H.-Y. Chen et al. / Tetrahedron 70 (2014) 2378e2382
2381
into the reference cell. 0.80 mM solutions of AMP, ADP, and ATP
were placed in a 250
The concentrations of
m
b
L continuously rotating (250 rpm) syringe.
-GCD and -biGCD were 0.05 mM. The ti-
b
trant and titrand concentrations were selected to ensure the cri-
terion 10<Ka[M]T<1000, in which [M]T is the total concentration of
Scheme 2. Synthetic scheme of L2.
host. The titration was conducted by adding 10
nucleotide solutions into the solutions of the
rivatives. The first 10 L aliquot injection was ignored to eliminate
the effect of solute diffusion across the syringe tip during the
equilibration period.33,34 Thereafter, the 10
L aliquot of the nu-
mL aliquots of the
b
-cyclodextrin de-
m
4.7. Synthesis of mono-6-deoxy-6-biguanidino-b-cyclodex-
m
trin (b-biGCD)
cleotide solutions was injected at an interval of 10 min. The signal
(heat flow) produced during each injection leveled off when the
guest became excessive. All titration experiments were carried out
at 298ꢀ0.1 K.35 The titration data were fitted with the Origin pro-
gram provided by VP-ITC instrument, the fitting formula was
shown as following equation:
b
-biGCD was prepared from the intermediate 6-monodeoxy-6-
monoamino- -cyclodextrin ( -ACD), which was first prepared
from -cyclodextrin ( -CD) through a series of reactions. To a so-
lution of -ACD (1.134 g, 1.000 mmol) in dry dimethylformamide
b
b
b
b
b
(DMF) was added diisopropylethylamine (DIEA) (2.5 mL,
0.120 mmol) followed by L2 (0.230 g, 1.220 mmol). The mixture was
stirred for 72 h at room temperature.36,37 At the end of the reaction,
15 mL of ether was added, and the resulting sticky solid product
was collected, washed using ether, and dried in vacuo to yield
0.235 g (19%) of crude product. The crude product was purified by
column chromatography on Sephadex G-25 using deionized water
as eluent. 0.18 g (16%) pale crystalline solid was precipitated from
acetone/ether.
2
3
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
ꢀ
ꢁ
2
NMtDHV0
Xt
1
Xt
1
4Xt
4
5
Q ¼
1þ
þ
ꢂ
1þ
þ
ꢂ
NMt
2
NMt NKMt
NMt NKMt
(1)
The value of Q above can be calculated at the end of the injection
and designated Q. V0¼active cell volume; Mt is bulk and free con-
centration of macromolecule in V0; Xt is bulk and free concentration
of ligand. Thus, the number of sites (N), the molar enthalpy changes
1H NMR (500 MHz, DMSO-d6, ppm) 7.93 (2H, d,
C
C
44H73N3O34N2H2), 2.00 (2H, s, C44H73N3O34N2H2), 6.57 (2H, s,
44H73N4O34NH2), 4.30e4.72 (20H, m, eOH), 4.88e4.82 (7H, m,
(D
H), and the inclusion compound association constants Ka were
obtained from this fitting process, and S was calculated based on
the value of H and Ka. The total heat produced during each in-
jection was obtained by integrating heat flows over time. The cal-
culated total heat ( cal) was plotted against the mole ratio of the
D
eCH); 13C NMR dH (500 MHz, DMSO-d6, ppm) 162.7 (1C, s,
D
C
43H75N2O34CN3), 162.1 (1C, s, C43H75N2O34CN3), 101.9 (7C, s, eCH),
59.8e77.9 (m, 34C, eCH2); MS (ESI, H2O, m/z) [MþH]þ calcd 1218.3,
found 1218.5. Anal. Calcd for (C44H75N5O34)$(H2O)8: C (38.80), H
(6.73), N (5.14). Found C (38.83), H (6.67), N (5.18) (Scheme 3).
m
titrant (guest) to the titrand (host). ‘Exo’ denoted the exothermic
reaction.
The free energy (
DG) of the binding reaction is determined via:
D
G ¼
D
H ꢂ T
D
S
(2)
in which
data.
DH and DS can be obtained from the fitting process of ITC
4.5. Molecular modeling
The semiempirical calculations were performed using the Mo-
lecular Docking Simulations 2.1 software combined with SYSBL 7.3
to study the hosteguest inclusion compounds. The molecular
structures were generated by the molecular builder provided in the
SYSBL 7.3 and optimized by means of Molecular Docking Simula-
tions. The initial distances between the key atoms of inclusion
compounds were summarized in Supplementary data.
4.6. Synthesis of N-amidino-amidinopyrazole-1 hydrochlo-
ride (L2)
DIEA (0.348 mL, 2.0 mmol) was added to 0.5 mL of DMF solution
of amidinopyrazole-1 hydrochloride L1 (0.293 g, 2.0 mmol). The
mixture was stirred for 64 h at room temperature.36 Then ether
(15 mL) was added, and the resulting sticky solid product was
collected by filtration, washed with ether, and dried in vacuum to
yield 0.34 g (90%) of crude product. Recrystallization of the crude
product from ethanol or ether yielded 0.13 g (42%) white crystalline
solid.
Scheme 3. Synthetic scheme of b-biGCD.
Caution: The perchlorate salts of organic compounds are po-
tentially explosive. These compounds must be prepared and han-
dled with great care!
1H NMR (500 MHz, DMSO-d6, ppm) 6.57 (1H, s, C5H7N5NH), 7.85
(1H, d, C5H7N5NH), 8.20 (2H, s, C5H6N5NH2), 8.35e8.37 (3H, m,
C3H3N5H5); MS (ESI, H2O, m/z) [MþH]þ calcd 152, found 153.15.
Anal. Calcd for C5H8N6$HCl$H2O: C (28.79), H (5.42), N (40.30).
Found C (28.83), H (5.38), N (40.26) (Scheme 2).
Acknowledgements
This work was supported by National Natural Science Founda-
tion of China (Nos., 21121061 and 21172274), the Doctoral Scientific