Molecular recognition in anisotropic media. Binding of alkylpyridines to amphiphilic zinc porphyrins incorporated in liposomal bilayer membranes

Article abstract of DOI:10.1039/b817241b

Two amphiphilic zinc porphyrins were incorporated into liposomal bilayer membranes, egg phosphatidylcholine (Egg PC) and dipalmitoylphosphatidylcholine (DPPC). Binding free energy of alkylpyridines to the zinc porphyrins linearly increased as the length o

Full text of DOI:10.1039/b817241b

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Molecular recognition in anisotropic media. Binding of alkylpyridines to
amphiphilic zinc porphyrins incorporated in liposomal bilayer membranes†
Ryosuke Murakami, Asuka Minami and Tadashi Mizutani*
Received 1st October 2008, Accepted 9th January 2009
First published as an Advance Article on the web 16th February 2009
DOI: 10.1039/b817241b
Two amphiphilic zinc porphyrins were incorporated into liposomal bilayer membranes, egg
phosphatidylcholine (Egg PC) and dipalmitoylphosphatidylcholine (DPPC). Binding free energy of
alkylpyridines to the zinc porphyrins linearly increased as the length of the alkyl chains of the guest
increased, showing that the guest was incorporated deep in the bilayer membranes in a hydrophobic
environment. Comparison of the free energy increase per CH2 group indicated that recognition of
3-alkylpyridines were favored over that of 4-alkylpyridines by dDDG◦ = 0.3–0.7 kJ mol-1, and this
preference was a◦ttributed to the anisotropy of the liposomal bilayer membranes. The binding was
exothermic (DH = -15 to -21 kJ mol-1) when the liposome was in a liquid crystalline phase, while it
was endothermic (DH◦ = 53 to 62 kJ mol-1) when the liposome was in a gel phase. Local disorder of
lipid molecules may be a driving force for binding in the latter case. Lipid bilayer membranes provide a
unique medium for molecular recognition, in which the anisotropy and entropy of the lipid molecules
add new features to binding.
experience different solvation interactions, which in turn influence
Introduction
their function and in particular their binding affinity and selec-
Liquid crystals are characterised by both mobility and order, as
tivity. In the present work, we have examined the recognition of
expected for a mesophase lying between that of a crystalline array
different guests by amphiphilic receptors embedded in liposomal
and that of an isotropic liquid, although the mobility of a liquid
bilayer membranes having accessible gel and liquid crystal phases.
crystalline material is closer to that of a liquid than a solid.1
The guests were alkylpyridines and the receptors amphiphilic
For a solute dissolved in a liquid crystal, solvation interactions
Zn(II) porphyrins, and the results have shown that there is indeed a
may be similar to those in an isotropic liquid, except in that they
significant influence of solvation anisotropy on binding selectivity.
are modified by directional order. Such anisotropic solvation is a
unique feature of liquid crystals and is a factor to be considered
in biological materials such as cell membranes.
We prepared amphiphilic zinc porphyrins 1 and 2 as receptors
and incorporated them in a liposomal membrane.6,7 We then
studied the binding of alkylpyridines and imidazoles to these
receptors, and found that the receptors showed different selectivity
toward positional isomers of alkylpyridines. The binding is
exothermic in the liquid crystalline state, while it is endothermic
in the gel phase, implying that binding mechanisms sensitively
reflect the changes in the molecular motions of lipid molecules.
We suggest that local melting of the lipid molecules upon guest
binding is one of the driving forces of molecular recognition.
Molecular recognition is a process in which ‘preorganisation’
of the reacting species can have an enormous influence upon the
selectivity of recognition, as expressed in the value of the relevant
equilibrium constant.2 Since solvation is a form of molecular
recognition, any degree of organisation or ordering in a liquid
phase or an interface is expected to have a significant influence
upon solvation, as appreciated in areas as diverse as medicine and
materials science.3 Studies of the influence of anisotropy within
liquid crystals on molecular recognition are, nonetheless, relatively
limited.4
Results and discussion
Lyotropic liquid crystals such as liposome membranes are
involved in many important biological processes, such as signal
transduction and protein translocation. Liposomes typically show
a transition between gel and liquid crystal phases differing with
respect to both the mobility and ordering of the membrane
constituents.5 Thus, molecules embedded within the membranes
Synthesis of amphiphilic zinc porphyrins and incorporation
into liposomes
Two amphiphilic zinc porphyrins 1 and 2 with either two or four
dodecyloxy groups attached on one side and carboxylate groups on
the other were prepared according to Scheme 1, and characterized
by 1H NMR and mass spectra. Several isomers were formed, but
the cis and trans isomers could be separated by silica gel column
chromatography. The diagnostic 1H NMR signals for the two
isomers are b-pyrrole protons appearing in the 8.8–9.4 ppm region
– the cis isomer shows two singlets and two doublets, while the trans
isomer two doublets due to their symmetry (see ESI†). A more
polar fraction, the cis isomer, was collected, and its structure was
Department of Molecular Science and Biochemistry, Faculty of Science
and Engineering, Doshisha University, Kyotanabe, Kyoto, 610-0321, Japan.
E-mail: tmizutan@mail.doshisha.ac.jp; Fax: +81 774 65 6794; Tel: +81 774
65 6623
† Electronic supplementary information (ESI) available: 1H NMR spectra
of 1, 10a, 10b, 11b, and UV-visible spectral data of the receptors
in liposomes and the host–guest complexes in liposomes. See DOI:
10.1039/b817241b
1
confirmed by H NMR. Egg phosphatidylcholine (Egg PC) and
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Scheme 1 Preparation of amphiphilic zinc porphyrins. Reagents: (a) K2CO3, acetonitrile; (b) propionic acid; (c) KOH; (d) Zn(OAc)2.
Table 1 Binding constantsa,band binding free energy of alkylpyridines to
zinc porphyrin 1 incorporated in Egg PC liposomes at 25 ◦C. n is the
carbon number in the alkyl group (pyridyl-(CH2)nH)
dipalmitoylphosphatidylcholine (DPPC) liposomes incorporating
the zinc porphyrins were prepared by probe-sonication of a
suspension of lipids and the porphyrin followed by purification
by gel filtration.
