Synthesis of cyclodextrins with carboxylic acids at the secondary rim and an evaluation of their properties as chemzymes for glycoside hydrolysis

Article abstract of DOI:10.1002/ejoc.201200404

Seven β-cyclodextrin-mono-, -di-or -tetra-O-acetic acids were prepared and investigated for their propertiesas catalysts of the hydrolysis of four nitrophenyl glycosides.

Full text of DOI:10.1002/ejoc.201200404

FULL PAPER
DOI: 10.1002/ejoc.201200404
Synthesis of Cyclodextrins with Carboxylic Acids at the Secondary Rim and an
Evaluation of Their Properties as Chemzymes for Glycoside Hydrolysis
[
a]
[a]
[a]
[a]
You Zhou, Emil Lindbäck, Lavinia G. Marinescu, Christian Marcus Pedersen, and
Mikael Bols*[
a]
Keywords: Enzyme models / Hydrolysis / Kinetics / Cyclodextrins / Synthetic methods
Seven β-cyclodextrin-mono-, -di- or -tetra-O-acetic acids
were prepared and investigated for their properties
as catalysts of the hydrolysis of four nitrophenyl glycos-
ides.
Introduction
the sugar protruding. Catalytic groups (carboxylic acids, cy-
anohydrin and so on) attached to the cyclodextrin rim can
The mild conditions, selectivity and speed of biochemical then potentially catalyze the cleavage reaction. Indeed,
processes greatly exceed ordinary chemical reactions. This cyclodextrin itself has been shown to be able to catalyze
efficiency is achieved exclusively through enzyme catalysis. hydrolysis of aryl glycosides. α-Cyclodextrin can accelerate
Enzymes use binding and proximity effects to achieve large the hydrolysis of 4-nitrophenyl glycosides by up to 6-fold at
2
3 [1]
[7]
rate enhancements (up to 10 ) for specific reactions and pH = 12.5, whereas it was shown that α- or β-cyclodextrin
substrates. It would be highly desirable if chemists could can accelerate the hydrolysis of 2-(deoxyglycosyl)pyrid-
[
8]
mimic the enzyme’s catalytic process and learn how to cre- inium salts at pH = 7. However, cyclodextrins where the
ate catalysts suited for otherwise impossible processes. Such C-6 carbon atoms have been oxidized to carboxylate groups
[
2]
[3]
chemzymes or enzyme mimics have been reported to were more efficient. At pH = 7–8 a hydrolysis rate increase
3
catalyze enzyme-like transformations from a variety of sub- (kcat/kuncat) of up to 10 was achieved with a compound
[
4]
strates.
that had two carboxylic acid groups at the A and D resi-
[
9]
Interestingly, glycoside hydrolysis is one enzymatic trans- dues. A compound with a single carboxylic acid group at
formation that is both biologically widespread and impor- the primary rim did, however, not catalyze hydrolysis. The
tant, and has some of the highest enzymatic rate enhance- catalysis by these compounds was believed to be caused by
[
1]
ments observed. It is also one of the enzymatic reactions electrostatic effects.
[5]
that has been subject to the most detailed study. Retaining
glycosidases have two carboxylic acids in the active site with
one acting as an acetal protonator and the other as a nu-
cleophile. Inverting glycosidases also have two carboxylic
acids with similar functions except that the nucleophile now
is a general base on a water molecule making the latter
nucleophilic. In the retaining enzymes the distance be-
tween the carboxylate groups is 5.0 Å, whereas it is 10.5 Å
in the inverting glycosidases. The detailed information avail-
able about glycosidases,[ and the stoichiometric simplicity
of the reaction catalyzed by these enzymes, makes the gly-
cosidase reaction ideal to mimic.
Because phenyl glycosides can bind to cyclodextrins with
the monosaccharide protruding from either face it may be
anticipated that carboxylic acid groups at the secondary rim
could also catalyze hydrolysis. Indeed, we recently ob-
[10]
served
catalysis from a per-O-methylated β-cyclodextrin
having a carboxymethyl or carboxyethyl group at the 2-OH
position, which was surprising because (1) a single carb-
oxylate group was sufficient for catalysis, (2) the corre-
sponding 6-O-carboxymethyl derivative was inactive, and
[
6]
6]
(3) previously the unmethylated 2-O-carboxymethyl-β-
cyclodextrin was found inactive (though on different glycos-
[8]
ide substrates). To obtain more insight into these puzzling
With cyclodextrin-based mimics of glycosidases progress
has been made.
an aromatic group of an aryl glycoside in the cavity with
results we have, in this paper, synthesized a full series of
mono-O-carboxymethyl-β-cyclodextrins with and without
permethylation as well as a di- and a tetra-O-carboxymethyl
compound (7–9 and 19–22; Figure 1). We find that the 2-
O-carboxymethyl-substituted cyclodextrins generally have a
very low or non-existing activity regardless of the position
of the catalytic group and were not able to confirm the level
of catalysis previously reported in 2-O-carboxymethyl-per-
O-methylcyclodextrin.
[
2,4]
The role of the cyclodextrin is to bind
[
a] Department of Chemistry, University of Copenhagen,
Universitetsparken 5, 2100 Copenhagen Ø, Denmark
E-mail: bols@chem.ku.dk
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201200404.
Eur. J. Org. Chem. 2012, 4063–4070
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Y. Zhou, E. Lindbäck, L. G. Marinescu, C. M. Pedersen, M. Bols
FULL PAPER
with RuCl /NaIO in aqueous acetonitrile gave acids 4–6
3
4
in 82–85% yield. Zemplen deacetylation with NaOMe in
methanol gave 7–9 in 91–97% yield (Scheme 2).
Scheme 2. Synthesis of mono-O-carboxymethyl-cyclodextrins 7–9.
Figure 1. O-Carboxymethylated cyclodextrins prepared in this
work.
Results and Discussion
Compounds 7–9 and 20–21 (Figure 1) were all prepared
by mono-allylation of β-cyclodextrin as outlined in Scheme
1
. The synthesis was based on previously reported allyl-
[
11]
ations.
β-Cyclodextrin was treated with allyl bromide in
aqueous NaOH, which gave a mixture of unreacted, mono-,
di- and polyallylated β-cyclodextrins. The monoallylated
fraction was separated from the unreacted and overreacted
fractions by chromatography on silica gel by using 2-prop-
anol/aqueous ammonia. After peracetylation of the mono-
allyl fraction with Ac O/Et N at elevated temperatures, it
Scheme 3. Synthesis of monools 12 and 13.
2
3
was possible to separate the isomers by chromatography to
give 1, 2 and 3 (eluted in that order) in 10, 12 and 26%
yield, respectively.
Scheme 1. Monoallylation of β-cyclodextrin.
