New synthesis of sn-1,2- and sn-2,3-O-diacylglycerols application to the synthesis of enantiopure phosphonates analogous to triglycerides: A new class of inhibitors of lipases

Article abstract of DOI:10.1002/(SICI)1099-0690(199907)1999:7<1671::AID-EJOC1671>3.0.CO;2-Z

Phosphonate compounds mimic the first transition state occurring during enzymatic carboxyester hydrolysis of natural substrates by forming a covalent bond with the catalytic serine. However, until now the organophosphorus compounds used in the inhibition studies more or less resembled a natural triglyceride substrate. In order to elucidate the interfacial activation and the mechanism of action of lipases, specific inhibitors need to be prepared. To achieve this goal, enantiomerically pure sn-1,2- and sn-2,3O- didecanoylglycerol compounds were prepared - starting from a C-4 chiral synthon, 3-buten-1,2-diol - and treated with n-pentylphosphonic dichloride and p-nitrophenol to afford the corresponding diastereomeric phosphonates, which were acylglycerol analogs. Subsequent separation of each of the phosphonate diastereomers A/B or ent-A/ent-B, performed by HPLC, led to four enantiopure stereoisomers that will be investigated as inhibitors of Human Pancreatic Lipase (HPL) and Human Gastric Lipase (HGL) using the monomolecular film technique.

Full text of DOI:10.1002/(SICI)1099-0690(199907)1999:7<1671::AID-EJOC1671>3.0.CO;2-Z

FULL PAPER
New Synthesis of sn-1,2- and sn-2,3-O-Diacylglycerols ؊
Application to the Synthesis of Enantiopure Phosphonates Analogous to
Triglycerides: A New Class of Inhibitors of Lipases
[a]
[a]
[b]
[a]
´
Frank Marguet, Jean-Francois Cavalier, Robert Verger, and Gerard Buono*
¸
Keywords: Asymmetric synthesis / “Chiron” / Enzymatic resolution / Diacylglycerophosphonates / Lipase inhibitor
Phosphonate compounds mimic the first transition state
occurring during enzymatic carboxyester hydrolysis of
natural substrates by forming a covalent bond with the
from a C-4 chiral synthon, 3-buten-1,2-diol – and treated
with n-pentylphosphonic dichloride and p-nitrophenol to
afford the corresponding diastereomeric phosphonates,
catalytic serine. However, until now the organophosphorus which were acylglycerol analogs. Subsequent separation of
compounds used in the inhibition studies more or less
resembled natural triglyceride substrate. In order to
elucidate the interfacial activation and the mechanism of
action of lipases, specific inhibitors need to be prepared. To
each of the phosphonate diastereomers A/B or ent-A/ent-B,
performed by HPLC, led to four enantiopure stereoisomers
that will be investigated as inhibitors of Human Pancreatic
Lipase (HPL) and Human Gastric Lipase (HGL) using the
a
achieve this goal, enantiomerically pure sn-1,2- and sn-2,3- monomolecular film technique.
O-didecanoylglycerol compounds were prepared – starting
we have focused our research on a new class of inhibitors
of lipases that involve a phosphonate reactive group in a
Introduction
The design and synthesis of specific inhibitors of various
animal and microbial lipases is of fundamental value for
understanding the mechanisms involved in the catalytic ac-
tivity of lipases. Furthermore, lipase inhibitors should have
potential in terms of useful applications in the fields of
chemistry, biochemistry, and medicine. Among the range
of lipase inhibitors known, phosphonate compounds are of
fundamental interest to better characterize the mechanism
of catalysis. As revealed by X-ray crystallography,[1] these
compounds mimic, in both their charge distribution and
geometry, the first transition state occurring during enzy-
matic carboxyester hydrolysis of natural substrates. They
are thus efficient inhibitors of lipases by formation of a co-
valent bond between the nucleophilic Oγ oxygen of the ac-
tive serine of the enzyme and the phosphorus atom.[1][2]
However, the organophosphorus compounds used in these
crystallographic studies to a large extent resemble a natural
triacylglycerol substrate. Despite the well recognized im-
portance of these enzymes, relatively little progress has been
made towards elucidation of their interfacial activation and
mechanism of action. In order to achieve this goal, specific
inhibitors need to be prepared.
triacylglycerol structure 1 (Figure 1).
Figure 1. Structures of 1,2- and 2,3-diacyl-sn-glycerophosphonates
analogous to triglycerides
In an attempt to further characterize the catalytic mecha-
nism of digestive lipases by using phosphorus inhibitors,[2]
The phosphonate moiety is located at an external posi-
tion, sn-1 or sn-3, of the glycerol backbone, with p-nitro-
phenoxy as a leaving group. We have intentionally retained
the two carbonyl ester linkages in order to mimic as closely
as possible the structure of acylglycerols (natural substrates
of lipases). These molecules have two chiral centers and, as
such, exist as four stereomers. Because of the presence of
the p-nitrophenoxy, which is a good leaving group, on the
phosphorus atom, the strategy for the synthesis of these di-
acylglycerophosphonates 1 requires the formation of the
[a]
`
´
Laboratoire de Synthese Asymetrique, ENSSPICAM, UMR
`
´
6516, “Synthese, Catalyse, Chiralite”,
avenue Escadrille Normandie-Niemen, F-13397 Marseille Cedex
20
Fax: (internat.) ϩ 33-4/91027776
E-mail: buono@spi-chim.u-3mrs.fr
Laboratoire de Lipolyse Enzymatique, UPR 9025, IFR 1 du
CNRS,
31, Chemin Joseph Aiguier, F-13402 Marseille Cedex 20
Fax: (internat.) ϩ 33-4/91715857
E-mail: verger@ibsm.cnrs-mrs.fr
[b]
Eur. J. Org. Chem. 1999, 1671Ϫ1678
WILEY-VCH Verlag GmbH, D-69451 Weinheim, 1999
1434Ϫ193X/99/0707Ϫ1671 $ 17.50ϩ.50/0
1671

F. Marguet, J.-F. Cavalier, R. Verger, G. Buono
FULL PAPER
phosphonate unit during the last step. The elaboration of
the diglyceride unit will then be achieved before the coup-
ling with the phosphorus moiety.
In this article, we describe a new synthesis of racemic
and chiral 1,2- and 2,3-diglycerides Ϫ starting from a C-4
synthon, 3-buten-1,2-diol Ϫ and the isolation of each of the
four organophosphorus stereoisomers 1, which are anal-
ogous to triglycerides.
Results and Discussion
We decided to synthesize a triglyceride analog, in which
the two acyl ester chains possess ten carbon atoms, in order
to perform specific inhibition tests using the monomolecu-
lar film technique.[3] Moreover, because inhibition of diges-
tive lipases is improved with short alkyl chains,[2] the length
of the saturated (R3) alkyl chain on the phosphorus atom
was fixed to five carbon atoms.
Scheme 1. Racemic synthesis of 1,2-diglyceride 9 by the benzylation
pathway. Reagents and conditions: (a) H2SO4, HgSO4, H2O, micro-
wave oven. Ϫ (b) BzlBr, NaH, Bu4NI, THV. Ϫ (c) Pivaloyl chloride,
CH2Cl2, Pyr, 0°C to room temp. Ϫ (d) O3, Me2S, CH2Cl2, Ϫ78°C.
Ϫ (e) NaBH4, CH2Cl2, EtOH, 0°C. Ϫ (f) Decanoic acid, DCC,
DMAP, Et2O. Ϫ (g) 5% Pd/C, H2, EtOH.
