Tunable pK of amino acid residues at the air-water interface gives an L-zyme (Langmuir Enzyme)

Article abstract of DOI:10.1021/ja053226g

Various amino acid-carrying amphiphiles were synthesized, and the pK values of the attached amino acid residues were investigated at the air-water interface and in aqueous vesicles using π-A isotherm measurements, ¹H NMR titration, and IR spect

Full text of DOI:10.1021/ja053226g

Published on Web 08/06/2005
Tunable pK of Amino Acid Residues at the Air-Water
Interface Gives an L-zyme (Langmuir Enzyme)
Katsuhiko Ariga,*,†,‡ Takashi Nakanishi,† Jonathan P. Hill,§ Makoto Shirai,‡
Minoru Okuno,‡ Takao Abe,‡ and Jun-ichi Kikuchi‡
Contribution from the Supermolecules Group, AdVanced Materials Laboratory, National Institute
for Materials Science (NINS), 1-1 Namiki, Tsukuba 305-0044, Japan, International Center for
Young Scientists (ICYS), National Institute for Materials Science (NIMS), 1-1 Namiki,
Tsukuba 305-0044, Japan, and Graduate School of Materials Science, Nara Institute of Science
and Technology (NAIST), 8916-5 Takayama-cho, Ikoma 630-0192, Japan
Received May 17, 2005; E-mail: ARIGA.Katsuhiko@nims.go.jp
Abstract: Various amino acid-carrying amphiphiles were synthesized, and the pK values of the attached
amino acid residues were investigated at the air-water interface and in aqueous vesicles using π-A isotherm
measurements, 1H NMR titration, and IR spectroscopy in reflection-adsorption mode. The ꢀ-amino group
of the Lys residue embedded at the air-water interface displays a significant pK shift (4 or 5 unit) compared
with that observed in bulk water, while the pK shift in aqueous vesicles was not prominent (ca. 1 unit).
Moreover, pK values of the amino acids at the air-water interface can be tuned simply by control of the
subphase ionic strength as well as by molecular design of the amphiphiles. A simple equation based on
the dominant contribution by the electrostatic energy to the pK shift reproduces well the surface pressure
difference between protonated and unprotonated species, suggesting a reduction in the apparent dielectric
constant at the air-water interface. Hydrolysis of a p-nitrophenyl ester derivative was used as a model
reaction to demonstrate the use of the Lys-functionalized monolayer. Efficient hydrolysis was observed,
even at neutral pH, after tuning of pK for the Lys residue in the monolayer, which is a similar case to that
occurring in biological catalysis.
Introduction
revealed that the pK values of the functional groups in enzymes
are perturbed by the proximity of neighboring charges and the
Highly selective and efficient material conversions conducted
by biomolecular catalysts (i.e., enzymes) originate from the
sophisticated organization of their amino acid residues. The
shape and functional groups of their substrates are recognized
in the reaction cavities of enzymes, which have been mimicked
by well-structured supermolecules called artificial enzymes.1
Another surprising characteristic of biomolecular catalysts can
be found in the fine-tuning of the physicochemical nature of
the amino acid residues. In particular, control of their dissocia-
tion behaviors is one of the key factors influencing a powerful
catalysis, which seems to be unusual in aqueous media under
ambient pH conditions. Investigations using X-ray crystal-
lography, molecular dynamics, and protein engineering have
hydrophobicity of the surrounding environment.2
A straightforward approach for pK control of the amino acids
is by modification of biomolecules using protein engineering
methods such as point mutation,3 which sometimes leads to
successful pK control of the desired residues. However, predic-
tion of the appropriate modification of the amino acid sequences
is not always easy. Rather than modifying the enzymes
themselves, supply of an appropriate environment to the target
residues by use of a molecular assembly could facilitate pK
(2) (a) Highbarger, L. A.; Gerlt, J. A.; Kenyon, G. L. Biochemistry 1996, 35,
41-46. (b) Lee, J. K.; Houk, K. N. Science 1997, 276, 942-945. (c) Barbas,
C. F., III; Heine, A.; Zhong, G.; Hoffmann, T.; Gramatikova, S.; Bjo¨rnestedt,
R.; List, B.; Anderson, J.; Stura, E. A.; Wilson, I. A.; Lerner, R. A. Science
1997, 278, 2085-2092. (d) Dyson, H. J.; Jeng, M.-F.; Tennant, L. L.; Slaby,
I.; Lindell, M.; Cui, D.-S.; Kuprin, S.; Holmgren, A. Biochemistry 1997,
36, 2622-2636. (e) Khare, D.; Alexander, P.; Antosiewicz, J.; Bryan, P.;
Gilson, M.; Orban, J. Biochemistry 1997, 36, 3580-3589. (f) Ryu, D. D.
Y.; Nam, D.-H. Biotechnol. Prog. 2000, 16, 2-16. (g) Bayfield, M. A.;
Dahlberg, A. E.; Schulmeister, U.; Dorner, S.; Barta, A. Proc. Natl. Acad.
Sci. U.S.A. 2001, 98, 10096-10101. (h) Oyelere, A. K.; Kardon, J. R.;
Strobel, S. A. Biochemistry 2002, 41, 3667-3675. (i) Nielsen, J. E.;
McCammon, J. A. Protein Sci. 2003, 12, 1894-1901.
(3) (a) Goldberg, J. M.; Swanson, R. V.; Goodman, H. S.; Kirsch, J. F.