Synthesis of the three possible carboxymethyl-substituted
β-cyclodextrins (7–9) then proceeded easily from 1, 2 or 3
by the procedure outlined in Scheme 2. Oxidative cleavage Scheme 4. Synthesis of per-O-methylated acids 19–22.
4
064 Eur. J. Org. Chem. 2012, 4063–4070
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Cyclodextrins with Carboxylic Acids Group as Potential Chemzymes
The O-methylated derivatives were prepared by using the and 13. Not surprisingly the 2-OH group, which is more
[
13]
selective demethylation strategy reported by the Sollogoub acidic,
is alkylated more readily under these conditions,
[
12]
group. Reaction of per-O-methyl-β-cyclodextrin with Di- and chromatographic separation gave 12 and 13 in 18 and
isobutylaluminium hydride (DIBAL) gave 2A,3B-diol (ac- 74% yield, respectively (Scheme 3). Sollogoub et al.[ have
cording to the Sollogoub nomenclature) in 51% yield also described more extensive demethylation of 10, and this
12]
(Scheme 3). Other products such as monools, triols and tet- chemistry was also tried. Reaction of 10 with 50 equiv. of
raols are also formed in this reaction, and unreacted start- DIBAL at room temperature gave the tetraol 14, which was
ing material is also present. Compound 11 was separated isolated in 30% yield after chromatography (Scheme 4).
by using chromatography and then treated with 1 equiv. of Many other products were also formed during this reaction.
MeI and NaH in THF to produce a mixture of monools 12 Compounds 11, 12, 13, and 14 were all O-allylated by using
Table 1. Kinetic data for the hydrolysis of the various glycoside substrates (4–25 mm) in phosphate buffer (500 mm, pH = 8.0) by various
catalysts (0.6 mm).
Eur. J. Org. Chem. 2012, 4063–4070
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Y. Zhou, E. Lindbäck, L. G. Marinescu, C. M. Pedersen, M. Bols
FULL PAPER
excess allyl bromide and potassium tert-butoxide in di- aryl glycoside hydrolysis reaction regardless of whether the
methyl sulfoxide (DMSO). This gave the expected mono-, remaining hydroxy groups are O-methylated or not.
di- or tetra-O-allylated products 15–18 in 71–87% yield.
Oxidation of 15–18 with RuCl /NaIO gave the correspond-
3
4
ing mono-, di- and tetracarboxylic acids 19–22 smoothly in
Experimental Section
63–79% yield.
General: Solvents were dried by means of a solvent purification
system. All reagents were used as received. Solvent evaporation was
carried out by using a rotary evaporator. Glassware used for water-
free reactions was dried at 130 °C for 2 h before use. Chromatog-
raphy columns were packed with silica gel 60 (230–400 mesh) as
the stationary phase. TLC plates (Merck, 60, F254) were visualized
The cyclodextrin acids 7–9 and 19–22 were tested for ca-
talysis of aryl glycoside hydrolysis. The hydrolysis of four
nitrophenyl glycosides (Table 1) at 60 °C and pH = 8.0 was
monitored with and without the presence of a cyclodextrin
derivative (0.6 mm) at various concentrations. The phos-
phate concentration was 0.5 m, and the native cyclodextrins
by spraying with cerium sulfate (1%) and molybdic acid (1.5%) in
1
gave no catalysis under these conditions. Where catalysis 10% H SO and heating until colored spots appeared. H NMR,
2
4
1
3
was observed the data were analyzed for saturation kinetics.
In this study the unprotected cyclodextrin derivatives 7–9 500 MHz instrument.
were found to be surprisingly poor catalysts (Table 1). Nei-
ther the 2-O-carboxymethyl (9) nor the 6-O-carboxymethyl β-cyclodextrin (2) and Per-O-acetyl-2
8) compounds showed any catalysis at pH = 8.0. The 3- a stirred solution of β-cyclodextrin hydrate (10.0 g, 8.8 mmol) and
C NMR and COSY spectra were recorded with a Bruker
A
A
Per-O-acetyl-3 -O-allyl-β-cyclodextrin (1), Per-O-acetyl-6 -O-allyl-
-O-allyl-β-cyclodextrin (3): To
A
(
O analogue 7 showed catalysis with 3 substrates, but only sodium hydroxide (3.87 g, 96.8 mmol) in water (44 mL), allyl brom-
ide (381 μL, 4.4 mmol) was slowly added, and the resulting emul-
sion was stirred at room temp. for 12 h. The mixture was then neu-
Michaelis–Menten kinetics with α-glucopyranoside. In this
case the substrate binding was rather poor (KM = 53 mm).
tralized with sulfuric acid, concentrated to dryness, and a mixture
The rate acceleration that occurs when the substrate is
of mono-allyl-β-cyclodextrin regioisomers was separated from β-
bound inside the cavity is kcat/kuncat = 80.
cyclodextrin and di- and polysubstituted β-cyclodextrin derivatives
The O-methylated cyclodextrin acids all displayed some
catalysis, though in many cases saturation kinetics could
not be observed, and in general the catalysis was poor. In
by chromatography on silica gel (elution mixture 1-PrOH/H
concd. aq. NH , 10:2.5:1). The resulting mixture of regioisomers
was peracetylated: acetic anhydride (20 mL) and triethylamine
2
O/
3
the cases where Michaelis–Menten catalysis was observed (20 mL) were added, and the mixture was stirred and heated to
kcat varied in the range 4.4–54ϫ10 s , which is lower 90 °C for 3 h. The reagents were then co-evaporated with toluene,
–
7 –1
[9]
than was observed with diacids. The highest rate accelera- and the residue was dissolved in EtOAc (400 mL), extracted with
HCl (1 m, 400 mL) and H O (3ϫ400 mL). Then the organic phase
was dried with MgSO and concentrated under reduced pressure.
Purification by using a silica gel column afforded compound 1
10%, 800 mg) as a white foam. R = 0.32 (CH Cl /MeOH, 30:1).