We began the synthesis with a C-4 synthon: 3-buten-1,2-
diol (3). This compound was obtained by isomerization of
(Z)-2-buten-1,4-diol (2) in an acidic medium catalysed by
mercuric ions.[4] Synthon 3 is important for the elaboration
of many building blocks.[5] It has been used as an inter-
mediate in the synthesis of pharmaceutical com-
pounds,[6a,6b] natural products[6c] as well as for the synthesis
of (2R,3R,5E)-2-hydroxy-3-methyl-5-heptenal (4).[7] This
latter compound is an important intermediate in the syn-
thesis of (MeBmt),[4][7] which is a nonnatural amino acid
constituting cyclosporine. (R)-(ϩ)- and (S)-(Ϫ)-1-tosyloxy-
3-buten-2-ol (12), both prepared from 3, are attractive chi-
ral precursors for the synthesis of chiral phosphonates anal-
ogous to triglycerides. Chiron 3 can be efficiently obtained
from compound 12 by various pathways: for instance, by a
kinetic resolution through a Sharpless asymmetric epoxida-
tion,[8a] or by an enzymatic esterification.[8b,8c] Moreover,
compound 3 can also be prepared from butadiene mono-
epoxide using the method recently described by Jacobsen
et al.[8d,8e]
5 and 6 could not be separated either by distillation nor by
silica gel chromatography, we employed selective chemical
reagents with respect to primary alcohol functions: TsCl,[9a]
PivCl,[9b] TrCl.[9c] Although the PivCl reacted much faster
than the other two (4 h for a complete conversion against
at least 12 h for TsCl or TrCl), the yields of alcohol 5 were
similar with all three reagents (75%). Subsequent ozonolysis
of recovered 1-benzyloxy-3-buten-2-ol (5) in the presence of
dimethyl sulfide, followed by sodium borohydride re-
duction, led to the 1,2-diol 7 in 84% yield. This compound
was then esterified to give 3-O-benzyl-1,2-di-O-decanoyl-
rac-glycerol (8) in 93% yield. The subsequent hydro-
genolysis of 8 with 5% Pd/C in ethanol gave the 1,2-di-
glyceride compound 9 in 90% yield, without the presence
of the 1,3-isomer.
The synthesis has been optimized in order to include a
regioselective benzylation step. Several methods were ex-
plored and the most interesting one is represented in
Scheme 2.
Racemic Synthesis
A synthetic method for the preparation of racemic 1,2-
diglycerides 9 is outlined in Scheme 1.
The first step requires the mono-protection of the pri-
mary alcohol function of 3 in order to elaborate the 1,2-
diacylglycerol unit. We employed benzyl bromide to avoid
the isomerization of 1,2-diglyceride into the 1,3-diglyceride
during cleavage of the protecting group. The benzylation of
diol 3 was not selective and led to a mixture of two isomers
in 52% yield: the secondary alcohol 5 and the primary al-
cohol 6 in a 70:30 ratio. Optimization of the reaction me-
dium (decrease of the temperature of the reaction, slower
addition of benzyl bromide) did not significantly improve
the selectivity of the reaction (80:20 ratio). Furthermore,
under these modified conditions, a marked decrease in the
degree of conversion of the reaction was observed with time
Scheme 2. Racemic synthesis of 1,2-diglyceride 9 by the benzyli-
dene acetal pathway. Reagents and conditions: (a) PhCHO, p-
TsCOH, PhMe, reflux. Ϫ (b) O3, Me2S, CH2Cl2, Ϫ78°C. Ϫ (c)
NaBH4, CH2Cl2, EtOH, 0°C. Ϫ (d) Decanoic acid, DCC, DMAP,
in comparison with the original compounds. Since alcohols Et2O. Ϫ (f) 5% Pd/C, H2, EtOH.
1672 Eur. J. Org. Chem. 1999, 1671Ϫ1678

New Synthesis of sn-1,2- and sn-2,3-O-Diacylglycerols
FULL PAPER
The reaction of diol 3 with benzaldehyde, catalysed by p- dium (entry 2) did not have any effect on the enantioselec-
TsOH, led to two epimers of the benzylidene acetal 10 in tivity of the enzymatic reaction. Nevertheless, when the re-
90% yield. Ozonolysis of 10, followed by sodium borohy- action was performed at a lower temperature (5°C), chiral
dride reduction, gave the alcohol 11 in 74% yield. Next, discrimination increased to give an E value of 59 (entry 3).
opening of the acetal ring with DIBALH in ether at 0°C
In contrast, enzymatic resolution of “tosylated alcohol”
led exclusively to compound 7 in 80% yield. The selective (R ϭ Ts) 12, performed under similar conditions as in the
formation of 7 can be rationalized in terms of initial forma- case of benzylic alcohol 5, was more enantioselective (Table
tion of aluminium alkoxide.[10] The aluminium atom plays 1, entries 4Ϫ5). At room temperature (entry 4), the enanti-
the role of a Lewis acid for the coordination to the neigh- omer ratio was 104 as compared to 18 in the previous case.
boring oxygen atom, which allows selective cleavage of the A decrease in the temperature of the reaction medium
cyclic acetal site to give the vicinal diol 7. Finally, as pre- (10°C) gave rise to an increase in E to 149, coinciding with
viously depicted in Scheme 1, acylation of 7 and subsequent an excellent chiral discrimination, for a conversion of only
hydrogenolysis led to 1,2-diglyceride 9.
21% (entry 5). Furthermore, in hexane/ether solution we
did not observe deactivation of the enzyme by acidic con-
taminants, which is in contrast to the work described by
Boaz et al.[8b]
Enantioselective Synthesis
Elaboration of Chiral 1,2- and 2,3-Diacylglycerols. 1,2-di-
acyl-sn-glycerols are easily available from the chiral pool,
starting from -mannitol,[19] but 2,3-diacyl-sn-glycerols are
not so easy to synthesize. With the method we have devel-
oped, both of these enantiomers are easy to prepare. As the
chiral recognition by the lipase PS was more marked with
1-tosyloxy-3-buten-2-ol (12) (Table 1), we decided to use
(S)-12 for the synthesis of 2,3-diacyl-sn-glycerols (R)-9, as
depicted in Scheme 3. In order to avoid formation of epoxy-
butene during the next step (Scheme 3-b), the alcohol func-
tion of (S)-12 was first acetylated to give compound (S)-13.
The tosylate group was then substituted with cesium acetate
in DMSO/DMF to form the diacetate (S)-14 in quantitative
yield. Saponification with methanolic K2CO3 produced the
chiral diol (S)-3 in 65% yield. Next, the chemical pathway
developed in Scheme 2 for racemic compounds led to 2,3-
O-didecanoyl-sn-glycerol [(R)-9].
Enzymatic resolution employing a lipase as a catalyst was
used in order to obtain optically pure compounds. Many
different alcohols can be resolved easily by this method.[11]
Transesterification of 3-buten-1,2-diol (3) as well as al-
coholysis in an organic medium of the corresponding di-
acetates catalysed with different lipases are highly regiose-
lective.[12] However, if lipases show only low enantioselec-
tivities during the monoacylation step,[13] the acylation of
the second alcohol group is more enantioselective than that
of the first alcohol group.[14] Moreover, transesterification
or hydrolysis catalysed by lipases of mono-protected diols
or corresponding acylated compounds, are performed with
high enantioselectivities.[15]
Given the above results, we decided to resolve with li-
pases the building blocks “benzylic alcohol” 5 or “tosylated
alcohol” 12 rather than the diol 3.[8c]
Resolution of Synthon C-4. We employed a lipase from
Pseudomonas sp, which shows a high selectivity for the hy-
drolysis of esters of secondary alcohols, as well as for the
corresponding esterification reactions.[16] All crude lipases
available from Pseudomonas sp have a stereochemical pref-
erence for the (R) configuration on the reactive center of
the secondary alcohol. This stereochemical preference has
led to a model for the active site.[17] With lipases, esterifi-
cations catalysed in organic solvents are often more enan-
tioselective than the corresponding hydrolysis reactions in
aqueous media.[18] Thus, we used irreversible esterification
reactions in organic media instead of hydrolysis reactions
(Equation 1).
In a same way, compound (R)-13, obtained by enzymatic
resolution of alcohol 12 by lipase PS (Equation 1), led to
the 1,2-O-didecanoyl-sn-glycerol [(S)-9] enantiomer.
