Influence of intramolecular hydrogen bonds in the enzyme-catalyzed regioselective acylation of quinic and shikimic acid derivatives

Article abstract of DOI:10.1016/j.tet.2006.03.074

Selective mono-functionalization of 3-epi, 4-epi-, and 5-epi quinic and shikimic acid derivatives has been accomplished by enzymatic acylation with Candida antarctica lipase A (CAL-A). We propose that the selectivity of this lipase is related to both the

Full text of DOI:10.1016/j.tet.2006.03.074

Tetrahedron 62 (2006) 5401–5410
Influence of intramolecular hydrogen bonds in the
enzyme-catalyzed regioselective acylation of
quinic and shikimic acid derivatives
*
´
Nuria Armesto, Susana Fernandez, Miguel Ferrero and Vicente Gotor
´
Departamento de Quımica Organica e Inorganica and Instituto Universitario de Biotecnologıa de Asturias,
´
´
´
Universidad de Oviedo, 33006-Oviedo, Asturias, Spain
Received 1 February 2006; revised 20 March 2006; accepted 22 March 2006
Available online 12 April 2006
Abstract—Selective mono-functionalization of 3-epi, 4-epi-, and 5-epi quinic and shikimic acid derivatives has been accomplished by
enzymatic acylation with Candida antarctica lipase A (CAL-A). We propose that the selectivity of this lipase is related to both the inherent
receptor selectivity and the degree of intramolecular hydrogen bonding in the ligand. Conformational analysis of the hydroxyl protons has
been carried out by 1H NMR spectroscopy. We have shown that exchange of the hydroxyl protons by acid catalysis provides a useful method
for the detection of intramolecular hydrogen bonds. The interpretation of exchange rates and coupling constants determines the direction of
the H-bonds as conditioned by the relative acceptor and donor properties of the hydroxyl groups. The selectivity of the acylation agrees fully
with the effectiveness of H-bonding networks in polyol compounds and with the higher reactivity of the equatorial hydroxyl groups.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
the primary 50-OH during the synthesis of 50-O-galactosyl-
ated nucleosides. Hydrogens of such primary hydroxyls
are shown to be intramolecularly hydrogen-bonded with
the heteroaromatic system present in the molecule, and con-
sequently proved to be resistant to hydrogen abstraction.5 In
another example, selective 2-O-benzylation of sucrose is due
to the persistence of an intramolecular hydrogen bond, which
makes the hydroxyl group at the 2-position the most readily
deprotonated in aprotic solvents.6 Hydrogen bonds are also
confirmed to play a significant role in the acidic behavior
of the various hydroxyl groups of the sucrose molecule.7
The relative reactivities in the DMAP-catalyzed acetylation
were successfully correlated with the calculated proton affin-
ity of each OH group in carbohydrates by Yoshida et al.8
They found that secondary OH groups were preferentially
acetylated in the presence of the primary OH group at
position 6. In contrast, the 6-O-acetylated product was the
major one in the absence of DMAP. In addition, molecular
recognition through hydrogen-bonding interactions is one
of the challenging goals in supramolecular chemistry.9
The selective functionalization of one particular hydroxyl
group among others of similar reactivity in polyhydroxylated
molecules is extremely difficult in organic synthesis. A major
problem is the lack of an efficient orthogonal protection–
deprotection strategy. In the carbohydrate chemistry, Wong
et al.1 have described the synthesis of a library of oligosac-
charides using a designed building-block with four selec-
tively removable protecting groups as acceptors for
glycosylation. On the other hand, regioselectivity in sugar
chemistry is often solved using an approach based on com-
plexation-induced activation of a particular OH group. For
this purpose, tin2 and boron3 reagents have been widely used.
In some cases certain hydroxyl groups have shown an anoma-
lous reactivity, with a secondary hydroxyl group being func-
tionalized instead of the usually more reactive primary
one.4 However, steric interactions within themselves are
not sufficient to explain this unusual reactivity. The exis-
tence of noncovalent intramolecular bonding interactions
involving hydroxyls is well recognized in carbohydrate
chemistry. The role of intramolecular hydrogen bonding
has been used as an explanation for the low reactivity of
Enzyme-catalyzed reactions are practical processes for the
selective modifications of polyol derivatives. The regio-
selectivity of enzyme catalysis has been exquisitely ex-
ploited in the fields of nucleosides,10 steroids,11 and
carbohydrates.12 Enzymes catalyze reactions under mild
conditions, diminish undesired side-reactions, and facilitate
product recovery. Furthermore, enzymes are proteins, and as
such they are completely biodegradable.
Supplementary data associated with this article can be found in the online
version, at doi:10.1016/j.tet.2006.03.074
* Correspondingauthor. Tel./fax:+34985103448;e-mail:vgs@fq.uniovi.es
0040–4020/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2006.03.074

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N. Armesto et al. / Tetrahedron 62 (2006) 5401–5410
ported for the natural isomer,13b the enzymatic transesterifi-
CO Me
2
MeO C OH
2
cation of methyl 3-epi-shikimate (5) was carried out at 20 ꢀC
with vinyl acetate as acylating agent and solvent, CAL-A,
3
4
3
5
5
4
˚
and in the presence of molecular sieves 4 A. CAL-A ex-
HO
OH
HO
OH
OR
OR
hibited excellent selectivity towards the 3-position, giving
rise to methyl 3-O-acetyl-3-epi-shikimate (6) in 88% yield
after 0.5 h. In addition, methyl 3,5-di-O-acetyl-3-epi-shiki-
mate was isolated as a minor compound (92:8 ratio 6:diacyl
derivative calculated by 1H NMR). To avoid the formation of
the diacyl derivative other solvents suitable for the enzy-
matic acylation of methyl shikimate such as tert-butylmethyl
ether (TBME), toluene, 1,4-dioxane, chloroform or THF
were used. In general, longer reaction times were achieved,
although CAL-A maintained a similar degree of selectivity.
1, R= H, Methyl Shikimate
2, R= Ac
3, R= H, Methyl Quinate
4, R= Ac
CO Me
2
MeO C OH
2
HO
OR
HO
OH
OR
OH
5
7
, R= H, Methyl 3-epi-Shikimate
, R= H, Methyl 3-epi-Quinate
6, R= Ac
8, R= Ac
If as seems, the selectivity of CAL-A is highly dependent on
the relative position of the hydroxyl groups, CAL-A would
also acylate the 3-position of methyl 3-epi-quinate (7) as it
did with methyl 3-epi-shikimate (5). This fact was corrobo-
rated when the latter was subjected to similar reaction con-
ditions as those described for methyl quinate. That is, with
vinyl acetate both as solvent and ꢀacylating agent, CAL-A,
CO Me
2
MeO C OH
2
HO
OH
HO
OH
OR
OR
˚
molecular sieves 4 A, and at 40 C. However, since 7 is
9, R= H, Methyl 4-epi-Shikimate
10, R= Ac
11, R= H, Methyl 4-epi-Quinate
12, R= Ac
a meso compound, positions 3 and 5 are prochirals. Thus,
CAL-A catalyzes the acylation of methyl 3-epi-quinate
toward the C-3 or C-5 position with 95% regioselectivity
(C-3 or C-5 vs C-4) in 4.5 h. Importantly, no traces of
CO Me
2
MeO C OH
2
1
4-O-acyl or diacyl derivatives were detected by H NMR
of the crude reaction mixture.
RO
OH
HO
OR
OH
OH
The enantiopurity of compound 8 was determined by deriva-
tization to the corresponding trans-acetal 17 (Scheme 1). In
addition, (ꢁ)-17 was synthesized by protection of trans-1,2-
diol in meso-methyl 3-epi-quinate and subsequent treatment
with acetic anhydride as described in Scheme 2.