4-Alkylpyridines
3-Alkylpyridines
n
K/M-1
-DG◦/kJ mol-1
n
K/M-1
-DG◦/kJ mol-1
Binding of alkylpyridines
1
2
3
5
800 110
16.6 0.3
19.1 0.07
20.9 0.2
26.0 0.07
1
2
4
6
590 65
1900 190
25000 1300 25.1 0.1
230000 18000 30.6 0.2
15.8 0.3
18.7 0.2
The binding constants of alkylpyridines in pH 7.1 Bis-Tris buffer
were determined by monitoring the absorbance in the Soret band
as a function of the concentrations of guest, followed by a least-
squares fit to the equilibrium equation for the formation of a 1 : 1
complex. Upon addition of the solution of 3-alkylpyridines or
4-alkylpyridines to the liposome solution incorporating 1, the
Soret band underwent a red-shift from 430 nm to 432 nm.8
Similarly the Soret band of 2 in liposome underwent a red-
shift from 431 nm to 433 nm upon addition of pyridines. In
the difference spectra, the minimum appeared at 425 nm and the
maximum at 434 nm. These spectral changes are similar to those
of binding of pyridines to zinc porphyrin, i.e., coordination of
pyridyl nitrogen to the zinc.9 The spectra no longer changes after
about 10 s. Thus the binding of the guest is relatively a fast process.
Binding constants of 3-alkylpyridines and 4-alkylpyridines to 1–
Egg PC liposome at 25 ◦C and 50 ◦C are listed in Tables 1 and 2,
respectively. Binding constants to 2–Egg PC liposome at 25 ◦C are
listed in Table 3. Binding constants to 1–DPPC liposome at 25 ◦C
and at 50 ◦C are listed in Tables 4 and 5, respectively. The binding
constants were determined three to four times for independently
prepared liposome solutions to check reproducibility. Compared
to the binding constants in uniform solutions, errors were larger,
particularly for DPPC at 25 ◦C. The standard errors of the means
are also shown in the tables. The transition temperature from
the gel phase to the liquid crystalline phase for DPPC has been
reported to be 41 ◦C,10 and thus the DPPC liposome is in the gel
phase at 25 ◦C and in the liquid crystalline phase at 50 ◦C. The Egg
PC liposome has a gel-to-liquid crystal transition temperature of
2240 60
4700 340
36800 980
a Averages of three to four independent determinations are shown. The
errors shown are standard errors of the mean. b The binding constant of
pyridine was 209 M-1.
Table 2 Binding constants aand binding free energy of alkylpyridines to
zinc porphyrin 1 incorporated in Egg PC liposomes at 50 ◦C. n is the
carbon number in the alkyl group (pyridyl-(CH2)nH)
4-Alkylpyridines
3-Alkylpyridines
n
K/M-1
-DG◦/kJ mol-1
n
K/M-1
-DG◦/kJ mol-1
1
2
3
5
340 96
1100 210
3000 360
15.7 0.8
18.8 0.5
21.5 0.3
1
2
4
6
310 45
1000 200
11000 2400 25.0 0.6
120000 10000 31.4 0.2
15.4 0.4
18.5 0.5
25000 3200 27.2 0.3
a Averages of three to four independent determinations are shown. The
errors shown are standard errors of the mean.
-10 ◦C, and thus it is in the liquid crystalline phase both at 25 ◦C
and 50 ◦C.
Recognition of alkyl chains and pyridyl nitrogen
Plots of the binding free energy against the alkyl chain length for
1–Egg PC and 2–Egg PC are shown in Fig. 1. The plots show
that the binding free energy increased linearly with increasing the
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Table 3 Binding constants aand binding free energy of alkylpyridines to
zinc porphyrin 2 incorporated in Egg PC liposomes at 25 ◦C. n is the
carbon number in the alkyl group (pyridyl-(CH2)nH)
energy of recognition of the alkyl group and the pyridyl nitrogen,
respectively. They are summarized in Table 6. As shown in Table 6,
the values of -dDG◦/dn for 4-alkylpyridines are in the range 2.3–
2.9 kJ mol-1. These values are similar to those reported for the
binding of 4-alkylpyridines to water-soluble zinc porphyrins: The
values of -dDG◦/dn are 2.2, 2.6, 3.2, and 2.6 kJ mol-1 per CH2 for
3, 4, 5, and 6, respectively (see Table 7). The results suggest that
the bilayer membrane and water-soluble porphyrins 3–6 provide a
similar hydrophobic environment around the guest.
4-Alkylpyridines
3-Alkylpyridines
n
K/M-1
-DG◦/kJ mol-1
n
K/M-1
-DG◦/kJ mol-1
1
2
3
5
450 33
1100 110
2600 600
15.1 0.2
17.4 0.2
19.5 0.6
1
2
4
6
410 19
14.9 0.1
17.7 0.1
23.2 0.03
28.5 0.2
1270 51
11800 140
99000 6700
22000 1500 24.8 0.2
a Averages of three to four independent determinations are shown. The
errors shown are standard errors of the mean.
Table 4 Binding constants aand binding free energy of alkylpyridines to
zinc porphyrin 1 incorporated in DPPC liposomes at 25 ◦C. n is the carbon
number in the alkyl group (pyridyl-(CH2)nH)
4-Alkylpyridines
3-Alkylpyridines
n
K/M-1
-DG◦/kJ mol-1
n
K/M-1
-DG◦/kJ mol-1
1
2
3
5
220 76
320 83
1200 190
9600 640
13.4 0.9
14.3 0.6
17.6 0.4
22.7 0.2
1
2
4
6
160 52
400 250
8000 550
12.6 0.8
14.8 1.5
22.3 0.2
27.3 0.1
60000 2300
a Averages of three to four independent determinations are shown. The
errors shown are standard errors of the mean.