The absolute configurations of the two enantiomers of
compound 9 were determined by chemical correlation (see
Experimental Section for details). The enantiomeric purity
of the tosylated alcohol (S)-12 after enzymatic resolution
was determined to be greater than 99% (measured by chiral
HPLC), since the 2,3-diglyceride (R)-9 was obtained with
92% ee (measured by 19F-NMR spectroscopy), which corre-
sponded to a 3% racemization. We can assume that this
racemization occurred by substitution of the tosylate group
during the reaction with cesium acetate. This racemization
could have occurred by an SNi reaction catalysed by the
cesium ion. The acetate group could then attack either at
the primary carbon, with retention of configuration [com-
pound (1), Equation 2], or at the secondary carbon, with
inversion of configuration [compound (2), Equation 2].
With “benzylic alcohol” 5 (R ϭ Bzl), the two enantio-
mers were poorly discriminated by lipase PS at room tem-
perature (Table 1, entries 1Ϫ2). The enantiomeric excess of
remaining substrate as well as the product formed were
moderate (eeS ϭ 0.70, eeP ϭ 0.79) for a conversion of 47%,
Synthesis of Compound 1
For the elaboration of phosphonates 1, which are anal-
which corresponds to an enantiomer ratio or E value[18c] of ogous to triglycerides, the synthesis was developed as for
18. The addition of molecular sieves into the reaction me- more simple alcohols but using alkylphosphonic dichlo-
Eur. J. Org. Chem. 1999, 1671Ϫ1678
1673

F. Marguet, J.-F. Cavalier, R. Verger, G. Buono
FULL PAPER
Table 1. Enzymatic Resolution of alcohols 5 and 12 with Lipase PS
X-ray diffraction, or NOE experiments did not allow us to
determine the absolute configuration at the phosphorus
atom of these triglyceride analogs. However, further experi-
ments are currently in progress in order to make this assign-
ment.
By convention, and until the absolute configuration at
phosphorus can be made, we have designated as isomer A
the one that resonates at lower fields in 31P NMR, and iso-
mer B the one at higher fields. Thus, 1,2-didecanoyl-sn-gly-
cerol led to two diastereomers named A and B, and 2,3-
didecanoyl-sn-glycerol to two other diastereomers named
ent-A and ent-B, enantiomers of A and B, respectively (Fig-
ure 1).
Scheme 3. Enantioselective synthesis of chieral 2,3-diglyderide (R)-
9. Reagents and conditions: (a) Acetyl chloride, CH2Cl2, Pyr, 0°C.
Ϫ (b) Caesium acetata, DMSO, DMF. Ϫ (c) K2CO3, MeOH, H2O.
Ϫ (d) PhCHO, p-TsCOH, PhMe, reflux. Ϫ (e) O3, Me2S, CH2Cl2,
Ϫ78°C. Ϫ (f) NaBH4, CH2Cl2, EtOH, 0°C. Ϫ (g) DIBALF, Et2O,
0°C to room tem. Ϫ (h) Decanoic acid, DCC, DMAP, Et2O. Ϫ (i)
5% Pd/C, H2, EtOH.
Conclusion
We have described the enantioselective synthesis of op-
tically pure 1,2- and 2,3-O-didecanoyl-sn-glycerophospho-
nates, which exist as two pairs of enantiomers, starting from
a chiral diol obtained by an irreversible enzymatic acylation
of (R,S)-12: (R)- and (S)-3, respectively. The strategy de-
picted in Scheme 3, which leads to enantiopure diglycerides
9, could also be applied to the synthesis of various mixed
ride.[2a] The alcohol group from 1,2- and 2,3-O-didecanoyl- diglyceride compounds.
sn-glycerol was first allowed to react with 1 equivalent of
Inhibition studies with each enantiomer A, ent-A, B, ent-
B on Human Pancreatic Lipase (HPL) and Human Gastric
Lipase (HGL) are under currently under way.
pentylphosphonic dichloride 15 in the presence of 1.1
equivalents of N-methylimidazole (NMI) in dichlorometh-
ane. In this case, only monosubstitution was observed. Sub-
sequent addition of 1 equivalent of p-nitrophenol in the
presence of 1.1 equivalents of NMI led to a mixture of two
diacylglycerophosphonate diastereomers (Equation 3), as
can be seen by 31P-NMR spectroscopy (two peaks sepa-
rated by 0.2 ppm). These diastereomers were separated by
HPLC (silica column): retention times (24.6 min and
27.3 min) were sufficient for a total separation of the two
stereomers.
Experimental Section
General Remarks: Unless stated otherwise, all starting materials
were obtained from commercial suppliers and were used without
further purification. Tetrahydrofuran (THF) was dried by distil-
lation from potassium/benzophenone under nitrogen. Diethyl ether
(ether) was distilled from sodium. Ethanol was removed from di-
chloromethane (CH2Cl2) by passing it through a silica column im-
mediately prior to use. N,N-Dimethylformamide (DMF) was dis-
Cyclodextrin inclusion complexes prepared to obtain
suitable crystals for elucidation of the stereochemistry by tilled under reduced pressure from barium oxide immediately prior
1674 Eur. J. Org. Chem. 1999, 1671Ϫ1678

New Synthesis of sn-1,2- and sn-2,3-O-Diacylglycerols
FULL PAPER
to use. Dimethylsulfoxide (DMSO) was dried with CaH2, then dis-
tilled under reduced pressure immediately prior to use. Reactions
involving phosphorus compounds were performed under an inert
atmosphere of dry nitrogen. Ϫ Flash-chromatography was per-
formed as described by Still et al.[21] using silica gel 60 (70Ϫ230
mesh, Merck). Thin-layer chromatography (TLC) was carried out
on silica gel plates (0.25 mm, Merck), and the following detection
methods were used: UV lamp (254 nm); PMA: TLC plate dipped
into a solution containing 5% phosphomolybdic acid in absolute
ethanol, and heated on a hot plate; PAA: TLC plate dipped into a
solution containing 3% p-anisaldehyde, 4% concentrated H2SO4,
Enantiomers of 13 were separated on a Chiralpak AS column
(Daicel Chemical Industries) eluted with hexane/2-propanol (90:10)
at 25°C. The (R) enantiomer eluted first. Retention times: t1
21.4 min; t2 ϭ 26.1 min.
ϭ
3-Buten-1,2-diol (3): A mixture of 20.0 g (227 mmol) of (Z)-2-
buten-1,4-diol (2), mercuric sulfate (80 mg) and concentrated sul-
furic acid (0.12 mL) in water (10 mL) was kept in a microwave oven
for 3Ϫ5 min (temperature of the reaction mixture was ca. 50°C).
After the usual workup procedure,[5a] 13.3 g (66%) of 3-buten-1,2-
diol (3) was isolated as a colorless liquid by fractional distillation
(bp 78Ϫ84°C/10 Torr). Ϫ 1H NMR (CDCl3) δ ϭ 5.9/5.7 (m, 1 H),
5.4/5.1 (m, 2 H), 4.2/4.0 (m, 3 H), 3.6/3.3 (m, 2 H). Ϫ 13C NMR
(CDCl3) δ ϭ 136.7, 116.6, 73.4, 66.2. Ϫ C4H8O2 (88.11): calcd. C
54.5, H 9.2; found, C 53.2, H 8.9.