Biochemistry 1991, 30, 305-312. (b) Balashov, S. P.; Govindjee, R.; Kono,
M.; Imasheva, E.; Lukashev, E.; Ebrey, T. G.; Crouch, R. K.; Menick, D.
R.; Feng, Y. Biochemistry 1993, 32, 10331-10343. (c) Gloss, L. M.; Kirsch,
J. F. Biochemistry 1995, 34, 3999-4007. (d) Griesbeck, O.; Baird, G. S.;
Campbell, R. E.; Zacharias, D. A.; Tsien, R. Y. J. Biol. Chem. 2001, 276,
29188-29194. (e) Harrison, M. D.; Yanagisawa, S.; Dennison, C.
Biochemistry 2005, 44, 3056-3064. (f) Seebeck, F. P.; Hilvert, D. J. Am.
Chem. Soc. 2005, 127, 1307-1312.
† Supermolecules Group, Advanced Materials Laboratory, National
Institute for Materials Science (NINS).
§ International Center for Young Scientists (ICYS), National Institute
for Materials Science (NIMS).
‡ Graduate School of Materials Science, Nara Institute of Science and
Technology (NAIST).
(1) (a) Breslow R. Science 1982, 218, 532-537. (b) Tabushi, I. Tetrahedron
1984, 40, 269-292. (c) Smith, J.; Ariga, K.; Anslyn, E. V. J. Am. Chem.
Soc. 1993, 115, 362-364. (d) Kneeland, D. M.; Ariga, K.; Lynch, V. M.;
Huang, C. Y.; Anslyn, E. V. J. Am. Chem. Soc. 1993, 115, 10042-10055.
(e) Komiyama, M. J. Biochem. 1995, 118, 665-670. (f) Murakami, Y.;
Kikuchi, J.; Hisaeda, Y.; Hayashida, O. Chem. ReV. 1996, 96, 721-758.
(g) Breslow, R.; Dong, S. D. Chem. ReV. 1998, 98, 1997-2011. (h) Khan,
A. R.; Forgo, P.; Stine, K. J.; D’Souza, V. T. Chem. ReV. 1998, 98, 1977-
1996. (i) Merlau, M. L.; Mejia, M. D. P.; Nguyen, S. T.; Hupp, J. T. Angew.
Chem., Int. Ed. 2001, 40, 4239-4242.
9
12074
J. AM. CHEM. SOC. 2005, 127, 12074-12080
10.1021/ja053226g CCC: $30.25 © 2005 American Chemical Society

Tunable pK for Lamgmuir Enzyme
A R T I C L E S
Chart 1. Structures of Amino Acid-Carrying Amphiphiles and
control, because pK perturbations have been reported at the
surfaces of micelles,4 lipid bilayer vesicles,5 self-assembled
monolayers,6 and Langmuir monolayers.7 However, the pK
behaviors at different interfaces have thus far been discussed
separately, and there has been no systematic interpretation.
One of us reported significant differences in molecular
interaction at molecular, microscopic, and macroscopic inter-
faces, in which the most efficient modification of the molecular
interaction was achieved at a macroscopic interface, such as
the air-water interface.8 On the basis of that knowledge, we
have recently investigated the pK behaviors of amino acid
residues (usually the ꢀ-amino group in the Lys residue, both at
the air-water interface and at a vesicle surface9) and have found
that the air-water interface provides an appropriate microen-
vironment for fine-tuning of pK and for an enzyme-like reaction.
Here, we report three important findings: (i) superior pK control
at the air-water interface when compared with a vesicle system;
(ii) pK tuning by 4 or 5 units by control of ionic strength in the
subphase and prediction of the interfacial dielectric nature by a
simple equation; (iii) demonstration of ester hydrolysis by the
pK-controlled Lys residue at the air-water interface. These
findings should lead to development of a novel concept for
artificial enzymes that is proposed to be referred to as the
Langmuir-enzyme (L-zyme).
Related Compounds
Experimental Section
1. Materials. Structures of the amino acid-carrying amphiphiles and
related molecules used in this research are shown in Chart 1. Syntheses
of N+C5Lys2C16,10 N+C5Asp2C16,11 and N+C5Ala2C16 have been
12
described previously. Compounds N+C1Lys2C16, N+C5Glu2C16, Suc2C16,
and AcHis2C16 were newly synthesized by coupling of a dialkylamine
(4) (a) Tokiwa, F.; Ohki, K. J. Phys. Chem. 1967, 71, 1824-1829. (b)
Yalkowsky, S. H.; Zografi, G. J. Colloid Interface. Sci. 1970, 34, 525-
533. (c) Whiddon, C. R.; Bunton, C. A.; So¨derman, O. J. Phys. Chem. B
2003, 107, 1001-1005. (d) Kanicky, J. R.; Shah, D. O. Langmuir 2003,
19, 2034-2038. (e) Roy, D.; Karmakar, R.; Mondal, S. K.; Sahu, K.;
Bhattacharyya, K. Chem. Phys. Lett. 2004, 399, 147-151.
(5) (a) Mille, M.; Vanderkooi, G. J. Colloid Interface Sci. 1977, 61, 475-
484. (b) Haines, T. H. Proc. Natl. Acad. Sci. U.S.A. 1983, 80, 160-164.
