Synthesis and properties of alkyl phosphorylcholine amphiphiles with a linear and an asymmetrically branched alkyl chain

Article abstract of DOI:10.1246/bcsj.78.1558

Alkyl phosphorylcholine amphiphiles bearing one linear chain and one asymmetrically branched alkyl chain were successfully synthesized using 2-chloro-2-oxo-1,3,2-dioxaphospholane in tetrahydrofuran or ethyl acetate. ¹H and ³¹PNMR studies revealed that the linear alkyl phosphorylcholines (C_n-PC) provide aqueous micelles in D₂O and reverse micelles in CDCl₃, while the branched alkyl phosphorylcholines (ISOFOL_n-PC) give vesicles in D₂O. The critical micelle concentrations (CMCs) of C_n-PC were measured by fluorescence dye solubilization methods: the CMCs of C₁₂-PC, C ₁₄-PC, C₁₆-PC, and C₁₈-PC were 1.6, 0.38, 0.16, and 0.11 mM, respectively, in water at 25 °C. The critical association concentrations (CACs) of ISOFOL_n-PC, ISOFOL_n-PC, and ISOFOL₂₄-PC were 0.068, 0.005, and 0.077 mM, respectively, in water at 25 °C. The vesicle size of ISOFOL_n-PC in aqueous solution was measured by the dynamic light scattering method. The mean diameter of ISOFOL_n-PC vesicles was approximately 30 nm and the size distribution was relatively monodisperse. The ISOFOL_n-PC vesicles formed were colloidally stable in water over the period of several weeks.

Full text of DOI:10.1246/bcsj.78.1558

1558 Bull. Chem. Soc. Jpn., 78, 1558–1564 (2005)
ꢀ 2005 The Chemical Society of Japan
Synthesis and Properties of Alkyl Phosphorylcholine Amphiphiles
with a Linear and an Asymmetrically Branched Alkyl Chain
ꢀ
Eui-Chul Kang, Shingo Kataoka, and Kenji Kato
Tsukuba Research Laboratory, NOF Corporation, 5-10 Tokodai, Tsukuba, Ibaraki 300-2635
Received July 13, 2004; E-mail: euichul kang@nof.co.jp
Alkyl phosphorylcholine amphiphiles bearing one linear chain and one asymmetrically branched alkyl chain were
successfully synthesized using 2-chloro-2-oxo-1,3,2-dioxaphospholane in tetrahydrofuran or ethyl acetate. 1H and
31P NMR studies revealed that the linear alkyl phosphorylcholines (Cn-PC) provide aqueous micelles in D2O and reverse
micelles in CDCl3, while the branched alkyl phosphorylcholines (ISOFOLn-PC) give vesicles in D2O. The critical mi-
celle concentrations (CMCs) of Cn-PC were measured by fluorescence dye solubilization methods: the CMCs of C12-PC,
C14-PC, C16-PC, and C18-PC were 1.6, 0.38, 0.16, and 0.11 mM, respectively, in water at 25 ꢁC. The critical association
concentrations (CACs) of ISOFOL16-PC, ISOFOL20-PC, and ISOFOL24-PC were 0.068, 0.005, and 0.077 mM, respec-
tively, in water at 25 ꢁC. The vesicle size of ISOFOLn-PC in aqueous solution was measured by the dynamic light scat-
tering method. The mean diameter of ISOFOLn-PC vesicles was approximately 30 nm and the size distribution was rel-
atively monodisperse. The ISOFOLn-PC vesicles formed were colloidally stable in water over the period of several
weeks.
Low molecular weight amphiphiles such as surfactants and
phospholipids have been widely used in many fields, such as
cosmetics, pharmaceuticals, and paints. There are classified
as anionic, cationic, nonionic, or zwitterionic surfactants ac-
cording to their polar hydrophilic head groups. During the past
four decades, many research groups have extensively studied
the fundamental and physicochemical properties of these am-
phiphiles with regard to the roles of molecular structure, polar
head groups and counter ions in aqueous solutions.1–3 Of those,
however, the studies of zwitterionic surfactants were relatively
fewer than those of other anionic, cationic, and nonionic sur-
factants. The phosphatidylcholine is one of the representative
zwitterionic amphiphiles, which most widely exist on cell
membranes.
branched alkyl chain.17 However, the asymmetrically branched
alkyl phosphorylcholines used in this study were synthesized
by the reaction of 2-chloro-2-oxo-1,3,2-dioxaphospholane
and a primary alcohol with an asymmetrically branched alkyl
chain. In this paper, the new synthesis and properties of alkyl
phosphorylcholines with one linear and one asymmetrically
branched alkyl chain are described in detail using NMR,
DSC, dynamic light scattering, fluorescence spectroscopy,
and static surface tension measurements.
