 6.0, 15.6 Hz, H-3 of
R-form), 6.84 (0.5H, dd, J = 6.0, 15.6 Hz, H-3 of S-form),
7.38–7.42 (6H, m, H-ph), 7.49–7.52 (4H, m, H-ph).
Bis[(-)-MTPA] Ester of Methyl Ester of 3 (3a) 1H-NMR
(CDCl3, 400 MHz) d: 0.86-1.61 (11H, m, H3-10 and
CH2 9 4), 3.52–3.57 (6H, m, OCH3 9 2), 3.74–3.76 (3H,
m, CO2CH3), 5.04–5.13 (1H, m, H-9), 5.51–5.60 (1H, m,
H-4), 5.85 (0.29H, d, J = 15.6 Hz, H-2 of 4R9R-form),
5.86 (0.21H, d, J = 15.6 Hz, H-2 of 4R9S-form), 5.96
(0.37H, d, J = 15.6 Hz, H-2 of 4S9R-form), 5.98 (0.13H,
d, J = 15.6 Hz, H-2 of 4S9S-form), 6.76 (0.29H, dd, J =
5.6, 15.6 Hz, H-3 of 4R9R-form), 6.78 (0.21H, dd, J = 5.6,
15.6 Hz, H-3 of 4R9S-form), 6.81 (0.37H, dd, J = 5.6,
123

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Lipids (2011) 46:263–270
15.6 Hz, H-3 of 4S9R-form), 6.83 (0.13H, dd, J = 5.6,
15.6 Hz, H-3 of 4S9S-form), 7.39–7.43 (6H, m, H-ph),
7.50–7.53 (4H, m, H-ph).
dd, J = 4.8, 15.6 Hz, Ha-2), 2.70 (1H, dd, J = 8.4,
15.6 Hz, Hb-2), 3.54 (3H, m, OCH3), 3.57 (3H, m, OCH3),
3.66 (3H, s, CO2CH3), 5.11–5.17 (1H, m, H-11), 5.44–5.49
(1H, m, H-3), 7.38–7.40 (6H, m, H-ph), 7.53–7.54 (4H, m,
H-ph).
(-)-MTPA Ester of Methyl Ester of 4 (4a) 1H-NMR
(CDCl3, 400 MHz) d: 0.88 (3H, t, J = 7.2 Hz, H3-10),
1.18–1.58 (12H, m, CH2 9 6), 2.58 (1H, dd, J = 4.8,
16.0 Hz, Ha-2), 2.65 (1H, dd, J = 8.0, 16.0 Hz, Hb-2),
3.56 (3H, m, OCH3), 3.59 (3H, s, CO2CH3), 5.48 (1H, m,
H-3), 7.37–7.43 (3H, m, H-ph), 7.52–7.54 (2H, m, H-ph).
The ratio of R to S was determined by the intensity of
the signals arising from both enantiomers.
Results
(?)-MTPA Ester of Methyl Ester of 4 (4b) 1H-NMR
(CDCl3, 400 MHz) d: 0.87 (3H, t, J = 7.2 Hz, H3-10),
1.20–1.67 (12H, m, CH2 9 6), 2.61 (1H, dd, J = 4.8,
16.0 Hz, Ha-2), 2.70 (1H, dd, J = 8.0, 16.0 Hz, Hb-2),
3.55 (3H, m, OCH3), 3.66 (3H, s, CO2CH3), 5.48 (1H, m,
H-3), 7.37–7.41 (3H, m, H-ph), 7.52–7.54 (2H, m, H-ph).
The total lipid fraction obtained from lyophilized royal
jelly powder (1 kg) was separated repeatedly by silica gel
column chromatography with various solvents to give the
crude fatty acid fraction. This fraction was further sepa-
rated with HPLC, and seven compounds 1–7 were isolated
in pure form (see ‘‘Experimental Procedure’’).
