Design and synthesis of a new series of amphiphilic peptide-β- cyclodextrins as phase transfer carriers for glucosamine

Article abstract of DOI:10.1016/j.tet.2010.06.075

A new series of amphiphilic β-cyclodextrins were designed and synthesized by grafting peptide chains on to all primary hydroxyl groups via ester bond formation. The desired amphiphilic structures have been produced from ester connection between the C-6 of β-cyclodextrin and the carboxyl group of N-acetylated resides: H₂N-Leu-COOH, H₂N-Leu-Gly-COOH, H₂N-Leu-Gly-Leu-COOH, and H₂N-Leu-Gly-Leu-Gly-COOH (3a-d). The synthetic pathway involves selective bromination of all primary hydroxyls of β-cyclodextrins and then substitution with the carboxylate moiety of the mentioned N-acetyl residues in the presence of DBU (1,8-diazabicyclo[5,4,0] undec-7-ene). The ability of the synthetic compounds for extraction and phase transfer of glucosamine, as a hydrophilic organic compound, was then studied. The results showed a considerable ability of these amphiphilic compounds for extraction and a selective tendency of 3c for phase transfer of glucosamine.

Full text of DOI:10.1016/j.tet.2010.06.075

Tetrahedron 66 (2010) 6754e6760
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Design and synthesis of a new series of amphiphilic peptide-
as phase transfer carriers for glucosamine
b-cyclodextrins
,
a
c
a
Seyed Mohammad Seyedi a, Hamid Sadeghian b , Atena Jabbari , Amir Assadi , Hamideh Momeni
*
a Department of Chemistry, School of Sciences, Ferdowsi University of Mashhad, Mashhad 917751436, Islamic Republic of Iran
b Department of Laboratory Sciences, School of Paramedical Sciences, Mashhad University of Medical Sciences, Mashhad 91389-13131, Islamic Republic of Iran
c Department of Medicinal Chemistry, School of Pharmacy and Pharmaceutical Sciences Research Center, Tehran University of Medical Sciences, Tehran, Islamic Republic of Iran
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 8 April 2010
Received in revised form 7 June 2010
Accepted 28 June 2010
Available online 3 July 2010
A new series of amphiphilic b-cyclodextrins were designed and synthesized by grafting peptide chains
on to all primary hydroxyl groups via ester bond formation. The desired amphiphilic structures have
been produced from ester connection between the C-6 of -cyclodextrin and the carboxyl group of
b
N-acetylated resides: H2NeLeueCOOH, H2NeLeu-GlyeCOOH, H2NeLeu-Gly-LeueCOOH, and H2NeLeu-
Gly-Leu-GlyeCOOH (3aed). The synthetic pathway involves selective bromination of all primary
hydroxyls of
b-cyclodextrins and then substitution with the carboxylate moiety of the mentioned
Keywords:
Peptide synthesis
DBU
Extraction
Molecular modeling
log D
N-acetyl residues in the presence of DBU (1,8-diazabicyclo[5,4,0]undec-7-ene). The ability of the syn-
thetic compounds for extraction and phase transfer of glucosamine, as a hydrophilic organic compound,
was then studied. The results showed a considerable ability of these amphiphilic compounds for
extraction and a selective tendency of 3c for phase transfer of glucosamine.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
development of biological receptors that may help to understand
the complicated mechanism of the molecular recognition by living
Cyclodextrins (CDs) are valuable compounds as drug carriers
due to their ability to form inclusion complexes.1 The potential
utilities of these compounds are solubilization,2 encapsulation,3
and transport of biologically active molecules.4 Amphiphilic cy-
clodextrin derivatives are very important in pharmaceutical ap-
plications based on their ability for self-organization in aqueous
media. Liposomes,5 nanoparticles,6 vesicles,7 and micellar aggre-
gates8 have been prepared from amphiphilic cyclodextrins to
generate the versatile carrier and delivery systems for hydrophilic
and lipophilic drugs. These compounds are tailored by grafting
various lengths of lipophilic chains on to the entire primary or
secondary hydroxyl groups of the glucopyranose units. Among
these derivatizations, grafting fatty-acids, -alcohols, and -amines
on to primary hydroxyl group of cyclodextrins are most common.6
The lipophilic chains of these structures would make an intimate
contact with biological membranes (Fig. 1).
cells and bacteria. While a large variety of amphiphilic CDs were
synthesized by modifying with long alkyl chains as hydrophobic
substituents, only a few works based on peptide functionalized CDs
were reported.6
In this study, a new series of amphiphilic b-cyclodextrin de-
rivatives were designed and synthesized by grafting peptide chains
on to the primary hydroxyl groups via ester bond formation. The
new structures are composed of various lengths of N-acylated li-
pophilic peptide chains on the primary hydroxyl groups. The ability
of the synthetic compounds for extraction and phase transfer of
glucosamine, a hydrophilic organic compound, was then studied.
