A chemoenzymatic access to D- and L-sphingosines employing hydroxynitrile lyases

Article abstract of DOI:10.1016/S0040-4020(99)01024-8

A chemoenzymatic access to D- or L-sphingosines is presented comprising of a total synthesis of the L-threo-isomer and formal syntheses of the other three isomers. Key to the development of a general synthetic strategy has been the use of enantiocomplementary hydroxynitrile lyases (Hnls) to yield an enantiomeric pair of starting materials. The (S)-Hnl from Hevea brasiliensis has been used to prepare L-threo-sphingosine in 14 steps and an overall 12% yield. Application of the (R)-Hnl from Prunus amygdaIus formally allows synthesis of D-threo- and D-erythro-sphingosines. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(99)01024-8

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 781–790
A Chemoenzymatic Access to d- and l-Sphingosines Employing
Hydroxynitrile Lyases
†
*
Dean V. Johnson, Ulfried Felfer and Herfried Griengl
¨
¨
Spezialforschungsbereich Biokatalyse, Institut fur Organische Chemie der Technischen Universitat Graz, Graz, A-8010 Austria
Received 15 October 1999; accepted 23 November 1999
Abstract—A chemoenzymatic access to d- or l-sphingosines is presented comprising of a total synthesis of the l-threo-isomer and formal
syntheses of the other three isomers. Key to the development of a general synthetic strategy has been the use of enantiocomplementary
hydroxynitrile lyases (Hnls) to yield an enantiomeric pair of starting materials. The (S)-Hnl from Hevea brasiliensis has been used to prepare
l-threo-sphingosine in 14 steps and an overall 12% yield. Application of the (R)-Hnl from Prunus amygdalus formally allows synthesis of
d-threo- and d-erythro-sphingosines. ᭧ 2000 Elsevier Science Ltd. All rights reserved.
Introduction
interest in the synthesis of bioactive compounds.8,9 These
enzymes are available with complementary selectivities, i.e.
the (R)-Hnls from Prunus amygdalus10 (PaHnl) and Linum
usitatissium11 yield (R)-cyanohydrins whilst the (S)-Hnls
from Hevea brasiliensis12 (HbHnl), Sorghum bicolor13 and
Manihot esculenta14 yield the corresponding (S)-cyano-
hydrins.
Sphingosines are long chain 2-amino-1,3 diols which exist
in four stereoisomeric forms. d-erythro-sphingosine has
been reported as an inhibitor of protein kinase-C1 (PKC),
an enzyme which mediates a number of cellular responses.1e
l-erythro- and the more commonly occurring d-erythro-
sphingosine are commercially available. Sphingosines are
the basic components of a much larger class of compounds
known as glycosphingolipids, which exhibit a number of
biological functions including HIV binding2 and ‘biological
markers’ for the detection of cancer.3 Furthermore, these
compounds are of considerable biological significance in
the area of cellular interactions.4 To date numerous
syntheses of sphingosines have been reported and they can
be divided into those prepared from chiral pool materials
(such as carbohydrates or l-serine), asymmetric reactions or
by chemoenzymatic means.5a The chemoenzymatic
methods are rarer.5 Of all the reports, the syntheses of
We wish to present a new method which allows access to
l-threo- and erythro-sphingosine using the HbHnl, as well
as the d-isomers of sphingosine using the PaHnl. In each
case the stereochemistry of the secondary hydroxyl center of
the sphingosine is determined by the selectivity of the Hnl
(Scheme 1). To date a chemoenzymatic method which
employs such a general synthetic sequence has yet to be
developed.
d-erythro-sphingosine by Randunz et al.,6a Polt et al.,6b Ito
Results and Discussion
et al.6c,d and Solladie-Cavallo et al. are noteworthy for
´
6e
l-Series sphingosines
their efficiency (35–50% yield) and brevity (4–6 steps).
Recently, Enders et al. have reported an elegant diastereo-
selective and enantioselective method to both d-erythro-
and l-threo-sphingosine.7
Enantiopure cyanohydrin (S)-2 (99% e.e., 90% yield) was
prepared from (E)-2-octenal (Scheme 2) employing the
(S)-selective HbHnl,15,16 followed by protection of the free
cyanohydrin. Transformation17 of the protected cyano-
hydrin (S)-2 gave the diastereomeric amine 3 (Scheme 2)
The high chemo- and stereoselectivity of biocatalysts has
promoted their use in the synthesis of natural products. In
this respect, production of enantiomerically pure cyano-
hydrins by hydroxynitrile lyases (Hnls) is of significant
1
(57% d.e. by H and 13C NMR spectra).18
Deprotection of 3 with tetrabutylammonium fluoride
1
(TBAF) yielded amine 4 (57% d.e. by H and 13C NMR
Keywords: hydroxynitrile lyases; sphingosines; selective coupling.
* Corresponding author. Present address: Institut fu¨r Organische Chemie
spectroscopy) and further treatment of the diastereomeric
mixture with 1,1-carbonyldiimidazole followed by sepa-
ration on silica gel column chromatography gave diastereo-
merically pure 5 and 6 (Ͼ99% e.e., Ͼ99% d.e.) (Scheme 3).
¨
der Technischen Universitat Graz, Stremayrgasse 16, A-8010 Graz,
Austria. Fax: ϩ43-316-8738740.
E-mail: dvj@orgc.tu-graz.ac.at
†
0040–4020/00/$ - see front matter ᭧ 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(99)01024-8

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D. V. Johnson et al. / Tetrahedron 56 (2000) 781–790
Scheme 1. Application of Hnls to the synthesis of the four isomers of C-18 sphingosine.
The configurations of the oxazolidinones 5 and 6 were
determined by 1H NMR spectroscopy, the trans-compound
5 displayed a coupling constant of 5.86 Hz between H1 and
H2, whilst the coupling constant for the cis-compound 6 was
8.02 Hz. These figures are consistent with those
previously reported for this type of cis- and trans-
oxazolidinones.17,19
The trans-oxazolidinone 5 was converted20 into the methyl
ester 7 without racemization. However the cis-oxazo-
lidinone 6 yielded some of the desired ester 8 (15%), but
predominantly the amide 9 (73%) which was interconverted
into 8 by prolonged reflux (7 days) in methanol under acidic
conditions (64% yield with 30% recovered starting material,
Scheme 3).
Scheme 2. Reagents: (a) Hnl from Hevea brasiliensis, HCN Refs. 16, 35, 100%. (b) tert-butyldiphenylsilyl chloride, imidazole, DMF, RT, 90%. (c) (i)
DIBALH, Et2O, 3 h, Ϫ70ЊC then NH4Br/MeOH (ii) benzylamine, 1.75 h, RT (iii) NaCN/NH4Br in MeOH, 2.25 h, Ϫ45ЊC, 93%.
Scheme 3. Reagents: (a) TBAF, THF, 20 min, RT, 100%. (b) 1,1-carbonyldiimidazole, CH2Cl2, 1 h, RT, 100%. (c) HCl(g), Et2O/MeOH then H2O, 10ЊC, 24 h,
88%. (d) MeOH, conc. HCl, reflux, 7 days, 64% with 30% recovered 9.

D. V. Johnson et al. / Tetrahedron 56 (2000) 781–790
783
Scheme 4. Reagents: (a) OsO4, N-methylmorpholino-N-oxide, acetone/pH 6.8 phosphate buffer, RT, 16 h, 93%. (b) NaIO4, acetone/water, RT, 16 h, 96%. (c)
C13H27CHI2 (21), CrCl2, DMF/THF, RT, 3 h, 32% (d) LiBH4, THF, 30 min, RT, 94%. (e) TIPSCl, DMF, 24 h, 0ЊC–RT, 96%. (f) Na/NH3(l), 5 min, Ϫ75ЊC,
70% (g) TBAF, THF, 1.25 h, 0ЊC, 94%. (h) 2N KOH, EtOH, 2 h, 85ЊC, 90%. (i) Ac2O, triethylamine, DMAP, 2 h, 0ЊC, 95%.