Bis[(-)-MTPA] Ester of Methyl Ester of 5 (5a) 1H-NMR
(CDCl3, 600 MHz) d: 1.25 (2H, d, J = 6.6 Hz, H3-10 of
3R9S-form), 1.33 (1H, d, J = 6.6 Hz, H3-10 of 3R9R-
form), 1.31–1.66 (10H, m, CH2 9 5), 2.55 (0.33H, dd,
J = 4.8, 16.2 Hz, Ha-2 of 3R9R-form), 2.56 (0.67H, dd, J =
4.8, 16.2 Hz, Ha-2 of 3R9S-form), 2.63 (0.33H, dd,
J = 7.8, 16.2 Hz, Hb-2 of 3R9R-form), 2.64 (0.67H, dd,
J = 7.8, 16.2 Hz, Hb-2 of 3R9S-form), 3.51–3.56 (6H, m,
OCH3 9 2), 3.59 (3H, m, CO2CH3), 5.10–5.14 (1H, m,
H-9), 5.43–5.48 (1H, m, H-3), 7.37–7.42 (6H, m, H-ph),
7.50–7.53 (4H, m, H-ph).
The molecular formula of 1, C10H18O4, was established
1
using high resolution FAB-MS. The H-NMR spectrum of
1 showed trans olefinic (dH 5.80 and 6.89), oxymethine (dH
3.57), and allylic (dH 2.21) groups together with non-
equivalent methylene signals. The 13C-NMR spectrum
showed one oxy methine (dC 73.1) and one oxy methylene
(dC 67.3) units together with carboxyl (dC 171.4) and
olefinic (dC 123.7 and 149.5) carbons. The COSY and
HMBC spectra showed significant correlation peaks
between H2-10/H-9, H-2/C-1, and H-3/C-1, respectively.
These findings revealed that 1 was 9,10-dihydroxy-2E-
decenoic acid. The absolute configuration of C-9 was
determined using the modified Mosher’s method.1
Bis[(?)-MTPA] Ester of Methyl Ester of 5 (5b) 1H-NMR
(CDCl3, 600 MHz) d: 1.24 (1H, d, J = 6.6 Hz, H3-10 of
3R9R-form), 1.32 (2H, d, J = 6.6 Hz, H3-10 of 3R9S-
form), 1.11–1.65 (10H, m, CH2 9 5), 2.58 (0.67H, dd,
J = 4.8, 16.2 Hz, Ha-2 of 3R9S-form), 2.59 (0.33H, dd,
J = 4.8, 16.2 Hz, Ha-2 of 3R9R-form), 2.68 (0.67H, dd, J =
7.8, 16.2 Hz, Hb-2 of 3R9S-form), 2.69 (0.33H, dd,
J = 7.8, 16.2 Hz, Hb-2 of 3R9R-form), 3.53–3.56 (6H, m,
OCH3 9 2), 3.66 (3H, m, CO2CH3), 5.08–5.13 (1H, m,
H-9), 5.42–5.48 (1H, m, H-3), 7.36–7.40 (6H, m, H-ph),
7.51–7.54 (4H, m, H-ph).
Methylation of 1 with diazomethane, followed by
treatment with (-)-MTPA chloride gave the (-)-MTPA
ester (1a). The 1H-NMR spectrum of 1a showed two sets of
signals in a ratio of ca 3:1 due to optical isomers. The Dd
values [d (-)-MTPA ester – d (?)-MTPA ester] of the
major characteristic H-2 and H-3 protons were ?0.011 and
?0.013 ppm, respectively, whereas those of Ha-10 and
Hb-10 were -0.025 and -0.076 ppm, respectively, indi-
cating the 9R configuration. Hence, 1 was revealed to be a
mixture of 9R,10- and 9S,10-dihydroxy-2E-decenoic acids
in a ratio of ca 3:1 (Fig. 1).
The high resolution FAB-MS of 2 showed an [M-H]-
ion peak at m/z 201.1118, consistent with the same
molecular formula, C10H18O4, as that of 1, and indicating
that 2 is an isomer of 1. The 1H-NMR spectrum of 2
showed similar trans olefinic (dH 5.97 and 6.92) signals
due to protons H-2 and H-3, and equivalent H2-10 (dH
Bis[(-)-MTPA] Ester of Methyl Ester of 6 (6a) 1H-NMR
(CDCl3, 600 MHz) d: 1.18–1.68 (14H, m, CH2 9 7), 1.25
(2.25H, d, J = 6.0 Hz, H3-12 of 3R11S-form), 1.33
(0.75H, d, J = 6.0 Hz, H3-12 of 3R11R-form), 2.57 (1H,
dd, J = 4.8, 15.6 Hz, Ha-2), 2.64 (1H, dd, J = 7.8,
15.6 Hz, Hb-2), 3.52 (3H, m, OCH3), 3.54 (3H, m, OCH3),
3.59 (3H, s, CO2CH3), 5.11–5.16 (1H, m, H-11), 5.45–5.49
(1H, m, H-3), 7.38–7.41 (6H, m, H-ph), 7.52–7.53 (4H, m,
H-ph).
