Synthesis and characterization of barnacle adhesive mimetic towards underwater adhesion

Article abstract of DOI:10.1246/cl.150311

A polyacrylamide including tetra-alanine units, hydroxy groups, and hexyl groups was synthesized as a mimetic of barnacle underwater adhesive proteins. The synthesized barnacle mimetic polymer was dissolved in water and subjected to a condensation reaction with hexylamine. Gelation through multiple hydrogen bonds of the oligo-alanine units was confirmed. The adhesive strength of the bonded substrate with the gel-forming barnacle mimetic polymer solution was demonstrated to be 402 kPa for poly(methyl methacrylate) (PMMA) plate adhesion by the tensile shear adhesion test.

Full text of DOI:10.1246/cl.150311

Received: April 5, 2015 | Accepted: May 4, 2015 | Web Released: May 13, 2015
CL-150311
Synthesis and Characterization of Barnacle Adhesive Mimetic towards Underwater Adhesion
Jin Nishida,1,2 Yuji Higaki,1,2 and Atsushi Takahara*1,2
JST, ERATO, Takahara Soft Interfaces Project, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395
1
2
Institute for Materials Chemistry and Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395
(
E-mail: takahara@cstf.kyushu-u.ac.jp)
A polyacrylamide including tetra-alanine units, hydroxy
groups, and hexyl groups was synthesized as a mimetic of
barnacle underwater adhesive proteins. The synthesized barnacle
mimetic polymer was dissolved in water and subjected to a
condensation reaction with hexylamine. Gelation through
multiple hydrogen bonds of the oligo-alanine units was
confirmed. The adhesive strength of the bonded substrate
with the gel-forming barnacle mimetic polymer solution was
demonstrated to be 402 kPa for poly(methyl methacrylate)
(PMMA) plate adhesion by the tensile shear adhesion test.
Figure 1. Schematic image of adhesion by barnacle mimetic
polymer.
Underwater adhesion is a fascinating and complex theme for
human technology. Marine sessile organisms such as mussels
and barnacles easily accomplish underwater adhesion. The well-
documented adhesion mechanism in mussels is based on mussel
adhesive proteins that have a high concentration of dihydroxy-
phenylalanine (DOPA).1 The catechol group in DOPA is
involved in the crosslinking reaction through aerobic oxidation,
and the anchor to metal oxide surfaces is accomplished by
hydrogen bonding or chelate bond formation.2 Based on these
findings, various artificial mussel adhesive protein mimetics
O
H
N
OH
N
H
O
O
n
x
NH2
PAAm-diAla: x=2, n / m = 1 / 20
PAAm-triAla: x=3, n / m = 1 / 14
PAAm-tetAla: x=4, n / m = 1 / 15
m O
,3
Figure 2. Polyacrylamides with oligo-alanine units.
4­8
were reported.
Barnacles are another well-known marine sessile organism.
Barnacles attach to material surfaces in seawater through the
formation of a proteinaceous adhesive cement.9 Because the
adhesive cement is insoluble, the adhesive materials and
adhesion mechanism have not been fully understood. Some
carboxy group of oligo-alanine units and hexylamine. Cross-
linking through the oligo-alanine unit assembly is triggered by
the reaction induced hydrophobic interaction. For the surface
interacting moieties, the hydrophilic hydroxy groups and the
hydrophobic hydrocarbon (hexyl) groups are incorporated into
the side group. The adhesive strength for the metal or plastic
plates with the gel-forming barnacle mimetic adhesive was
examined by the tensile shear adhesion test.
The target polymer was synthesized by radical copolymer-
ization of an acrylamide with the oligo-alanine unit and
acrylamide monomers. The protected oligo-alanine unit was
synthesized through conventional methods, adopting 9-fluore-
nylmethyloxycarbonyl (Fmoc) and tert-butyl ester (OtBu)
protecting groups (Scheme S1, Supporting Information). The
Fmoc protecting group at the amine group was removed, and
a polymerizable acrylamide group was introduced by con-
densation reaction with an activated acrylamide derivative
(Scheme S2). In the case of di- and tri-alanine type monomers,
the protecting group was removed and radical polymerization
was performed (Scheme S3). In the case of tetra-alanine type
monomers, the protecting group was removed after polymeriza-
tion (Scheme S4). Polyacrylamides that consist of acrylamides
with the oligo-alanine unit and acrylamide (PAAm-diAla,
PAAm-triAla, and PAAm-tetAla) are shown in Figure 2.
