68
C. Gervaise et al. / Biochimie 94 (2012) 66e74
31P NMR Hahn Echo experiments with high power proton
0.353 mmol, 1 eq) and diisopropylethylamine (60 mL, 0.353 mmol,
decoupling were performed at magnetic fields of 7.0 T or 11.7 T. The
static NMR experiments (31P resonance frequency of 121.49 MHz)
were done on a Bruker ASX-300 spectrometer (Bruker, Wissembourg,
France) using a 7 mm CP-MAS probe and the MAS NMR experiments
were realized on a Bruker DRX 500 (Bruker, Wissembourg, France) at
a frequency of 202.47 MHz with a 4 mm CP-MAS probe. A stable
spinning rate was obtained at 4.7 kHz. The 90ꢃ pulse length was set to
1 eq) were added. The reaction mixture was stirred at 0 ꢃC for 1 h and
at room temperature for 1 h. The solvent was removed by evaporation
under reduced pressure. Then, diethyl ether was added to precipitate
diisopropylethylamine which was eliminated by filtration. The
solvent was removed by evaporation under reduced pressure and
the residue was purified by column chromatography on silica gel
(CH2Cl2/MeOH 98:2) with 35% yield.
7
m
s at 121.49 MHz and 7.5
ms at 202.47 MHz. A 40
ms delay was used
1H NMR (500 MHz, CDCl3)
5.14e5.09 (m, 7H, H1, HI); 4.00e3.75 (m, 13H, H5, HV, H6a); 3.95
d (ppm): 5.37e5.31 (m, 4H, Hdiene);
between the 90ꢃ pulse and the 180ꢃ pulse. Respectively 38,900 and
2600 scans were acquired for static and MAS experiments. The
recycling time was set to 5 s. Line broadenings of 100 Hz and 10 Hz
were applied respectively for static and MAS experiments. All
measurements were performed at 25 ꢃC. The chemical shifts were
referenced relative to external H3PO4.
0
0
0
(t, 4H, H1 , J1 ,2 ¼ 7 Hz); 3.66e3.44 (m, 20H, H3, HIII, H4, HIV, H6b);
3.63 (s, 18H, O(6)CH3); 3.50 (s, 21H, O(3)CH3); 3.38 (s, 21H, O(2)CH3);
3.30 (m, 2H, HVIa, HVIb); 3.18 (m, 6H, H2, HII); 3.11 (dd, 1H, HII,
0
0
0
JI,II ¼ 9.5 Hz, JII,III ¼ 3 Hz); 2.00 (m, 8H, H8 , H11 ); 1.64 (m, 8H, H2 ,
0
0
0
0
H3 ); 1.28 (m, 40H, CH2); 0.88 (t, 6H, H18 , J17 ,18 ¼ 7 Hz).
13C NMR (125 MHz, CDCl3)
d
(ppm): 130,4, 130,1 (4C, C]C);
2.4. Surface tension measurement
99.7e99.1 (7C, C1, CI); 82.8e81.8 (14C, C2, CII, C3, CIII); 80.9e80.5 (7C,
C4, CIV); 71.4e71.0 (13C, C5, CV, C6); 66.7 (2C, C7, CI); 61.9e61.6 (6C,
O(6)CH3); 59.6e59.3 (7C, O(3)CH3); 59.0e58.7 (7C, O(2)CH3); 42.5
Surface tension (
g
) was measured at 20 ꢃC for each solution,
M for example
0 0 0
(1C, CVI); 31.0, 30.9 (4C, C2 , C3 ); 33.0e23.1 ( 20C, CH2); 27.6 (4C, C8 ,
obtained by dilution of a mother solution S0 (83.2
m
0
0
with phosphate buffer solution) in the range S0/2, S0/4, S0/6, S0/16,
S0/32, S0/64 and S0/128, after attaining thermal and area equilib-
rium (more than 12 h) using the Willielmy method (Tensimat N3
Prolabo tensiometer). The critical micellar concentration value
C11 ); 14.5 (2C, C18 ) Elemental analysis C98H180NO37P, H2O found C,
58.35; H, 8.84, N, 0.67 requires C, 58.46; H, 9.11, N, 0.70.
HRMS (ESI): m/z: 2017.1956 ([M þ Na]þ, (C98H180NO37NaP)
requires 2017.1870).
(CMC) was performed using graph
the molar concentration of the solution.
g
¼ f(log C), in which C indicates
2.7.2. 6I-Amido- -alanyl-(6-Di-oleylphosphoramidyl)-6I-deoxy-
b
2I,3I-di-O-methyl-hexakis(2IIeVII,3IIeVII,6IIeVII-tri-O-methyl)-
cyclomaltoheptaose 5
2.5. Calibration curve of carboxyfluorescein fluorescence intensity
in water
Lyophilized 6I-Amido- -alanyl-6I-deoxy-2I,3I-di-O-methyl-hex-
b
akis(2IIeVII,3IIeVII,6IIeVII-tri-O-methyl)-cyclomaltoheptaose (100 mg,
From a mother solution of 0.1 mg/mL of carboxyfluorescein in
water a dilution was done to obtain a 10ꢁ6 mg/mL solution. Then,
from this preparation, ten dilutions were prepared from 10ꢁ7 to
10ꢁ6 mg/mL. Measurements of fluorescence are obtained from
these different dilutions using a fluorimeter (VersaFluorÔ Fluo-
rometer BIO-RAD) to trace the calibration curve of carboxy-
fluorescein fluorescence intensity in water (R2 ¼ 0.994). The
excitation filter used was 485e495 nm and the emission filter was
505e515 nm.
