Synthesis of 2,3-O-dibenzyl-6-O-sulfobutyl-α and β cyclodextrins: new chiral surfactants for capillary electrophoresis

Article abstract of DOI:10.1016/j.tetlet.2015.05.057

Abstract Amphiphilic sodium hexakis(2,3-O-dibenzyl-6-O-sulfobutyl)cyclomaltohexaose and sodium heptakis(2,3-O-dibenzyl-6-O-sulfobutyl)cyclomaltoheptaose were synthesized in four steps. Pyrene fluorescence studies indicate micelle formation at 90 μM in water for both modified cyclodextrins (CDs). Both CDs offer potential as chiral micellar selectors for enantiomeric separations using capillary electrophoresis. Sodium heptakis(2,3-O-dibenzyl-6-O-sulfobutyl)cyclomaltoheptaose baseline resolved fluorescent cyanobenzylindole (CBI) derivatives of d/l-serine at concentrations of 50-200 μM.

Full text of DOI:10.1016/j.tetlet.2015.05.057

Accepted Manuscript
Synthesis of 2,3-dibenzyl-6-sulfobutyl-α and β cyclodextrins: new chiral sur-
factants for capillary electrophoresis
James A. McKee, Thomas K. Green
PII:
S0040-4039(15)00866-7
DOI:
http://dx.doi.org/10.1016/j.tetlet.2015.05.057
TETL 46329
Reference:
Tetrahedron Letters
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13 May 2015
18 May 2015
Please cite this article as: McKee, J.A., Green, T.K., Synthesis of 2,3-dibenzyl-6-sulfobutyl-α and β cyclodextrins:
new chiral surfactants for capillary electrophoresis, Tetrahedron Letters (2015), doi: http://dx.doi.org/10.1016/
j.tetlet.2015.05.057
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James A. McKee, Thomas K. Green
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1
Tetrahedron Letters
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Synthesis of 2,3-dibenzyl-6-sulfobutyl-α and β cyclodextrins: new chiral
surfactants for capillary electrophoresis
James A. McKee a,*, Thomas K. Greena,b,*
aDepartment of Chemistry and Biochemistry, University of Alaska Fairbanks, Fairbanks, AK, 99775-5940
bInstitute of Arctic Biology, University of Alaska Fairbanks, Fairbanks, AK, 99775-5940
ARTICLE INFO
ABSTRACT
Amphiphilic sodium hexakis (2,3-O-dibenzyl-6-O-sulfobutyl) cyclomaltohexaose and
sodium heptakis (2,3-O-dibenzyl-6-O-sulfobutyl) cyclomaltoheptaose were synthesized in
four steps. Pyrene fluorescence studies indicate micelle formation at 90 µM in water for
both modified cyclodextrins (CDs). Both CDs offer potential as chiral micellar selectors
for enantiomeric separations using capillary electrophoresis. Sodium heptakis (2,3-O-
dibenzyl-6-O-sulfobutyl ) cyclomaltoheptaose baseline resolved fluorescent
Article history:
Received
Received in revised form
Accepted
Available online
Keywords:
cyclodextrin
cyanobenzylindole (CBI) derivatives of D/L-serine at concentrations of 50-200 µM.
micelle
enantioseparation
capillary electrophoresis
2015 Elsevier Ltd. All rights reserved
.
Introduction
synthesize sodium hexakis (2,3-O-dibenzyl-6-O-sulfobutyl)
cyclomaltohexaose (5a, Scheme 1) and sodium heptakis (2,3-O-
dibenzyl-6-O-sulfobutyl) cyclomaltoheptaose (5b, Scheme 1) for this
purpose. These cyclodextrin derivatives present 12 and 14 aromatic
moieties, respectively, on one side of the cyclodextrin while 6 and 7
anionic sulfobutylethers, respectively, protrude from the other side of
the molecule. The synthesis is described here and, as shown below,
both CD derivatives form micelles with critical micelle
Cyclodextrins (CDs) are chiral oligosaccharide macrocycles
composed of 6, 7, and 8 α-1, 4-D-glucopyranose units classified as
α-, β-, and γ-cyclodextrin, respectively. CDs command great interest
not only because of complexation ability of the native macrocycle,
but due to possession of two secondary hydroxyls and one primary
hydroxyl per glucopyranose unit. Each face of the pocket opening
can be tailored with different moieties. Synthetic modification
strategies are varied and have been well documented.1-3
concentrations (CMCs) of ~ 90 µM.
Undersubstitution of these CD hydroxyls is common due to the harsh
conditions typically required for full substitution and the extensive
purification necessary for isolation of single cyclodextrin isomers.4
Selectively modified cyclodextrins that are undersubstituted are
characterized by an average degree of substitution.
