Stereochemistry of 10-sulfoxidation catalyzed by a soluble Δ9 desaturase

Article abstract of DOI:10.1039/b921753c

The stereochemistry of castor stearoyl-ACP Δ⁹ desaturase-mediated 10-sulfoxidation has been determined. This was accomplished by ¹⁹F NMR analysis of a fluorine-tagged product, 18-fluoro-10-thiastearoyl ACP S-oxide, in combination with a chiral solvating agent, (R)-AMA. Sulfoxidation proceeds with the same stereoselectivity as hydrogen removal from the parent stearoyl substrate. These data validate the use of thia probes to determine the stereochemistry and cryptoregiochemistry of desaturase-mediated oxidations.

Full text of DOI:10.1039/b921753c

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Stereochemistry of 10-sulfoxidation catalyzed by a soluble D9 desaturase†
Amy E. Tremblay,a Nigel Tan,a Ed Whittle,b Derek J. Hodgson,c Brian Dawson,c
Peter H. Buist*a and John Shanklin*b
Received 19th October 2009, Accepted 5th December 2009
First published as an Advance Article on the web 7th January 2010
DOI: 10.1039/b921753c
The stereochemistry of castor stearoyl-ACP D9 desaturase-mediated 10-sulfoxidation has been
determined. This was accomplished by 19F NMR analysis of a fluorine-tagged product,
18-fluoro-10-thiastearoyl ACP S-oxide, in combination with a chiral solvating agent, (R)-AMA.
Sulfoxidation proceeds with the same stereoselectivity as hydrogen removal from the parent stearoyl
substrate. These data validate the use of thia probes to determine the stereochemistry and
cryptoregiochemistry of desaturase-mediated oxidations.
results2,9 point to abstraction of the C-10 HR substrate as the
Introduction
initial event for soluble D9 desaturase oxidation of the parent
The desaturase family of enzymes catalyze the highly selective,
stearoyl substrate. A critical test of this interpretation would be to
determine the stereochemistry of soluble D9 desaturase-mediated
10-sulfoxidation. This is a nontrivial task in the case of soluble
desaturases since typically only nanomole amounts of enzymatic
product are available for analysis. In this paper, we report on how
our recently developed 19F NMR methodology,10,11 in combination
with the appropriate chiral NMR solvating agent, was used to
solve this problem.
O2-dependent, dehydrogenation of lipidic substrates and play
critically important roles in a diverse array of biological functions.1
These include cold acclimation, biodefence and biosignaling.
Desaturases, like the closely related hydroxylases, are capable of
attacking a variety of unactivated C–H bonds. A prototypical
example of this intriguing biological chemistry is the pro R-
selective 9,10-desaturation of long-chain fatty acid derivatives
by soluble plant stearoyl ACP D9 desaturases (Scheme 1A).2 An
identical reaction, which is also pro R-selective, is catalyzed by
ubiquitous, membranous D9 desaturases as exemplified by the
yeast stearoyl CoA desaturase.3 The soluble and membrane-bound
subclasses of desaturases are not structurally related with respect
to primary sequence, but are thought to share a common, non-
heme diiron catalytic core.4
A critically important mechanistic issue that relates to all
desaturases involves determining the order of hydrogen removal—
the site of initial oxidation (cryptoregiochemistry).5 In the case
of membrane-bound D9 desaturases, it was readily determined,
through the use of KIE methodology, that C9–H cleavage
preceded C10–H bond rupture.5 This result was corroborated
by showing that the putative diiron dioxo oxidant oxygenated
a 9-thia substrate analogue more efficiently than the corre-
sponding 10-thiaoctadecanoyl CoA ester.6 The notion that the
enzymatic oxidant is located near C-9 was further strengthened
by demonstrating that the stereochemistry of yeast (membranous)
D9 desaturase-mediated sulfoxidation matched that of H-removal
for the parent reaction.7 In contrast, it has been found that
soluble castor stearoyl ACP D9 desaturase sulfoxidizes S-10
substrate analogues efficiently (Scheme 1B) whereas S-9 analogues
primarily undergo chain cleavage with this enzyme, presumably
by C–H activation at C-10.8 Taken together, these and other
Results and discussion
The stereochemical analysis of desaturase-mediated sulfoxidation
requires a determination of the absolute configuration of the
sulfoxy product. Previously, enantiomers of fatty acid sulfox-
ides could be distinguished using 1H or 13C NMR analysis
in combination with a chiral solvating reagent, (S)-(+)-MPAA.
