Defining the Catechol-Cation Synergy for Enhanced Wet Adhesion to Mineral Surfaces

Article abstract of DOI:10.1021/jacs.6b03453

Mussel foot proteins (Mfps) exhibit remarkably adaptive adhesion and bridging between polar surfaces in aqueous solution despite the strong hydration barriers at the solid-liquid interface. Recently, catechols and amines - two functionalities that account

Full text of DOI:10.1021/jacs.6b03453

Communication
pubs.acs.org/JACS
Defining the Catechol−Cation Synergy for Enhanced Wet Adhesion
to Mineral Surfaces
Michael V. Rapp,†,⊥ Greg P. Maier,‡,⊥ Howard A. Dobbs,† Nicholas J. Higdon,‡ J. Herbert Waite,*,§
Alison Butler,*,‡ and Jacob N. Israelachvili*,†,∥
‡
§
†Department of Chemical Engineering, Department of Chemistry and Biochemistry, Molecular, Cellular, and Developmental
∥
Biology, and Materials Department, University of California, Santa Barbara, California 93106, United States
S
* Supporting Information
We recently demonstrated that synergistic interactions
ABSTRACT: Mussel foot proteins (Mfps) exhibit
remarkably adaptive adhesion and bridging between
polar surfaces in aqueous solution despite the strong
hydration barriers at the solid−liquid interface. Recently,
catechols and aminestwo functionalities that account for
>50 mol % of the amino acid side chains in surface-
priming Mfpswere shown to cooperatively displace the
interfacial hydration and mediate robust adhesion between
mineral surfaces. Here we demonstrate that (1) synergy
between catecholic and guanidinium side chains similarly
promotes adhesion, (2) increasing the ratio of cationic
amines to catechols in a molecule reduces adhesion, and
(3) the catechol−cation synergy is greatest when both
functionalities are present within the same molecule.
between catechol and Lys groups promote adhesion to a wet
mineral surface, rationalizing the high Lys composition of
interfacial Mfps.21 To reduce the complexity of studying full
proteins, we measured the adhesive interactions of a bacterial
siderophorecyclic trichrysobactin,22 an iron-chelating small
molecule (1053 g/mol) comprising catecholic 2,3-dihydrox-
ybenzoic acid (DHBA) and Lys functionalitiesand synthetic
siderophore analogues assembled around the tris(2-
aminoethyl)amine (Tren) scaffold, such as Tren-Lys-Cam
(TLC) (Figure 1A). Through direct force measurements with
a surface forces apparatus (SFA), we determined that the
siderophore and its analogues mediate robust adhesion between
two mica surfaces by displacing hydrated salt ions from the
surface with their cationic Lys groups, allowing catechols to
form bidentate bonds to the underlying aluminosilicate surface.
Removing either catechol or amine functionalities from the
analogues significantly reduced or eliminated adhesion,
respectively.21
We report herein the nanoscale adhesive properties of new
siderophore analogues that further explore the synergy between
catechols and cationic moieties in wet mineral surface priming.
The guanidinium cation in Arg displaces surface salt and
promotes adhesion but is less effective than Lys. Doubling the
number of Lys groups per molecule (from three to six) while
retaining the same number of catechol groups (three) decreases
the overall adhesion between surfaces, as the ratio of catechol
binding groups to total molecular area decreases. Finally, we
demonstrate that comixtures of two separate molecules that
contain only catechols (appended to a Tren core) and only
amines do not recreate the same adhesion synergy as the
intramolecular configuration, suggesting that the adsorbate
geometry and configurational entropy contribute significantly
to the adhesion. Overall, these results suggest a rationale for the
molecular compositions of Mfp adhesion priming proteins and
offer design criteria for functional bioadhesives and coatings.