). H NMR (500 MHz, CDCl ): δ =
), 5.49 (dd, J = 10.1, 8.9 Hz, 1 H, 3-
), 5.14–5.03 (m, 8 H, 1-H,
), 4.81–4.67 (m, 7 H, 2-H), 4.61–4.46 (m, 8 H, 6-H,
tion was seen with diacid 19, where a rate acceleration of
05 was observed for the hydrolysis of 4-nitrophenyl α-d-
2
1
4
glucopyranoside. This obviously means that having two
carboxylate groups is better than one, and that the likely
cause for this is the higher probability (in 19) that the sub-
strate will bind close to an acid. On the other hand 22,
which has four acid groups, does not work. Catalysis was
(
f
2
2
1
[α]
D
= +124.1 (c = 1.0, CHCl
3
3
5
.96–5.88 (m, 1 H, CH=CH
2
H), 5.36–5.25 (m, 6 H, 3-H, CH=CH
CH=CH
2
2
seen but not involving the cyclodextrin cavity, and it is pos- OCH CH=CH ), 4.38 (dd, J = 12.5, 3.7 Hz, 1 H, 6-H), 4.30–4.04
2
2
sible that having four methylcarboxylate groups at the pri- (m, 13 H, 6-H, OCH CH=CH , 5-H), 3.90 (dd, J = 9.6, 1.6 Hz, 1
2
2
mary rim is too many to allow effective binding of these H, 5-H), 3.80 (dd, J = 10.0, 8.8 Hz, 1 H, 3-H), 3.77–3.65 (m, 6 H,
4
-H), 3.58 (t, J = 9.2 Hz, 1 H, 4-H), 2.14–1.98 (m, 60 H, OAc)
substrates. It is obvious from models that there is no steric
hindrance in 22 as the carboxymethyl groups are small, and
there is plenty of space at the secondary face when four (or
even more) of these groups are attached. Therefore, it ap-
pears more likely that it is electrostatic effects that make the
productive binding of the substrate unfavorable in the case
of 22.
1
3
ppm. C NMR (126 MHz, CDCl
3
): δ = 171.03, 170.96, 170.87,
70.77, 170.71, 170.69, 170.66, 170.61, 170.52, 170.51, 170.49,
70.40 (C=O), 169.73, 169.68, 169.66, 169.59, 169.53, 169.48
), 116.01 (CH=CH ), 97.73, 97.48, 97.19,
6.73, 96.47, 96.38 (C-1), 81.09 (C-4), 77.66 (C-4), 77.34 (C-3),
6.67 (C-4), 75.81 (C-4), 75.74 (C-4), 74.87 (OCH CH=CH ),
2.17, 72.01, 71.46, 71.12, 70.97, 70.86, 70.82, 70.67, 70.61, 70.50,
1
1
(
C=O), 135.63 (CH=CH
2
2
9
7
7
2
2
More specifically the catalysis by 20, which was pre- 70.33, 69.84, 69.78, 69.68, 69.36 (C-2, C-3, C-5), 62.85, 62.64, 62.53
viously reported to give a kcat value of 2.7ϫ10 s , was (C-6), 21.27, 21.16, 21.11, 20.96, 20.91, 20.85 (CH ) ppm. HRMS
[
10]
–5 –1
3
+
found in this study to give a 50 times lower value. Repeating (MALDI-TOF): calcd. for
C
85
H
114
O
55Na 2037.602; found
2037.794.
the synthesis of 20 by using the method described in that
paper, which was oxidation of the allyl group in two steps
Further elution afforded compound 2 (12%, 966 mg) as a white
by ozone and sodium chlorate,[ did not change the value. foam. R = 0.27 (CH Cl /MeOH, 30:1). The analytical data for 2
10]
f 2 2
Thus, we could not confirm our preliminary results with 20. are in agreement with those reported previously.[
11]
Further elution afforded compound 3 (26%, 2.1 g) as a white foam.
R
f
= 0.21 (CH
2
Cl
2
/MeOH, 30:1). [α]
D
= +111.5 (c = 1.0, CHCl
3
).
),
Conclusions
1
H NMR (500 MHz, CDCl
3
): δ = 5.82–5.74 (m, 1 H, CH=CH
2
Our results consistently show that a β-cyclodextrin with 5.34–5.14 (m, 9 H, 3-H, CH=CH ), 5.09–4.98 (m, 6 H, 1-H), 4.86
2
an O-carboxymethyl group is a rather poor catalyst of the (d, J = 3.3 Hz, 1 H, 1-H), 4.83–4.71 (m, 6 H, 2-H), 4.58–4.46 (m,
4066
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Cyclodextrins with Carboxylic Acids Group as Potential Chemzymes
7
3
H, 6-H), 4.30–4.15 (m, 7 H, 6-H), 4.13–4.07 (m, 6 H, 5-H), 4.02– 2A-O-Carboxymethyl-β-cyclodextrin (9): Compound 6 (500 mg,
.92 (m, 3 H, 5-H, OCH CH=CH ), 3.71–3.57 (m, 7 H, 4-H), 3.27 0.247 mmol) was subjected to the procedure described for the prep-
2
2
(
dd, J = 10.0, 3.3 Hz, 1 H, 2-H), 2.09–1.97 (m, 60 H, OAc) ppm.
aration of 7 to give 9 (280 mg, 97%). The analytical data for 9 are
in agreement with those reported previously.
13
[8]
C NMR (126 MHz, CDCl
70.60, 170.52, 170.46, 170.43, 170.39, 170.28, 169.66, 169.42,
69.40, 169.37, 169.19 (C=O), 134.45 (CH=CH ), 117.81
), 98.45, 97.25, 97.10, 96.92, 96.67, 96.61, 96.52 (C-1),
8.42 (C-4), 77.83 (C-2), 77.75, 76.94, 76.69, 76.65, 76.33 (C-4),
2.74 (C-3), 72.21 (OCH -CH=CH ), 71.39, 70.90, 70.82, 70.74,
0.60, 70.53, 70.47, 70.16, 69.98, 69.61, 69.54, 69.49, 69.25 (C-3, C-
, C-5), 63.01, 62.56 (C-6), 21.02, 20.93, 20.81 (CH ) ppm. HRMS
55Na 2037.602; found
3
): δ = 171.05, 170.96, 170.80, 170.74,
1
1
Heptakis-2,3,6-tri-O-methyl-β-cyclodextrin (10): β-Cyclodextrin
2
(
2
12 g, 10.57 mmol) was dissolved in DMSO (300 mL). NaH (60%,
0 g, 528 mmol, 50 equiv.) was added, and the mixture stirred at
room temp. under nitrogen for 1 h. Iodomethane (33 mL,
28 mmol, 50 equiv.) was then added dropwise at 0 °C. The mixture
was stirred overnight, slowly warming to room temp. The reaction
was quenched by adding H O (around 500 mL) at 0 °C, and then
extracted with Et O (5ϫ300 mL). The combined organic extracts
were washed with brine, dried with MgSO , filtered, and the solvent
was removed in vacuo. The residue was subjected to flash
chromatography (CHCl /MeOH, 60:1) to give 10 (13.5 g, 90%).
(CH=CH
2
7
7
7
2
2
2
5
3
2
+
(MALDI-TOF): calcd. for
C
85
H
114
O
2
2
037.670.
