Deuterium NMR Used to Indicate a Common Mechanism for the Biosynthesis of Ricinoleic Acid by Ricinus communis and Claviceps purpurea

Article abstract of DOI:10.1021/ja038814d

Previous studies have shown that ricinoleic acid from castor bean oil of Ricinus communis is synthesized by the direct hydroxyl substitution of oleate, while it has been proposed that ricinoleate is formed by hydration of linoleate in the ergot fungus Claviceps purpurea. The mechanism of the enzymes specific to ricinoleate synthesis has not yet been established, but hydroxylation and desaturation of fatty acids in plants apparently involve closely related mechanisms. As mechanistic differences in the enzymes involved in the biosynthesis of natural products can lead to different isotopic distributions in the product, we could expect ricinoleate isolated from castor or ergot oil to show distinct ²H distribution patterns. To obtain information concerning the substrate and isotope effects that occur during the biosynthesis of ricinoleate, the site-specific natural deuterium distributions in methyl ricinoleate isolated from castor oil and in methyl ricinoleate and methyl linoleate isolated from ergot oils have been measured by quantitative ²H NMR. First, the deuterium profiles for methyl ricinoleate from the plant and fungus are equivalent. Second, the deuterium profile for methyl linoleate from ergot is incompatible with this chemical species being the precursor of methyl ricinoleate. Hence, it is apparent that 12-hydroxylation in C. purpurea is consistent with the biosynthetic mechanisms proposed for R. communis and is compatible with the general fundamental mechanistic similarities between hydroxylation and desaturation previously proposed for plant fatty acid biosynthesis.

Full text of DOI:10.1021/ja038814d

Published on Web 02/21/2004
Deuterium NMR Used To Indicate a Common Mechanism for
the Biosynthesis of Ricinoleic Acid by Ricinus communis and
Claviceps purpurea
Isabelle Billault,* Peter G. Mantle, and Richard J. Robins
Contribution from the Laboratoire d’Analyse Isotopique et Electrochimique de M e´ tabolismes,
CNRS UMR6006, UniVersity of Nantes, BP 92208, 44322 Nantes, France, and the
Department of Biochemistry, Imperial College of Science, Technology, and Medicine,
London SW7 2AY, U.K.
Received October 1, 2003; E-mail: isabelle.billault@chimbio.univ-nantes.fr
Abstract: Previous studies have shown that ricinoleic acid from castor bean oil of Ricinus communis is
synthesized by the direct hydroxyl substitution of oleate, while it has been proposed that ricinoleate is
formed by hydration of linoleate in the ergot fungus Claviceps purpurea. The mechanism of the enzymes
specific to ricinoleate synthesis has not yet been established, but hydroxylation and desaturation of fatty
acids in plants apparently involve closely related mechanisms. As mechanistic differences in the enzymes
involved in the biosynthesis of natural products can lead to different isotopic distributions in the product,
2
we could expect ricinoleate isolated from castor or ergot oil to show distinct H distribution patterns. To
obtain information concerning the substrate and isotope effects that occur during the biosynthesis of
ricinoleate, the site-specific natural deuterium distributions in methyl ricinoleate isolated from castor oil
and in methyl ricinoleate and methyl linoleate isolated from ergot oils have been measured by quantitative
2
H NMR. First, the deuterium profiles for methyl ricinoleate from the plant and fungus are equivalent. Second,
the deuterium profile for methyl linoleate from ergot is incompatible with this chemical species being the
precursor of methyl ricinoleate. Hence, it is apparent that 12-hydroxylation in C. purpurea is consistent
with the biosynthetic mechanisms proposed for R. communis and is compatible with the general fundamental
mechanistic similarities between hydroxylation and desaturation previously proposed for plant fatty acid
biosynthesis.
Introduction
Ricinoleic acid is also produced in other organisms, notably
10
the pathogenic ergot fungus, ClaViceps purpurea. This fungus,
which can be responsible for the devastating destruction of cereal
crops, produces and accumulates ricinoleic acid in the sclerotia.
In contrast to the situation in castor oil plants, it has been
proposed that biosynthesis of ricinoleic acid in the fungus
9
Ricinoleic acid (12-OH, C18:1∆ ) is an attractive precursor
for industrial synthesis. The presence of the 12-hydroxyl
substituent and the ∆ desaturation makes it amenable as a
9
precursor to several potentially useful derivatives,1 while the
ease of cultivating the castor oil plant Ricinus communis makes
it simple to obtain large quantities of this product. Ricinoleic
acid constitutes about 85-90% of the triglyceride fatty acids
found in castor bean oil.
-3
12
11,12
involves the hydration of the ∆ bond of linoleic acid.
Such
a difference in synthetic mode is surprising in view of the
relatively uncommon occurrence of this fatty acid. However,
hydration of an existent olefinic bond in a fatty acid is a
recognized mechanism of hydroxylation. Thus, in a pseudomonad,
The enzymatic mechanism capable of stereo- and regio-
selectively introducing a hydroxyl at C12 of oleic acid (â to
the desaturation) has evoked considerable interest. Studies in
R. communis have indicated that ricinoleic acid is synthesized
by the direct hydroxylation of oleic acid esterified specifically
as the phosphatidylcholine ester.5
13
oleic acid is converted to 10-hydroxystearic acid, while linoleic
14
acid is also hydroxylated at the 10 position. Thus, it is
important to establish whether the enzymatic apparatus for this
biosynthesis in the fungus is indeed more comparable to that
in the bacterium than that in the plant.