1
Differential shielding effects on H or 13C reporter atoms could
be evaluated using a Pirkle-type binding model12 that had been
validated with synthetic chiral reference standards (Fig. 1A).7,13
This approach was feasible because milligram amounts of analyte
could be produced using a convenient in vivo desaturating system.6
However, to analyze the nanomole amounts of sulfoxide produced
by in vitro stearoyl ACP D9 desaturase oxidations, use of 1H-
decoupled 19F NMR and a fluorine-tagged substrate appeared
to be the method of choice. This methodology features several
advantages, namely, the lack of naturally occurring interferences,
inherently high sensitivity and wide chemical shift range. In-
deed, we have been able to monitor the sulfoxidation of 18-
fluoro-thiaoctadecanoyl ACP derivatives at the nanomole level
of detection.8b,e To apply our stereochemical methodology in
this case, we require that the shielding effects of a Pirkle-type
solvating agent (Fig. 1A) extend out to the CH2F reporting group
located several carbons away from the sulfoxy center. A systematic
study was carried out that involved testing four chiral H-bonding
aromatic compounds (Fig. 1B) with a set of analytes of varying
chain length, (CH3(CH2)nCH2S(O)(CH2)7CO2Me. We chose to use
9-sulfoxy fatty acids in these experiments since the corresponding
9-thia precursors were readily synthesized from commercially
available 8-bromooctanoic acid. As is clear from Fig. 1C, the
aDepartment of Chemistry, Carleton University, 1125 Colonel By Drive,
Ottawa, Ontario, Canada K1S 5B6
bBrookhaven National Laboratory, Department of Biology, Upton, NY
11973, USA
cCentre for Biologics Research, Biologics and Genetic Therapies Directorate,
Health Canada, Tunney’s Pasture, Ottawa, Ontario, Canada K1A 0L2
† Electronic supplementary information (ESI) available: Experimental
details and characterization data. See DOI: 10.1039/b921753c
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Scheme 1 (A) D9 Desaturation of stearoyl ACP. (B) D9 Desaturase-mediated sulfoxidation of 10-thiastearoyl ACP.
ability to use the terminal methyl group to discriminate between
sulfoxide enantiomers (dD) falls off uniformly as a function of
increasing distance between the reporting atom and sulfoxy center.
Importantly, it appeared that use of (S) or (R)- AMA ((9-anthryl)
methoxy acetic acid) would offer the best chance of success in the
stereochemical analysis of an enzymatically produced 18-fluoro-
10-sulfoxide (n = 6, where n + 1 = number of methylene groups
between reporter group and sulfoxy centre).
The absolute configuration of bio-1 was inferred by spiking the
biological sample with an approximately equimolar amount of
racemic 1 (Fig. 2C). The resonance of highest intensity, belonging
to bio-1, can be assigned to the S enantiomer15 by application
of the Pirkle-type binding model combined with our recent
discovery11 that 19F NMR resonances, unlike 1H NMR resonances,
are shifted downfield under the influence of the proximal aromatic
ring of aromatic acids such as MPAA and AMA (Fig. 4).
Sufficient quantities of sulfoxy analyte were obtained by scaling
up (15¥) a typical stearoyl ACP D9 desaturase assay in order to
convert approximately 500 nmol of 18-fluoro-10-thiaoctadecanoyl
ACP to the corresponding sulfoxide (Scheme 2). The product was
isolated from the enzymatic mixture as the primary alcohol (bio-
1, 18-fluoro-10-thiaoctadecan-1-ol S-oxide) by methylene chloride
extraction after reductive (NaBH4) workup. Evaporation of the
combined methylene chloride extracts yielded a residue that was
Further support for our stereochemical assignment was ob-
tained by synthesizing samples of (R)- and (S)-1 as authen-
tic reference samples. These were synthesized from 1-bromo-8-
fluorooctane (Scheme 3) using the method of Alcudia et al.16 The
key intermediate in this synthetic sequence was diastereomerically
enriched DAG-(S)- or (R)-w-fluorobutanesulfinates (de = 66 and
82%, respectively) (DAG = diacetone-D-glucose). The absolute
configuration of the sulfinates was confirmed by a comparison
1
1
taken up in CDCl3 (1 mL) and analyzed by H-decoupled 19F
of the chiroptical and NMR data (characteristic H resonances
NMR, and shown to exhibit a signal with adequate signal to
noise, at the expected chemical shift (-218.375 ppm, reference
standard14) (Fig. 2A). The appearance of this 19F resonance did not
change (Fig. 2B) upon the addition of (R)-AMA (50 mg ml-1)—an
amount, which in a parallel trial experiment, resulted in induced
nonequivalence (dD = 0.005 ppm) of the 19F resonances for a
racemic, synthetic sample of 114 (Fig. 3A). These results indicated
that the enzymatic product, bio-1, was of high enantiomeric purity.
(H2) and 13C resonances (C1–C4) for these compounds with that
of literature values reported for the two diastereomeric DAG
n-propylsulfinates16 and DAG n-butylsulfinates.17 Treatment of
DAG-(S)- or (R)-w-fluorooctanesulfinates with 3 equivalents of
8-OTBS-1-nonylmagnesium bromide yielded, after deprotection,
enantiomerically enriched samples of (R)- and (S)-1 respectively.