1
1% acetic acid in 95% ethanol, and heated on a hot plate. Ϫ H-
NMR spectra were obtained on a Bruker AC 200 using CDCl3 as
solvent and tetramethylsilane (TMS) as an internal standard. 13C-
NMR spectra were obtained at 50.36 MHz. 31P-NMR spectra were
obtained on a Bruker AC 100 at 40.54 MHz (85% H3PO4 aqueous
solution was used as an external standard) and 19F-NMR spectra
at 94.22 MHz (CFCl3 was used as an external standard). Spectral
splitting patterns are designated as s, singlet; d, doublet; t, triplet;
q, quadruplet; m, multiplet. Ϫ Elemental analyses were performed
at the Service Commun de Microanalyse, University of Aix-Mar-
1-Benzyloxy-3-buten-2-ol (5): A solution of 3-buten-1,2-diol (3)
(3.60 g, 41 mmol) in anhydrous THF (50 mL) at 0°C was added to
a stirred suspension of sodium hydride (1.0 g, 43 mmol) in anhy-
drous THF (200 mL) under nitrogen. The reaction mixture was
cooled to Ϫ30°C. Tetrabutylammonium iodide (320 mg, 0.9 mmol)
was added followed by a solution of benzyl bromide (7.50 g,
44 mmol) in anhydrous THF (100 mL). After stirring for 20 h, the
reaction mixture was neutralized with water (40 mL) and then the
pH adjusted with concentrated HCl to give pH 7. The aqueous
layer was saturated with NaCl and extracted with CH2Cl2 (3ϫ 40
mL). The organic layers were combined, dried with Na2SO4, fil-
tered, and concentrated. The residue was purified by flash chroma-
tography eluting with pentane/EtOAc (7:3) to give 3.8 g (52%) of a
mixture of 1-benzyloxy-3-buten-2-ol (5) (Rf ϭ 0.44, 30% EtOAc in
pentane, PAA) and 2-benzyloxy-3-buten-1-ol (6) (Rf ϭ 0.40, 30%
EtOAc in pentane, PAA) in an 80:20 ratio.
seille III, France. All compounds were characterized by a combi-
20
nation of 1 H, 13 C, 31P NMR, elemental analyses and [α]D
.
Absolute Configuration: The derivatization of 1,2-diacylglycerol 9
with a chiral derivative agent (o-fluorophenoxy lactic acid)[22] al-
lows us to differentiate, by 19F-NMR spectroscopy, the two dia-
stereomers. As -mannitol leads specifically to 1,2-O-diacyl-sn-gly-
cerol, it is possible to assign to each NMR signal the absolute con-
figuration of the sn-2 carbon of the glycerol backbone, and in this
way to determine the absolute configuration of the asymmetric
center formed during the enantioselective step.
Separation of the Two Isomers: to a solution of the two isomers 5
and 6 (240 mg, 1.35 mmol) in CH2Cl2 (4.0 mL) and pyridine (1.0
mL) was added pivaloyl chloride (82 mg, 0.7 mmol) at 0°C. After
stirring the mixture at room temperature for 15 h, CH2Cl2 (10 mL)
was added and the solution was washed with 10% K2CO3 (2ϫ 5
mL), saturated CuSO4 (2ϫ 5 mL), saturated NH4Cl (5 mL), and
brine (5 mL). The organic layer was dried with Na2SO4, filtered,
and concentrated. The residue was purified by flash chromatogra-
phy eluting with pentane/EtOAc (7:3) to give 150 mg (63%) of 1-
benzyloxy-3-buten-2-ol (5) (Rf ϭ 0.44, 30% EtOAc in pentane,
PAA). Ϫ 1H NMR (CDCl3) δ ϭ 7.5 (m, 5 H), 6.1/7.8 (m, 1 H),
5.5/5.3 (m, 2 H), 4.7 (s, 2 H), 4.5 (m, 1 H), 3.7/3.4 (m, 2 H), 3.2 (d,
1 H). Ϫ 13C NMR (CDCl3) δ ϭ 137.7, 136.7, 128.3, 127.7, 116.1,
74.0, 73.2, 71.3. Ϫ C11H14O2 (178.23): calcd. C 74.1, H 7.9; found,
C 74.6, H 8.2.
The lipase PS has a stereopreference for the (R) enantiomer with
each synthon 5 and 12. This is in agreement with the studies under-
taken with crude lipases from Pseudomonas sp., which all have a
stereochemical preference for the (R) configuration on the reactive
center of the secondary alcohol.
Preparation of the Diastereomeric Esters for ee Measurements of
Alcohols 5 and 9: The appropriate alcohol (0.175 mmol) and o-
fluorophenoxy lactic acid (48.2 mg, 0.262 mmol) were dissolved in
anhydrous ether (2 mL) and the mixture cooled to 0°C. DCC
(54.2 mg, 0.262 mmol) and DMAP (3.2 mg, 0.026 mmol) in ether
(3 mL) were added. The cooling bath was removed and the mixture
was stirred for 12 h. The solution was then carefully filtered
through a pressed cotton plug and diluted with ether (5 mL). The
solution was washed successively with saturated NH4Cl (5 mL),
10% NaHCO3 (2ϫ 5 mL), water (5 mL), and brine (5 mL). The
organic layer was dried with MgSO4, filtered, and concentrated.
The crude product (yield 80Ϫ90%) was analysed by 19F-NMR
spectroscopy.
1-Benzyloxypropan-2,3-diol (7): A solution of 1-benzyloxy-3-buten-
2-ol (5) (640 mg, 3.6 mmol) in CH2Cl2 (80 mL) was introduced into
an ozonolysis tube. The solution was cooled to Ϫ78°C and ozone
was bubbled into the solution. After 15 min, the reaction was a
deep blue color, which was an indication of saturation with ozone.
The solution was then purged at Ϫ78°C with nitrogen and Me2S
(660 µL) was added. The cooling bath was removed and the solu-
tion was stirred for 1 h. The mixture was then concentrated to an
approximate volume of 50 mL, and ethanol (20 mL) was added.
The solution was cooled to 0°C and sodium borohydride (120 mg,
3.1 mmol) was added. After 20 min, solvents were removed and the
residue was dissolved in methanol (10 mL) and CH2Cl2 (20 mL).
After stirring for 1 h, CH2Cl2 (40 mL) was added and the organic
layer was washed with brine (3ϫ 10 mL), dried with Na2SO4, fil-
tered, and concentrated. The residue was purified by flash chroma-
tography eluting with EtOAc to give 550 mg (84%) of 1-benzyloxy-
propan-2,3-diol (7) (Rf ϭ 0.43, EtOAc, PMA). Ϫ 1H NMR
Enantiomeric Purity: Analysis of enantiomeric purity was deter-
mined by NMR in the presence of a chiral derivative agent or by
HPLC with chiral columns.
(a) NMR Analysis was performed on compounds 5 and 9, onto
which was added o-fluorophenoxy lactic acid.[22] The two dia-
stereomers formed were analysed and quantified by NMR (1 H, 13
C, 19F). The fluorine resonance of each enantiomer is separated by
9.8 Hz for 5 and 15.3 Hz for 9.
(b) HPLC. Enantiomers of 12 were separated on a Chiracel OD-H
column (J. T. Baker) with hexane/2-propanol (98:2) as eluent, at
25°C. The (R) enantiomer eluted first. Retention times: t1
34.0 min; t2 ϭ 38.6 min.
ϭ
Eur. J. Org. Chem. 1999, 1671Ϫ1678
1675

F. Marguet, J.-F. Cavalier, R. Verger, G. Buono
FULL PAPER
(CDCl3) δ ϭ 7.3 (s, 5 H), 4.5 (s, 2 H), 3.8 (m, 1 H), 3.6/3.4 (m, 4
tion was stirred for 1 h. The mixture was then evaporated to an
H), 2.9 (s, 2 H). Ϫ 13C NMR (CDCl3) δ ϭ 137.7, 128.5, 127.8, approximate volume of 50 mL and ethanol (50 mL) was added.
73.5, 71.6, 70.8, 63.9. Ϫ C10H14O3 (182.22): calcd. C 65.9, H 7.7;
found, C 66.1, H 7.5.