4-7
,8,9
(
(
(
1) Lee, S. L.; Cheng, H. Y.; Chen, W. C.; Chou, C. C. Process Biochem.
991, 33, 453-459.
2) Fabritius, D.; Sch a¨ fer, H. J.; Steinb u¨ chel, A. Appl. Microbiol. Biotechnol.
998, 50, 573-578.
3) Kim, H. J.; Kuo, T. M.; Hou, C. T. J. Ind. Microbiol. Biotechnol. 2000,
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1991, 280, 507-514.
1
(9) Lin, J.-T.; Lew, K.; Chen, J.; Iwasaki, Y.; McKeon, T. A. Lipids 2000, 35,
481-486.
1
(10) Mantle, P.; Nisbet, L. J. Gen. Microbiol. 1976, 93, 321-334.
(11) Morris, L.; Hall, S.; James, A. Biochem. J. 1966, 100, 29c-30c.
(12) Morris, L. J. Biochem. J. 1970, 118, 681-693.
(13) Niehaus, W. G.; Kisic, A.; Torkelson, A.; Bednarczyk, D. J.; Schroepfer,
G. J. Biol. Chem. 1970, 245, 3802-3809.
(14) Schroepfer, G.; Niehaus, W. G.; McCloskey, J. A. J. Biol. Chem. 1970,
245, 3795-3801.
2
4, 167-172.
(4) Morris, L. Biochem. Biophys. Res. Commun. 1967, 29, 311-315.
(5) Moreau, R.; Stumpf, P. Plant Physiol. 1981, 67, 672-676.
(6) Galliard, T.; Stumpf, P. J. Biol. Chem. 1966, 241, 5806-5812.
(7) Smith, M.; Jonsson, L.; Stymne, S.; Stobart, K. Biochem. J. 1992, 287,
1
41-144.
3250
9
J. AM. CHEM. SOC. 2004, 126, 3250-3256
10.1021/ja038814d CCC: $27.50 © 2004 American Chemical Society

Deuterium NMR To Indicate Mechanism for Biosynthesis
A R T I C L E S
The mechanisms of the enzymes involved in ricinoleate
synthesis have not yet been established. However, it is proposed
that the 12-hydroxylase that acts on oleate is both genetically
the action of two different enzymes should possess different
2
H distributions. Especially important in this context is the low
2
1
( H/ H) ratio at the C-13 position of linoleate compared with
the C-13 of oleate.
and mechanistically similar to the long-chain fatty acid desatu-
rase enzymes.1
5-19
This hypothesis is strongly supported by
studies in which the alteration of key amino acids in the active
1
2
center of oleate ∆ -desaturase leads to substantial 12-hydrox-
ylase activity.1 The current mechanistic model for desaturation,
9
9
based on studies of the X-ray structure of a soluble ∆ -desaturase
20
isolated from R. communis and of the reaction kinetics of a
wide range of desaturases,1
6,21
invokes a high-valent diiron
center and molecular oxygen. Essentially, a slow activation step
involves the removal of pro(R) hydrogen and the creation of a
short-lived radical. In desaturation, a rapid syn disproportion-
ation results in the loss of the R-hydrogen and olefinic bond
formation. Alternatively, attack by a putative iron-bound hy-
droxyl will lead to hydroxylation of the position from which
the initial hydrogen was abstracted. The insertion of the hydroxyl
4
with retention of configuration supports this proposal.
In contrast, hydratases, which add water directly to an olefinic
bond, are mechanistically dissimilar to the fatty acid desaturases.
The desaturase mechanism involves molecular oxygen as
substrate, whereas oxygen is introduced from water by hydratase
activity.
Due to different sensitivities to heavy isotopes, these distinct
mechanisms are likely to leave characteristic patterns in the
residual H levels of ricinoleic acid derived from different
With the objective of establishing whether a distinction exists
between the biosynthetic mechanisms used by the fungal
pathogen and the crop plant, the site-specific natural abundance
2
origins. This pattern will reflect both that of the starting substrate
and the kinetic isotope effects intrinsic to the enzymes involved.
As each individual reaction acts on different positions in the
substrate, this gives rise to a specific nonstatistical isotope
2
isotope fractionations in H of ricinoleate isolated from both
2
sources have been measured by quantitative H NMR. In
2
1
particular, we have focused on the relative values of ( H/ H)12
22
2
1
distribution in the final product.
It has been shown by quantitative H NMR spectroscopy that
and ( H/ H)13 in each species. This is the first report in which
2
1
2
natural abundance ( H/ H) ratios have been effectively used
directly to deduce similarity or dissimilarity in enzyme mech-
anisms.
2
the distribution of H at natural abundance in the fatty acids
2
3,24
proposed as precursors for ricinoleic acid is nonstatistical.
Thus, measurement of the site-specific natural isotope fraction-
Materials and Methods
2
9
ation in H of methyl oleate 8 (C18:1∆ ) isolated from plants
2
Materials. Castor seed oil from R. communis was obtained from
independent origins: Sigma-Aldrich (castor 1, lot: 85H0439, 1995)
and Rh oˆ ne-Poulenc (castor 2, lot: A28457, 2000). The sample of ergot
oil produced by the fungus C. purpurea was isolated from selected
mycelium cultured axenically on a liquid nutrient medium in such a
way as to differentiate and proliferate a compact purple-pigmented tissue
has shown a strong impoverishment in H at site 9 but not at
9
site 10 of the ∆ -ethylenic bond. Similarly, methyl linoleate
C18:2∆9 ) shows a strong impoverishment at positions 9 and
,12
(
13 but not at positions 10 and 12. While the exact cause of
these impoverishments is yet to be firmly established, they can
reasonably be attributed to a combined influence of the initial
25
form that closely resembled that of typical parasitic ergots. The tissue
2
H contents of the hydrogens of the substrates involvedsstearate
contained approximately 30% (w/w) of oil. This was extracted in
chloroform/methanol (2:1), stored anaerobically, and refrigerated.