13, R= H, Methyl 5-epi-Shikimate
14, R= Ac
15, R= H, Methyl 5-epi-Quinate
16, R= Ac
Figure 1. Structures of natural (1 and 3), 3-epi (5 and 7), 4-epi (9 and 11),
and 5-epi (13 and 15) shikimic and quinic acid derivatives and their regio-
selective enzymatic acetylated products (2, 4, 6, 8, 10, 12, 14, and 16) with
CAL-A using vinyl acetate both as acylating agent and solvent.
Chiral HPLC analysis of (ꢁ)-17 and derivative of 17 ob-
tained from the enzymatic reaction revealed an enantiomeric
excess of 60% (see page S2 in Supplementary data). The as-
signment of the relative configuration of 17 was determined
by comparison of the sign of the optical rotation of our syn-
thesized derivative of 17 and that of the enantiomerically
pure derivative prepared according to Scheme 2.15 This
was corroborated by chiral HPLC analysis of both com-
pounds, which clearly establishes the configuration
(1R,3S,4S,5R) for our compound. This means that acetyla-
tion of methyl 3-epi-quinate takes place with moderate
selectivity towards 3-position. We did not try to find the
optimal conditions for asymmetrization of 7 since it was
not the aim of this study.
In our ongoing research focused on the synthesis of quinic
and shikimic acid derivatives, we carried out the regioselec-
tive enzymatic acylation of methyl shikimate (1, Fig. 1) and
methyl quinate (3) with Candida antarctica lipase A (CAL-
A).13 This lipase allowed the selective acylation of the C-4
hydroxyl group, giving rise to a variety of ester derivatives
of both acids, including the cinnamate analogues. Previous
studies on the enzymatic acylation of quinic and shikimic
acid derivatives have been described by Guyot et al.14a and
Danieli et al.14b Although, the use of enzymes in synthesis
is common, the basis of their selectivity is not well under-
stood. The goals of this study are both to regioselectively
prepare the valuable monoacyl analogues of the 3-epi,
4-epi, and 5-epi quinic and shikimic acid derivatives, and
to identify the molecular basis of the regioselectivity of
CAL-A towards these compounds as a function of the degree
of intramolecular hydrogen bonding within the ligand.
MeO2C OH
OH
MeO2C OH
MeO2C
O
a
b
HO
OH
OAc
OAc
HO
OH
OH
MeO
2. Results and discussion
O
OMe
8
7
2.1. Regioselective enzymatic acylation of methyl 3-epi,
4-epi, and 5-epi quinic and shikimic acid derivatives
17
(a) CAL-A, CH2=CHOAc, sieves 4 Å, 40 °C, 4.5 h; (b) butane-
2,3-dione, CH(OMe)3, ( )-CSA, MeOH, 65 °C, 2 h
2.1.1. Enzymatic acylation of (L)-methyl 3-epi-shikimate
(5) and meso-methyl 3-epi-quinate (7).15 As previously re-
Scheme 1.

N. Armesto et al. / Tetrahedron 62 (2006) 5401–5410
5403
MeO2C OH
MeO2C
HO
MeO2C
O
OH
OH
A
B
a
b
HO
HO
OH
CO2Me
OH
H5
H4
HO
O
OAc
OH
OH
HO
MeO
MeO
O
O
OH
H3
CO2Me
H5
H3
H4
OMe
OMe
7
13
( )-18
( )-17
Figure 2. Chair conformations of methyl 5-epi-shikimate in MeOH-d4 (A)
and in CDCl3 (B).
MeO2C
O
OH
MeO2C
MeO2C
HO
OH
OH
ref. 14
b
axial one (A, Fig. 2). On the other hand, OH-3 and OH-5
are in axial orientation and OH-4 is in equatorial one, which
are the proposed conformation in CDCl3 (B, Fig. 2). In that
conformation, strong 1,3-diaxial intramolecular hydrogen
bonds are present and as a result this should be the favored
conformation in nonhydrogen bonding solvents.
OAc
O
OH
OH
MeO
MeO
O
O
OH
OMe
OMe
3
(+)-18
(+)-17
(a) butane-2,3-dione, CH(OMe)3, ( )-CSA, MeOH, 65 °C, 2 h;
(b) Ac2O, DMAP, Py, CH2Cl2, 0 °C, 4 h
A comparison of the acylation of 13 in hydrogen (acetone,
vinyl acetate) and nonhydrogen (CH2Cl2, CHCl3) bonding
solvents has been performed. No changes in the regioselec-
tivity were observed with any of the solvents tested.
Scheme 2.
2.1.2. Enzymatic acylation of (L)-methyl 4-epi-shikimate
(9) and (L)-methyl 4-epi-quinate (11).16 We extended the
aforementioned methodology to 4-epi derivatives. First, we
studied the acylation reaction of methyl 4-epi-shikimate
(9) with vinyl acetate at 20 ꢀC in the presence of molecular
sieves, and catalyzed by CAL-A. The transesterification
takes place in 1.5 h with total selectivity towards the C-4 hy-
droxyl group, methyl 4-O-acetyl-4-epi-shikimate (10) being
isolated exclusively in 80% yield after flash chromato-
graphy.
Finally, selective acylation at the C-4 position of methyl 5-
epi-quinate (15) was successfully accomplished using
CAL-A at 40 ꢀC for 4.5 h. As with the 5-epi-shikimate
derivative, methyl 5-epi-quinate showed different conforma-
tion in MeOH-d4 and CDCl3 solvents. Protons H-6a and
H-2a exhibited in MeOH-d4 a 3JHHz12.1 Hz corresponding
to an axial–axial vicinal coupling to H-3 and H-5, in addition
to a large geminal coupling (2JHHz12.1 Hz). Therefore,
OH-3 and OH-5 are in equatorial orientation and OH-4 in
axial one (A, Fig. 3). On the other hand, in CDCl3 the value
of this vicinal coupling constant is z3.8 Hz, which is con-
sistent with an axial position for OH-3 and OH-5, and an
equatorial orientation for OH-4 (B, Fig. 3).
Similarly, methyl 4-epi-quinate (11) was subjected to reac-
tion with CAL-A under identical conditions as 7. However,
the presence of corresponding 1,5-lactone 19 (see below,
Fig. 8D) was observed and a mixture of acylated derivatives
was obtained. Formation of the lactone has been previously
observed in the attempted recrystallization of 11. To avoid
this by-product, the reaction was carried out in the absence
of molecular sieves since these remove the MeOH and shift
the equilibrium towards the lactone. Also, the temperature
was decreased from 40 to 20 ꢀC. In these conditions,
CAL-A furnished exclusively the 4-O-acetyl derivative 12
after 24 h.
A
B
HO
OH
OH
OH
CO2Me
OH
H4
HO
H5
HO
OH
H5
H3
H3
CO2Me
H4
15
Figure 3. Chair conformations of methyl 5-epi-quinate in MeOH-d4 (A) and
in CDCl3 (B).
2.1.3. Enzymatic acylation of (L)-methyl 5-epi-shikimate
(13) and meso-methyl 5-epi-quinate (15).17 When the reac-
tion was done with methyl 5-epi-shikimate (13), CAL-A
showed total selectivity towards the C-5 hydroxyl group,
a 100% conversion being achieved after 0.3 h at 20 ꢀC.
Although different solvents were tested (acetone, vinyl ace-
tate, CH2Cl2, and CHCl3) CAL-A maintains the excellent
regioselectivity; methyl 4-O-acetyl-5-epi-quinate (16) in all
cases being isolated.