Table 5 Binding constantsa and binding free energy of alkylpyridines to
zinc porphyrin 1 incorporated in DPPC liposomes at 50 ◦C. n is the carbon
number in the alkyl group (pyridyl-(CH2)nH)
4-Alkylpyridines
3-Alkylpyridines
n
K/M-1
-DG◦/kJ mol-1
n
K/M-1
-DG◦/kJ mol-1
1
2
3
5
290 60
910 30
2300 180
23000 1800 27.0 0.2
15.2 0.6
18.3 0.1
20.8 0.2
1
2
4
6
300 55
1000 130
12000 650
109000 3100 31.1 0.08
15.3 0.5
18.6 0.3
25.2 0.1
a Averages of three to four independent determinations are shown. The
errors shown are standard errors of the mean.
Comparison of the values of -dDG◦/dn of 3-alkylpyridines
with those of 4-alkylpyridines indicates that the recognition
free energy of the alkyl chain at the 3-position was greater
than that of the alkyl chain at the 4-position both in receptor
1 and receptor 2. The selectivity of 3-alkylpyridines over
4-alkylpyridines of 1 (-DdDG◦/dn) is higher (0.6–0.7 kJ mol-1)
than that of 2 (0.3 kJ mol-1) at 25 ◦C. At 50 ◦C, however,
the selectivity of◦3-alkylpyridines over 4-alkylpyridines of 1 was
reduced (-DdDG /dn = 0.2–0.3 kJ mol-1). The receptors 1 and 2
can recognize the alkyl chains of the guest, and the recognition
occurs more effectively for 3-alkylpyridines than 4-alkylpyridines.
Different affinity for alkyl groups at the 3-position and the
4-position suggests that anisotropic packing of lipid molecules
in the bilayer membrane affects the energy of hydrophobic
interactions (see Fig. 2).
The recognition parameter of the pyridyl nitrogen, -DG◦ (n =
0), is determined by both the basicity of the nitrogen and the
hydrophilicity/hydrophobicity around the zinc ion. Larger values
of -DG◦ (n = 0) of 4-ethylpyridine than those of 3-ethylpyridine
are thus attributed to the greater basicity of the former guest
(see Table 10). The partition of guest between the aqueous phase
and the liposomal bilayer membrane also affects the binding
Fig. 1 Plot of the free energy of binding to 1–Egg PC and 2–Egg PC at
25 ◦C against the number of CH2 groups in the alkyl group of guest.
alkyl chain length of the guest. The slope of the lines, -dDG◦/dn,
and the intercept, -DG◦ (n = 0), are a measure of the free
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a
Table 6 The binding free energy increment per CH2 group and the extrapolated binding free energy to n = 0, as determined from the slope and the
intercept of the plot. Typical examples of the plots are shown in Fig. 1
Entry
Host
Liposome
Guest
T/◦C
(-dDG◦/dn)/kJ mol-1
-DG◦at n = 0/kJ mol-1
Phase
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
2
2
Egg PC
Egg PC
DPPC
3-Alkylpyridines
4-Alkylpyridines
3-Alkylpyridines
4-Alkylpyridines
3-Alkylpyridines
4-Alkylpyridines
3-Alkylpyridines
4-Aalkylpyridines
3-Alkylpyridines
4-Alkylpyridines
25
25
25
25
50
50
50
50
25
25
2.98 0.07
2.35 0.09
3.0 0.2
12.9 0.3
14.2 0.3
9.4 0.7
10.3 0.8
12.2 0.02
12.9 0.09
12.2 0.1
12.3 0.1
12.2 0.1
12.6 0.3
LCb
LC
Gel
Gel
LC
LC
LC
LC
LC
LC
DPPC
2.5 0.3
Egg PC
Egg PC
DPPC
DPPC
Egg PC
Egg PC
3.20 0.004
2.86 0.03
3.18 0.04
2.92 0.04
2.72 0.03
2.41 0.09
10
a Errors are standard deviations of the regression analysis. b LC: liquid crystalline.
Table 7 Incremental free energy per CH2 group in the guest alkyl group
8 in Table 6) indicates that recognition of the basic nitrogen of
the pyridine ring is stronger in the liquid crystalline phase. The
variable temperature UV-visible absorption spectra of a solution
of 1–DPPC liposome are shown in Fig. 3. As the temperature
is lowered, the Soret band maximum is shifted to a longer
wavelength, suggesting that water coordinates to the zinc at lower
temperatures in the gel phase. Therefore this explains the lower
values of -DG◦ (n = 0) in the gel phase.
(-dDG◦/dn)/kJ mol-1
Ref.
4-Alkylpyridine, Egg PC
3-Alkylpyridine, Egg PC
4-Alkylpyridine, 3
4-Alkylpyridine, 4
4-Alkylpyridine, 5
4-Alkylpyridine, 6
Phase transfer from water
to hexane
2.35 0.09
2.98 0.07
2.2
2.6
3.2
2.60 0.05
3.85
This work
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31
31
12
Unpublished
13
Fig. 2 A schematic representation of 3-hexylpyridine bound to 1 in
DPPC.
Fig. 3 UV-visible spectra of 1–DPPC solution (pH 7.1 Bis-Tris buffer) in
the temperature range of 25 to 50 ◦C.
free energy. The hexadecane/water partition coefficients were
reported to be 1.3 (3-methylpyridine), 1.0 (4-methylpyridine),
5.8 (3-ethylpyridine), and 4.7 (4-ethylpyridine).11 These partition
coefficients suggest that 3-alkylpyridines are more distributed
in the liposomal bilayer membranes than 4-alkylpyridines. This
factor may contribute the difference between 3-alkylpyridines and
4-alkylpyridines of -DG◦ (n = 0), but not to the difference of
-dDG◦/dn. Comparison of 1 and 2 (entries 1 and 2 versus 9 and 10
in Table 6) shows that 1 showed better recognition of the pyridyl
nitrogen and better discrimination between the 3-alkylpyridines
and 4-alkylpyridines than 2. Packing of the alkyl chains in the
liposomal bilayer membranes may be different between the two
receptors. For instance, both receptors caused local disorder in
the packing of lipid molecules to different extent. This could
affect the binding behavior of the receptors. Comparison of the
two parameters, -dDG◦/dn and -DG◦ (n = 0), between the liquid
crystalline phase and the gel phase (entries 3 and 4 versus 7 and
Hydrophobic interactions in the bilayer membrane
It is interesting to compare the values of -dDG◦/dn to the
unitary free energy of transfer of hydrocarbon molecules from
a hydrocarbon solvent to an aqueous medium, which was studied
in detail by Hermann23 as well as Tanford and coworkers.24 Since
the origin of hydrophobic interactions is the interfacial energy
between the hydrophobic surface of solute and water, we can
2
˚
compare the free energy changes per 1 A increment/decrement
of the interfacial area. Hermann reported that the free energy for
transfer of saturated hydrocarbons from hydrocarbon to water is
proportional to the surface area of the cavity created by the solute
in the aqueous solution; the numerical value of the proportionality
2
constant is estimated to be 138 J mol-1 per A . Tanford et al.
