Interactions of some modified mono- and bis-β-cyclodextrins with bovine serum albumin

Article abstract of DOI:10.1016/j.bmc.2005.08.002

Two mono-substituted β-cyclodextrins and two bridged bis-β-cyclodextrins, that is, mono(6-(2-aminoethylamino)-6-deoxy)-β- cyclodextrin (1), mono(6-(2-(2-aminoethylamino)ethylamino)-6-deoxy)-β- cyclodextrin (2), ethylene-1,2-diamino bis-6-(6-deoxy-β-cyclodextrin) (3), and iminodiethylene-2,2′-diamino bis-6-(6-deoxy-β-cyclodextrin) (4), were prepared from β-cyclodextrin. Their binding ability with bovine serum albumin as a model protein was investigated through proton magnetic resonance (¹H NMR), ultraviolet visible spectroscopy (UV-vis), circular dichroism (CD), and fluorescence spectroscopy. In the ¹H NMR spectra of the modified cyclodextrins, the resolution of proton signals decreases after the addition of BSA. From the UV and CD spectra, it is found that both the UV absorption and the α-helix content of BSA increase with the concentration of the modified cyclodextrins. The protein-ligand interactions cause a fluorescence quenching. The quenching constants are determined using the Stern-Volmer equation to provide an observation of the binding affinity between modified cyclodextrins and BSA. All these results indicate that the modified cyclodextrins can interact with BSA and the bridged bis(β-cyclodextrin)s (3 and 4) have much stronger interactions than the mono-substituted β-cyclodextrins (1 and 2). The strong binding stability of bis-cyclodextrins should be attributed to the cooperative effect of two adjacent cyclodextrin moieties. Job's plot shows that the complex stoichiometries of BSA to the modified cyclodextrins were 1:4 for 1 and 2, as well as 1:3 for 3 and 4, respectively.

Full text of DOI:10.1016/j.bmc.2005.08.002

Bioorganic & Medicinal Chemistry 14 (2006) 131–137
Interactions of some modified mono- and bis-b-cyclodextrins
with bovine serum albumin
Hui Gao, Yi-Nong Wang, Yun-Ge Fan and Jian-Biao Ma*
Key Laboratory of Functional Polymer Materials, Ministry of Education, Institute of Polymer Chemistry,
Nankai University, Tianjin 300071, China
Received 5 June 2005; revised 31 July 2005; accepted 1 August 2005
Available online 23 September 2005
Abstract—Two mono-substituted b-cyclodextrins and two bridged bis-b-cyclodextrins, that is, mono(6-(2-aminoethylamino)-6-de-
oxy)-b-cyclodextrin (1), mono(6-(2-(2-aminoethylamino)ethylamino)-6-deoxy)-b-cyclodextrin (2), ethylene-1,2-diamino bis-6-(6-de-
oxy-b-cyclodextrin) (3), and iminodiethylene-2,20-diamino bis-6-(6-deoxy-b-cyclodextrin) (4), were prepared from b-cyclodextrin.
Their binding ability with bovine serum albumin as a model protein was investigated through proton magnetic resonance (1H
1
NMR), ultraviolet visible spectroscopy (UV–vis), circular dichroism (CD), and fluorescence spectroscopy. In the H NMR spectra
of the modified cyclodextrins, the resolution of proton signals decreases after the addition of BSA. From the UV and CD spectra, it
is found that both the UV absorption and the a-helix content of BSA increase with the concentration of the modified cyclodextrins.