2. Results and discussion
Our interest in the design and synthesis of
b-CD peptide de-
rivatives emerges from the work of Imamura et al. in which prep-
Furthermore, bilayer vesicles composed of amphiphilic CDs
have been shown to bind specific guests to recognize molecular
signals.9,10 This suggests that amphiphilic CDs could be used for the
aration of amphiphilic compounds from per-amino-b-CD using
peptide chemistry was reported.11 The desired amphiphilic struc-
tures have been produced from ester linkage between all the C-6 of
b-CD and the carboxyl group of N-acetylated resides:
H2NeLeueCOOH, H2NeLeu-GlyeCOOH, H2NeLeu-Gly-LeueCOOH,
and H2NeLeu-Gly-Leu-GlyeCOOH (Scheme 1).
* Corresponding author at: Department of Laboratory Sciences, School of Para-
medical Sciences, Mashhad University of Medical Sciences, Mashhad, 91857-63788,
Islamic Repubic of Iran. Tel.: þ98 05117610111; fax: þ98 05117628088; e-mail
address: sadeghianh@mums.ac.ir (H. Sadeghian).
In this molecular designing,
L-leucine (Leu), the most lipophilic
amino acid, was selected for extension of the cavity of
b
-CD and
0040-4020/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.06.075

S.M. Seyedi et al. / Tetrahedron 66 (2010) 6754e6760
6755
Figure 1. Schematic presentation of amphiphilic b-CD situated in bilayer membrane.
OH
Br
OOCR
O
O
O
NBS, PPh3, DMF
70-80oC, 3-4h
RCOOH / DBU
HO
HO
HO
OH
OH
OH
O
O
O
7
7
7
1
2
3a-d
O
O
O
H
N
H
N
COO
N
H
N
H
N
H
COO
O
O
a
(68%)
d (54%)
RCOO :
O
O
O
H
N
H
N
COO
N
H
N
H
N
H
COO
O
O
b
(62%)
c (67%)
Scheme 1. General procedure for the synthesis of amphiphilic b-CDs (3aed).
organizing an external lipophilic structure. Due to the inhibiting
effect of pH on the amphiphilic property of the compounds, the
N-acetyl form of the aforementioned residues was used. To prevent
steric hindrance adjust to leucine, Gly was selected as the preferred
connector of the two residues.
peptide b. Reaction of Isobutyl chloroformate (IBCF) with b in the
presence of DBU afford to formation of the related carbonate.
Directly reaction of the carbonate with leucine methyl ester pro-
duces the compound b0. Alkali hydrolysis of b0 led to formation of
N-acetyl peptide c. N-acetyl tetrapeptide d was synthesized form c
according to the procedure reported for b. It was found that yield of
ester hydrolysis increased when NaOH(aq)/acetone, NaOH/MeOH,
and NaOH/EtOH were used for di, tri, and tetrapeptide, respectively
(Scheme 2). On the basis of the mentioned synthetic pathway, four
amphiphilic compounds, 3aed, were synthesized (Scheme 1).
Binding behavior was studied using glucosamine as a guest
molecule. The relative binding affinity of 3aed toward glucosamine
was measured using a phase extraction method.16 The decrease in
concentration of aqueous glucosamine after shaking for 12 h with
The synthetic pathway involves selective bromination of all pri-
mary hydroxyl groups of
b
-CD12,with the N-bromosuccinamide
(NBS) and triphenylphosphine (PPh3), then substitution with the
carboxylate moiety of the mentioned N-acetyl residues in the
presence of DBU (1,8-Diazabicyclo[5,4,0]undec-7-ene).13 Peptide
chains were synthesized via reaction of isobutyl carbonate of the
N-acetyl residue with methyl or ethyl ester of the desired amino
acid14 and finally C-terminal ester hydrolysis (Scheme 2). The car-
bonate was formed in situ via reaction of the DBU-functionalized
carboxyl group of residue with isobutyl chloroformate (IBCF).14 In
comparison with triethylamine,15 using DBU led to an increase in
peptide yield with less by-product formation. Reaction of IBCF with
N-acetyl leucine (a) in the presence of 1,8-diazabicyclo[5,4,0]undec-
7-ene (DBU) produces a corresponding carbonate. In situ conden-
sation of the carbonate with glycine ethyl ester produces the
compound (a0). Alkali hydrolysis of a0 lead to formation of N-acetyl
a lipid phase (octanol) containing 3aed and
and reported as extraction content (%E).