4
A
Per-O-acetyl-3 -O-carboxymethyl-β-cyclodextrin (4): Compound 1
375 mg, 0.186 mmol) was dissolved in acetonitrile (4 mL), and a
(
3
saturated aqueous solution of sodium periodate (4 mL), an aque-
ous ruthenium(III) chloride solution (5%, 50 μL) was added, and
the reaction mixture was stirred at room temp. The reaction was
The analytical data for 10 are in agreement with those reported
previously.[
14]
2A,3B-Dihydroxy-per-O-methyl-β-cyclodextrin (11): A solution of
monitored by using TLC (CHCl
ance of the starting material (around 3 h), the mixture was ex-
tracted with CHCl (3ϫ15 mL). The collected CHCl extracts were
washed with Na (1%, 3ϫ30 mL) to remove traces of Ru salts
and dried with MgSO . The solvent was evaporated and the residue
purified by chromatography on a silica gel column (elution mixture
CHCl /MeOH, 20:1, 10:1) to give 4 (310 mg, 82%). R = 0.31
CHCl /MeOH, 10:1). The analytical data for 4 are in agreement
3
/MeOH, 10:1). After disappear-
DIBAL-H in toluene (1.0 m, 19.44 mmol, 9 equiv., 19.44 mL) was
added to a solution of compound 10 (3.09 g, 2.16 mmol) in anhy-
drous toluene (77.5 mL) at 0 °C under nitrogen. Then the mixture
was stirred at 0 °C for 18 h under nitrogen. Aqueous HCl (1 m) was
carefully added dropwise and the mixture was stirred vigorously at
room temperature for 10 min. The toluene phase was separated and
the aqueous layer was extracted with ethyl acetate (3ϫ60 mL). The
combined organic extracts were washed with brine, dried with
3
3
2 2 5
S O
4
3
f
(
3
[11]
with those reported previously.
MgSO
4
, filtered, and the solvent was removed in vacuo. The residue
Cl /MeOH, 40:1) to
A
Per-O-acetyl-6 -O-carboxymethyl-β-cyclodextrin (5): Compound 2
was subjected to flash chromatography (CH
2
2
(
402 mg, 0.199 mmol) was subjected to the procedure described for
the preparation of 4 to give 5 (345 mg, 85%). R = 0.48 (CHCl
MeOH, 10:1). The analytical data for 5 are in agreement with those
give 11 (1.55 g, 51%). The analytical data for 11 are in agreement
with those reported previously.[
12]
f
3
/
2A-Hydroxy-per-O-methyl-β-cyclodextrin (12) and 3A-Hydroxy-per-
O-methyl-β-cyclodextrin (13): NaH (60%, 18.16 mg, 0.454 mmol,
1.0 equiv.) was added to a solution of 11 (636 mg, 0.454 mmol) in
anhydrous THF (60 mL) at 0 °C under nitrogen. After stirring at
[
11]
reported previously.
A
Per-O-acetyl-2 -O-carboxymethyl-β-cyclodextrin (6): Compound 3
(
849 mg, 0.422 mmol) was subjected to the procedure described for
the preparation of 4 to give 6 as a white foam (710 mg, 83%).
= 0.41 (H O/2-propanol/EtOAc, 1:3:3). [α] = +113.7 (c = 1.0,
). H NMR (500 MHz, CDCl ): δ = 5.36–5.22 (m, 7 H, 3- room temperature under nitrogen for a further 12 h. CH
H), 5.10–5.00 (m, 7 H, 1-H), 4.79–4.69 (m, 6 H, 2-H), 4.55–4.45 added dropwise to quench the reaction, and the solvent was re-
m, 7 H, 6-H), 4.30–3.94 (m,16 H, 6-H, 5-H, OCH COOH), 3.77– moved in vacuo. The residue was dissolved in CH Cl , washed with
.58 (m, 7 H, 4-H), 3.38 (dd, J = 10.1, 2.8 Hz, 1 H, 2-H), 2.10– brine, dried with MgSO , filtered and the solvent removed in vacuo.
Purification on a silica column afforded compound 12 as a white
foam (18%, 120 mg); R = 0.34 (EtOAc/MeOH, 10:1). Further elu-
70.40 (OAc), 170.08 (COOH), 169.69, 169.60, 169.51 (OAc), tion afforded compound 13 as a white foam (74%, 477 mg); R
0 °C for 1 h, CH
The reaction mixture was stirred at 0 °C for 6 h and then kept at
OH was
3
I (0.454 mmol, 1.0 equiv., 0.028 mL) was added.
R
CHCl
f
2
D
1
3
3
3
(
2
2
2
3
1
1
1
9
7
7
4
13
.98 (m, 60 H, OAc) ppm. C NMR (126 MHz, CDCl
3
): δ =
70.83, 170.79, 170.75, 170.66, 170.58, 170.51, 170.43, 170.41,
f
f
=
9.85, 97.28, 97.06, 96.78, 96.53 (C-1), 78.88 (C-2), 78.49, 78.04,
7.34, 77.02, 76.55, 76.34, 76.21 (C-4), 72.62, 71.01, 70.93, 70.67,
0.59, 70.52, 70.36, 70.28, 70.01, 69.93, 69.76, 69.63, 69.52 (C-3,
0.22 (EtOAc/MeOH, 10:1). The analytical data for 12 and 13 are
in agreement with those reported previously.[
15]
2A,3 ,2 ,3 -Tetrahydroxy-per-O-methyl-β-cyclodextrin
B
D E
(14):
C-2, C-5), 68.22 (OCH
2
TOF): calcd. for C84
2
COOH), 62.84, 62.76, 62.63, 62.38 (C-6),
DIBAL-H (160 mmol, 50 equiv., 1 m in toluene, 160 mL) was
added to a stirred solution of permethylated cyclodextrin 10
1.17, 20.97, 20.91, 20.83, 20.77 (OAc) ppm. HRMS (MALDI-
+
H
112
O57Na 2055.576; found 2055.520.