˚
reported that the value falls between 83 and 105 J mol-1 per A at
2
˚
25 ◦C. Assuming the solvent accessible surface area of a CH2
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-1
group is 31.8 A, the values of 2.3 kJ mol and 3.0 kJ mol-1
Table 10 pKa values of pyridines and imidazoles
˚
of -dDG◦/dn correspond to the proportionality constants of
Amine
pKa
Ref.
hydrophobic interactions of 72 and 94 J mol-1 per A , respectively.
2
˚
The proportional constants obtained from the solubility data
and those obtained from the host–guest association constants are
close to each other, suggesting that the alkyl group of guest is
in a hydrophobic environment similar to that of a hydrocarbon
when bound to receptors 1 and 2 embedded in the liposomal
membranes.25
Pyridine
5.32
6.02
6.02
5.66
5.70
6.99
7.02
7.21
16
17
17
18
17
19
20
21
4-Methylpyridine
4-Ethylpyridine
3-Methylpyridine
3-Ethylpyridine
Imidazole
N-Methylimidazole
N-Ethylimidazole
Binding of imidazoles as a measure of local environment around
the binding site
binding to water-soluble porphyrin receptors with a hydrophobic
binding site. Porphyrin receptors 1 and 2 incorporated in the
liposomes showed the ratios between the porphyrin receptor
having a hydrophobic binding site (entry 1) and hydrophilic zinc
tetracarboxyphenylporphyrin 7 (entry 6). Therefore, in liposomes,
the bound imidazole should be close to the interface between the
aqueous layer and the alkyl chain layer, where water molecules
can penetrate to some extent. The ratios increase in the order 1–
DPPC, 2–Egg PC, and 1–Egg PC. The order is the same as the
increasing values of -DG◦ (n = 0): 10.3 for 1–DPPC, 12.6 for 2–
Egg PC, and 14.2 kJ mol-1 for 1–Egg PC at 25 ◦C. Although the
parameter -DG◦ (n = 0) reflects the hydrophilicity around the zinc
ion and the pyridine-to-imidazole binding constant ratios reflect
the hydrophilicity near the amino nitrogen of imidazole, these two
parameters show a similar behavior. In toluene, the ratios are low
(entry 7, Table 9), and this should be ascribed to the electronic
effects, since imidazoles are more basic than pyridines (Table 10).
The binding constants of N-methylimidazole and N-ethyl-
imidazole were determined similarly in pH 7.1 Bis-Tris buffer
at 25 ◦C, and are listed in Table 8. Imidazole has two nitrogen
atoms and the imino nitrogen is bound to the zinc while the
amino nitrogen is strongly hydrated in water. If the binding site
is hydrophobic, hydration of the amino nitrogen is energetically
unfavorable, since water molecules are forced to enter the hy-
drophobic binding site. Thus, the ratio of binding constant of
pyridine to imidazole should be a good measure of hydrophobicity
of the binding site. If the binding site is hydrophobic, binding of
hydrated imidazole would be strongly prohibited, leading to a large
ratio of the binding constant of pyridine to imidazole. The ratios
are listed in Table 9 along with other relevant data reported for
similar systems. In entries 1–3 in Table 9, large ratios are listed for
Table 8 Binding constants of N-alkylimidazole to 1–Egg PC and
2–Egg PC in pH 7.1 phosphate buffer at 25 ◦C. Binding constants of
alkylpyridines are also listed for comparison
Binding mechanisms in the liquid crystalline phase and in the gel
phase of liposomes
Host
Guest
K/M-1
Ref.
The temperature dependence of the binding constants of 4-
ethylpyridine and 3-ethylpyridine to 1 incorporated either in
DPPC liposome or Egg PC liposome is shown in Fig. 4. The
binding of 4-ethylpyridine to 1 incorporated in Egg PC liposome
is characterized by a negative enthalpy change for the temperature
range 25–60 ◦C (Table 11). The Egg PC liposome is in the
1–Egg PC
2–Egg PC
4-Methylpyridine
N-Methylimidazole
4-Methylpyridine
4-Ethylpyridine
N-Methylimidazole
N-Ethylimidazole
4-Methylpyridine
4-Ethylpyridine
N-Methylimidazole
N-Ethylimidazole
Pyridine
800 110
This work
This work
This work
This work
This work
This work
31
31
31
31
14
14
15
15
86
6
450 33
1100 110
64
2
100 17
38400
103 000
234
303
69
21
3300
46000
3
7
8
Imidazole
Pyridine
N-Methylimidazole
Table 9 The ratio of binding constants of 4-methylpyridine to N-methyl-
imidazole as a measure of hydrophilicity/hydrophobicity around the zinc
ion at 25 ◦
C
Entry Host K(pyridine)/K(imidazole) Media
Ref.
1
2
3
4
5
6
7
3
5
6
1
2
7
8
164
20.1
20
Water
Water
Water
Egg PC This work
Egg PC This work
Water
Toluene 15
31
12
1
Unpublished work
9
1
7.0 0.6
3.3a
14
Fig. 4 van’t Hoff plot in the temperature range 25–60 ◦C for the binding
of 4-ethylpyridine and 3-ethylpyridine to 1 incorporated in either DPPC
or Egg PC liposomes. The three phases of the DPPC liposome, La¢, Pb¢,
and Lb¢, are shown. ᭹: 4-ethylpyridine and 1–Egg PC, ᭺: 4-ethylpyridine
and 1–DPPC, and ꢀ: 3-ethylpyridine and 1–DPPC.