The trans-ester 7 was the diastereomer that possessed the
correct stereochemistry for conversion to the desired
l-threo-sphingosine. Cleavage of the heptenyl moiety of 7
was achieved in two steps by employing first, osmium tet-
roxide/NMO (to yield a mixture of diols 10)21 then sodium
metaperiodate in THF/water (overall 90% for the two
steps).22 The resultant aldehyde 11 was always freshly
prepared for use in the ensuing coupling reaction (Scheme 4).
Compound 21 was coupled27a,28 with the aldehyde 11 to
yield two products in an overall yield of 32%. The selec-
tivity proved to be good yielding a 31:1 ratio of compounds
12 and 13 in favor of the desired (E)-isomer (Scheme 4).29
This is in contrast to the Wittig couplings performed by
Sugawara et al.30 or Hudlicky et al.5a which proceeded
with a preference for (Z)-bond formation. The geometric
isomers (12 and 13) were separable by silica gel column
chromatography and their geometry was confirmed by
1
Reformation of the alkene (cf. 7) to its corresponding penta-
decyl form was then required. Employing the modified
Wittig conditions of Schlosser,23 which yield high (E)-selec-
tivity in alkene formation, attempts at coupling a phospho-
nium salt (tetradecyltriphenylphosphonium bromide)24 to
aldehyde 11 failed. Such conditions gave almost exclusive
formation of an (E)-alkene in the synthesis of d-erythro-
sphingosine by Schmidt and Zimmermann.25 The aldehyde
11 proved to be too sensitive to these basic reaction con-
ditions and only a complex mixture of products resulted
which were not fully characterized. Hence, we sought an
alternative method which would convert an aldehyde to an
(E)-alkene. Recently, the use of geminal dichromium
reagents, prepared from chromium dichloride and corre-
sponding geminal diiodo compound,26 have been reported
for selective (E)-alkene formation27 as well as a mild
alternative to the Wittig reaction.28 Thus, the required
1,1-diiodo compound 21 was prepared (25% yield) using
commercially available tetradecanal 20, hydrazine hydrate
and iodine (Scheme 5).26a
analyzing the coupling constants of their H NMR spectra.
Chiral HPLC analysis of 12 revealed that no racemization of
the aldehyde had occurred during the reaction. Conversion
of the ester 12 to the alcohol 14 was achieved using lithium
borohydride in THF (94%) (Scheme 6). The alcohol was
then converted to the triisopropylsilyloxyether 15, a suitable
protecting group for the ensuing basic deprotection of the
nitrogen.31 Treatment of 15 with sodium in liquid ammonia
at low temperature (Ϫ78ЊC, 5 min) cleanly removed the
N-benzyl group. Deprotection of the triisopropylsilyl
(TIPS) group in 16 proceeded smoothly with TBAF to
yield the oxazolidinone 17. Subsequent basic hydrolysis32
Scheme 5. Reagents: (a) (i) Hydrazine hydrate, Et2O; (ii) triethylamine, I2,
25%.

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D. V. Johnson et al. / Tetrahedron 56 (2000) 781–790
Scheme 6. Application to d-erythro-sphingosine.
Scheme 7. Reagents: (a) (R)-Hnl from P. amygdalus, HCN, 3ЊC; (b) tert-butyldiphenylsilylchloride, imidazole, DMF, RT, 30%.
of 17 yielded the desired l-threo-sphingosine (l-18) whose
spectral characteristics were consistent with those
previously reported.7,33 The integrity of the structural
assignment was supported by conversion of l-18 to the
known triacetate 19.7,33a The preparation of the cis-oxazo-
lidinone 8 (Scheme 3) followed by application of the same
chemistry shown in Scheme 4 represents a formal synthesis
of l-erythro-sphingosine (l-22) (Scheme 6).
have also developed a general synthetic method which
allows the formal synthesis of the other three stereoisomers
of the sphingosine family.
Experimental
General
1H and 13C NMR were recorded on Varian Gemini 200 MHz
and Bruker MSL 300 MHz instruments in CDCl3 (unless
otherwise stated). Chemical shifts are relative to TMS (d
0.00) with CHCl3 as internal standard [d 7.23 (1H) and d
76.90 (13C)]. FT-IR (in n/cmϪ1) were recorded on a Bomen
Michelson 100 instrument as a neat film on a NaCl disc
unless otherwise stated. Optical rotations were measured
using a Perkin–Elmer 341 instrument at 589 nm (Na line)
in a 1 dm cuvette. Melting points were determined on a
Bu¨chi melting point apparatus and are uncorrected.
Elemental analysis was made on EA 1108 CHN-analyzer.
HPLC enantiomeric separations were performed using a
Jasco 880-PU pump and a Jasco 875 UV/VIS detector
connected to a CHIRACEL OD-H chiral HPLC column
(25 cm×0.46 cm). For (R)- or (S)-2 a mobile phase con-
taining 99.75:0.25, heptane:isopropanol was employed and
75:25 heptane:isopropanol with 1% (v/v) triethylamine for
compounds 5–7, and 12. All chromatographic runs were
performed with a flow rate of 0.5 mL/min and detection at
254 nm. Tetrahydrofuran was distilled from sodium benzo-
phenone ketyl, methanol and dichloromethane from calcium
hydride, and DMF from dibutyltin dilaurate and desmopur-
15 (150 mg of each/100 mL DMF). Diethyl ether was dried
over calcium chloride, distilled and stored over sodium
wire. HCl(g) was dried by passing it through a flask contain-
ing concentrated sulfuric acid. NH3(g) was dried by passing it
through a column of solid potassium hydroxide and after
condensation at Ϫ40ЊC was distilled at atmospheric
pressure. (E)-2-octenal (1) was distilled under reduced
pressure prior to use. In all preparations sodium sulfate
d-Series sphingosines
In order to access the d-series sphingosines, a hydroxynitrile
lyase with a stereochemical preference to yield (R)-cyano-
hydrins was required (Scheme 1). Two such Hnls, those
from L. usitatissium and P. amygdalus (PaHnl) have
found biocatalytic applications for the production of
(R)-cyanohydrins.9 However, the wide substrate tolerance
and cheap source material (almonds) of the PaHnl makes
this the preferred catalyst for (R)-cyanohydrin formation.
This catalyst34 transformed (E)-2-octenal (1) into its corre-
sponding cyanohydrin, subsequent derivatization with tert-
butyldiphenylsilyl chloride yielded (R)-(2) (30% yield, 96%
e.e., Scheme 7).
The preparation of (R)-2 represents a formal synthesis of the
two d-series stereoisomers of sphingosine, since only the
application of the same chemical steps and reagents outlined
in Schemes 2–4 are required to convert (R)-(2) into the
isomers d-19 and d-22 (Scheme 7).