The ability of 5b to act as a chiral selector in capillary
electrophoresis is demonstrated with a fluorescent derivative of D/L-
serine. D/L-serine was a chosen as a test analyte because of the
biological significance of D-serine as a neuromodulator16 and the
challenge it presents in chiral separation among CBI-D/L-amino
acids.17 Specifically, the D/L-serine is derivatized with naphthalene-
2,3-dicarboxaldehyde and cyanide under basic conditions18 to form a
fluorescent cyanobenz[f]isoindole -D/L-serine (CBI-D/L-serine)
derivative. We demonstrate that chiral resolution is achieved with
50-200 µM of 5b in the background electrolyte.
CDs and modified CDs have been used extensively as chiral
selectors in capillary zone electrophoresis. In micellar electrokinetic
chromatography, the buffer contains a micelle-forming surfactant as
a pseudo-stationary phase.5 The analytes differentially partition into
the micelle and are separated. For chiral separation, a micelle-
forming surfactant such as sodium dodecyl sulfate is typically used
with the chiral cyclodextrin added to establish a secondary
equilibrium with the micelle. Sometimes, a single chiral surfactant
such as a bile salt is employed.6
Results and Discussion
Sodium hexakis (2,3-O-dibenzyl-6-O-sulfobutyl) cyclomalto-
hexaose (5a) and sodium heptakis (2,3-O-dibenzyl-6-O-sulfobutyl)
cyclomaltoheptaose (5b) were synthesized according to Scheme 1.
The primary hydroxyl groups were readily protected using standard
procedures.2 tert-Butylammonium iodide (TBAI) in catalytic
amounts has been used to quantitatively benzylate sterically hindered
hydroxyls on a glucose derivatives at room temperature in the
presence of excess sodium hydride in tetrahydrofuran (THF).19 This
Our interest has been in developing micelle-forming amphiphilic
cyclodextrins that can be used as chiral surfactants in capillary
electrophoresis. A number of amphiphilic CDs have been previously
synthesized, including both ionic and nonionic CDs.7-15 As far as we
are aware, amphiphilic CDs which form micelles have not been used
in capillary electrophoresis studies. The intention of this work was to

2
Tetrahedron
concept was adapted to perbenzylate the secondary hydroxyls of
cyclodextrins by using 0.01 equivalents of TBAI, two equivalents of
benzyl bromide, excess sodium hydride in THF and heating the
reaction to reflux for two days. Larger amounts of benzyl bromide
were required for complete benzylation if the reaction was performed
at room temperature. The addition of TBAI was found to be critical
for the synthesis of both 3a and 3b; benzylation in its absence,
regardless of amount of benzyl bromide or time allotted, was found
to produce only undersubstituted benzyl derivatives as determined by
NMR spectroscopy. We hypothesize that the requirement of TBAI is
the catalytic in situ formation of benzyl iodide, with its better leaving
group. Ammonium fluoride was chosen over the more traditional
tetrabutylammonium fluoride as a deprotection agent due to ease of
workup; 4a and 4b are soluble in chloroform whereas ammonium
fluoride is not, thus eliminating the need for column chromatography
of 4a and 4b.
environment. 24 The values of the I1/I3 ratio were used to estimate the
hydrophobicity of the aggregate microenvironment using an
empirical scale of the relative band intensities of pyrene in different
solvents.25 Initial I1/I3 values at the CMC in Figure 1 indicate a
methanol/methylene chloride-like environment inside the aggregates
but with the local polarity approaching benzyl alcohol as the
concentration of amphiphile increases.25 This apparent change in the
hydrophobicity of the interior with concentration past the CMC may
be due to activity effects and/or greater exclusion of water from the
interior as concentration of monomer increases. The CMCs of both
5a and 5b are determined to be approximately 90 µM.
Figure 1. Determination of CMC of 5a using pyrene fluorescent molecular
2 µM.
Scheme 1: Synthesis of amphiphilic cyclodextrin derivatives (n = 6,7)
probe. CMC = 90
Complete sulfoalkylation of primary hydroxyls in tetrahydrofuran
(THF) has been shown previously to be problematic.20 Kirschner and
Green were able to synthesize single isomer 2,3-dialkyl-6-
sulfoalkylated cyclodextrin derivatives using a 3-fold molar excess
of 18-crown-6 ether in addition to alkanesultone and potassium
hydride in THF.20 18-crown-6 ether, by complexing with potassium
counterion, helps to solubilize the increasing anionic CD. This
procedure, while successful, requires addition of large amounts of
18-crown-6 ether, followed by ion-exchange chromatography and
numerous extractions for its removal. We find that addition of 1,4
butanesultone (3 eq per hydroxyl), and excess sodium hydride in dry
DMF, with mild heating, allows for complete sulfobutylation to 5a
and 5b. The products were conveniently purified by ultrafiltration.