b-CD was determined
Aqueous solutions of the glucosamine (5 mM) (pH 7.4) were
separately extracted with octanol (lipid phase), containing 3aed
and
was extracted effectively and without selectivity by 3aed but no
significant extraction was observed for -CD (Table 1). If it be
b-CD, (5 mM) and compared with the control. The glucosamine
b

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S.M. Seyedi et al. / Tetrahedron 66 (2010) 6754e6760
O
O
NaOH (aq) / Acetone
1) DBU / IBCF
H
N
OH
COOEt
N
H
N
H
H N
2
COOEt
2)
O
O
a
a'
O
O
O
1) DBU / IBCF
H2N COOMe
2)
NaOH / MeOH
H
H
N
N
COOH
N
N
H
N
COOMe
H
H
O
O
b
(97%)
b'
O
O
1) DBU / IBCF
O
O
H
H
N
H
N
N
COOEt
N
N
COOH
N
N
H
2) H2N
COOEt
H
H
H
O
O
O
c
(77%)
c'
O
O
NaOH / EtOH
H
N
H
N
COOH
N
H
N
H
O
O
d
(71%)
Scheme 2. Synthetic pathway of peptides aed.
Table 1
½host ꢀ Gluꢁ ¼ ½Gluꢁaqðbefore extractionÞꢀ½Gluꢁaqðafter extractionÞ
Extraction and phase transferring percentage of glucosamine (%E and %T, re-
spectively) by compounds 3aed
and
Case
%E
%T
½Gluꢁ ¼ ½Gluꢁtotalꢀ½host ꢀ Gluꢁand½hostꢁ
¼ ½hostꢁtotalꢀ½host ꢀ Gluꢁ
3a
3b
3c
3d
CD
Ctr.
24.4ꢂ0.9
23.8ꢂ2.2
28.6ꢂ1.8
26.3ꢂ1.2
0.40ꢂ0.2
0.21ꢂ0.1
1.62ꢂ0.3
1.50ꢂ0.2
3.49ꢂ0.3
1.63ꢂ0.4
0.74ꢂ0.1
0.43ꢂ0.1
The observed glucosamine binding constants (Kb) of 3aed are
outlined in (Table 2).
Table 2
Octanol/water distribution coefficient (log D), experimental free energy of binding
(D DGb)
Gob), binding constant (Kb) and average of estimated free energy of binding (
for compounds 3aed
assumed that glucosamine (Glu) equivalently (1:1) interacts with
the host molecules, the equilibrium constant of extraction could be
defined as the binding constant (Kb):
Compd
D
Gb
D
Gbo
Kb
84.2
80.8
110.8
95.6
log D
3a
3b
3c
3d
ꢀ5.81ꢂ0.13
ꢀ6.17ꢂ0.22
ꢀ5.55ꢂ0.26
ꢀ6.11ꢂ0.29
ꢀ2.62
ꢀ2.60
ꢀ2.79
ꢀ2.70
1.17ꢂ0.07
1.05ꢂ0.04
1.57ꢂ0.05
1.11ꢂ0.09
Kb ¼ ð½hostꢀGluꢁÞ=ð½Gluꢁ½hostꢁÞ hostþGlu%hostꢀGlu
(1)
In the other experiment, phase transfer of glucosamine by 3aed
and -CD was determined. In this work the tendency of each
compound for glucosamine transferring between two aqueous
By considering the insolubility of glucosamine in octanol and
the very low water solubility of the hosteGlu complex, it was as-
sumed that:
b

S.M. Seyedi et al. / Tetrahedron 66 (2010) 6754e6760
6757
phases, separated by octanol, was measured. Two stirred aqueous
phases (pH 7.4), in which one of them (primary phase) contained
glucosamine (25 mM), were connected through octanol, containing
To explore the origin of these effects, the octanol/water distribu-
tion coefficient (log D) of 3aed was measured using shake flask
method (Table 2).17 Byconsideringthelog Dvalues, thephasetransfer
results of 3aed could be rationalized. It was concluded that more li-
pophilic host molecule (3c: log D 1.57) has a higher capability for
transferring glucosamine in determined period time.
test compound (3aed and b-CD; 5 mM), without any solvent dif-
fusion (Fig. 2). After 24 h the glucosamine concentration of the
intact aqueous phase (secondary phase), was determined. The ratio
of glucosamine concentration of the primary and secondary phases
was recorded as phase transferring extent (%T).
To complete the study, the binding affinity of glucosamine toward
3aed was calculated and reported as estimated binding free energy
(
D
Gb). In this section, the 3D structures of 3aed were modeled via
grafting the desired residues on all primary hydroxyls of -CD crystal
structure followed by geometry optimization (PM3 methods). In the
modeled molecules, residues are aligned above the -CD ring and the
cylindrical structure shape is observed in which the outer part is
hydrophobic while the head ( -CD ring) possess the hydrophilic
b
b
b
nature. The structurewasstabilizedbycrossintermolecularhydrogen
bonds of amide groups. The binding affinity was estimated in Auo-
DockTools software using autodock4.0 program.18 100 docked con-
formers of glucosamine were generated in ADT software for each of
3aed. The detailed assessment of all 100 docked models revealed as
followings: 100% of docking results had nearly identical orientations
Octanol
Phase I
in the
3b, 68% of docking results had nearly identical orientations in the
-CD ring and 32% in the pocket formed by the residues with average
Gb of ꢀ6.13 and ꢀ6.25 kcal/mol, respectively; for 3c, 58% of docking
results had nearly identical orientations in -CD ring and 42% in the
mentioned pocket with average
Gb of ꢀ5.62 and ꢀ5.45 kcal/mol,
respectively, and finally for 3d, 69% of docking results had nearly
identical orientations in -CD ring and 31% in the mentioned pocket
with average
b
-CD ring with average
D
Gb of ꢀ5.81 kcal/mol for 3a; while for
Phase II
b
D
Magnet
b
Figure 2. Schematic presentation of the apparatus used for phase transfer experiment.
