On the mechanism of the unexpected facile formation of meso-diacetate products in enzymatic acetylation of alkanediols

Article abstract of DOI:10.1021/jo026652i

The mechanism of the unexpected facile formation of meso-diacetate previously observed in the enzymatic resolution of dl/meso mixtures of 2,4-pentanediol and 2,5-hexanediol with Candida antarctica lipase B has been elucidated. It was found that the formation of meso-diacetate proceeds via different mechanisms for the two diols. Enzyme-catalyzed acylation of AcO-d₃ labeled (R)-monoacetates of meso-2,4-pentanediol and meso-2,5-hexanediol and analysis of the mesodiacetates obtained show that the former reaction proceeds via intramolecular acyl migration while the latter occurs via direct S-acylation of the alcohol. For the (R)-monoacetate of (R,S)-2,4-pentanediol the intramolecular acyl migration was fast and therefore direct S-acylation by the external acyl donor is suppressed. For the hexanediol monoacetate the rate ratio (pseudo E value) between (5R,2R)- and (5R,2S)-5-acetoxy-2-hexanol was experimentally determined to be k_R,R/k_R,S = 25, which is about 10-20 times lower than the E value for 2-pentanol and 2-octanol. In a preliminary experiment it was demonstrated that facile acyl migration in the 1,3-diol derivative can be utilized to prepare syn-1,3-diacetoxynonane (β90% syn) in high enantioselectivity (β99% ee) via a chemoenzymatic dynamic kinetic asymmetric tyransformation of a meso/dl mixture of 1,3-nonanediol.

Full text of DOI:10.1021/jo026652i

On th e Mech a n ism of th e Un exp ected F a cile F or m a tion of
m eso-Dia ceta te P r od u cts in En zym a tic Acetyla tion of Alk a n ed iols
Michaela Edin and J an-E. Ba¨ckvall*
Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University,
SE-106 91 Stockholm, Sweden
jeb@organ.su.se
Received November 4, 2002
The mechanism of the unexpected facile formation of meso-diacetate previously observed in the
enzymatic resolution of dl/meso mixtures of 2,4-pentanediol and 2,5-hexanediol with Candida
antarctica lipase B has been elucidated. It was found that the formation of meso-diacetate proceeds
via different mechanisms for the two diols. Enzyme-catalyzed acylation of AcO-d3 labeled
(R)-monoacetates of meso-2,4-pentanediol and meso-2,5-hexanediol and analysis of the meso-
diacetates obtained show that the former reaction proceeds via intramolecular acyl migration while
the latter occurs via direct S-acylation of the alcohol. For the (R)-monoacetate of (R,S)-2,4-
pentanediol the intramolecular acyl migration was fast and therefore direct S-acylation by the
external acyl donor is suppressed. For the hexanediol monoacetate the rate ratio (pseudo E value)
between (5R,2R)- and (5R,2S)-5-acetoxy-2-hexanol was experimentally determined to be kR,R/kR,S
) 25, which is about 10-20 times lower than the E value for 2-pentanol and 2-octanol. In a
preliminary experiment it was demonstrated that facile acyl migration in the 1,3-diol derivative
can be utilized to prepare syn-1,3-diacetoxynonane (>90% syn) in high enantioselectivity (>99%
ee) via a chemoenzymatic dynamic kinetic asymmetric transformation of a meso/dl mixture of 1,3-
nonanediol.
SCHEME 1. DKR of Diols
In tr od u ction
As part of our ongoing program on the combined
enzyme- and transition metal-catalyzed dynamic kinetic
resolution (DKR) of various substrate classes,1 a proce-
dure for diols was recently reported.2 Despite the excel-
lent enantiomeric excess obtained, this process showed
moderate to low diastereoselectivity for certain diols,
which gave also the meso-compound containing an S-
acylated hydroxyl group (anti-Kazlauskas product)
(Scheme 1).2 This is unexpected since Candida antarctica
lipase B (CALB) usually exhibits a high preference for
(R)-alcohols,1,3 assuming the order of preference of the
R-hydroxyl substituents agrees with large-small; CALB-
catalyzed acylations normally lead to product formation
in strict agreement with Kazlauskas’ rule.4
proposed to account for the unexpected facile acylation
of the (S)-alcohol function: (i) an intramolecular acyl
transfer from the (R)-acetate to the (S)-alcohol in the
monoacylated (R,S)-diol with subsequent enzyme-cata-
lyzed acylation of the (R)-hydroxyl group released (cf.
path A, Scheme 2) and (ii) direct acylation of the (S)-
alcohol due to a lower enantioselectivity for the mono-
acylated diol (cf. path B, Scheme 2).
Because of the unexpectedly large proportion of meso-
diacetate produced in the dynamic kinetic resolution of
acyclic diols, we decided to study the mechanism of the
reaction. In the present work we have employed deute-
rium labeling and shown that the pentanediol derivative
follows the first pathway (via intramolecular acyl trans-
fer), whereas the hexanediol proceeds via the second
pathway (direct acylation).
Also, in a report on the kinetic resolution (KR) of diols
using the same enzyme, considerable amounts of the
meso-diacetate were produced employing 2,4-pentanediol
as the substrate.5 Two possible mechanisms have been
(1) (a) Huerta, F. F.; Minidis, A. B. E.; Ba¨ckvall, J .-E. Chem. Soc.
Rev. 2001, 30, 321. (b) Persson, B. A.; Larsson, A. L. E.; Le Ray, M.;
Ba¨ckvall, J .-E. J . Am. Chem. Soc. 1999, 121, 1645. (c) Larsson, A. L.
