Hetero-bifunctional γ-cyclodextrins having dansylcysteine and tosyl groups at two adjacent sugar units: synthesis and determination of regio-chemistry

Article abstract of DOI:10.1016/j.tetlet.2007.03.003

Ditosylation of two adjacent 6-positions of γ-cyclodextrin, subsequent mono-substitution with l-cysteine and final condensation with dansyl chloride afford the title compounds whose clockwise and counterclockwise isomers are separated from each other and

Full text of DOI:10.1016/j.tetlet.2007.03.003

Tetrahedron Letters 48 (2007) 3267–3271
Hetero-bifunctional c-cyclodextrins having dansylcysteine and
tosyl groups at two adjacent sugar units: synthesis
and determination of regio-chemistry
Hua Yu,a,b Yuji Makino,a Makoto Fukudome,a Ru-Gang Xie,b
De-Qi Yuana,* and Kahee Fujitaa,*
aDepartment of Molecular Medicinal Sciences, Graduate School of Biomedical Sciences, Nagasaki University,
Bunkyo-machi 1-14, Nagasaki 852-8521, Japan
bSchool of Chemistry, Sichuan University, Chengdu 610064, Sichuan, China
Received 8 February 2007; revised 28 February 2007; accepted 1 March 2007
Available online 4 March 2007
Abstract—Ditosylation of two adjacent 6-positions of c-cyclodextrin, subsequent mono-substitution with L-cysteine and final con-
densation with dansyl chloride afford the title compounds whose clockwise and counterclockwise isomers are separated from each
other and their regio-chemistry is unambiguously determined by a new strategy based on the combination of enzyme degradation of
the hetero-bifunctional c-cyclodextrin to the corresponding disubstituted maltotriose and fragmentation analysis of the latter by
PSD-MS technique.
Ó 2007 Elsevier Ltd. All rights reserved.
Cyclodextrins (CDs) have been attracting worldwide
interests in various fields relating host–guest recogni-
tion.1 However, their fine functionalization is difficult
and presents the bottleneck for molecular design based
on them. The hetero-bifunctional CDs not only effect
a three point recognition of asymmetric species, but also
exercise strong asymmetric induction in catalyzing the
chemical transformations such as transamination reac-
tions.2 However, these hetero-bifunctional CDs are very
difficult to access. Methods for the preparation of
hetero-bifunctional CDs are extremely scarce although
some methodologies have become available for mono-
or mono-facial functionalization.3 For b-CD, several
methods have been reported for the hetero-bifunctional-
ization, including random stepwise substitution with
different reactants4 and selective introduction of two
different functional groups to two specific positions.5
For c-CD, which has a wider cavity and possesses quite
different binding properties from b-CD, the only
attempt to the introduction of two different groups
was made by Hamada who substituted one tosylate
group of 6A,6X-ditosyl-c-CDs with a chromophore.6 Ex-
cept for the C2 symmetrical 6A,6E-ditosyl-c-CD, each of
the other three ditosyl-c-CDs generates a pair of clock-
wise and counterclockwise isomers which are hard to
separate from each other, and the clockwise–counter-
clockwise isomeric mixtures were employed in molecular
sensing. To the best of our knowledge, structurally well-
characterized pure unsymmetrical hetero-bifunctional c-
CDs have not yet been reported. Herein, we describe the
syntheses of pure hetero-bifunctional c-CDs bearing a
tosyl and a N-dansyl-L-cysteine residues at the two
adjacent 6-positions and the application of MALDI-
TOFMS in the determination of regio-chemistry. These
bifunctional c-CDs may serve as important intermedi-
ates for further functionalization.
The synthetic approach is depicted in Scheme 1. 6A,6B-
Ditosylate 1 of c-CD was synthesized by reacting c-CD
with tosyl chloride in pyridine7 and separated from the
other regio-isomers by chromatography on a preparative
ODS-column. Treatment of ditosylate 1 with L-cysteine
in DMF/H2O alkaline solution gave 6B-cysteine-6A-
tosyl-c-CDs (2) and its counterclockwise regio-isomer 3
Keywords: Cyclodextrin; Modification; Sulfonylation; MALDI-
TOFMS.