0.067 mmol, 1 eq) and dioleylphosphite (39 mg, 0,067 mmol, 1 eq)
were dissolved in anhydrous dichloromethane (350
atmosphere. At 0 ꢃC, a spatula of molecular sieves 3 Å, bromotri-
chloromethane (66 L, 0.674 mmol, 10 eq) and diisopropylethylamine
(117 L, 0.674 mmol, 10 eq) were added. The reaction mixture was
mL) under argon
m
m
stirred at 0 ꢃC for 1 h and at room temperature for 1 h. The solvent
was removed by evaporation under reduced pressure. Then, diethyl
ether was added to precipitate diisopropylethylamine which was
eliminated by filtration. The solvent was removed by evaporation
under reduced pressure and the residue was purified by column
chromatography on silica gel (CH2Cl2/MeOH 98:2) to give 5 with the
yield of 66%.
2.6. Calibration curve absorbance of scopolamine derivatives
Different solutions of scopolamine and N-methyl-scopolamine
were prepared from 10ꢁ5 mol/L to 10ꢁ4 mol/L. Measurements of
absorbance are obtained from these solutions using a UVeVisible
spectrophotometer (Cary 50 Bio, Varian) to trace the calibration
curve of scopolamine derivatives absorbance in different aqueous
media: NaCl 0.9% (R2 ¼ 0.994 for scopolamine and N-methyl-
1H NMR (500 MHz, CDCl3)
d (ppm): 6.19 (s, 1H, NHCO); 5.34 (m,
0
0
0
4H, Hdiene); 5.16e5.07 (m, 7H, H1, HI); 3.95 (t, 4H, H1 , J1 ,2 ¼ 9.5 Hz);
3.97e3.75 (m, 13H, H5, HV, H6a); 3.69e3.45 (m, 20H, H3, HIII, H4, HIV,
H6b); 3.63 (s, 18H, O(6)CH3); 3.51e3.49 (s, 21H, O(3)CH3); 3.38 (s,
0
21H, O(2)CH3); 3.21e3.16 (m, 2H, H19 ); 3.19 (dd, 7H, H2, HII,
0
J1,2 ¼ JI,II ¼ 15.5 Hz, J2,3 ¼ JII,III ¼ 5.5 Hz); 2.37 (t, 2H, H20
,
scopolamine) Glucose 5% (R2
¼
0.990 for scopolamine and
J19 e20 ¼ 11 Hz); 2.01e1.97 (m, 8H, H8 , H11 ); 1.67e1.62 (m, 4H, H2 ,
0
0
0
0
0
R2 ¼ 0.997 N-methyl-scopolamine), phosphate buffer (R2 ¼ 0.995
for scopolamine and R2 ¼ 0.981 N-methyl-scopolamine). For
measurements on entrapment of scopolamine derivatives in
nanoparticles, a blank has been done with nanoparticles alone in
the aqueous media. The wavelengths used were 197 nm for
scopolamine and 202 nm for N-methyl-scopolamine.
H3 ); 1.25 (m, 40H, CH2); 0.87 (t, 6H, H18 ,, J17 -18 ¼ 10.5 Hz).
0
0
0
0
13C NMR (125 MHz, CDCl3)
d
(ppm): 171.6 (1C, NHCO); 130.4,
130.2 (4C, C]C); 99.5e99.1 (7C, C1, CI); 82.3e80.7 (21C, C2, CII, C3,
0
CIII, C4, CIV); 71.8e70.2 (14C, C5, CV, C6, CVI); 66.9 (2C, C1 ); 62.0 (6C, O
0
0
C(6)CH3); 59.5e58.7 (14C, O(3)CH3, O(2)CH3); 38.2 (2C, C19 , C20 );
0
32.3e23.1 (20C, CH2); 14.5 (2C, C18 ).
Elemental analysis C101H185 N2O38P, H2O found C, 57.93; H, 8.89,
N, 1.35 requires C, 58.19; H, 9.04, N, 1.34.
2.7. Synthesis and product characterization
HRMS (ESI): m/z: 2088.2278 ([M þ Na]þ, (C101H185N2O38NaP)
requires 2088.2241).
2.7.1. 6-Di-oleylphosphoramidyl-6I-deoxy-2I,3I-di-O-methyl-
hexakis(2IIeVII,3IIeVII,6IIeVII-tri-O-methyl)-cyclomaltoheptaose 4
Lyophilized 6I-Amino-6I-deoxy-2I,3I-di-O-methyl-hexakis(2IIeVII
,
2.7.3. 6-Di-stearylphosphoramidyl-6I-deoxy-2I,3I-di-O-methyl-
hexakis(2IIeVII,3IIeVII,6IIeVII-tri-O-methyl)-cyclomaltoheptaose 6
3
IIeVII,6IIeVII-tri-O-methyl)-cyclomaltoheptaose (500 mg, 0.353 mmol,
1 eq) and dioleylphosphite (206 mg, 0.353 mmol,1 eq) were dissolved
in anhydrous dichloromethane (1 mL) under argon atmosphere. At
Lyophilized 6I-Amino-6I-deoxy-2I,3I-di-O-methyl-hexakis(2IIeVII
,
3
IIeVII,6IIeVII-tri-O-methyl)-cyclomaltoheptaose (100 mg, 0.071 mmol,
1 eq) and distearylphosphite (83 mg, 0.141 mmol, 2 eq) were
0 ꢃC, a spatula of molecular sieves 3 Å, bromotrichloromethan (35
mL,