E.; Persson, B. A.; Ba¨ckvall, J .-E. Angew. Chem., Int. Ed. Engl. 1997,
36, 1211.
(2) Persson, B. A.; Huerta, F. F.; Ba¨ckvall, J .-E. J . Org. Chem. 1999,
64, 5237.
(3) Anderson, E. M.; Larsson, K. M.; Kirk, O. Biocatal. Biotransform.
1998, 16, 181.
(4) Kazlauskas, R. J .; Weissfloch, A. N. E.; Rappaport, A. T.; Cuccia,
L. A. J . Org. Chem. 1991, 56, 2656.
(5) Mattson, A.; O¨ hrner, N.; Hult, K.; Norin, T. Tetrahedron:
Asymmetry 1993, 4, 925.
Resu lts a n d Discu ssion
By deuterium labeling of the acetate group in (R)-
monoacetate of the meso-diol it would be possible to
10.1021/jo026652i CCC: $25.00 © 2003 American Chemical Society
Published on Web 02/15/2003
2216
J . Org. Chem. 2003, 68, 2216-2222

Formation of meso-Diacetate Products
SCHEME 2. Deu ter iu m La belin g to Distin gu ish betw een Acyl Tr a n sfer a n d Dir ect En zym a tic Acyla tion
SCHEME 3. Ster eoselective P r ep a r a tion of
SCHEME 4. Ster eoselective P r ep a r a tion of
(R)-Mon oa ceta tes of m eso-2,4-P en ta n ed iol
(R)-Mon oa ceta tes of m eso-2,5-Hexa n ed iol
SCHEME 5
differentiate between the intramolecular acyl transfer
pathway (path A, Scheme 2) and the direct acylation
route (path B, Scheme 2). In the intramolecular pathway
the deuterated acetoxy group would be transferred from
the (R)-alcohol to the (S)-alcohol group via a cyclic
intermediate. The (R)-alcohol function released would be
rapidly acylated via the enzyme and give diacetate
deuterated in the S-position. The pathway via direct
acylation would give a diacetate in which deuterium is
retained in the R-position.
A. P r ep a r a tion of Sta r tin g Ma ter ia ls a n d Refer -
en ce Com p ou n d s. Pure meso-diol 1 was obtained by
flash chromatography of the commercially available dl/
meso-diol. Enzymatic acylation of 1 employing CALB and
acyl donor 4 with careful monitoring of the reaction (TLC)
gave monoacetate 2 in high selectivity. Analogously,
stereoselective monoacylation of diol 1 by CALB and
deuterium-labeled acyl donor 5 afforded deuterated
monoacetate 3 (Scheme 3).
The nonlabeled and labeled (R)-monoacetates of meso-
hexanediol were prepared in analogy with the pentane-
diols. Pure meso-diol 6 was prepared from a commercially
available dl/meso-diol (dl/meso ∼1:1) by converting the
isomers into cyclic sulfites as described in the literature,6
followed by separation and hydrolysis. CALB-catalyzed
acylation of diol 6 with acyl donor 4 gave monoacetate 7,
while the same reaction employing labeled acyl donor 5
furnished monoacetate 8 (Scheme 4).
tively. The high R-selectivity of CALB in esterification
as well as in hydrolysis of esters was taken advantage
of. Reaction of the commercial diol with enzyme and acyl
donor 4 overnight afforded diacetates 10 and unchanged
(S,S)-diol 9, easily separable by flash chromatography.
Diacetates 10 were isolated in a ratio of (R,R)/(R,S) ∼
63:37. The significant production of (R,S)-diacetate can
most likely be explained by the direct anomalous S-
acylation found for monoacetates of 2,5-hexanediol (vide
infra). This reaction gave also a monoacetate fraction
containing mainly the (R)-monoacetate of the (R,S)-diol
((R,S)/(S,S) ) 95:5). Since this fraction was not used for
further studies, it was discarded and its structure omitted
in the scheme (Scheme 5). Optically pure monoacetate 7
was instead prepared as depicted in Scheme 4 (vide
supra). Chemical acetylation of 9 afforded monoacetate
11 (Scheme 5). Enzymatic hydrolysis of diacetate fraction
10 furnished (R,R)-diol 12, which was chemically trans-
formed into monoacetate 13 (Scheme 6). Although
chemoenzymatic routes to the pure enantiomers of hex-
anediol have been reported, our approach to combine
enzymatic esterification and hydrolysis is to the best of
our knowledge a novel approach toward these structures.
The stereoisomeric monoacetates 11 and 13 were also
prepared from the commercially available dl/meso-2,5-
hexanediol mixture via diol 9 and diacetates 10, respec-
(6) Caron, G.; Kazlauskas, R. J . Tetrahedron: Asymmetry 1994, 5,
657.
J . Org. Chem, Vol. 68, No. 6, 2003 2217

Edin and Ba¨ckvall
SCHEME 8. An a lysis of Deu ter a ted Dia ceta tes
SCHEME 6
SCHEME 7. Resu lts fr om En zym a tic Acyla tion of
Mon oa ceta tes
SCHEME 9. Resu lts fr om En zym a tic Acyla tion of
Mon oa ceta tes
B. Stu d ies of En zym a tic Acyla tion of th e (R)-
Mon oa ceta te of (R,S)-2,4-P en ta n ed iol. To distinguish
between the two mechanisms outlined in Scheme 2 we
studied the enzymatic acylation of specifically deuterated
monoacetate 3 and nondeuterated monoacetate 2 with
acyl donor 4 and 5, respectively. Enzyme-catalyzed
acetylation of (R)-monoacetate 3, specifically trideuter-
ated in the acetyl group, with CALB and nondeuterated
acyl donor 4 gave diacetate 14, which now has the
deuterium at the (S)-acetate and no deuterium in the
original (R)-acetate (Scheme 7, eq 1). Analogously, en-
zymatic acetylation of nondeuterated (R)-monoacetate 2
with deuterated acyl donor 5 afforded diacetate 15, where
the deuterated acetoxy group of the acyl donor ends up
in the R-position (Scheme 7, eq 2).