The protein–ligand interactions cause a fluorescence quenching. The quenching constants are determined using the Stern–Volmer
equation to provide an observation of the binding affinity between modified cyclodextrins and BSA. All these results indicate that
the modified cyclodextrins can interact with BSA and the bridged bis(b-cyclodextrin)s (3 and 4) have much stronger interactions
than the mono-substituted b-cyclodextrins (1 and 2). The strong binding stability of bis-cyclodextrins should be attributed to the
cooperative effect of two adjacent cyclodextrin moieties. JobÕs plot shows that the complex stoichiometries of BSA to the modified
cyclodextrins were 1:4 for 1 and 2, as well as 1:3 for 3 and 4, respectively.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
focused on amino acid and oligopeptide recognition by
CyDs and their derivatives.4–7 However, there have been
only a few studies due to about protein recognition by
CyDs or modified CyDs as ligands8,9 of the complexity
of protein secondary and ternary structures.10 Bovine
serum albumin (BSA), an easily obtained protein with
well-known properties, is often used as a model pro-
tein.11 Binding of several different categories of small
molecules to BSA has been studied using spectroscopy
techniques to elucidate the details of protein structures
and binding mechanisms.12–15
Numerous problems have to be solved before the prac-
tical use of therapeutic peptides and proteins on account
of their chemical and enzymatic instabilities, poor
absorption through biological membranes, rapid plasma
clearance, peculiar dose–response curves, and immuno-
genicity.1 Among the various inorganic and organic
compounds, cyclodextrins (CyDs) and their derivatives
may serve as carriers for therapeutically important pep-
tides, proteins, and oligonucleotides.2 CyDs are a class
of cyclic oligosaccharides with six to eight a-glucose
units linked by a-1,4-glucose bonds. They have well-
defined chemical structures with many sites for chemical
modification or conjugation, availability of different
cavity sizes, low toxicity, certain hydrophilicity, and
protection effect of the included/conjugated drugs from
biodegradation.2,3 In recent years, many researches were
Most proteins, including BSA, have hydrophobic resi-
dues, such as alkyl and aromatic groups, ionic groups,
such as –NH3 and –COOꢀ, hydrophilic polar groups
+
such as –OH and –NH2, etc. Those groups can act as
binding sites with ligand molecules that have similar
groups. In this study, –NH3 as the ionic group and
+
–NH2 as the polar group different from –OH on CyDs
molecules were introduced to b-cyclodextrin (b-CyD).
Additionally, two b-CyD molecules were coupled
together with an amino-containing spacer to obtain
some b-CyD derivatives with high binding ability to
protein. In our laboratory, two mono-substituted
Keywords: b-Cyclodextrin; Bovine serum albumin; Protein; Coopera-
tive interaction; Inclusion complex; Stoichiometry.
*
Corresponding author. Tel.: +86 22 23507746; fax: +86 22
23502749; e-mail: jbma@nankai.edu.cn
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.08.002

132
H. Gao et al. / Bioorg. Med. Chem. 14 (2006) 131–137
OH
D2O on the Varian UNITY-plus 400 spectrometer at
400 MHz.
OH
O
OH
2.2.2. Preparation of bis-b-CyD derivatives 3 and 4. Amino
b-CyD derivatives 1 and 2 were allowed to react with
excess 6-OTs-b-CyD in DMF when stirring in nitrogen
atmosphere at 80 ꢁC for 3 days to obtain bis-b-CyD
derivatives 3 and 4.17 The crude products were purified
by precipitation from a mass of acetone and then by chro-
matography on a CM Sephadex C-25 ionic column, as
well as Sephadex G-25 column. The yield of 3 and 4 was
21% and 25%, respectively. Their structures were validat-
ed by 1H NMR spectra in D2O and elemental analyses.
O
HO
7
OTs
OH
H2N
N
NH2
n
TsCl / CH3CN
alkaline aqua
H
n=0, 1, DMF , 70 o
C
6-OTs-β-CD
NH
N
NH2
n
H
2.3. Measurements of the interactions of b-CyD deriva-
tives with BSA
N
H
N
N
6-OTs-β -CD
n
H
H
N2 , DMF , 80 o
C
2.3.1. 1H NMR measurements. 1H NMR spectra of CyD
derivatives were recorded from a Varian UNITY-plus
400 spectrometer at 400 MHz in the presence and ab-
sence of BSA. Water suppression was achieved with a
presaturation pulse, prior to acquisition. The stock
BSA solutions were freshly prepared by dissolving
BSA in D2O. The molecular weight of BSA, 66 700,
was used to estimate the molar ratios of the CyD deriv-
atives to BSA. The concentration of the modified CyDs
was 2.30 mM and that of BSA was varied from 0.12 to
0.46 mM.