*
Corresponding authors. E-mail addresses: deqiyuan@nagasaki-u.
ac.jp; fujita@nagasaki-u.ac.jp
0040-4039/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2007.03.003

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H. Yu et al. / Tetrahedron Letters 48 (2007) 3267–3271
OH
O S
3
O S
3
OH
TsCl
O
O
O
A
O
L-cysteine
Pyridine
O
B
O
O
O
HO
OH
8
HO
OH HO
OH
HO
OH
6
1
( + other isomers )
HO C
2
HO C
2
NH
2
NH
H
2
H
S
S
O S
3
O S
3
OH
OH
O
O
A
O
O
O
A
O
O
O
B
O
B
O
O
O
+
HO
OH HO
OH
HO
OH HO
OH
HO
OH
HO
OH
6
6
2
3
Dansyl chloride
N
N
O
S
O
O
S
HO C
2
NH
H
HO C
2
O
NH
S
O S
3
S
OH
O S
3
H
OH
O
O
A
O
O
O
O
+
B
O
O
A
O
O
O
B
O
HO
OH HO
OH
HO
OH
HO
OH HO
OH
HO
OH
6
6
4
5
H
O
H
NH
O
S
O
S
O
O
N
H
O
N
S
O
S
O
A
A
N
B
B
endo-topology
exo-topology
Scheme 1.
as an unseparable mixture. Sulfonylation of the amino
group with dansyl chloride in alkali aqueous CH3CN
solution and subsequent chromatography on a reversed-
phase column allowed the separation of the two regio-iso-
mers 4 (with shorter retention time) and 5 (with longer
retention time) in 36% and 25% yields, respectively.
2.31 ppm, respectively, in integration ratios of 8/6/3.
In the aromatic region, 6 doublets were recognized at
d 8.46 (d, 3J = 8.52 Hz, 1H), 8.22 (d, 3J = 8.52 Hz,
3
3
2H), 8.15 (d, J = 7.14 Hz, 1H), 7.76 (d, J = 8.32 Hz,
3
3
2H), 7.43 (d, J = 8.32 Hz, 1H), 7.22 (d, J = 7.42 Hz,
1H) and two triplets overlapped around d 7.55 (2H).
In addition to the basic chemical shift pattern of cyclo-
dextrin,8 the 13C NMR spectrum of compound 4 dem-
onstrated 14 peaks for the aromatic carbons (d 110–
160 ppm), and the carboxylic carbon, a- and b-carbons
of cysteine moiety, and the methyl carbons of dansyl
and tosyl groups were observed at d 170.9, 57.5, 37.6,
45.0 and 21.0 ppm, respectively. A sugar carbon reso-
Hetero-bifunctional c-CDs 4 and 5 share the molecular
ions at m/z 1809 ([M+H]+) and 1831 ([M+Na]+) in their
TOFMS spectra and displayed similar NMR spectra.
The 1H NMR spectrum of 4 demonstrated the anomeric
protons of c-CD, methyl protons of dansyl and methyl
protons of tosyl moieties at ca. d 4.87, 2.85 and

H. Yu et al. / Tetrahedron Letters 48 (2007) 3267–3271
3269
nates at very high field (d 33.8), which can be assigned to
the thio-substituted C6. These observations are consis-
tent with the 1/1/1 ratio of c-CD/tosyl/dansyl-L-cys-
teine. Similar results were also obtained with
compound 5. However, the NMR spectra are powerless
towards the regio-chemistry of hetero-bifunctional c-
CDs 4 and 5.
by TOFMS which displayed the pseudo-molecular ion
at m/z 1019 (M+Na). The PSD-MS spectrum of 7 was
very simple (Fig. 1). Apart from the mother peak at
m/z 1019, only three fragment ions were observed at
m/z 847, 520 and 350 as the sodium adducts. The former
two fragment ions were generated by the one-site cleav-
ages of type-a and type-b, respectively, while the latter
one at m/z 350 in much lower abundance corresponds
to the two-site cleavage of both type-a and type-b.12
The type-b cleavage strongly evidences the sugar se-
quence of 7 and therefore its precursor 4. Based on this
assignment, the other isomer 5 should take the reversed
arrangement for the two functional groups.