This substitution reaction is known to proceed with inversion of
configuration at the sulfinyl centre of the DAG-alkylsulfinates.16
With reference standards of known absolute configuration in
hand, we were able to simulate the 19F NMR spectrum of the spiked
biological sample (Fig. 2C) using samples of known enantiomeric
composition. The 1H-decoupled 19F NMR spectrum of (R)-3 and
(S)-1 (2 : 1 ratio) or (R)-3 and (S)-1 (1 : 2 ratio), to which (R)-AMA
(4 equivalents) was added, are shown in Fig. 3B and C, respectively.
A comparison of these spectra with the spectrum shown in Fig. 2C
clearly demonstrates that enzymatically produced 1 bears the S
configuration.
Conclusions
1. The enantioselectivity of soluble castor D9 stearoyl ACP-
mediated 10-sulfoxidation matches that of hydrogen removal for
the parent reaction (compare Scheme 1A and 1B). This result
validates the use of thia probes to report on the stereochemistry
Scheme 2 Desaturase-mediated sulfoxidation of 18-fluoro-10-thiaoc-
tadecanoyl ACP.
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1
Fig. 1 (A) Pirkle-type complexation model featuring differential shielding effects of (S)-(+)-MPAA on H NMR signals of a methyl reporter group;
(B) various chiral solvating agents that are useful in probing stereochemistry of fatty acid sulfoxides; (C) magnitude of induced non-equivalence as a
function of the number of methylene units (n) between the reporting group and C10 of 9-sulfoxide.
Fig. 2 1H-decoupled 19F NMR signal of (A) enzymatically generated sample of 18-fluoro-10-thiaoctadecan-1-ol (bio-1); (B) (bio-1) + (R)-AMA (50 mg
mL-1); (C) (bio-1) spiked with racemic 1 + (R)-AMA (50 mg mL-1).
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Fig. 3 1H-decoupled 19F NMR signal of (A) racemic 18-fluoro-10-thiaoctadecan-1-ol (racemic-1); (B) (R)-1 : (S)-1, 2 : 1 mixture + (R)-AMA; (B) (R)-1 :
(S)-1, 1 : 2 mixture + (R)-AMA.
involving chiral NMR solvating agents were carried out using
a Bru¨ker AMX 400 spectrometer while the corresponding work
1
using H-decoupled 19F NMR was accomplished at 282.4 MHz
on the Bru¨ker Avance 300 spectrometer. 1H-decoupled 19F NMR
spectra of biological samples were recorded on a Bru¨ker AM
400 instrument at 376.5 MHz; H and 13C chemical shifts are
1
Fig. 4 Pirkle-type binding model showing collision complex between
(R)-AMA and enantiomers of 18-fluoro-10-thia-octadecan-1-ol.
expressed in ppm (d) and are referenced to tetramethylsilane. 19
F
NMR chemical shifts (ppm) are referenced to external CFCl3. J-
values are reported in Hertz (Hz). The spectra of all samples were
recorded as dilute CDCl3 solutions.
of desaturase-mediated reactions and gives valuable information
on the topology of the active site.
Mass spectra of synthetic intermediates were obtained by
GC/MS using a Kratos 1H mass spectrometer coupled to a
HP 5980 Series 2 gas chromatograph equipped with a J. &
W. DB-5 capillary column (30 m ¥ 0.21 mm), temperature
programmed from 120 to 320 ◦C at 10 ◦C min-1. GC-MS analysis
of the enzymatic reaction (disappearance of starting material)
was carried out using a HP5973 mass spectrometer coupled to a
HP6890 GC equipped with a SP2340 capillary column (60 m ¥
0.2◦5 mm), temperature programmed from 100 to 160 ◦C at
25 C min-1 and from 160 to 240 ◦C at 10 ◦C min-1.
2. 1H-decoupled 19F NMR in combination with the use of
fluorine-tagged substrates and the appropriate chiral solvating
agent constitutes powerful methodology for stereochemical anal-
ysis of in vitro desaturase-mediated sulfoxidation at the nanomole
level of detection (~1000 fold increase in sensitivity). This approach
may have application to other enzymatic transformations in cases
where fluorine tagging is feasible.
3. The scope and limitations of Pirkle-type NMR solvating
agents in probing sulfoxide stereochemistry have been delineated.
Thus, it appears that reagents bearing phenyl, 2-naphthyl or 9-
anthryl substituents are able to induce nonequivalence of reporter
groups that are situated up to 7 carbons from the sulfinyl centre.
Use of solvating agents with larger aromatic ring systems would
potentially extend the range at which the fluorine tag can function.
Flash chromatography with silica gel (230-400 mesh) was used
to purify all intermediates and substrates. Visualization of UV-
inactive materials on silica gel TLC was accomplished by a
combination of water spray or I2 vapor as appropriate.