D
Among the tested compounds, a modest phase transfer was
observed for 3c, while the experiments with 3a,b, and d showed
worse results (Table 1). Phase transfer of glucosamine by b-CD was
significantly lower.
b
DGb of ꢀ6.14 and ꢀ6.08 kcal/mol, respectively (Fig. 3)
(Table 2). Due to the orientation mentioned above, the docked
conformers displayed hydrogen bonds with hydroxyl and amides of
Figure 3. All of the docked models of glucosamine in the cavity of 3d. The below figure represents the cross view of docked models in 3d cavity.

6758
S.M. Seyedi et al. / Tetrahedron 66 (2010) 6754e6760
the sugars and residues in 3aed. Considering
D
Gob¼ꢀRT ln Kb and Eq.
docked conformers of glucosamine structures with lowest-energy.
(1), the high similarity between experimental and theoretical results
After docking procedure in AD4, docking results were submitted to
Accelrys DS Visualizer v2.0.1.734722 for further evaluations. The
results of docking processing (ΔGb: Estimated Free Energy of
Binding) are outlined in (Table 2).
(D
Gob and
Docking results of 3aed showed that the binding free energy of
glucosamine for all of the host molecules is almost equal and the
docked models are mainly located in -CD cavity. This could explain
DGb, respectively) was anticipated.
b
why the extraction contents and Kb of 3aed are similar.
4.3. Assessment of extraction and phase transfer ability
Phase transfer properties of 3aed is mainly depend on the ex-
ternal lipophilic character of their cavity while the host molecules
have similarbindingaffinity toward glucosamine, the morelipophilic
one (3c), would transfer higher quantities of the guest molecule.
The extraction content of the synthetic host molecules and b-
CD were assayed by 12 h shaking a 2 mL solution of the glucos-
amine (5 mM) in water (pH 7.4dphosphate buffer) with 2 mL
solution of host molecule (5 mM) in octanol at 25 ꢃC. In the ex-
periment, shaking of glucosamine solution with intact octanol was
set as a control. The experiment was done four times for each of
the host molecules and control. The concentration of glucosamine
in aqueous phase was measured by using the spectrophotometric
method reported by Rondle et al.23 in which acetylacetone and
Ehrlich’s reagent used as chromogenic materials. The extracted
quantity of glucosamine as same as the Gluehost concentration is
equal to:
3. Conclusion
In summary, we have designed and synthesized a new series
of hydrophobic peptide
b-cyclodextrins as a phase transfer
carrier for glucosamine. These types of cage-molecules could be
a precursor for designing and synthesis of other similar mole-
cules as carriers for desired biological compounds in the future.
Considering the external hydrophobicity and terminal hydro-
philicity of these molecular structures, it would be hypothesized,
that the natural capacity of these compounds to locate in the
bilayer membrane of the cells, act as a channel for transferring
special compounds.
½Gluꢁðbefore extractionÞꢀ½Gluꢁðafter extractionÞand extraction
contentð%TÞ ¼ 100 ꢄ ½Glu ꢀ hostꢁ=½Gluꢁtotal
Phase transferring extent (%T) of compounds 3aed was de-
termined by using the apparatus shown in (Fig. 2).24 In Figure 2,
phase I (10 mL) is aqueous solution of glucosamine (25 mM) with
pH 7.4 (phosphate buffer) and phase II (30 mL) is only aqueous
solution of phosphate buffer (pH 7.4). The octanol phase (30 mL)
containing the host molecule (5 mM) contacts with both aqueous
phases. In control test, the octanol phase had no host compound.
After stirring of the aqueous phases (24 h), the glucosamine con-
centration in phase II was measured according to the Rondle
method. The experiment was repeated three times for each host
molecules and control. %T equal to:
4. Experimental
4.1. Chemicals and instruments
Melting points were recorded on an Electrothermal type 9100
melting point apparatus. 1H NMR (500 MHz) and 13C NMR
(125 MHz) were obtained by using a Bruker Avance DRX-500
Fourier transformer spectrometer. Chemical shifts are reported in
parts per million (d) downfield from tetramethylsilane (TMS). The
IR spectra were obtained on a 4300 Shimadzu Spectrometer. Ele-
mental analysis was obtained on a Thermo Finnigan Flash EA
microanalyzer. All spectrophotometric measurements were carried
out using an Agilent 8453 spectrophotometer.