The solution was cooled to 0°C and sodium borohydride (108 mg,
2.6 mmol) was added. After 20 min, the solvents were removed and
the residue was dissolved in methanol (10 mL) and CH2Cl2 (20
mL). After stirring for 1 h, CH2Cl2 (50 mL) was added and the
organic layer was washed with brine (10 mL), dried with Na2SO4,
filtered, and concentrated. The residue was purified by flash chro-
matography eluting with pentane/ether (5:5) to give 680 mg (74%)
of 1,2-benzylidene glycerol (11) (Rf ϭ 0.15, 50% ether in pentane,
1,2-O-Didecanoyl-3-O-benzyl-rac-glycerol (8): To a solution of l-
benzyloxypropan-2,3-diol (7) (1.35 g, 7.4 mmol) in pentane (60
mL) was added successively capric acid (2.48 g, 14.4 mmol),
DMAP (180 mg, 22 mmol), DCC (3.06 g, 14.8 mmol) and the re-
sulting suspension was stirred for 12 h. The solid was removed by
filtration through celite and the filtrate was concentrated under re-
duced pressure. The residue was purified by flash chromatography
eluting with pentane/ether (9:1) to give 3.40 g (93%) of 1,2-O-dide-
canoyl-3-O-benzyl-rac-glycerol (8) (Rf ϭ 0.30, 10% ether in pen-
tane, PMA). Ϫ 1H NMR (CDCl3) δ ϭ 7.31 (s, 5 H, C6H5), 5.24
(app. quintet, 1 H, J ϭ 3.9 Hz, sn-2 CH), 4.54 (s, 2 H, CH2ϪC6H5),
1
PMA). Ϫ H NMR (CDCl3) δ ϭ 7.5/7.3 (m, 5 H), 5.9 (s, 0.5 H),
5.8 (s, 0.5 H), 4.4/3.6 (m, 5 H), 2.7 (t, 0.5 H), 2.6 (t, 0.5 H). Ϫ 13C
NMR (CDCl3)
δ
ϭ
137.7Ϫ137.0, 129.5Ϫ129.2, 128.3,
125.6Ϫ126.3, 104.2Ϫ103.7, 76.9Ϫ76.5, 66.9Ϫ66.7, 63.1Ϫ62.5. Ϫ
C10H12O3 (180.20): calcd. C 66.6, H 6.7; found, C 66.1, H 6.9.
4.35 and 4.20 (dd, 1 H, J ϭ 3.9 Hz and J ϭ 11.9 Hz, and, dd, 1 1-Benzyloxypropan-2,3-diol (7): To a solution of DIBALH (1 /
H, J ϭ 6.3 Hz and J ϭ 11.9 Hz, CH2ϪOϪCO), 3.58 (d, 2 H, J ϭ CH2Cl2, 5 mmol) in anhydrous ether (10 mL) was added dropwise
5.2 Hz, CH2ϪOϪBzl), 2.32 (t, 2 H, J ϭ 7.6 Hz, CH2ϪCO) and at 0°C a solution of 1,2-benzylidene glycerol (11) (300 mg,
2.28 (t,
2
H,
J
ϭ
7.6 Hz, CH2ϪCO), 1.60 (m,
4
H, 1.67 mmol) in anhydrous ether (3 mL). The mixture was stirred for
4 h at room temperature and then methanol (2 mL) and saturated
CH2ϪCH2ϪCO), 1.40/1.10 [m, 24 H, (CH2)6], 0.88 (t, 6 H, J ϭ
6.0 Hz, CH3). Ϫ 13C NMR (CDCl3) δ ϭ 173.4, 173.1, 137.8, 128.5, NH4Cl (10 mL) were added. After 15 min, the solution was centri-
127.8, 127.7, 73.3, 70.1, 68.3, 62.7, 34.4, 34.1, 31.9, 29.5, 29.3, 29.2, fuged and the solvents removed. Ether (10 mL) was added and the
25.0, 24.9, 22.7, 14.1. Ϫ C30H50O5 (490.72): calcd. C 73.4, H 10.3;
found, C 72.9, H 9.9.
aqueous layer was extracted with CH2Cl2 (2ϫ 10 mL). The organic
layers were combined, dried with Na2SO4, filtered, and concen-
trated to give 240 mg (80%) of 1-benzyloxypropan-2,3-diol (7)
1,2-O-Didecanoyl-rac-glycerol (9): 1,2-O-didecanoyl-3-O-benzyl-
rac-glycerol (8) (265 mg, 0.54 mmol) was dissolved in 95% ethanol
(12 mL) containing 5% Pd/C (210 mg), and the mixture was stirred
under an atmosphere of H2 at room temperature. After 1 h, the
catalyst was removed by filtration through celite and the filtrate was
1
(Rf ϭ 0.43, EtOAc, PMA). Ϫ H NMR (CDCl3) δ ϭ 7.3 (s, 5 H),
4.5 (s, 2 H), 3.8 (m, 1 H), 3.6/3.4 (m, 4 H), 2.9 (s, 2 H). Ϫ 13C
NMR (CDCl3) δ ϭ 137.7, 128.5, 127.8, 73.5, 71.6, 70.8, 63.9. Ϫ
C10H14O3 (182.22): calcd. C 65.9, H 7.7; found, C 66.1, H 7.5.
concentrated under reduced pressure with a water bath temperature 1-Tosyloxy-3-buten-2-ol (12): To a solution of 3-buten-1,2-diol (3)
below 20°C. The residue was purified by flash chromatography
eluting with pentane/ether (6:4) to give 195 mg (90%) of 1,2-O-
didecanoyl-rac-glycerol (9) (Rf ϭ 0.25, 40% ether in pentane,
(2.0 g, 23 mmol) in CH2Cl2 (30 mL) and pyridine (20 mL) was
added p-TsOH (4.7 g, 25 mmol). The mixture was stirred for 2 d at
room temperature. CH2Cl2 (100 mL) was then added and the solu-
tion was washed with 2% HCl (3ϫ 30 mL), saturated CuSO4 (5ϫ
1
PMA). Ϫ H NMR (CDCl3) δ ϭ 5.08 (m, 1 H, J ϭ 4.8 Hz, sn-2
CH), 4.32 and 4.20 (dd, 1 H, J ϭ 4.4 Hz and J ϭ 11.9 Hz, and dd, 30 mL), 10% K2CO3 (2ϫ 30 mL), and brine (30 mL). The solution
1 H, J ϭ 5.7 Hz and J ϭ 11.9 Hz, CH2ϪOϪCO), 3.73 (t, 2 H, J ϭ
5.5 Hz, CH2ϪOH), 2.35 (t, 2 H, J ϭ 7.3 Hz, CH2ϪCO) and 2.32
was centrifuged and the organic layer was dried with Na2SO4, fil-
tered, and concentrated. The residue was purified by flash chroma-
(t, 2 H, J ϭ 7.3 Hz, CH2ϪCO), 2.27 (s, 1 H, OH), 1.62 (m, 4 H, tography eluting with pentane/ether (7:3) to give 3.80 g (68%) of 1-
CH2ϪCH2ϪCO), 1.40/1.10 [m, 24 H, (CH2)6], 0.88 (t, 6 H, J ϭ
tosyloxy-3-butene (12) (Rf ϭ 0.27, 30% ether in pentane, PAA). Ϫ
6.1 Hz, CH3). Ϫ 13C NMR (CDCl3) δ ϭ 173.4, 173.7, 72.1, 62.2, 1H NMR (CDCl3) δ ϭ 7.85 (d, 2 H, J ϭ 8 Hz), 7.40 (d, 2 H, J ϭ
61.2, 34.0, 31.8, 29.4, 29.2, 29.1, 24.8, 22.6, 14.0. Ϫ C23H44O5 8 Hz), 5.9/5.7 (m, 1 H), 5.5/5.3 (m, 2 H), 4.4 (m, 1 H), 4.1/3.9 (m,
(400.60): calcd. C 68.9, H 11.1; found, C 69.1, H 10.8.
2 H), 2.7 (s, 1 H), 2.5 (s, 3 H). Ϫ 13C NMR (CDCl3) δ ϭ 145.1,
134.7, 132.6, 130.0, 128.0, 118.1, 73.0, 70.4, 21.7. Ϫ C11H14O4S
(242.29): calcd. C 54.5, H 5.8, S 13.3; found, C 54.2, H 6.3, S 13.1.