General Methods. H NMR (500 MHz) and 13C NMR (125 MHz)
spectra were recorded with a Bruker DRX 500 spectrometer. Chemical
for oleate and oleate for linoleatesand isotope effects associated
9
12
1
with the ∆ - and ∆ -desaturases. Hence, it can be predicted
that ricinoleate formed from these two different substrates by
3
shifts are given relative to TMS in CDCl . Flash chromatography was
(
(
(
(
(
(
15) Shanklin, J.; Cahoon, E. B. Annu. ReV. Plant Physiol. Plant Mol. Biol.
998, 49, 611-641.
16) Behrouzian, B.; Buist, P. H. Prostaglandins Leukotrienes Essent. Fatty Acids
performed with silica gel Normasil (40-63 µm, Prolabo). Petroleum
ether was distilled before use. Reaction was followed by gas chroma-
1
tography on a HP-5 capillary column (30 m × 0.32 mm, film thickness
2
003, 68, 107-112.
17) Van de Loo, F. J.; Broun, P.; Turner, S.; Somerville, C. Proc. Natl. Acad.
Sci. U.S.A. 1995, 92, 6743-6747.
0.52 µm); carrier gas, He, 1.2 mL‚min ; split 1:40; FID temp, 280
-1
-
1
°
C; thermal gradient 100 °C initially for 1 min, ramped at 10 °C min
18) Broun, P.; Shanklin, J.; Whittle, E.; Somerville, C. Science 1998, 282,
-
1
1
315-1317.
to 240 °C for 1 min, ramped at 10 °C min to 280 °C for 1 min, then
19) Broadwater, J. A.; Whittle, E.; Shanklin, J. J. Biol. Chem. 2002, 277,
5613-15620.
20) Lindqvist, Y.; Huang, W. J.; Schneider, G.; Shanklin, J. EMBO J. 1996,
-1
ramped at 10 °C min to 298 °C, and held at 298 °C for 2 min.
1
2
H NMR Spectroscopic Measurements. The samples were previ-
ously characterized by their H and C NMR spectra. The H NMR
spectroscopy was carried out as previously described on a Bruker DRX
500 spectrometer with a dual probe ( H/ H) 10 mm in diameter
operating at 76.7 MHz and fitted with a F field-frequency-locking
1
5, 4081-4092.
1
13
2
(
21) Behrouzian, B.; Buist, P. H. Phytochem. ReV. 2003, 2, 103-111.
22) Robins, R.; Billault, I.; Duan, J.-R.; Guiet, S.; Pionnier, S.; Zhang, B.-L.
Phytochem. ReV. 2003, 87-102.
(
1
2
(23) Billault, I.; Guiet, S.; Mabon, F.; Robins, R. ChemBioChem 2001, 2, 425-
19
4
31.
(24) Duan, J.-R.; Billault, I.; Mabon, F.; Robins, R. ChemBioChem 2002, 3,
52-759.
7
(25) Mantle, P. Trans Br. Mycol. Soc. 1969, 52, 381-392.
J. AM. CHEM. SOC.
9
VOL. 126, NO. 10, 2004 3251

A R T I C L E S
Billault et al.
device.23 Acquisition temperature: 310 K. The internal reference used
three steps) of 2 and 0.59-0.70 g (75-90% for three steps) of 3. H-
1
was pyridine or N,N,N′,N′-tetramethylurea (TMU). The isotopic ratio
and 13C NMR (CDCl
3
) spectra of 2 were identical to those reported
2
1
23
of pyridine ( H/ H)pyr was calibrated relative to TMU calibrated relative
previously.
2
6
to the V.SMOW scale. Sample preparation: 1, 0.90 g; mass pyridine
0.19 g; mass CHCl ) 0.87 g; mass CCl ) 2.4 g; volume C
0 µL. 2 and 4: 0.60-0.75 g (depending on product); mass pyridine
0.21 g; mass CHCl ) 1.20 g; mass CCl ) 2.40 g; volume C
0 µL. Sample preparation of 5, 6, and 7 was as described previously.
(
R) 1,1′-Bis(methoxy)-3-acetylnonane 4. To a solution of 3 (0.59-
.70 g, 2.89-3.43 mmol) in pyridine (8 mL) was added acetic anhydride
1.5 mL, 15.4 mmol). After 15 h at ambient temperature under agitation,
the mixture was evaporated and coevaporated with toluene (3 × 30
mL) to give quantitatively 4. H NMR (CDCl ): δ 4.93 (1H, m, H-3)
)
3
4
6 6
F )
0
(
6
)
6
3
4
6 6
F )
23
1
3
2
1
The ( H/ H)
and Pref are the stoichiometric numbers of hydrogen at site i and in the
reference, S and Sref are the areas of the signals, and M , m
ref are the molecular weight and mass of the sample and the reference,
respectively.
i
ratios of samples were calculated from eq 1, where P
i
4.36 (1H, dd, J ) 5.0 and 6.2 Hz, H-1), 3.25 and 3.27 (2 × 3H, 2s,
2 × OMe), 1.98 (3H, s, COCH ), 1.80 (1H, ddd, J ) 14.1, 7.5 and 5.0
3
i
S
S
and Mref
,
Hz, H-2), 1.75 (1H, ddd, J ) 14.1, 6.2 and 4.5 Hz, H-2′), 1.49 (2H, m,
1
3
m
H-4), 1.30-1.15 (8H, m, 4 CH ), 0.82 (3H, t, J ) 7.0 Hz). C NMR
(CDCl ): δ 170.6 (COOCH ), 102.0 (C-1), 71.1 (C-3), 53.3 and 52.5
2
3
3
(
2 × OCH
3
2 3 3
), 37.4-21.2 (6 CH and COOCH ), 14.1 (CH ).