The only method available for the determination of the
regio-chemistry of hetero-bifunctional CD derivatives
includes enzymatic conversion of the macrocycle to lin-
ear oligosaccharide and fragmentation analysis of the
latter by EI-MS.9 However, the EI-MS measurement
requires the acylation of all the hydroxyl groups of the
oligosaccharides, which greatly increases the molecular
weight of the analytes and makes fragmentations very
complicated and difficult to define in some cases. Since
TOFMS is powerful in sequencing bio-macromole-
cules,10 we attempted to apply it in the structural deter-
mination of disubstituted CD derivatives. Combination
of the amylase-promoted degradation of 4 and the post-
source decay (PSD) TOFMS measurement11 ensures a
convenient and unambiguous assignment of the struc-
ture of 4 (Scheme 2). Compound 4 was decomposed to
maltotriose 6 by the enzyme action of a-amylase and
the latter, though good for PSD measurements at this
stage, was reduced to 7 in order to remove the anomer
equilibrium and facilitate the NMR analysis. The
reduced triose structure of compound 7 was confirmed
Because it is the first attempt to apply PSD-MS in the
determination of the regio-chemistry of disubstituted
CDs, the reliability of the above assignment is recon-
firmed independently by NMR analysis of compound
7. Thanks to the effect of the substituents, the reso-
nances spread out widely both in the 1H and 13C
NMR spectra of 7, and all the signals relating the two
substituted glucose residues can be assigned with the
aid of 2D NMR measurements (Fig. 2). Both the C3
(d 83.9 ppm) of the glucitol residue, which is linked to
the middle glucose residue by an oxygen atom, and the
C4 (d 79.5 ppm) of the tosylglucoside appeared in the
normal region (around d 80 ppm) of the C4 carbons of
CD derivatives. However, the C4 of the dansylcys-
teine-modified glucose residue demonstrated ca. 8 ppm
N
N
O
S
NH
H
a
O
O
S
NH
H
HO C
2
O
HO C
2
S
O S
3
OH
O S
3
NaBH
α-amylase
S
4
OH
OH
4
O
O
O
A
OH
HO
HO
O
O
B
O
A
O
HO
O
O
OH
B
HO
OH
OH HO
OH
HO
OH
HO
OH HO
OH
6
7
b
Scheme 2. Amylase-promoted degradation of the hetero-bifunctional c-CD 4 and the fragmentation patterns of 7 observed in the PSD-MS spectrum.
Figure 1. Post-source decay mass (PSD-MS) spectrum of compound 7.

3270
H. Yu et al. / Tetrahedron Letters 48 (2007) 3267–3271
Figure 2. The 1H, 13C, and HMBC NMR spectra of triose 7 in DMSO-d6 (only the sugar part is shown). The inset highlights the important
information employed in the sequence determination.
upfield shift, and resonated at d 71.8 ppm, a chemical
shift within the typical range of the hydroxyl-substituted
secondary carbons of saccharides. These observations
imply that compound 7 should have the dansylcys-
teine-modified glucose as the terminal residue located
at the non-reducing end, which is in consistence with
the result of PSD-MS measurement. Heteronuclear mul-
tiple bond correlation (HMBC13) experiments were fur-
ther carried out to confirm the saccharide sequence as it
can provide long range H–C hetero-nuclei correlations
between the O-bridged positions of two adjacent sugar
residues. As shown in Figure 2, the C4 of the tosylglu-
cose (d 79.5 ppm) strongly correlated to the anomeric
H1 proton of dansylcysteine-glucose (d 4.63 ppm) while
its own H1 (d 4.79 ppm) coupled with the C3 of the gluc-
itol residue that is generated by reduction of the glucose
unit at the reducing end of 6. Since these signals are all
clearly separated from each other and do not overlap
with any other ones, the observed multiple bond C–H
correlations clarify unambiguously the sugar sequence
of 7 and strongly support the results of PSD-MS.
and 5 was undertaken to provide an efficient approach
to strictly control the topology of the functional CDs.14
Experimental
1H and 13C NMR spectra were determined with a Varian
500 MHz NMR spectrometer by setting the frequency at
1
500 MHz for H spectra and at 125 MHz for 13C spec-
tra. MALDI-TOFMS spectra (positive) were recorded
on Applied Biosystems Voyager System 6336 spectrom-
eter using 2,5-dihydroxybenzoic acid as a matrix for
reflector mode and a-cyano-2-hydroxycinnamic acid as
a matrix for PSD mode.