0.072b
a The ratio of binding constants of pyridine to imidazole. b The ratio of
binding constants of pyridine to N-methylimidazole.
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Table 11 Enthalpy and entropy changes in binding to 1 incorporated in
of the phase transition from the gel phase to the liquid crystalline
phase of the DPPC liposome has been reported to be 124 J
K-1 mol-1,28 and thus the large entropic gain of 260 J K-1 mol-1
can be attributed to the local phase transition around the guest
molecule, if we assume that one guest molecule causes local melting
of two lipid molecules.
liposomal membrane determined by the van’t Hoff plota
DH◦,
DS◦, J K-1
Liposome Phase T/◦C Guest
kJ mol-1
mol-1
DPPC
DPPC
DPPC
Egg PC
Lb¢
La¢
La¢
La¢
25–35 3-Ethylpyridine
62 13 260 50
41–60 3-Ethylpyridine -19.0
41–60 4-Ethylpyridine
25–50 4-Ethylpyridine
6
2
2
-1 23
-15
-22
11
6
7
-8
Conclusions
a Enthalpy changes and entropy changes were determined by the least
square line fit to the data shown in Fig. 4. Standard deviations of DH◦ and
DS0 Were calculated according to the following equations.22
Binding of alkylpyridines to amphiphilic zinc porphyrins incorpo-
rated in the liposomal bilayer membrane occurs more favorably
when the alkyl group of the guest is longer. The binding is driven
enthalpically in the liquid crystalline phase and entropically in the
gel phase. We suggest that local melting of lipid molecules around
the porphyrin–guest complex may drive the binding in the gel
phase. 3-Alkylpyridines are favored over 4-alkylpyridines due to
the anisotropic packing of lipid molecules. These features suggest
that the dynamics of the lipid molecules, as reflected in the entropic
term, play a major role in the affinity and selectivity of binding in
liposomal bilayer membranes.
n
n
n
2
2
xi2
ˆ
ˆ
y - y
y - y
(
)
(
)
i
i
i
i
Â
Â
Â
i=1
i=1
i=1
sDH ∞ = R
, sDS ∞ = R
n
n
2
2
(n - 2)
x - x
n(n - 2)
x - x
(
)
(
)
i
i
Â
Â
i=1
i=1
where R is the gas constant, n is the number of data points, xi is T-1, yi is
lnK, and
n
xi
Â
DH∞xi DS∞
i=1
x =
,
yi = -
+
.
Experimental section
n
R
R
Measurements
liquid crystalline state in this temperature range. Binding to 1
incorporated in the DPPC liposome was much more complex,
and three different mechanisms seem to be operating depending
on the temperature. The binding in the liquid crystalline phase is
characterized by a negative enthalpy changes, where the binding
constant decreases as increasing temperature. On the other hand,
the binding in the gel phase is characterized by a positive enthalpy
changes, where the binding is tighter at higher temperature. The
phase transition of the DPPC liposome has been thoroughly
studied in the literature.26 The gel phase (Lb¢) of DPPC is
characterized by all-trans alkyl chains with their long axes tilted to
the bilayer normal. Upon heating to 34.2 ◦C, the Pb¢ phase appears,
which is characterized by the absence of any hydrocarbon chain
tilt to the bilayer normal, and the onset of the rigid-body rotation
of the lipid molecule along their long axes. Further heating of the
Pb¢ phase to 41.4 ◦C results in the gel-to-liquid crystal (La¢) phase
transition. In the liquid crystalline phase, La¢, there is the onset
of trans–gauche isomerization in the acyl chains and fast lateral
diffusion of the lipid molecules.
UV/Visible spectra were recorded on a Shimadzu Multispec-
1500 spectrophotometer. Egg phosphatidylcholine (Egg PC) was
purchased from NOF corporation (COATSOME NC-50). It has
a C12–C22 saturated fatty acid at the glycerol a-position and a
C12–C20 unsaturated fatty acid at the glycerol b-position.
Egg PC host
Porphyrin 1 0.09 mg (8.4 ¥ 10-8 mol) and egg phosphatidylcholine
(Egg PC) 0.08 g (1.0 ¥ 10-4 mol) were dissolved in CHCl3. The
solution was evaporated, and dried in vacuo. To the thin lipid film
was added 20 mL of 0.083 M Bis-Tris buffer (pH = 7.1), and
the mixture stirred at room temperature for 30 min. After the
suspension was sonicated for five minutes five times at 0 ◦C, it
was centrifuged for 30 min. In order to isolate a small unilamellar
vesicle, a supernatant was separated by gel-permeation chromatog-
raphy on Sepharose-4B (collecting unturbid clear fraction). After
the clear solution was left overnight at 0 ◦C, it was used as the Egg
PC host.
The binding behavior corresponds well to the phase changes
in the DPPC liposome, displaying different binding mechanisms
in each phase. The negative enthalpy changes observed for the
binding in the liquid crystalline phase have been reported for
binding of nitrogeneous bases to zinc porphyrin both in water and
in organic solvents.14,27 Since the major driving force of binding is
the Lewis acid (zinc)–Lewis base (nitrogeneous base) interactions,
the exothermic process is easily rationalized. The positive enthalpy
changes observed for the binding in the gel phase had never been
reported, and it is quite unusual. The binding in the gel phase
is driven by a positive entropy term, and therefore the system
should become more random as the guest binds to the receptor.
One possible explanation is that incorporation of the guest causes
disorder in the packing of the acyl chains of lipid molecules,
accounting for the positive entropic changes. The entropy change
DPPC host
Porphyrin 1 0.09 mg (8.4 ¥10-8 mol) and dipalmitoylphosphatidyl-
choline (DPPC) 0.04 g (5.5 ¥ 10-5 mol) were dissolved in CHCl3.