SCHEME 10. An a lysis of Deu ter a ted Dia ceta tes
These results unambiguously show that an intra-
molecular acetyl migration accounts for the formation of
the (S)-acetate. Apparently, the acyl migration in the syn
isomer (R,S) is relatively fast in this case, which is
consistent with the observation2 that there was more
meso-diacetate than (R,R)-product in the combined ru-
thenium- and enzyme-catalyzed DKR of the dl/meso-diol
(dl/meso ∼1:1). In the latter study the relative amount
of meso configuration in the product was higher than in
that of the starting material, suggesting that the acyl
migration is faster than the ruthenium-catalyzed epimer-
ization.
The analysis of diacetates 14 and 15 and determination
of the location of deuterium is difficult to make by
conventional methods. These pseudo meso compounds are
formally enantiomers of one another and differ only in
the deuterium labeling. To analyze these compounds we
took advantage of the fact that lipases can hydrolyze
meso-diacetates with high R-selectivity (Kazlauskas’ rule
considered). Enzymatic hydrolysis of diacetate 14 in a
buffered aqueous solution afforded 16 (Scheme 8). It was
found that the optical rotation of 16 had the opposite sign
to that of the starting monoacetate 3. The analogous
enzymatic hydrolysis of 15 furnished 17. Analysis of
monoacetates 16 and 17 by 1H NMR and MS showed that
the former is deuterated in the acetoxy group whereas
the latter is nondeuterated. A small amount of nondeu-
terated acetate (ca. 13%) in 16 and deuterated acetate
(ca 8%) in 17 was observed. This shows that the reaction
proceeds to ∼90% via intramolecular acyl migration (path
A, Scheme 2) and to ∼10% via the direct acyl transfer
pathway (path B, Scheme 2).
The presence of three deuteriums in the acetoxy group
of compound 16 (Scheme 8, eq 1) establishes that di-
acetate 14 has the d3-acetate in the S-position. Further-
more, the lack of deuterium in product 17 shows that the
isotope incorporation is at the (R)-acetate in diacetate 15
(Scheme 8, eq 2).
These results prove that the enzyme-catalyzed forma-
tion of the meso-diacetate from meso-2,4-pentanediol
proceeds via mechanism A, where the second acylation
is preceded by an intramolecular acyl transfer (Scheme
2). This acyl transfer releases the fast-reacting (R)-
alcohol, which is subsequently rapidly acylated by the
enzyme. A related acyl transfer has been observed in
lipase-catalyzed hydrolysis of diacetates of meso-1,3-diols
(2-substituted 1,3-propane diols).7 Analysis of mono-
acetates 16 and 17 by chiral GC revealed a slightly lower
enantiomeric excess (ee 90-92%) compared to that of the
starting materials 3 and 2, respectively (94% ee). This is
probably due to a minor contribution from nonselective
hydrolysis by the enzyme, or to intramolecular acyl
migration (path A) in 16 and 17 during the reaction.
C. Stu d ies of En zym a tic Acyla tion of th e (R)-
Mon oa ceta te of (R,S)-2,5-Hexa n ed iol. To make the
analogous study of a 1,4-related diol, deuterium-labeled
(7) Liu, K. K.-C.; Nozaki, K.; Wong, C.-H. Biocatalysis 1990, 3, 169.
2218 J . Org. Chem., Vol. 68, No. 6, 2003

Formation of meso-Diacetate Products
monoacetate 8 of meso-hexanediol was acetylated in the
presence of the enzyme. Interestingly, this substrate
furnished diacetate 18, which now has the labeling
retained in the R-position (Scheme 9, eq 1). Likewise,
acetylation of nondeuterium-labeled monoacetate 7 under
the same reaction conditions, using the deuterated acyl
donor, gave diacetate 19 where the deuterated acetoxy
group from the acyldonor is found in the S-position of
the diacetate (Scheme 9, eq 2).
Diacetates 18 and 19 were analyzed again by R-
selective hydrolysis with use of CALB in aqueous phos-
phate buffer (Scheme 10). The formation of (S)-monoac-
etates 20 and 21 was confirmed by the optical rotation,
which now had the opposite sign to that of the starting
(R)-monoacetates 8 and 7. This unambiguously confirms
the R-selectivity of the enzyme hydrolysis. Analysis of
monoacetates 20 and 21 by 1H NMR and MS showed that
the former is nondeuterated and that the latter is
trideuterated in the acetoxy group. The lack of deuterium
in product 20 (Scheme 10, eq 1) establishes that the label
was in the R-position of diacetate 18. Analogously, the
presence of deuterium in monoacetate 21 (Scheme 10,
eq 2) shows that the deuterated acetate was in the
S-position of diacetate 19.
F IGURE 1. Ra te of a cetyla tion of (a ) a lcoh ol 13 a n d (b)
a lcoh ol 7.
SCHEME 11. Rela tive Ra tes of Ster eoisom er ic
Mon oa ceta tes
These results prove that mechanism B operates for the
hexanediol and that the meso-diacetate is obtained
through a direct enzymatic acylation of the (S)-alcohol.