(
4.57 g, 3.2 mmol) in anhydrous toluene (40 mL) at room temp. un-
3A-O-Carboxymethyl-β-cyclodextrin (7): Compound 4 (300 mg,
.148 mmol) was dissolved in a solution of NaOMe in MeOH
0.1 m, 6 mL), and the solution was stirred at room temp. overnight.
der nitrogen and stirred for 3 h. The solution was cooled to 0 °C,
the reaction quenched with aqueous HCl (1 m), and the mixture
was stirred vigorously at room temperature for 30 min. The toluene
phase was collected and the aqueous phase extracted with ethyl
acetate (3ϫ200 mL). The combined organic phases were washed
0
(
Then water (6 mL) was added, the solution was neutralized with
Dowex 120 in H+ form, and the solvents were evaporated. The
solid was dissolved in water (10 mL), the treatment with Dowex
was repeated once more, and 7 was obtained by freeze-drying
with brine, dried with MgSO
vacuo. The residue was subjected to flash chromatography
Cl /CH OH, 30:1) to give 14 as white foam (1.3 g, 30%). The
4
, and the solvent was removed in
(165 mg, 95%). The analytical data for 7 are in agreement with (CH
2
2
3
[11]
those reported previously.
analytical data for 14 are in agreement with those reported pre-
viously.[
12]
6A-O-Carboxymethyl-β-cyclodextrin (8): Compound 5 (320 mg,
.158 mmol) was subjected to the procedure described for the prep-
A
B
0
2 ,3 -Diallyl-per-O-methyl-β-cyclodextrin
(15):
3 3
(CH ) COK
aration of 7 to give 8 (172 mg, 91%). The analytical data for 8 are
(176 mg, 1.57 mmol, 10 equiv.) was added to a solution of 11
(220 mg, 0.157 mmol) in anhydrous DMSO (2 mL) at room temp.
in agreement with those reported previously.[
11]
Eur. J. Org. Chem. 2012, 4063–4070
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
4067

Y. Zhou, E. Lindbäck, L. G. Marinescu, C. M. Pedersen, M. Bols
71.49 (C-6), 71.16, 71.05, 71.01 (C-5), 61.66, 61.63, 61.59, 61.56
FULL PAPER
under nitrogen. After stirring for 5 min, allyl bromide (2.36 mmol,
0
.2 mL, 20 equiv.) was added, and the mixture was stirred at room
temp. The reaction was monitored by using TLC (CH Cl /MeOH,
5:1). The product has a larger R value than the starting com-
[OCH
3 3
(C3)], 59.10, 59.09 [OCH (C6)], 58.87, 58.74, 58.65, 58.63,
2
2
58.59 [OCH (C2)] ppm. HRMS (MALDI-TOF): calcd. for
3
1
f
C
65
H
114
O
35Na+ 1477.703; found 1477.721.
pound. After disappearance of the starting compound and the
mono-allylation compound, water was carefully added to quench
2A,3 ,2 ,3 -Tertraallyl-per-O-methyl-β-cyclodextrin (18): (CH
B
D
E
3
)
3
-
COK (1.077 g, 9.6 mmol, 30 equiv.) was added to a solution of 14
439 mg, 0.32 mmol) in anhydrous DMSO (10 mL) at room temp.
under nitrogen. After stirring for 5 min, allyl bromide (9.6 mmol,
.108 mL, 40 equiv.) was added, and the mixture was stirred at
room temp. The reaction was monitored by using TLC (CH Cl
MeOH, 15:1) and HRMS (MALDI-TOF). The product has a
larger R value than the starting compound. After 3 h, water was
carefully added to quench the reaction, and the mixture was ex-
tracted with CH Cl (3ϫ20 mL). The collected CH Cl extracts
were washed with brine, dried with MgSO , and the solvent was
removed in vacuo. The residue was subjected to flash chromatog-
raphy (CH Cl /CH OH, 200:1) to give 18 as white foam (410 mg,
4%). R = 0.62 (EtOAc/CH OH, 10:1). [α] = +123.4 (c = 1.0,
). 1H NMR (500 MHz, CDCl
CHCl ): δ = 6.10–6.02 (m, 2 H,
), 5.97–5.90 (m, 2 H, A,DCH=CH
CH=CH ), 5.29–5.23 (m, 4 H,
), 5.17–5.06 (m, 11 H, 7ϫ 1-H,
the reaction, and the mixture was extracted with CH
2
Cl
extracts were washed with brine,
, and the solvent was removed in vacuo. The
Cl /CH OH,
= 0.46 (EtOAc/
). H NMR (500 MHz,
): δ = 6.09–6.02 (m, 1 H, 3BCH = CH
), 5.98–5.90 (m, 1 H,
2
(
2 2
(3ϫ10 mL). The collected CH Cl
dried with MgSO
4
1
residue was subjected to flash chromatography (CH
00:1) to give 15 as white foam (203 mg, 87%). R
2
2
3
2
2
/
1
f
1
3 D 3
CH OH, 10:1). [α] = +51.7 (c = 0.4, CHCl
f
CDCl
3
2
2
ACH = CH
3B
2A
2
), 5.30–5.23 (m, 2 H, CH=CH
2
,
CH=CH
), 4.52 (dd, J = 12.2,
2
), 4.27 (dd, J = 12.2, 5.7 Hz, 1 H,
2
), 5.16–
2
2
2
2
3
B
, 2ACH=CH
5
.08 (m, 9 H, 1-H, CH=CH
.7 Hz, 1 H, 3BOCH
CH=CH
), 4.20–4.12 (m, 2 H, OCH
dd, J = 10.6, 3.4 Hz, 1 H, 6-H), 3.87–3.77 (m, 13 H, 6-H, 5-H),
2 2
4
5
2
3B
2A
OCH
2
CH=CH
2
2 2
CH=CH ), 3.93
2
2
3
(
8
f
3
D
B
3.69–3.48 [m, 57 H, 3-H, OCH
3
(C3), 4-H, 6Ј-H, 3-H, OCH
(C6)], 3.33 (dd, J = 9.6, 3.5 Hz,
3
3
3
(
1
C2)], 3.38–3.37 [m, 21 H, OCH
H, 2-H), 3.23–3.18 (m, 6 H, 2-H) ppm. C NMR (126 MHz,
3
B,E
A,D
2
2
A
13
B,E
CH=CH
CH=CH
2
,
,
CH=CH
2
2A
CDCl
3
): δ = 136.49 (3BCH = CH
2
), 135.51 ( CH = CH
2
), 117.09
A,D
B,E
B,E
2
CH=CH
2
), 4.53 (m, 2 H, OCH
2
CH=CH
), 4.19–4.11 (m, 4 H,
), 3.94 (dd, J = 10.5, 3.2 Hz, 2 H, 6-H), 3.85–
.68 (m, 14 H, 5ϫ 6-H, 7ϫ 5-H, 2ϫ 3-H), 3.67–3.48 [m, 49 H,
ϫ 4-H, 5ϫ OCH (C-3), 7ϫ 6-H, 5ϫ 3-H, 5ϫ OCH (C-2)],
.38–3.30 [m, 23 H, 7ϫ OCH (C-6), 2ϫ 2-H], 3.23–3.17 (m, 5 H,
ϫ 2-H) ppm. C NMR (126 MHz, CDCl ): δ = 136.52, 136.49
CH=CH ), 117.06, 117.04
), 115.97, 115.94 ( CH=CH ) 99.17, 99.16, 99.05,
2
), 4.26
(
2ACH = CH
2
), 115.93 ( CH = CH
3B
2
), 99.11, 99.10, 99.04, 99.02
B,E
(
2 2
dd, J = 12.3, 5.7 Hz, 2 H, OCH CH=CH
(C-1), 82.23, 82.20, 82.16, 82.12, 82.08, 81.98, 81.91, 81.88, 81.84
A,D
2 2
OCH CH=CH
(
C-2, C-3), 80.60, 80.59, 80.30, 80.08, 79.98, 79.89, 79.85, 79.79,
3
7
3
5
B
A
3B
7
9.76 (C-4, C-3,
C-2), 74.76
), 71.60, 71.45 (C-6), 71.18, 71.08, 70.95 (C-5),
1.69, 61.56, 61.51 [OCH (C3)], 59.12 [OCH (C6)], 58.70, 58.55
2 2
( OCH CH=CH ), 72.05
3
3
2
AOCH
(
2 2
CH=CH
3
6
3
3
13
3
[
C
OCH
3
(C2)] ppm. HRMS (MALDI-TOF): calcd. for
35Na 1503.719; found 1503.719.