(c) Swairjo, M. A.; Seaton, B. A.; Roberts, M. F. Biochim. Biophys. Acta
1994, 1191, 354-361. (d) Rostovtseva, T. K.; Aguilella, V. M.; Vodyanoy,
I.; Bezrukov, S. M.; Parsegian, V. A. Biophys. J. 1998, 75, 1783-1792.
(e) Silvander, M.; Hansson, P.; Edwards, K. Langmuir 2000, 16, 3696-
3702. (f) Apel, C. L.; Deamer, D. W.; Mautner, M. N. Biochim. Biophys.
Acta 2002, 1559, 1-9. (g) Lee. S.; De´saubry, L.; Nakatani, Y.; Ourisson,
G. C. R. Chimie 2002, 5, 331-335.
(6) (a) Creager, S. E.; Clarke, J. Langmuir 1994, 10, 3675-3683. (b) Troughton,
E. B.; Bain, C. D.; Whiteside, G. M.; Nuzzo, R. G.; Allara, D. L.; Porter,
M. D. Langmuir 1988, 4, 365-385. (c) Bain, C. D.; Whitesides, G. M.
Langmuir 1989, 5, 1370-1378. (d) God´ınez, L. A.; Castro, R.; Kaifer, A.
E. Langmuir 1996, 12, 5087-5092. (e) Hu, K.; Bard, A. J. Langmuir 1997,
13, 5114-5119. (f) Sugihara, K.; Shimazu, K.; Uosaki, K. Langmuir 2000,
16, 7101-7105. (g) Kakiuchi, T.; Iida, M.; Imabayashi, S.; Niki, K.
Langmuir 2000, 16, 5397-5401. (h) Munakata, H.; Kuwabata, S. Chem.
Commun. 2001, 1338-1339. (i) Schweiss, R.; Welzel, P.; Knoll, W.;
Werner, C. Chem. Commun. 2005, 256-258.
to the C-terminal and a polar headgroup to the N-terminal of the
corresponding amino acid residue. The detailed descriptions of the
synthesis are summarized in Supporting Information. Hexadecanoic acid
4-nitrophenyl ester (PNPC16) was purchased from Sigma. Water used
for the subphase was distilled using an Autostill WG220 (Yamato) and
deionized by a Milli-Q Lab (Millipore). Spectroscopic grade benzene
and ethanol (Wako Pure Chem.) were used as the spreading solvents.
High-purity sulfuric acid, sodium hydroxide, and sodium sulfate were
used as received.
2. π-A Isotherm Measurement. π-A isotherms were measured
using an FSD-300 computer-controlled film balance system (USI
System). A solution (benzene/ethanol (70/30 v/v)) of the amphiphile
(ca. 1 mM, and ca. 100 µL) was spread on the subphase at 20.0 ( 0.2
°C and compressed at a rate of 0.2 mm s-1 (or 20 mm2 s-1 based on
area). The subphase pH was adjusted by addition of the minimum
amount of aqueous sulfuric acid or sodium hydroxide with fluctuation
of subphase pH during a single measurement being less than 0.2 pH
units because of the shielded atmosphere. The ionic strength of the
subphase was adjusted with sodium sulfate.
(7) (a) Bagg, J.; Haber, M. D.; Gregor, H. P. J. Colloid Interface Sci. 1966,
22, 138-143. (b) Oishi, Y.; Takashima, Y.; Suehiro, K.; Kajiyama, T.
Langmuir 1997, 13, 2527-2532. (c) Esker, A. R.; Zhang, L.-H.; Olsen, C.
E.; No, K.; Yu H. Langmuir, 1999, 15, 1716-1724. (d) Kanicky, J. R.;
Poniatowski, A. F.; Mehta, N. R.; Shah, D. O. Langmuir 2000, 16, 172-
177. (e) Maierhofer, A. P.; Brettreich, M.; Burghardt, S.; Vostrowsky, O.;
Hirsch, A.; Langridge, S.; Bayerl, T. M. Langmuir 2000, 16, 8884-8891.
(f) Dynarowicz-Latka, P.; Cavalli, A.; Oliveira, O. N., Jr. Thin Solid Films
2000, 360, 261-267.
(8) Onda, M.; Yoshihara, K.; Koyano, H.; Ariga, K.; Kunitake, T. J. Am. Chem.
Soc. 1996, 118, 8524-8530.
(9) Preliminary results were reported in Ariga, K.; Abe, T.; Kikuchi, J. Chem.
Lett. 2000, 82-83.
3. Evaluation of pK Values. Dissociation constants (pK) at the air-
water interface were evaluated from the pH dependence of the apparent
molecular areas. The apparent total molecular area (Aobsd) is given,
assuming the absence of condensation effects, with the molecular areas
AB and ABH of the unprotonated and protonated species respectively,
and the B content of x, where B is NH2, COO-, or imidazole free base.
(10) Kikuchi, J.; Takashima, T.; Nakano, H.; Hie, K.; Etoh, H.; Noguchi, Y.;
Suehiro, K.; Murakami, Y. Chem. Lett. 1993, 553-556.
(11) Murakami, Y.; Hisaeda, Y.; Ogawa, A.; Miyajima, T.; Hayashida, O.; Ohno,
T. Tetrahedron Lett. 1993, 34, 863-866.
(12) Murakami, Y.; Nakano A.; Yoshimatsu, A.; Uchitomi, K.; Matsuda, Y. J.
Am. Chem. Soc. 1984, 106, 3613-3623.
9
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A R T I C L E S
Ariga et al.