In contrast to other epi-isomers, 1H NMR of the 5-epi-shiki-
mate derivative revealed different half-chair conformations
in MeOH-d4 and CDCl3. Change from MeOH-d4 to CDCl3
resulted in an upfield shift of H-4 and a downfield shift of
H-5 (see pages S47 and S55 in Supplementary data). This
is in line with exchanges in the axial and equatorial posi-
tions. Moreover, the coupling constants of H-5 are very sig-
nificant, indicating an axial position in MeOH-d4 (3JHH
9.6 Hz) and an equatorial position in CDCl3 (3JHH 5.0 Hz).
Thus, in MeOH-d4 solution, triol 13 prefers a conformation
with OH-3 and OH-5 in equatorial orientation and OH-4 in
2.2. Conformational analysis of intramolecular hydro-
gen bonds and their influence in the enzymatic catalysis
In order to explain the regioselectivity exhibited by CAL-A
in quinic and shikimic acid derivatives, we next study the
role of intramolecular hydrogen bonds between vicinal alco-
hols within each ligand.
IR and 1H NMR spectroscopy are common methods to deter-
mine intra- and intermolecular H-bonding. IR allows direct

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N. Armesto et al. / Tetrahedron 62 (2006) 5401–5410
observation of the free and hydrogen-bonded hydroxyl
stretch resonances (nOH), which are subsequently used to de-
termine the extent of intramolecular hydrogen bonding.18
Since our molecules possess several OH groups this tech-
nique is not adequate due to the superposition of the OH
bands. NMR spectra potentially provide more useful infor-
mation in the form of coupling constants, chemical shifts,
temperature coefficients, and NOEs, although in order to ob-
tain this data the suppression of intermolecular exchange of
hydroxyl groups is necessary. In these conditions, vicinal
coupling constants (3J) for hydroxyl protons calculated by
1H NMR can provide important structural information in
A
B
H5
H
H5
CO2Me
CO2Me
H
O
H
O
O H
O
O
H
O
H
H3
H3
H4
H4
1
C
D
H5
H
H5
O
H
O
OH
CO2Me
H
H
O
H
O
O
H
CO2Me
O
H
O
H3
H4
H3
H4
3
terms of hydrogen-bonding. Since the magnitude of J de-
3
pends on the dihedral angle, according to the Karplus equa-
tion19 derived for hydroxyl protons,20 the values of 3JCH,OH
can predict the orientation of the hydroxyl groups.
Figure 5. Clockwise and reverse clockwise orientations of methyl shikimate
(A and B) and methyl quinate (C and D) hydrogen-bonding networks.
For the detection of intramolecular hydrogen bonds sample
dilution is a powerful method.21 The limiting chemical shifts
and the concentrations required for fast exchange are charac-
teristically different for protons that are intramolecularly hy-
drogen-bonded. Intramolecular H-bonds can also be detected
by a weak dependence of the chemical shift of OH groups
upon the temperature.22 Due to the low solubility of our sub-
strates in chloroform or methylene chloride (commonly used
deuterated solvents), whichare nonhydrogen-bonding, acon-
centration dependent hydroxyl proton exchange is not ade-
quate. Similarly, since a conformational equilibrium is
present in the target molecules, a dependence upon the tem-
perature of the OH signals in 1H NMR is not suitable. These
facts oblige us to study the exchange process by acid cataly-
sis. As a consequence, rigorous removal of acid and water
from sample, solvent, and NMR tube should be controlled.
To the best of our knowledge, it is the first time that hydro-
gen bond interactions are determined by exchange of the
hydroxyl protons with acid in an organic solvent solution.
1 exists preferentially in the chair conformation indicated in
Figure 5 (A and B). To assign the spectra and determine the
structures, 1H–1H homonuclear correlation experiments
have been performed. The analysis of the spectrum of 1 pro-
vides the coupling constants of the hydroxyl groups:
3
3
3JH-3,OH-3¼4.7 Hz, JH-4,OH-4¼5.5 Hz, and JH-5,OH-5
¼
3.5 Hz. Comparison of spectra A–C in Figure 4 suggests
that the signal corresponding to OH-5 is altered first, since
it appears as a singlet instead of a doublet. Spectrum C shows
that the next hydroxyl to loose its coupling is OH-3, whereas
OH-4 still maintains certain residual coupling. These
results of hydroxyl exchange indicated an acidity order of
OH-5>OH-3>OH-4.
There are two possible orientations for the hydrogen bonds
in the cyclohexene ring of 1 (A and B, Fig. 5). The hydroxyl
OH-5 forms a weak H-bond with the adjacent hydroxyl OH-
4 both are in equatorial position. Diequatorial trans-1,2-di-
ols are expected to form weaker intramolecular bonds. The
strong hydrogen bond is formed by OH-3 and OH-4, which
are in cis orientation. Equatorial–axial vicinal diols in six-
membered rings have stronger intramolecular hydrogen
bonds than equatorial–equatorial diols. On the other hand,
OH-3 is more acidic than OH-4 and this suggests that the ax-
ial OH-3 and not the equatorial OH-4 acts preferentially as
H-acceptor.22b Thus, the directional hydrogen-bonding
network is that indicated in Figure 5B. In addition, an equa-
torial OH group in a six-membered ring system can be
2.2.1. Study of hydrogen bonds network in methyl shiki-
mate (1). After careful preparation of the NMR samples (see
Section 4) the rate of exchange of the hydroxyl protons with
the solvent is slow enough, because of which the coupling
1
pattern is detectable. Figure 4 shows the H NMR spectra
of methyl shikimate (1) in CDCl3 solution before (spectrum
A) and after addition of subsequent portions of 0.05 mM tri-
fluoroacetic acid in CDCl3 solution (spectra B and C). Ester
Figure 4. 1H NMR spectra (300 MHz) of 1 in CDCl3 solution (2 mM): (A) in absence of acid; (B) first change observed after acid addition; and (C) second
change observed after acid addition.

N. Armesto et al. / Tetrahedron 62 (2006) 5401–5410
5405
functionalized preferentially in the presence of secondary
axial partners.
hydroxyl groups in the enzyme-catalyzed acetylation reac-
tion. The positive charge in the transition state is stabilized
through two consecutive efficient hydrogen bonds. The
OH-5 group does not effectively participate in the hydro-
gen-bonding network (trans-diequatorial hydrogen bond be-
tween OH-5 and OH-4), and acetylation at the OH-3 reduces
the extent of charge delocalization (through only one
effective hydrogen bond). Interestingly, in the presence of
CAL-A, the regioselectivity of the acetylation of 3 agrees
fully with cooperative H-bond effects and by a higher reac-
tivity of the equatorial groups, methyl 4-O-acetylquinate (4)
being obtained exclusively.13a
According to the points mentioned above, when OH-4 re-
sults acylated the positive charge in the transition state is de-
localized through a strong intramolecular hydrogen bond. In
contrast, OH-5 forms a less-efficient hydrogen bond to delo-
calize the charge. If these parameters have an influence in the
transition state of the enzymatic acylation, the major regio-
isomer formed should be the 4-O-acetylated derivative. In
fact, acylation of 1 catalyzed by CAL-A takes place with
excellent selectivity at OH-4.13b
2.2.2. Study of hydrogen bonds network in methyl qui-
nate (3). The H NMR spectra of methyl quinate (3) in
2.2.3. Study of hydrogen bonds network in methyl 3-epi-
shikimate (5). The 1H NMR spectra of methyl 3-epi-shiki-
mate (5) in MeOH-d4, acetone-d6, or CDCl3 solutions
show the chair conformation indicated in Figure 7A, in
1
CDCl3 or MeOH-d4 solution shows that the preferred chair
conformation is close to the one observed for methyl shiki-
mate (C and D, Fig. 5). Compound 3 is characterized by
3
which the large coupling constants of H-4 (dd, JHH¼9.8,
3
3
3
two large JH-3,OH-3¼8.6 Hz and JH-4,OH-4¼9.1 Hz, and
a smaller 3JH-5,OH-5¼2.5 Hz (A, Fig. 6). Comparison of the
1H NMR spectra A–C (Fig. 6) reveal that the order of acidity
of the hydroxyl groups is OH-5>OH-4>OH-3.