Certain interfacial priming proteins secreted from mussels,
such as Mfp-3f in Mytilus edulis,23 contain stoichiometric
compositions of Arg and Dopa, which parallel the high
compositions of Lys and Dopa found in other variants, such
as Mfp-5 in Mytilus californianus.4 To determine whether Arg is
functionally similar to Lys in synergy with catechol in wet
adhesion, we synthesized a siderophore analogue, Tren-Arg-
ater undermines polymer adhesion to surfaces. Water
Wand hydrated salt ions strongly bind to hydrophilic
surfaces (e.g., minerals, metals, oxides, fabrics, and biological
interfaces) to form a thin hydration film that impedes the
intimate contact between the polymer and the surface that is
necessary for durable adhesion.1−3 To surmount this obstacle,
wet adhesives and coatings must displace the hydration layer,
bond to the underlying surface, and resist deterioration. Marine
mussels routinely accomplish this feat on intertidal rocks with a
quick-curing blend of intrinsically disordered proteins known as
mussel foot proteins (Mfps).4,5 Of the >15 known Mfps, two
vanguard proteinsMfp-3 and Mfp-5are deposited first to
prime the wet surface and form the interfacial bridge that
couples the rest of the holdfast to the surface.4,6 Mfp-3 and -5
both consist of unique amino acid compositions, containing
20−30 mol % 3,4-dihydroxyphenylalanine (Dopa), a catecholic
amino acid, with stoichiometric levels of cationic residues that
are primarily lysine (Lys) and arginine (Arg). Dopa and
cationic residues commonly occur in adjacent positions along
the protein backbone. Over the past decade, the multifaceted
catecholic functionality of Dopa7which adheres to polar
surfaces through hydrogen or coordination bonds,8 and
chelates metals or covalently cross-links to form cohesive
glues9,10has led to a surge of mussel-inspired adhesives and
coatings for applications such as medical or dental adhe-
sives,11−13 self-healing hydrogels,14,15 biopolymer scaffolds,16,17
and biocompatible coatings.18−20 However, until recently the
role of cationic residues in mussel adhesives has been poorly
understood and underutilized in bioinspired adhesives.
Received: April 4, 2016
© XXXX American Chemical Society
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DOI: 10.1021/jacs.6b03453
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Journal of the American Chemical Society
Communication
smooth and well-studied interface of mica allows for molecular-
level insights into the adhesive mechanisms of adsorbates. Mica
possesses a negative structural charge at surface Al sites and
strongly adsorbs hydrated cations (e.g., K+) and water to form a
tightly bound hydration layer that is characteristic of most polar
surfaces in solution.1,24,25 In aqueous environments, robust
attachment to surfaces is contingent upon displacement of this
hydration layer and binding of molecules to the underlying
surface.
In a buffer that mimics the solution imposed by mussels
during plaque deposition (150 mM KNO3 + 50 mM acetate,
pH 3.3),26 a hydration layer with DT = 13
1 Å formed
between mica surfaces at 10 mN/m compression (Figure 1B,
black circles). Upon separation of the two surfaces, only a weak
van der Waals adhesion force was measured.2 However, after
nanomolar amounts of TAC were injected into the gap solution
between the surfaces (resulting concentration of 200 μM) and
equilibrated, the thickness of the intervening layer decreased to
12 1 Å, and a large adhesion force, Fad = −43 6 mN m−1,
was measured upon separation after 10 min in contact, which
increased slightly with additional contact time (Figure 1C). The
measured adhesion forces between two crossed-cylinder
surfaces in SFA experiments were converted into adhesion
energies using the Johnson−Kendall−Roberts theory (Ead
=
Fad/1.5πR).27 Overall, the decrease in the thickness of the
intervening layer between mica surfaces, the increase in
adhesion, and the sharply vertical shape of the force curve are
consistent with a monolayer of TAC that bridges the mica
surfaces and mediates adhesion.2
The force−distance profiles confirm that the guanidinium
cations in TAC displace hydrated salt layers and promote
adhesive synergy with catechols at mineral surfaces, similar to
the amine cations in TLC yet with subtle differences. First,
TAC demonstrates a critical adsorption concentration (CAC)
approximately 10 times higher than that of TLC before the
molecules adsorb to the mica interface (Figure 1C). Moreover,
TAC mediates adhesion that is only 50−60% of the maximum
TLC adhesion. The intervening TAC film thickness (12 1 Å)
is comparable to that of TLC (9 1 Å), indicating comparable
adsorption densities. We conclude that the bulkier structure
and delocalized charge of guanidinium28 decrease the
magnitude of the cation’s electrostatic interaction with the
negatively charged sites on mica, as compared with amine. This
effect is analogous to the more favorable adsorption free energy
of K+ (smaller radius) compared with Cs+ (larger radius) to
mica.3 This conclusion further indicates that the cationic
residues Lys and Arg contribute significantly to the equilibrium
adhesion energy, rather than solely catechol.
