Imidazolyl cyclodextrins: Artificial serine proteases enabling regiospecific reactions

Article abstract of DOI:10.1002/anie.200701156

(Chemical Equation Presented) Remote control: The imidazolyl group in a series of imidazolyl cyclodextrins guides the sulfonylation of a specific hydroxy group in the presence of many others to effect a regioselective 1,22 remote functionalization (see st

Full text of DOI:10.1002/anie.200701156

Communications
DOI: 10.1002/anie.200701156
Cyclodextrins
Imidazolyl Cyclodextrins: Artificial Serine Proteases Enabling
Regiospecific Reactions**
De-Qi Yuan,* Yumika Kitagawa, Keisuke Aoyama, Toshihiko Douke, Makoto Fukudome, and
Kahee Fujita*
Inspired by the high selectivity and high efficiency of
enzymes, scientists have made tremendous efforts in creating
synthetic molecules that mimic the basic structural and
catalytic features of enzymes.[1,2] Breslow and co-workers
demonstrated that artificial enzymes can target a single
functionality even in the presence of other more reactive
functionalities without protective groups.[3] Wulff et al. pre-
pared some imprinted polymer-based artificial enzymes that
have strong catalytic activity that clearly surpasses the
corresponding catalytic antibodies.[4] Many other artificial
enzymes have also displayed interesting selectivity and
remarkable rate acceleration.[1,2,5] However, little workhas
been done to demonstrate that only the specifically located
functionalities in the presence of many others could actively
take part in the catalysis, which is the general feature of
natural enzymes. Chymotrypsin uses the Asp102-COOÀ/
His57-Im/Ser195-OH triad in catalyzing the hydrolysis of
peptide substrates and the Ser195-OH group is acylated
during the first step.[6] This enzymatic function has attracted a
long-standing interest and has been extensively mimicked.[1,2]
Cyclodextrins (CDs) remarkably accelerate (by a factor of up
to six million) the hydrolysis of nitrophenyl esters by a
mechanism closely related to that of proteases.[7] The acyl
group is transferred to the secondary side of the CD, but it is
not known whether the 2-OH or 3-OH group attacks the ester
first.[8] Imidazolyl CDs demonstrate improved catalytic abil-
ity, whereas in this case, nothing is known about where the
acyl group is transferred to.[9] On the other hand, it still
represents a great challenge to functionalize CDs by intro-
ducing two or more different functional groups with high
selectivity.[10] These drawbacks have hampered the rational
design of more sophisticated artificial enzymes. Herein we
demonstrate that among the many hydroxy groups of
imidazolyl CDs, only the one at a specific position is engaged
in the catalysis, which enables the establishment of a novel
efficient strategy for the hetero-bifunctionalization and
-trifunctionalization of CDs.
6-Imidazolyl-b-CD (1b) catalyzes the hydrolysis of p-
nitrophenyl acetate. HPLC of the reaction mixture revealed
the formation of a new CD species with a longer retention
time than that of 1b. Structural analysis indicated that the
product is the isomerically pure monoacetate 2b (Scheme 1),
Scheme 1. Regioselective acylation of 1b; n=0–5.
thus implying that one of the 20 hydroxy groups can receive
the acyl group of the substrate. Detailed NMR analysis
(Figure 1S in the Supporting Information) revealed the
acetylation of one primary hydroxy group (6-OH), but it did
not allow the specification of that site.
Considering the significance of CD sulfonates in the
functionalization of CDs, we examined the reaction of 1b with
mesitylenesulfonyl chloride to elucidate the cooperation
between the imidazolyl and the hydroxy groups of CDs as
well as to develop a new concept for the hetero-bifunction-
alization or -multifunctionalization of CDs. The reaction was
carried out at room temperature in a phosphate buffer at pH 8
containing 33% dimethylformamide (DMF). The sulfonate
3b, among the estimated maximum 20 isomeric monosulfo-
nates, was recognized and isolated in 13% yield together with
unreacted 1b (63%, Scheme 2). No obvious sulfonylation was
observed with b-CD under these conditions. Structure
determination of 3b indicated the sulfonylaton of the 6E-OH
group, implying that only the 6E-OH group is actively engaged
in the reaction although the 6D-OH and 6E-OH groups are
similarly located with respect to the imidazolyl moiety.[11]
These results emphasize that precise geometrical matching
is required for the cooperation between the hydroxy and
imidazolyl groups.