The products were fully characterized by 1H, 13C, COSY, and
HMQC NMR spectroscopy as well as ESI-MS.
We found that 5b was effective as a chiral selector in capillary
electrophoresis studies of fluorescent CBI-derivatives of D,L amino
acids. Electrophoresis was performed in reverse polarity mode
(anode on detector side) at -25 kV with low pH (2.00) phosphate
buffer. Under this condition, electroosmotic flow is minimized and
highly charged anionic 5b migrates toward the detector. CBI-amino
acids may interact through their hydrophobic naphthalene group to
form inclusion complexes in the CD cavity and/or through
interaction with the benzyl groups of the secondary rim of the CD.
CBI-amino acids can also interact through H-bonding with the CD.
Strong complex formation should result in sweeping of the CBI-
amino acids toward the detector.
Average migration times of fluorescent CBI-D/L-serine pair were
observed to decrease with increasing concentration of 5b. This result
is expected since, as concentration of 5b increases, the equilibrium
shifts toward complex and, given that the complex is negatively
charged (-7), the CBI-D-serine is driven toward the detector more
rapidly. Apparent electrophoretic mobilities (average of CBI-D/L-
serine pair), µi, were calculated from the migration times according
to the equation26
Pyrene fluorescence has been used to determine CMCs of calixarene-
based surfactants.21 We used a modified procedure from Lopez-Diaz
et al.22 to determine the CMCs of 5a and 5b. Pyrene solution in
methanol (1.25 µL of 2 mM) was pipetted into vials and blown dry
with nitrogen. Aqueous solutions of 5a or 5b (2.5 mL) ranging from
5 µM to 5 mL were subsequently added and stirred resulting in a
pyrene concentration of 1 µM. The emission spectrum of pyrene
was obtained using an excitation wavelength of 320 nm. The
emission range was set between 350 and 450 nm. Emission
intensities were recorded at 373 nm for 1st vibration peak (I1) and
384 nm for 3rd vibrational peak (I3).22
⁄
ꢁė
/
µi = d
=
(1)
Ĝ
Ġ
⁄
ꢂ ꢁħ
ꢀ
where ꢃ$ is electrophoretic velocity (cm/s), E is field strength
(V/cm), ͆ is length of capillary to the detector, ͆ is the total length,
ꢄ
is the migration time, and V is the applied voltage (V). Mobility
/
ͨ
(
depends on charge/size ratio. To obtain actual mobilities, apparent
mobilities should be corrected for both (1) electroosmotic flow and
(2) viscosity changes in the background electrolyte due to increasing
concentration of 5b. At pH 2.00, however, electroosmotic flow is
virtually abolished and is too small to measure. Also, we find that
viscosity changes are negligible over the concentration range of 5b
(0-1 mM). Thus the apparent mobilities are the actual mobilities in
this study.
The ratio of I1/I3 in the emission spectrum of pyrene changes in
response to the solvent polarity; the I1/I3 ratio is a reflection of the
local structure in the vicinity of the probe. A change in local dipole
moment indicates the equilibrium partitioning from an aqueous
environment to a more hydrophobic one.23 Hydrophobic molecules
(e.g. pyrene) have a greater affinity for the hydrophobic micellar
core than the hydrophilic bulk solution. A plot of the (I1/I3) ratio
against the log of surfactant concentration produces a sigmoidal
shaped curve. The CMCs of the amphiphilic cyclodextrin derivatives
were determined from the sharp changes in the slopes as pyrene
transitions from an increasing less polar to a micelle hydrophobic
Actual mobilities as a function of [5b] are shown in Figure 2.
Mobility of the analyte increases with [5b] as the equilibrium
concentration of charged complex increases (higher charge/size

3
ratio). Maximum mobility is reached at ~800 - 1000 µM of 5b,
which indicates nearly complete complexation of the CBI-serine pair
in this concentration range.
In contrast, we obtain baseline chiral resolution of CBI-D/L-serine
with 50 µM of 5b in the background electrolyte. The ability of 5b to
resolve CBI-D/L-serine enantiomers at such low concentrations must
be considered in the context of intermolecular interactions. First, it is
clear that the 5b has unusually high binding to the serine derivatives.