The possibility of a direct chemical acetylation was ruled
out since no reaction could be observed in the absence of
enzyme, neither on the isomeric mixture of 2,5-hex-
anediols nor on the pure (R)-monoacetate. In addition,
no acyl migration could be detected when monoacetate 8
was heated in toluene at 70 °C for 24 h in a control
experiment.
3-hydroxybutanoate (E ) 31),10 where presumably the
ester group leads to an additional interaction with the
enzyme.
D. Kin etic Stu d ies of En zym a tic Acyla tion of 2,5-
Hexa n ed iol Mon oa ceta tes. Because of the unexpected
facile direct enzymatic acylation of the (S)-alcohol func-
tion of the (R)-monoacetate of meso-2,5-hexanediol it was
of interest to study the kinetics of the process. The
relatively easy enzymatic acylation of the (S)-alcohol of
this monoacetate suggests that there is an additional
interaction between the enzyme and the acetoxy group
of the 5-acetoxy-2-hexanol. This interaction could facili-
tate acylation of the latter diol monoacetate and lead to
a much lower rate ratio (pseudo E value) between the
(S)-2-ol and the (R)-2-ol of the monoacetate, compared
to that of the parent 2-hexanol. It was therefore of
interest to determine the relative rate of the (2R)- and
(2S)-alcohols when there is an acetate in the 5-position.
Kinetic studies of the diastereomeric (2R,5R)-5-acetoxy-
2-hexanol and (2S,5R)-5-acetoxy-2-hexanol were per-
formed and the rates were compared for the approximate
first-order acetylation reactions. This gave a pseudo E
value of 25 for the enzyme (Scheme 11 and Figure 1).
This value is at least 1 order of magnitude smaller than
that for 2-hexanol.8 It is interesting to note that a related
moderate enantioselectivity with CALB was observed for
tert-butyl 4-hydroxypentanoate (E ) 10)9 and ethyl
The effect of a neighboring ester group was further
studied by examining whether the configuration of the
neighboring acetate would influence the rate of the
acylation of the alcohol. Remote recognition of a stereo-
genic carbon atom away from the reaction site has been
reported for hydrolytic enzymes in enantioselective hy-
drolysis11 and transesterification.12 The relative rates of
(2S,5R)-alcohol 7 (vide supra) and (2S,5S)-alcohol 11
were compared and in this case the kinetic studies
resulted in a pseudo E value of 2.2 (Scheme 11 and Figure
S1 available as Supporting Information). Comparison of
the pseudo ER,R/R,S ) 25 with the pseudo ER,S/S,S ) 2.2,
indicates a small but significant influence of the config-
uration at the acetoxy bearing carbon atom.13
E. Syn th etic Ap p lica tion of th e Acyl Migr a tion
Mech a n ism . The fact that the acetyl migration is fast
in syn-1,3-diol monoacetates, as shown for 2 and 3, could
(10) Magnusson, A.; Hult, K.; Holmquist, M. J . Am. Chem. Soc.
2001, 123, 4354.
(11) (a) Mizuguchi, E.; Achiwa, K. Tetrahedron: Asymmetry 1993,
4, 2303 and references therein. (b) Hughes, D. L.; Bergan, J . J .; Amato,
J . S.; Bhupathy, M.; Leazer, J . L.; McNamara, J . M.; Sidler, D. R.;
Reider, P. J .; Grabowski, E. J . J . J . Org. Chem. 1990, 55, 6252.
(12) Ors, M.; Morcuende, A.; J ime´nez-Vacas, M. I.; Valverde, S.;
Herrado´n, B. Synlett 1996, 449.
(13) To investigate the origin of the effect, the CALB-catalyzed
acylation rate of (S,S)-diol monoacetate 11 was compared with the rate
of monoacylation of (S,S)-diol 9 itself. It was found that they were
acylated with the same rate (pseudo E ) 1). The plot obtained is
available as Supporting Information. This finding suggests that the
electronegativity of the neighboring group is of importance.
(8) Rotticci, D.; Hæffner, F.; Orrenius, C.; Norin, T.; Hult, K. J . Mol.
Catal. B: Enzymol. 1998, 5, 267.
(9) Runmo, A.-B. L.; Pa`mies, O.; Faber, K.; Ba¨ckvall, J .-E. Tetra-
hedron Lett. 2002, 43, 2983.
J . Org. Chem, Vol. 68, No. 6, 2003 2219

Edin and Ba¨ckvall
standard, and coupling constants (J ) are given in Hz and
without sign. Enantiomeric and diastereomeric excess were
determined by analytical gas chromatography employing a CP-
Chirasil-Dex CB column for all monoacetates as well as for
diol 1, and a J W Cyclodex-B column for diacetate 10. Optical
rotations, [R]D, were measured at the sodium D line and at
ambient temperature. Mass spectra were recorded at an
ionizing voltage of 30 eV (EI). All reactions were carried out
under argon atmosphere in flame-dried glassware, except for
those reactions utilizing a water buffer as solvent, which were
run in air. Unless otherwise noted, reagents are commercially
available and were used without further purification. Toluene
was distilled from calcium hydride and stored over 4 Å
molecular sieves. Methylene chloride (CH2Cl2) was distilled
from calcium hydride prior to use. Aqueous 0.1 M phosphate
buffer pH 7.5 was prepared in a standard fashion with KH2-
PO4 and 1 M NaOH. Acyl donor 4 was prepared according to
a literature procedure,1b and acyl donor 5 was prepared by the
same method employing acetyl chloride-d3.14 Solvents used for
extractions and chromatography were of technical grade and
were distilled before use. The CALB (Candida Antarctica
lipase B) used was the immobilized commercially available
Novozym 435.