1
Bis[(?)-MTPA] Ester of Methyl Ester of 6 (6b) 1H-NMR
(CDCl3, 600 MHz) dH: 1.38–1.65 (14H, m, CH2 9 7), 1.25
(0.75H, d, J = 6.0 Hz, H3-12 of 3R,11R-form), 1.33
(2.25H, d, J = 6.0 Hz, H3-12 of 3R11S-form), 2.61 (1H,
To establish whether the modified Mosher’s method is applicable to
compounds possessing a 1,2-diol, we firstly examined this method
using 3-(octadecyloxy)-1,2S-propanediol. The Dd values of the
corresponding (-)- and (?)-MTPA esters were consistent with those
predicted for S configuration.
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Lipids (2011) 46:263–270
267
Fig. 1 Structures of chiral fatty
acids 1–6
OH
9
O
O
O
O
O
R:S 1:1
1
3
5
4
HO
HO
2
4
6
10
OH
OH
OH
OH
10
R:S 3:1
OH
OH
OH
R:S 1:1
4
9
OH
OH
3R
R:S 2:1
OH
OH
OH
OH
OH
O
9
11
3R
3R
R:S 1:2
R:S 1:3
Fig. 2 1H-NMR spectrum of
H-2 of 3a and anisotropic
effects of two MTPA groups
OMTPA
MTPAO
H-2
MeO
COOMe
MeO
H3C
COOMe
4R
9R
shielding
(strong)
shielding
(weak)
F3C
F3C
O
O
H
H
OMTPA
MTPAO
MeO
MeO
MeOOC
CH3
MeOOC
4S
9S
deshielding
(strong)
deshielding
(weak)
F3C
F3C
O
O
H
H
4S,9R
4R,9R
4R,9S
4S,9S
PPM
6.00
5.95
5.90
5.85
5.80
3.54) signals together with an oxy methine (dH 4.22) group
coupled with H-3. These findings revealed that 2 was a
positional isomer of 1; that is, 2 differed in the position of
the secondary hydroxyl group placed at C-4 instead of C-9
as in 1, and hence the structure was determined to be 4,
10-dihydroxy-2E-decenoic acid. Methylation of 2 with
diazomethane followed by esterification with (-)-MTPA
chloride gave the (-)-MTPA ester (2a). The 1H-NMR
spectrum of 2a showed, similar to that of 1a, two sets of
signals assignable to optical isomers, but, in a ratio of ca.
1:1. From these findings, it was determined that 2 was a
mixture of 4R,10- and 4S,10-dihydroxy-2E-decenoic acids,
in a ratio of ca. 1:1 (Fig. 1).
Compound 3 (C10H18O4) was found, using two dimen-
1
sional H-NMR (COSY and HMBC) spectroscopic analy-
ses, to be another isomer, 4,9-dihydroxy-2E-decenoic acid.
The 1H-NMR spectrum of (-)-MTPA ester (3a) showed, in
contrast to those of 1a and 2a, four sets each of signals due
to protons H-2 and H-3, indicating that 3a is a mixture of
four isomers (4R9S, 4S9R, 4R9R, and 4S9S). As can be
seen in Fig. 2, the olefinic signal for proton (H-2) of the
4R9R isomer should appear at the highest field because of
the anisotropic effect of the two phenyl groups of the
MTPA esters (strong shielding by the near 4R-MTPA?