Experimental
Materials. 1-Dodecanol (C12-OH), 1-tetradecanol (C14-OH),
1-hexadecanol (C16-OH), and 1-octadecanol (C18-OH) were pur-
chased from Tokyo Kasei Co., Tokyo, Japan. 2-Hexyl-1-decanol
(ISOFOL16), 2-octyl-1-dodecanol (ISOFOL20), and 2-decyl-1-
tetradecanol (ISOFOL24) were purchased from Sasol Germany
GmbH, Brunsbuettel, Germany. 2-Chloro-2-oxo-1,3,2-dioxaphos-
pholane and L-ꢀ-dipalmitoyl phosphatidylcholine (DPPC) are our
products (Nippon Oil & Fats Co., Ltd., Tokyo, Japan). Tetra-
hydrofuran, ethyl acetate, and acetonitrile were purchased from
Kanto Chemicals Co., Tokyo, Japan, as dehydrated grade solvents.
Triethylamine and diisopropylamine were purchased from Wako
Pure Chemicals Co., Tokyo, Japan. Trimethylamine was pur-
chased from Tokyo Teisan Co., Ltd., Tokyo, Japan. All other
solvents and chemicals were used without further purification.
Synthesis of Linear Alkyl Phosphorylcholines (Cn-PCs).
The synthetic procedure of linear alkyl phosphorylcholine was
as follows, in general. A solution of 10.0 g (70.0 mmol) of 2-
chloro-2-oxo-1,3,2-dioxaphospholane in 20 mL of dry ethyl ace-
tate was added dropwise to a mixture of 13.04 g (70.0 mmol) of
1-dodecanol and 7.08 g (70.0 mmol) of diisopropylamine in 100
mL of dry ethyl acetate at 0 ꢁC under vigorous stirring. After
Various polymers having a phosphorylcholine polar group
were synthesized and studied.4–9 Several phosphorylcholine
polymers,
2-(methacryloyloxy)ethyl
phosphorylcholines
(MPCs), have shown the enhanced biocompatibility of the
substrate surfaces which are coated by them.6–9 Several phos-
phorylcholine amphiphiles with a linear alkyl chain were also
synthesized,10–12 and their physicochemical and biological
properties were investigated in regard to the critical micelle
concentration (cmc),13,14 the solubility in aqueous and organic
media,12,15 the interaction of phospholipase A2,16 and antimi-
crobial properties.14 The phosphorylcholine amphiphiles bear-
ing a single symmetrically branched alkyl chain were also
studied by Overmars and his co-workers.17 These symmetrical-
ly branched alkyl phosphorylcholine amphiphiles exhibited the
formation of stable vesicles with diameters of 30–100 nm, as
was confirmed by electron microscopy, fluorescence depolari-
zation measurements, and differential scanning calorimetry
(DSC). These symmetrically branched alkyl phosphorylcho-
lines were synthesized by reaction of 2-chloro-1,3,2-dioxa-
phospholane and the secondary alcohol of a symmetrically
ꢁ
the addition was completed, stirring was continued at 0 C for 1
h and at room temperature for another 1 h under nitrogen atmo-
sphere. The diisopropylamine hydrochloride that precipitated
Published on the web August 4, 2005; DOI 10.1246/bcsj.78.1558

E.-C. Kang et al.
Bull. Chem. Soc. Jpn., 78, No. 8 (2005) 1559
was filtered off. The filtrate was evaporated under reduced pres-
sure up to half of the content. Each concentrated solution of 1-
(2-oxo-1,3,2-dioxaphospholan-2-yloxy) dodecane and 150 mL of
dry acetonitrile was placed into a glass pressure bottle. After the
For the purification of 2-hexyldecyl phosphorylcholine
(ISOFOL16-PC), diethyl ether was used, and for that of 2-octyl-
dodecyl phosphorylcholine (ISOFOL20-PC), tetrahydrofuran/
acetonitrile was employed.
ꢁ
mixture was cooled down to ꢂ20 C, 8.3 g (0.14 mol) of anhy-
1H NMR (CD3OD) ꢁ for ISOFOL16-PC: 0.89 (t, 6H, CH3), 1.37
(m, 24H, (CH2)5, (CH2)7), 1.58 (m, 1H, OCH2CH), 3.23 (s, 9H,
þN(CH3)3), 3.62 (m, 2H, CH2Nþ), 3.77 (t, 2H, CH2OP), 4.24
(br, 2H, POCH2) and for ISOFOL20-PC: 0.89 (t, 6 H, CH3) 1.31
(m, 32 H, (CH2)7, (CH2)9) 1.58 (m, 1 H, OCH2CH), 3.22 (s,
9H, þN(CH3)3), 3.62 (m, 2H, CH2Nþ), 3.77 (t, 2H, CH2OP),
4.24 (br, 2H, POCH2). 31P NMR ꢁ for ISOFOL16-PC: 1.24; and
for ISOFOL20-PC: 1.24. MS (FAB) m=z: 408 (MHþ): Anal. Calcd
for C21H47NPO4 (MHþ); and 464 (MHþ): Anal. Calcd for
C25H55NPO4.