3 n=0
4 n=1
1 n=0
2 n=1
Scheme 1. Syntheses of the modified b-CyDs 1–4.
b-CyDs and two bridged bis-b-CyDs, that is, mono
(6-(2-aminoethylamino)-6-deoxy)-b-cyclodextrin (1), mono-
(6-(2-(2-aminoethylamino)ethylamino)-6-deoxy)-b-cyclo-
dextrin (2), ethylene-1,2-diamino bis-6-(6-deoxy-b-cyclo-
dextrin) (3), and iminodiethylene-2,20-diamino bis-6-(6-
deoxy-b-cyclodextrin) (4), were prepared from b-CyD,
as shown in Scheme 1.16,17 And their binding ability
with bovine serum albumin as a model protein was
investigated using proton magnetic resonance (1H
NMR), ultraviolet visible spectroscopy (UV–vis), circu-
lar dichroism (CD), and fluorescence spectroscopy.
2.3.2. UV–vis measurements. The UV absorption spectra
were recorded at 25 0.1 ꢁC on the U-3010 spectropho-
tometer (HITACHI, Japan) with 1 · 1 · 4 cm quartz
cell. The concentration of BSA was kept at
5.25 · 10ꢀ5 M and that of 1–4 was 0, 2 · 10ꢀ5
,
4 · 10ꢀ5, 6 · 10ꢀ5, 8 · 10ꢀ5, 1 · 10ꢀ4, and 2 · 10ꢀ4 M
in phosphate buffer solutions (PBS, pH 7.4, 0.1 M),
respectively.
2. Materials and methods
2.1. Materials
2.3.3. CD measurements. The far-UV CD measurements
of the samples in PBS were performed at 25 0.1 ꢁC in
the range of 190–260 nm on the Jasco J-715 spectropo-
larimeter (JASCO, Tokyo, Japan) with a 1 cm light path
length quartz cylindrical cuvette. The following parame-
ters were set: resolution = 0.2 nm; speed = 100 nm/min;
bandwidth = 1 nm; response = 1 s; and sensitivity =
20 mdeg. The average of three scans was calculated. The
concentration of BSA was 0.3 mg/mL and the ratio of
modified CyDs to BSA was 5:1, 25:1, and 50:1,
respectively.
BSA in electrophoresis purity (fatty acid free) was pur-
chased from Institute of Hematology & Hospital of
Blood Diseases, Chinese Academy of Medical Sciences,
and used without further purification. b-CyD of reagent
grade (Shanghai Reagent Factory, China) was recrystal-
lized twice from water and dried in vacuo at 95 ꢁC for
24 h prior to use. N,N0-Dimethylformamide (DMF)
was dried over calcium hydride for 2 days and then dis-
tilled under a reduced pressure before use. D2O is of the
isotopic purity with 99.9%, Cambridge Isotope Labora-
tories, Inc.
2.3.4. Fluorescence measurements. 0.25 mL of modified
CyDs solutions was added to 3.0 mL of 10 lM BSA
solution in PBS to reach final concentration of 0, 10,
25, 50, 100, and 200 lM, respectively. After 10 min,
fluorescence measurements were carried out at
25 0.1 ꢁC on a fluorescence instrument (Jobin Yvon,
Fluromax-P) with a 1 cm cell. The excitation wavelength
was 283 nm and the emissions were scanned from 303 to
440 nm for all samples. The slits of excitation and emis-
sion monochromators were adjusted to 3 nm.