2
1
2
1
Pref × m × M × S
H
ref
s
i
H
9,12-Octadecadienoic Acid Methyl Ester (Syn. Methyl Linoleate)
)
×
(
)
(
)
5. The solution was synthesized from the same sample of ergot oil as
used to isolate 1. The mixture of non-oxygenated FAMEs recovered
from silica chromatography (5.8 g) was subjected to silica-Ag
chromatography. The isolated 5 was chemically modified as described
previously to give 9,9′-bis(phenylthio)nonanoic acid methyl ester 6 and
methyl 1,1′-bis(phenylthio)hexane 7. The structure and purity of these
P × m × M × S
H i
i
s
ref
ref
H ref
The calculation of S
performed using a curve-fitting algorithm (Perch NMR software,
University of Kuopio, Finland).
i
and Sref for the monodeuterated isomers was
Chemical Synthesis. (R)-12-Hydroxy-9-octadecenoic acid Methyl
Ester (syn. Methyl Ricinoleate) 1 was synthesized from both castor
and ergot oils. Oil (10.0 g) in a solution of sodium hydroxide in
methanol (150 mL, 20 g‚L ) was heated at reflux for 1 h. The mixture
was diluted by 100 mL of MeOH, and then a solution of boron
trifluoride in methanol (33 mL; 50% w/w) was added. The mixture
was heated for an additional 45 min and then cooled. At ambient
temperature, hexane (300 mL) was added, and the mixture was kept
under agitation for 30 min. A saturated NaCl aqueous solution was
added, both phases were separated, and the aqueous phase was extracted
1
13
23,24
products were checked by H and C NMR spectroscopy.
Results
-1
2
2
H Distribution in Methyl Ricinoleate 1. Access to H
isotopic data for methyl ricinoleate 1 isolated from castor oil
or ergot oil was carried out in three steps: (1) transmethylation
of oil, (2) separation of 1 from other FAMEs, and (3) chemical
modification. Two unrelated samples of castor oil (castor 1,
castor 2) and one sample of ergot oil (ergot) were treated in
exactly the same way.
with CHCl
dried (MgSO
fatty acid methyl esters (FAMEs). Flash chromatography on silica gel
3
. The combined organic phases were washed with water,
4
), filtered, and evaporated to give 9.2-10.3 g of mixed
Transmethylation of oil samples (10 g) gave a mixture of
FAMEs (ca. 10 g) from which methyl ricinoleate 1 was isolated
by silica gel chromatography. The quantities obtained repre-
sented yields >95%, based on the quantity of 1 in the starting
oil estimated by GC after transmethylation (data not shown).
2
(
column: 40 × 5.0 cm ; solvent gradient: petroleum ether (40-70°)/
Et O: 95/5 to 50/50) gave 1 (castor 1, 8.6 g; castor 2, 8.8 g; ergot oil;
.6 g) and a mixture of non-oxygenated FAMEs (castor 1, 1.0 g; castor
2
2
2
(
2
(
1
, 1.0 g; ergot oil: 5.8 g). H NMR (CDCl ) of 1: δ 5.50 and 5.35
3
2H, m, H-9 and H-10), 3.61 (3H, s, COOMe), 3.55 (1H, m, H-12),
.24 (2H, t, J ) 7.5 Hz, H-2), 2.15 (2H, t, J ) 7.0 Hz, H-11), 1.99
2H, q, J ) 6.0 Hz, H-8), 1.60-1.35 (4H, m, H-13 and H-3), 1.33-
2
The H NMR spectrum of isolated 1 proved inadequate to
2
1
2
1
2
1
obtain information on the ( H/ H)9, ( H/ H)10, ( H/ H)12, and
( H/ H)13 ratios due to the overlapping of resonance signals.
To reveal masked isotopic data, a chemical modification was
applied. The cleavage of the olefinic bond of 1 gave two
aldehydes, which were directly protected to give products 2 and
1
3
2
1
1
.15 (16H, m, CH
2
), 0.83 (3H, t, J ) 7.0 Hz). C NMR (CDCl
), 133.4 and 125.3 (C-9 and C-10), 71.6 (C-12), 51.5
), 36.9-22.7 (13 CH ), 14.1 (CH ).