7.8 Hz) and H-5 (ddd, JHH¼9.7, 9.7, 6.2 Hz) suggest an
axial disposition (see pages S44 and S51 in Supplementary
data).
The exchange of hydroxyl protons of alcohols after addition
of acid to NMR sample implies an acidity order of OH-
4zOH-5>OH-3 (see pages S62 and S63 in Supplementary
data). The two hydrogen bonds are between trans-diequato-
rial OH groups, and, evidently, none of them are involved in
a strong intramolecular H-bond. The fact that OH-3 is the
lesser acidic hydroxyl, and thus a H-bond donor, indicates
the direction of the H-bonds (Fig. 7A). The enzymatic acyl-
ation of 5 led almost exclusively to 3-acetyl derivative 6.
This is in keeping with the more efficient hydrogen bond
Since we have an additional hydroxyl proton in the molecule
(OH-1) for which 1H NMR spectrum does not provide cou-
pling information (it is a tertiary alcohol), acidity does not
indicate the direction of the hydrogen bonds. For that, vici-
nal coupling constants were employed. As previously men-
3
tioned, the value of JCH,OH depends on the dihedral angle
between the hydroxyl proton and the hydrogen of the vicinal
3
carbon. JCH,CH values of 11–15 Hz are characteristic of
axial–axial vicinal couplings, while the presence of electro-
negative atoms give rise to 3J values of 8–10 Hz. According
3
to the Karplus equation, the large JH-4,OH-4¼9.1 Hz evi-
dences a predominant conformation in which the O–H bond
is almost anti to the C–H bond (dihedral angle close to
150–180ꢀ).23 The same interpretation can be applied to
OH-3, suggesting that the direction of the hydrogen bonds is
that which is indicated in Figure 5D. OH-3 and OH-1 form
a strong 1,3-diaxial hydrogen bond. Also, the hydrogen-
bonding between OH-4 and OH-3 is effective. Thus, the
OH-4 should exhibit much higher reactivity than the other
A
B
H
5
H
3
H
5
H
H
H
OH
CO2Me
CO2Me
O
3
O
H
O
H
O
H
O
4
H
O
4
H
H
5
7
Figure 7. Hydrogen-bonding orientations of methyl 3-epi-shikimate (A)
and methyl 3-epi-quinate (B).
Figure 6. 1H NMR spectra (300 MHz) of 3 in CDCl3 solution (2 mM): (A) in absence of acid; (B) first change observed after acid addition; (C) second change
observed after acid addition.

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N. Armesto et al. / Tetrahedron 62 (2006) 5401–5410
between OH-3 and OH-4 due to the pseudoequatorial posi-
tion of OH-3.
stabilized through the most efficient hydrogen bond. In prac-
tice, CAL-A catalyzes the acylation at OH-4 with total selec-
tivity, the 4-O-acetyl derivative 10 being isolated with
excellent yield.
2.2.4. Study of hydrogen bonds network in methyl 3-epi-
quinate (7). As with the shikimate derivative, analysis of
the coupling constants of meso-methyl 3-epi-quinate (7)
evidences the preferred chair conformation, shown in
Figure 7B. Large coupling constant values are observed
2.2.6. Study of hydrogen bonds network in methyl 4-epi-
quinate (11). The hydroxyl exchange study of (ꢂ)-methyl
4-epi-quinate (11) was more difficult than in previous cases
because of the low solubility of this derivative in CDCl3, and
even in CD2Cl2. The 1H NMR of 11 in CDCl3 shows the cou-
pling constants of two hydroxyls (3JCH,OH¼8.7 Hz and
3JCH,OH¼3.1 Hz), which were assigned by COSY spectrum
as OH-3 and OH-4, respectively, (page S69 in Supplemen-
tary data). Taking into account that proton spectra in
CDCl3, MeOH-d4, and acetone-d6 evidence the chair confor-
mation indicated in Figure 8B, the small value of the cou-
pling constant for OH-4 indicates that the axial OH-4, and
not the equatorial OH-5, acts preferentially as H-acceptor.
In addition, the large value of 3JH-3,OH-3 suggests a dihedral
angle of 180ꢀ, showing the donor character of OH-3, and
compatible with a strong 1,3-diaxial H-bond (see Fig. 8B).
In accordance with this, the acylation should take place at
OH-3, although this hydroxyl is in axial configuration. How-
ever, CAL-A exhibited total selectivity towards OH-4. This
is in keeping with the chair conformation similar to that pre-
viously shown for methyl 4-epi-shikimate (Fig. 8C). In this
case, an efficient hydrogen bond between OH-5 and the
oxygen of the ester group increases the hydrogen-bonding
network. In addition, OH-4 is in equatorial orientation.
Similarly, enzymatic acetylation of the corresponding lac-
tone 19 (Fig. 8D), which is restricted in this chair conforma-
tion, also afforded exclusively the 4-O-acetyl derivative 20
(Fig. 9).
2
3
for axial H-2 and H-6 (dd, JHH¼12.5 Hz, JHH¼12.5 Hz),
and thus H-3 and H-5 are in axial orientation.
The three secondary OH groups of 7 are involved in weak
intramolecular H-bonds. Hydroxyl exchange is similar,
although the H-bond formed by OH-3 (or OH-5) seems to
be more stable (see pages S64 and S65 in Supplementary
data). In addition, the two possible orientations of the hy-
droxyl network are equivalent (Fig. 7B), since the molecule
possesses a plane of symmetry.
Acetylation of 7 catalyzed by CAL-A led to exclusive
acylation at OH-3 or OH-5. The larger value of the coupling
constant for OH-3 (or OH-5) (3JH-3,OH-3¼3.7 Hz and
3JH-4,OH-4¼2.9 Hz) indicates a more adequate dihedral angle
for effective hydrogen-bonding.
2.2.5. Study of hydrogen bonds network in methyl 4-epi-
shikimate (9). The analysis of (ꢂ)-methyl 4-epi-shikimate
(9) by 1H NMR in several deuterated solvents (CDCl3,
MeOH-d4, and acetone-d6) determines the chair conforma-
tion indicated in Figure 8A. The coupling constants of H-5
with the two H-6 protons show values ꢃ5 Hz, which indicate
an equatorial position for H-5. This was corroborated by
3JHH¼w7.8 Hz between H-3 and H-4, which is in agreement
with an axial–pseudoaxial disposition for both protons.
The study of hydroxyl exchange indicates an acidity order of
OH-5>OH-3zOH-4 (see pages S66 and S67 in Supplemen-
tary data). The analysis of the acidity order determines the
direction of the hydrogen bonds. Since OH-5 is the most
acidic group, it is an acceptor, and as a consequence the di-
rection of the hydrogen bonds network is as shown in
Figure 8A. Thus, the enzymatic acylation of 9 should
proceed regioselectively towards the 4-position: OH-4 is
equatorial, and the positive charge in the transition state is
H
4
H
5
AcO
HO
OH
O
C
H3
O
20
Figure 9.
