Steric-control for the enantioselective hydrolysis of amino acid esters in hybrid membrane systems.

Article abstract of DOI:10.1248/cpb.51.224

The enantioselective hydrolysis of the amino acid esters, p-nitrophenyl-N-dodecanoyl-D(L)-phenylalaninates (C(12)-D(L)-Phe-PNP) catalyzed by active tripeptide, N-(benzyloxycarbonyl)-L-phenylalanyl-L-histidyl-L-leucine (Z-PheHisLeu) in the presence of coag

Full text of DOI:10.1248/cpb.51.224

2
24
Notes
Chem. Pharm. Bull. 51(2) 224—226 (2003)
Vol. 51, No. 2
Steric-Control for the Enantioselective Hydrolysis of Amino Acid Esters in
Hybrid Membrane Systems
a
b
b
b
,a,b
Osamu TANOUE, Hideaki ICHIHARA, Koichi GOTO, Yo k o M ATSUMOTO, and Ryuichi UEOKA*
a
b
Department of Applied Life Science, Sojo University; and Division of Applied Chemistry, Graduate School of Sojo
University; 4–22–1 Ikeda, Kumamoto 860–0082, Japan.
Received November 5, 2002; accepted November 26, 2002
The enantioselective hydrolysis of the amino acid esters, p-nitrophenyl-N-dodecanoyl-D(L)-phenylalaninates
C -D(L)-Phe-PNP) catalyzed by active tripeptide, N-(benzyloxycarbonyl)-L-phenylalanyl-L-histidyl-L-leucine (Z-
(
1
2
PheHisLeu) in the presence of coaggregates (hybrid membranes) composed of native phospholipid, L-a-dimyris-
toylphosphatidylcholine (DMPC) and nonionic surfactant, polyoxyethylene (8) lauryl ether (C (EO) ) was easily
12
8
controlled by regulating the reaction temperature and changing the composition of coaggregates. Furthermore,
excellent correlations were observed between the enantioselectivity in the hydrolysis of C -D(L)-Phe-PNP cat-
12
alyzed by Z-PheHisLeu in the presence of coaggregates and physical properties of hybrid membranes. It is as-
sumed that catalytic activities of tripeptide catalyst in hybrid membranes should be regulated by changing the
microenvironments of reaction fields.
Key words enantioselective hydrolysis; hybrid membrane; stereochemical control
Stereoselective reactions have attracted considerable atten- enantioselective hydrolysis of C -D(L)-Phe-PNP catalyzed by
1
2
tion in connection with understanding the origins of the Z-PheHisLeu in the hybrid membranes composed of phos-
1
—5)
stereoselectivity observed with proteolytic enzymes.
the course of our study on the stereoselective hydrolysis 7.4. The relations between the enantioselectivity and the mi-
deacylation) of amino acid and dipeptide esters with the croenvironment of the reaction fields are discussed.
In pholipid (DMPC) and nonionic surfactant (C (EO) ) at pH
12 8
(
functional molecular assemblies composed of synthetic sur-
factants and reactive species, we emphasized that the stereo- Results and Discussion
chemical (enantiomeric and diastereomeric substrates) con-
The composition dependence of coaggregates on the enan-
trol could be attained by changing the composition of the co- tioselective hydrolysis of C -D(L)-Phe-PNP catalyzed by
1
2
6
—8)
9)
aggregates
and regulating ionic strength and tempera- Z-PheHisLeu was investigated in hybrid membranes of
1
0,11)
ture.
attained at the optimum composition of coaggregate.