weak shielding by the far 9R-MTPA). The signal observed
at the second highest field could be assigned as proton H-2
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268
Lipids (2011) 46:263–270
Fig. 3 1H-NMR spectrum of 5a
3R,9S
0.08 ppm
0.01 ppm
3R,9S
3R,9R
3R,9R
PPM
PPM
1.325
1.300
1.275
1.250
2.600
2.575
2.550
2.525
Ha-2
H3-10
MTPAO
MTPAO
O
R/S
OMe
Ha-2
H -10
3
strong effect
weak effect
of the 4R9S isomer (strong shielding by the near 4R-
MTPA ? weak deshielding by the far 9S-MTPA). Again,
employing the modified Mosher’s method as described
above, olefinic signals due to the four isomers were suc-
cessively discriminated, and thus 3 was revealed to be a
mixture of 4S9R-, 4R9R-, 4R9S-, and 4S9S-dihydroxy-2E-
decenoic acids, in a ratio of ca. 9:7:5:3. On comparison
with those of 2a, the Dd values for protons H-2 and H-3
were consistent with the assignment of configurations
(Fig. 4).
in contrast to that of 3a, showed two signals for protons
H2-2, and accordingly 5 was regarded as being a mixture of
enantio- and/or diastereoisomers. We then prepared the
(?)-MTPA ester (5b). If compound 5 was a mixture of
enantiomers [(3R9R and 3S9S) or (3R9S and 3S9R)], the
chemical shifts of all signals arising from the (?)- and (-)-
MTPA esters would reverse each other, and similar to those
of 2a, the Dd values would have the opposite sign (Fig. 4).
However, the signals in the spectra of 5a and 5b gave
different chemical shifts. Based on these findings, com-
pound 5 was revealed to be a mixture of diastereomers. The
anisotropic effect of the phenyl group of the MTPA ester
decreases with increasing numbers of methylene groups
Analysis of the NMR and FAB-MS spectra of 4
(C10H20O3) revealed the structure to be 3-hydroxydecanoic
acid. The 1H-NMR spectrum of (-)-MTPA ester (4a)
showed, unlike those of 1a–3a, signals due to one optical
isomer. We then prepared the (?)-MTPA ester (4b). The
Dd value for protons H2-2 showed that the configuration at
C-3 was R (Fig. 4).
1
between it and the chiral center. In the H-NMR spectrum
of 5a, the difference in the chemical shifts for the Ha-2
group (0.01 ppm) was less than that for the H3-10 group
(0.08 ppm) (Fig. 3). Therefore it was determined that the
C-9 position has two (R and S) configurations, while the
C-3 carbon has only one. Since the Dd values for the H2-2
protons in both the (?)- and (-)-MTPA esters showed the
same minus sign, the configuration of the C-3 position
could be assigned as R. On the basis of the results obtained
above, compound 5 was found to be a mixture of 3R9R-
and 3R9S-dihydroxydecanoic acids, in a ratio of ca. 1:2.
1
The H-NMR spectrum of 5 (C10H20O4) showed two
oxy methine signals (d 3.71 and 3.97), and similar to that of
4, no signals due to olefinic protons. One oxy methine was
coupled with the terminal methyl, H3-10, and the other was
coupled with the nonequivalent protons, H2-2. From this
information, it was clear that the two hydroxyl groups were
located at the C-3 and C-9 positions, and hence the struc-
ture of 5 was defined as 3,9-dihydroxydecanoic acid. To
clarify the absolute configuration, 5 was converted into the
1
The H-NMR spectrum of 6 was quite similar to that
of 5, including signals due to two methines and one
terminal methyl group. Its negative ion FAB-MS showed
1
(-)-MTPA derivative (5a). The H-NMR spectrum of 5a,
123

Lipids (2011) 46:263–270
269
1
9S
MTPAO
Fig. 4 Dd values of H-NMR
chemical shift difference (ppm)
for MTPA esters
9R
OMTPA
O
O
O
O
O
O
+0.013
−0.034
MTPAO
MTPAO
OMe
OMe
OMe
OMe
OMe
OMe
−0.104
+0.011
−0.025
−0.076
MTPAO
9R
MTPAO
4R
9S
OMTPA
−0.074
−0.013
−0.135
−0.011
+0.025
+0.076
MTPAO
9S
MTPAO
4R
1a
+0.074
+0.135
MTPAO
9R
MTPAO
4S
O
O
+0.034
−0.055
MTPAO
MTPAO
OMe
OMe
+0.104
−0.123
MTPAO
MTPAO
4S
4R
3a
+0.055
+0.123
3R
MTPAO
MTPAO
4S
O
2a
OMe
−0.032
−0.051
+0.008
4a/4b
11R
3R
9R
3R
MTPAO
MTPAO
MTPAO
O
O
MTPAO
O
O
OMe
OMe
OMe
OMe
−0.047
−0.058
+0.086
−0.031
−0.050
+0.078
9S
MTPAO
3R
MTPAO
11S
MTPAO
3R
MTPAO
−0.009
−0.031
−0.070
−0.031
−0.050
−0.078
5a/5b
6a/6b
an [M-H]- ion peak at m/z 231, which was 28 mass units
more than that of 5, while in the 13C-NMR spectrum, two
additional methylene signals were observed. From these
findings, 6 was revealed to be 3,11-dihydroxydodecanoic
acid. The absolute configurations of the two secondary
hydroxy groups were determined in the same manner as
described for 5. With the Dd values and signs of the
diagnostic signals, similar to those of 5, the C-3 position
was determined to be the R configuration, while the C-11
carbon was found to have both forms (Fig. 4). Accord-
ingly, 6 was identified as a mixture of the isomers,
3R11R and 3R11S in a ratio of 1:3.