Furthermore, we previously observed that the selectivity of
CAL-A was independent of the solvent employed in the
acylation of 5-epi-derivatives, despite both compounds
having different conformations in different solvents. These
facts suggest that the enzyme active site fits the most effec-
tive hydrogen-bonding network conformation, since the
later better stabilizes the transition state.
A
B
H4
H5
H
H
H5
O
O
O
H
O
CO2Me
O
H4
H
CO Me
2
O
H
H
H3
H
H3
O
2.2.7. Study of hydrogen bonds network in methyl 5-epi-
shikimate (13). The H NMR spectrum of 13 in CDCl3
9
11
1
exhibits well-resolved OH signals (3JH-3,OH-3¼9.9 Hz,
C
D
H4
H4
3
3JH-4,OH-4¼6.3 Hz and JH-5,OH-5¼5.5 Hz). Hydroxyl ex-
H5
H5
O
change after acid addition provided an acidity order of
OH-5>OH-4>OH-3 (see pages S70 and S71 in Supplemen-
tary data). Thus, OH-5 should be the acceptor of OH-3, the
two hydroxyl groups being involved in the molecule’s stron-
gest hydrogen bond, which is almost 1,3-diaxial. This obser-
vation is consistent with the large coupling constant value
of OH-3, suggesting a dihedral H–C–O–H angle close to
O
H
O
O
OH
OH
H
H
H
O
3 H
O
C
C
O
OMe
H
H3
O
11
19
Figure 8. Hydrogen bond networks of methyl 4-epi-shikimate (A), methyl
4-epi-quinate (B and C), and methyl 4-epi-quinate lactone derivative 19 (D).

N. Armesto et al. / Tetrahedron 62 (2006) 5401–5410
5407
A
B
A
H
H
H
Asp
Asp
O
O
O
H
CO2Me
H
O
H
O
H
H5
O
-
-
CO
CO
2
2
CO2Me
His
His
H5
O
+
H
H
H3
H3
N
N
H4
H4
Ser
R
Ser
N
N
13
15
H
O
H
O
O
Figure 10. Hydrogen bonds network of methyl 5-epi-shikimate (A) and
methyl 5-epi-quinate (B).
O
O
1
1
R
R O
R
-
TI-2
180ꢀ. The coupling constants of OH-4 and OH-5 also cor-
roborated the direction of the hydrogen bonds indicated in
Figure 10A.
B
Asp
Asp
-
-
CO
CO
2
2
His
His
+
H
H
Thus, if acylation takes place at the equatorial OH-4 group,
the transition state is stabilized through an effective hydro-
gen bond network. However, we isolated exclusively the 5-
O-acyl derivative. This result would be explained due to
acyl migration from position 4 to 5 (acyl migrations were
previously observed for methyl O-acylshikimate derivatives
in certain conditions13). To confirm this fact, we tried to pre-
pare the corresponding 4-O-acyl analogue by independent
synthesis. On doing so, 3,5-di-O-TBDMS protection of
methyl 5-epi-shikimate and subsequent acetylation of the
OH-4 group afforded the 4-acetyl-3,5-di-O-TBDMS deriva-
tive. All attempts to obtain methyl 4-O-acetyl-5-epi-shiki-
mate, after silyl deprotection, gave rise to a mixture of
3-, 4-, and 5-O-acetyl derivatives, in which the 5-O-acyl
derivative was the major compound.
N
N
Ser
Ser
R
N
N
O
δ+
O
H
O
H
δ-
δ+
δ
+
H
O
R
O
H
O
-
O
O
Figure 11. (A) Tetrahedral intermediate (TI-2) in the serine mechanism ca-
talysis. (B) General representation of TI-2 in which positive charge is delo-
calized through hydrogen-bonding networks in quinic and shikimic acid
derivatives.
phile attacks the latter intermediate, assisted by the catalytic
histidine, yielding a second tetrahedral intermediate (TI-2).
This intermediate collapses to expel the acylated product
and gives the free enzyme.
2.2.8. Study of hydrogen bonds network in methyl 5-epi-
quinate (15). Finally, we also studied the hydrogen bonds in
methyl 5-epi-quinate (15). The exchange of the hydroxyl
protons of alcohols by acid shows the hydrogen bonds to
be of similar strength. As previously mentioned, in CDCl3
the chair conformation is indicated in Figure 10B. The
We propose that transition state hydrogen-bonding plays an
important role in enzyme selectivity, and can be understood
as one result of the effectiveness of hydrogen-bonding net-
works (the original hydrogen bond network from the enzyme
conjugates with intramolecular hydrogen bonding in the
ligand). It is worth noting that a web of hydrogen-bonding
interactions links the substrate binding sites to the catalytic
triad. Two key hydrogen bonds in the TI-2 are that from
His to the oxygen of Ser, and that with the OR1 group (A,
Fig. 11). In the case of quinic and shikimic acid derivatives,
the positive charge in the transition state can also be delocal-
ized through a hydrogen-bonding network present in the
polyhydroxylated molecule (B, Fig. 11). The acylation reac-
tion takes place preferentially at the hydroxyl group that par-
ticipates in the more effective hydrogen-bonding network.
JCH,OH values for hydroxyl groups (3JH-3,H-5,OH-3,OH-5
¼
3
7.6 Hz and JH-4,OH-4¼8.2 Hz) imply that the O–H bond
is almost anti to the C–H bond and confirm that OH-1,
OH-3, and OH-5 are involved in 1,3-diaxial hydrogen bond-
ing. As methyl 3-epi-quinate, this is a meso form and the two
possible orientations of the hydroxyl network are equivalent.
Since equatorial hydroxyls are more reactive than axials, the
acylation reaction should take place towards that position. In
fact, the enzymatic process proceeds selectively at the OH-4
group.
Acylation of quinic and shikimic acid derivatives was also
carried out in the presence of DMAP as catalyst and in
1 mM concentration in CHCl3, suitable conditions for effi-
cient intramolecular hydrogen bonding.8 In some cases,
the regioselectivity of the acetylation agrees with the H-
bonding scheme shown and a rise in the reactivity of certain
OHs was observed. However, the reaction is not as selective
as the process catalyzed by CAL-A and a mixture of acylated
products was obtained.
In view of these results, we propose that the cooperative
effect of hydrogen bonds is responsible for the enzyme–
substrate interactions, and leads to the selectivity detected.
Unusually strong hydrogen bonds, known as low barrier
hydrogen bonds (LBHB), have emerged as a new rationale
for the exceptional catalytic abilities of some enzymes.24 It
has been suggested that these LBHB are significant contrib-
utors to the intermediate stabilization and catalytic power of
the enzyme.
The catalytic mechanism of lipases is based on a ‘catalytic
triad’ composed of a nucleophilic serine residue activated
by a hydrogen bond in relay with histidine and aspartate or
glutamate. The interaction between the enzyme and the ac-
ylating agent yields the acyl–enzyme intermediate. A nucleo-
3. Summary
Selective acylated derivatives of 3-epi, 4-epi, and 5-epi-iso-
mers of quinic and shikimic acids have been efficiently syn-
thesized in a one-step enzymatic transesterification reaction

5408
N. Armesto et al. / Tetrahedron 62 (2006) 5401–5410
with vinyl acetate as acyl donor and C. antarctica lipase A as
catalyst. These analogues are useful as chiral building blocks
and as optically active synthetic precursors for natural prod-
ucts. A study of the exchange rate of hydroxyl protons by 1H
NMR spectroscopy has been performed to determine the
strength of the intramolecular H-bonds in these substrates.
In addition, vicinal coupling constants JCH,OH provide struc-
tural information in terms of dihedral H–O–C–H angles and
evidence the directional hydrogen-bonding network. We
have established that selectivity of C. antarctica lipase A
is related to both the inherent receptor selectivity and the
degree of intramolecular hydrogen bonding in the ligand.