Especially, an extremely high enantioselectivity was DMPC/C (EO) , as shown in Fig. 1. The concentration of
1
2
8
6
)
C (EO) is expressed as mol% of total lipids. Both the sec-
1
2
8
However, the values of k and k (the first-order rate con- ond-order rate constants (ka,obsd) and enantioselectivity
t
s
L
D
a,obsd
stants with and without the peptide catalyst, respectively) are (ka,obsd/k
) values increased as the C (EO) concentration
12 8
fairly close for the enantioselective hydrolysis in the aggre- was raised in the range of 10—30 mol%. No clear solution
gate systems of synthetic surfactants. Then, it is impossible was obtained in the C (EO) concentration range over
1
2
8
to disregard the errors of k and k values when we discuss 30 mol% (phase separation occurred in this region), but high
t
s
catalytic activities of the artificial membranes bound enzyme second-order rate constants and enantioselectivity were ob-
on the basis of ka,obsd values.
In this study, we employed native phospholipids instead of
synthetic surfactants for the hydrolysis of amino acid esters
1
2)
12)
(
C -D(L)-Phe-PNP) with tripeptide (Z-PheHisLeu) in the
12
coaggregate systems. The advantage of this experiment
should enable to evaluate of the catalytic activities directly on
the basis of ka,obsd values because of the large difference of
the k and k values, that is, the k /k ratios were above 30 and
t
s
t
s
4
folds for the L- and D-substrates, respectively.
Recently, in the course of our study on artificial mem-
branes composed of native phospholipids, very useful knowl-
edges for medical engineering applications in vitro and in
vivo have been obtained. For example, artificial lipid mem-
branes were used as a drug carrier in drug delivery systems,
such as 1,3-bis(2-chloroethyl)-1-nitrosourea encapsulated in
hybrid liposomes (artificial membranes) which should be ef-
1
3)
fective against malignant glioma in vitro and in vivo.
Moreover, the artificial membranes themselves were found to
be useful as a new type of anticancer drug without side ef-
1
4,15)
fects.
In this study, we report on the remarkable effects (compo-
sition of hybrid membranes and reaction temperature) on the
*
To whom correspondence should be addressed. e-mail: ueoka@life.sojo-u.ac.jp
© 2003 Pharmaceutical Society of Japan

February 2003
225
Fig. 3. Temperature Dependence of Rate Constants (ka,obsd) and Enantiose-
L
D
a,obsd
lectivity (ka,obsd/k
) for the Hydrolysis of C -D(L)-Phe-PNP Catalyzed by
Fig. 1. Composition Dependence of Rate Constants (ka,obsd) and Enantio-
12
L
D
Z-PheHisLeu in 70 mol% DMPC/30 mol% C (EO) Hybrid Membranes at
selectivity (ka,obsd/k
) for the Hydrolysis of C -D(L)-Phe-PNP Catalyzed
12 8
a,obsd
12
pH 7.4 and mϭ0.01
by Z-PheHisLeu in DMPC/C (EO) Hybrid Membranes at 25 °C and
1
2
8
Ϫ3
Ϫ4
[DMPC]ϭ1.0ϫ10 M, [Z-PheHisLeu]ϭ2.0ϫ10 M, [C -D(L)-Phe-PNP]ϭ1.0ϫ
mϭ0.01
12
Ϫ5
1
0
M.
Ϫ3
Ϫ4
[
DMPC]ϭ1.0ϫ10 M, [Z-PheHisLeu]ϭ2.0ϫ10 M, [C -D(L)-Phe-PNP]ϭ1.0ϫ
M.
12
Ϫ5
1
0
tant to increase the enantioselectivity for the hydrolysis of
C -D(L)-Phe-PNP (Fig. 2B). These observations suggest that
1
2
the size and the fluidity of hybrid membranes should change
upon the addition of C (EO) and result in a large enhance-
1
2
8
ment of enantioselectivity at the C (EO) concentration
1
2
8
range of 20—30 mol%. From these results, it is clear that the
catalytic activity could be controlled by changing the compo-
sition ratio of DMPC and C (EO) in hybrid membranes
1
2
8
systems and the high enantioselectivity in the artificial mem-
branes was attained on the verge of phase separation region
as well as the enhancement of catalytic activity in the native
enzymes.