IR and NMR Measurements. IR spectra with KBr were
recorded with a Fourier transform infrared spectrometer (FTIR-
7300, Jasco Co., Tokyo, Japan). 1H NMR (270 MHz) studies were
carried out in D2O (deuterated water), CD3OD (deuterated meth-
anol), and CDCl3 (deuterated chloroform) by using a JEOL JNM-
EX270 spectrometer. The chemical shifts were recorded as parts
per million (ppm) with a reference to residual solvent resonance.
31P NMR (109 MHz) studies were made with the same spectrom-
eter using phosphoric acid as the external standard. Line widths
were measured as the full width at half-height maximum intensity.
The concentration of NMR samples was approximately 2.0 wt %
in all experiments. The ISOFOLn-PC samples were prepared in
deuterated water by sonication.
drous trimethylamine was added, and the reaction was carried
ꢁ
out at 70 C for 12 h. After 12 h, the reaction mixture was again
ꢁ
cooled down to ꢂ20 C to precipitate dodecyl phosphorylcholine
(C12-PC). The precipitates were separated by filtration, and dis-
solved in a small amount of ethanol. C12-PC was reprecipitated
by pouring the C12-PC/ethanol solution into an excess amount
ꢁ
of ethyl acetate. Subsequent drying in vacuum for 24 h at 50 C
yielded 9.1 g (37%) of a hygroscopic white solid.
1H NMR (CDCl3) ꢁ 0.88 (t, 3H, CH3), 1.25 (m, 18H, (CH2)9),
1.57 (m, 2H, OCH2CH2), 3.41 (s, 9H, þN(CH3)3), 3.77–3.84 (m,
4H, CH2Nþ, CH2OP), 4.29 (br, 2H, POCH2). 31P NMR ꢁ 0.61.
MS (FAB) m=z: 352 (MHþ). Anal. Calcd for C17H39NPO4.
For the synthesis and purification of tetradecyl phosphorylcho-
line (C14-PC), hexadecyl phosphorylcholine (C16-PC), and octa-
decyl phosphorylcholine (C18-PC), tetrahydrofuran was used for
ethyl acetate as the reaction solvent. C14-PC was purified by etha-
nol/ethyl acetate. C16-PC was purified by ethanol/diethyl ether.
For the purification of C18-PC, tetrahydrofuran was used.
1H NMR (CDCl3) ꢁ for C14-PC: 0.88 (t, 3H, CH3), 1.25 (m,
22H, (CH2)11), 1.58 (m, 2H, OCH2CH2), 3.41 (s, 9H, þN(CH3)3),
3.76–3.84 (m, 4H, CH2Nþ, CH2OP), 4.27 (br, 2H, POCH2); for
C16-PC: 0.88 (t, 3H, CH3), 1.25 (m, 26H, (CH2)13), 1.57 (m,
2H, OCH2CH2), 3.40 (s, 9H, þN(CH3)3), 3.76–3.83 (m, 4H,
CH2Nþ, CH2OP), 4.27 (br, 2H, POCH2); and for C18-PC: 0.88
(t, 3H, CH3), 1.25 (m, 30H, (CH2)15), 1.57 (m, 2H, OCH2CH2),
3.40 (s, 9H, þN(CH3)3), 3.76–3.83 (m, 4H, CH2Nþ, CH2OP),
4.27 (br, 2H, POCH2). 31P NMR ꢁ for C14-PC: 0.58; for C16-
PC: 0.54; and for C18-PC: 0.54. MS (FAB) m=z: 380 (MHþ):
Anal. Calcd for C19H43NPO4, 408 (MHþ): Anal. Calcd for
C21H47NPO4, and 436 (MHþ): Anal. Calcd for C23H51NPO4.