2.2. Synthesis of b-CyD derivatives
2.2.1. Preparation of amino b-CyD derivatives 1 and 2.
Mono(6-O-(p-toluenesulfonyl))-b-cyclodextrin (6-OTs-
b-CyD) was prepared by the reaction of p-tosyl chloride
with b-CyD in alkaline aqueous solution.18,19 6-OTs-b-
CyD reacted with freshly distilled ethyldiamine and
diethylenetriamine16 to afford the amino derivatives of
b-CyD 1 and 2 in ca. 70% yield. Their purities were
detected by thin-layer chromatography (TLC) and their
To establish the stoichiometry of the complexes, JobÕs
experiments were also performed by means of fluores-
1
structures were elucidated by H NMR spectroscopy in

H. Gao et al. / Bioorg. Med. Chem. 14 (2006) 131–137
133
cence spectroscopy. The method of continuous varia-
tions6,20,21 was employed as follows: the total mole con-
centrations of modified CyDs and BSA were held
constant (10ꢀ5 mol), while the mole fraction of modified
CyDs varied from 0 to 1.0. After being incubated for 1 h
at 25 ꢁC, its fluorescence emission intensity was then
measured as described above. Duplicate fluorescence
intensity readings were taken to ensure reproducibility.
A JobÕs plot of fluorescence emission intensity difference
versus the mole fraction of modified CyDs was used to
determine the stoichiometry of binding from the ratio
of mole fraction of modified CyDs to BSA.
ened effect may be attributed to the external viscosity
of protein solution.23 Instead, at the concentrations of
BSA less than 0.23 mM, as studied by Sułkowska
et al.,24 the protons of the small molecule stabilized by
the interactions with the protein can be identified if it
has a longer correlation time and gives a much-broad-
ened spectral resonance signal. Therefore, there must
be some interactions between the modified CyDs and
BSA. These interactions, including hydrophobic interac-
tions, hydrogen bonds, and electrostatic interactions,
considerably restrict the protons and change the proton
signals in their NMR spectra. The high loss of signal res-
olution for the NMR spectra of the compounds 3 and 4
takes place as compared with that of 1 and 2 in the pres-
ence of BSA. The reason is undoubtedly the cooperative
effect of their two hydrophobic cavities located near
each other in the compounds 3 and 4 as the Bis-CyDs.
3. Results and discussion
NMR spectroscopy is increasingly used to elucidate
interactions of CyDs with biopolymers.8 Although the
current resolution of one-dimensional NMR spectra is
not high enough to locate the protons of BSA due to
BSA has a maximum UV/Vis absorption at ca. 280 nm
and this property can be used to determine BSA concen-
tration in a proper concentration range. The typical UV
spectra changes of 1–4 are shown in Figure 2. The UV
absorbance in the spectrum of BSA increases gradually
with the increased concentration of CyDs 1–4, and the
degree of absorption intensity change is influenced with
the modified CyDs in the order of 4 > 3 > 2 > 1. The re-
sults indicate that the solvation shell and the intramolec-
ular or intermolecular association of the chromophoric
groups on BSA macromolecule may be partly or totally
destroyed in the presence of the modified CyDs. The
inclusion and other complex interactions of the modified
CyDs with BSA lead to an altered solute–environment
interaction. When the concentrations of 1–4 are more
than 2 · 10ꢀ4 M (approximately 4 times of the BSA con-
centration) in a phosphate buffer (PBS), the change of
the BSA adsorption spectra can hardly be observed
again, indicating that the highest equivalence is achieved
at the molar ratio of 1:4 for BSA to the modified CyDs.
1
the complicated structure of BSA, the H NMR signal
changes of the modified CyDs in the absence and pres-
ence of BSA were studied to monitor their interactions.
The modified b-CyDs possess the outer-surface (H-2
and H-4) and inner-surface (H-3 and H-5) protons, as
well as the H-6 protons situated at the primary side of
the CyDs cavity.8,22 The adsorption of proton signals
is illustrated in Figure 1. The H-1 proton signal at d
4.9 ppm is of no significance because it is influenced by
the water suppression at d 4.8 ppm. On addition of
BSA, the chemical shift of every proton signal in the
NMR spectra of compounds 1–4 almost remains un-
changed. The resolution loss of proton signals of modi-
fied CyDs is observed and its loss extent is enhanced
with the increased BSA concentration. When the con-
centration of BSA reaches up to 0.46 mM, the broad-
CD spectra may provide some information on the sec-
ondary structures of proteins, nucleic acids and the
binding of ligands to these types of macromolecules.25
The binding of the modified CyDs to those biopolymers
was also confirmed by CD spectra. Final spectra may be
presented in mean residue ellipticity, [h]:
hM
10CLNr
½hꢁ ¼
.