SCHEME 12. DYKAT of a 1,3-Diol
be used for synthetic transformations via a dynamic
kinetic asymmetric transformation (DYKAT). For a 1,3-
diol where only one of the alcohol functions (the least
sterically hindered) can be enzymatically acylated under
DYKAT conditions to give (R)-acetate, the other alcohol
would be intramolecularly acylated via acyl migration.
Because the acyl migration is favored in the syn-diol, the
acetate formed via intramolecular acylation will be of
S-configuration (see Scheme 12). The released (R)-alcohol
will subsequently be rapidly acylated to give the syn-
diacetate. Under dynamic conditions, where the alcohol
functions will be epimerized by e.g. a metal catalyst, a
racemic diastereomeric mixture of a 1,3-diol could be
transformed into one enantiomer of the diacetate. In
preliminary studies a racemic syn/anti mixture (syn/anti
∼1:1) of 2,4-nonanediol (25) was treated with ruthenium
catalyst 27, CALB, and acyl donor 4 to give enantiomeri-
cally pure (>99% ee) syn-diacetate 26 (syn/anti 90:10) in
58% yield after 72 h. This shows that a dynamic process
operates involving enzymatic resolution coupled with
ruthenium-catalyzed epimerization and syn-acyl migra-
tion. In this process control of the absolute stereochem-
istry at both chiral centers is obtained. Optimization of
this reaction and studies on its scope and limitations will
be reported in due course.
m eso-2,4-P en ta n ed iol (1).15 Flash chromatography (Et2O)
of commercial dl/meso-2,4-pentanediol gave pure meso-2,4-
pentanediol (1) (>99% de) as a pale yellow, highly viscous oil
about to crystallize. 1H NMR (300 MHz, CDCl3) δ 4.05 (m, 2H),
3.03 (br s, 2H), 1.53 (m, 2H), 1.20 (d, J ) 6.3, 6H); 13C NMR
(75 MHz, CDCl3) δ 68.7, 46.1, 24.0.
m eso-2,5-Hexa n ed iol (6). Commercial dl/meso-2,5-hexane-
diol (dl/meso ∼1:1) was converted into cyclic sulfites according
to a literature procedure.6 After 1 h at room temperature the
reaction mixture was concentrated on silica. Flash chroma-
tography (pentane/Et2O 10:1) gave a pure fraction of meso-
1
sulfite, as confirmed by H NMR.6
This sulfite (2.74 g, 16.7 mmol) was refluxed in 2 M NaOH
(60 mL) for 1 h and the reaction mixture was allowed to cool
to room temperature. The solution was saturated with solid
NaCl and extracted with EtOAc (5 × 40 mL). The combined
organic phases were concentrated in vacuo on silica and
subjected to flash chromatography with a stepwise eluent
gradient (pentane/EtOAc 1:1 f 0:1). Removal of the solvent
in vacuo gave 1 (1.76 g, 89%) as white crystals. Its NMR
spectra were in agreement with those previously reported.15b
The 13C NMR spectrum contained only signals corresponding
to the meso-diol; no traces of the racemic diol were detected.15b
Gen er a l P r oced u r e for En zym a tic Mon oa cyla tion of
m eso-Diols: (2S,4R)-4-(2,2,2-Tr id eu ter ioa cetoxy)-2-p en -
ta n ol (3). To a solution of diol 1 (0.398 g, 3.82 mmol) and acyl
donor 5 (1.01 g, 5.82 mmol) in toluene (11.5 mL) was added
CALB (0.229 g). The Schlenk flask was evacuated and filled
with argon and this procedure was repeated three times before
it was sealed and the mixture was stirred at room tempera-
ture. After 33 min the reaction mixture was filtered and the
enzyme was washed with Et2O. The filtrate was evaporated
under vacuo and flash chromatography (stepwise gradient of
pentane/Et2O 1:1 f 1:2) of the residue afforded 3 (0.507 g, 89%)
Con clu sion s
We have been able to distinguish between the two
previously proposed mechanisms for the formation of
anti-Kazlauskas products observed in CALB-catalyzed
acylation of 2,4-pentanediol and 2,5-hexanediol. Deute-
rium-labeling studies show that the former diol gives a
monoacetate that is prone to undergo intramolecular acyl
transfer (Mechanism A, Scheme 2), whereas the latter
substrate is diacylated in a direct enzymatic fashion
(Mechanism B, Scheme 2). For the hexandiol monoac-
etates 7 and 8 a neighboring effect of the acetate reduces
the substrate specificity of the enzyme. The facile acyl
migration in monoacylated 1,3-diols was applied to
DYKAT of nonane-1,4-diol to give the enantiomerically
pure syn-diacetate.
as a pale yellow oil about to crystallize. [R]24 -1.6 (c 3.0,
D
CHCl3); ee 94%; 1H NMR (300 MHz, CDCl3) δ 5.04 (app sextet,
J ) 6.3, 1H), 3.89 (br app sextet, J ) 6.2, 1H), 2.00 (m, 0.4H),
1.83 (app dt, J ) 7.9, 14.0, 1H), 1.59 (app dt, J ) 5.1, 14.0,
1H), 1.26 (d, J ) 6.2, 3H), 1.21 (d, J ) 6.2, 3H); 13C NMR (75
MHz, CDCl3) δ 170.7, 69.4, 65.6, 45.2, 23.7, 20.9 (m), 20.3.