Synthesis of Branched Alkyl Phosphorylcholines
(ISOFOLn-PCs). The synthetic procedure of branched alkyl
phosphorylcholine was as follows, in general. A solution of 10.0
g (70.0 mmol) of 2-chloro-2-oxo-1,3,2-dioxaphospholane in 30
mL of dry tetrahydrofuran was added dropwise to a mixture of
24.78 g (70.0 mmol) of 2-decyltetradecanol and 7.1 g (70ꢁ.0 mmol)
of triethylamine in 140 mL of dry tetrahydrofuran at 0 C under
vigorous stirrinꢁg. After the addition was completed, stirring was
continued at 0 C for 1 h and at room temperature for another 1
h under nitrogen atmosphere. The triethylamine hydrochloride
that precipitated was filtered off. The filtrate was evaporated under
reduced pressure up to half of the content. Each concentrated so-
lution of 1-(2-oxo-1,3,2-dioxaphospholan-2-yloxy) 2-decyltetra-
decane and 150 mL of dry acetonitrile was placed into a glass
pressure bottle. After the mixture was cooled down to ꢂ20 ꢁC,
8.3 g (0.14 mol) of anhydrous trimethylamine was added, and
Vesicle Size Measurements. The ISOFOLn-PC samples (20.0
mg) were suspended in water (10.0 mL) under stirring for 30 min
ꢁ
at 50 C. The resulting suspension was sonicated using a probe-
type sonifier (Branson Sonifier 250) for 10 min at 40 W. The size
of ISOFOLn-PC vesicles was measured by NICOMP 380ZLS
Particle Sizer with DPSS laser (wavelength, 532 nm).
Static Surface Tension Measurements. Aqueous ISOFOLn-
PC solutions were prepared by dissolving the ISOFOLn-PC in
Milli-Q water to the desired concentration. The static surface
tension of aqueous ISOFOLn-PC was measured with a Surface
Tensiometer CBVP-A3 by the Wilhelmy plate technique (Kyowa
Interface Sci. Co., Tokyo, Japan).
Fluorescence Measurements. Aqueous Cn-PC solutions were
prepared by dissolving the Cn-PC in Milli-Q water to the desired
concentration. The fluorescence dye solubilization method was
employed to determine the onset of surfactant micellization
(CMC). A stock solution of 1.0 mM pyrene in THF was prepared.
A 10 mL aliquot of the pyrene/THF stock solution was added to
2.0 mL of Cn-PC solution, so that the final Cn-PC solution con-
tained 0.5% v/v THF and 0.005 mM pyrene. The solution was left
in the dark to equilibrate for 30 min before the fluorescence mea-
surement. Fluorescence spectra were recorded on a Hitachi F-
3010 fluorescence spectrometer. The excitation wavelength was
330 nm, and the slit widths were set at 5.0 nm (excitation) and
3.0 (emission). The I1=I3 ratio of the pyrene emission was taken
as the ratio of the emission intensity at 373 nm to that at 384
nm. The temperature of the water-jacketed cell holder was con-
trolled with an Omron circulating controller.
ꢁ
the reaction was carried out at 70 C for 12 h. After 12 h, the re-
action mixture was again cooled down to ꢂ20 ꢁC to precipitate 2-
decyltetradecyl phosphorylcholine (ISOFOL24-PC). The precipi-
tates were separated by filtration. ISOFOL24-PC was purified by
tetrahydrofuran and acetone. Subsequent drying in vacuum for
24 h at 50 ꢁC yielded 15.2 g (41.8%) of a hygroscopic white solid.
1H NMR (CD3OD) ꢁ 0.89 (t, 6H, CH3), 1.29 (m, 40H, (CH2)9,
(CH2)11), 1.58 (m, 1H, OCH2CH), 3.22 (s, 9H, þN(CH3)3), 3.62
(m, 2H, CH2Nþ), 3.77 (t, 2H, CH2OP), 4.23 (br, 2H, POCH2).
31P NMR ꢁ 1.25. MS (FAB) m=z: 520 (MHþ). Anal. Calcd for
C29H63NPO4.
Results and Discussion
Syntheses of several alkyl phosphorylcholine amphiphiles
with linear and branched alkyl chains succeeded: C12-
OH, C14-OH, C16-OH, C18-OH, ISOFOL16, ISOFOL20, and
ISOFOL24 with 2-chloro-2-oxo-1,3,2-dioxaphospholane in tet-

1560 Bull. Chem. Soc. Jpn., 78, No. 8 (2005)
Properties of Alkyl Phosphorylcholines
1
rahydrofuran or ethyl acetate (Scheme 1).
The H NMR spectra and line width of the major peaks of
C12-PC in various solvents are shown in Fig. 2 and Table 1.
In D2O, the peaks due to the choline methyl group were rela-
tively sharp at 3.14 ppm, whereas the peaks at 0.78 ppm (meth-
yl) and 1.19 ppm (methylene) assigned to the dodecyl group
protons were broad. This broadening suggests that the motion
of the corresponding protons is restricted, providing evidence
for the formation of a sort of aggregate via association of
the dodecyl chains. But this aggregation disappears when
C12-PC is dissolved in methanol. The spectrum of C12-PC in
CD3OD appeared as sharp peaks for all the proton signals.
On the other hand, in CDCl3, the peaks attributed to the dodec-
yl protons appeared as sharp peaks, while the peaks due to the
trimethylammonium protons of choline group were observed
as a broad peak with a downfield shift in comparison with
the locations in CD3OD. The peaks attributed to the methylene
protons of choline group at 3.57 ppm in D2O were observed as
broad and partially overlapping peaks at 3.81 ppm in CDCl3.