Aobsd ) (1 - x)AB + xABH
(1)
Combining eq 1 with dissociation constant, K ) ([B] + [H+])/[BH]
results in eq 2.
(ABH - Aobsd
)
log
) pH - pK
(2)
[
]
(Aobsd - AB)
The pK value of the N+C5Lys2C16 monolayer was also evaluated
using reflection-absorption infrared (RA-IR) spectra of a single
monolayer transferred at 20 mN m-1 by the horizontal drawing-up
method13 on a gold-deposited glass slide (prepared with a VPC-260
vapor-deposition apparatus (ULVAC)), using an FT-IR spectrometer
Magna 560 (Nicolet) equipped with an MCT detector. The pK value
was estimated from the pH-dependence of the intensity of the N-H
vibration at 3250 cm-1 relative to that of the CH2 vibration at 2925
cm-1, which is insensitive to pH change.
Figure 1. π-A Isotherms on pure water at 20 °C: (a) Suc2C16; (b)
AcHis2C16; (c) N+C5Lys2C16; (d) N+C5Glu2C16; (e) N+C5Ala2C16; (f)
N+C5Asp2C16; (g) N+C1Lys2C16.
For pK determination in the vesicular state, N+C5Lys2C16 was
dispersed in D2O (10 mM) using a vortex mixer for 2 min, followed
by sonication (30 W, 3 min). DCl or NaOD was added to the aqueous
vesicle, and the solution was equilibrated to a stable pD for 2 h. The
addition of 0.4 to the reading from the pH meter gave the corrected
pD.14 1H NMR spectra were measured using a JEOL JNM-270
spectrometer.
4. Evaluation of PNPC16 Hydrolysis. A mixed monolayer of the
amino acid-carrying amphiphiles (N+C5Lys2C16 or N+C5Ala2C16) and
Figure 2. (A) (a) Chemical shifts (δ) of ꢀ-methylene protons of N+C5-
Lys2C16 in aqueous vesicles at various pH conditions; (b) molecular areas
at 20 mN m-1 of N+C5Lys2C16 in monolayer on water at various pH
conditions. (B) Linear plots of the data presented in (A): (a) X ) δ for
vesicular system; (b) X ) molecular area of the monolayer system. In both
the plots, Xobsd, XNH3, and XNH2 represent the corresponding parameters of
the observed samples, the value for the protonated species, and the value
for the unprotonated species, respectively. The intercepts of the abscissa
provide the pK values.
PNPC16 (2:1 in molar ratio) was spread and compressed to 20 mN m-1
.
The hydrolysis of PNPC16 was evaluated from the decrease in
absorbance at 300 nm ascertained by surface reflection-absorption
spectroscopy, because the hydrolyzed product p-nitrophenolate group
escapes deep into the subphase. The amount of unreacted p-nitrophenyl
group was quantified on the basis of the calibration curve of PNPC16
at 300 nm, and conversion of the PNPC16 hydrolysis could be calculated.
The surface-reflective UV-vis spectra were measured using a photo-
diode array-equipped spectrometer (Otsuka Electronics, model MCPD-
7000).
of electrostatic repulsion between protonated amine groups. The
pH profile of the molecular areas at 20 mN m-1 (Figure
2A(b)) was converted to the linear plot (Figure 2B(b)), resulting
in a significantly shifted pK of 5.1. Similar pK values of 5.3
and 5.2 were obtained from the molecular areas at 5 and 35
mN m-1, respectively. Conversely, π-A isotherms of the Ala-
functionalized amphiphile, N+C5Ala2C16, did not show any
changes in molecular area in the pH range from 2 to 12.
The monolayer of another Lys-functionalized amphiphile,
N+C1Lys2C16, was also investigated, giving results in pK values
of 6.1, 6.1, and 6.0 at 5, 20, and 35 mN m-1, respectively (see
Table 1). Similarly, large pK shifts at the air-water interface
were confirmed for the two kinds of Lys amphiphiles. More
direct evidence for this pK shift was obtained by protonation
of the amino group and IR analyses for the monolayers
transferred to a gold-coated glass slide. Peaks at around 3250
cm-1, mostly originating from ν(N-H), intensified as pH
increased, while the sharp peak for νas(CH2) at 2925 cm-1 was
unchanged (Figure 3A). IR absorbance intensities at 3250 cm-1
relative to those at 2925 cm-1 were converted to a linear plot
(Figure 3B), giving a pK value of 6.1, which is identical with
that determined from the π-A isotherm measurements.
Results and Discussion
1. Enhanced pK Shifts at the Air-Water Interface
Compared with Vesicular System. π-A isotherms of the
amphiphiles at 20 °C on pure water are summarized in Figure
1. All of the isotherms except Suc2C16 display an expanded
profile. To select an interfacial medium suitable for efficient
pK control, we compared pK values both at the air-water inter-
face and at an aqueous vesicle surface. As a model amphiphile,
N+C5Lys2C16 was used, which can form a bilayer vesicle in
bulk water or a Langmuir monolayer. First, the dissociation
property of the aqueous bilayer vesicle of N+C5Lys2C16 was
monitored by observation of the chemical shift of the ꢀ-meth-
1
ylene protons in the H NMR spectra (Figure 2A(a)), and its
linear plot provides a pK value of 9.4 (Figure 2B(a)). The
correlation coefficient close to unity (0.99) in the plot indicates
that the bilayer system has a single apparent pK value. Since
the pK of the aqueous Lys side chain is 10.5, the pK observed
in an aqueous vesicle has a shift of only ca. 1 pK unit.