(2S,4R)-4-Acet oxy-2-p en t a n ol (2). The same procedure
was used to give a colorless oil of 2. Yield 83%. ee 94%; 1H
Exp er im en ta l Section
Gen er a l Meth od s. Analytical TLC was performed with use
of aluminum plates precoated with silica gel 60 F254 and
detected with UV light and phosphomolybdic acid (5% (w/v)
in EtOH). Flash chromatography was carried out on 60 Å (35-
70 µm) silica gel. 1H NMR and 13C NMR spectra were recorded
at 400 or 300 MHz and at 100 or 75 MHz, respectively.
Chemical shifts (δ) are reported in ppm, using the residual
solvent peak in CDCl3 (δH 7.26 and δC 77.0) as internal
(14) Acetyl-d3 chloride, 99+ atom % D, was purchased from Aldrich
company. However, 1H NMR showed the bottle to contain about 35%
CD2HCOCl, which is why a proton signal from the acetyl group in the
deuterated monoacetates is reported.
(15) (a) Gordillo, B.; Herna´ndez, J . Org. Prep. Proced. Int. 1997, 29,
195. (b) Ikeda, H.; Sato, E.; Sugai, T.; Ohta, H. Tetrahedron 1996, 52,
8113.
2220 J . Org. Chem., Vol. 68, No. 6, 2003

Formation of meso-Diacetate Products
NMR (300 MHz, CDCl3) δ 5.04 (app sextet, J ) 6.4, 1H), 3.89
(app sextet, J ) 6.3, 1H), 2.04 (s, 3H), 1.84 (app dt, J ) 7.7,
14.4, 1H), 1.59 (app dt, J ) 5.0, 14.4, 1H), 1.26 (d, J ) 6.4,
3H), 1.21 (d, J ) 6.3, 3H); 13C NMR (100 MHz, CDCl3) δ 170.7,
69.5, 65.5, 45.1, 23.7, 21.3, 20.2.
tane/Et2O 1:2 f EtOAc) gave 12 as white crystals (0.286 g,
37%). [R]23D -35.4 (c 9.0, CHCl3) (lit.16 [R]25D -35.7 (c 1, CHCl3),
lit.18 [R]25 -39.6 (c 1, CHCl3)); the 1H NMR and 13C NMR
D
spectra were in accordance with those previously reported.15b,16
(2R,5R)-5-Acetoxy-2-h exa n ol (13). 13 was prepared from
12 as described for the preparation of compound 11 from 9.
Isolated as a pale yellow oil, partly crystallized. Yield 45%
(>99% ee, 99% de). 1H NMR (400 MHz, CDCl3) δ 4.91 (app
sextet, J ) 6.2, 1H), 3.80 (br m, 1H), 2.02 (s, 3H), 1.76-1.38
(m, 4H), 1.22 (d, J ) 6.4, 3H), 1.19 (d, J ) 6.2, 3H); 13C NMR
(100 MHz, CDCl3) δ 170.8, 70.8, 67.6, 34.7, 32.0, 23.5, 21.3,
19.9.
(2S,5R)-5-Acetoxy-2-h exa n ol (7). Isolated as a pale yellow
oil. Yield 81%. [R]29 +9.1 (c 1.00, CHCl3) (lit.16 [R]25 +9.6 (c
D
D
1
1, CHCl3)); ee >99%; H NMR (400 MHz, CDCl3) δ 4.90 (app
sextet, J ) 6.2, 1H), 3.79 (app sextet, J ) 6.2, 1H), 2.03 (s,
3H), 1.62 (m, 2H), 1.46 (m, 2H), 1.22 (d, J ) 6.4, 3H), 1.19 (d,
J ) 6.2, 3H); 13C NMR (100 MHz, CDCl3) δ 170.8, 71.0, 67.6,
34.8, 32.1, 23.5, 21.3, 19.9.
Gen er a l P r oced u r e for Acyla tion of Diol Mon oa ce-
ta tes: (2S,4R)-4-Acetoxy-2-(2,2,2-tr id eu ter ioa cetoxy)p en -
ta n e (14). To a stirred solution of diol monoacetate 3 (0.302
g, 2.02 mmol) and acyl donor 4 (0.521 g, 3.05 mmol) in toluene
(6.1 mL) was added CALB (0.121 g). The flask was evacuated
and filled with argon and this procedure was repeated three
times before it was sealed and then heated in an oil bath at
70 °C. The reaction mixture was stirred for 18.5 h and then
allowed to cool to room temperature. The mixture was filtered
and the enzyme was washed with Et2O (100 mL). The filtrate
was washed with 1 M NaOH (3 × 40 mL). The washings were
back-extracted with Et2O (2 × 60 mL) and the combined
organic phases were washed with water and brine, dried
(MgSO4), and concentrated. Flash chromatography (stepwise
gradient of pentane/Et2O 5:1 f 3:1) furnished 14 as a pale
yellow oil (0.299 g, 77%). 1H NMR (300 MHz, CDCl3) δ 4.97
(app sextet, J ) 6.2, 2H), 2.03 (s, 3H), 1.98 (m, 1H), 1.63 (m,
1H), 1.23 (d, J ) 6.2, 6H); 13C NMR (100 MHz, CDCl3) δ 170.5,
68.0, 67.9, 41.8, 21.3, 20.8 (m), 20.1.
(2S,5R)-5-(2,2,2-Tr id eu ter ioa cetoxy)-2-h exa n ol (8). Iso-
lated as a pale yellow oil. Yield 83%. [R]23D +8.0 (c 1.27, CHCl3);
1
ee >99%; H NMR (400 MHz, CDCl3) δ 4.90 (app sextet, J )
6.2, 1H), 3.79 (app sextet, J ) 6.2, 1H), 2.00 (m, 0.4H), 1.63
(m, 2H), 1.46 (m, 2H), 1.22 (dd, J ) 6.2, 1.5, 3H), 1.19 (dd, J
) 5.9, 1.5, 3H); 13C NMR (100 MHz, CDCl3) δ 170.9, 71.0, 67.7,
34.9, 32.1, 23.5, 20.8 (m), 19.9.