The IR spectra of alkyl phosphorylcholines are shown in
Fig. 1. The absorption peaks characteristic of the alkyl chains
appeared at 2924 cmꢂ1, 2854 cmꢂ1 (C–H stretching), and 1468
cmꢂ1 (C–H bending). The absorption bands at 1247 cmꢂ1
(P=O stretching), 1089 cmꢂ1 (C–O–P stretching), and 970
cmꢂ1 (Nþ(CH3)3 stretching) are attributed to the existence
of phosphorylcholine groups. The alkyl phosphorylcholines
did not exhibit an absorption band at 1738 cmꢂ1 (C=O stretch-
ing) in comparison with those of DPPC. All other absorptions
coincided with the structure of DPPC.
O
CH2
CH2
O
O
Cl
P
O
P
CH2
CH2
O
O
R O
R OH
THF or EtOAc
O
P
CH3
TMA
MeCN
OCH2CH2N CH3
CH3
R O
O
g
a
b
c
d
e
f
O
P
OCH2CH2N(CH3)3
CH3(CH2)9CH2CH2O
R: (CH2)xCH3
CH2CH(CH2)nCH3
(CH2)mCH3
x = 11,13,15,17
O
m = 7, n = 5 (ISOFOL16)
9,
11,
7 (ISOFOL20)
9 (ISOFOL24)
(a)
H2O
g
b
Scheme 1. Synthetic route of alkyl phosphorylcholine am-
phiphiles.
a
c
e
d f
(a)
(b)
(c)
(b)
(c)
g
b
H2O
MeOH
a
d
f
e
c
TMS
g
b
a
d,f
c
e
4000
3000
2000
Wavenumber / cm
1600
1000
600
-1
ppm
Fig. 1. IR spectra of (a) dodecyl phosphorylcholine
(C12-PC), (b) 2-decyltetradecyl phosphorylcholine
(ISOFOL24-PC), and (c) L-ꢀ-dipalmitoyl phosphatidyl-
choline (DPPC).
5
4
3
2
1
0
Fig. 2. 1H NMR spectra of dodecyl phosphorylcholine
(C12-PC) in (a) D2O, (b) CD3OD, and (c) CDCl3 at 25 ꢁC.
Table 1. 1H NMR Chemical Shifts (ppm) and Line WidthsaÞ (Hz) of C12-PC (with Reference to
Fig. 2)
Solvent
D2O
a
b
c
d
e
f
g
0.78
1.19
(10.8 Hz)
1.28
(4.1 Hz)
1.25
(4.3 Hz)
1.51
3.75
4.16
3.57
3.14
(2.2 Hz)
3.21
(1.7 Hz)
3.41
(4.2 Hz)
CD3OD
CDCl3
0.89
0.88
1.61
1.57
3.86
3.81
4.24
4.29
3.62
3.81
a) Line widths were measured as the full width at half-height maximum intensity.

E.-C. Kang et al.
Bull. Chem. Soc. Jpn., 78, No. 8 (2005) 1561
Thus, the motion of the choline group of phosphorylcholine is
highly restricted in CDCl3. This indicated that the phosphoryl-
choline moieties are confined in a restricted environment,
while the alkyl chains can rotate freely. The phosphorus peak
of phosphorylcholine in C12-PC appeared as a sharp peak at
0.53, 1.09, and 0.61 ppm in D2O, CD3OD, and CDCl3, respec-
Table 2. In CD3OD, the signals of the choline group and of
the 2-decyltetradecyl group were relatively sharp. In CDCl3,
the 2-decyltetradecyl signals were also sharp, while the peaks
of choline methyl protons were observed as broad at a down-
field location. This also indicates that the phosphorylcholine
moieties locate in a restricted environment, while the alkyl
chains can rotate relatively freely. On the other hand, in
D2O, the trimethylammonium protons of the choline group
at 3.15 ppm and the 2-decyltetradecyl protons at 0.83 ppm
(methyl) and 1.23 ppm (methylene) all were broad. The peaks
at 3.81 ppm assigned to the methylene protons of the 2-decyl-
tetradecyl group in CD3OD appeared as a broad band and
overlapped with peaks observed at 3.58 ppm in D2O. This re-
sult indicates that the phosphorylcholine moieties and the alkyl
chain are both in restricted microenvironments.
1
tively (Fig. 4a). H and 31P NMR of other linear alkyl phos-
phorylcholines, C14-PC, C16-PC, and C18-PC, also showed
spectra similar to those of C12-PC. Results of 1H and 31P NMR
indicate that the linear alkyl phosphorylcholines would form
aqueous micelle in D2O and reverse micelle in CDCl3.