Gelation tests were carried out with the aqueous polymer
solution to confirm the crosslinking network of the alanine unit.
The polyacrylamides were dissolved in a basic solution through
interesting features for the barnacle adhesive have been reported:
i) Crosslinking points are not covalent bonds.1
0,11
It is expected
that amyloid-like aggregates of self-assembled proteins produce
the insoluble and tough crosslinking network. ii) Proteins that
localize at the adhesive/adherent interface contain hydrophilic
12
and hydrophobic amino acid residues that interact with
1
universal substrates.
In the present study, we designed a polyacrylamide as a new
artificial barnacle adhesive protein mimetic consisting of two
components: i) a self-assembly motif for polymer crosslinking
and ii) surface anchoring moieties for attachment to universal
substrates (Figure 1). For the self-assembly motif, we adopted
oligo-alanine units, which exist in natural materials such as the
1
3,14
crystalline phase of spider silk
complexes.
and insoluble amyloid protein
1
5,16
The strong interaction of multiple hydrogen
bonds and molecular packing lead to the remarkable self-
assembly. There are many reports on the construction of ordered
structures from oligo-alanine containing chemicals, based on
their superior self-assembling property.1
7­20
Therefore, the oligo-
alanine unit is expected to be a good candidate for the
self-assembled crosslinking motif. For self-assembling ability
enhancement, an alkyl chain was introduced at the end of
the alanine unit through condensation reaction of the terminal
Chem. Lett. 2015, 44, 1047–1049 | doi:10.1246/cl.150311
© 2015 The Chemical Society of Japan | 1047

O
O
O
(b)
H
H
N
H
N
hexylamine /
N
N
N
OH
H
H
WSC / HONSu
O
O
O
n
NH2
(a)
mO
n / m = 1 / 15
(c)
H
N
O
H
O
H
O
b
N
b
N
Hexylamine/
HONSu / WSC
N
N
N
H b
H b
O
H
a
nO
O
butylamine /
WSC / HONSu
5
wt% aqueous solution
NH2
of PAAm-tetAla
mO
n / m = 1 / 15
Figure 3. Gelation tests for tetra-alanine type polymer
PAAm-tetAla) aqueous solutions. (a) 5 wt % PAAm-tetAla
HOD
(
aqueous solution, (b) 5 wt % PAAm-tetAla aqueous solution
with hexylamine and condensation reagents (WSC-HONSu),
(c) 5 wt % PAAm-tetAla aqueous solution with butylamine and
PAAm-tetAla
WSC-HONSu.
H O
2
DMSO
PAAm-tetAla after
reaction with
hexylamine
b
a
deprotonation of the carboxy group at the oligo-alanine terminal,
and then were protonated by adding hydrochloric acid solution.
Under these conditions, no gelation was observed in any type of
alanine unit. However, the tetra-alanine type polymer (PAAm-
tetAla) solution became opaque (Figure 3a). This result suggests
that the aggregation of the tetra-alanine units exists in the
solution, but the network structure is not enough for gelation.
To enhance the interaction of the tetra-alanine group, we
introduced a hydrophobic group at the end of the alanine unit.
Dehydration condensation reaction of the carboxy groups in
PAAm-tetAla with alkyl amines (butylamine or hexylamine)
was performed by means of water-soluble carbodiimide and N-
hydroxysuccinimide (WSC-HONSu). The PAAm-tetAla solu-
tion was gelated by the reaction with hexylamine after 4 h
1
Figure 4. H NMR spectra of PAAm-tetAla before and after
1
reaction with hexylamine. H NMR measurements were carried
out at 298 K in DMSO-d6.
O
O
O
H
N
H
N
H
N
N
H
N
H
OH
O
O
O
n
H
N
(Figure 3b), but gelation was not observed by the reaction with
mO
butylamine even after 24 h (Figure 3c). The complimentary
interaction of the amide bonds in oligo-alanine units is not
sufficient for self-assembly in aqueous solution, and the hydro-
phobic part is necessary for assembly. Hydrophobic interaction
promotes the aggregation of tetra-alanine units, and the hexyl
groups are suitable for the gelation trigger.