In the formula, h is the measured ellipticity (mdeg), M is
the molecular weight of the protein, C is the BSA con-
centration (mg/mL), L is the pathlength of the cell
(cm), and Nr is the number of amino acids of the protein
molecules.26
At first, CD spectra were analyzed using a curve-fitting
program software, CDPro, as described by Sreerama
and Woody27 to obtain the secondary structural con-
tents of the proteins. However, the low signal-to-noise
ratio below 195 nm implies a poor quality of the spectra
recorded in the region and the results are not reliable.
So, the a-helix content of proteins may be estimated
according to the following equation:28
Figure 1. The 1H NMR spectra of the CyD derivatives 1–4 (2.3 mM)
in the absence (1–4, from A to D) of and the presence (1–4, from E to
H) of BSA (0.23 mM).

134
H. Gao et al. / Bioorg. Med. Chem. 14 (2006) 131–137
1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
A
C
f
f
a
a
260
265
270
275
280
285
290
260
265
270
275
280
285
290
λ/nm
λ/nm
1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
B
D
f
f
a
a
260
265
270
275
280
285
290
260
265
270
275
280
285
290
λ/nm
λ/nm
Figure 2. The UV/Vis spectral changes of BSA (5.25 · 10ꢀ5 M) in the presence of 1 (A), 2 (B), 3 (C), and 4 (D) with concentrations of 0, 2 · 10ꢀ5
4 · 10ꢀ5, 6 · 10ꢀ5, 8 · 10ꢀ5, 1 · 10ꢀ4, and 2 · 10ꢀ4 M (from a to f) in the PBS buffer.
,
½hꢁmrd ꢀ 4000
%a-helix content ¼
;
33000 ꢀ 4000
where [h]mrd is the mean molar ellipticity per residue at
208 nm (deg cm2 dmolꢀ1).
Typical CD spectra of BSA in the presence of the repre-
sentative modified CyD, 4, are shown in Figure 3. As
expected, the strong double minimums, respectively, at
220 and 209 nm suggest that the a-helix structure exists
in BSA25 and no apparent peak shift occurs in the pres-
ence of the modified CyDs. The intensity of the double
minimums reflects more than 50% of a-helical structure
in BSA. The binding of the modified CyDs to BSA re-
sults in a deepening of the trough at 208 nm, which is
due to an increase in the helix content of BSA with
the conformational changes. The estimated percentages
of a-helix contents from CD spectra are listed in Table
1. It is measured that the native BSA at pH 7.4 in the
PBS buffer has 60.5% of a-helix, close to the value of
59 2% reported previously.29,30 In parallel to the UV
absorbance change trend, the percentage of a-helix con-
tent also increases in the order of 4 > 3 > 2 > 1 when
these modified CyDs are added. The interactions of
Figure 3. The CD spectra of BSA (0.3 mg/mL) upon addition of
compounds 4 (a: 0, b: 0.15, c: 0.75, and d: 1.5 mg/mL) in the PBS
buffer.
the modified CyDs with BSA can weaken the BSA sol-
vation with the water molecules and the self-association
within the chain of BSA, so that a much tighter second-
ary structure of a-helix becomes dominant in BSA.

H. Gao et al. / Bioorg. Med. Chem. 14 (2006) 131–137
135
Table 1. The a-helix percentages in BSA (0.03 mg/mL) upon addition
of compounds 1–4 with concentrations of 0.15 mg/mL (a), 0.75 mg/mL
(b), and 1.5 mg/mL (c)
possibly affects the overall three-dimensional structure
of peptides and proteins.1 In the studies of a 17-mer heli-
cal model peptide, Chakrabartty et al.32 assessed the
aromatic side-chain contribution to far-UV CD by
substituting an alanine at the N-terminus of the helix
with aromatic residues. Nevertheless, the complicated
compositions of BSA make the substitution method
unfeasible. As a whole, it is conceivable that besides
the aromatic contribution, CD changes contribute to
the backbone conformations of BSA.