Interestingly, 1H and 31P NMR spectra of the branched alkyl
phosphorylcholines exhibited chemical shifts slightly different
from those of the linear alkyl phosphorylcholines. The
1H NMR spectra and line width of the major peaks of
ISOFOL24-PC in various solvents are shown in Fig. 3 and
Figure 4b shows the 31P NMRꢁspectra of ISOFOL24-PC in
D2O, CD3OD, and CDCl3 at 25 C. The phosphorus peak of
the phosphorylcholine of ISOFOL24-PC observed at 1.24
ppm in CD3OD was sharp, similarly to the case of C12-PC.
However, in D2O, the phosphorus peak of ISOFOL24-PC ap-
peared as a broad and symmetric peak with a peak width of
38.2 Hz at 0.54 ppm. This also indicates that the molecular
motion of the phosphorylcholine moieties of ISOFOL24-PC
O
g
a
b
c
d
e
f
OCH2CH2N(CH3)3
P
CH3(CH2)11CHCH2O
O
CH3(CH2)8CH2
1
in D2O is more highly restricted than that of C12-PC. H and
(a)
H2O
g
(a)
(b)
In D O
2
e
d,f
(b)
(c)
g
b
H2O
MeOH
a
In
CD OD
3
d f
e
c
In CDCl
3
ppm
ppm
ppm
5
4
3
2
1
0
2
1
0
2
1
0
Fig. 3. 1H NMR spectra of 2-decyltetradecyl phosphoryl-
choline (ISOFOL24-PC) in (a) D2O, (b) CD3OD, and (c)
Fig. 4. 31P NMR spectra of (a) dodecyl phosphorylcholine
(C12-PC) and (b) 2-decyltetradecyl phosphorylcholine
ꢁ
ꢁ
CDCl3 at 25 C.
(ISOFOL24-PC) in D2O, CD3OD, and CDCl3 at 25 C.
Table 2. 1H NMR Chemical Shifts (ppm) and Line WidthsaÞ (Hz) of ISOFOL24-PC (with Reference
to Fig. 3)
Solvent
D2O
a
b
c
d
e
f
g
0.83
1.23
(31.5 Hz)
1.29
(3.9 Hz)
1.22
(6.4 Hz)
—
3.58
4.18
3.58
3.15
(9.0 Hz)
3.22
(1.7 Hz)
3.41
(4.4 Hz)
CD3OD
CDCl3
0.90
0.85
1.60
1.52
3.81
3.89
4.28
4.31
3.65
3.71
a) Line widths were measured as the full width at half-height maximum intensity.

1562 Bull. Chem. Soc. Jpn., 78, No. 8 (2005)
Properties of Alkyl Phosphorylcholines
O
g
a
b
c
d
e
f
At 25 °C in CD OD
OCH2CH2N(CH3)3
P
CH3(CH2)7CHCH2O
3
O
CH3(CH2)4CH2
At 70 °C
H2O
g
b
At 70 °C in D O
a
2
e
d f
c
At 45 °C
H2O
At 25 °C in D O
2
H2O
At 25 °C
2
1
0
-1
Fig. 6. 31P NMR spectra of 2-hexyldecyl phosphorylcho-
line (ISOFOL16-PC) under different conditions.
d,f
ppm
5
4
3
2
1
0
Fig. 5. 1H NMR spectra of 2-hexyldecyl phosphorylcholine
(ISOFOL16-PC) in D2O at different temperatures.
(a)
(b)
(c)
(d)
31P NMR spectra of other branched alkyl phosphorylcholines,
ISOFOL16-PC (peak width of phosphorus peak, 17.2 Hz) and
ISOFOL20-PC (peak width of phosphorus peak, 28.7 Hz), were
also similar to those of ISOFOL24-PC. Generally, the phos-
phorus peak of the conventional egg PC liposomes exhibits a
broad and asymmetric peak in D2O.18,19 The shape of the peak
is characteristic of the lamellar structure of lipid bilayers.
31P NMR spectrum of the ISOFOL24-PC in this work was also
similar to those of the egg PC liposomes.18,19 These NMR re-
sults would suggest the formation of vesicles via self-associa-
tion of asymmetrically branched alkyl chains in D2O.
To understand the molecular motion in solution in more de-
tail, NMR studies were carried out at different temperatures
over 25–70 ꢁC. Figures 5 and 6 show results of 1H and
31P NMR spectra of ISOFOL16-PC measured at different tem-
peratures. With increasing temperature, the methyl protons at
0.79 ppm and the methylene protons at 1.19 ppm of the 2-hex-
yldecyl group, and the trimethylammonium protons at 3.14
ppm of the choline group became sharper and more intense, in-
dicating an increase in the motion of both the phosphorylcho-
line moieties and the 2-hexyldecyl chains. We noted a broad
peak that would be attributable to the methylene protons (sig-
nal, d) of the 2-hexyldecyl and that overlapped with the meth-
ylene protons (signal, f) of the choline group at 3.53 ppm. This
2
1
0
-1
Fig. 7. 31P NMR spectra of (a) C12-PC, (b) ISOFOL16-PC,
(c) ISOFOL20-PC, and (d) ISOFOL24-PC in D2O at 25 ꢁC.
creasing temperature.