B,E
A,D
(
(
CH=CH
CH=CH
2
), 135.52, 135.50
(
2
+
67
H
116
O
A,D
B,E
2
2
2A-O-Allyl-per-O-methyl-β-cyclodextrin (16): Compound 12
120 mg, 0.085 mmol) was subjected to the procedure for the prepa-
ration of 15 to give 16 (87 mg, 71%). R = 0.33 (EtOAc/2-propanol,
99.03, 98.99 (C-1), 82.19, 82.13, 82.06, 81.95, 81.88, 81.80 (C-2, C-
(
3), 80.30, 80.03, 79.97, 79.92, 79.81, 79.79, 79.76, 79.62 (C-2Ј, C-4,
B,E
f
C-3Ј), 74.82 ( OCH
(
2
CH=CH
, C-6, C-6Ј) 71.22, 71.20, 70.98, 70.96, 70.92,
70.89, 70.84 (C-5), 61.73, 61.71, 61.66, 61.63, 61.61, 61.44, 61.42
, 1-H), [OCH (C-3)], 59.24, 59.22, 59.10, 59.09, 58.99, 58.96, 58.84, 58.81,
.07–5.05 (m, 1 H, 1-H), 4.23 (dd, J = 12.9, 5.8 Hz, 1 H, 58.71, 58.69, 58.59, 58.57 [OCH (C-6), OCH (C-2)] ppm. HRMS
CH=CH ), 4.12 (dd, J = 12.9, 5.3 Hz, 1 H, OCH CH=CH ), (MALDI-TOF): calcd. for
.89–3.79 (m, 14 H, 6-H, 5-H), 3.66–3.56 [m, 35 H, OCH (C3), 4- 1555.706.
H, 6Ј-H], 3.54–3.49 [m, 25 H, 3-H, OCH (C2)], 3.38–3.37 [m, 21
H, OCH (C6)], 3.33 (dd, J = 9.6, 3.5 Hz, 1 H, 2-H), 3.20–3.18 (m,
H, 2-H) ppm. C NMR (126 MHz, CDCl
2
), 72.00, 71.95, 71.64, 71.52, 71.39
1
A,D
1
0:1). [α]
D
= +140.9 (c = 1.0, CHCl
): δ = 5.99–5.91 (m, 1 H, CH=CH
), 5.15–5.12 (m, 7 H, CH=CH
3
). H NMR (500 MHz,
2 2
OCH CH=CH
CDCl
3
2
), 5.33 (dd, J = 17.3,
1.7 Hz, 1 H, CH=CH
2
2
3
5
3
3
+
OCH
2
2
2
2
71 120
C H O35Na 1555.750; found
3
3
3
2A,3 -O-Dicaboxymethyl-per-O-methyl-β-cyclodextrin (19): Com-
B
3
pound 15 (500 mg, 0.338 mmol) was dissolved in acetonitrile
13
6
3
): δ = 135.46
), 99.51 ( C-1), 99.13 (C-1), 82.19,
2.11, 82.01, 81.96, 81.91, 81.84 (C-2, C-3), 80.55, 80.40, 80.32,
(
9 mL), then saturated aqueous sodium periodate (9 mL) and 5%
A
2 2
(CH=CH ), 116.85 (CH=CH
aqueous ruthenium(III) chloride (100 μL) were added, and the re-
action mixture was stirred at room temp. The reaction was moni-
8
8
6
A
0.23, 79.91 (C-4, C-2), 71.87 (OCH
), 71.15, 71.10, 71.04, 70.97 (C-5), 61.76, 61.73, 61.65, 61.59
(C3)], 59.12 [OCH (C6)], 58.77, 58.74, 58.63, 58.61 [OCH
C2)] ppm. HRMS (MALDI-TOF): calcd. for C65
477.703; found 1477.385.
2 2
CH=CH ), 71.60, 71.52 (C-
tored by using TLC (CHCl
the starting compound, the mixture was extracted with CHCl
3ϫ15 mL). The collected CHCl extracts were washed with 1%
Na (3ϫ30 mL) to remove traces of Ru salts and dried with
MgSO . The solvent was evaporated, and the product was purified
by chromatography on silica gel [CH Cl /MeOH, 30:1 (1% formic
acid)] to give 19 (405 mg, 79%). R = 0.10 (H O/2-propanol/EtOAc,
1:3:3). [α] = +114.7 (c = 1.0, CHCl
): δ = CDCl ): δ = 5.22 (d, J = 3.3 Hz, 1 H, 1-H), 5.19 (d, J = 3.6 Hz, 1
), 5.26 (dd, J = 17.3, 1.8 Hz, 1 H,
), 5.15–5.10 (m, 8 H, CH=CH , 1-H), 4.49 (dd, J = 11.9,
.7 Hz, 1 H, OCH CH=CH ), 4.26 (dd, J = 11.9, 5.7 Hz, 1 H,
OCH CH=CH ), 3.92 (dd, J = 10.6, 3.8 Hz, 1 H, 6-H), 3.87–3.77 17.1 Hz, 1 H, OCH
m, 13 H, 6-H, 5-H), 3.70–3.55 [m, 33 H, 3-H, OCH
3
/MeOH, 10:1). After disappearance of
3
[OCH
3
3
3
(
3
114
H O
35Na+
(
1
2 2 5
S O
4
3A-Allyl-per-O-methyl-β-cyclodextrin (17): Compound 13 (369 mg,
0
1
2
2
.261 mmol) was subjected to the procedure for the preparation of
5 to give 17 (297 mg, 78%). R = 0.39 (EtOAc/CH OH, 10:1).