,9′-Bis(methoxy)nonanoic Acid Methyl Ester 2 and (R)-1,1′-Bis-
methoxy)-3-hydroxynonane 3. To a solution of 1 (1.20 g, 3.80 mmol)
/H O (22 mL/0.22 mL) was added, at room
temperature, N-methyl-morpholine oxide (0.64 g, 5.40 mmol), and then
a solution of OsO in 2-methyl-2-propanol (0.53 mL, 2.5%) was added
3
) of 1:
δ 174.4 (COOCH
3
(
COOCH
3
2
3
14
9
(
3
. These were separated by silica gel chromatography. To
in a mixture of CHCl
3
2
2
1
2
facilitate the ( H/ H)i measurement of 3 by H NMR, the
alcoholic function was protected to give 4 (Scheme 1). Samples
of methyl ricinoleate 1 (castor 1, castor 2, and ergot) and their
respective compounds 2 and 4 were submitted to quantitative
4
slowly. After 24 h, solvent was evaporated, then coevaporated with
toluene (3 × 30 mL) to give 1.5 g of a yellow solid. The residue was
2
2
H NMR. Figure 1 shows the H NMR spectra obtained from
4
taken up in MeOH (80 mL), and then NaIO was added. After 15 h,
methyl ricinoleate 1 and derivatives 2 and 4 obtained from castor
oil (castor 1). In these spectra, the area of the H NMR signal
the reaction mixture was filtered to provide a mixture of aldehydes in
MeOH. To this solution was added TsOH‚H O (0.87 g, 4.60 mmol),
2
2
and the solution was heated at 40 °C. After 90 min, the solution was
cooled to room temperature, and molecular sieves (3 Å, 10 g) were
added. After 15 h, the mixture was filtered and evaporated, and the
is directly proportional to the number of monodeuterated
2
1
isotopomers present. The site-specific isotopic ratio ( H/ H) is
i
defined in the following equation and expressed in parts per
million (ppm):
residue was taken up in Et
NaHCO (1%). The aqueous phases were extracted with Et
combined organic phases washed with water, dried over (MgSO
filtered, and then evaporated to give a mixture of 2 and 3. Flash
chromatography on silica gel using petroleum ether (40-70°)/Et O (80/
0 to 50/50) as solvent gradient provided 0.64-0.80 g (72-90% for
2
O and washed with an aqueous solution of
O, and the
),
3
2
4
2
H
H
Di
N2H,i
)
)
(
1
)
H
P N
1H
i
2
i
i
2
where N2H,i is the number of monodeuterated isotopomers of
type i, N1H is the number of fully protonated molecules, and Pi
is the number of equivalent hydrogen atoms at site i.
(
26) Gonfiantini, R.; Stichler, W.; Rozanski, K. In IAEA, Standards and
Intercomparison Materials for Stable Isotopes of Light Elements; IAEA:
Vienna, 1995; Vol. IAEA-Techdoc-825, pp 13-29.
3252 J. AM. CHEM. SOC.
9
VOL. 126, NO. 10, 2004

Deuterium NMR To Indicate Mechanism for Biosynthesis
A R T I C L E S
2
2
2
Figure 1. H NMR spectra of methyl ricinoleate 1 and products 2 and 4 isolated from castor oil. (A) H NMR spectrum of methyl ricinoleate 1. (B) H
2
NMR spectrum of 2. (C) H NMR spectrum of 4. To maintain continuity, the sites are numbered following their position in methyl ricinoleate 1.
Scheme 1
2
1
2
1
The measured ( H/ H)i values for methyl ricinoleate 1 (castor
, castor 2, and ergot samples) and derivatives 2 and 4 are
experiments is very satisfactory. Standard errors for ( H/ H)i
values presented in Tables 1-3 are acceptable, with most being
1
2
1
presented by carbon number in Tables 1-3. Data for methyl
ricinoleate 1 from ergot are the means of two independent
transesterifications and NMR acquisitions (Table 3). Data from
derivatives 2 and 4 from castor 2 and ergot samples are the
means of two independent chemical modifications and NMR
acquisitions (Tables 2 and 3). The reproducibility of the
less than 5 ppm. Most ( H/ H)i values were determined with
less than 5% deviation, even when results were the mean of
two independent manipulations.
During chemical modification, a risk exists that isotopic
fractionation may be introduced due to kinetic and thermody-
namic isotopic effects intrinsic to the manipulation. A number
J. AM. CHEM. SOC.
9
VOL. 126, NO. 10, 2004 3253

A R T I C L E S
Billault et al.
2
1
a
Table 1. ( H/ H)i Values (in ppm) of Methyl Ricinoleate 1 (Castor 1) and Corresponding Derivatives 2 and 4
carbon n°
castor 1
SD
2
3
4−7
8
9
10
11
12
COOMe
13
14−17
13−17
18
139.9b
5.7
97.5
1.0
128.2c
2.1
139.9b
5.7
105.0d
0.7
105.0d
0.7
139.9b
5.7
124.8e
1.9
124.8e
1.9
128.2c
2.1
127.8
2.0
f
g
130.2g
2.0
2
/4
SD
measured or derived
143.9
2.3
130.2
2.0
139.0
2.1
79.3
2.5
125.0
6.1
108.9
3.1
122.1
1.0
125.3
1.6
117.6
1.1
119.0
0.8
126.7
0.5
140i
97
h
139j
163h
79
j
125k
109k
122k
125j
118k
119k
128i
a
(2
1
H/ H)i values were calculated as described in the Materials and Methods section. Three acquisitions were made for each sample. For each acquisition,
(2
H/ H)i values were calculated for resonance at 8.5 and 7.0 ppm of the internal reference, pyridine. SD is the standard deviation for each value. Sites 2,
1
b
c
d
8
, and 11 all resonate at the same frequency. Sites 4-7 and 13-17 all resonate at the same frequency. Sites 9 and 10 resonate at the same frequency.
e
f
Sites 12 and OMe resonate at the same frequency. To maintain continuity, the sites in 4 are numbered following their position in methyl ricinoleate 1.
g
Sites 3 and 8 resonate at the same frequency. h Calculated ( H/ H) value. Direct values from 1. Direct values from 2. k Direct values from 4. l SD is the
2
1
i
j
standard deviation calculated from six values obtained for two independent chemical and NMR experiments.