The solution was completely evaporated, and dried in vacuo. To
the thin lipid film was added 20 mL of 0.083 M Bis-Tris buffer
(pH = 7.1), and the mixture stirred at 50 ◦C (above the main phase
transition temperature of DPPC) for 30 min. After the suspension
was sonicated for five minutes five times at 50 ◦C, it was centrifuged
for 30 min. In order to isolate a small unilamellar vesicle, a
supernatant was separated by gel-permeation chromatography
on Sepharose-4B (collecting the clear fraction). After this clear
solution was left overnight at room temperature, it was used as
DPPC host.
1442 | Org. Biomol. Chem., 2009, 7, 1437–1444
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Synthesis of porphyrin 1
1. A solution of 11a (63.5 mg, 5.93 ¥ 10-5 mol) and
Zn(OAc)2-saturated methanol (30 mL) in CHCl3 was stirred
at room temperature for 1 h in dark. The solution was evap-
orated, and the residue was dissolved in CHCl3. The CHCl3
layer was washed with HCl (0.05 M, twice) and pure water
(twice). The solution was dried over Na2SO4, and evaporation
of the solvent afforded 5,10-di(4-dodecyloxyphenyl)-15,20-di(4-
carboxylphenyl)porphyrin zinc complex as a purple solid (46.1 mg,
68.4%). 1H NMR (400 MHz, pyridine-d5): d = 0.89 (t, J = 6.8, 6H;
CH3), 1.30 (m, 32H; CH2), 1.64 (m, 4H; CH2), 1.99 (m, 4H; CH2),
4.28 (t, J = 6.4 Hz, 4H; CH2), 7.49 (d, J = 8.4 Hz, 4H; phenyl),
8.40 (d, J = 8.4 Hz, 4H; phenyl), 8.51 (d, J = 8.0 Hz, 4H; phenyl),
8.85 (d, J = 8.4 Hz, 4H; phenyl), 9.19 (overlapped singlet and
doublet, 4H, pyrrole), 9.35 (overlapped singlet and doublet, 4H,
pyrrole); MS (TOF): m/z = 1137 [M + H]+; UV-visible spectrum:
lmax = 427 nm, e = 3.12 ¥ 105 M-1cm-1 (chloroform).
9a. This was prepared by
a
reported procedure29. p-
Hydroxybenzaldehyde (4.39 g, 0.036 mol), dodecyl bromide
(8.25 mL, 0.037 mol), potassium carbonate (6.01 g, 0.15 mol)
and acetonitrile 60 mL were placed in a 300 mL flask fitted with a
reflux condenser and a mechanical stirrer. The mixture was then
heated and stirred at reflux for 12 h. After the mixture was cooled
to room temperature, it was transferred to a separating funnel,
treated with water (150 mL), and extracted with hexane (150 mL).
The organic layer was washed twice with NaOH (10%) and finally
with water until neutrality was reached. The organic solution,
after dried over Na2SO4, was concentrated in a rotary evaporator,
giving p-dodecyloxybenzaldehyde as a yellow oil (9.60 g, 91.8%).
1H NMR and mass spectra were in agreement with those reported
in the literature.30 1H NMR (400 MHz, CDCl3): d (ppm) = 0.87
(t, J = 7.2 Hz, 3H; CH3), 1.36 (m, 16H; CH2), 1.46 (m, 2H; CH2),
1.80 (m, 2H; CH2), 4.06 (t, J = 6.8 Hz, 2H; CH2), 7.00 (d, J =
9.2 Hz, 2H; phenyl), 7.82 (d, J = 9.2 Hz, 2H; phenyl), 9.82 (s, 1H;
CHO); MS (FAB): m/z = 291 [M + H]+.
Synthesis of porphyrin 2
Compound 2 was prepared in a similar manner to that of 1, except
that the metallation was followed by hydrolysis.
10a. Compound 9a (17.88 g, 0.063 mol) and 4-formylbenzoic
acid methyl ester (10.34 g, 0.063 mol) were dissolved in propionic
acid (225 mL). The solution was heated to 120 ◦C with continuous
stirring. Pyrrole (8.82 mL, 0.12 mol) was slowly added to the
heated solution, and heating was continued at 120 ◦C for 30 min
in dark. After the solution was cooled to room temperature,
it was left to stand overnight. After the purple crystalline
product was washed with MeOH, 10a and the trans isomer
were separated from other isomers by column chromatography
(SiO2, dichloromethane–hexane = 3 : 2). Further column chro-
matographic separation (SiO2, dichloromethane–hexane = 3 : 2)
afforded 10a as a purple solid (0.22 g, 0.67%). 1H NMR (500 MHz,
CDCl3): d (ppm) = -2.78 (s, 2H; NH), 0.90 (t, J = 6.9 Hz, 6H;
CH3), 1.23–1.63 (m, 36H; CH2), 1.90 (m, 4H; CH2), 4.12 (s, 6H;
CH3), 4.26 (t, J = 6.9 Hz, 4H; CH2), 7.30 (d, J = 8.4 Hz, 4H;
phenyl), 8.12 (d, J = 8.4 Hz, 4H; phenyl), 8.31 (d, J = 7.7 Hz, 4H;
phenyl), 8.44 (d, J = 7.6 Hz, 4H; phenyl), 8.91 (overlapped singlet
and doublet, 4H, pyrrole), 8.79 (overlapped singlet and doublet,
4H, pyrrole); MS (TOF): m/z = 1099 [M + H]+.
9b. 1H NMR (400 MHz, CDCl3): d (ppm) = 0.87 (t, J =
6.9 Hz, 6H; CH3), 1.25 (m, 36H; CH2), 1.84 (m, 4H; CH2), 4.06
(m, 4H; CH2), 6.94 (d, J = 9.9 Hz, 2H; phenyl), 7.38–7.55 (m, 2H;
phenyl), 9.82 (s, 1H; CHO); MS (FAB): m/z = 475 [M + H]+.