). H NMR (500 MHz, CDCl
f
2
1
f
3
D
3
). H NMR (500 MHz,
1
[α]
D
= +155.5 (c = 1.0, CHCl
3
3
3
6.09–6.01 (m, 1 H, CH=CH
2
H, 1-H), 5.14 (d, J = 3.2 Hz, 2 H, 1-H), 5.12–5.10 (m, 2 H, 1-H),
5.08 (d, J = 3.4 Hz, 1 H, 1-H), 4.63 (d, J = 17.6 Hz, 1 H, OCH2-
CH=CH
5
2
2
2
2
COOH), 4.45 (d, J = 17.7 Hz, 1 H, OCH
COOH), 4.31 (d, J = 17.0 Hz, 1 H, OCH
3
(C3), 4-H, COOH), 4.04 (dd, J = 10.5, 2.1 Hz, 1 H, 6-H), 3.95 (dd, J = 10.6,
2
COOH), 4.38 (d, J =
2
2
2
2
-
A
(
A
6
Ј-H], 3.53–3.47 [m, 27 H, 3-H, OCH
(C6)], 3.22–3.17 (m, 7 H, 2-H) ppm. 1 C NMR (126 MHz, H, 1ϫ B3-H, 1ϫ OCH
): δ = 136.28 (CH=CH ), 116.02 (CH=CH ), 99.21, 99.09, 6Ј-H, 5ϫ OCH (C-3), 5ϫ 3-H, 6ϫ OCH
8.82 (C-1), 82.20, 82.15, 81.96, 81.89 (C-2, C-3), 80.54, 80.51, 22 H, 7ϫ OCH (C-6), 1ϫ 2-H], 3.22–3.18 (m, 6 H, 6ϫ 2-H)
0.34, 80.26, 79.94 (C-4, AC-3), 74.85 (OCH ppm. 13C NMR (126 MHz, CDCl
CH=CH ), 71.59, ): δ = 172.33 (COOH), 171.51
3
(C2)], 3.37–3.36 [m, 21 H, 3.7 Hz, 1 H, 6-H), 3.90–3.70 [m, 17 H, 5ϫ 6-H, 7ϫ 5-H, 1ϫ 3-
3
OCH
CDCl
9
8
3
3
(C-3)], 3.69–3.46 [m, 52 H, 7ϫ 4-H, 7ϫ
(C-2)], 3.42–3.36 [m,
3
2
2
3
3
3
2
2
3
4068
www.eurjoc.org
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2012, 4063–4070

Cyclodextrins with Carboxylic Acids Group as Potential Chemzymes
COOH), 99.64, 99.48, 99.46, 99.43, 99.27, 99.26, 99.12, 99.10,
(
OCH
3.96–3.92 (m, 2 H, 6-H), 3.89 (dd, J = 10.8, 3.9 Hz, 1 H, 6-H), 3.69
[s, 3 H, 1ϫ OCH (C-3)], 3.67 [s, 3 H, 1ϫ OCH (C-3)], 3.54 [s, 3
(C-3)], 3.52 [m, 6 H, 2ϫ OCH (C-3)], 3.50 [s, 3 H,
(C-2)], 3.49 [s, 3 H, 1ϫ OCH (C-2)], 3.45 [s, 3 H, 1ϫ
(C-2)], 3.42 [m, 6 H, 1ϫ OCH (C-2)], 3.81–3.36 (m, 32 H,
4ϫ 6-H, 7ϫ 5-H, 7ϫ 4-H, 7ϫ 6Ј-H, 7ϫ 3-H), 3.33–3.22 [m, 25
2 2
COOH), 4.21 (dd, J = 17.1, 3.9 Hz, 2 H, OCH COOH),
9
8
8.89, 98.87, 98.79 (C-1), 82.72, 82.49, 82.33, 82.05, 81.99, 81.94,
1.87, 81.68, 81.62, 81.31, 81.19, 80.78, 80.67, 80.55, 80.24, 79.79
3
3
(
C-2, C-3, C-4), 71.88, 71.64, 71.50, 71.39, 71.34, 71.28, 71.22, H, 1ϫ OCH
1.15, 71.01, 70.80, 70.42 (C-6, C-5, OCH COOH), 68.69 (OCH 1ϫ OCH
COOH), 62.44, 61.80, 61.71, 61.68, 61.60, 61.57, 61.41, 61.25, 61.22 OCH
OCH (C-3)], 59.22, 59.14, 59.11, 59.05, 58.68, 58.66, 58.52, 58.49,
8.47, 58.42, 58.40 [OCH (C-6), OCH (C-2)] ppm. HRMS
MALDI-TOF): calcd. for 39Na 1539.667; found
539.729.
3
3
7
2
2
-
3
3
3
3
[
3
1
3
5
(
3
3
H, 7ϫ OCH
NMR (126 MHz, CDCl
(COOH), 99.82, 99.66, 99.64, 99.50, 99.47, 99.16, 98.88, 98.87,
3
(C-6), 4ϫ 2-H], 3.14–3.08 (m, 3 H, 2-H) ppm.
C
+
C
65
H
112
O
3
): δ = 172.12, 172.10, 171.44, 171.41
1
9
8
7
7
6
5
8.76 (C-1), 82.71, 82.57, 81.93, 81.82, 81.59, 81.49, 81.44, 81.36,
1.08, 80.90, 80.47, 80.44, 80.14, 80.03, 79.87 (C-2, C-3, C-4),
2.12, 72.02, 71.94, 71.54, 71.45, 71.38, 71.33, 71.10, 71.04, 70.94,
2A-O-Carboxymethyl-per-O-methyl-β-cyclodextrin (20): Compound
6 (42.2 mg, 0.029 mmol) was subjected to the procedure for the
preparation of 19 to give 20 as a white foam (31 mg, 73%). R
.29 (H O/2-propanol/EtOAc, 1:3:3). [α] = +69.2 (c = 0.7,
): δ = 5.18 (d, J = 3.6 Hz, 1
H, 1-H), 5.14 (dd, J = 8.8, 3.7 Hz, 2 H, 1-H), 5.10 (dd, J = 4.6,
1
f
=
0.88, 70.52, 70.45 (C-6, C-5, OCH
2.53, 62.48, 61.66, 61.64, 61.43, 61.18 [OCH
9.05, 58.83, 58.80, 58.43, 58.39, 58.20 [OCH
2
COOH), 68.71 (OCH
(C-3)], 59.23, 59.11,
(C-6), OCH (C-2)]
43Na 1627.647;
2
COOH),
0
2
1
D
3
3 3
CHCl ). H NMR (500 MHz, CDCl
3
3
+
ppm. HRMS (MALDI-TOF): calcd. for C67
found 1627.891.