Figure 7 shows the 31P NMR spectra of linear and branched
alkyl phosphorylcholines, C12-PC, ISOFOL16-PC, ISOFOL20-
PC, and ISOFOL24-PC in D2O at 25 ꢁC. The phosphorus peak
of alkyl phosphorylcholines in D2O was observed at 0.53–0.63
ppm. Though the phosphorus peaks of linear alkyl phosphoryl-
cholines all appeared relatively sharper and symmetric, those
of all branched alkyl phosphorylcholines appeared broader
ꢁ
broad peak at 3.53 ppm at 25 C separated into two peaks at
ꢁ
3.53 and 3.62 ppm above 45 C. In 31P NMR spectrum, the
phosphorus peak also shifted from 0.63 ppm at 25 ꢁC to
ꢁ
1.23 ppm at 70 C and slightly sharpened (Fig. 6). These re-
sults indicate that the mobilities of the phosphorylcholine moi-
eties and the alkyl chains both significantly increase with in-

E.-C. Kang et al.
Bull. Chem. Soc. Jpn., 78, No. 8 (2005) 1563
1.7
1.6
1.5
1.4
1.3
1.2
1.1
-40
-30
-20
-10
0
10
20
-5
-4
-3
-2
-1
Temperature / °C
log[ Alkyl phosphorylcholine ]
Fig. 8. DSC curve of ISOFOL24-PC obtained on heating.
Fig. 9. Changes in I1=I3 ratio of pyrene emission in aque-
ous solution as a function of the logarithm of alkyl phos-
phorylcholine concentration at 25 ꢁC: C12-PC ( ); C14-PC
and unsymmetric. With the branched alkyl phosphorylcho-
lines, the phosphorus peak became gradually broader with in-
creasing alkyl chain length. These results indicate that the mo-
bility values of the branched alkyl phosphorylcholines are sig-
nificantly restricted in water compared to those of unbranched
alkyl phosphorylcholines. This difference would be related
with the solution structures of different aggregates such as mi-
celle and vesicle.
(
); C16-PC ( ); C18-PC ( ).
80
70
60
50
40
30
20
10
Thermograms of ISOFOLn-PC were recorded on a differen-
tial scanning calorimeter (DSC; Seiko DSC-210) at a heating
rate of 5 ꢁC/min (Fig. 8). The phase transition temperature be-
tween the gel and the liquid-crystal phase was determined
from the peak temperature of the main endothermic transition.
The phase transition temperature of ISOFOL16-PC was not ob-
ꢁ
served in the temperature range of ꢂ70 to 20 C. The phase
-8
-7
-6
-5
-4
-3
-2
-1
transition temperatuꢁres of ISOFOL20-PC and ISOFOL24-PC
were ꢂ63 and ꢂ21 C, respectively.
log[ Alkyl phosphorylcholine ]
The sizes of the branched alkyl phosphorylcholine aggre-
gates in aqueous solution were measured by the dynamic light
scattering method. The mean diameters of ISOFOL20-PC and
ISOFOL24-PC were approximately 32:1 ꢃ 4:9 and 29:0 ꢃ 3:8
nm, and were relatively monodisperse. The ISOFOLn-PC ag-
gregates were colloidally stable in water over several weeks.
The result also suggests that the branched alkyl phosphoryl-
cholines form aggregates larger in size than those of the un-
branched alkyl phosphorylcholines.
Fig. 10. Static surface tension in aqueous solution as a
function of the logarithm of alkyl phosphorylcholine con-
centration at 25 ꢁC: C12-PC ( ); ISOFOL16-PC ( );
ISOFOL20-PC ( ); ISOFOL24-PC ( ).
above the CMC. The CMC was determined from the inflection
of the I1=I3 values vs alkyl phosphorylcholine concentration
curves. The CMC of Cn-PC decreased with an increase of
the hydrophobic alkyl chain length: the CMCs of C12-PC,
C14-PC, C16-PC, and C18-PC werꢁe 1.6, 0.38, 0.16, and 0.11
mM, respectively, in water at 25 C. In the CMC of C12-PC
and C14-PC, the CMC decreased to approximately a half when
one methylene group is added in the hydrophobic alkyl chain.