H
N
OH
l
O
PHydAAm-tetAla:n / m / l = 1 / 0 / 12
PHydAAm-Hex-tetAla:n / m / l = 1 / 1 / 10
1
Figure 5. Barnacle adhesive protein mimetic polymers.
To clarify the degree of substitution, the H NMR spectrum
of the hydrophobized polymer was measured. The formed gel
was purified by dialysis. After lyophilization, the obtained
polymer was dissolved in DMSO-d6, and the H NMR spectrum
was measured (Figure 4). The degree of substitution was
estimated from the ratio of methylene groups in the alkyl chain
signal (0.8 ppm) and the methine groups in the oligo-alanine
complementally multiple hydrogen bonds in tetra-alanine units
are required for gelation. The repeating number of the oligo-
alanine unit and terminal alkyl chain length would correlate with
the adhesive strength of the adhesives.
1
Another function of barnacle cement proteins, the surface
anchoring moiety, was introduced through the adoption of
hydroxy and hexyl groups in the polyacrylamide. The tetra-
alanine-containing monomer was copolymerized with hydroxy-
ethylacrylamide and hexylacrylamide. We obtained the tetra-
alanine type polymer with the hydroxy group (Figure 5,
PHydAAm-tetAla) or hydroxy and hexyl groups (Figure 5,
PHydAAm-Hex-tetAla). Gelation and the adhesive properties of
these copolymers were confirmed by the gelation test and the
tensile shear adhesion test, respectively. Aqueous solutions of
PHydAAm-tetAla or PHydAAm-Hex-tetAla were prepared with
condensation reagents (WSC-HONSu) and a curing reagent
(hexylamine). The solution viscosity gradually increased, and a
gel was obtained after 4 h (Figure S6). The induction time for
(4.2 ppm). The integration ratio of methylene to methine was
2.7/4, and the degree of substitution was calculated to be 0.9.
A gelation test was carried out with a polyacrylamide with
shorter oligo-alanine units for the confirmation of the effect of
oligo-alanine groups. The tri-alanine type polymer (PAAm-
triAla) was subjected to a condensation reaction with hexyl-
amine under the same conditions as the PAAm-tetAla system. In
this case, no gelation was observed even after 24 h (Figure S5).
The 10 wt % aqueous solution of the PAAm-triAla system was
more viscous than the 5 wt % solution as a result of the increase
in the association constant; however, the solution could not
accomplish the gelation. Both the hydrophobic interaction by
the hexylamide terminal and the coherent interaction through
1048 | Chem. Lett. 2015, 44, 1047–1049 | doi:10.1246/cl.150311
© 2015 The Chemical Society of Japan

Table 1. Tensile shear adhesion strength of adhered stainless
steel or PMMA plates by gelation of barnacle mimetic polymer
aqueous solution
group. Adhesion of stainless steel plates and PMMA plates was
accomplished through the gelation of the aqueous polymer
solution. We believe that very efficient adhesives can be
designed by tuning the polymer structure, including the mo-
lecular weight and composition of the monomer units based on
our barnacle mimetic molecular design. This barnacle adhesive
protein mimetic is useful for aqueous underwater adhesives, and
it would provide insights for barnacle adhesive cement.
Tensile shear adhesion strength/kPa
PHydAAm-tetAla gel
PHydAAm-Hex-tetAla gel
Stainless steel
PMMA
24
91
21
402
Supporting Information is available electronically on J-STAGE.
gelation was almost the same as an adhesive without anchoring
groups. Adhesion tests were carried out with stainless steel or
poly(methyl methacrylate) (PMMA) plates. The adhesive solu-
tion including polyacrylamide (PHydAAm-tetAla or PHydAAm-
Hex-tetAla), condensation reagents, and curing reagent was
sandwiched between the plates. Samples were kept at room
temperature, under high relative humidity (more than 90%). The
adhesion strength of the stainless steel plates or PMMA plates
was measured using a tensile shear adhesion tester (Table 1).