100 ꢄ ½Gluꢁphase II=½Gluꢁtotal
4.2. Molecular modeling and docking study
4.3.1. N-Acetyl-
11 mmol) was added dropwise to a solution of N-acetyl-
L
-leucylglycine (b). Isobutyl chloroformate (1.4 mL,
-leucine
L
4.2.1. Structure optimization. 3D structures of compounds 3aed
were simulated by grafting desired residues on all primary
hydroxyl of the X-ray structure of b-CD and minimized under semi-
empirical PM3 method (Convergence limit¼0.01; Iteration
limit¼50; rms gradient¼0.1 kcal/mol; FletchereReeves optimizer
algorithm) in HyperChem7.5.19
Crystal structure of b-CD complexed with cyclodextrin-binding
proteinwasretrievedfromRCSBProteinDataBank(PDBentry:2ZYN).
(a) (1.73 g,10 mmol) and 1.6 mL of DBU in 50 ml chloroform while
stirring in ice-water. After removing the ice-bath, the reaction
mixture was stirred at room temperature for 1 h. Glycine ethyl ester
(1.39 g, 10 mmol) was then added and stirring continued for 2 h.
The resulting solution was washed with 1 M NaOH (2ꢄ30 ml), 1 M
HCl (2ꢄ30 mL), and water (1ꢄ30 mL). The organic layer was dried
over anhydrous magnesium sulfate. After removing the solvent
under reduced pressure, the residue was recrystallized from hex-
ane to give N-acetyl-
-leucylglycine ethyl ester (a0) (1.41 g, 82%).
L
4.2.2. Molecular docking. Automated docking simulation was
implemented to dock glucosamine into the cavity of 3aed with
AutoDockTools 4.0 version 1.5.418 using Lamarckian genetic algo-
rithm.20 This method has beenpreviously shown to produce binding
models similar to the experimentally observed models.14,21 The
torsion angles of the ligands were identified, hydrogens were added
to the macromolecule, bond distances were edited and solvent pa-
rameters were added to the enzyme 3D structure. Partial atomic
charges were then assigned to the macromolecule as well as ligands
(Gasteiger for the ligands and Kollman for the host molecules).
The regions of interest of the host molecules were defined by
considering all of the molecular structure in the grid box. The
docking parameter files were generated using Genetic Algorithm
and Local Search Parameters (GALS) while number of generations
was set to 100. The docked complexes were clustered with a root-
mean-square deviation tolerance of 0.5 Å. Autodock generated 100
White crystal, mp: 223e225 ꢃC; 1H NMR: (CD3OCD3)
d
0.85e0.93 (m, 6H, 2CH3), 1.20 (t, J¼7 Hz, 3H, OCH2CH3), 1.50e1.70
(m, 3H, CHCH2CH(CH3)2), 1.91 (s, 3H, CH3-acetyl), 3.90 (d, J¼6 Hz,
2H, NCH2CO), 4.15 (q, J¼6 Hz, 2H, OCH2CH3), 4.40e4.53 (m, 1H,
NCHCO), 7.30 (m, 1H, NH), 7.60 (m, 1H, NH).
A suspension of a0 (1 g, 3.8 mmol) in 6 ml of NaOH 1 N and ac-
etone (4 mL) was stirred at room temperature for 2 h, and after
removing acetone, acidified with HCl 6 M. The precipitated crystals
were collected and washed with water. The crystals were recrys-
tallized from ethyl acetate and oven dried to give N-Acetyl-
L-leu-
cylglycine (b) (0.97 g, 97%).
White crystal, mp: 176e178 ꢃC; 1H NMR: (DMSO-d6)
d 0.85e0.9
(m, 6H, 2CH3), 1.4e1.6 (m, 3H, CHCH2CH(CH3)2), 1.80 (s, 3H, CH3-
acetyl), 3.70 (d, J¼6 Hz, 2H, NCH2CO), 4.30 (q, J¼6 Hz 1H, NCHCO),
8.00 (d, J¼9 Hz, 1H, NH), 8.20 (t, J¼6 Hz, 1H, NH), one exchangeable
proton is missing.

S.M. Seyedi et al. / Tetrahedron 66 (2010) 6754e6760
6759
Anal. Calcd for C10H18N2O3: C, 52.17; H, 7.83; N, 12.17. Found: C,
52.34; H, 7.80; N, 12.22.
1.53e1.62 (m, 2H, 2CHCH2CH(CH3)2), 1.82 (s, CH3-acetyl), 3.6e3.75
(m, 4H, 2NCH2CO), 4.17e4.20 (m, 1H, NCHCO), 4.28e4.32 (m, 1H,
NCHCO), 7.74 (d, J¼8.4 Hz, 1H, NH), 8.05e8.09 (dd, J1¼7.4 Hz,
J2¼7.4 Hz, 1H, NH), 8.1e8.24 (m, 2H, 2NH), 12.5 (br s, 1H, COOH)
Anal. Calcd for C18H32N4O6: C, 53.98; H, 8.05; N, 13.99. Found: C,
53.72; H, 7.99; N, 14.01.