All reagents and starting materials for organic synthesis were
purchased from Sigma-Aldrich and used without purification. 8-
Thiooctanoic acid was prepared from 8-bromooctanoic acid as
previously described.18 Tetrahydrofuran (THF) and diethyl ether
(Et2O) were freshly distilled from Na–benzophenone ketyl. All
air- and moisture-sensitive reactions were performed under N2.
Organic extracts were typically dried by gravity filtration through
anh. Na2SO4, and solvents were evaporated in vacuo on a Bu¨chi
RE 111 Rotavapor.
Experimental
General methods
1
Routine H and 13C NMR spectra were obtained at 300.2 and
75.5 MHz respectively on a Bru¨ker Avance 300 spectrometer.
Some 13C NMR spectra were recorded at 53.6 MHz on a Gemini
200 spectrometer as indicated. Preliminary 1H NMR experiments
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Scheme 3 Synthesis of (R)- and (S)-18-fluoro-10-thiaoctadecan-1-ol.
Chiral solvating reagents, MPAA, Naproxen and TFAE, are
available from Sigma-Aldrich. (R)- and (S)-AMA were kindly
provided by Dr Jose Abad (IIQAB. CSIC. Barcelona).
13C-NMR (75 MHz, CDCl3): 84.12 (d, J = 163.1), 33.90, 32.75,
30.35 (d, J = 19.2), 29.03, 28.63, 28.05, 25.08 (d, J = 5.4). 19F
NMR (282.4 MHz, CDCl3): -218.30.
All buffers and salts, NADH, BSA and other biochemicals were
purchased from Sigma-Aldrich. Protein concentrations were mea-
sured by the method of Bradford19 with the use of bovine serum
albumin as standard protein. The purification of castor stearoyl-
ACP D9 desaturase and required cofactors and the synthesis of
substrate ACP derivatives has been previously described.20 The
ACP used was Spinach ACP isoform I.20c
Thioacetic acid S-(8-fluoro-octyl) ester. To a solution of potas-
sium thioacetate (1.08 g, 9.5 mmol) in anh. DMF (25 ml) was
added 1-bromo-8-fluorooctane (1.97 g, 9.3 mmol) dissolved in
DMF (15 ml) under N2 at room temperature. After stirring at
room temperature for 15 h, the reaction mixture was diluted with
CH2Cl2 (100 ml), washed with dH2O (5 ¥ 60 ml), sat. NaCl (75 ml),
dried (Na2SO4) and roto-evaporated. The resulting oil was then
partitioned between hexanes (2 ¥ 20 ml) and dH2O (20 ml), and
the combined organic layers were dried (Na2SO4) and evaporated
to give the title compound (1.87 g, 9.1 mmol, 97%) as an amber oil.
Synthesis of diastereomerically enriched fluorine-tagged alkyl
sulfinates
TLC (EtOAc–hexane 15 : 85): Rf 0.54. IR (film): 2932, 2857, 1693
1-Bromo-8-fluorooctane. To (diethylamino)sulfur trifluoride
(DAST) (2.57 ml, 3.16 g, 19.6 mmol) at room temperature under
N2, was added 8-bromo-1-octanol (1.68 ml, 2.05 g, 9.8 mmol)
dropwise. The solution was heated to 50 ◦C for 4 h with stirring,
quenched with ice water (150 ml) and extracted with CH2Cl2 (3 ¥
50 ml). The combined extracts were washed with sat. NaHCO3
(3 ¥ 30 ml) and H2O (50 ml), then dried (Na2SO4) and filtered
through Florisil. The solvent was removed by roto-evaporation to
obtain the title compound (1.97 g, 9.3 mmol, 95%) as an amber
liquid. TLC (EtOAc–hexane 15 : 85): Rf 0.63. B.p. 120 ◦C (22.5 mm
Hg) (lit 118–120 ◦C (22.5 mm Hg)21). IR (film): 2933, 2857, 1464,
-1
=
(C O), 1464, 1430, 1354, 1135, 1047, 1003, 956, 725, 627 cm .
1H-NMR (300 MHz, CDCl3): 4.41 (dt, JHF = 47.3, JHH = 6.2,
2H), 2.85 (t, J = 7.3, 2H), 2.31 (s, 3H), 1.5-1.8 (m, 4H), 1.23–1.45
(m, 8H). 13C-NMR (75 MHz, CDCl3): 195.94, 84.09 (d, J = 163.2),
30.58, 30.33 (d, J = 19.3), 29.43, 29.06, 29.02, 28.93, 28.63, 25.05
(d, J = 5.4). 19F NMR (282.4 MHz, CDCl3): -218.27.