Next, the pK value of the same lipid composing a Langmuir
monolayer was evaluated on the basis of the molecular area
change in their π-A isotherms under pH control. The molecular
area of the N+C5Lys2C16 monolayer in an expanded phase in
the whole pH region at 20 °C increased as pH decreased because
A comparison of pK values in an aqueous vesicle and in a
Langmuir monolayer indicates that a remarkable difference
exists between these environments. Several researchers have
investigated microenvironments at the air-water interface using
chromophores to demonstrate a decrease in the dielectric
constant.15 A hydrophobic environment at the water surface
(13) Oishi, Y.; Kuri, T.; Takashima, Y.; Kajiyama, T. Chem. Lett. 1994, 1445-
1446.
(14) Tatsumoto, K.; Martell, A. E. J. Am. Chem. Soc. 1981, 103, 6203-6208.
9
12076 J. AM. CHEM. SOC. VOL. 127, NO. 34, 2005

Tunable pK for Lamgmuir Enzyme
A R T I C L E S
Table 1. Observed pK Values for Amino Acid and Related Residues at the Air-Water Interface (20 °C)
functional
group
ionic
surface pressure
functional
group
ionic
surface pressure
1
1
lipid
strength
(mN m-
)
pK
lipid
strength
(mN m-
)
pK
Monolayer
N+C5Lys2C16
N+C5Lys2C16
N+C5Lys2C16
N+C5Lys2C16
N+C5Lys2C16
N+C5Lys2C16
N+C5Lys2C16
N+C5Lys2C16
N+C1Lys2C16
N+C1Lys2C16
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
0
0
0
5
20
35
35
35
5
20
35
5
5.3
5.1
5.2
7.3
9.5
9.8
10.5
9.8
6.1
6.1
N+C1Lys2C16
N+C1Lys2C16
N+C1Lys2C16
N+C5Asp2C16
N+C5Asp2C16
N+C5Asp2C16
N+C5Asp2C16
N+C5Glu2C16
Suc2C16
NH2
NH2
NH2
COOH
COOH
COOH
COOH
COOH
COOH
imidazolyl
0
35
35
35
5
20
35
35
35
5
6.0
7.9
9.6
5.4
5.3
5.3
5.8
4.6
10.0
3.6
0.0001
0.01
0
0
0
0.1
0
0
0.0001
0.001
0.1
0.1
0.1
0
0
20
AcHis2C16
0
35
Vesicle
C+C5Lys2C16
NH2
0
-
9.4
interaction between these interfaces was experimentally dem-
onstrated by Onda et al. who reported that the binding constants
between guanidinium and phosphate ions at the air-water
interface are 103-104 times larger than those at the surface of
an aqueous bilayer.8 This report also supports an enhanced
electrostatic effect at the air-water interface. Interestingly, the
difference in guanidinium/phosphate binding constant in Onda’s
work corresponds roughly to the pK difference of the Lys
amphiphile between the two interfaces, although the molecular
interactions examined are opposing. Some literatures reported
differences of hydrogen bonding capability between the air-
water interface and the micelle surface, which may be also
related with the above-mentioned phenomena.18 Very recently,
we found a significant pK difference in phosphoric acid
dissociation between the Langmuir monolayer and the aqueous
vesicle, where the former interface induces a larger shift than
that at the latter one, and their difference is ca. 4 units.19
The experimental and theoretical information listed here
indicates that a more efficient pK shift can be obtained at the
air-water interface than in aqueous vesicles. This may be caused
by enhanced molecular interactions at the air-water interface
where influence of the hydrophobic phase would be effectively
communicated to the interfacial molecular species probably
because of the smoothness of the interface.
2. Tuning of pK Values at the Air-Water Interface. Table
1 summarizes the pK values for the Langmuir monolayers
investigated. The pK values changed significantly, depending
on ionic strength of the subphase, while the influence of the
surface pressure can be neglected. As plotted in Figure 4, the
Lys pK values in both the N+C5Lys2C16 and N+C1Lys2C16
monolayers increase as ionic strength increases and approach
closely the value reported in a bulk aqueous medium. The effect
of electric repulsion between the trimethylammonium group and
Lys ammonium or between Lys ammonium groups themselves
would be shielded by addition of salts. Our most important
finding is that the pK of the Lys residue can be tuned by ca. 4
or 5 units simply by changing the ionic strength of the aqueous
subphase.
Figure 3. (A) Reflection-absorption infrared (RA-IR) spectra of the
N+C5Lys2C16 monolayer transferred onto a gold-covered glass slide: (a)
at pH 4.4; (b) at pH 6.5; (c) at pH 10.3. (B) Linear plots for the pK
determination using IR intensities (absorbances) at 3250 cm-1 relative to
those at 2925 cm-1, where Iobsd, INH3, and INH2 represent the corresponding
values of the observed films, the value for the protonated species, and the
value for the unprotonated species, respectively. The intercept of the abscissa
provides the pK value.