4.3.2. N-Acetyl-
L-leucylglycyl-L-leucine (c). Isobutyl chloroformate
(1.4 mL, 11 mmol) was added dropwise to a solution of N-acetyl-
L-
leucylglycine (b) (2.30 g, 10 mmol) and DBU (1.6 mL) in chloroform
(50 mL) while stirring in ice-water. After removing the ice-bath, the
reaction mixture was stirred at room temperature for 1 h. Leucine
methyl ester (1.81 g, 10 mmol) was then added and stirring con-
tinued for 3 h. The resulting solution was washed with 1 M NaOH
(2ꢄ30 ml), 1 M HCl (2ꢄ30 ml), and water (1ꢄ30 ml). The organic
layer was dried over anhydrous magnesium sulfate. After removing
the solvent under reduced pressure, the residue was recrystallized
4.4. General procedure for the synthesis of 3aed
Compound heptakis (6-bromo-6-deoxy)-b-cyclodextrin (2) was
synthesized according to the method reported in the literature.12 A
mixture of compound 2 (0.50 g, 0.32 mmol), one of the peptides
aed (4.0 mmol) and DBU (0.64 mL, 4.2 mmol) in DMF (10 mL) was
heated at 70e80 ꢃC for 12e48 h (for a and b 12 h and for c and
d 48 h). After cooling, the reaction mixture was then poured in to
a solution of saturated NaCl (40 mL). The precipitated solids were
collected and washed with water and potassium carbonate 5% and
then oven dried to give products 3aed.
from ethyl acetate/hexane to give N-acetyl-
L
-leucylglycyl-
L-leucine
methyl ester (b0) (1.58 g, 69%).
White crystal, mp: 133e135 ꢃC; 1H NMR: (CD3OCD3)
d
0.80e0.97
(m, 12H, 4CH3), 1.5e1.7 [m, 6H, 2CHCH2CH(CH3)2], 1.91 (s, 3H, CH3-
acetyl), 3.60 (s, 3H, OCH3), 3.70e3.90 (m, 2H, NCH2CO), 4.20e4.40
(m, 1H, NCHCO), 7.40e7.53 (m, 2H, 2NH), 7.70e7.85 (br m, 1H, NH).
Anal. Calcd for C17H31N3O5: C, 57.12; H, 8.74; N, 11.76. Found: C,
56.49; H, 8.70; N, 11.77.
4.4.1. Heptakis [6-O-(N-acetyl-
L
-leucyl)]-
b
-cyclodextrin (3a). White
A suspension of b0 (0.89 g, 2.5 mmol) in solution of NaOH (2 g) in
methanol (30 mL) was stirred at room temperature for 2 h. Then
water (10 mL) was added and after removing of methanol, acidified
with HCl 6 M. The precipitated crystals were collected and washed
with water. The crystals were recrystallized from acetone and oven
solid (0.34 g, 68%), mp: 197e198 ꢃC; 1H NMR: (DMSO-d6)
d
0.86 (d,
J¼6 Hz, 42H, 14CH3), 1.45e1.55 (m, 14H, 7CHCH2CH(CH3)2),
1.60e1.66 (m, 7H, 7CHCH2CH(CH3)2), 1.85 (s, 21H, CH3-acetyl),
3.40e3.48 (m, 14H, H-2, H-4), 3.60e3.69 (m, 7H, H-3), 3.80e3.89
(m, 7H, H-5), 4.03e4.10 (m, 7H, 7NCHCO), 4.22e4.28 (m, 14H, 2H-
6), 4.80e4.85 (m, 7H, H-1), 5.89e5.96 (m, 14H, OH-2 and OH-3),
dried to give N-Acetyl-L-leucylglycyl-L-leucine (c) (0.68 g, 77%).
White crystal, mp: 174e175 ꢃC; 1H NMR: (DMSO-d6) 0.80e0.87
(m, 12H, 4CH3), 1.4e1.48 (m, 4H, 2CHCH2CH(CH3)2), 1.54e1.62 (m,
2H, 2CHCH2CH(CH3)2), 1.82 (s, 3H, CH3-acetyl), 3.62e3.73 (m, 2H,
NCH2CO), 4.14e4.22 (m, 1H, NCHCO), 4.26e4.32 (m, 1H, NCHCO),
7.94e8.04 (m, 1H, 2NH), 8.05e8.07 (m, 1H, NH), 8.16e8.32 (m, 1H,
NH), one exchangeable proton is missing.