2
3
8-Fluorooctanesulfinyl chloride. Thioacetic acid S-(8-fluoro-
octyl) ester (1.84 g, 8.9 mmol) and acetic anhydride (0.84 ml,
0.91 g, 8.9 mmol) were combined in a 3-neck RBF equipped
with a magnetic stirrer bar and N2 inlet. The reaction vessel was
cooled to -10 ◦C and sulfuryl chloride (1.44 ml, 2.40 g, 17.8 mmol)
was added dropwise over a half hour. Stirring was continued for
another half hour at -10 ◦C. The solution was roto-evaporated to
1
1247, 1047, 1002, 724, 645 cm-1. H-NMR (300 MHz, CDCl3):
4.43 (dt, 2JHF = 47.3, 3JHH = 6.2, 2H), 3.41 (t, J = 6.8, 2H), 1.86
3
(p, J = 7.1, 2H), 1.69 (dm, JHF = 24.9, 2H), 1.34-1.50 (m, 8H).
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give the title compound (1.88 g, crude) as an amber oil: 4.44 (dt,
2JHF = 47.3, 3JHH = 6.1, 2H), 3.39 (m, 2H), 1.93 (m, 2H), 1.6-1.8
(m, 2H), 1.3-1.6 (m, 8 H). 13C-NMR (75 MHz, CDCl3): 84.08 (d,
J = 163.3), 64.43, 30.32 (d, J = 19.3), 29.05, 28.86, 28.25, 25.08
(d, J = 5.2), 22.22. 19F NMR (282.4 MHz, CDCl3): -218.40.
dH2O (10 ml). The combined organic phases were washed with sat.
NaCl (10 ml), dried (Na2SO4) and concentrated in vacuo to yield
the title compound (2.97 g, 8.8 mmol, 85%) as a pale oil. TLC
1
(EtOAc–hexane 15 : 85): Rf 0.67; H-NMR (300 MHz, CDCl3):
3.60 (t, J = 6.6, 2 H), 3.42 (t, J = 6.8, 2 H), 1.86 (m, 2 H), 1.47-
1.59 (m, 2 H), 1.32 (m, 10H), 0.89 (s, 9 H), 0.05 (s, 6 H). 13C-NMR
(75 MHz, CDCl3): 63.29, 34.03, 32.86, 29.42, 29.33, 29.72, 28.17,
31.60, 25.77, 18.90, -5.24.
1,2:5,6-Di-O-isopropylidene-a-D-glucofuranosyl
(+)-(R)-8-
fluorooctanesulfinate. To diacetone-D-glucose (0.77 g, 2.9 mmol)
◦
in dry THF (12 ml) under N2 gas at -78 C was added pyridine
(0.29 ml, 3.5 mmol) followed by 8-fluorooctanesulfinyl chloride
(3.52 mmol). The mixture was stirred for 3 h (-78 ◦C). The
reaction was quenched dropwise with water (5 ml) then dissolved
in DCM (50 ml). The resulting mixture was washed with 5%
HCl (50 ml), 2% NaHCO3 (50 ml), and sat. NaCl (50 ml), then
dried over Na2SO4 and concentrated by roto-evaporation. The
title compound was obtained by flash chromatography (40%
Et2O–hexanes), to obtain a thick, pale oil (0.79 g, 1.8 mmol, 62%,
dr R : S: 91 : 9). [a]D21 = +1.39 deg cm3 g-1 dm-1(c 1.10, acetone).
TLC (EtOAc–hexane 1 : 1): Rf 0.70. IR (film): 2987, 2935, 2859,
1457, 1374, 1256, 1217, 1165, 1136, 1076, 1023, 955, 887, 837,
754, 730, 687 cm-1. 1H-NMR (300 MHz, CDCl3): 5.91 (d, J = 3.5,
tert-Butyl-[10-(8-fluoro-octane-1-(S)-sulfinyl)-decyloxy]-dime-
thylsilane. Freshly ground magnesium turnings (0.14 g,
5.9 mmol) were covered with dry THF (1.5 ml) under N2 gas. To
this mixture, was added (9-bromo-nonyloxy)-tert-butyldimethyl-
silane (1.49 g, 4.4 mmol) in dry THF (7 ml). The resulting mixture
was refluxed for 7 h and then cooled to room temperature.
In another vessel, the (R)-DAG sulfinate ester prepared above
(0.33 g, 0.77 mmol) was dissolved in toluene (16 ml) and cooled to
0 ◦C. To this solution was added a portion of the Grignard reagent
(4.5 ml, 2.6 mmol) and the reaction mixture was stirred for 30 min
at 0 ◦C, allowed to warm to room temperature and stirred for
24 h. The solution was quenched with sat. NH4Cl (10 ml) and then
extracted with DCM (2 ¥ 10 ml). The combined organic phases
were dried over Na2SO4 and concentrated by roto-evaporation.
The title compound was obtained by flash chromatography (60%
EtOAc–hexanes) to obtain a white solid (0.27 g, 0.61 mmol, 80%).