112
H O
3
.8 Hz, 2 H, 1-H), 5.07 (d, J = 3.5 Hz, 1 H, 1-H), 4.97 (d, J =
.4 Hz, 1 H, 1-H), 4.45 (d, J = 16.5 Hz, 1 H, OCH COOH), 4.20
COOH), 3.98 (dd, J = 10.2, 3.1 Hz, 1
H, 6-H), 3.90–3.75 (m, 12 H, 6-H, 5-H), 3.72–3.45 [m, 61 H, 5-H,
-H, OCH (C-3), 6Ј-H, OCH (C-2), 3-H], 3.39–3.34 [m, 22 H,
(C-6), 2-H], 3.21–3.17 (m, 6 H, 2-H) ppm. 1 C NMR
OCH
126 MHz, CDCl ): δ = 170.83 (COOH), 99.63, 99.54, 99.52, 99.20,
3
2
Procedure for Determining the Rate of Hydrolysis: Each assay was
performed on samples (1 mL) prepared from aqueous solutions of
the appropriate nitrophenyl glycoside (0.5 mL) at different concen-
trations mixed with phosphate buffer (0.5 m, 0.5 mL) containing
either 7–9 or 19–22 (0.58 mm) or nothing as control. The reactions
were monitored at 59 °C by using UV absorption at 400 nm and
typically monitored for 12 h. Rates were calculated from the slope
of the progress curve of each reaction. Uncatalysed rates were ob-
tained directly from the control samples. Catalyzed rates were cal-
culated by subtracting the uncatalysed rate from the rate obtained
from the appropriate cyclodextrin-containing sample. The cata-
lyzed rates were used to construct a Hanes plot ([S]/V vs. [S]) from
(d, J = 16.4 Hz, 1 H, OCH
2
4
3
3
3
3
(
3
9
8
4
7
6
5
9.15, 98.94, 98.91 (C-1), 82.62, 82.36, 82.14, 82.04, 81.95, 81.90,
1.77, 81.61, 81.33, 80.79, 80.63, 80.38, 79.93, 79.55 (C-2, C-3, C-
), 72.09, 71.98 (C-6), 71.88 ( C-5), 71.66, 71.58 (C-6), 71.25, 71.04,
A
A
0.97, 70.88 (C-5), 70.61 ( C-6), 69.78 (OCH
2
COOH), 61.71,
(C-3)], 59.39, 59.36, 59.30,
9.26, 59.16, 59.11, 59.01, 58.92, 58.48, 58.45, 58.34, 58.33 [OCH
(C-2)] ppm. HRMS (MALDI-TOF): calcd. for
37Na+ 1495.677; found 1495.597.
1.64, 61.58, 61.55, 61.40, 61.20 [OCH
3
3
(C-6), OCH
3
which Km and Vmax were determined. kcat was calculated as Vmax
/
C
64 112
H O
[cyclodextrin]; kuncat was determined as the slope from a plot of
3A-O-Carboxymethyl-per-O-methyl-β-cyclodextrin (21): Compound
7 (305 mg, 0.21 mmol) was subjected to the procedure for the
preparation of 19 to give 21 as a white foam (221 mg, 1%). R
.31 (H O/2-propanol/EtOAc, 1:3:3). [α] = +122.3 (c = 1.0,
): δ = 5.71 (d, J = 3.4 Hz, 1
H, 1-H), 5.14–5.13 (m, 2 H, 1-H), 5.10–5.08 (m, 3 H, 1-H), 5.05
d, J = 3.7 Hz, 1 H, 1-H), 4.58 (d, J = 17.6 Hz, 1 H, OCH COOH),
COOH), 4.11 (dd, J = 10.7, 2.8 Hz,
H, 6-H), 3.87–3.67 (m, 15 H, 6-H, 5-H, A4-H, A3-H), 3.66–3.44
(C-3), 6Ј-H, OCH (C-2), 3-H], 3.40–3.33
(C-6)], 3.29 (dd, J = 9.8, 3.4 Hz, 1 H, 2-H), 3.23
V
uncat vs. [S]. The following extinction coefficients were determined
1
–1
–1
and used in the calculations: ε = 17.04 mm cm (pH = 8.0, 59 °C,
f
=
4-nitrophenolate), ε = 2.28 mm cm (pH = 8.0, 59 °C, 2-nitro-
–1
–1
0
2
1
D
phenolate).
3 3
CHCl ). H NMR (500 MHz, CDCl
Supporting Information (see footnote on the first page of this arti-
cle): H and C NMR spectra of compounds.
1
13
(
2
4.45 (d, J = 17.6 Hz, 1 H, OCH
2
1
Acknowledgments
[
m, 58 H, 4-H, OCH
m, 21 H, OCH
dd, J = 9.4, 3.5 Hz, 1 H, 2-H), 3.19–3.14 (m, 5 H, 2-H) ppm.
NMR (126 MHz, CDCl ): δ = 172.45 (COOH), 99.80, 99.50, 99.41,
9.31, 99.00, 98.95, 98.91 (C-1), 82.69, 82.66, 82.54, 82.29, 82.21,
3
3
[
3
We thank the Free Danish National Research Council (FNU) and
the Lundbeck Foundation for financial support.
1
3
(
C
3
9
8
8
[
[
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0.48, 80.40, 80.30, 80.25 (C-2, C-3, C-4, C-3, AC-4), 71.82
A
A
(OCH
2
COOH), 71.72, 71.67 (C-6), 71.54 ( C-5), 71.48 (C-6), 71.37,
[
A
7
6
5
1.18, 71.10, 71.05, 71.01, 70.89 (C-5), 70.55 ( C-6), 61.96, 61.73,
1.70, 61.57, 61.54, 61.52 [OCH (C-3)], 59.17, 59.11, 59.09, 59.03,
8.94, 58.73, 58.66, 58.55, 58.51, 58.13 [OCH (C-6), OCH (C-2)]
37Na 1495.677;
3
3
3
+
ppm. HRMS (MALDI-TOF): calcd. for C64
found 1495.691.
112
H O
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cedure for the preparation of 19 to give 22 as a white foam (118 mg,
3%). R = 0.44 (H O/2-propanol/EtOAc, 1:2:2, 0.5% formic acid).
α] = +112.1 (c = 1.0, CHCl
.17 (d, J = 3.4 Hz, 1 H, 1-H), 5.09 (t, J = 3.5 Hz, 2 H, 1-H), 5.04
B
D
E
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[
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2
-
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2
COOH), 4.37–4.34 (m, 2 H, OCH COOH), 4.31–4.27 (m, 2 H,
Eur. J. Org. Chem. 2012, 4063–4070
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
4069

Y. Zhou, E. Lindbäck, L. G. Marinescu, C. M. Pedersen, M. Bols
FULL PAPER
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Received: March 29, 2012
Published Online: June 6, 2012
4070
www.eurjoc.org
© 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2012, 4063–4070