Conclusions
In conclusion we have developed a novel chemoenzymatic
method, which allows the total synthesis of l-threo-
sphingosine. By exploiting two hydroxynitrile lyases with
complementary enantioselectivites (i.e. those from the
HbHnl and PaHnl), the same achiral precursor (i.e. (E)-2-
octenal) and the same order of chemical steps/reagents we

D. V. Johnson et al. / Tetrahedron 56 (2000) 781–790
785
was used as the drying reagent. All other solvents and
reagents were obtained from commercial suppliers and
used as purchased. Petroleum ether refers to fraction of
boiling point 60–80ЊC. All products were purified by silica
gel column chromatography using Merck Kieselgel 60 F254
(230–400 mesh). TLC plates were run on silica gel Merck
60 F254, compounds were visualized by spraying with Mo-
reagent (NH4)6Mo7O24·4H2O (100 g/L), Ce(SO4)2·4H2O
(4 g/L) in H2SO4 (10% v/v) or KMnO4 reagent KMnO4
(2.5 g/L), Na2CO3 (20 g/L) in H2O. A recombinant hydro-
xynitrile lyase, which is homologous to the natural enzyme
from Hevea brasiliensis, was prepared by overexpression in
Pichia pastoris and a crude cytosolic extract35 was used for
the biotransformation of 5. A defatted sample of PnHnl was
prepared according to the literature procedure.36
7.35–7.51 (m, 6H), 7.63–7.75 (m, 4H); 13C NMR
(50 MHz) d 14.05, 19.31, 22.50, 26.71, 28.23, 31.28,
31.81, 63.59, 118.72, 124.71, 127.89, 128.02, 130.30,
130.43, 131.67, 132.15, 132.17, 135.82, 135.87, 136.90.
(ϩ)-(2R/S,3S,4E)-2-benzylamino-3-[(tert-butyldiphenyl-
silyl)oxy]dec-4-enenitrile (3). To a solution of (S)-2
(11.7 g, 30 mmol) in Et2O (240 mL) under an argon atmos-
phere at Ϫ70ЊC was added DIBALH (1.0 M in hexane,
75 mL, 75 mmol, 2.5 equiv.) over a 45 min period. Stirring
at this temperature was continued for a further 3 h. Then,
ammonium bromide (7.31 g, 57 mmol, 2.5 equiv.) in
methanol (120 mL) was added slowly and the mixture was
allowed to warm to room temperature (RT). Subsequently,
benzylamine (16.4 mL, 150 mmol, 5 equiv.) was added and
stirring continued for a further 100 min after which the
solution was cooled to Ϫ45ЊC and a mixture of sodium
cyanide (4.41 g, 90 mmol, 3 equiv.) and ammonium
bromide (8.78 g, 90 mmol, 3 equiv.) in methanol
(180 mL) was added dropwise. The mixture was stirred
for 100 min at Ϫ45ЊC then 1 h at RT, poured into a flask
containing water (500 mL) and Et2O (1000 mL), and stirred
for 15 min. The biphasic mixture was separated and the
aqueous layer was extracted with Et2O (2×250 mL). The
organic extracts were combined, dried, filtered and evapo-
rated under reduced pressure. Purification using flash
chromatography [98:2, petroleum ether:EtOAc with 1%
(v/v) triethylamine] yielded the amine 3 (14.00 g, 93%) as
a colorless oil: d.e. 57%, Rf 0.27 (95:5, petroleum ether:
Et2O); [a]2D0ˆϩ24.2 (c 4.35, MeOH); IR (cmϪ1) 2938,
2857, 2227 (w), 1427, 1110, 975, 739, 703; 1H NMR
(200 MHz) 0.85 (t, 3H, Jˆ6.3 Hz), 0.97–1.39 (m, 6H),
1.15 (s, 9H), 1.63 (bs, 1H), 1.90 (m, 2H), 3.40 (d, 0.2H,
Jˆ3.34 Hz), 3.49 (d, 0.8 H, 4.52 Hz), 3.98 (2×d, 1H,
Jˆ13.2 and 13.0 Hz), 4.02 (2×d, 1H, Jˆ13.2 and
13.0 Hz), 4.22 (dd, 0.8H, Jˆ8.05 and 4.57 Hz), 4.36 (dd,
0.2H, Jˆ7.5 and 3.3 Hz), 5.29–5.68 (m, 2H), 7.05–7.49 (m,
11H), 7.57–7.77 (m, 4H); 13C NMR (50 MHz) 14.05, 19.31,
22.50, 26.73, 26.94, 27.04, 28.20, 28.39, 31.32, 31.96,
32.11, 51.38, 55.93, 56.87, 74.53, 75.19, 118.37, 118.86,
126.75, 127.53, 127.79, 128.08, 128.21, 128.29, 128.35,
128.43, 128.59, 129.79, 129.96, 130.01, 133.20, 133.25,
133.30, 135.83, 135.99, 136.15, 136.39, 137.55, 138.27;
EIMS m/z (relative intensity) 483 (12), 453 (29), 426 (92),
365 (68), 336 (10), 309 (20), 199 (100), 183 (17), 135 (40),
91 (84), 78 (19), 57 (8); HRMS calcd for C33H42N2SiO:
510.3067, found: 510.3058.
(ϩ)-(2S,3E)-2-[(tert-butyldiphenylsilyl)oxy]non-3-eneni-
trile [(S)-2]. (E)-2-octenal (1) (10.0 g, 79.2 mmol) was
treated with HCN (10 equiv.) and the Hnl from Hevea
brasiliensis as catalyst using a reported method16,35 and
then derivatized with tert-butyldiphenylchlorosilane
(24.3 g, 88.5 mmol, 1.1 equiv.) and imidazole (10.2 g,
150 mmol, 1.9 equiv.).37 After work-up, the crude product
was purified by column chromatography using a gradient
mixture of petroleum ether:Et2O (98:2–90:10) as the eluent
which yielded (S)-2 (27.6 g, 90%) as a colorless oil:
e.e. Ͼ99%, Rf 0.42 (95:5, petroleum ether:Et2O);
[a]2D0ˆϩ25.6 (c 4.85, MeOH); IR (cmϪ1) 2937, 2857,
1
2227 (w), 1427, 1110, 975, 702; H NMR (200 MHz), d
0.9 (t, 3H, Jˆ7.0 Hz), 1.11 (s, 9H), 1.19–1.38 (m, 6H),
1.99 (m, 2H), 4.77 (d, 1H, Jˆ6.3 Hz), 5.50 (dd, 1H,
Jˆ15.2 and 6.4 Hz), 5.65 (dt, 1H, Jˆ15.2 and 6.6 Hz),
7.37–7.52 (m, 6H), 7.65–7.77 (m, 4H); 13C NMR
(50 MHz) d 14.04, 19.29, 22.49, 26.69, 28.21, 31.27,
31.80, 63.58, 118.72, 124.69, 127.85, 128.00, 130.28,
130.41, 131.75, 132.17, 135.81, 135.86, 136.89; EIMS m/z
(relative intensity) 391 (3), 334 (100), 307 (11), 264 (19),
239 (11), 209 (21), 199 (52), 181 (10), 109 (15), 78 (11), 57
(9); HRMS calcd for C25H33NSiO: 391.2331, found:
391.2340.
(Ϫ)-(2R,3E)-2-[(tert-butyldiphenylsilyl)oxy]non-3-eneni-
trile [(R)-2]. To a cooled solution (5ЊC) of (E)-2-octenal (1)
(504 mg, 4 mmol) in tert-butylmethyl ether (MTBE) was
added rehydrated (R)-Hnl from Prunus amygdalus36 (2 g)
then HCN (0.54 g, 0.78 mL, 20 mmol) and the mixture was
vigorously stirred using an overhead mechanical stirrer.
After 26 h, a further portion of (R)-Hnl was added (2 g).