would suppress protonation of the ꢀ-amino group of Lys because
of destabilization of charged species in the nonpolar medium
through increased electrostatic potentials. On the basis of the
nonlinear Poisson-Boltzmann equation Smart and McCammon
estimated the pK value of a long-chain alkylamine at the air-
water interface and demonstrated that a distance of a few
angstroms above the water surface results in a shift of several
units in the pK of the amino group.16 According to their
estimation, the distance between the polar head and the Lys
amino group in N+C5Lys2C16, as estimated by molecular
modeling (0.8-1.4 nm), corresponds to a positioning of the Lys
amino group appropriate for a significant shift of the pK. The
importance of interface structure on electrostatic interaction was
similarly estimated by Sakurai et al. on the basis of quantum
chemical calculations in which the interaction between cationic
guanidinium and anionic phosphate could be significantly
strengthened by the influence of the hydrophobic lipid phase.17
If the water-lipid interface is more disordered as expected for
aqueous vesicles, the influence of the hydrophobic phase would
not be effectively communicated to functional groups embedded
at the interface. In fact, the difference in efficiency of molecular
(15) (a) Grieser, F.; Thistlethwaite, P.; Triandos, P. J. Am. Chem. Soc. 1986,
108, 3844-3846. (b) Petrov, J. G.; Mo¨bius, D. Langmuir 1989, 5, 523-
528. (c) Petrov, J. G.; Mo¨bius, D. Langmuir 1996, 12, 3650-3656. (d)
Matsui, J.; Mitsuishi, M.; Miyashita, T. Macromolecules 1999, 32, 381-
386.
The pK values of various amino acids can be also tuned by
molecular design. The pK of the N+C1Lys2C16 monolayer is
lower by ca. 1 unit than that of the N+C5Lys2C16 monolayer.
(16) Smart J. L.; McCammon, J. A. J. Am. Chem. Soc. 1996, 118, 2283-2284.
(17) (a) Sakurai, M.; Tamagawa, H.; Inoue, Y.; Ariga, K.; Kunitake, T. J. Phys.
Chem. B 1997, 101, 4810-4816. (b) Sakurai, M.; Tamagawa, H.; Furuki,
T.; Inoue, Y.; Ariga, K.; Kunitake, T. Chem. Lett. 1995, 1001-1002. (c)
Tamagawa, H.; Sakurai, M.; Inoue, Y.; Ariga, K.; Kunitake, T. J. Phys.
Chem. B 1997, 101, 4817-4825.
(18) (a) Lovelock, B.; Grieser, F.; Healy, T. W. J. Phys. Chem. 1985, 89, 501-
507. (b) Hall, R. A.; Hayes, D.; Thistlethwaite, P. J.; Grieser, F. Colloid
Surf. 1991, 56, 339-356.
(19) Ariga, K.; Yuki, H.; Kikuchi, J.; Dannemuller, O.; Albrecht-Gary, A.-M.;
Nakatani, Y.; Ourisson, G. Langmuir 2005, 21, 4578-4583.
9
J. AM. CHEM. SOC. VOL. 127, NO. 34, 2005 12077

A R T I C L E S
Ariga et al.
ai
1
kT
π )
+
(4)
∑
( )( )
ri r2
A - Ao
Differences in surface pressure between protonated (πBH) and
unprotonated species (πB) at a given molecular area are given
by eq 5.
ai
1
πBH - π ) ∆
(5)
∑
B
( )
ri r2
( )
[
]
If the difference in surface pressure between protonated and
unprotonated species is dominated by electrostatic interaction
contributions, the energy term in (C/ꢀ) × (1/r) becomes the main
attribute in ∑ terms, where ꢀ is the dielectric constant and C is
the constant depending on the two-dimensional geometries of
amphiphiles. This assumption simplifies the eq 5 with constant
term C′ as follows.
Figure 4. Effect of ionic strength of the aqueous subphase on pK values
of the lysine residue at 20 °C: (a) N+C5Lys2C16; (b) N+C1Lys2C16.
The Lys residue in the N+C1Lys2C16 monolayer may locate in
a more hydrophilic environment than that in the N+C5Lys2C16
monolayer because of the short spacer between the Lys group
and the polar head. The pK behaviors of N+C5Asp2C16 and
N+C5Glu2C16, which contain COOH as a dissociation group,
are somewhat different from those observed for the Lys-based
amphiphiles. The pK values obtained lie within a range from
4.6 to 5.8 and are similar to those of carboxylates in water. In
addition, an increase in the ionic strength did not significantly
change the pK value. In these monolayers, destabilization of
the carboxylate group might be compensated by charge attraction
from the cationic polar head. In sharp contrast, the monolayer
of Suc2C16, which contains no cationic headgroup, showed a
significant pK shift (10.0), where the effect of strong destabi-
lization of the charged species at the air-water interface
becomes apparent. Similarly, AcHis2C16 does not contain a
charged headgroup, and its monolayer displays a large decrease
in pK of its imidazolyl group compared with the corresponding
value observed in bulk water. All the results obtained here
suggest the crucial role of electrostatic interactions for pK shifts
at the air-water interface.
Evaluation of the apparent dielectric constant at the air-water
interface provides indispensable information for further tuning
of the surface pK. We adopted a simplified model for this
purpose although many detailed but complicated equations have
been thus far proposed.20 In analogy with the van der Waals
equation in two dimensions, the following equation holds
between surface pressure π and molecular area A with excluded
area (Ao), Boltzmann constant (k), and absolute temperature
(T).21
C
1
πBH - πB )
(6)
(7)
3
( )( )
ꢀ
r
C′
ꢀ
(πBH - πB)A3/2
)
This consideration was applied to the actual π-A isotherms
of N+C1Lys2C16 in the protonated (Figure 5A(a)) and unpro-
tonated states (Figure 5A(b)). A plot of (πNH3 - πNH2)A3/2 as a
function of molecular area (Figure 5B) becomes constant
between the lift-off pressure and the collapse pressure. Influence
of a double-layer potential on surface acidic environment has
been discussed on the basis of Gouy-Chapmann theory.22
Consideration of the surface pressure difference based on the
Gouy-Chapmann theory gives eq 8, which contains the
constants (D and D′) and bulk concentration of ions (c).21
D′
πBH - πB ) Dc1/2 cosh sinh-1
-1
(8)
1/2
{
(
)}
[
]
Ac
Davies proposed a simplification of this term to 2kT/A.19a
However, a plot of (πNH3 - πNH2)A does not show the constant
behavior as effectively as a (πNH3 - πNH2)A2 plot (Figure 5C).