The temperature effect on the enantioselective hydrolysis
of C -D(L)-Phe-PNP catalyzed by Z-PheHisLeu was investi-
1
2
gated in the hybrid membranes of 70 mol% DMPC/30 mol%
C (EO) , as shown in Fig. 3. It is noteworthy that the tem-
1
2
8
perature dependence of both the second-order rate constants
and enantioselectivity for the hydrolysis of C -D(L)-Phe-PNP
1
2
L
D
were bell-shaped with a maximum (ka,obsd/k a,obsdϭ15) at
5 °C. From this result, Tc of the hybrid membranes of
2
Fig. 2. Composition Dependence of Apparent Hydrodynamic Diameter
(
A) and Fluorescence Polarizations of DPH and tma-DPH (B) in 70 mol% DMPC/30 mol% C (EO) could be estimated ki-
12 8
1
6)
DMPC/C (EO) Hybrid Membranes at 25 °C and mϭ0.01
netically to be ca. 25 °C, though it was impossible to esti-
mate Tc of these hybrid membranes on the basis of differen-
tial scanning calorimetry (DSC) method.
12
8
Ϫ3
[
DMPC]ϭ1.0ϫ10 M.
tained on the verge of phase separation.
Interestingly, the temperature dependence of d value was
hy
The hydrodynamic diameter (d ) of the hybrid membranes also bell-shaped with a maximum at 25 °C (Fig. 4A). On the
hy
was evaluated by dynamic light scattering (dls). The d value other hand, the P values observed from tma-DPH and DPH
hy
was sharply increased in the C (EO) concentration range of decreased along with the elevation of temperature, as shown
1
2
8
2
0—30 mol%, and interestingly, the enantioselectivity for the in Fig. 4B. However, the inflection in the correlation of P
hydrolysis of C -D(L)-Phe-PNP was also extremely enhanced against temperature was observed around 25 °C. This result
1
2
in the same region of coaggregate composition (Fig. 2A). On indicates that the fluidity of the hybrid membranes of
the other hand, the fluorescence polarizations (P) observed 70 mol% DMPC/30 mol% C (EO) is delicately changed
1
2
8
from 1-[(4-trimethylammonio)phenyl]-6-phenyl-1,3,5-hexa- around 25 °C, that is somewhat higher than the phase transi-
triene iodide (tma-DPH) placed in the pseudo-hydrophobic tion temperature (Tc; 23 °C) of pure DMPC membrane esti-
domain near the membrane surface and DPH placed in the mated on the basis of DSC method. These results suggest
inner hydrophobic domain gently decreased as the C (EO)
that the enantioselective hydrolysis of C -D(L)-Phe-PNP
12
8
12
concentration was raised. These results indicate that the en- could be controlled by regulating the temperature of hybrid
hancement of fluidity of hybrid membranes should be impor- membranes systems, and the highest enantioselectivity was

2
26
Vol. 51, No. 2
for the hydrolytic cleavage (hydrolysis) of C -D(L)-Phe-PNP with and with-
1
2
out a nucleophile and [nucleophile] indicates the initial nucleophile concen-
0
tration.
The dynamic light-scattering measurements were performed with
BROOKHAVEN BI-90 particle sizer and a He–Ne laser light source (Spec-
tra-Physics Model 127-35). The hydrodynamic diameter (d ) was calculated
hy
by Stokes–Einstein relation, Eq. 2,
dhyϭkT/3phD
(2)
where k is Boltzmann’s constant, T is absolute temperature, h is the solvent
viscosity, and D is the diffusion coefficient.