TLC (EtOAc–hexane 75 : 25): Rf 0.30. IR (KBr): 3434, 2927, 2854,
1636, 1471, 1388, 1361, 1256, 1103, 1017, 934, 837, 776, 726,
1H), 4.78 (d, J = 3.5, 1H), 4.71 (d, J = 1.2, 1H), 4.43 (dt, 2JHF
=
47.3, 3JHH = 6.1, 2H), 3.96-4.17 (m, 4H), 2.81 (2dt, J = 7.8, 13.4,
2H), 1.70 (m, 2H), 1.28-1.47 (m, 10H), 1.50 (s, 3H), 1.41 (s, 3H),
1.32 (s, 3H), 1.30 (s, 3H). 13C-NMR (75 MHz, CDCl3): 112.40,
109.42, 105.35, 84.07 (d, J = 163.1), 83.84, 83.05, 80.93, 72.14,
67.70, 57.86, 30.33 (d, J = 19.3), 29.09, 28.93, 28.66, 26.86, 26.74,
26.19, 25.29, 25.10 (d, J = 5.3), 21.03. 19F NMR (282.4 MHz,
CDCl3): -218.36 EI-MS: m/z 423 (27 [M - 15]+), 365 (3), 249 (7),
185 (19), 170 (14, [FC8H16SO]+), 127 (28, [C6H7O3]+), 101 (100,
[C5H9O2]+), 85 (13), 59 (20), 43 (39, [C2H3O]+); HR-EI-MS m/z
423.1851 ([M–CH3]+, C19H32O7FS requires 423.1853).
1
2
663 cm-1. H-NMR (300 MHz, CDCl3): 4.42 (dt, JHF = 47.3,
3JHH = 6.1, 2H), 3.58 (t, J = 6.6, 2H), 2.64 (m, 4H), 1.68-1.84
(m, 4H), 1.58-1.84 (m, 2H), 1.22-1.54 (m, 20H), 0.88 (s, 9H), 0.03
(s, 6H). 13C-NMR (75 MHz, CDCl3): 84.09 (d, J = 163.2), 63.26,
52.47, 52.38, 52.38, 32.82, 30.32 (d, J = 19.2), 29.33, 29.30, 29.15,
29.08, 28.94, 28.87, 28.76, 25.97, 25.97, 25.97, 25.74, 25.07 (d,
J = 5.3), 22.60, 22.58, 18.36, -5.27, -5.27. 19F NMR (282.4 MHz,
CDCl3): -218.36. EI-MS: m/z 419 (2 [M - OH]+), 379 (100, [M–t-
Bu]+), 359 (3), 199 (3), 101 (7), 89 (3), 75 (24), 69 (16), 55 (16), 41
(10).
1,2:5,6-Di-O-isopropylidene-a-D-glucofuranosyl (-)-(S)-8-fluoro-
octanesulfinate. Obtained as for 1,2:5,6-di-O-isopropylidene-a-
D-glucofuranosyl-(+)-(R)-8-fluorooctanesulfinate, except for the
solvent (toluene) and the base (i-Pr2NEt). (30% yield, dr R : S:
17 : 83). [a]2D1 = -36.2 deg cm3 g-1 dm-1 (c 1.20, acetone). TLC
(EtOAc–hexane 1 : 1): Rf 0.67. IR (film): 2988, 2934, 2859, 1714,
tert-Butyl-[10-(8-fluoro-octane-1-(R)-sulfinyl)-decyloxy]-dime-
thylsilane. Obtained as for tert-butyl-[10-(8-fluoro-octane-1-(S)-
sulfinyl)-decyloxy]-dimethylsilane, using (S)-DAG sulfinate ester
(69% yield). Structural data identical for that of tert-butyl-[10-(8-
fluoro-octane-1-(S)-sulfinyl)-decyloxy]-dimethylsilane.
1
1457, 1373, 1256, 1220, 1165, 1135, 1075, 1022, 830. H-NMR
(300 MHz, CDCl3): 5.91 (d, J = 3.6, 1H), 4.74 (d, J = 2.3, 1H),
4.60 (d, J = 3.7, 1H), 4.43 (dt, 2JHF = 47.3, 3JHH = 6.1, 2H), 3.98-
4.32 (m, 4H), 2.78 (m, 2H), 1.60-1.79 (m, 2H), 1.30-1.48 (m, 10H),
1.51 (s, 3H), 1.43 (s, 3H), 1.34 (s, 3H), 1.31 (s, 3H). 13C-NMR
(75 MHz, CDCl3): 112.46, 109.24, 104.97, 84.08 (d, J = 163.1),
83.61, 80.38, 79.21, 72.40, 66.71, 57.37, 30.32 (d, J = 19.5), 29.06,
28.92, 28.64, 26.74, 26.71, 26.27, 25.19, 25.10 (d, J = 5.3), 21.26.