Preparation of hetero-bifunctional c-CDs 4 and 5
L-Cysteine hydrogen chloride (27 mg) and 6A,6B-ditosyl-
c-CD7 (620 mg) were dissolved in DMF (3 mL), and a
0.58 M aqueous Na2CO3 solution (10 mL) was added.
After being stirred at rt for 2 h, the reaction mixture
was neutralized, diluted with water, membrane filtered
and chromatographed on a reversed-phase Lobar col-
umn (Rp-18, size C). Elution of the column with a gra-
dient from water to 25% aqueous ethanol solution (a
total 2 L) afforded 2 and 3 as an unseparable mixture
(135 mg, 22.4 %) together with the recovery of the unre-
acted ditosylate (400 mg, 64.5%). TOFMS: m/z 1554
(M+H), 1576 (M+Na). The mixture of 2 and 3
(207 mg) was dissolved in 0.08 M aqueous NaHCO3
solution (10 mL), and an acetonitrile solution (10 mL)
containing dansyl chloride (36.2 mg) was added. The
In summary, we have described for the first time the syn-
theses of isomerically pure hetero-bifunctional c-CDs. A
new method is established to determine the regio-chem-
istry of hetero-bifunctional CDs by the combination of
enzyme-promoted degradation reaction and PSD-MS
measurement, which is thought to be powerful and gen-
erally applicable in the structural determination of CD
derivatives bearing two or more substituents. Hetero-
bifunctional CDs 4 and 5 may serve as important inter-
mediates for further functionalization. As an on-going
project, the intramolecular displacement reaction of 4

H. Yu et al. / Tetrahedron Letters 48 (2007) 3267–3271
3271
mixture was stirred at rt for 30 min, and then it was neu-
tralized, concentrated, precipitated with acetone and fil-
tered. The precipitates were collected, dissolved in water
and chromatographed (Lobar column, Rp-18, size C)
after membrane filtration. Elution of the column with
water (1 L) and a gradient from water to 30% aqueous
ethanol solution (a total 2 L) enabled the separation of
4 and 5.
J = 12.60, 4.35 Hz, 1H; cys-b), 2.36 (s, 6H; Ts-CH3),
2.27 ppm (dd, J = 14.20, 7.10 Hz, 1H; 6B). 13C NMR
(DMSO-d6, TMS int.): d 170.9 (COOH), 151.2, 144.6,
135.9, 132.2, 129.9, 129.2, 129.0, 127.8, 127.7, 127.6,
123.3, 119.1, and 115.0 (Ar); 100.9 (1B), 100.2 (1A),
83.9 (3glucitol), 79.5 (4A), 72.9 (3B), 72.5 (2B), 72.3 (5B),
71.8 (4B), 71.4 (2A), 71.2, 70.1 (3A), 69.9 (6A), 68.0
(5A), 62.6, and 61.9 (1 and 6glucitol), 57.2 (cys-a), 45.0
(NCH3), 36.5 (cys-b), 33.6 (6B), 21.0 (Ts-CH3) ppm.
Compound 4: eluted faster, 86.3 mg, 36%. TOFMS: m/z
1809 ([M+Na]+). 13C NMR (DMSO-d6, TMS int.): d
170.9, 151.1, 144.7, 136.3, 132.3, 130.0, 129.1, 128.9,
127.7, 127.5, 127.4, 123.1 119.1, 114.8, 101.6, 101.5,
101.3, 84.1, 81.4, 80.9, 80.8, 80.6, 80.3, 72.8, 72.6, 72.4,
72.2, 72.0, 71.3 69.5, 68.8, 59.8, 57.5, 45.0, 37.6, 33.8,
21.0 ppm. Compound 5: eluted slower, 60 mg, 25%.