We found a satisfactory correlation between reactivity of
hydroxyl groups and effectiveness of the corresponding
hydrogen-bonding network.
for 5, 4.5 h for 7, 1.5 h for 9, 24 h for 11, 0.3 h for 13, and
4.5 h for 15. The mixture was filtered, and the organic sol-
vent was evaporated. Then, the reaction crude was subjected
to flash chromatography (gradient eluent 50–80% Et2O/
CH2Cl2 for 6; gradient eluent EtOAc–3% MeOH/EtOAc
for 8; 40% acetone/CH2Cl2 for 10; 45% acetone/CH2Cl2
for 12; 20% acetone/CH2Cl2 for 14; and gradient eluent
EtOAc–5% MeOH/EtOAc for 16) to give 6 (white solid,
88% yield), 8 (colorless oil, 84% yield), 10 (colorless oil,
80% yield), 12 (colorless oil, 82% yield), 14 (colorless oil,
88% yield), and 16 (colorless oil, 85% yield).
4.2.1. Methyl 3-O-acetyl-3-epi-shikimate (6). Rf (Et2O):
0.13; mp: 106–107 ꢀC; IR (NaCl): y 3406, 2952, 2921,
1
1719, 1654, 1438, 1374, 1240, and 1072 cmꢂ1; H RMN
(300 MHz, CDCl3): d 2.17 (s, 3H, OAc), 2.30 (dddd, 1H,
2
H6a , | JHH|17.8, 3JHH 9.8, |4JHH|3.7, 5JHH 3.1 Hz), 2.95(ddd,
0
2
1H, H6e , | JHH| 17.7, 3JHH 5.7, |4JHH| 1.1 Hz), 3.71 (dd, 1H,
0
4. Experimental
3
H4, JHH 10.0, 8.0 Hz), 3.78 (s, 3H, OMe), 3.87 (ddd, 1H,
4.1. General spectroscopy and experimental data
H5, 3JHHw10.0,w10.0, 5.9 Hz), 5.43 (m, 1H, H3), and 6.62
(m, 1H, H2) ppm; 13C RMN (75.5 MHz, CDCl3): d 21.0
(C10), 31.7 (C6), 52.2 (OMe), 69.5 (C5), 74.7, 74.9
(C3+C4), 130.4 (C1), 134.4 (C2), 166.0 (C7), and 171.5
(C9) ppm; [a]2D0 +38 (c 1.07, CHCl3); MS (ESI+, m/z): 253
[(M+Na+), 100%] and 269 [(M+K+), 6%]; Anal. Calcd (%)
for C10H14O6: C, 52.16; H, 6.13. Found: C, 52.2; H, 6.1.
C. antarctica lipase A (CAL-A, chirazyme L-5, c-f, lyophi-
lized, 25 U/g using 1-phenylacetate) was obtained from
Roche. TLC chromatograms were visualized by heating af-
ter spraying with a 5% aqueous sulfuric acid solution con-
taining cerium sulfate (1%) and molybdophosphoric acid
(2.5%). Column chromatography was performed over silica
˚
60 A (230–400 mesh) except for 10, 12, 14, and 20 in which
4.2.2. Methyl 3-O-acetyl-3-epi-quinate (8). Rf (EtOAc):
0.18; IR (KBr): y 3396, 2956, 2927, 1732, 1435, 1371, 1259,
˚
˚
silica 60 A (32–63 mm) pH 7 was used. Molecular sieves 4 A
were dried at 180 ꢀC in a vacuum over 2 h. Methyl 3-epi-,
4-epi-, and 5-epi-shikimate and quinate derivatives were
1
1130, 1067, and 1036 cmꢂ1; H RMN (300 MHz, acetone-
d6): d 1.76 (ddd, 2H, H6a+H2a, |2JHH| 12.9, 3JHH 11.7, |2JHH
5.7 Hz), 1.98 (s, 3H, OAc), 2.01–2.1 (m, 2H, H6e+H2e, under
|
dried in a high vacuum (10ꢂ5 mbar) before use. H and
1
13C NMR signals assignment is based on selective homo-
nuclear and heteronuclear decoupling experiments.
solvent), 2.9 (br s, 1H, OH), 3.40 (dd, 1H, H4, JHH w9.2,
3
w9.2 Hz), 3.71 (s, 3H, OMe), 3.83 (ddd, 1H, H5, 3JHH 11.6,
3
9.0, 4.6 Hz), and 5.02 (ddd, 1H, H3, JHH 11.6, 9.6,
Chiral HPLC analysis were performed using a chiralcel
OD-H column, a flow of 0.5 mL/min, 5% EtOH/hexane as
eluent, and at 35 ꢀC. Sample concentration was 0.5 mg/mL.
For (ꢁ)-17, tR 12.7 and 13.9 min.
4.8 Hz) ppm; 13C RMN (75.5 MHz, acetone-d6): d 21.7
(C10), 39.4, 42.0 (C2+C6), 53.3 (OMe), 70.7 (C5), 73.7
(C3), 74.8 (C1), 78.8 (C4), 171.5 (C9), and 176.2 (C7) ppm;
[a]2D0 (compound from the enzymatic reaction) +19 (c 1.37,
MeOH); [a]2D0 (compound enantiomerically pure) +31 (c
1.31, MeOH); MS (ESI+, m/z): 271 [(M+Na+), 100%], 287
[(M+K+), 7%], and 287 [(M+H+), 1%]; Anal. Calcd (%) for
C10H16O7: C, 48.39; H, 6.5. Found: C, 48.4; H, 6.6.
1
4.1.1. Sample preparation for H NMR hydroxyl ex-
change studies. CDCl3 was filtered over anhydrous
K2CO3, which serves to deacidify and partially dry the sol-
vent. CDCl3 was stored under N2 over K2CO3 and powdered
ꢀ
˚
4 A molecular sieves (predried under vacuum at 180 C dur-
ing 2 h) to completely remove the water. Syringes and NMR
tubes were dried on a vacuum line at room temperature. The
samples were prepared by dissolving a portion of the quinic
and shikimic acid derivative in CDCl3 to make a 2 mM solu-
tion. For the hydroxyl exchange study successive portions of
0.05 mM trifluoroacetic acid solution in CDCl3 were added
to the NMR tube and corresponding 1H NMR spectra
(300.13 MHz) were recorded.
4.2.3. Methyl4-O-acetyl-4-epi-shikimate(10). Rf (CH2Cl2):
0.13; IR (NaCl): y 3420, 2950, 1710, 1655, 1430, 1358, 1250,
and 1182 cmꢂ1; 1H RMN (300 MHz, CDCl3): d 2.16 (s, 3H,
2
3
0
OAc), 2.58 (dd, 1H, H6a , | JHH| 18.5, JHH 4.5 Hz), 2.68
0
(dddd, 1H, H6e , | JHH| 18.6, JHH 4.4, |4JHH| w2.2, JHH
w2.2 Hz), 3.78 (s, 3H, OMe), 4.28 (ddd, 1H, H5, JHH 4.5,
2
3
5
3
3
4.3, 2.4 Hz), 4.59 (m, 1H, H3), 4.91 (dd, 1H, H4, JHH 6.9,
2.1 Hz), and 6.84 (m, 1H, H2) ppm; 13C RMN (75.5 MHz,
CDCl3): d 21.1 (C10), 31.1 (C6), 52.1 (OMe), 66.4 (C5), 67.3
(C3), 76.8 (C4), 128.6 (C1), 136.9 (C2), 166.7 (C9), and
171.4 (C7) ppm; [a]2D0 ꢂ56 (c 0.86, CHCl3); MS (ESI+, m/z):
253 [(M+Na+), 100%] and 231 [(M+H+), 3%]; Anal. Calcd
(%) for C10H14O6: C, 52.17; H, 6.13. Found: C, 52.2; H, 6.1.