8.22e8.24 (m, 7H, 7NH). 13C NMR (DMSO-d6)
d 21.56, 22.63, 23.28,
24.91(CH3 and CH isobutyl, CH3 acetyl), 40.39 (CH2 isobutyl), 50.66
(HNCHCO), 63.63, 69.59, 72.67, 73.18 (C6, C5, C3, and C2), 81.94 (C4),
102.65 (C1),169.99 (CONH),172.66 (COO). IR (KBr disc)
n 1738 (C]O
ester) and 1682 (C]O amide) cmꢀ1
.
Anal. Calcd for C98H161N7O49: C, 52.99; H, 7.31; N, 4.41. Found: C,
52.70; H, 7.10; N, 4.25.
Anal. Calcd for C16H29N3O5: C, 55.98; H, 8.45; N, 12.24. Found: C,
55.77; H, 8.49; N, 12.20.
4.4.2. Heptakis [6-O-(N-acetyl-
White solid (0.31 g, 62%), mp: 210e212 ꢃC; 1H NMR: (DMSO-d6)
0.84 (d, 42H, 14CH3), 1.35e1.50 (m, 14H, 7CHCH2CH(CH3)2),
1.53e1.63 (m, 7H, 7CHCH2CH(CH3)2), 1.84 (s, 21H, 7CH3-acetyl),
3.40e3.46 (m, 14H, H-2, H-4), 3.58e3.64 (m, 7H, H-3), 3.79e3.87
(m, 21H, H-5, and 7NCH2CO), 4.19e4.31 (m, 21H, 2H-6, 7NCHCO),
4.80e4.90 (m, 7H, H-1), 5.80e6.00 (m, 14H, OH-2, and OH-3),
7.91e8.00 (m, 7H, 7NH), 8.25e8.39 (m, 7H, 7NH). 13C NMR (DMSO-
L-leucylglycyl)]-b-cyclodextrin (3b).
4.3.3. N-Acetyl-
formate (1.4 mL, 11 mmol) was added dropwise to a solution of N-
acetyl- -leucylglycyl- -leucine (c) (3.43 g, 10 mmol) and of DBU
(1.6 mL) in chloroform (50 mL) while stirring in ice-water. After
removing the ice-bath, the reaction mixture was stirred at room
temperature for 1 h. Glycine ethyl ester (1.81 g, 10 mmol) was then
added and stirring continued for 3 h. The resulting solution was
washed with 1 M NaOH (2ꢄ30 mL), 1 M HCl (2ꢄ30 mL), and water
(1ꢄ30 mL). The organic layer was dried over anhydrous magnesium
sulfate. After removing of the solvent under reduced pressure, the
L-leucylglycyl-L-leucylglycine (d). Isobutyl chloro-
d
L
L
d6)
d 21.97, 22.94, 23.49, 24.62 (CH3 and CH isobutyl, CH3 acetyl),
41.03, 41.27 (CH2 isobutyl, HNCH2CO), 51.16 (HNCHCO), 63.84,
69.33, 72.64, 73.28 (C6, C5, C3, and C2), 81.95 (C4), 102.67 (C1),
residue was recrystallized from hexane to give N-acetyl-
glycyl-
-leucine methyl ester (c0) (2.16 g, 63%).
White crystal, mp: 140e141 ꢃC; 1H NMR: (DMSO-d6, ppm)
L
-leucyl-
169.74, 169.92 (CONH), 173.28 (COO). IR (KBr disc)
n 1731 (C]O
L
ester) and 1680 (C]O amide) cmꢀ1
.
d
0.81
Anal. Calcd for C112H182N14O56: C, 51.33; H, 7.00; N, 7.48. Found:
C, 51.96; H, 7.19; N, 7.81.
(d, J¼6.5 Hz, 6H, 2CH3), 0.86 (d, J¼6.5 Hz, 6H, 2CH3), 1.14 (t, J¼10 Hz,
3H, OCH2CH3), 1.39e1.47 (m, J¼7.3 Hz, 4H, 2CHCH2CH(CH3)2),
1.55e1.6 (m, J¼7 Hz, 2H, 2CHCH2CH(CH3)2), 1.82 (s, 3H, CH3-acetyl),
3.65e3.79 (m, 4H, 2NCH2CO), 4.05 (q, J¼7.1 Hz, 2H, OCH2CH3),
4.16e4.22 (m, 1H, NCHCO), 4.25e4.32 (m, 1H, NCHCO), 7.78 (d,
J¼8.35 Hz, 1H, NH), 8.03 (dd, J¼10, 5.35 Hz, 1H, NH), 8.19e8.22 (m,
1H, NH), 8.4 (t, J¼8 Hz, 1H, NH).