19F NMR (282.4 MHz, CDCl3): -218.37 EI-MS: m/z 423 (31 [M -
15]+), 367 (4). 365 (3), 249 (3), 185 (17), 127 (30, [C6H7O3]+), 101
(100, [C5H9O2]+), 85 (12), 59 (19), 43 (44, [C2H3O]+); HR-EI-MS
m/z 423.1858 ([M–CH3]+, C19H32O7FS requires 423.1853).
10-(8-Fluoro-octane-1-(S)-sulfinyl)-decan-1-ol. To a solution
of (S)-OTBS-sulfoxide (18 mg, 0.041 mmol) in MeOH (1.4 ml) at
0 ◦C was added (1S)-(+)-10-camphorsulfonic acid (2 mg, 9 mmol)
with stirring. The resulting solution was stirred for 45 min. The
reaction was quenched with sat. NaHCO3 (3.5 ml) and extracted
with DCM (3 ¥ 2 ml). The extracts were dried (Na2SO4) and
dried under a gentle flow of N2 to obtain the title compound as
a white solid (11 mg, 0.033 mmol, 81%). TLC (EtOAc–hexane
75 : 25): Rf 0.03. IR (KBr): 3437, 2923, 2850, 1636, 1468, 1417,
1063, 1018, 932, 879, 727 cm-1. 1H-NMR (300 MHz, CDCl3): 4.43
(9-Bromo-nonyloxy)-tert-butyldimethylsilane.
9-Bromo-1-
nonanol (2.33 g, 10.4 mmol), tert-butyldimethylsilyl chloride
(1.85 g, 12.3 mmol) and imidazole (1.85 g, 27.1 mmol) were
combined and DMF (2.6 ml) was added. The mixture was stirred
at room temperature under N2 overnight. The resulting cloudy
yellow mixture was diluted with ether (10 ml) and washed with
sat. NaCl (10 ml). The organic phase was evaporated in vacuo and
the resulting oil was partitioned between hexanes (3 ¥ 10 ml) and
2
3
(dt, JHF = 47.3, JHH = 6.1, 2H), 3.63 (t, J = 6.5, 2H), 2.65 (m,
4H), 1.28-1.84 (m, 26H). 13C-NMR (75 MHz, CDCl3): 84.14 (d,
J = 163.0), 62.95, 52.44, 52.39, 52.39, 32.73, 30.33 (d, J = 19.3),
29.25, 29.25, 29.09, 29.09, 28.95, 28.82, 28.76, 25.67, 25.08 (d, J =
5.2), 22.59, 22.59. 19F NMR (282.4 MHz, CDCl3): -218.34. EI-MS
(pk intensity, fragment): m/z 305 (72 [M - OH]+), 157 (27), 143
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Org. Biomol. Chem., 2010, 8, 1322–1328 | 1327
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(20, [C9H18OH]+), 83 (33), 69 (100), 55 (78), 41 (36). HR-EI-MS
J. Biol. Chem., 2009, 284, 18559; P. H. Buist, Nat. Prod. Rep., 2004,
21, 249; P. H. Buist, Chemistry of Fatty Acid Desaturases, Wiley
Encyclopedia of Chemical Biology, 2008, Vol. 2, p. 1, John Wiley &
Sons, Hoboken, NJ.
2 B. Behrouzian, C. K. Savile, B. Dawson, P. H. Buist and J. Shanklin,
J. Am. Chem. Soc., 2002, 124, 3277.
m/z 305.2319 ([M - OH]+, C17H34OFS requires 305.2314).
10-(8-Fluoro-octane-1-(R)-sulfinyl)-decan-1-ol. Obtained as
for (S) sulfoxide, using (R)-OTBS sulfoxide (83% yield). The
analytical data for this compound is identical to the other
enantiomer.
3 G. J. Schroepfer and K. Bloch, J. Biol. Chem., 1965, 240,
54.
4 B. G. Fox, J. Shanklin, J. Y. Ay, T. M. Loehr and J. Sanders-Loehr,
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B. G. Fox and E. Munck, Proc. Natl. Acad. Sci. U. S. A., 1997, 94,
2981.
D9 Desaturase-mediated sulfoxidation of
18-fluoro-10-thia-octadecanoyl-ACP
5 P. H. Buist and B. Behrouzian, J. Am. Chem. Soc., 1996, 118,
Incubation of 18-fluoro-10-thia-octadecanoyl-ACP with D9 desat-
urase was carried out at room temperature and reaction progress
was monitored for disappearance of starting material by GC-
MS analysis of silylated aliquots (0.1 ml TMCS + BSTFA).