The fluorescence spectra were measured on a Hitachi F-2000 spectropho-
tometer. The emissions at 434 nm originating from 1-[(4-trimethylammo-
nio)phenyl]-6-phenyl-1,3,5-hexatriene iodide tma-DPH and at 430 nm origi-
nating from 1,6-diphenyl-1,3,5-hexatriene (DPH) were monitored upon exci-
tations at 360 and 357 nm, respectively. The fluorescence polarizations (P)
of tma-DPH and DPH were measured after the sonication of the hybrid
membrane solutions and calculated by Eq. 3,
Pϭ(I ϪC I )/(I ϩC I )
(3)
vv
f
vv
vv
f vh
where I is the fluorescence intensity and the subscripts v and h refer to the
Fig. 4. Temperature Dependence of Apparent Hydrodynamic Diameter orientations, vertical and horizontal, respectively, for the excitation and ana-
A) and Fluorescence Polarizations of DPH and tma-DPH (B) in 70 mol% lyzer polarizers in this sequence: e.g., Ivh indicates the fluorescence intensity
DMPC/30 mol% C (EO) Hybrid Membranes at pH 7.4 and mϭ0.01
measured with a vertical excitation polarizer and a horizontal analyzer polar-
izer. C is the grating correction factor, given by I /I .
(
12
8
Ϫ3
[
DMPC]ϭ1.0ϫ10 M.
f
hv hh
Acknowledgments This work was supported in part by a Grant-in-Aid
for Science Research from the Ministry of Education, Science and Culture
of Japan (No. 14350439).
observed around the phase transition temperature.
Conclusion
References and Notes
The stereochemical control for the enantioselective hydrol-
ysis of amino acid esters could be established by changing
the composition of coaggregates and by regulating the reac-
tion temperature. In particular, a very interesting feature of
this study was that the remarkably large enhancement of the
enantioselectivity could be attained between stable and un-
stable regions (around the phase separation and/or the phase
transition temperature) of the artificial membranes.
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(
Experimental
Materials The enantiomeric substrates were prepared from N-(benzyl-
oxycarbonyl)-D(L)-phenylalanine by the esterification of the COOH group
with p-nitrophenol as described in ref. 1. Satisfactory analytical data were
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Tetrahedron Lett., 40, 2129—2132 (1999).
2
3
obtained for C -D-Phe-PNP: mp 108.0—108.2 °C; [a] ϩ10.8° (cϭ2,
12
D
CHCl ). Anal. Calcd for C H N O : C, 69.21; H, 7.74; N, 5.98. Anal.
3
27 36
2
5
Found: C, 69.07; H, 7.67; N, 5.96. C -L-Phe-PNP: mp 107.0—107.5 °C;
1
2
2
3
[
a] Ϫ10.8° (cϭ2, CHCl ). Anal. Found: C, 68.98; H, 7.77; N, 5.96.
D
3
The sample solutions were prepared by dissolving both DMPC and
C (EO) in tris(hydroxymethyl)aminomethane (Tris)-KCl buffer with the
12
8
12) The enantioselectivity for the hydrolysis of C -D(L)-Phe-PNP by Z-
12
sonication (BRANSONIC Model B2200 aparatus, 80W) at 45 °C for 60 min
and filtered through a 0.45 mm filter.
Rates of p-nitrophenol liberation from p-nitrophenyl esters were measured 13) Kitamura I., Kochi M., Matsumoto Y., Ueoka R., Kuratsu J., Ushio Y.,
PheHisLeu was highest among the enantiomeric substrates and the
peptide catalysts employed in our study, as mentioned in ref. 6.
at 400 nm with a Hitachi 150-20 UV spectrophotometer. The reaction
Cancer Res., 56, 3986—3992 (1996).
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rate constant (ka,obsd) for the hydrolysis of an ester substrate was evaluated by
Eq. 1,
Pharm. Bull., 18, 1456—1458 (1995).
15) Imamura C., Kemura Y., Matsumoto Y., Ueoka R., Biol. Pharm. Bull.,
2
0, 1119—1121 (1997).
ka,obsdϭ(k Ϫk )/[nucleophile]
(1)
t
s
0
1
6) Kunitake T., Okahata Y., J. Am. Chem. Soc., 99, 3860—3861 (1977).
where k and k refer, respectively, to the observed first-order rate constants
t
s