In the case of stainless steel, the adhesion strength through the
gelation of PHydAAm-tetAla or PHydAAm-Hex-tetAla solution
was almost equal, whereas there was a large difference in the
adhesion strength of PMMA plates. For PMMA plate adhesion,
PHydAAm-tetAla shows adhesion strength similar to that of
stainless steel. On the other hand, PHydAAm-Hex-tetAla
exhibits adhesion strength for PMMA plates 20 times higher
than that of stainless steel. In the case of stainless steel, partial
dewetting of the polymer gel was observed at the failure faces
both in PHydAAm-tetAla and PHydAAm-Hex-tetAla systems,
whereas the polymer gel of PHydAAm-Hex-tetAla entirely
wetted and remained the failure faces of PMMA plates without
dewetting. The hydrophobic hexyl groups contribute to the
cohesive interaction with PMMA surfaces and adhesive poly-
mers, and prevent dewetting during gel formation. The differ-
ence in adhesion strength is attributed to the homogeneity and
defects in the adhesive interfaces.
References
1
2
3
4
5
6
7
J. H. Waite, Int. J. Adhes. Adhes. 1987, 7, 9.
J. H. Waiter, Ann. N. Y. Acad. Sci. 1999, 875, 301.
J. H. Waite, Integr. Comp. Biol. 2002, 42, 1172.
H. Yamamoto, J. Chem. Soc., Perkin Trans. 1 1987, 613.
M. Yu, T. J. Deming, Macromolecules 1998, 31, 4739.
H. Lee, B. P. Lee, P. B. Messersmith, Nature 2007, 448, 338.
C. R. Matos-Pérez, J. D. White, J. J. Wilker, J. Am. Chem.
Soc. 2012, 134, 9498.
8
9
J. Nishida, M. Kobayashi, A. Takahara, ACS Macro Lett.
2013, 2, 112.
G. Walker, J. Mar. Biol. Assoc. U. K. 1972, 52, 429.
10 K. Kamino, K. Inoue, T. Maruyama, N. Takamatsu, S.
Harayama, Y. Shizuri, J. Biol. Chem. 2000, 275, 27360.
11 K. Kamino, Biofouling 2010, 26, 755.
12 K. Kamino, J. Adhes. 2010, 86, 96.
13 A. H. Simmons, C. A. Michal, L. W. Jelinski, Science 1996,
271, 84.
14 J. Kümmerlen, J. D. van Beek, F. Vollrath, B. H. Meier,
Macromolecules 1996, 29, 2920.
15 M. Gasset, M. A. Baldwin, D. H. Lloyd, J. M. Gabriel, D. M.
Holtzman, F. Cohen, R. Fletterick, S. B. Prusiner, Proc. Natl.
Acad. Sci. U.S.A. 1992, 89, 10940.
16 S. E. Blondelle, B. Forood, R. A. Houghten, E. Pérez-Payá,
Biochemistry 1997, 36, 8393.
In conclusion, as a barnacle adhesive protein mimetic, a new
polymer for underwater adhesion was designed and synthesized,
and the adhesive property was demonstrated. The polymer
includes two essential functions of barnacle adhesive proteins.
One is crosslinking ability based on self-assembly though
complementary hydrogen bonds (not covalent bonds). The other
is affinity to the adherent through both polar and van der Waals
interactions. We confirmed that the polymer aqueous solution
causes gelation by aggregation of oligo-alanine units through
hydrogen bonds with the assistance of the hydrophobic terminal
17 N. Yamada, K. Ariga, M. Naito, K. Matsubara, E. Koyama,
J. Am. Chem. Soc. 1998, 120, 12192.
18 P. H. J. Kouwer, M. Koepf, V. A. A. Le Sage, M. Jaspers,
A. M. van Buul, Z. H. Eksteen-Akeroyd, T. Woltinge, E.
Schwartz, H. J. Kitto, R. Hoogenboom, S. J. Picken, R. J. M.
Nolte, E. Mendes, A. E. Rowan, Nature 2013, 493, 651.
19 S. E. Paramonov, H.-W. Jun, J. D. Hartgerink, J. Am. Chem.
Soc. 2006, 128, 7291.
20 S. Szela, P. Avtges, R. Valluzzi, S. Winkler, D. Wilson, D.
Kirschner, D. L. Kaplan, Biomacromolecules 2000, 1, 534.
Chem. Lett. 2015, 44, 1047–1049 | doi:10.1246/cl.150311
© 2015 The Chemical Society of Japan | 1049