TOFMS: m/z 1809 ([M+Na]+). 13C NMR (DMSO-d6,
TMS int.): d 169.3, 151.2, 145.4, 137.7, 129.5, 129.0,
128.9, 128.3, 128.0, 127.8, 125.5, 123.4, 119.3, 114.9,
102.4, 101.7, 101.2, 86.2, 82.1, 81.1, 81.0, 80.7, 80.4,
73.2, 72.9, 72.8, 72.7, 72.6, 72.5, 72.3, 72.2, 72.0, 71.9,
70.1, 69.9, 60.0, 59.8, 59.4, 57.9, 44.9, 33.8, 33.1,
20.7 ppm.
Acknowledgement
We are indebted to Japan Maize Products Co. Ltd. for
the generous gift of b-CD.
References and notes
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Preparation and reduction of maltotriose 6
A mixture of 2 (100 mg) and a-amylase (EC 3.2.1.1 from
Aspergillus oryzae, Sigma) (100 mg) in 0.1 M acetate
buffer (pH 5.5, 10 mL) containing 0.02 M CaCl2 was
allowed to stand for 3 days at 40 °C. After being
neutralized with NaOH solution and heated for 10 min
in a boiling water-bath, the mixture was membrane-
filtered (cellulose acetate, 0.8 lm), diluted to 250 mL
with water and chromatographed on a reversed-phase
Lobar column (Rp-18, size C, eluted with a gradient
of 20–50% aqueous EtOH) to afford a major product
6 (29 mg, 52%). TOFMS: m/z 1017 ([M+Na]+).
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Compound
6 (30 mg) and NaBH4 (30 mg) were
dissolved in water (3 mL) and the resultant mixture
was stirred at rt for 1 h. The reaction solution was then
subjected to dilution with 25% ethanol, membrane filtra-
tion and chromatography on a reversed-phase Lobar
column (Rp-18, size C). Elution of the column with
water (1 L) and a gradient of 10–30% aqueous ethanol
(a total 2 L) afforded compound 7 (23 mg, 76.5%).
TOFMS: m/z 1019 ([M+Na]+). H NMR (DMSO-d6,
1
TMS int.): d 8.45 (d, 3J = 8.47 Hz, 1H), 8.25 (d,
3J = 8.70 Hz, 1H), 8.20 (d, J = 7.10 Hz, 1H), 7.74 (d,
3
3J = 8.30 Hz, 2H), 7.61 (m, 2H), 7.40 (d, J = 8.30 Hz,
3
3
2H), and 7.25 (d, J = 7.10 Hz, 1H) (Ar–H); 5.65 (m,
2H), 5.43 (d, 3J = 6.87 Hz, 1H), ca. 5.4 (v br, 1H),
3.25 (br, 1H), 4.97 (d, 3J = 4.81 Hz, 1H), 4.56 (d,
3J = 3.89 Hz, 1H), and 4.52 (m, 2H) (OH); 4.79 (d,
3J = 3.66 Hz, 1H; 1A), 4.63 (d, J = 3.66 Hz, 1H; 1B),
3
0
11. Spengler, B. J. Mass Spectrom. 1997, 32, 1019–1036.
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4.44 (dd, J = 10.86, 3.10 Hz, 1H; 6A ), 4.18 (d,
J = 9.39 Hz, 1H; 6A), 4.04 (m, 1H; 5A), 3.64–3.57 (m,
4H; 3A, 3glucitol, 2H of glucitol), 3.45–3.32 (m, 7H; 5B,
cys-a, 5H of glucitol), 3.29–3.12 (m, 4H; 3B, 4A, 2A,
2B), 2.96 (t, 3J = 9.38 Hz, 1H; 4B), 2.83 (s, 6H;
14. Yu, H.; Yuan, D.-Q.; Makino, Y.; Fukudome, M.; Xie,
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B0
N(CH3)2), 2.81–2.77 (m, 2H; cys-b0, 6 ), 2.72 (dd,