After 42 h the enzyme was removed by filtration and
extracted with MTBE (2×40 mL). The conversion was
estimated to be 30% (by TLC). The organic layers were
combined, dried, filtered and concentrated in vacuo. The
crude product was then treated with tert-butyldiphenyl-
chlorosilane (550 mg, 2.00 mmol, 0.5 equiv.) and imidazole
(163 mg, 2.40 mmol, 0.6 equiv.) as previously described.37
After work-up, the silyl ether was purified by column
chromatography eluting with a gradient mixture of petro-
leum ether:Et2O (98:2–90:10) to yield (R)-2 (430 mg, 30%)
as a colorless oil: e.e. 96%, Rf 0.42 (95:5, petroleum ether:
Et2O); [a]2D0ˆϪ27.2 (c 3.90, MeOH); 1H NMR (200 MHz)
d 0.9 (t, 3H, Jˆ7.0 Hz), 1.11 (s, 9H), 1.21–1.37 (m, 6H),
1.96–2.06 (m, 2H), 4.78 (d, 1H, Jˆ7.05 Hz), 5.50 (dd, 1H,
Jˆ15.3 and 6.3 Hz), 5.72 (dt, 1H, Jˆ15.3 and 6.4 Hz),
(ϩ)-(2R/S,3S,4E)-2-benzylamino-3-hydroxydec-4-eneni-
trile (4). Tetrabutylammonium fluoride (1.0 M in THF,
21.9 mL, 21.9 mmol, 1.02 equiv.) was added dropwise to
a cooled (5ЊC) solution of 3 (10.6 g, 21.5 mmol) in THF
(445 mL). The mixture was allowed to warm to RT and
stirred for a further 1.5 h after which it was poured into a
mixture of EtOAc:saturated sodium bicarbonate solution
(550 mL:190 mL) and stirred for further 15 min. The
aqueous layer was saturated with sodium chloride, then
separated and extracted with EtOAc (2×200 mL). The
organic extracts were combined, dried, filtered and evapo-
rated under reduced pressure. Purification using flash chro-
matography and a gradient eluent mixture [80:20, petroleum
ether:EtOAc with 1% (v/v) triethylamine to 50:50 petro-
leum ether:EtOAc with 1% (v/v) triethylamine] yielded

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D. V. Johnson et al. / Tetrahedron 56 (2000) 781–790
the amine 4 (5.86 g, 100%) as a colorless oil: d.e. 58%, Rf
0.24 (80:20, petroleum ether:EtOAc); [a]2D0ˆϩ34.5 (c 5.19,
MeOH); IR (cmϪ1) 3436, 2937, 2247 (w), 1697, 1459, 1257,
(3.00 g, 10.06 mmol) in methanol:Et2O (15 mL:225 mL) was
bubbled dry HCl(g) for 10 min. The mixture was allowed to
stand at 10ЊC for 24 h after which it was further cooled to
4ЊC. Water (5 mL) was added and stirring continued for
20 min. The aqueous layer was separated, extracted with
Et2O (3×60 mL). The organic layers were combined,
washed with 5% sodium bicarbonate solution, dried, filtered
and concentrated under reduced pressure. Purification using
flash chromatography (80:20, petroleum ether:EtOAc)
yielded 7 (2.93 g, 88%) as a colorless oil: e.e. Ͼ99%, Rf
0.34 (80:20, petroleum ether:EtOAc); [a]2D0ˆϪ70.92
(c 5.35, MeOH); IR (cmϪ1) 2936, 2864, 1757, 1407, 1214,
1
1121, 980, 739, 699; H NMR (200 MHz, d4-MeOH), 0.92
(t, 3H, Jˆ6.27 Hz), 1.22–1.53 (m, 6H), 2.03–2.19 (m, 2H),
3.33 (bs, 0.5H), 3.05 (2×d, 1H), 3.92 (q, 2H, Jˆ13.1 Hz),
4.13–4.30 (m, 1H), 5.58 (dd, 1H, Jˆ15.21 and 7.29 Hz),
5.75–5.95 (m, 1H), 7.21–7.47 (m, 5H); 13C NMR (50 MHz,
d4-MeOH) 14.48, 23.62, 29.83, 32.52, 33.31, 33.35, 52.40,
56.65, 57.30, 73.14, 73.59, 119.52, 119.84, 128.56, 128.81,
129.57, 129.63, 135.80, 137.38, 139.88; EIMS m/z (relative
intensity) 245 (6), 199 (79), 181 (5), 146 (100), 127 (7), 120
(20), 91 (62), 78 (5), 57 (10); HRMS calcd for C17H24N2O:
272.1889, found: 272.1878.
1
989, 704; H NMR (200 MHz) 0.88 (t, 3H, Jˆ6.27 Hz),
1.16–1.45 (m, 6H), 1.97–2.13 (m, 2H), 3.72 (s, 3H), 3.75
(d, 1H, Jˆ5.13 Hz), 4.22 (d, 1H, Jˆ15.0 Hz), 4.87 (2×d,
2H, Jˆ15.2 and 11.9 Hz), 5.40 (dd, 1H, Jˆ15.2 and
6.96 Hz), 5.85 (dt, 1H, Jˆ15.3 and 6.78 Hz), 7.18–7.41
(m, 5H); 13C NMR (50 MHz) 14.03, 22.48, 28.29, 31.26,
32.01, 47.30, 52.84, 61.80, 76.64, 125.27, 128.20, 128.43,
128.91, 134.59, 137.38, 156.97, 169.47; EIMS m/z (relative
intensity) 331 (5), 287 (6), 272 (56), 228 (61), 182 (10), 106
(11), 91 (100), 65 (5); HRMS calcd for C19H25NO4:
331.1784, found: 331.1782.
(Ϫ)-(E,4S,5S)-3-benzyl-4-cyano-5-(1-heptenyl)oxazolidin-
2-one (5) and (Ϫ)-(E,4R,5S)-3-benzyl-4-cyano-5-(hept-1-
enyl)oxazolidin-2-one (6). To a stirred solution of the dia-
stereomeric mixture 4 (5.66 g, 20.80 mmol) in CH2Cl2
(60 mL) under an argon atmosphere was added a solution
of 1,1-carbonyldiimidazole (6.74 g, 41.6 mmol, 2 equiv.) in
CH2Cl2 (60 mL) and stirring was continued for 1 h. To this
was added a mixture of CH2Cl2:water (500 mL:200 mL),
the biphasic mixture separated and the aqueous layer
extracted with CH2Cl2 (2×200 mL). The organic extracts
were combined, dried, filtered and evaporated under
reduced pressure. Purification by column chromatography
(90:10, cyclohexane:EtOAc to 80:20 cyclohexane:EtOAc)
yielded, in order of elution, pure 5 (5.22 g, 84%) as a
colorless oil and pure 6 (963 mg, 16%) as a pale yellow oil.
(Ϫ)-(E,4S,5S)-3-benzyl-5-(1-heptenyl)-4-methoxycarbonyl-
oxazolidin-2-one (8). Through a cooled (3ЊC) solution of 6
(914 mg, 3.06 mmol) in methanol:Et2O (0.25 mL:3.8 mL)
was bubbled dry HCl(g) for 10 min. The mixture was
allowed to stand at 10ЊC for 24 h after which a further
portion of methanol (2 mL) was added and the mixture
stirred for further 3 h at 5ЊC. Water (5 mL) was added and
stirring continued for 45 min. The aqueous layer was
separated and extracted with EtOAc (3×70 mL). The
organic layers were combined, dried, filtered and concen-
trated under reduced pressure. Recrystallization from
acetone yielded 9 (780 mg, 73%) as a white solid. Rf 0.40
(50:50, petroleum ether: EtOAc); Mp 185–186ЊC,
[a]2D0ˆϪ36.78 (cˆ3.00, DMSO); IR (cmϪ1) 3381, 3215,
(Ϫ)-(E,4S,5S)-3-benzyl-4-cyano-5-(1-heptenyl)oxazolidin-
2-one (5). d.e.Ͼ99%, e.e. Ͼ99%, Rf 0.42 (80:20, petroleum
ether:EtOAc); [a]D20ˆϪ73.4 (c 3.05, MeOH); IR (cmϪ1
)
2935, 2860, 2247 (w), 1768, 1446, 1398, 1197, 970, 703;
1H NMR (200 MHz) 0.88 (t, 3H, Jˆ6.23 Hz), 1.15–1.50 (m,
6H), 1.98–2.18 (m, 2H), 3.93 (d, 1H, Jˆ5.86 Hz), 4.15 (d,
1H, Jˆ15.0 Hz), 4.98 (2×d, 2H, Jˆ15.0 and 13.4 Hz), 5.35
(dd, 1H, Jˆ15.3 and 7.65 Hz), 5.97 (dt, 1H, Jˆ15.2 and
6.82 Hz), 7.22–7.53 (m, 5H); 13C NMR (50 MHz, CDCl3)
13.99, 22.44, 28.09, 31.25, 32.04, 47.26, 51.21, 76.94,
114.82, 123.07, 128.59, 128.83, 129.30, 133.77, 140.26,
155.68; EIMS m/z (relative intensity) 254 (3), 150 (100),
106 (31), 91 (89), 65 (6); HRMS calcd for C18H22N2O2:
298.1681, found: 298.1690.