In addition, plotting of logarithms of πNH3 - πNH2 against A
shows a slope of 1.49 with a correlation coefficient of 0.995
(Figure 5D). These results prove the viability of eq 7.
Plots based on eq 7 provide relative values of the apparent
dielectric constant (ꢀ). Figure 6 shows values calculated for
the N+C5Lys2C16 and N+C1Lys2C16 monolayers at various
ionic strengths. These dielectric constants are values relative to
those observed for the N+C5Lys2C16 monolayer with ionic
strength of 0.1 where the observed pK value is close to that of
amino groups in bulk water. If we assume that dielectric constant
at high ionic strength equals that of bulk water (80 at 20 °C),
dielectric constants at the surfaces of N+C5Lys2C16 and
N+C1Lys2C16 monolayers on pure water are estimated to be
52 and 66, respectively. These values are in good agreement
with those estimated experimentally by Grieser et al.15a and
Petrov and Mo¨bius.15c However, determination of absolute
(π - π*)(A - Ao) ) kT
(3)
The term π* represents the contribution of molecular interac-
tion to the surface pressure and has dimensions of energy per
area. The energy term from molecular interaction may be given
generally by a polynomial expression of the averaged molecular
distance (r), giving the general expression of π* as ∑(ai/ri)(1/
r2). Therefore, surface pressure π is given by eq 4.
(20) (a) Davis, J. T. J. Colloid Sci. 1956, 11, 377-390. (b) Caspers, J.;
Goormaghtigh, E.; Ferreira, J.; Brasseur, R.; Vandenbranden, M.; Ruyss-
chaert, J.-M. J. Colloid Interface Sci. 1983, 91, 546-551. (c) Petrov, J.
G.; D.; Mo¨bius, D. Langmuir 1990, 6, 746-751. (d) Ruckenstein, E.; Li,
B. J. Phys. Chem. B 1998, 102, 981-989. (e) Fainerman, V. B.; Vollhardt,
D. J. Phys. Chem. B 2003, 107, 3098-3100. (f) Rusanov, A. I. J. Chem.
Phys. 2004, 120, 10736-10747.
(22) (a) Taylor, D. M.; Oliveira, O. N., Jr.; Morgan, H. Chem. Phys. Lett. 1989,
161, 147-150. (b) Teppner, R.; Haage, K.; Wantke, D.; Motschmann, H.
J. Phys. Chem. B 2000, 104, 11489-11496. (c) Cavalli, A.; Dynarowicz-
Latka, P.; Oliveira, O. N., Jr.; Feitosa, E. Chem. Phys. Lett. 2001, 338,
88-94.
(21) Gaines, G. L. Insoluble Monolayers at Liquid-Gas Interfaces; Interscience
Publishers: New York, 1966.
9
12078 J. AM. CHEM. SOC. VOL. 127, NO. 34, 2005

Tunable pK for Lamgmuir Enzyme
A R T I C L E S
Figure 7. (A) Surface absorption spectra of the mixed monolayer of N+C5-
Lys2C16 and PNPC16 (2:1 in molar ratio) at pH 5.1 and at ionic strength of
0 (20 °C, 20 mN m-1): (a) 0 s; (b) after 120 s. (B) Time-course of
conversion of the PNPC16 hydrolysis within the mixed monolayer [lipid
X:PNPC16 with 2:1 in molar ratio] (20 °C, 20 mN m-1): (a) lipid
X ) N+C5Lys2C16 at pH 5.1 and at ionic strength of 0; (b) lipid X )
N+C5Lys2C16 at pH 8.0 with ionic strength of 0; (c) lipid X ) N+C5Lys2C16
at pH 8.0 and at ionic strength of 0.1; (d) lipid X ) N+C5Ala2C16 at pH
8.0 and at ionic strength of 0.
Figure 5. (A) π-A Isotherms of N+C1Lys2C16 (20 °C): (a) at pH 3; (b)
at pH 11.0. (B) Plot of the parameter in eq 7 [(πNH3 - πNH2) × A3/2] as a
function of the molecular area using the data presented in (A). (C) Plots of
the parameter in (a) [(πNH3 - πNH2) × A] and (b) [(πNH3 - πNH2) × A2] as
a function of the molecular area using the data presented in (A). (D) Linear
relation between log(πNH3 - πNH2) and -log A where the slope and inter-
cept are 1.49 and 0.59, respectively, with the correlation coefficient of
0.995.