Compounds 1–3 and 5 are the first hydroxy fatty acids
isolated from royal jelly, although 5 has already been
detected in human urine by Tserng et al. [8]. Compound 6,
was recently isolated from royal jelly, by Melliou and co-
workers [9], however, the absolute configuration remains
unclear. Similar to 9-HDA in royal jelly, the chiral acids
obtained in this study are revealed to be mixtures of
enantio- or diastereo-isomers (Fig. 4).
It should be noted that the absolute configurations of the
secondary hydroxy groups located at C-3 of the chiral acids
are all the R form, while those at positions C-4, C-9, and
C-11 have both forms.
A recent study of the genome sequencing of the hon-
eybee, A. mellifera, demonstrated that the species has more
odorant receptors than other insects, such as Drosophila
melanogaster, Anopheles gambiae, and Bombyx mori,
indicating that the honeybee lifestyle involves enhanced
pheromone communication [10].
Discussion
Seven hydroxy fatty acids were isolated in the pure state
from the royal jelly of honeybees (Apis mellifera).
123

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Lipids (2011) 46:263–270
3. Johnston NC, Law JH, Weaver N (1965) Metabolism of
9-ketodec-2-enoic acid by worker honeybees (Apis mellifera L.).
Biochemistry 4:1615–1621
4. Noda N, Umebayashi K, Nakatani K, Miyahara K, Ishiyama K
(2005) Isolation and characterization of some hydroxy fatty and
phosphoric acid esters of 10-hydroxy-2-decenoic acid from the
royal jelly of honeybees (Apis mellifera). Lipids 40:833–838
5. Kusumi T, Ooi T, Ohkubo Y, Yabuuchi T (2006) The modified
Mosher’s method and sulfoximine method. Bull Chem Soc Jpn
79:965–980
Considering the above information together with the
relationship between 9-ODA and 9-HDA, it is believed that
the chiral acids isolated in the present study are precursors
of unknown semiochemicals, and that their corresponding
oxo-forms may play physiologically important functions
and roles in the hierarchy of honeybee colonies. We are
now synthesizing these oxo-derivatives in order to examine
the biological activities of these compounds.
6. Ohtani I, Kusumi T, Kashman Y, Kakisawa H (1991) High-field
FT NMR application of Mosher’s method. The absolute config-
urations of marine terpenoids. J Am Chem Soc 113:4092–4096
7. Ono M, Yamada F, Noda N, Kawasaki T, Miyahara K (1993)
Determination by Mosher’s method of the absolute configuration
of mono- and dihydroxyfatty acids originated from resin glyco-
sides. Chem Pharm Bull 41:1023–1026
Acknowledgments The authors wish to thank Dr. K. Hashimoto,
Institute for Bee Products & Health Science, for supplying the royal
jelly. This study was supported in part by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Culture, Sports, Science
and Technology of Japan.
8. Tserng K-Y, Jin S-J (1991) Metabolic origin of urinary
3-hydroxydicarboxylic acids. Biochemistry 30:2508–2514
9. Melliou E, Chinou I (2005) Chemistry and bioactivity of royal
jelly from Greece. J Agric Food Chem 53:8987–8992
10. The honeybee genome sequencing Consortium (2006) Insights
into social insects from the genome of the honeybee Apis melli-
fera. Nature 443:931–949
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2. Butler CG (1954) The method and importance of the recognition
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