1,2-Benzylidene-3-buten-1,2-diol (10): 3-buten-1,2-diol (3) (500 mg,
5.7 mmol) was dissolved in toluene (10 mL) containing benzal-
dehyde (600 mg, 6.3 mmol) and a catalytic amount of p-TsOH
(20 mg). The mixture was refluxed for 3 h. The solution was then
concentrated and the residue was distilled with a bulb-to-bulb oven
to give 900 mg (90%) of an epimeric mixture of 1,2-benzylidene-3-
Lipase-Mediated Acylation of Alcohol 5: To a solution of 1-benzyl-
oxy-3-buten-2-ol (5) (150 mg, 0.84 mmol) in hexane (10 mL) was
added successively vinyl acetate (300 mg, 3.36 mmol) and lipase PS
(75 mg). The mixture was vigorously stirred for 4 h at room tem-
buten-1,2-diol (10) (bp 110Ϫ112°C/7.6·10Ϫ3 Torr). Ϫ Rf ϭ 0.56 perature. The reaction was then stopped by filtration through celite.
1
and 0.62, 10% EtOAc in pentane, PAA. Ϫ H NMR (CDCl3) δ ϭ
The solvent was removed and the residue was purified by flash
7.5/7.1 (m, 5 H), 6.0/5.7 (m, 2 H), 5.4/5.1 (m, 2 H), 4.6/4.5 (m, 1 chromatography eluting with pentane/EtOAc (8:2) to give 84 mg
H), 4.3/4.1 (m, 1 H), 3.8/3.5 (m, 1 H). Ϫ 13C NMR (CDCl3) δ ϭ (45%) of acetate of 5 (79% ee by 19F NMR) and 82 mg (55%) of
137.8Ϫ137.1, 135.5Ϫ135.3, 129.3Ϫ129.1, 128.4, 126.6Ϫ126.4,
alcohol 5 (70% ee by 19F NMR). Ϫ Rf ϭ 0.50 for acetate and 0.18
118.5Ϫ118.1, 104.4Ϫ103.8, 78.4Ϫ77.4, 70.4Ϫ70.0. Ϫ C11H12O2 for alcohol, 20% EtOAc in pentane, PAA. Ϫ 1H NMR (CDCl3)
(176.22): calcd. C 75.0, H 6.9; found, C 75.2, H 6.8.
δ ϭ 7.3 (s, 5 H), 6.0/5.7 (m, 1 H), 5.6/5.4 (m, 2 H), 5.2 (m, 1 H),
4.6 (s, 2 H), 3.6 (d, 2 H, 5.5 Hz), 2.0 (s, 3 H). Ϫ 13C NMR (CDCl3)
δ ϭ 170.1, 137.9, 133.3, 128.3, 127.7, 127.6, 117.9, 73.1, 71.2, 21.1.
Ϫ C13H16O3 (220.26): calcd. C 70.9, H 7.3; found C 69.7, H 6.9.
1,2-Benzylideneglycerol (11): In an ozonolysis tube was introduced
1,2-benzylidene-3-buten-1,2-diol (10) (900 mg, 5.1 mmol) in
CH2Cl2 (80 mL). The solution was cooled to Ϫ78°C and ozone
was bubbled into the solution. After 15 min, the reaction was a
deep blue color, which was an indication of saturation with ozone.
Lipase-Mediated Acylation of Alcohol 12: To a solution of 1-tosyl-
oxy-3-buten-2-ol (12) (1.0 g, 4.13 mmol) in hexane/ether (1:1, 50
The solution was then purged at Ϫ78°C with nitrogen and Me2S mL) was added successively vinyl acetate (1.44 g, 16.5 mmol) and
(750 µL) was added. The cooling bath was removed and the solu-
lipase PS (500 mg). The mixture was vigorously stirred for 3 h at
1676
Eur. J. Org. Chem. 1999, 1671Ϫ1678

New Synthesis of sn-1,2- and sn-2,3-O-Diacylglycerols
FULL PAPER
room temperature. The reaction was then stopped by filtration
through celite. The solvent was removed and the residue was puri-
pentane/EtOAc (8:2) (Rf ϭ 0.30, 20% EtOAc in pentane, PMA).
The product was dissolved in ether (10 mL) and washed with 5%
fied by flash chromatography eluting with pentane/ether (7:3) to K2CO3 (5ϫ 3 mL), saturated NH4Cl (3 mL), and brine (3 mL).
give 570 mg (2 mmol) of acetate 13 (95% ee by HPLC using Chiral- The organic layer was dried with MgSO4, filtered, and concentrated
pak AS, eluent: 10% iPrOH in hexane) and 490 mg (2.03 mmol) of under reduced pressure to give 175 mg (22%) of O-[(1,2-dideca-
alcohol 12 (84% ee by HPLC using Chiracel OD-H, eluent: 2%
noyl)glyceryl] O-(p-nitrophenyl) n-pentylphosphonate (1). Ϫ 31P
1
iPrOH in hexane). Ϫ Rf ϭ 0.43 for 13 and 0.27 for 12, 30% ether NMR (CDCl3) δ ϭ 30.4, 30.2. Ϫ H NMR (CDCl3) δ ϭ 8.24 (d,
in pentane, PAA. Ϫ 1H NMR (CDCl3) δ ϭ 7.7 (d, 2 H, J ϭ 8 Hz),
2 H, J ϭ 9.1 Hz, H-Car), 7.37 (d, 2 H, J ϭ 9.1 Hz, H-Car), 5.23
7.3 (d, 2 H, J ϭ 8 Hz), 5.7/5.5 (m, 1 H), 5.4/5.1 (m, 3 H), 4.0 (m, (app. quintet, 1 H, J ϭ 5.0 Hz, sn-2 CH), 4.35/4.20 (m, 2 H,
2 H), 2.4 (s, 3 H), 1.9 (s, 3 H). Ϫ 13C NMR (CDCl3) δ ϭ 169.8, CH2ϪOϪCO), 4.20/4.05 (m, 2 H, CH2ϪOϪPO), 2.29 (t, 4 H, J ϭ
145.1, 132.8, 131.2, 130.0, 128.0, 119.8, 71.5, 69.8, 21.7, 20.9. Ϫ 7.5 Hz, CH2ϪCO), 1.92 (m, 2 H, CH2ϪPO), 1.62 (m, 6 H,
C13H16O5S (284.33): calcd. C 54.9, H 5.7, S 11.3; found, C 55.2, H
5.5, S 11.5.
CH2ϪCH2ϪCO and CH2ϪCH2ϪPO), 1.40/1.05 (m, 28 H), 0.88
(m, 9 H, CH3). Ϫ 13C NMR (CDCl3) δ ϭ 173.0, 172.6, 155.3 (d,
J ϭ 8.5 Hz), 144.6, 125.5, 120.9 (d, J ϭ 4.2 Hz), 69.4 (d, J ϭ
5.8 Hz), 64.2, 61.6, 34.0, 33.9, 32.4 (d, J ϭ 17.2 Hz), 31.8, 29.3,
29.2, 29.1, 25.7 (d, J ϭ 139.3 Hz), 24.7, 22.6, 22.0, 21.7 (d, J ϭ
7.2 Hz), 14.0, 13.6. Ϫ C34H58NO9P (655.81): calcd. C 62.3, H 8.9,
N 2.1, P 4.7; found, C 62.5, H 8.6, N 1.9, P 5.2.
(S)-1-Tosyloxy-2-acetyloxy-3-butene [(S)-13]: To a solution of 1-to-
syloxy-3-buten-2-ol [(S)-12] (440 mg, 1.81 mmol) in CH2Cl2 (6 mL)
at 0°C was added pyridine (180 mg, 2.1 mmol) and acetyl chloride
(180 mg, 2.1 mmol). The mixture was stirred for 30 min. The solu-
tion was then concentrated and ether (5 mL) was added. After fil-
tration and concentration, the residue was purified by flash chro-
matography eluting with pentane/ether (7:3) to give 480 mg (93%)
of (S)-1-tosyloxy-2-acetyloxy-3-butene [(S)-13] (Rf ϭ 0.43, 30%
ether in pentane, PAA). Ϫ 1H NMR (CDCl3) δ ϭ 7.7 (d, 2 H, J ϭ
8 Hz), 7.3 (d, 2 H, J ϭ 8 Hz), 5.7/5.5 (m, 1 H), 5.4/5.1 (m, 3 H),
4.0 (m, 2 H), 2.4 (s, 3 H), 1.9 (s, 3 H). Ϫ 13C NMR (CDCl3) δ ϭ
169.8, 145.1, 132.8, 131.2, 130.0, 128.0, 119.8, 71.5, 69.8, 21.7, 20.9.