10b. 1H NMR (500 MHz, CDCl3): d (ppm) = -2.78 (s, 2H;
NH), 0.84 (t, J = 6.9 Hz, 6H; CH3), 0.90 (t, J = 6.9 Hz, 6H;
CH3), 1.20–2.04 (m, 80H; CH2), 4.12–4.31 (m, 14H), 8.30 (d, J =
7.7 Hz, 4H; phenyl), 8.44 (d, J = 7.7 Hz, 4H; phenyl), 7.23–7.26
(m, 2H; phenyl), 7.70 (dd, J = 2.3, 9.9 Hz, 2H; phenyl), 7.78 (d,
J = 2.3 Hz, 2H; phenyl), 8.78 (overlapped singlet and doublet, 4H,
pyrrole), 8.93 (overlapped singlet and doublet, 4H, pyrrole); MS
(TOF): m/z = 1468 [M + H]+.
11b. 1H NMR (500 MHz, CDCl3): d = 0.82 (t, J = 6.9 Hz,
6H; CH3), 0.89 (t, J = 6.9 Hz, 6H; CH3), 1.19–2.04 (m, 80H; CH2),
4.09–4.30 (m, 14H; CH2), 7.23–7.25 (m, 2H; phenyl), 7.71 (dd, J =
2.3, 9.9 Hz, 2H; phenyl), 7.76 (d, J = 2.3 Hz, 2H; phenyl), 8.29 (d,
J = 7.7 Hz, 4H; phenyl), 8.42 (d, J = 7.7 Hz, 4H; phenyl), 8.87
(overlapped singlet and doublet, 4H, pyrrole), 9.03 (overlapped
singlet and doublet, 4H, pyrrole); MS (TOF): m/z = 1530
[M + H]+.
11a. Hydrolysis of the ester groups was carried out according
to a published procedure.31 10a (0.13 g, 1.18 ¥ 10-4 mol) was dis-
solved in a solution prepared by mixing THF (54 mL), methanol
(10.8 mL), and KOH (0.99 g, 1.76 ¥ 10-2 mol) under Ar. After being
stirred at room temperature for 48 h, the solution was evaporated.
HCl (0.5 M) was added until neutrality, followed by ethyl acetate
(40.5 mL). The organic layer was washed twice with water and
NaCl (13%), and dried over Na2SO4. Evaporation of the solvent
and purification by preparative TLC (pyridine) afforded 5,10-di-
(4-dodecyloxyphenyl)-15,20-di(4-carboxylphenyl)porphyrin as a
2. 1H NMR (500 MHz, CDCl3): d = 0.84 (t, J = 6.9 Hz, 6H;
CH3), 0.90 (t, J = 6.9 Hz, 6H; CH3), 1.21–2.20 (m, 80H; CH2), 4.13
(t, J = 6.9 Hz), 4.31 (t, J = 6.9 Hz), 7.22–7.29 (m, 2H; phenyl),
7.71 (d, J = 9.9 Hz, 2H; phenyl), 7.79 (s, 2H; phenyl), 8.36 (d, J =
7.7 Hz, 4H; phenyl), 8.55 (d, J = 7.7 Hz, 4H; phenyl), 8.87 (s, 2H),
8.92 (d, J = 4.6 Hz, 2H), 9.03 (s, 2H), 9.05 (d, J = 4.6 Hz, 2H);
MS (TOF): m/z = 1502 [M + H]+; Elemental analysis: C, 71.10;
H,7.88; N, 3.49. Calc. for C94H124N4O8Zn·5H2O: C, 70.85; H, 7.84;
N, 3.49%.
1
purple solid (0.10 g, 81.6%). H NMR (500 MHz, pyridine-d5):
d = -2.30 (s, 2H; NH), 0.88 (t, J = 6.9, 6H; CH3), 1.28 (m, 32H;
CH2), 1.65 (m, 4H; CH2), 2.02 (m, 4H; CH2), 4.28 (t, J = 6.9 Hz,
4H; CH2), 7.53 (d, J = 7.6 Hz, 4H; phenyl), 8.36 (d, J = 7.9 Hz,
4H; phenyl), 8.51 (d, J = 6.9 Hz, 4H; phenyl), 8.86 (d, J = 6.9 Hz,
4H; phenyl), 9.09 (overlapped singlet and doublet, 4H, pyrrole),
9.23 (overlapped singlet and doublet, 4H, pyrrole); MS (TOF):
m/z = 1071 [M + H]+; Elemental analysis: C, 74.61; H,7.38; N,
4.63. Calc. for C70H78N4O6·3H2O: C, 74.70; H, 6.99; N, 4.98%.
References
1 P. J. Collings, Liquid Crystals: Nature’s Delicate Phase of Matter,
Princeton University Press, 1990.
2 (a) D. J. Cram, Angew. Chem. Int. Ed. Engl., 1986, 25, 1039; (b) D. J.
Cram, Angew. Chem. Int. Ed. Engl., 1988, 27, 1009.
This journal is
The Royal Society of Chemistry 2009
Org. Biomol. Chem., 2009, 7, 1437–1444 | 1443
©

View Article Online
3 (a) S. M. K. Davidson and S. L. Regen, Chem. Rev., 1997, 97, 1269;
(b) E. L. Doyle, C. A. Hunter, H. C. Phillips, S. J. Webb and N. H.
Williams, J. Am. Chem. Soc., 2003, 125, 4593.
4 For recent study on protein-membrane interactions, see: B. Jeppesen,
C. Smith, D. F. Gibson and J. F. Tait, J. Biol. Chem., 2008, 283, 6126.
5 I. Voszka, Z. Szabo, G. Csik, P. Maillard and P. Grof, J. Photochem.
Photobiol. B, 2005, 79, 83.
11 M. H. Abraham, G. S. Whiting, R. Fuchs and E. J. Chambers, J. Chem.
Soc. Perkin Trans. 2, 1990, 291.
12 T. Mizutani, K. Kozake, K. Wada and S. Kitagawa, Chem. Commun.,
2003, 2918.
13 M. H. Abraham, J. Am. Chem. Soc., 1982, 104, 2085.
14 T. Mizutani, K. Wada and S. Kitagawa, J. Am. Chem. Soc., 1999, 121,
11425–11431.