Compound
Helix
BSA
60.5
60.7
59.4
61.0
61.1
62.6
63.4
61.9
64.1
66.9
63.4
65.3
68.4
BSA-1 (a)
BSA-1 (b)
BSA-1 (c)
BSA-2 (a)
BSA-2 (b)
BSA-2 (c)
BSA-3 (a)
BSA-3 (b)
BSA-3 (c)
BSA-4 (a)
BSA-4 (b)
BSA-4 (c)
Meanwhile, protein fluorescence has been used as a sen-
sitive detector for the conformational change studies in
the ternary protein structures. BSA is a single-chain
transporting protein of 585 amino acids with 17 disulfide
bridges, two tryptophanes (Trp 135 and Trp 214), and
one free thiol at Cys 34. It is known that the fluorescence
of aqueous tryptophan-containing proteins arises almost
from their tryptophanyl residues.24 The fluorescence
emission spectrum of BSA at an excitation of 283 nm
shows kmax at 349 nm. Addition of each CyD derivative
induces a loss in fluorescence intensity due to the
quenching effect at various degrees. Typical fluorescence
quenching spectra of BSA in the presence of compounds
However, the CD spectrum is not due solely to the poly-
peptide backbone and its conformation. Other UV-
active chromophores, such as the aromatic groups of
tyrosine and phenylalanine, may contribute to CD of
proteins and polypeptides.31 Aromatic amino acids are
known to complex with CyDs. Such an interaction
A
1.20
1.15
1.10
1.05
1.00
700
600
500
400
300
200
100
0
a
g
0
50
100
150
200
[Q](µm)
300
320
340
360
380
400
420
440
λ
/nm
B
1.20
1.15
1.10
1.05
1.00
700
600
500
400
300
200
100
0
a
g
0
50
100
150
200
[Q](µm)
300
320
340
360
380
400
420
440
λ/nm
Figure 4. Fluorescence spectral changes of BSA (10 lM) with an excitation wavelength of 283 nm upon addition of compounds 2 (A), 4
(B) (0–200 lM, from a to g) in the PBS buffer. The inset is the corresponding Stern–Volmer plot (n = 5).

136
H. Gao et al. / Bioorg. Med. Chem. 14 (2006) 131–137
2 and 4 are shown in Figure 4. The fluorescence intensity
decreases with an increase in the concentration of the
added compounds. As a conclusion, the interactions
between the CyD derivatives and BSA contribute to
the alteration of BSA ternary structure near the binding
site.
redshift in maximum emission wavelength is then
aroused as a result of the change in the local tryptophan
microenvironment. In case 4 is sticky to BSA, then it is
imperative to consequently rearrange the tryptophan
microenvironment in BSA.33
To establish a stoichiometry, a double reciprocal curve
is first plotted36 for F/DF (where DF = F0 ꢀ F) versus
1/[modified CyD] employing the data in Figure 4. Non-
linearity from the double reciprocal plots indicates that
higher-order complexes are involved.
Due to the interactions of compounds 1–4 with BSA, a
quenching of fluorescence takes place and the Stern–
Volmer analysis allows us to estimate their quenching
constants. Fluorescence quenching was carried out by
measuring the fluorescence intensities at 349 nm as a
function of the concentration of each compound:
Finally, in the concentration range examined, JobÕs plot
shows the maximum at a molar fraction of 0.75 (1 and 2)
and 0.8 (3 and 4), indicating that the complex stoichiom-
etries of BSA to the modified CyDs are 1:4 for both 1
and 2, as well as 1:3 for both 3 and 4, respectively. Rep-
resentative JobÕs plot for the complex of 2/BSA and 4/
BSA are shown in Figure 5. When considering the
molecular dimension, several CyDs derivatives may be
involved in forming a complex with BSA. The size of
the bridged CyDs is larger than that of mono-substitut-
ed CyDs. Therefore, four molecules of the mono-substi-
tuted CyDs (1 and 2) can bind with one molecule of
F 0=F ¼ 1 þ Kq½Qꢁ;
where F0 and F are, respectively, fluorescence intensities
in the absence and presence of quencher, Kq is the Stern–
Volmer quenching constant, and [Q] is the concentration
of the quencher. The equation assumes a linear plot of
F0/F versus [Q] and the slope equals Kq.12 Kq values of
the tryptophan quenching, expressing tryptophan acces-
sibility to the quencher,33 are listed in Table 2. The ex-
tent of quenching is higher for the bridged CyDs than
for the mono-substituted CyDs. The cooperative effects
should play an important role in the interactions of BSA
with the modified CyDs as mentioned above. The Kq of
diethylenetriamino-substituted CyDs (2 or 4) was a little
more than the corresponding ethylenediamino-substitut-
ed ones (1 or 3). This may be caused by the electrostatic
interactions owing to the more amino groups in com-
pounds 2 and 4 than in 1 and 2.