(2S,5S)-2,5-Hexa n ed iol (9) a n d Dia ceta tes 10. To a
stirred solution of dl/meso-hexanediol ∼1:1 (0.303 g, 2.56
mmol) and acyl donor 4 (1.31 g, 7.69 mmol) in toluene (8 mL)
was added at room temperature CALB (0.154 g). The Schlenk
flask was evacuated and filled with argon and the procedure
was repeated three times before the flask was sealed. The
reaction mixture was stirred at room temperature overnight
and was filtered. The enzyme was washed with Et2O and the
filtrate was concentrated in vacuo. Flash chromatography with
pentane/EtOAc (stepwise gradient 1:1 f 0:1) provided un-
changed alcohol 9 (0.064 g, 21%) as white crystals. [R]24D +34.4
1
(c 9.0, CHCl3) (lit.17 [R]25 +35.1 (c 9.5, CHCl3)); the H NMR
D
and 13C NMR spectra are consistent with those previously
reported15b,16 and the 13C NMR shows no traces of diastereo-
isomeric meso-diol.15b From this column was also isolated a
fraction of pure monoacetate (0.159 g, 39%) as a colorless oil.
ee >99%; de 89%; (R,S)/(S,S) ) 95:5.
(2R ,4S )-4-Ace t oxy-2-(2,2,2-t r id e u t e r ioa ce t oxy)p e n -
1
ta n e (15). A pale yellow oil. Yield 73%. H NMR (400 MHz,
CDCl3) δ 4.95 (app sextet, J ) 6.2, 2H), 2.01 (s, 3H), 1.97 (app
dt, J ) 7.2, 14.5, 1H), 1.61 (app dt, J ) 5.9, 14.2, 1H), 1.24 (d,
J ) 6.3, 6H); 13C NMR (100 MHz, CDCl3) δ 170.4, 67.9, 67.9,
41.8, 21.3, 20.8 (m), 20.0.
The fastest running fractions from the above column
contained diacetate, acyl donor 4, and p-chlorophenol in a
mixture. These fractions were concentrated in vacuo, then
diluted with Et2O (25 mL) and washed with 1 M NaOH (3 ×
10 mL). The aqueous phases were extracted with Et2O (2 ×
20 mL) and the combined organic layers were washed with
water and brine. Drying (MgSO4), concentration, and flash
chromatography (pentane/Et2O stepwise gradient 7:1 f 3:1)
gave diacetate 10 (0.186 g, 36%) as a pale yellow oil. (R,R)/
(R,S) ∼ 63:37.
(2R ,5S )-5-Ace t oxy-2-(2,2,2-t r id e u t e r ioa ce t oxy)h e x-
a n e (18). Isolated as needle-shaped crystals. Yield 74%. 1H
NMR (300 MHz, CDCl3) δ 4.86 (m, 2H), 2.02 (s, 3H), 2.00 (m,
0.4H), 1.55 (m, 4H), 1.20 (d, J ) 6.3, 6H); 13C NMR (100 MHz,
CDCl3) δ 170.6, 70.6, 70.6, 31.8, 21.3, 20.8 (m), 19.9.
(2S ,5R )-5-Ace t oxy-2-(2,2,2-t r id e u t e r ioa ce t oxy)h e x-
a n e (19). Isolated as needle-shaped crystals. Yield 77%. 1H
NMR (300 MHz, CDCl3) δ 4.87 (m, 2H), 2.03 (s, 3H), 2.00 (m,
0.3H), 1.55 (m, 4H), 1.20 (d, J ) 6.3, 6H); 13C NMR (100 MHz,
CDCl3) δ 170.7, 70.7, 70.6, 31.8, 21.3, 20.9 (m), 19.9.
Gen er a l P r oced u r e for R-Selective Hyd r olysis of Di-
acetates: (2R,4S)-4-(2,2,2-Tr ideu ter ioacetoxy)-2-pen tan ol
(16). A mixture of diacetate 14 (182 mg, 0.95 mmol) and CALB
(29 mg) in a 0.1 M phosphate buffer pH 7.5 (10 mL) was stirred
at room temperature for 9 h. The solution was filtered and
the enzyme was washed with EtOAc. Solid NaCl was added
to saturate the water phase and the layers were separated.
The aqueous phase was extracted with EtOAc (4 × 20 mL)
and the combined extracts were washed with brine, dried
(MgSO4), and concentrated. Flash chromatography (pentane/
Et2O 1:2) gave 16 as a pale yellow oil (98 mg, 69%). [R]22D +2.0
(c 3.0, CHCl3); ee 92%; 1H NMR (300 MHz, CDCl3) δ 5.03 (app
sextet, J ) 6.4, 1H), 3.88 (m, 1H), 2.03 (s, 0.5H), 2.00 (m, 0.3H),
1.83 (app dt, J ) 7.7, 14.3, 1H), 1.58 (app dt, J ) 5.5, 14.2,
1H), 1.25 (d, J ) 6.3, 3H), 1.20 (d, J ) 6.2, 3H); 13C NMR (100
MHz, CDCl3) δ 170.7, 69.4, 65.5, 45.1, 23.7, 20.9 (m), 20.3; MS
(EI) m/z (rel) 150 (2.5, M+ + 1), 71 (84), 64 (100), 63 (70), 61
(41).