On the other hand, the change of CMC between C16-PC and
C18-PC was very small. These results are agreement with the
experimental values of ionic and nonionic surfactants.23
Figure 10 shows the surface tension curves of alkyl phos-
phorylcholine in aqueous solution as a function of log alkyl
phosphorylcholine concentration at 25 ꢁC. The surface tension
decreased with an increase of alkyl phosphorylcholine concen-
tration. Especially, the surface tension of ISOFOL20-PC and
ISOFOL24-PC dramatically decreased with an increase of al-
kyl phosphorylcholine concentrations. The critical association
concentration (CAC) of ISOFOLn-PC was determined from
the inflection of the surface tension values vs alkyl phospho-
rylcholine concentration curves. The CACs of ISOFOL16-
PC, ISOFOL20-PC, and ISOFOL24-PC were 0.068, 0.005,
and 0.077 mM, respectively, in water at 25 ꢁC. The association
The CMC of Cn-PC was studied by the fluorescence dye sol-
ubilization method. The fluorescence dye solubilization meth-
ods, based upon changes in fluorescence intensity of a dye
upon incorporation into micelles, are among the most sensitive
and convenient assays for CMC.20,21 The solvent polarity
dependence of the pyrene emission is expressed in terms of
the ratio, I1 (373 nm)/I3 (384 nm) of the intensities of the
(0,0) band (I1) to that of the (0,2) band (I3) of the emission.
The CMC of surfactants can be obtained from measurements
of the changes in I1=I3 ratio of the intensity of the pyrene emis-
sion as a function of surfactant concentration. The values typ-
ically range from about 1.9 in polar solvents to about 0.6 in hy-
drocarbons.21,22 Figure 9 shows the changes of the I1=I3 ratio
of pyrene emission in aqueous solution as aꢁfunction of log al-
kyl phosphorylcholine concentration at 25 C. The I1=I3 ratio
of pyrene sharply decreased with an increase of alkyl phos-
phorylcholine concentration, and leveled off (ꢄ1:21). This in-
dicated that the pyrene is solubilized in the hydrophobic inte-
rior as the alkyl phosphorylcholine concentrations increase

1564 Bull. Chem. Soc. Jpn., 78, No. 8 (2005)
Properties of Alkyl Phosphorylcholines
and N. Nakabayashi, J. Biomed. Mater. Res., 26, 1543 (1992).
8 A. L. Lewis, P. D. Hughes, L. C. Kirkwood, S. W.
Leppard, R. P. Redman, L. A. Tolhurst, and P. W. Stratford,
concentrations of the branched alkyl phosphorylcholines were
lower than those of the unbranched alkyl phosphorylcholines
in aqueous media.
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To characterize these aggregates formed in aqueous media
as spherical micelle, mesophase, or vesicle, Tanford24 and
Israelachvili25,26 proposed a concept of packing parameter.
9
The critical packing parameter (CPP) is defined as v=ao lc,
ꢅ
where v is the hydrocarbon chain volume, ao is the optimal
cross-sectional surface area per head group, and lc is the crit-
ical chain length of the alkyl chains.24 The amphiphiles form
micelles when the CPP-value is below 0.5, while they form
vesicles with the CPP-value is between 0.5 and 1.0. We calcu-
lated the CPP-value of alkyl phosphorylcholines with the val-
ꢀ 2
ues of 62.0–71.7 A for hydrated choline head group of phos-
phatidylcholine vesicle.25,26 The calculated CPP-values are
0.29–0.34 for the linear alkyl phosphorylcholines and 0.61–
0.71 for the branched alkyl phosphorylcholines; such values
are consistent with the micelle-forming properties in the linear
alkyl phosphorylcholines and the vesicle-forming properties in
the branched alkyl phosphorylcholines, respectively. These re-
sults are consistent with the present results of NMR, DSC, dy-
namic light scattering, fluorescence spectroscopy, and static
surface tension measurements for the alkyl phosphorylcholine
amphiphiles.
17 F. J. J. Overmars, J. B. F. N. Engberts, and W. D. Weringa,
Recl. Trav. Chim. Pays-Bas, 113, 293 (1994).
18 J. Seelig, Biochim. Biophys. Acta, 515, 105 (1978).
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21 K. Kalyanasundaram and J. K. Thomas, J. Am. Chem. Soc.,
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22 H. Ringsdorf, J. Venzmer, and F. W. Winnik, Macromole-
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23 A. Kitahara and K. Aoki, ‘‘Introduction to Colloid and
Surface Chemistry,’’ 3rd ed, Hirokawa Publishing Co., Tokyo
(1987), Chap. 4, p. 75.
24 C. Tanford, J. Phys. Chem., 76, 3020 (1972).
25 J. N. Israelachvili, D. J. Mitchell, and B. W. Ninham,
J. Chem. Soc., Faraday Trans. 2, 1976, 1525.
26 J. N. Israelachvili, S. Marcelja, and R. G. Horn, Q. Rev.
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7