20
15
10
5
A slight redshift in emission maximum wavelength is
also observed in BSA fluorescence when the solution
of 4 is added, suggesting that the binding of 4 is accom-
panied by changes in the dielectric environment of at
least one of the two indole rings in BSA.34,35 It is known
that a shift of maximum emission wavelength corre-
sponded to a polarity change around the chromophore
residues. A redshift always indicates that tryptophan
residues are, on average, more exposed to the solvent,
whereas a blueshift is a consequence of transferring
tryptophan residues into a more hydrophobic environ-
ment. In this study, the redshift can be explained as
the result of change in the higher structures of BSA.
Since the interaction of BSA with the modified CyDs re-
sults in a microenvironment change of BSA backbone
and its residues, the increased a-helix content (as shown
in Table 1) indicates a tightening secondary structure
and hence the ternary structure of BSA should be bro-
ken to some extent. While the ternary structure of
BSA is changed, the tryptophan residue may be exposed
to the outside, owing to the affinity of compound 4. The
0
0.0
0.2
0.4
0.6
0.
Fraction of 2
30
25
20
15
10
5
Table 2. The Stern–Volmer quenching constants of tryptophan residue
fluorescence in the presence of compounds 1–4
Compound
Stern–Volmer constant, Kq[Mꢀ1
]
0
0.0
0.2
0.4
0.6
0.8
1.0
1
2
3
4
305 14
485 27
893 26
994 44
Fraction of 4
Figure 5. Continuous variation plot of the 2/BSA ([2] + [BSA] =
10ꢀ5 M) and 4/BSA system ([4] + [BSA] = 10ꢀ5 M) in PBS.

H. Gao et al. / Bioorg. Med. Chem. 14 (2006) 131–137
137
11. Spahr, P. F.; Edsall, J. T. J. Biol. Chem. 1964, 239, 850.
BSA, while three bridged CyDs (3 and 4) can complex
with one BSA.
12. Perez-Rodrigrez, M.; Attwood, D.; Ruso, J. M.; Taboada,
P.; Varela, L. M.; Mosquera, V. Phys. Chem. Chem. Phys.
2001, 3, 1655.
13. Narazaki, R.; Maruyama, T.; Otagiri, M. BBA-Protein
Struct. M. 1997, 1338, 275.
4. Conclusion
14. Chadborn, N.; Bryant, J.; Bain, A. J.; OÕShea, P. Biophys.
J. 1999, 76, 2198.
15. Reynolds, J. A.; Herbert, S.; Polet, H.; Steinhardt, J.
Biochemistry 1967, 6, 937.
The interactions of BSA with the modified CyDs were
observed, owing to the formation of complex on the
basis of H NMR, UV, CD, and emission fluorescence
1
spectroscopy. Analysis of the helix content and Stern–
Volmer quenching constant indicates that the complex-
ation abilities of compounds 1–4 were in the order of
4 > 3 > 2 > 1. For the stoichiometry, BSA can complex
with the modified CyDs at a ratio of 1:4 (for 1 and 2)
and 1:3 (for 3 and 4).
16. May, B. L.; Kean, S. D.; Easton, C. J.; Lincoln, S. E.
J. Chem. Soc., Perkin Trans. 1 1997, 997, 3157.
17. Liu, Y.; You, C. C.; Li, B. Chem. Eur. J. 2001, 7, 1281.
18. Petter, R. C.; Salek, J. S.; Sikorski, C. T.; Kumaravel, G.;
Lin, F. T. J. Am. Chem. Soc. 1990, 112, 3860.