(2S,5S)-5-Acetoxy-2-h exa n ol (11). To a stirred solution
of diol 9 (52 mg, 0.44 mmol), DMAP (cat.), and Et3N (0.123
mL, 0.88 mmol) in CH2Cl2 (5 mL) at room temperature was
added Ac2O (44 µL, 0.46 mmol) dropwise over 1 h. The reaction
mixture was stirred at room temperature for 5 h, and then
poured into 1 M HCl (5 mL). The layers were separated and
the aqueous phase was extracted with CH2Cl2. The combined
organic phases were washed with saturated aq Na2CO3, water,
and brine. Drying (MgSO4) followed by flash chromatography
(pentane/Et2O 1:2) furnished 24 mg (46%) of 11 as a colorless
oil (>99% ee, 99% de). The spectral data were identical with
those of its enantiomer 13.
(2R,5R)-2,5-Hexa n ed iol (12). Enzyme (0.197 g) was added
to a stirred emulsion of diacetate fraction 10 (1.33 g, 6.56
mmol) in a 0.1 M phosphate buffer pH 7.5 (60 mL) and the
reaction mixture was stirred at room temperature for 17 h.
The reaction mixture was then filtered through a filter paper
and the enzyme was washed with EtOAc. The aqueous phase
was saturated with solid NaCl before the layers were sepa-
rated and the aqueous phase extracted with EtOAc (4 × 40
mL). The combined organic phases were washed with brine,
dried (MgSO4), and concentrated. Flash chromatography (pen-
(2R,4S)-4-Acetoxy-2-p en ta n ol (17). A pale yellow oil.
Yield 45%. ee 92%; 1H NMR (400 MHz, CDCl3) δ 5.03 (m, 1H),
3.89 (m, 1H), 2.03 (s, 3H), 1.83 (app dt, J ) 7.8, 14.3, 1H),
1.58 (app dt, J ) 5.0, 14.3, 1H), 1.26 (d, J ) 6.3, 3H), 1.20 (d,
(16) Kim, M.-J .; Lee, I. S. J . Org. Chem. 1993, 58, 6483.
(17) Serck-Hanssen, K.; Sta¨llberg-Stenhagen, S.; Stenhagen, E. Ark.
Kemi 1953, 5, 203.
(18) Burk, M. J .; Feaster, J . E.; Harlow, R. L. Tetrahedron: Asym-
metry 1991, 2, 569.
J . Org. Chem, Vol. 68, No. 6, 2003 2221

Edin and Ba¨ckvall
J ) 6.2, 3H); 13C NMR (75 MHz, CDCl3) δ 170.7, 69.5, 65.7,
45.2, 23.7, 21.4, 20.3; MS (EI) m/z (rel) 147 (0.7, M+ + 1), 91
(13), 71 (45), 64 (13), 61 (100).
En zym a tic Acyla tion of Mon oa ceta tes for Kin etic
Deter m in a tion s. To a solution of 13 (34 mg, 0.21 mmol) and
4 (0.109 g, 0.64 mmol) in toluene-d8 (0.64 mL) was added
CALB (13 mg). The tube was evacuated and argon filled three
times and was then rocked during the reaction, which was
(2R,5S)-5-Acetoxy-2-h exa n ol (20). Isolated as a pale
yellow oil (23 mg, 84%). [R]24 -8.8 (c 1.10, CHCl3); ee >99%
D
1
1
(chiral GC); H NMR (400 MHz, CDCl3) δ 4.90 (app sextet, J
monitored by H NMR spectroscopy.
) 6.2, 1H), 3.79 (app sextet, J ) 6.2, 1H), 2.03 (s, 3H), 1.63
(m, 2H), 1.46 (m, 2H), 1.21 (d, J ) 6.2, 3H), 1.19 (d, J ) 6.2,
3H); 13C NMR (75 MHz, CDCl3) δ 170.8, 71.0, 67.8, 34.9, 32.2,
23.6, 21.4, 20.0; MS (EI) m/z (rel) 161 (62, M+ + 1), 101 (100),
83 (48), 67 (80), 61 (47).
Ackn owledgm en t. Financial support from the Swed-
ish Research Council and the Swedish Foundation for
Strategic Research is gratefully acknowledged.
(2R,5S)-5-(2,2,2-Tr ideu ter ioacetoxy)-2-h exan ol (21). Iso-
Su p p or tin g In for m a tion Ava ila ble: Graphical presenta-
tion of the rates obtained from kinetic measurements of (S,S)-
diol monoacetate 11 and (S,S)-diol 9 and 1H NMR and 13C
NMR spectra of compounds 2, 3, 7, 8, and 13-21. This
material is available free of charge via the Internet at
http://pubs.acs.org.
lated as a colorless oil. Yield 55%. [R]29 -9.5 (c 1.30, CHCl3);
D
1
ee >99%; H NMR (400 MHz, CDCl3) δ 4.90 (app sextet, J )
6.3, 1H), 3.79 (m, 1H), 2.00 (m, 0.4H), 1.63 (m, 2H), 1.45 (m,
2H), 1.22 (d, J ) 6.2, 3H), 1.19 (d, J ) 6.2, 3H); 13C NMR (75
MHz, CDCl3) δ 170.8, 70.9, 67.8, 34.9, 32.2, 23.6, 20.9 (m), 20.0;
MS (EI) m/z (rel) 164 (36, M+ + 1), 101 (100), 85 (38), 83 (43),
67 (71).
J O026652I
2222 J . Org. Chem., Vol. 68, No. 6, 2003