1
2924, 2856, 1732, 1677, 1427, 1228, 968, 704; H NMR
(300 MHz, d6-DMSO) 0.89 (t, 3H, Jˆ6.59 Hz), 1.21–1.42
(m, 6H), 2.01–2.08 (m, 2H), 3.35 (s, 0.75H), 3.83 (d, 1H,
Jˆ15.44 Hz), 4.11 (d, 1H, Jˆ8.51 Hz), 4.76 (d, 2H,
Jˆ15.4 Hz), 5.12 (t, 1H, Jˆ8.48 Hz), 5.42 (dd, 1H,
Jˆ15.3 and 8.48 Hz), 5.92 (dt, 1H, Jˆ15.3 and 6.75 Hz),
7.29–7.44 (m, 5H), 7.61 (bs, 0.5H); 13C NMR (50 MHz,
CDCl3) 13.79, 21.82, 27.71, 30.54, 31.43, 46.34, 60.43,
75.78, 123.12, 127.52, 127.68, 128.60, 136.08, 138.28,
157.51, 168.05, 168.11; Anal. calcd for C18H24N2O3: C,
68.33; H, 7.65; N, 8.85; Found: C, 68.35; H, 7.60; N, 8.77.
(Ϫ)-(E,4R,5S)-3-benzyl-4-cyano-5-(1-heptenyl)oxazolidin-
2-one (6). d.e.Ͼ99%, e.e. Ͼ99%, Rf 0.30 (80:20, petroleum
ether:EtOAc); [a]D20ˆϪ67.7 (c 5.49, MeOH); IR (cmϪ1
)
1
2919, 2243 (w), 1756, 1453, 1411, 1204, 972, 702; H
NMR (200 MHz) 0.88 (t, 3H, Jˆ6.59 Hz), 1.13–1.50 (m,
6H), 2.07–2.21 (m, 2H), 4.10 (d, 1H, Jˆ14.8 Hz), 4.35 (d,
1H, Jˆ8.02 Hz), 4.90 (d, 1H, Jˆ8.06 Hz), 5.00 (d, 1H,
Jˆ14.8 Hz), 5.68 (dd, 1H, Jˆ15.2 and 8.06 Hz), 6.05 (dt,
1H, Jˆ15.2 and 6.77 Hz), 7.28–7.50 (m, 5H); 13C NMR
(50 MHz) 14.01, 22.45, 28.11, 31.23, 32.19, 47.45, 51.52,
74.86, 113.33, 121.64 128.66, 128.80, 129.29, 134.05,
141.90, 155.83; EIMS m/z (relative intensity) 298 (3), 254
(17), 150 (100), 197 (10), 106 (39), 91 (81), 65 (6); HRMS
calcd for C18H22N2O2: 298.1681, found: 298.1687.
The mother liquor was concentrated under reduced pressure
and purified using column chromatography to yield 8
(150 mg, 15%) as a pale yellow oil Rf 0.36 (80:20, petro-
leum ether:EtOAc); [a]2D0ˆϪ45.29 (c 1.10, CH2Cl2); IR
1
(cmϪ1) 2926, 2856, 1759, 1439, 1214, 981, 704; H NMR
(200 MHz) 0.86 (t, 3H, Jˆ6.60 Hz), 1.20–1.36 (m, 6H),
1.97–2.08 (m, 2H), 3.67 (s, 3H), 4.00 (d, 1H,
Jˆ14.89 Hz), 4.12 (d, 1H, Jˆ8.98 Hz), 4.91 (d, 1H,
Jˆ14.89 Hz), 4.96 (t, 1H, 8.00 Hz), 5.30 (dd, 1H, Jˆ15.3
and 7.81 Hz), 5.89 (dt, 1H, Jˆ15.3 and 6.64 Hz), 7.20–7.34
(m, 5H); 13C NMR (50 MHz) 14.00, 22.47, 28.33, 31.20,
32.14, 47.44, 52.30, 61.12, 75.70, 121.78, 128.25, 128.43,
128.61, 128.95, 135.08, 139.37, 157.48, 168.28; EIMS m/z
(Ϫ)-(E,4R,5S)-3-benzyl-5-(1-heptenyl)-4-methoxycarbonyl-
oxazolidin-2-one (7). Through a cooled (3ЊC) solution of 5

D. V. Johnson et al. / Tetrahedron 56 (2000) 781–790
787
(relative intensity) 287 (4), 272 (22), 228 (30), 182 (9), 106
(11), 91 (100), 65 (5); HRMS calcd for C19H25NO4:
331.1784, found: 331.1785.
15.26, 15.35, 20.73, 47.42, 47.54, 47.77, 52.84, 54.88,
57.37, 57.50, 57.66, 57.76, 60.45, 63.29, 63.68, 77.09,
77.33, 77.47, 78.40, 89.99, 95.19, 95.42, 96.62, 96.72,
128.43, 128.70, 128.90, 129.30, 157.73, 170.98, 171.26,
197.09.
Compound 9 was converted into the desired ester 8 by the
following procedure: A solution of 9 (650 mg, 1.85 mmol)
in methanol (270 mL) and concentrated hydrochloric acid
(3 mL) was heated at reflux for 7 days. The mixture was
cooled to RT, concentrated to approximately one quarter
of its original volume, diluted with EtOAc (250 mL) and
washed with water (2×30 mL) and then brine (30 mL).
Silica gel column chromatography using a gradient elution
(petroleum ether:EtOAc, 5:1 to neat EtOAc) yielded in
order of elution 8 (400 mg, 64%) and 9 (200 mg, 30%).
(Ϫ)-(E,4R,5S)-3-benzyl-4-methoxycarbonyl-5-(1-penta-
decenyl)oxazolidin-2-one (12). All apparatus was covered
in aluminum foil to exclude light. To a stirred suspension of
CrCl2 (1.83 g, 14.9 mmol, 9.3 equiv.) under an argon atmos-
phere at RT in THF (36 mL) was added DMF (1.14 mL,
14.9 mmol, 9.3 equiv.). After 30 min a solution containing
iodoalkane 21 (1.68 g, 3.73 mmol, 2.3 equiv.) and aldehyde
11 (441 mg, 1.59 mmol) in THF (8 mL) was added and
stirring continued for 1.5 h. The mixture was then diluted
with EtOAc (30 mL) and poured into water (80 mL). Solid
NaCl was added to the point of saturation and the aqueous
layer extracted with EtOAc (3×40 mL). The combined
extracts were dried, filtered and concentrated under reduced
pressure. Purification using flash chromatography (95:5–
90:10–85:15, petroleum ether:EtOAc) yielded in order of
elution pure 12 (220 mg, 31%) as a colorless oil which
solidified upon storage in a refrigerator and the correspon-
ding pure cis-isomer 13 (7 mg, 1%).