2
1
a
Table 2. ( H/ H)i Values (in ppm) of Methyl Ricinoleate 1 (Castor 2) and Corresponding Derivatives 2 and 4
carbon n°
2
3
4−7
8
9
10
11
12
COOMe
13
14−17
13−17
18
castor 2
SD
expt 1: 2/4
SD
expt 2: 2/4
SD
137.4b
5.0
145.7
4.9
142.7
0.2
98.6
1.0
129.6c
0.4
140.4
2.4
137.0
0.7
137.4b
5.0
103.4d
0.8
82.4
2.8
79.1
0.9
103.4d
0.8
132.7
3.4
118.7
2.2
137.4b
5.0
104.0
2.7
102.5
2.3
123.5e
1.3
130.8
2.8
123.1
4.5
123.5e
1.3
128.4
2.8
125.5
1.2
129.6c
0.4
131.9
1.0
127.2
0.7
124.0
2.2
f
f
g
133.0g
2.8
133.0
2.8
114.3
1.6
123.8
1.7
119.2
1.3
122.1
1.2
g
126.9g
0.9
126.9
0.9
means 2/4
SD
measured or derived
144.2
3.5
130.0
3.8
99
138.7
2.5
130.0
3.8
80.7
2.6
81
125.7
8.1
103.2
2.4
126.9
5.4
127.0
2.1
119.0
5.4
120.7
2.0
125.6
2.3
l
144i
h
139j
161h
j
126k
103k
127k
127j
119k
121k
132i
a
For footnotes, see Table 1.
2
1
a
Table 3. ( H/ H)i Values (in ppm) of Methyl Ricinoleate 1 (Ergot) and Corresponding Derivatives 2 and 4
carbon n°
2
3
4−7
8
9
10
11
12
COOMe
13
14−17
13−17
18
expt 1: ergot
SD
expt 2: ergot
SD
129.3b
3.4
125.4
105.2
5.3
106.1
1.9
119.6c
0.5
120.2
129.3b
3.4
90.6d
3.0
90.6d
3.0
129.3b
3.4
126.4e
1.0
126.4e
1.0
119.6c
0.5
120.2
133.9
0.4
139.7
1.1
136.8
3.3
128.7
1.6
b
c
125.4b
2.2
88.6d
2.7
88.6d
2.7
125.4b
2.2
129.7e
2.3
129.7e
2.3
c
2.2
127.4
0.6
119.9
0.6
119.9
b
c
127.4b
3.1
89.6d
2.8
50.2
5.2
39.4
1.5
89.6d
2.8
131.4
0.7
122.9
4.5
127.4b
3.1
92.6
1.8
99.7
6.4
128.1e
2.2
127.4
5.3
120.6
3.5
128.1e
2.2
127.3
0.7
130.0
1.3
c
means ergot
SD
105.7
3.6
3.1
137.9
1.4
133.7
4.3
0.6
128.0
0.7
130.1
2.2
0.6
f
g
122.8g
0.4
expt 1: 2/4
122.8
0.4
123.7
1.7
130.3
2.0
115.6
1.5
118.4
0.6
SD
expt 2: 2/4
f
g
120.0g
1.8
120.0
1.8
126.3
2.7
SD
means 2/4
SD
measured or derived
135.8
3.7
121.4
1.9
129.0
1.9
121.4
1.9
44.8
6.8
45
127.1
5.5
96.1
5.7
96
124.0
5.5
128.7
1.8
127.0
4.0
117.0
1.8
127.5
2.4
l
136j
106i
129j
137h
j
127k
k
124k
129h
127j
117k
137i
a
For footnotes, see Table 1.
2
1
2
1
Table 4. Comparison between ( H/ H)i Values in ppm measured
on Methyl Ricinoleate 1 (Castor 1, Castor 2, and Ergot) and their
Corresponding Derivatives 2 and 4
exception was the ( H/ H)18 of the ergot sample, for which 1
gives a value 7% higher than that measured for 4 (Table 4).
This difference is probably due to the presence in the ergot
sample of 1 of a small impurity that possesses the same chemical
shift as that of site 18 in methyl ricinoleate 1 and which is
removed during purification of 4. However, as all other values
are in good agreement, it can be concluded that the chemical
modification of 1 to 2 and 4 has not introduced aberrant
comparison
2, 8, 11
4−7/13−17
9, 10
18
castor 1 (M)
products (P)
139.9
138.6
1.3
137.4
136.3
1.2
127.4
123.0
128.2
127.7
0.5
129.6
128.5
1.1
119.9
123.4
-3.5
105.0
102.2
2.9
103.4
103.2
0.2
89.6
86.0
3.6
127.8
126.7
1.1
131.9
125.6
6.3
136.8
127.5
9.3
a
∆
(M - P)
castor 2 (M)
products (P)
a
∆
(M - P)
2
1
(
H/ H)i values.
By this procedure, the ( H/ H)i for a number of sites was
directly determined. Other sites may be obtained by calculation,
such as the ( H/ H)8, which may be calculated by using the value
( H/ H)3,8 measured in 2, and the value for ( H/ H)3 measured
ergot (M)
a
products (P)
2
1
∆
(M - P)
4.3
2
1
a
(2
1
2
1
2
1
H/ H)2,8,11 for products was calculated from ( H/ H)2 and ( H/ H)11
directly measured in 2 and 4, respectively, and ( H/ H)8 calculated from
H/ H)3 in 1 and ( H/ H)3,8 measured in 2.