Ϫ C13H16O5S (284.33): calcd. C 54.9, H 5.7, S 11.3; found, C 55.2,
H 5.5, S 11.5.
Separation of Diastereomers A/B (or ent-A/ent-B): Separation of
diastereomers A/B (or ent-A/ent-B) was performed by HPLC using
a silica column (250 ϫ 10 mm, particle size: 5 µm) from S.F.C.C.
under semi-preparative conditions:
Ϫ room temperature (25°C)
Ϫ solvent: heptane/2-propanol (99.05/0.95)
Ϫ UV detection at λ ϭ 234 nm
Ϫ flow rate: 3 mL/min
15 injections of 10 µL each (125 mg/mL) led to the recovery of
both enantiomers: t1 (A or ent-A) ϭ 24.5 min, 31P NMR: δ ϭ 30.4.
Ϫ t2 (B or ent-B) ϭ 27.3 min, 31P NMR: δ ϭ 30.2.
(S)-1,2-Diacetyloxy-3-butene [(S)-14]: To a solution of 1-tosyloxy-
2-acetyloxy-3-butene [(S)-13] (100 mg, 0.35 mmol) in DMSO/DMF
(4:1, 4 mL) under nitrogen was introduced cesium acetate (140 mg,
0.70 mmol). The mixture was stirred for 3 d at room temperature.
Water (10 mL) was then added and the mixture was extracted with
ether (3ϫ 20 mL). The organic layer was dried with MgSO4, fil-
tered, and concentrated. The residue was purified by flash chroma-
tography eluting with pentane/ether (7:3) to give 60 mg (99%) of
(S)-1,2-diacetyloxy-3-butene [(S)-14] (Rf ϭ 0.30, 30% ether in pen-
Optical rotations were measured using a PerkinϪElmer 241 MC
polarimeter and gave the following values:
[α(A)]20 ϭ ϩ7.45 (c ϭ 0.5, CH2Cl2)
D
[α(ent-A)]20 ϭ Ϫ7.25 (c ϭ 0.4, CH2Cl2)
D
[α(B)]20 ϭ ϩ1.28 (c ϭ 0.5, CH2Cl2)
D
[α(ent-B)]20 ϭ Ϫ1.25 (c ϭ 0.4, CH2Cl2)
D
1
tane, PAA). Ϫ H NMR (CDCl3) δ ϭ 5.9/5.7 (m, 1 H), 5.5 (m, 1
H), 5.4/5.2 (m, 2 H), 4.3 (m, 1 H), 4.1 (m, 1 H), 2.1 (s, 1 H), 2.0
(s, 1 H). Ϫ 13C NMR (CDCl3) δ ϭ 170.6, 170.0, 132.3, 118.8, 72.0,
64.7, 21.0, 20.7. Ϫ C8H12O4 (172.18): calcd. C 55.8, H 7.0; found,
C 56.1, H 7.3.
Acknowledgments
This research was carried out under the BIOTECH G-Lipase (con-
tract BIO2-CT94Ϫ3041) Program of the European Union. We
thank Amano for generous gift of lipase PS. We are indebted to Pr.
C. Roussel (ENSSPICAM) for helpful discussions and providing us
with AS and OD-H columns. We are grateful to Dr. A. Heumann
(CNRS, UMR 6516) for providing us with the chiral derivative
agent (o-fluorophenoxy lactic acid).
(S)-3-Buten-1,2-diol [(S)-3]: To a stirred solution of 1,2-diacetyloxy-
3-butene [(S)-14] (270 mg, 1.57 mmol) in methanol/water (96:4, 7
mL) was added potassium carbonate (480 mg, 3.5 mmol). The mix-
ture was stirred for 1 h and then filtered through celite. The reac-
tion was neutralized to pH 7. After filtration, the mixture was con-
centrated and the residue was purified by flash chromatography
eluting with EtOAc to give 90 mg (65%) of (S)-3-buten-1,2-diol
[1] [1a]
U. Derewenda, A. M. Brzozowski, D. M. Lawson, Z. S.
[1b]
1
Derewenda, Biochemistry 1992, 31, 1532Ϫ1541. Ϫ
A. M.
[(S)-3] (Rf ϭ 0.45, EtOAc, PMA). Ϫ H NMR (CDCl3) δ ϭ 5.9/
Brzozowski, Z. S. Derewenda, U. Derewenda, G. Dodson, D.
5.7 (m, 1 H), 5.4/5.1 (m, 2 H), 4.2/4.0 (m, 3 H), 3.6/3.3 (m, 2 H).
M. Lawson, J. P. Turkenburg, F. Bjorkling, B. Huge-Jernsen, S.
13C NMR (CDCl3) δ ϭ 135.7, 115.5, 73.4, 55.2. Ϫ C4H8O2
[1c]
Ϫ
A. Patkar, L. Thim, Nature 1991, 351, 491Ϫ494. Ϫ
M.
(88.11): calcd. C 54.5, H 9.2; found, C 53.2, H 8.9.
Cygler, P. Grochulsi, R. J. Kazlauskas, R. D. Schrag, F. Bou-
thillier, B. Rubin, A. N. Serreqi, A. K. Gupta, J. Am. Chem.
[1d]
O-[(1,2-Didecanoyl)glyceryl] O-(p-nitrophenyl) n-pentylphosphonate
(1): Under nitrogen, a mixture of (S)-1,2-O-didecanoyl-sn-glycerol
[(S)-9] (500 mg, 1.25 mmol), N-methylimidazole (125 mg,
1.5 mmol) and CH2Cl2 (10 mL) was slowly added dropwise to a
solution of pentylphosphonic dichloride 15 (237 mg, 1.25 mmol) in
CH2Cl2 (10 mL). The reaction was then stirred for a further 30 min.
A solution of p-nitrophenol (174 mg, 1.25 mmol), N-methylimida-
zole (125 mg, 1.5 mmol) and CH2Cl2 (8 mL) was then slowly added
dropwise. The solvent was removed under reduced pressure and the
crude product was purified by flash chromatography eluting with
Soc. 1994, 116, 3180Ϫ3186. Ϫ
S. Longhi, M. L. M. Man-
nesse, H. M. Verheij, G. H. de Haas, M. Egmond, E. Knoops-
Mouthuy, C. Cambillau, Protein Science 1997, 6, 275Ϫ2762. Ϫ
[1e]
M.-P. Egloff, F. Marguet, G. Buono, R. Verger, C. Cambil-
lau, H. van Tilbeurgh, Biochemistry 1995, 34, 2751Ϫ2762.
[2] [2a]
F. Marguet, C. Cudrey, R. Verger, G. Buono, Biochim. Bio-
phys. Acta 1994, 1210, 157Ϫ166. Ϫ [2b] M.-P. Egloff, S. Ransac,
F. Marguet, E. Rogalska, H. van Tilbeurgh, G. Buono, C. Cam-
´
billau, R. Verger, Oleagineux, Corps Gras et Lipides 1995, 2,
[2c]
52Ϫ67. Ϫ
S. Ransac, F. Carriere, E. Rogalska, R. Verger, F.
Marguet, G. Buono, E. Pinho Melo, J. M. S. Cabral, M.-P. Eg-
loff, H. van Tilbeurgh, C. Cambillau, The Kinetics, Specificities
Eur. J. Org. Chem. 1999, 1671Ϫ1678
1677

F. Marguet, J.-F. Cavalier, R. Verger, G. Buono
FULL PAPER
and Structural Features of Lipases in NATO-ASI Series H: Cell
biology, vol. 96 (Ed.: J. A. F. Op den Kamp), Springer-Verlag,
Berlin, 1996; pp. 265Ϫ304.
thesis. A Comprehensive Handbook, vol. IϪII, VCH Publishers,
New York, 1995.