4.4.3. Heptakis [6-O-(N-acetyl-
(3c). White solid (0.33 g, 67%), mp: 197e199 ꢃC; 1H NMR: (DMSO-
d6)
L-leucylglycyl-leucyl)]-b-cyclodextrin
d
0.85 (d, J¼10 Hz, 84H, 28CH3), 1.43e1.59 (m, 42H, 14CHCH2CH
(CH3)2), 1.84 (s, 21H, 7CH3-acetyl), 3.35e3.38 (m, 14H, H-2 and H-3,
H-4), 3.64e3.89 (m, 28H, H-3 and H-5, and 7NHCH2CO), 4.1e4.32
(m, 28H, H-6, and 14NCHCO), 4.81e4.87 (m, 7H, H-1), 5.89e6.06
(m, 14H, OH-2, and OH-3), 8.00e8.31 (m, 21H, 21NH). 13C NMR
Anal. Calcd for C20H36N4O6: C, 56.06; H, 8.47; N, 13.07.Found: C,
55.79; H, 8.44; N, 12.99.
A suspension of c0 (1.07 g, 2.5 mmol) in solution of NaOH (2 g) in
ethanol (30 mL) was stirred at room temperature for 2 h. Then
water (10 mL) was added and after removing of ethanol, acidified
with HCl 6 M. The precipitated crystals were collected and washed
with water. The crystals were recrystallized from acetone and oven
(DMSO-d6) d 21.60, 22.00, 22.09, 22.92, 23.31, 23.44, 24.60 (CH3 and
CH isobutyl, CH3 acetyl), 40.96, 40.23 (CH2 isobutyl, HNCH2CO
covered by DMSO pick), 50.53, 51.74, 52.00 (HNCHCO), 63.66, 69.51,
72.61, 73.26 (C6, C5, C3, and C2), 81.76 (C4), 102.69 (C1), 169.36,
170.14, 172.40 (CONH), 173.03 (COO). (KBr disc)
n 1733 (C]O ester)
dried to give N-Acetyl-
L
-leucylglycyl-
L
-leucine (d) (0.76 g, 71%).
and 1681 (C]O amide) cmꢀ1
.
White crystal, mp: 170e171 ꢃC; 1H NMR: (DMSO-d6, ppm)
Anal. Calcd for C154H259N21O63: C, 54.20; H, 7.65; N, 8.26. Found:
C, 53.13; H, 7.27; N, 8.15.
d
0.80e0.87 (m, 12H, 4CH3), 1.40e1.48 (m, 4H, 2CHCH2CH(CH3)2),

6760
S.M. Seyedi et al. / Tetrahedron 66 (2010) 6754e6760
4.4.4. Heptakis [6-O-(N-acetyl-
L
-leucylglycyl-
L
-leucylglycyl)]-
b
-cy-
References and notes
clodextrin (3d). White solid (0.27 g, 54%), mp: 200e201 ꢃC; 1H
1. Uekama, K.; Hirayama, F.; Irie, T. Chem. Rev. 1998, 98, 2045e2076.
2. Duchene, D. News Trends in Cyclodextrin and Derivatives; Edition de Santé: Paris,
1991.
NMR: (DMSO-d6)
d
0.87 (d, J¼6.5 Hz, 84H, 28CH3), 1.44e1.6 (m,
42H,14CHCH2CH(CH3)2),1.84 (s, 21H, 7CH3-acetyl), 3.33 (m,14H, H-
2, and H-4), 3.61e3.84 (m, 42H, H-3 and H-5, and 14NCH2CO),
4.21e4.32 (m, 28H, 2H-6, and 14NCHCO), 4.85e4.93 (m, 7H, H-1),
5.81e5.96 (m, 14H, OH-2, and OH-3), 7.8e8.3 (m, 28H, 28NH). 13C
3. Szejtli, J. Med. Res. Rev. 1994, 14, 353e386.
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1999, 318, 82e90.
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NMR (DMSO-d6)
d 21.84, 22.09, 22.57, 22.90, 23.42, 23.58, 24.61
(CH3 and CH isobutyl, CH3 acetyl), 40.57, 41.00, 41.21, 42.45 (CH2
isobutyl, HNCH2CO), 51.12, 51.85, 52.00 (HNCHCO), 63.66, 69.58,
72.56, 73.33 (C6, C5, C3, and C2), 81.90 (C4), 102.53 (C1), 169.17,
170.05, 170.35, 170.66 (CONH), 173.11 (COO). (KBr disc)
n 1735 (C]O
ester) and 1681 (C]O amide) cmꢀ1
.
Anal. Calcd for C168H280N28O70: C, 52.94; H, 7.30; N, 10.29.
Found: C, 52.36; H, 7.40; N, 9.96.
Acknowledgements
We express our sincere gratitude to Dr. Hossein. Orafaie for
reviewing the manuscript. The results described in this paper were
part of a M.Sc. student thesis proposal.
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Supplementary data
Copies of 1H NMR and 13C NMR spectra for compounds b, c, d,
and 3aed. Supplementary data associated with this article can be
found in online version at doi:10.1016/j.tet.2010.06.075. These data
include MOL files and InChIKeys of the most important compounds
described in this article.
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