The reaction mixture consisted of castor D9 desaturase dimer
(175 mg ml-1, 20 ml), Anabaena vegetative ferredoxin (2 mg ml-1,
300 ml), maize root NADPH:ferredoxin reductase (0.25 mg ml-1,
150 ml), 18-fluoro-10-thia-octadecanoyl-ACP (3.5 mg ml-1, 1.6 ml)
in a total volume of 7.5 ml of buffer. The reaction was initiated by
the addition of NADPH (21 mg l-1, 1.5 ml) and allowed to continue
for 30 min. At this time, additional aliquots of ferredoxin (150 ml),
reductase (75 ml), NADPH (750 ml), desaturase (6 ml) were added
and the reaction allowed to continue for a further 30 min. The
reaction was terminated with the addition of THF (1.5 ml) and
the thioester linkage was reduced to the corresponding primary
alcohol with the addition of NaBH4 (9 mg). The mixture was
diluted with water (3 ml) and extracted with dichloromethane
(4 ¥ 6 ml). The phases were separated by centrifugation and the
combined organics were evaporated under a steady stream of N2
6295.
6 P. H. Buist, H. G. Dallmann, R. R. Rymerson, P. M. Seigel and P.
Skala, Tetrahedron Lett., 1988, 29, 435.
7 P. H. Buist and D. M. Marecak, J. Am. Chem. Soc., 1992, 114,
5073.
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401; (b) B. Behrouzian, D. Hodgson, C. K. Savile, B. Dawson, P. H.
Buist and J. Shanklin, Magn. Reson. Chem., 2002, 40, 524; (c) C. E.
Rogge and B. G. Fox, Biochemistry, 2002, 41, 10141; (d) R. D. White
and B. G. Fox, Biochemistry, 2003, 42, 7828; (e) A. E. Tremblay, P. H.
Buist, D. Hodgson, B. Dawson, E. Whittle and J. Shanklin, Magn.
Reson. Chem., 2006, 44, 629.
9 B. G. Fox, K. S. Lyle and C. E. Rogge, Acc. Chem. Res., 2004, 37,
421.
10 K. Y. Y. Lao, D. J. Hodgson, B. Dawson and P. H. Buist, Bioorg. Med.
Chem. Lett., 2005, 15, 2799.
11 A. E. Tremblay, K. Y. Y. Lao, D. J. Hodgson, B. Dawson and P. H.
Buist, Bioorg. Med. Chem. Lett., 2009, 19, 5146.
12 W. H. Pirkle, S. D. Beare and R. L. Muntz, Tetrahedron Lett., 1974, 15,
2295.
13 P. H. Buist, D. Marecak, H. L. Holland and F. M. Brown, Tetrahedron:
Asymmetry, 1995, 6, 7.
14 A sample of racemic 1 was prepared by LiAlH4 reduction of the
corresponding methyl ester that was available from a previous study:
D. J. Hodgson, K. Y. Y. Lao, B. Dawson and P. H. Buist, Helv. Chim.
Acta, 2003, 86, 3688.
15 It should be noted that the presence of the fluorine tag in 1 results in
a change in priority of the pendant alkyl group (C8–C18) relative to
the nonfluorinated compound. This results in assignment of the (S)-
configuration to the sulfoxy centre of bio-1 whereas the corresponding
non-fluorinated parent compound is R configured.
16 I. Ferna´ndez, N. Khiar, J. M. Llera and F. Alcudia, J. Org. Chem., 1992,
57, 6789.
17 F. Daligault, A. Arbore´, C. M. Nugier-Chauvin and H. Patin,
Tetrahedron: Asymmetry, 2004, 15, 917.
18 P. H. Buist, H. G. Dallmann, R. R. Rymerson and P. M. Seigel,
Tetrahedron Lett., 1987, 28, 857.
19 M. M. Bradford, Anal. Biochem., 1976, 72, 248.
20 (a) J. M. Studts and B. G. Fox, Protein Expression Purif., 1999,
16, 109; (b) J. Shanklin, Protein Expression Purif., 2000, 19, 319;
(c) J. A. Broadwater and B. G. Fox, Protein Expression Purif., 1999, 15,
314.
and the residue was diluted with 1 ml of CDCl3 for analysis by 19
NMR.
F
Acknowledgements
This work was supported by an NSERC PGS1 (to A. T), NSERC
USRA (to N. T.); N.S.E.R.C. Grant OGP-2528 (to P. H. B.); by the
Office of Basic Energy Sciences of the United States Department
of Energy (to J. S.). We are grateful Dr Jose Abad (IIQAB, CSIC,
Barcelona, SP) for initially providing us with samples of (S)- and
(R)-AMA and procedures for their preparation.
Notes and references
1 J. Shanklin and E. B. Cahoon, Annu. Rev. Plant Physiol. Plant Mol.
Biol., 1998, 49, 611; J. Shanklin, J. E. Guy, G. Mishra and Y. Lindqvist,
21 F. L. M Pattison, J. Org. Chem., 1956, 21, 748–53.
1328 | Org. Biomol. Chem., 2010, 8, 1322–1328
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The Royal Society of Chemistry 2010
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