(Ϫ)-(4R,5R)-3-benzyl-5-(1,2-dihydroxyheptyl)-4-methoxy-
carbonyloxazolidin-2-one (10). Ester 7 (1.63 g, 4.92 mmol)
was suspended in phosphate buffer (0.1 M, pH 6.8, 8.1 mL)
to which was added acetone (82 mL), then N-methylmor-
pholino-N-oxide (1.15 g, 9.84 mmol, 2 equiv.) followed by
two crystals of osmium tetroxide and stirred for 16 h at RT.
Sodium sulfite (3.70 g, 29.3 mmol, 5.9 equiv.) was added
and stirring was continued for 30 min. The mixture was
concentrated to one half of its original volume, diluted
with EtOAc (120 mL) and the organic phase washed with
water (2×10 mL), followed by brine (15 mL). The
combined organic extracts were dried, filtered and con-
centrated under reduced pressure. The crude residue was
purified using flash chromatography (99:1–99:2,
CH2Cl2:methanol) to yield 10 (1.66 g, 93%) as a white
solid. Mp 115–116ЊC; Rf 0.37 (50:50 petroleum ether:
EtOAc); [a]2D0ˆϪ42.44 (c 1.43, MeOH); IR (cmϪ1) 3403
(Ϫ)-(E,4R,5S)-methyl-3-benzyl-4-oxycarbonyl-5-(1-penta-
decenyl)oxazolidin-2-one (12). e.e. Ͼ99%, Rf 0.15 (85:15,
petroleum ether: EtOAc); [a]2D0ˆϪ53.09 (c 1.34, MeOH);
Mp. 31–32ЊC; IR (cmϪ1) 2923, 2852, 1764, 1753, 1445,
1
1407, 1203, 988, 704; H NMR (200 MHz) 0.88 (t, 3H,
Jˆ6.78 Hz), 1.25 (bs, 22H), 1.98–2.10 (m, 2H), 3.72 (s,
3H), 3.57 (d, 1H, Jˆ5.13 Hz), 4.21 (d, 1H, Jˆ15.0 Hz),
4.86 (2×d, 2H, Jˆ15.2 and 11.7 Hz), 5.39 (ddt, 1H,
Jˆ15.2, 6.98 and 1.3 Hz), 5.85 (dt, 1H, Jˆ15.2 and
8.43 Hz), 7.18–7.40 (m, 5H); 13C NMR (50 MHz) 14.16,
22.73, 28.51, 28.64, 28.94, 29.00, 29.11, 29.40, 29.47,
29.60, 29.70, 30.11, 31.96, 32.06, 47.29, 52.83, 61.80,
76.65,125.23, 128.18, 128.28, 128.42, 128.90, 135.10,
137.40, 157.18, 169.64; EIMS (EI) m/z (relative intensity)
384 (15), 340 (19), 228 (11), 106 (18), 91 (100), 44 (11);
HRMS calcd for C27H41NO4: 443.3036, found: 443.3043;
Anal. calcd for C27H41NO4.1H2O: C, 72.11; H, 9.77; N,
2.93; Found: C, 72.04; H, 9.74; N, 2.96.
1
(w), 2937 (w), 1758, 1743, 1415, 1227, 705; H NMR
(200 MHz, d6-acetone) 0.88 (t, 3H, Jˆ6.35 Hz), 1.20–1.57
(m, 8H), 3.45–3.57 (m, 1H), 3.67 (2×s, 4H), 4.22–4.38 (m,
3H), 4.61–4.74 (m, 2H), 7.28–7.34 (m, 5H); 13C NMR
(50 MHz, d6-acetone) 14.42, 23.39, 26.14, 26.23, 32.67,
33.79, 34.45, 47.71, 47.82, 52.92, 59.29, 70.73, 71.78,
73.95, 75.53, 77.01, 77.46, 128.53, 128.60, 129.00,
129.57, 129.52, 136.75, 137.15, 171.19, 183.49; EIMS m/z
(relative intensity) 365 (15), 306 (8), 262 (5), 234 (12), 205
(6), 220 (12), 176 (55), 130 (10), 106 (9), 91 (100), 65 (4);
HRMS calcd for C19H27NO6: 365.1838, found: 365.1838;
Anal. calcd for C19H27NO6: C, 62.58; H, 7.60; N, 3.77;
Found: C, 62.63; H, 7.67; N, 3.78.
(Ϫ)-(Z,4R,5S)-3-benzyl-4-methoxycarbonyl-5-(1-penta-
decenyl)oxazolidin-2-one (13). Rf 0.21 (80:20, petroleum
ether:EtOAc); [a]D20ˆϪ3.70 (c 0.60, CHCl3); 1H NMR
(200 MHz, CDCl3) 0.90 (t, 3H, Jˆ6.54 Hz), 1.24 (bs,
22H), 1.98–2.10 (m, 2H), 3.72 (s, 3H), 3.57 (d, 1H,
Jˆ5.13 Hz), 4.22 (d, 1H, Jˆ15.0 Hz), 4.90 (d, 1H,
Jˆ14.9 Hz), 5.21 (dd, 1H, Jˆ8.84 and 5.18 Hz), 5.36
(ddt, 1H, Jˆ10.3 and 8.92 Hz), 5.73 (dt, 1H, Jˆ10.5 and
7.32 Hz), 7.24–7.32 (m, 5H); 13C NMR (50 MHz) 14.17,
22.74, 27.83, 29.21, 29.30, 29.40, 49.11, 29.59, 29.60,
29.69, 29.70, 30.11, 31.96, 32.06, 47.39, 52.83, 62.25,
76.06, 125.11, 128.21, 128.43, 128.46, 128.94, 137.96,
137.40, 157.18, 169.64.