[12]
P. Cesti, A. Zaks, A. M. Klibanov, Appl. Biochem. Biotechnol.
1985, 11, 401.
[3] [3a]
R. Verger, G. H. de Haas, Chem. Phys. Lipids 1973, 10,
127Ϫ136. Ϫ [3b] G. Pieroni, R. Verger, J. Biol. Chem. 1979, 254,
A. J. M. Janssen, A. J. H. Kundler, B. Zwanenburg, Tetra-
[13] [13a]
´
[3c]
[13b]
´
´
M.-A. Mbappe, S. Sicsic,
[13c]
10090Ϫ10094. Ϫ
G. Pieroni, R. Verger, Eur. J. Biochem.
hedron 1991, 47, 7409Ϫ7416. Ϫ
[3d]
´
1983, 132, 639Ϫ644. Ϫ
Y. Gargouri, G. Pieroni, F. Ferrato,
Tetrahedron: Asymmetry 1993, 4, 1035Ϫ1040. Ϫ
L. Poppe,
R. Verger, Eur. J. Biochem. 1987, 169, 125Ϫ129.
L. Novak, M. Kajtar-Pedery, C. Szantay, Tetrahedron: Asym-
metry 1993, 4, 2211Ϫ2218.
[14] [14a]
[4] [4a]
A. V. Rama Rao, M. K. Gurjar, D. S. Bose, R. R. Devi, J.
[4b]
Org. Chem. 1991, 56, 1320Ϫ1321. Ϫ
H. H. Szmant, in: Or-
B. Herradon, S. Cueto, A. Marcuende, S. Valverde, Tetra-
[14b]
ganic Building Blocks of the Chemical Industry, Wiley Intersci-
ence, New York, 1989, p 258.
hedron: Asymmetry 1993, 4, 845Ϫ864. Ϫ
F. Theil, J.
Weidner, S. Ballschuh, A. Kunath, H. Schik, Tetrahedron Lett.
[5] [5a]
[14c]
A. V. Rama Rao, D. S. Bose, M. K. Gurjar, T. Ravindrana-
than, Tetrahedron 1989, 45, 7031Ϫ7040. Ϫ [5b] Natural product:
S. Hanessian, in: Total Synthesis of Natural Products: the “Chi-
ron” approch, Pergamon Press, Oxford, 1983.
1993, 34, 305Ϫ306. Ϫ
F. Theil, S. Ballschuh, A. Kunath,
[14d]
H. Schik, Tetrahedron: Asymmetry 1991, 2, 1031Ϫ1034. Ϫ
F. Theil, J. Weidner, S. Ballschuh, A. Kunath, H. Schik, J. Org.
Chem. 1994, 59, 388Ϫ393.
[15] [15a]
[6] [6a]
D. Bianchi, A. Bosetti, P. Cesti, P. Golini, Tetrahedron Lett.
R. L. Pederson, K. K.-C. Liu, J. F. Rutan, L. Chen, C.-H.
[15b]
[6b]
1992, 33, 3231Ϫ3234. Ϫ
W. L Nelson, J. E. Wennerstrom,
Wong, J. Org. Chem. 1990, 55, 4897Ϫ4901. Ϫ
C.-S. Chen,
[15c]
[6c]
S. R. Sankar, J. Org. Chem. 1977, 42, 1006Ϫ1012. Ϫ
R. A.
Y.-C. Liu, Tetrahedron Lett. 1989, 30, 7165Ϫ7168. Ϫ
M.-J.
[15d]
Johnson, K. B. Sharpless, in: Catalytic Asymmetric Synthesis
(Ed.: I. Ojima), VCH Publishers, New York, 1993, p. 103 and
p. 227.
Kim, Y. K. Choi, J. Org. Chem. 1992, 57, 1605Ϫ1607. Ϫ
U. Goergens, M. P. Schneider, J. Chem. Soc., Chem. Commun.
[15e]
ˆ
R. Chenevert, R. Gagnon, J. Org.
1991, 18, 1066Ϫ1068. Ϫ
[7]
MeBmt: (4R)-4-[(E)-2-butenyl]-4,N-dimethyl--threonine.
Chem. 1993, 58, 1054Ϫ1057.
[16]
W. Boland, C. Frobl, M. Lorenz, Synthesis 1991, 1049Ϫ1072.
[17] [17a]
R. J. Kazlauskas, A. N. E. Weissfloch, A. T. Rapport, L.
[17b]
A. Cuccia, J. Org. Chem. 1991, 56, 2656Ϫ2665. Ϫ
C. R.
Johnson, A. Golebiowski, T. K. McGill, D. H. Steensma, Tetra-
hedron Lett. 1991, 32, 2597Ϫ2600. Ϫ [17c] M.-J. Kim, H. J. Cho,
J. Chem. Soc., Chem. Commun. 1992, 19, 1411Ϫ1413. Ϫ [17d] K.
Burgess, L. D. Jennings, J. Am. Chem. Soc. 1991, 113,
6129Ϫ6139.
[18] [18a] C.-S. Chen, S. H. Wu, G. Girdaukas, C. J. Sih, J. Am. Chem.
Figure 2. Structures of synthon 4 and MeBmt
Soc. 1987, 109, 2812Ϫ2817. Ϫ [18b] C.-S. Chen, C. J. Sih, Angew.
[8] [8a]
[18c]
C. Neagu, T. Hase, Tetrahedron Lett. 1993, 34, 1629Ϫ1630.
Chem. Int. Ed. Engl. 1989, 28, 695Ϫ707. Ϫ
C. J. Sih, S. H.
Ϫ [8b] N. W. Boaz, R. L. Zimmerman, Tetrahedron: Asymmetry
Wu, Resolution of Enantiomers via Biocatalysis, in: Topics of
Stereochemistry, vol. 19 (Eds: E. L. Eliel, S. H. Wilen), Wiley
Interscience Publication, New York, 1989, pp 63Ϫ125.
1994, 5, 153Ϫ156. Ϫ [8c] F. Marguet, Ph. D. Dissertation, Univ.
[8d]
of Aix-Marseille III, France, 1994. Ϫ
E. N. Jacobsen, CHI-
[19] [19a]
REX Third International Conference on Process Development
Chemistry, 1997. Ϫ [8e] M. Tokunaga, J. F. Larrow, F. Kakinchi,
E. N. Jacobsen, Science 1997, 277, 936Ϫ938.
J. Kuszmann, E. Tomori, P. Dvortsak, Carbohydr. Res.
[19b]
1984, 132, 178Ϫ200. Ϫ
K. Yamanchi, F. Une, S. Tabata,
M. Kinoshita, J. Chem. Soc., Perkin Trans. I 1986, 765Ϫ770.
F. Marguet, I. Douchet, J.-F. Cavalier, G. Buono, R. Verger,
Colloids and Surfaces B: Biointerfaces 1999, 13, 37Ϫ45.
W. C. Still, M. Kahu, A. Mitra, J. Org. Chem. 1978, 43,
2923Ϫ2925.
[9] [9a]
[20]
[21]
[22]
L. H. Fieser, M. Fieser, in Reagents for Organic Synthesis,
[9b]
Vol. I, Wiley, New York, 1967, p 1179. Ϫ
S. E. Webber, Synthesis 1986, 453Ϫ461. Ϫ
O. Herandez, Tetrahedron Lett. 1979, 20, 95Ϫ98.
S. Takano, M. Akiyama, K. Ogasawara, Chem. Pharm. Bull.
1984, 32, 791Ϫ794.
K. C. Nicolaou,
S. K. Chandary,
[9c]
[10]
[11]
A. Heumann, R. Faure, J. Org. Chem. 1993, 58, 1276Ϫ1279.
Received January 18, 1999
K. Dranz, H. Waldman, in: Enzyme Catalysis in Organic Syn-
[O99027]
1678
Eur. J. Org. Chem. 1999, 1671Ϫ1678