[*] Prof. Dr. D.-Q. Yuan, Y. Kitagawa, K. Aoyama, T. Douke,
Dr. M. Fukudome, Prof. Dr. K. Fujita
Department of Molecular Medicinal Sciences
Graduate School of Biomedical Sciences
Nagasaki University
Nagasaki 852-8521 (Japan)
Fax: (+81)95-819-2423
E-mail: deqiyuan@nagasaki-u.ac.jp
fujita@nagasaki-u.ac.jp
[**] The authors are grateful to Japan Maize Products Co. Ltd. for a
generous gift of cyclodextrins.
Further information about the geometrical requirements
for the cooperation between the hydroxy and imidazolyl
groups was obtained from the reaction of the a and g ana-
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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Scheme 2. Regioselective sulfonylations of 1; Mess=mesitylenesul-
fonyl.
logues 1a and 1g under similar conditions (Scheme 2).
Compound 1a gave the mesitylenesulfonate 3a as the only
isolable product, which was converted to the C2-symmetrical
6A,6D-diimidazolyl-a-CD by treatment with imidazole (Fig-
ure 2S in the Supporting Information). The reaction of 1g
afforded 6A-imidazolyl-6F-mesitylenesulfonyl-g-CD (3g). The
common regiochemistry of 3a–3g revealed that the imida-
zolyl group always cooperates with the third 6-OH group on
the anomeric side of the imidazolylglucoside residue while the
number of sugar units on the other side may vary. Obviously
the imidazolyl group not only recognizes the hydroxy groups
by distance (spatial specificity), but also differentiates hy-
droxy groups located in a clockwise–counterclockwise rela-
tionship (orientation specificity). Similar recognition (“vec-
torial recognition”) was also performed by the carboxyl group
of a cysteine unit flexibly linked to g-CD.[12]
A plausible mechanism for the sulfonyl transfer may
include the sulfonylation of the imidazolyl group, which
delivers the sulfonyl group to the 6E-OH group of 1b to effect
a regioselective 1,22 remote functionalization, although two
other hydroxy groups (6D-OH and 2E-OH) are also situated
apart from the imidazolyl moiety by the same number of
single bonds as the 6E-OH group is.[13] The specificity of the
reaction may stem from the intrinsic structure of the
imidazolyl CDs. It is documented that an imidazolyl group
attached to a methylene carbon atom of a CD induces strong
shielding effects on the first subunit on the anomeric side
while it does not significantly influence the other side.[14]
These literature data together with the CPK model indicate
that in the preferential conformation around the C5 and
C6 atoms of the imidazolylglucoside residue, the imidazolyl
group is gauche to O5 and anti to C4 (Figure 1). This
structural information suggests that the sulfonyl group should
be delivered along the anomeric side of the 6-imidazolylglu-
coside residue but never along the opposite side. The possible
binding of the substrate in the CD cavity may also play an
important role because the sulfonylation did not occur when
the reaction medium was switched from water to DMF, which
disfavors the hydrophobic binding.
Figure 1. a) The preferential conformation around the C5 and
C6 atoms of the imidazolylglucoside residue suggested by the CPK
model and NMR spectroscopy. The imidazolyl group is gauche to O5
and anti to C4. b) The structure of the possible reaction intermediate
of 1b generated by energy minimization using an MM2 force field.
Only the 6E-OH group is located near the sulfonyl S atom on the rear
side of the imidazolyl N atom; S yellow, N blue, O red, C gray, H white.
treated with imidazole to give the corresponding 6A-imida-
zolyl-6X-iodo-b-CDs 4.[15] Reactions of 4b, 4c, 4 f, and 4g with
mesitylenesulfonyl chloride were undertaken under similar
conditions to that used for 1 (Table 1). In each case, one
product was recognized and isolated in moderate to good
yield and unreacted 4 could be recovered. Because it was
difficult to separate 4d from 4e, they were used as a mixture
in the sulfonylation. In this case, only 4d reacted to generate
one sulfonate. The sulfonyl group is attached to the 6E-OH
group with no exception (Schemes 5S–8S and Figures 5S–7S
in the Supporting Information). When the 6E-OH group is
modified (4e), the sulfonylation does not occur.