2
1
2
1
2
1
2
1
2
1
(
2
1
in 1. The measured or calculated ( H/ H)i values for the three
methyl ricinoleate 1 samples are compared in Figure 2A.
of internal checks must be performed to confirm that no
significant artifactual fractionation has occurred. Thus, the ( H/
H)i values obtained from compounds 2 and 4 may be compared
with the ( H/ H)i values measured for the intact methyl
ricinoleate 1 samples (Table 4). Data from methyl ricinoleate 1
compare favorably with the data from the respective products
2
2H Distribution in Methyl Linoleate 5. Methyl linoleate 5
was isolated from the same ergot oil sample as the C. purpurea
1 treated above. Following separation from 1 by silica chro-
matography, pure 5 was isolated by silica-Ag chromatography
and cleavage fragments 6 and 7 were obtained by chemical
1
2
1
23
2
1
2
and 4, with a difference ranging from 0.5 to 5.0%. The only
modification (Scheme 2). The ( H/ H)i data obtained from the
3254 J. AM. CHEM. SOC.
9
VOL. 126, NO. 10, 2004

Deuterium NMR To Indicate Mechanism for Biosynthesis
A R T I C L E S
Scheme 2
methyl linoleate 5 from ergot can be directly compared with
methyl ricinoleate 1 from the same source. The low level of
methyl oleate 8 in castor oil (3% total FAMEs) and the presence
7
9
11
in ergot oil of a mixture of C16:1∆, C18:1∆ , and C18:1∆
2
1
makes it impractical to measure directly the ( H/ H)i values for
methyl oleate from these sources.
Nevertheless, from these data, a number of conclusions can
be drawn.
2
It is clear that the distributions of H along the fatty acid
chain of ricinoleate 1 from castor and ergot samples are
nonstatistical. Methyl ricinoleate 1 isolated from castor oil shows
9
the same profile at the ∆ -desaturation as previously seen for
two sources of methyl oleate 8 isolated from different plant
2
1
2
1
2
1
origins, in that the ( H/ H)8 > ( H/ H)10 . ( H/ H)9. From this
it can be concluded that the hydroxylation of methyl oleate 8
2
at the 12 position has not significantly influenced the H-
distribution profile found in 1. Methyl ricinoleate 1 isolated from
ergot oil shows the same qualitative profile, with the notable
2
1
2
1
2
1
difference that the ( H/ H)8 ≈ ( H/ H)10 . ( H/ H)9. Whether
this is a significant difference in terms of mechanism remains
to be established. It could reflect the different sensitivity to H
2
of a combination of factors: (a) the reductases of the FAS
complex, which would result in a different pro-S-H/pro-R-H
9
ratio, (b) the ∆ -desaturase, (c) the hydrogen removed by the
9
∆
-desaturase (removal of the pro-S-H rather than the pro-R-
H), or (d) an influence of the hydroxylation mechanism.
A key observation in terms of mechanistic comparisons is
2
1
that the ( H/ H)13 of 1 from C. purpurea shows the same
relationship to other sites in the molecule as it does in 1 from
2
1
R. communis (Figure 2A). That is, its ( H/ H) ratio does not
correspond to that defined for the C13 position in any methyl
2
3,24,27
linoleate so far examined,
including that reported here for
2
1
2
1
ergot oil (Figure 2B). In all these cases, ( H/ H)13 , ( H/ H)12.
2
1
2
1
Rather, its ( H/ H) ratio is similar to that of values determined
Figure 2. Distribution of ( H/ H)i values at different sites in fatty acids
isolated from R. communis and C. purpurea oils. (A) Methyl ricinoleate 1
isolated from castor 1 (‚‚‚), castor 2 (red - - -) or ergot (- - -). (B) Methyl
ricinoleate 1 (- - -) and methyl linoleate 5 (red - - -) from ergot.
2
3,24,27
for saturated positions.
Thus, it can be concluded as
extremely improbable that linoleic acid is the precursor of
ricinoleic acid in C. purpurea. It could be argued that the
mechanism of hydration is associated with an overall kinetic
isotope effect that restores the observed value to that of the
2
H NMR spectra of 5-7 are summarized in Table 5. The
2
nonstatistical distribution of H is essentially as found previously
2
for other samples of methyl linoleate.2
3,24
2
1
original methylenic positions. As hydration involves an sp to
Notably, the ( H/ H)i
3
sp conversion, known to be associated with an inverse
values at the C9 and C13 positions are markedly depleted in
2
8,29
2
secondary isotope effect,
this would act to increase the value
H relative to other uneven carbon positions. A similar validation
2
1
of the ( H/ H)13 in the product. However, the introduction of
the H derived from water will show a normal primary isotope
effect and select against introducing H at C-13 of ricinoleate.
that preparation had not led to fractionation was carried out as
for methyl ricinoleate 1.
2
Discussion
The procedure described here has, for the first time, given
access to a large number of ( H/ H)i values for ricinoleic acid
from two different origins: plant and fungus. Furthermore,
(27) Guiet, S.; Robins, R. J.; Lees, M.; Billault, I. Phytochemistry 2003, 64,
227-233.
2
1
(
28) Garrett, C.; Wataya, Y.; Santi, D. Biochemistry 1979, 18, 2798-2804.
(29) Barr, P.; Robins, M.; Santi, D. Biochemistry 1983, 22, 1696-1703.
J. AM. CHEM. SOC. VOL. 126, NO. 10, 2004 3255
9

A R T I C L E S
Billault et al.