19. Liu, Y.; Yang, Y. W.; Cao, R.; Song, S. H.; Zhang, H. Y.;
Wang, L. H. J. Phys. Chem. B 2003, 107, 14130.
20. Ingham, K. C. Anal. Biochem. 1975, 68, 660.
21. Chen, C. H.; Battaglioli, G.; Martin, D. L.; Hobart, S. A.;
Colon, W. BBA 2003, 1645, 63.
Acknowledgments
22. Liu, Y.; Chen, G. S.; Zhang, H. Y.; Song, H. B.; Ding, F.
Carbohydr. Res. 2004, 339, 1649.
23. Chatham, J. C.; Forder, J. R. BBA-Gen. Subjects 1999,
1426, 177.
24. Sułkowska, A.; Bojko, B.; Rownicka, J.; Sułkowski, W.
Biopolymers 2004, 74, 256.
Authors acknowledge the National Natural Science
Foundation of China for the financial support (Pro-
ject No.: 20174018). We thank Prof. Ping-Chuan
Sun and Hai Lin for their measurement of 1H
NMR spectra.
25. Deep, S.; Ahluwalia, J. C. Phys. Chem. Chem. Phys. 2001,
3, 4583.
26. Peng, Z. G.; Hidajat, K.; Uddin, M. S. Colloids Surf., B
2004, 33, 15.
References and notes
1. Irie, T.; Uekama, K. Adv. Drug Delivery Rev. 1999, 36, 101.
2. Uekama, K.; Hirayama, F.; Irie, T. Chem. Rev. 1998, 98,
2045.
3. Nicolazzi, C.; Abdou, S.; Collomb, J.; Marsura, A.;
Finance, C. Bioorg. Med. Chem. 2001, 9, 275.
4. Ugawa, T.; Nishikawa, S. J. Phys. Chem. A 2001, 105,
4248.
27. Sreerama, N.; Woody, R. W. Anal. Biochem. 2000, 287,
252.
28. Greenfield, N. J.; Fasman, G. D. Biochemistry 1969, 8,
4108.
29. Serro, A. P.; Bastos, M.; Pessoa, J. C.; Saramago, B.
J. Biomed. Mater. Res. A 2004, 70, 420.
30. Norde, W.; Giacomelli, C. E. J. Biotechnol. 2000, 79, 259.
31. Manning, M. C.; Woody, R. W. Biochemistry 1989, 28,
8609.
5. Liu, Y.; You, C. C.; Zhang, H. Y.; Zhao, Y. L. Eur. J.
Org. Chem. 2003, 5, 1415.
6. Liu, Y.; Yang, Y. W.; Chen, Y.; Ding, F. Bioorg. Med.
Chem. 2005, 13, 963.
7. Liu, Y.; Yang, Y. W.; Song, Y.; Zhang, H. Y.; Ding, F.;
Wada, T.; Inoue, Y. ChemBioChem 2004, 5, 868.
8. Schneider, H. J.; Hacket, F.; Rudiger, V. Chem. Rev. 1998,
98, 1755.
32. Chakrabartty, A.; Kortemme, T.; Padmanabhan, S.;
Baldwin, R. L. Biochemistry 1993, 32, 5560.
33. Klajnert, B.; Bryszewska, M. Bioelectrochemistry 2002, 55,
33.
34. Yuan, T.; Weljie, A. M.; Vogel, H. J. Biochemistry 1998,
37, 3187.
9. Dotsikas, Y.; Loukas, Y. L. J. Pharm. Biomed. 2002, 29,
487.
35. Cui, F. L.; Fan, J.; Li, J. P.; Hu, Z. D. Bioorg. Med. Chem.
2004, 12, 151.
10. Peczuh, M. W.; Hamilton, A. D. Chem. Rev. 2000, 100,
2479.
36. Furusaki, E.; Ueno, Y.; Sakairi, N.; Nishi, N.; Tokura, S.
Carbohydr. Polym. 1996, 29, 29.