4.2. Enzymatic acylation of methyl 3-epi-, 4-epi-, and 5-
epi-shikimate and quinate derivatives. General proce-
dure for the synthesis of 6, 8, 10, 12, 14, and 16
In a standard procedure, vinyl acetate (0.75 mL) was added
to an Erlenmeyer flask that contained 5, 7, 9, 11, 13, or 15
4.2.4. Methyl 4-O-acetyl-4-epi-quinate (12). Rf (EtOAc):
0.2; IR (KBr): y 3402, 2946, 1733, 1447, 1434, 1237, 1128,
1
and 1091 cmꢂ1; H RMN (300 MHz, MeOH-d4): d 1.70
˚
(0.069 mmol), CAL-A (13 mg), and 4 A molecular sieves
(13 mg) under nitrogen. The suspension was shaken at
3
(dd, 1H, H2e, |2JHH| 13.8, JHH 6.6 Hz), 1.87 (dd, 1H, H6e,
3
250 rpm at 20 ꢀC (except for 7 and 15 at 40 ꢀC) for 0.5 h
|2JHH| 13.3, JHH 3.4 Hz), 2.11 (s, 3H, H10), 2.28–2.36

N. Armesto et al. / Tetrahedron 62 (2006) 5401–5410
5409
(m, 2H, H2a+H6a), 3.75 (s, 3H, OMe), 4.11 (1H, m, H3), 4.23
(ddd, 1H, H5, 3JHHw6.4,w6.4, 4.1 Hz), and 4.89 (dd, 1H, H4,
3JHH 6.4, 2.9 Hz) ppm; 13C RMN (75.5 MHz, MeOH-d4):
d 19.6 (C10), 36.7 (C2), 38.6 (C6), 51.38 (OMe), 63.8 (C5),
66.7 (C3), 74.1 (C1), 75.7 (C4), 171.1 (C9), and 174.0
(C7) ppm; [a]2D0 ꢂ19 (c 0.48, H2O); MS (ESI+, m/z): 271
[(M+Na)+, 100%] and 249 [(M+H)+, 10%]; Anal. Calcd
(%) for C10H16O7: C, 48.39; H, 6.5. Found: C, 48.2; H, 6.5.
CDCl3): d 1.30, 1.31 (2s, 6H, 2Me), 1.83 (dd, 1H, H2a,
3
|2JHH| 12.9, JHH 11.2 Hz), 1.88–1.99 (m, 2H, H6a+H6e),
2.08 (s, 3H, OAc), 2.18 (m, 1H, H2e), 3.26, 3.29 (2s, 6H,
3
OMe), 3.70 (dd, 1H, H4, JHH w9.9, w9.9 Hz), 3.80 (s, 3H,
OMe), 4.08 (ddd, 1H, H5, 3JHH 11.7, 9.9, 5.1 Hz), and 5.17
3
(ddd, 1H, H3, JHH 11.2, 10.1, 5.1 Hz) ppm; 13C RMN
(75.5 MHz, CDCl3): d 17.6, 17.63 (C13+C14), 21.0 (C18),
37.3, 38.5 (C2+C6), 53.1 (OMe), 47.5, 47.8 (C15+C16),
65.3 (CH), 69.2 (CH), 72.9 (CH), 73.2 (C1), 99.5, 99.54
(C9+C10), 170.1 (C17), and 175.1 (C7) ppm; MS (ESI+,
m/z): 385.1 [(M+Na+), 100%] and 401.1 [(M+K+), 33%];
Anal. Calcd (%) for C16H26O9: C, 53.03; H, 7.23. Found:
C, 52.9; H, 7.2.
4.2.5. Methyl 5-O-acetyl-5-epi-shikimate (14). Rf (Et2O):
0.18; IR (NaCl): y 3422, 2955 1718, 1654, 1438, 1372,
1259, and 1036 cmꢂ1; 1H RMN (300 MHz, CDCl3): d 2.13
0
0
(s, 3H, OAc), 2.52–2.78 (m, 2H, H6a +H6e ), 3.78 (s, 3H,
OMe), 4.12 (m, 1H, H4), 4.42 (m, 1H, H3), 5.07 (ddd, 1H,
3
H5, JHH 8.2, 6.0, 2.0 Hz), and 6.80 (m, 1H, H2) ppm; 13C
4.4. Methyl (1S,3R,4R,5S,20S,30S)-1,3-dihydroxy-4,5-
[2,3-dimethoxybutan-2,3-di(yloxy)]cyclohexan-1-
carboxylate [( )-18]
RMN (75.5 MHz, CDCl3): d 21.1 (C10), 26.0 (C6), 52.1
(OMe), 67.5 (C3), 68.5 (C4), 70.9 (C5), 128.6 (C1), 137.5
(C2), 166.3 (C7), and 170.3 (C9) ppm; [a]2D0 ꢂ60 (c 0.5,
MeOH); MS (ESI+, m/z): 253 [(M+Na)+, 100%] and 231
[(M+H)+, 5%]; Anal. Calcd (%) for C10H14O6: C, 52.17;
H, 6.13. Found: C, 52.2; H, 6.1.
The same procedure as previously described for 17 (from 8)
afford (ꢁ)-18 starting from 7. The crude was purified by
flash chromatography (70% EtOAc/hexane). White solid.
Yield: 91%. Spectroscopical data in agreement with the
enantiomerically pure compound previously described.15,25
Rf (20% MeOH/EtOAc): 0.6; 1H NMR (300 MHz,
4.2.6. Methyl 4-O-acetyl-5-epi-quinate (16). Rf (10%
MeOH/EtOAc): 0.30; IR (KBr): y 3387, 2957, 1727, 1651,
1455, 1435, 1258, 1128, and 1045 cmꢂ1
;
1H RMN
CDCl3): d 1.3, 1.34 (2s, 6H, Me), 1.92 (dd, 1H, H6a, |2JHH
|
13.4, JHH 10.1 Hz), 2.08 (m, 3H, H6e, H2a, H2e), 3.26 (2s,
6H, OMe), 3.59 (dd, 1H, H4, JHH 8.6, 3.2 Hz), 3.77 (s,
(300 MHz, acetone-d6): d 1.81 (dd, 2H, H6a+H2a, |2JHH
|
3
3
3
w12.0, JHH w12.0 Hz), 2.09 (s, 3H, OAc), 2.18 (dd, 2H,
3
3
H6e+H2e, |2JHH| 11.9, JHH 4.6 Hz), 3.71 (s, 3H, OMe),
3.80 (m, 2H, H3+H5), 3.99 (d, 2H, OH3+OH5, JHOH
3H, OMe), 4.18 (m, 1H, H3), and 4.28 (ddd, 1H, H5, JHH
3
10, 8.6, 4.5 Hz) ppm; 13C NMR (75.5 MHz, CDCl3):
d 17.5, 17.7 (C11+C12), 37.3 (CH2), 38.5 (CH2), 47.8, 52.8
(C13+C14), 62.3 (CH), 69.0 (CH), 72.6 (CH), 75.7 (C1),
99.6, 100.2 (C9+C10), and 174.1 (C]O) ppm.