This is evident from Figure 2, where the average mobility reaches a
plateau at or near 800 µM of 5b, indicative of nearly complete
complexation with CBI-D/L-serine at this concentration. The
binding constant, K, assuming 1:1 interaction between 5b and
analyte, was determined by nonlinear curve fitting of the following
equation31
Chiral resolution (Rs) reaches a maximum of ~2 (baseline resolution
is 1.50) at concentrations of 50-200 µM of 5b with a migration times
of 6-9 min. An electropherogram using 100 µM of 5b is illustrated
in Figure 3. A number of other chiral CBI-amino acids were
baseline-resolved at equally low concentrations.
ͮ[Ėꢆʞꢇꢈʟ
ꢅ$ Ɣ [
(2)
ę
ͥͮꢆʞꢇꢈʟ
where ꢅ$ is the calculated mobility, ꢅꢉ is the saturated mobility, ꢅ! is
the mobility with no CD. Both K and ꢅꢉ are treated as adjustable
parameters in the fit.
The treatment yields a K of 5510 560 M-1 and ꢅꢉ of 4.89 0.11 x
10-4 cm2 V-1 s-1. In contrast, a similar treatment of the mobility curve
of randomly sulfated β-CD (average degree of sulfation of 9) yields a
K of 81 3 M-1 and ꢅꢉ of 3.91 0.05 x 10-4 cm2 V-1 s-1
(see
Supplementary Data). The much stronger binding of 5b to serine is
most probably due to its well-defined and extended hydrophobic
cavity, with its sulfobutyl groups on the primary face and the benzyl
groups on the secondary face. The randomly sulfated β-CD has
sulfate groups on both faces of the CD and lacks the extended
hydrophobic cavity.
Figure 2. Electrophoretic mobilities, µi (cm2 V-1 s-1) and chiral resolution
(Rs) of CBI-D/L-serine pairs versus concentration of 5b. Capillary
electrophoresis: 50 µm id, 68 cm total, 48 cm to detector, -25 kV, 25 mM
phosphate, pH 2.0, 410 nm LIF detection.
Wren and Rowe developed a model relating enantiomer mobility to
the concentration of CD chiral selector.32 Penn et al. extended the
treatment33 and showed that maximum mobility difference (related to
resolution) occurs when the CD concentration equals the inverse of
the average binding constant, K. For 5b, this corresponds to 180
µM, consistent with the maximum in Rs achieved at 50-200 µM
observed in Figure 2. For randomly sulfated β-CD, maximum
mobility difference is calculated to be 12 mM, consistent with that
optimized experimentally.17
Conclusion
In summary, we have synthesized, for the first time, amphiphilic 2,3-
dibenzyl-6-sulfobutyl-α and β-cyclodextrins, sodium salts (5a and
5b). Both molecules form micelles and exhibit CMCs of about 90
µM. 5b is shown to bind strongly to fluorescent CBI-D/L-serine
derivatives while providing chiral resolution at low concentrations.
These new CDs have potential as chiral surfactants in capillary
electrophoresis studies that employ fluorescence detection.
Figure 3. Resolution of CBI-D/L-serine using 100 µM of 5b. See Figure 2
for CE conditions.
Most chiral separations of fluorescently-tagged amino acids employ
a CD or modified CD in combination with a micelle-forming
surfactant such as sodium dodecyl sulfate.16 Typically, the CD is
neutral and the surfactant is negatively charged. The analyte
distributes between the CD, the micelle and the aqueous phase to
provide separation. In most circumstances, the CD and surfactant
are in used concentrations greater than 1 mM. For example, CBI-
D/L-serine enantiomers have been separated using 30 mM β-CD/60
mM chiral sodium taurocholate27 and 10 mM γ-CD/50 mM sodium
dodecylsulfate.28 Other CBI/-DL-amino acids have been separated
with hydroxylpropyl-β-CD/50 mM sodium dodecylsulfate, but this
mixture failed to resolve the serine enantiomers.29
Acknowledgements
This study made use of the NIH / NIGMS Biomedical Mass
Spectroscopy Resource at Washington University in St. Louis MO,
which is supported by National Institutes of Health \ National
Institute of General Medical Sciences Grant # 8P41GM103422
Supplementary Data
Supplementary data, including the experimental section, NMR,
MS and binding constant data, can be found in the online version
at
In some cases, the use of a surfactant is not required. Quan et al.
employed combination of chiral selectors in 20 mM hydroxylpropyl-
γ-CD/15% (w/v) D-(+)-glucose to provide resolution of the serine
enantiomers.30 We have optimized resolution using 10 mM
commercially-available randomly sulfated β-CD in reverse polarity
mode at low pH.17 All of these examples to point to the usual
requirement of high concentrations of cyclodextrin (mM range) in
the background electrolyte, either with or without surfactant.
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