6 For interaction of amphiphilic porphyrins and related tetrapyrroles
with liposomes, see: (a) T. Katagi, T. Yamamura, T. Saito and Y.
Sasaki, Chem. Lett., 1982, 417; (b) J. H. Fuhrhop and T. Lehmann,
Liebigs Ann. Chem., 1984, 1057; (c) D. Brault, C. Vever-Bizet and T.
Le Doan, Biochim. Biophys. Acta, 1986, 857, 238; (d) M. Rotenberg,
R. Margalit and G. S. Wise, Biochim. Biophys. Acta, 1987, 905, 173;
(e) J. T. Groves and R. Neumann, J. Am. Chem. Soc., 1989, 111, 2900;
(f) H. Mihara, K. Nishino, R. Hasegawa and T. Fujimoto, Chem. Lett.,
1992, 1805; (g) T. Komatsu, K. Arai, H. Nishide and E. Tsuchida,
Chem. Lett., 1993, 1949; (h) D. Brault, C. Vever-Bizet and K. Kuzelova,
J. Photochem. Photobiol. B, 1993, 20, 191; (i) B. C. Patterson and J. K.
Hurst, Langmuir, 1993, 9, 16; (j) J. H. van Esch, M. C. Feiters, A. M.
Peters and R. J. M. Nolte, J. Phys. Chem., 1994, 98, 5541; (k) Y. S.
Kang and L. Kevan, J. Phys. Chem., 1994, 98, 4389; (l) L. Roitman, B.
Ehrenberg, Y. Nitzan, V. Kral and J. L. Sessler, Photochem. Photobiol.,
1994, 60, 421; (m) C. Schell and H. K. Hombrecher, Chem. Eur. J.,
1999, 5, 587; (n) Y. Hisaeda, E. Ohshima and M. Arimura, Colloids and
Surfaces A, 2000, 169, 143; (o) K. Iida, H. Kiriyama, A. Fukai, W. N.
Konings and M. Nango, Langmuir, 2001, 17, 2821; (p) T. Komatsu,
M. Moritake and E. Tsuchida, Chem. Eur. J., 2003, 9, 4626; (q) F. M.
Engelmann, S. V. O. Rocha, H. E. Toma, K. Araki and M. S. Baptista,
Int. J. Pharm., 2007, 329, 12.
7 T. Mizutani and D. G. Whitten, J. Am. Chem. Soc., 1985, 107, 3621.
8 A referee suggested that the pKa of water coordinated to the zinc should
be estimated from the spectral changes in a pH titration experiment.
The Soret band l max of 1 incorporated in EggPC was unchanged when
the pH of the solution was changed from 7 to 12, indicating that the
pKa value is higher than 12. For the values of pKa of zinc porphyrins,
see: E. Mikros, A. Gaudemer and R. Pasternack, Inorg. Chim. Acta,
1988, 153, 199; H. Imai, K. Misawa, H. Munakata and Y. Uemori,
Chem. Lett., 2001, 688.
15 H. Imai, S. Nakagawa and E. Kyuno, J. Am. Chem. Soc., 1992, 114,
6719.
16 R. W. Green, Aust. J. Chem., 1969, 22, 721.
17 H. C. Brown and X. R. Mihm, J. Am. Chem. Soc., 1955, 77, 1723.
18 E. F. G. Herington, Discuss. Faraday Soc., 1950, 9, 26.
19 S. P. Datta and A. K. Grzybowski, J. Chem. Soc. Phys. Org., 1966,
136.
20 W. Pfleiderer, G. Ritzmann, K. Harzer and J. C. Jochims, Chem. Ber.,
1973, 106, 2982.
21 B. Lenarcik and B. Barszcz, Pol. J. Chem., 1979, 53, 963.
22 J. N. Miller and J. C. Miller, Statistics and Chemometrics for Analytical
Chemistry, Prentice-Hall Publishing Inc., 2000.
23 R. B. Hermann, Proc. NatI. Acad. Sci. USA, 1977, 74, 4144; R. B.
Hermann, J. Phys. Chem., 1972, 76, 2754; R. B. Hermann, J. Phys.
Chem., 1975, 79, 163.
24 J. A. Reynolds, D. B. Gilbert and C. Tanford, Proc. Nat. Acad. Sci.
USA, 1974, 71, 2925.
25 For recent studies on recognition of alkyl groups, see: M. P. Schramm
and J. Rebek, Jr., Chem. Eur. J., 2006, 12, 5924.
26 R. N. A. H. Lewis, N. Mak and R. N. McElhaney, Biochemistry, 1987,
26, 6118, and references cited therein.
27 (a) J. R. Miller and G. D. Dorough, J. Am. Chem. Soc., 1952, 74, 3977;
(b) H. Ogoshi, T. Mizutani, T. Hayashi and Y. Kuroda, in ‘Porphyrins
and Metalloporphyrins as Receptor Models in Molecular Recognition’,
ed. K. Kadish, K. M. Smith and R. Guillard, San Diego, 2000.
28 N. Poklar, J. Fritz, P. Macek, G. Vesnaver and T. V. Chalikian,
Biochemistry, 1999, 38, 14999.
29 N. Spreti, L. Brinchi, R. Germani, M. V. Mancini and G. Savelli,
Eur. J. Org. Chem., 2004, 3865.
30 M. C. Lensen, J. A. A. W. Elemans, S. J. T. van Dingenen, J. W. Gerritsen,
S. Speller, A. E. Rowan and R. J. M. Nolte, Chem. Eur. J., 2007, 13,
7948.
9 T. Mizutani, K. Wada and S. Kitagawa, J. Org. Chem., 2000, 65, 6097.
10 M. C. Phillips, R. M. Williams and D. Chapman, Chem. Phys. Lipids,
1969, 3, 234.
31 H. Iwamoto, T. Mizutani and K. Kano, Chem. Asian J., 2007, 2,
1267.
1444 | Org. Biomol. Chem., 2009, 7, 1437–1444
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The Royal Society of Chemistry 2009
©