Figure 8. Conversion of the PNPC16 hydrolysis within the mixed monolayer
[N+C5Lys2C16: PNPC16 with 2:1 in molar ratio] after a reaction time of 90
s under various pH conditions (20 °C, 20 mN m-1).
as a function of reaction time (Figure 7B). The N+C5Lys2C16
monolayer promoted the PNPC16 hydrolysis at pH 5.1 (a) and
pH 8.0 (b), while the N+C5Ala2C16 monolayer did not show
any hydrolytic activity (d). The latter fact suggests that hydroxyl
anions in the vicinity of the quaternary ammonium headgroup,
if any, do not contribute to the hydrolysis, and the lysine amino
groups play an indispensable role in the hydrolysis. Interestingly,
increase of ionic strength (0.1) drastically suppressed the PNPC16
hydrolysis (c). Lower population of free amine under this
condition leads to inactivation of the N+C5Lys2C16 catalyst,
because the pK value of the lysine residues at the interface are
shifted to 10.5 at the ionic strength of 0.1 (see Table 1).
Figure 6. Effect of ionic strength on apparent dielectric constants at the
monolayer surface: (a) N+C5Lys2C16; (b) N+C1Lys2C16. These dielectric
constants are values relative to that observed for the N+C5Lys2C16
monolayer at an ionic strength of 0.1.
values of interfacial dielectric constants still needs careful
consideration as seen in discussions in some pioneering reports.23
3. Ester Hydrolysis Catalyzed by pK-Tuned Amino
Groups. Basic amino groups are able to catalyze hydrolysis of
esters, but their protonated forms are not. Therefore, pK shifts
of the amino groups to the neutral pH region are indispensable
for ester hydrolyses under biological conditions and are com-
monly accomplished in enzyme systems. Tuning of the pK of
Lys residues at the air-water interface could mimic this
biological activity.
A model system contains hexadecanoic acid 4-nitrophenyl
ester (PNPC16) as hydrolysis substrate in the monolayer of
N+C5Lys2C16 as the catalyst. Changes of surface-absorption
spectra, upon dissolution of p-nitrophenyl residue, were moni-
tored for evaluation of the hydrolysis of PNPC16 (see typical
example in Figure 7A). Conversion of the hydrolysis was plotted
The PNPC16 hydrolysis catalyzed by N+C5Lys2C16 was
investigated under various pH conditions, and the conversion
at 90 s was plotted as a function of subphase pH (Figure 8).
The maximum conversion was observed at around pH 5
although the data were scattered. Because the pK value of the
Lys residue at the corresponding condition is 5.1, coexistence
of free amine and protonated ammonium seems to more
effectively promote the reaction. Cooperative action of NH2 and
+
NH3 groups, where electrostatic interaction between ester
+
carbonyl oxygen and NH3 group assists nucleophilic attack
by the NH2 group at the ester carbonyl carbon (see Figure 9),
may play an important role in the PNPC16 hydrolysis in the
present case.
(23) (a) Demchak R. J.; Fort, T., Jr. J. Colloid Interface Sci. 1974, 46, 191-
202. (b) Vogel, V.; Mo¨bius, D. Thin Solid Films 1988, 159, 73-81.
9
J. AM. CHEM. SOC. VOL. 127, NO. 34, 2005 12079

A R T I C L E S
Ariga et al.
Figure 9. Plausible mechanism of the PNPC16 hydrolysis by lysine residues at the air-water interface.
Conclusion
of other reactions is now under investigation for generalization
of this concept.
The results obtained impressively illustrate that the air-water
interface is an appropriate medium for control of pK of amino
acid residues, where the pK values can be tuned within the range
of 4 or 5 units simply by adjusting the ionic strength in the
subphase. The origin of the effective pK shift at the air-water
interface when compared with the aqueous vesicle medium is
still unclear, but the smooth and undisturbed nature of the air-
water interface may enhance the influence of the low dielectric
phase on functional groups embedded at the air-water interface.
The hydrolysis by amino groups over the neutral pH region,
which is commonly seen in biological catalysts, was demon-
strated through pK control at the air-water interface. Rich
possibilities in design of amino acid-carrying amphiphiles and
the free mixing nature of Langmuir monolayers should lead to
development of a more sophisticated catalysis as seen in enzyme
reaction pockets. Elaborate functions, that can be referred to as
a “Langmuir enzyme (L-zyme)” will be realized as the end result
of the concept proposed in this research,24 and demonstration
Acknowledgment. The contents described in this paper are
partly supported by Grant-in Aid for Scientific Research on
Priority Areas (No. 17036070 “Chemistry of Coordination
Space”) from Ministry of Education, Science, Sports, and
Culture, Japan.
Supporting Information Available:
Syntheses of
N+C1Lys2C16, N+C5Glu2C16, Suc2C16, and AcHis2C16. This
material is available free of charge via the Internet at
http://pubs.acs.org.
JA053226G
(24) Some reports demonstrated catalysis and related reaction using Langmuir
monolayers and Langmuir-Blodgett films. (a) Tollner, K.; Popovitz-Biro,
R.; Lahav, M.; Milstein, D. Science 1997, 278, 2100-2102. (b) Abatti,
D.; Zaniquelli, M. E. D.; Iamamoto, Y.; Idemori, Y. M. Thin Solid Films
1997, 310, 296-302. (c) Fukuda, K.; Shibasaki, Y.; Nakahara, H.; Liu,
M.-H. AdV. Colloid Interface Sci. 2000, 87, 113-145. (d) Kumar, J. K.;
Oliver, J. S. J. Am. Chem. Soc. 2002, 124, 11307-11314.
9
12080 J. AM. CHEM. SOC. VOL. 127, NO. 34, 2005