3
6.6 Hz), 4.82 (s, 1H, OH1), and 5.27 (dd, 1H, H4, JHH
w2.4, w2.4 Hz) ppm; 13C RMN (75.5 MHz, acetone-d6):
d 21.2 (C10), 39.4 (C2+C6), 52.5 (OMe), 66.91 (C3+C5),
73.58 (C1), 75.0 (C4), 171.0 (C9), and 175.3 (C7) ppm; MS
(ESI+, m/z): 271 [(M+Na)+, 100%]; Anal. Calcd (%) for
C10H16O7: C, 48.39; H, 6.5. Found: C, 48.4; H, 6.4.
4.5. (1S,3R,4S,5R)-1,3,4-Trihydroxycyclohexan-1,5-
carbolactone (19)
4.3. Methyl (1S,3R,4R,5S,20S,30S)-3-acetoxy-4,5-[2,3-di-
methoxybutan-2,3-di(yloxy)]-1-hydroxycyclohexan-1-
carboxylate (17)
This compound was obtained in the attempted recrystalliza-
tion of 11. Rf (EtOAc): 0.35; mp: 148–150 ꢀC; IR (KBr):
y 3396, 3311, 2997, 2963, 1765, 1445, 1362, 1256, 1122,
1
and 1062 cmꢂ1; H NMR (300 MHz, MeOH-d4): d 1.96
3
A solution of 8 (20 mg, 0.081 mmol) in MeOH (1 mL) was
treated with trimethyl orthoformate (43 mL, 0.39 mmol),
2,3-butanedione (14 mL, 0.165 mmol), and camphorsulfonic
acid (0.93 mg, 0.004 mmol). The mixture was refluxed un-
der nitrogen for 2 h. The resulting solution was treated
with powdered NaHCO3 (spatula tip) and filtered. The fil-
trate was concentrated and subjected to flash chromato-
graphy (gradient eluent 50–80% Et2O/CH2Cl2) to afford
17 as a colorless oil (89% yield).
(dd, 1H, H2a, |2JHH| 11.9, JHH 10.6 Hz), 2.27 (d, 1H, H6a,
3JHH 11.5 Hz), 2.42–2.29 (m, 1H, H2e), 2.63 (ddd, 1H,
3
H6e, |2JHH| 11.7, JHH 6.7, |4JHH| 3.5 Hz), 3.68–3.86 (m,
3
2H, H3+H4), and 4.79 (d, 1H, H5, JHH 6.6 Hz) ppm; 13C
NMR (75.5 MHz, MeOD-d4): d 41.9 (C2), 42.3 (C6), 71.0
(C3), 74.1 (C1), 76.1 (C4), 80.5 (C5), and 179.5 (C7) ppm;
MS (ESI+, m/z): 371 [(2M+Na)+, 100%] and 197
[(M+Na)+, 70%]; [a]2D0 ꢂ94 (c 0.51, MeOH); Anal. Calcd
(%) for C7H10O5: C, 48.28; H, 5.79. Found: C, 48.2; H, 5.8.
4.3.1. Synthesis of ( )-17. Acetic anhydride (0.3 mL,
0.312 mmol) was added to a mixture of (ꢁ)-18 (50 mg,
0.156 mmol), pyridine (63 mL, 0.78 mmol), and DMAP
(1 mg, 1.56 mmol) in CH2Cl2 (2 mL) at 0 ꢀC. After 4 h at
0 ꢀC, the mixture was partitioned between CH2Cl2 and
H2O. The aqueous phase was reextracted with CH2Cl2 and
the combined organic layers were dried over Na2SO4. Filtra-
tion and concentration afforded a crude, which was purified
by flash chromatography (gradient eluent 50–80% Et2O/
CH2Cl2) to give (ꢁ)-17 (80% yield).
4.6. (1S,3R,4S,5R)-4-Acetoxy-1,3-dihydroxycyclohex-
ane-1,5-carbolactone (20)
Using the general procedure for enzymatic acylation previ-
ously described yield 20 (colorless oil, 70% yield) from
19. The reaction mixture was stirred at 40 ꢀC for 4.5 h.
The crude material was purified by flash chromatography
(40% acetone/CH2Cl2). Rf (EtOAc): 0.31; IR (NaCl):
y 3358, 2995, 2971, 1771, 1440, 1453, 1360, 1258, 1122,
1
and 1067 cmꢂ1; H NMR (300 MHz, MeOH-d4): d 2.03
3
(dd, 1H, H2a, |2JHH| 12.3, JHH 11.2 Hz), 2.31 (s, 3H,
Rf (20% Et2O/CH2Cl2): 0.29; IR (NaCl): y 3448, 2954, 1739,
OAc), 2.38 (d, 1H, H6a, |2JHH| 11.7 Hz), 2.39 (m, 1H, H2e),
1
3
1438, 1370, 1240, and 1132 cmꢂ1; H RMN (300 MHz,
2.67 (ddd, H6e, |2JHH| 11.7, JHH 6.7, |4JHH| 3.5 Hz), 3.98

5410
N. Armesto et al. / Tetrahedron 62 (2006) 5401–5410
(ddd, 1H, H3, 3JHH 11.0, 8.3, 7.3 Hz), 4.89 (dd, 1H, H5, 3JHH
6.7, 1.1 Hz), and 4.96 (dd, 1H, H4, JHH 8.3, 1.1 Hz) ppm;
11. Ferrero, M.; Gotor, V. Biocatalytic Synthesis of Steroids.
In Stereoselective Biocatalysis; Patel, R. N., Ed.; Dekker:
New York, NY, 2000; Chapter 20, pp 579–631.
12. (a) Gijsen, H. J. M.; Qiao, L.; Titz, W.; Wong, C.-H. Chem. Rev.
1996, 96, 443–473; (b) Riva, S. Carbohydrate Polyesters as Fat
Substitutes; Akoh, C. E., Swanson, B. G., Eds.; Dekker: New
York/Basel, 1994; pp 37–64; (c) Drueckhammer, D. G.;
Hennen, W. J.; Pederson, R. L.; Barbas, C. F., III; Gautheron,
C. M.; Krach, T.; Wong, C.-H. Synthesis 1991, 499–525.
3
13C NMR (75.5 MHz, MeOH-d4): d 20.9 (C9), 41.6 (C2),
42.0 (C6), 68.2 (C3), 73.8 (C1), 77.3 (C5), 78.1 (C4), 172.3
(C8), and 178.9 (C7) ppm; [a]2D0 ꢂ116 (c 0.48, MeOH);
MS (ESI+, m/z): 239 [(M+Na)+, 100%]; Anal. Calcd (%)
for C9H12O6: C, 50.0; H, 5.59. Found: C, 50.1; H, 6.0.
Acknowledgements
´
13. (a) Armesto, N.; Ferrero, M.; Fernandez, S.; Gotor, V. J. Org.
Chem. 2003, 68, 5784–5787; (b) Armesto, N.; Ferrero, M.;
This work has been supported by grants from Ministerio de
´
Fernandez, S.; Gotor, V. J. Org. Chem. 2002, 67, 4978–4981.
´
Educacion y Ciencia (MEC) (Spain; Project CTQ-2004-
04185). S.F. thanks MEC for a personal grant (Ramon y
Cajal Program). We thank Robin Walker for the english
language revision of the final manuscript.
14. (a) Guyot, B.; Gueule, D.; Pina, M.; Graille, J.; Farines, V.;
Farines, M. Eur. J. Lipid Sci. Technol. 2000, 102, 93–96; (b)
Danieli, B.; De Bellis, P.; Barzaghi, L.; Carrea, G.; Ottolina,
G.; Riva, S. Helv. Chim. Acta 1992, 75, 1297–1304.
´
15. Both compounds have been synthesized as described in:
´
Armesto, N.; Ferrero, M.; Fernandez, S.; Gotor, V. Tetrahedron
Lett. 2000, 41, 8759–8762.
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