(Ϫ)-(4R,5R)-3-benzyl-5-formyl-4-methoxycarbonyloxa-
zolidin-2-one (11). The diol 10 (200 mg, 0.547 mmol) was
suspended in water (4 mL) to which was added acetone
(10 mL) followed by NaIO4 (195 mg, 0.915 mmol)
portion-wise. The mixture was stirred for 16 h at RT then
concentrated to approximately one quarter of its original
volume under reduced pressure, then solid NaCl was
added to the point of saturation and extracted with EtOAc
(3×30 mL). The combined organic extracts were dried
filtered and concentrated under reduced pressure. The
crude residue was purified using flash chromatography
(70:30, EtOAc:petroleum ether) to yield 11 (144 mg,
96%) as a colorless oil: Rf 0.22 (30:70, petroleum ether:
(Ϫ)-(E,4S,5S)-3-benzyl-4-(hydroxymethyl)-5-(1-penta-
decenyl)oxazolidin-2-one (14). To a stirred solution of
ester 12 (205 mg, 0.463 mmol) in THF (1.5 mL) at RT
was added LiBH4 (20 mg, 0.926 mmol, 2 equiv.). After
20 min the mixture was cooled on an ice bath to 5ЊC and
1
EtOAc); [a]2D0ˆϪ1.0 (c 0.8, CHCl3); H NMR (200 MHz,
d6-acetone) 1.07 (m, 2H), 3.84–3.81 (m, 3H), 4.19–4.31
(m, 2H), 4.42–4.49 (m, 1H), 4.69–4.85 (m, 1.5 H), 5.66–
5.81 (m, 0.5H), 13C NMR (50 MHz, d6-acetone)14.41,

788
D. V. Johnson et al. / Tetrahedron 56 (2000) 781–790
methanol (1.5 mL) was added. After stirring for a further
15 min, Amberlite IR-120-Hϩ resin (which had been
washed with MeOH) was added portion-wise until the
basic solution became neutral. The mixture was filtered
and the solvent removed under reduced pressure. The
residue was re-suspended in methanol (10 mL) which was
then removed under reduced pressure, a procedure which
was repeated a further two times in order to remove boric
acid as its methyl ester. Purification using flash chroma-
tography (70:30, petroleum ether:EtOAc) yielded 14
(180 mg, 94%) as a colorless oil: Rf 0.36 (60:40, petroleum
15 (144 mg, 0.25 mmol) in dry Et2O (1.35 mL) added. After
5 min phosphate buffer (0.5 M, pH 6.8, 2.8 mL) was added
quickly and stirring continued for another 10 min until the
blue color had disappeared. The mixture was allowed to
warm to RT. After 1 h, water (15 mL) was added and the
reaction mixture was extracted with EtOAc (3×20 mL), the
organic extracts were combined, dried, filtered and
concentrated under reduced pressure. Purification by flash
chromatography using a gradient mixture of petroleum
ether:EtOAc (90:10–80:20) containing 1% (v/v) triethyl-
amine yielded 16 (84 mg, 70%) as a colorless oil: Rf 0.42
(70:30, petroleum ether:EtOAc); [a]2D0ˆϪ48.9 (c 2.05,
MeOH); IR (cmϪ1) 3264 (w), 2926, 2859, 1756, 1121,
ether:EtOAc); [a]D20ˆϪ71.27 (c 1.53, MeOH); IR (cmϪ1
)
1
3494, 2920, 2850, 1730, 1712, 1430, 1128, 972, 704; H
NMR (200 MHz) 0.88 (t, 3H, Jˆ6.85 Hz), 1.25 (bs, 22H),
1.98–2.05 (m, 2H), 2.60 (bs, 1H), 3.32 (dt, 1H, Jˆ6.67 and
3.41 Hz), 3.50 (dd, 1H, Jˆ12.48 and 2.90 Hz), 3.75 (dd, 1H,
Jˆ12.35 and 3.17 Hz), 4.32 (d, 1H, Jˆ15.28 Hz), 4.69 (d,
1H, Jˆ15.30 Hz), 4.79 (t, 1H, Jˆ7.12 Hz), 5.40 (dd, 1H,
Jˆ15.32 and 7.83 Hz), 5.83 (dt, 1H, Jˆ15.27 and 6.74 Hz),
7.27–7.38 (m, 5H); 13C NMR (50 MHz,) 14.23, 22.81,
28.84, 29.25, 29.48, 29.57, 29.69, 29.79, 32.05, 32.25,
46.68, 59.76, 62.34, 76.85, 126.01, 128.14, 129.08,
136.38, 137.57, 157.78; EIMS m/z (relative intensity) 415
(3), 384 (61), 340 (46), 280 (5), 150 (9), 106 (11), 91 (100),
44 (12); HRMS calcd for C26H41NO3: 415.3087, found:
415.3082.
1
991 (w); H NMR (200 MHz) 0.88 (t, 3H, Jˆ6.85 Hz),
1.05 (bs, 21H), 1.25 (bs, 22H), 2.01–2.11 (m, 2H), 3.59–
3.36 (m, 1H), 3.69–3.72 (m, 2H), 4.71 (dd, 1H, Jˆ5.38 and
1.95 Hz), 5.54 (dd, 1H, Jˆ15.32 and 7.51 Hz), 5.82 (dt, 1H,
Jˆ15.4 and 6.60 Hz); 5.96 (bs, 1H); 13C NMR (50 MHz)
11.88, 14.15, 17.95, 22.73, 28.74, 29.15, 29.39, 29.50,
29.62, 29.71, 31.96, 32.12, 59.97, 64.78, 79.80, 126.37,
136.70, 159.11; EIMS m/z (relative intensity) 438 (30),
408 (33), 394 (100), 250 (25), 216 (7), 172 (12), 131 (42),
103 (25), 75 (13), 43 (30); HRMS calcd for C28H55NO3Si:
481.3951, found: 481.3951.
(Ϫ)-(E,4S,5S)-4-hydroxymethyl-5-(1-pentadecenyl)oxa-
zolidin-2-one (17). To
a solution of 16 (70 mg,
(Ϫ)-(E,4R,5S)-3-benzyl-5-(1-pentadecenyl)-4-(tri-iso-pro-
pylsilyloxymethyl)oxazolidin-2-one (15). To a solution of
imidazole (45 mg, 0.662 mmol, 2 equiv.) in DMF (50 mL)
at 0ЊC was added tri-isopropylchlorosilane (95 mg,
0.496 mmol, 1.5 equiv.). After stirring for 20 min, 14
(137 mg, 0.331 mmol) in DMF (200 mL) was added
dropwise and the mixture was allowed to warm to RT and
stirring continued for 24 h. The mixture was diluted with
water (5 mL), extracted with CH2Cl2 (3×15 mL) and the
organic extracts were combined, washed with brine solution
(5 mL), dried, filtered and concentrated under reduced
pressure. Purification using flash chromatography (95:5–
90:10, petroleum ether:EtOAc) yielded 15 (184 mg, 96%)
as a colorless oil: Rf 0.31 (90:10, petroleum ether:EtOAc);
[a]2D0ˆϪ36.30 (c 2.03, MeOH); IR (cmϪ1) 2926, 2858,
0.145 mmol) in THF (200 mL) at 0ЊC under an argon atmos-
phere was added TBAF (1.0 M in THF, 152 mL,
1.05 equiv.) dropwise over 5 min. Stirring was continued
for 75 min. The solution was neutralized by the portion-
wise addition of Amberlite IR-120-Hϩ resin. The resin
was removed by filtration and washed with EtOAc
(3×2 mL) and the solvent removed by evaporation under
reduced pressure. Purification using flash chromatography
[CH2Cl2:methanol, 95:5ϩ1% (v/v) triethylamine] yielded
17 (44 mg, 94%) as a white solid whose spectral character-
istics were consistent with those previously reported:30 Rf
0.23 (CH2Cl2:methanol, 95:5); Mp 101–102ЊC, (Lit. 104–
105ЊC),30 [a]D20ˆϪ63.9 (c 2.05, CHCl3), [Lit.34 Ϫ32.0 (c
0.5, CHCl3)]; Anal. calcd for C19H35NO3·1H2O: C, 70.51;
H, 11.46; N, 4.01; Found: C, 70.38; H, 11.41; N, 4.02.
1
1754, 1115, 970 (w); H NMR (200 MHz) 0.88 (t, 3H,
Jˆ6.85 Hz), 1.05 (bs, 21H), 1.25 (bs, 22H), 1.97–2.06 (m,
2H), 3.26–3.33 (m, 1H), 3.69 (dq, 2H, Jˆ10.74 and
4.64 Hz), 4.15 (d, 1H, Jˆ15.14 Hz), 4.72 (t, 1H,
Jˆ6.35 Hz), 4.84 (d, 1H, Jˆ15.14 Hz), 5.40 (dd, 1H,
Jˆ15.35 and 7.40 Hz), 5.79 (dt, 1H, Jˆ15.14 and
6.65 Hz), 7.27–7.38 (m, 5H); 13C NMR (50 MHz) 11.90,
14.17, 17.98, 22.74, 28.80, 29.14, 29.41, 29.51, 29.62,
29.72, 31.98, 32.14, 46.45, 61.65, 61.84, 77.08, 126.43,
127.85, 128.05, 128.79, 136.21, 136.52, 158.14; EIMS m/z
(relative intensity) 528 (60), 484 (20), 379 (25), 340 (20),
307 (5), 262 (16), 174 (7), 131 (62), 91 (100), 75 (35), 65
(20), 44 (40); HRMS calcd for C35H61NO3Si: 571.4421,
found: 571.4428.
l-threo-sphingosine: (Ϫ)-(E,2S,3S)-2-aminooctadec-4-ene-
1,3-diol [(l)-18]. A mixture of 17 (20 mg, 61.5 mmol) in
ethanol (0.48 mL) and 2N NaOH (0.48 mL) was heated at
85ЊC for 2.5 h. The mixture was then cooled to RT, diluted
with Et2O (10 mL) and washed with 2N NaOH (3×2 mL)
and then brine solution (2 mL). The organic layer was dried,
filtered and concentrated in vacuo. The crude residue was
purified by flash chromatography [CH2Cl2:methanol,
90:10ϩ1% (v/v) NH4OH (25% in H2O)] to yield (l)-18
(16 mg, 90%) as a white solid whose spectral characteristics
were identical with those previously reported.7,33a,b Mp 84–
85ЊC, (Lit.7: 86–87ЊC); [a]D20ˆϪ2.40 (c 0.8, CHCl3), [Lit.7
Ϫ2.83 (c 1.0, CHCl3)].