2
1
Table 5. ( H/ H)i Values (in ppm) of Methyl Linoleate 5 (from Ergot Oil) and Corresponding Derivatives 6 and 7
carbon n°
2
3
4−6
7
8
9
10
3.5
143l
11
3.8
120i
12
3.5
143l
13
14
15
4.9
16, 17
4.9
18
4.8
COOMe
4.3
Me linoleate 5a
133.9 118.1 120.6c 120.6c 130.3d 104.5e 104.5e 120.1 104.5e 104.5e 130.3d 120.6c 120.6c 132.7 135.0
SD
4.2
142.3 117.2 130.8 117.2 130.1h
1.6 3.7 2.1 3.7
118i 131j 116l
3.8
4.9
4.9
4.2
3.5
52.8
3.2
3.5
78.3 114.2 119.8 100.8 127.9 132.1
7.0 2.3 1.5 1.0 2.1 5.5
114k 120k 101k 133i
4.2
b
f
g
g
derivatives 6/7
SD
measured or derived 134i
130j
53
j
78
k
a
(2
1
H/ H)i values were calculated as described in the Materials and Methods section. Three acquisitions were made for each sample except for linoleate
2
1
with two acquisitions. For 5, ( H/ H)i values were calculated for resonance at 8.5 and 7.0 ppm of the internal reference, pyridine, on each acquisition. SD
is the standard deviation calculated from four values. b For 6 and 7, ( H/ H)i values were calculated for resonance at 2.8 ppm of the internal reference, TMU,
2
1
c
d
on each acquisition. SD is the standard deviation calculated from three values. Sites 4-7 and 15-17 all resonate at the same frequency. Sites 8 and 14
resonate at the same frequency. Sites 9, 10, 12, and 13 resonate at the same frequency. To maintain continuity, the sites in 7 are numbered following their
e
f
g
h
i
j
position in methyl linoleate 5. Sites 3 and 8 resonate at the same frequency. Mean of two repetitions. Direct values from 1. Direct values from 6.
k
Direct values from 4. l Calculated ( H/ H) value.
2
1
The primary effect will override any weaker secondary effect,
and thus hydration of the ∆ bond will not lead to relative
desaturases from animals and fungi, which are membrane-bound
12
33,34
2
proteins.
Yet, from the similarity of the H profile established
2
2
1
enrichment in H. Hence, it can be predicted that the ( H/ H)13
of 1 derived by hydration of linoleate would retain impoverish-
ment at the 13 position. However, as the C13 is not impover-
ished, this is not the case. In addition, as the linoleate from C.
for 1 obtained from castor and ergot (Figure 2A), it would appear
that the two activities are mechanistically close. Notably, both
show depletion in the C9 and not in the C10 position. Further
studies of other fungal oils will help establish whether this
profile is general to this class of organism.
2
1
purpurea has ( H/ H)13 ) 78 ppm, the hydrogen added to C-13
to form a methylenic site in ricinoleate would need to have a
In conclusion, the results presented in this paper demonstrate
three points. First, the data strongly support the conclusion that
ricinoleic acid is biosynthesized by R. communis and C.
purpurea by a common mechanism. It is evident that oleate
and not linoleate is the substrate for hydroxylation, thus
correcting a long-standing anomaly in the literature. Second,
2
1
(
1
H/ H) value of about 180 ppm to give the measured value of
27 ppm (Table 3 and Figure 2B), well in excess of that for
water from southern England (ca. 150 ppm).
The last important deduction concerns the 12-(R)-hydroxyl-
ation step. During the formation of both oleate and 12-(R)-
ricinoleate in castor seed, it is the pro-R hydrogen of the
2
1
2
1
because ( H/ H) ≈ ( H/ H) in all samples studied, no
1
0
12
4
,15,30
methylenic sites 10 and 12 that is abstracted.
(
As shown
measurable secondary kinetic isotope effect on the position C12
associated with hydroxylation can be detected that is distinct
from any effect occurring in desaturation. This observation is
in agreement with the current proposal that 12-(R)-hydroxylation
2
1
2
1
2
1
Tables 1 and 2), ( H/ H)10 ≈ ( H/ H)12 < ( H/ H)8 in all
samples of 1. A strong isotope effect has been reported for the
9
12
membrane-bound ∆ - and ∆ -desaturases at the 9 and 12
1
9
positions, respectively, with no detectable isotope effect at the
and desaturation are mechanistically close. Third, it is shown
0 and 13 positions.2
1,31,32
Because the mechanism of the
2
1
that a detailed analysis of the natural abundance H distribution
enzyme involved in the 12-(R)-hydroxylation of oleate in R.
in similar chemical species can be used to predict biosynthetic
mechanisms.
communis is considered mechanistically close to these en-
zymes,1
6,17,19
this implies either that no secondary effect is
Acknowledgment. This publication is dedicated to the
memory of Fran c¸ oise Mabon.
associated with the hydroxylation or that the mechanisms in
the two sources do show some kinetic dissimilarities.
9
The soluble stearoyl-ACP ∆ -desaturase from higher plants
JA038814D
9
shows negligible sequence homology with the stearoyl-ACP ∆ -
(
33) Shanklin, J.; Somerville, C. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 2510-
(
(
(
30) Schroepfer, G.; Bloch, K. J. Biol. Chem. 1965, 240, 54-63.
2514.
31) Buist, P. H.; Behrouzian, B. J. Am. Chem. Soc. 1996, 118, 6295-6296.
32) Buist, P. H.; Behrouzian, B. J. Am. Chem. Soc. 1998, 120, 871-876.
(34) Mekhedov, S.; de Ilarduya, O. M.; Ohlrogge, J. Plant Physiol. 2000, 122,
389-401.
3256 J. AM. CHEM. SOC.
9
VOL. 126, NO. 10, 2004