Sequencing macrocyclic molecules such as cyclic peptides
and cyclic carbohydrates requires chemical or enzymatic ring
opening and subsequent spectroscopic analysis.[16] Structural
characterization of the intact cyclic molecules is extremely
difficult, even with the current state-of-the-art MS methods,
because the multiple and indiscriminate ring-opening clea-
vages occur to generate superimposing random ions, thus
making the interpretation difficult.[17] We have succeeded in
straightforward sequencing of the hetero-trifunctional CDs
by MALDI-PSD MS (PSD = post source decay). Compounds
5b and 5g are taken to illustrate the novel strategy. They were
converted into the corresponding di(3,6-anhydro) species 6b
and 6g, respectively, before PSD measurements in order to
avoid the complexity caused by any side-chain cleavage. Both
6b and 6g fragmented and preferentially generated a series of
On the basis of this enzyme-like catalysis, we developed
an efficient methodology for the hetero-trifunctionalization
of CDs. The 6A,6X-diiodo-b-CDs (X = B, C, or D) were
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Table 1: Selective sulfonylations of bifunctional CDs 4.[a]
Reactant
Position of I
Product
Yield [%]
Recovery of 4 [%]
Figure 2. MALDI-PSD mass spectra of 6b and 6g (left column), and
the expected fragments relating the imidazolyl tri-, tetra-, and penta-
saccharide segments of all the dianhydro-b-CDs that can be derived
from 4b and 4g (right column). The expected relative abundance was
deduced on an assumption of indiscriminate cleavage at any two
glycosidic linkages. The capital letters denote the locations of the
anhydro structure. The symbols * and ” denote the presence and
absence, respectively, of fragments that are important for differentia-
tion of the regioisomers.
4b
Rb
5b
5c
57
22
28
40
42
–
38
4c
Rc
4d+4e[b]
Rd +Re
Rf
5d[c]
5 f
35 (mainly 4e)
31
46
4 f
4g
Rg
5g
[a] One of the Rb–Rg groups is iodide, all others are OH. [b] A mixture was
used. [c] Only one product was formed.
pound 6g demonstrated a fragmentation pattern that is
in good agreement with that calculated for the EG
dianhydro species, which is characterized by the forma-
tion of the fragment [MHÀ2(anhydroGlc)ÀGlc]+ at
m/z 699 and the absence of the fragments [MHÀ2(an-
hydroGlc)]+ (m/z 861) and [MHÀ4Glc]+ (m/z 501).
These observations indicated that both 6b and 6g, and
therefore 5b and 5g, should have a modified E residue.
The assignment was confirmed by the routine method
employing enzymatic ring opening and subsequent MS
analysis.
The regiospecific reaction, which mimics well the
acyl transfer process of proteases, not only provides very
important information for the design of more sophisti-
cated artificial enzymes and receptors but also presents
an excellent example of controlling efficiently the reaction
selectivity by the combination of covalent and noncovalent
weakinteractions. The new concept described herein for the
regiospecific hetero-bifunctionalization or -trifunctionaliza-
tion of CDs may lead to a breakthrough in the selective
modification of CDs. The first successful straightforward
sequencing of the CD derivatives by mass spectrometry
should be of significance in the structural characterization of
cyclic carbohydrates, and it is expected to add considerable
impact to the structural analyses of a wide range of biological
and synthetic macrocyclic molecules.
PSD ions containing the imidazolyl group with considerable
abundance (Figure 2, left). For each compound, a library of
the calculated fragmentation patterns, consisting of five
di(3,6-anhydro)-b-CD candidates that can be derived from
the precursor molecule 4b or 4g, was built by assuming that
the cleavage occurs randomly at any two glycosidic linkages
(Figure 2, right; the capital letters denote the locations of the
anhydro moieties). Close resemblance was recognized
between the PSD spectrum of 6b and the expected spectrum
of the BE dianhydro species, both characterized by the
formation of the fragment [MHÀ2(anhydroGlc)À2Glc]+
(m/z 537) and the lackof the fragments [ MHÀ2(anhy-
droGlc)ÀGlc]+ (m/z 699) and [MHÀ3Glc]+ (m/z 663). Com-
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General procedure for the sulfonylation of imidazolyl CDs: A
solution of mesitylenesulfonyl chloride in DMF (0.6m, 4 mL) was
added to a pH 8.0 phosphate buffer solution (1m, 8 mL) containing an
imidazolyl CD (10 mm), and the resulting mixture was stirred at room
temperature. When the pH dropped below 7, the reaction mixture
was subjected to chromatography on a reversed-phase Lobar column
(Rp-18, eluted with a gradient composed of water and ethanol) to
afford the monosulfonate and the unreacted imidazolyl CD. All
sulfonate products were characterized with MS and NMR spectros-
copy and sequenced by MALDI-PSD mass spectrometry and/or by
the combination of enzymatic ring-opening reactions and MS
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[11] The sugar units are numbered with the capital letters in the
clockwise direction by starting from the imidazolylglucoside
unit.
Received: March 16, 2007
Published online: May 16, 2007
Keywords: biomimetic synthesis · cyclodextrins ·
.
host–guest systems · mass spectrometry · selectivity
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