(Ϫ)-(E,4R,5S)-5-(1-pentadecenyl)-4-(tri-iso-propylsilyloxy-
methyl)oxazolidin-2-one (16). To a flask containing freshly
distilled liquid ammonia under an argon atmosphere at
Ϫ65ЊC was added sodium (132 mg, 5.74 mmol, 23 equiv.)
and stirring continued for 30 min. The blue–bronze colored
solution was then further cooled to Ϫ75ЊC and a solution of
l-threo-sphingosine-N,O,O-triacetate (19). The compound
was prepared according to the method reported by Enders et
al.7 which afforded a colorless solid (16 mg, 95%) whose
spectral characteristics were identical to those previously
reported.7,33a Mp 42–43ЊC, (Lit.7: 41–42ЊC); [a]2D0ˆϩ8.35
(c 0.8, CHCl3), [Lit.7: ϩ8.78 (c 1.2, CHCl3)].

D. V. Johnson et al. / Tetrahedron 56 (2000) 781–790
789
9. For a review in this area: Johnson, D.V.; Griengl, H.; Adv.
Biochem. Eng. Biotechnol., 1998, 63, 31.
10. (a) Brussee, J.; Loos, W. T.; Kruse, C. G.; van der Gen, A.
Tetrahedron 1990, 46, 979. (b) Effenberger, F.; Ziegler, T.;
Fo¨rster, S. Angew. Chem. 1987, 99, 491; Angew. Chem. Int. Ed.
Engl. 1987, 26, 458.
1,1-Di-iodotetradecane (21). This compound is light sensi-
tive and all apparatus should be covered in aluminum foil to
exclude light. Compound 21 was prepared using the pro-
cedure of Pross et al.26a employing tetradecanal (10 g,
47.1 mmol), hydrazine hydrate (10.61 g, 212 mmol,
4.5 equiv.) and triethylamine (126 mL, 904 mmol,
19 equiv.). Purification using column chromatography and
neat hexane as the eluant yielded 21 (4.12 g, 22%) as a pale
11. Albrecht, J.; Jansen, I.; Kula, M.-R. Biotechnol. Appl.
Biochem. 1993, 17, 191.
red oil which solidified upon storage at Ϫ20ЊC: IR (cmϪ1
)
´
12. Schmidt, M.; Herve, S.; Klempier, N.; Griengl, H. Tetra-
2922, 2852, 1460, 1121, 1088, 721, 620, 545 (w); 1H NMR
(200 MHz) 0.88 (t, 3H, Jˆ6.23 Hz), 1.16–1.55 (m, 22H),
2.33 (m, 2H), 5.11 (t, 1H, Jˆ6.59 Hz, CHI2); 13C NMR
(50 MHz) 14.19, 22.75, 27.68, 29.41, 29.54, 29.63, 29.70,
29.73, 31.88, 31.98, 48.40.
hedron 1996, 52, 7833.
13. Kiljunen, E.; Kanerva, L. T. Tetrahedron: Asymmetry 1996, 7,
1105.
¨
14. Forster, S.; Roos, J.; Effenberger, F.; Wajant, H.; Sprauer, A.
Angew. Chem. 1996, 108, 493; Angew. Chem. Int. Ed. Engl., 35,
437.
15. (E)-2-Hexadecenal was selected as the initial substrate for the
HbHnl biotransformation as it contains the 15 carbons which make
up the hydrophobic portion of the sphingosine. However to date,
this aldehyde has not been transformed enantioselectively in either
an optimized aqueous12 or biphasic16 system.
Acknowledgements
The authors wish to acknowledge Ms Carina Illaszewicz, Dr
¨
Lothar Brecker and Dr Hansjorg Weber for recording the
¨
16. Griengl, H.; Klempier, N.; Pochlauer, P.; Schmidt, M.; Shi, N.;
NMR spectra and Dr Romano V. A. Orru for his useful
discussions during this work. Michael Schmidt is acknowl-
edged for the preparation of the corresponding cyanohydrin
of silyl compound (S)-2. Dr Wolfgang Kern is thanked for
recording the IR spectra, Dr Robert Saf for recording the MS
spectra, Mrs Gabriele Koberwein from Karl-Franzens
University Graz for performing elemental analysis and DI
Antonina Zabelinskaja-Mackova for preparing the HCN. Dr
Philipp Hadwiger is acknowledged for his critical proof-
reading of this manuscript and Dr Christoph Marschner
for his advice with the chromium coupling reaction.
Zabelinskaja-Mackova, A. A. Tetrahedron 1998, 54, 14477.
17. Zandbergen, P.; Brussee, J.; van der Gen, A.; Kruse, C. G.
Tetrahedron: Asymmetry 1992, 3, 769.
18. Variation of the amount of hydrogen cyanide used in the reac-
tion (1–6 equiv.) or of the temperature at which the cyanide addi-
tion was performed (Ϫ78ЊC to RT) showed no positive
improvement on the resultant diastereoselectivity of the amine 3.
19. Dondoni, A.; Fantin, G.; Fogagnolo, M.; Pedrini, P. J. Org.
Chem. 1990, 55, 1439.
20. Warmerdam, E. G. J. C.; van den Nieuwendijk, A. M. C. H.;
Kruse, C. G.; Brussee, J.; van der Gen, A. Recl. Trav. Chim. Pays.
Bas. 1996, 115, 20.
21. A 3:1 diastereomeric mixture of diols 10 was obtained. This
mixture was inseparable by column chromatography and the
stereochemical preference for this reaction was not determined.
22. When a one-step oxidative cleavage procedure of 7 using low
temperature (Ϫ78ЊC, CH2Cl2) ozonolysis was performed a slightly
lower yield for the formation of the aldehyde was obtained (82%).
23. (a) Schlosser, M.; Christmann, K. F. Angew. Chem. 1966, 78,
115; Angew. Chem. Int. Ed. Engl. 1966, 5, 126. (b) Schlosser, M.;
Christmann, K. F. Liebigs Ann. Chem. 1967, 708, 1. (c) Wenger, R.
M. Helv. Chim. Acta 1983, 66, 2308.
24. Garigipati, R. S.; Freyer, A. J.; Whittle, R. R.; Weinreb, S. M.
J. Am. Chem. Soc. 1984, 106, 7861.
25. (a) Schmidt, R. R.; Zimmermann, P. Tetrahedron Lett. 1986,
27, 481. (b) Zimmermann, P.; Schmidt, R. R. Liebigs Ann. Chem.
1988, 663.
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D. V. Johnson et al. / Tetrahedron 56 (2000) 781–790
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30
Reaction time (min)
Yield(%)a
a RˆH
5
10
30
76
91
b RˆTBDMS
c RˆTIPS
15
10
5
c RˆTIPS
a In all experiments the starting material was fully consumed.