To observe the effect of increasing the number of cationic
groups per molecule on the adhesion and surface affinity, we
synthesized a siderophore analogue, Tren-Lys-Lys-Cam
(TLLC), that doubles the ratio of lysine cations to catechols
(Figures 2A and S5−S9). SFA force−distance measurements
were performed with TLLC using the same procedure as for
TAC. Similar interaction profiles and adhesion forces were
measured with TLLC (Figure 2B); the thickness of the
intervening layer between the mica surfaces decreased to DT =
Figure 1. (A) Structures of the siderophore analogues TLC and TAC.
(B) SFA force−distance interactions for two mica surfaces in aqueous
buffer (150 mM KNO3 + 50 mM acetate, pH 3.3) (black circles) and
in 200 μM TAC (green circles). Open circles represent measurements
during the approach of the two surfaces, while solid circles represent
measurements during separation. The inset depicts the surfaces as they
interact throughout the measurement. (C) TLC- and TAC-mediated
adhesion forces, Fad, and energies, Ead, required to separate two mica
surfaces in aqueous solution, as functions of the time the surfaces were
left in adhesive contact. Error bars represent one standard deviation.
Cam (TAC), in which the Lys residues of TLC are replaced
with Arg (Figure 1A; synthesis and characterization are shown
in Figures S1−S4 in the Supporting Information). A SFA was
used to measure the radius-normalized force (F/R) versus
distance (D) profile for two cleaved mica surfaces during
approach, compression, and separation in a buffered solution of
TAC (Figure 1B). Mica is a molecularly smooth aluminosilicate
mineral (Si:Al ratio of 3:1) that serves as an ideal model for the
shale and clay minerals to which mussels commonly attach. The
12 1 Å, and a comparable adhesion force, Fad = −47
mN/m, was measured after 10 min in contact.
9
The lower adhesion energy measured with TLLC is further
evidence of the importance of catechols in robust adhesion.
Doubling the number of Lys groups increases the molecule’s
projected area at a surface yet keeps the number of catechol
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DOI: 10.1021/jacs.6b03453
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adhesive forces that were identical to those measured in
solutions of only TLB (Figure S11C-i). Additional adhesion
measurements were performed on mixtures of TC and amine
compounds (tetramethylamine, lysine, isopropylamine, aniline,
1,3-diaminopropane, diethylenetriamine, TREN, and 2,4,6-
triethyl-1,3,5-benzenetrimethanamine [TEBMA]); however,
no evidence of synergy or adhesion was measured in any of
these mixtures. In mixtures with TC and TREN or TEBMA,
the highly charged amine compounds adsorbed on the mica
surface, but no influence from TC was observed (Figure S11C-
ii,iii). The inability to recreate the adhesive performance of
TLC with mixtures of singly functionalized molecules suggests
that the molecular geometry and configurational entropy30
upon adsorption contribute significantly to the surface
phenomena of the siderophore analogues.
The specific binding mode of 2,3-dihydroxycatechol to the
surface and the resulting geometry of the siderophore
analogues are not known. The first hydroxyl pKa of the 2,3-
dihydroxycatechol in TLC or TAC is expected to be similar to
the pKa of the analogous siderophores chrysobactin (2,3-
dihydroxybenzoyl-D-Lys-L-Ser; pKa = 6.73) and vanchrobactin
((2,3-dihydroxybenzoyl-D-Arg-L-Ser; pKa = 6.79);31 thus, at pH
3.3 the catechols in TLC, TAC, and TLLC are fully protonated.
By virtue of single-molecule atomic force microscopy measure-
ments, Li et al.32 suggested that Dopa adsorbs on mica via
bidentate hydrogen bonds. Alternatively, catechols may form
mononuclear bidentate coordination complexes with mica’s
alumina sites,33−35 but this interaction is unconfirmed at mica’s
crystalline surface. Similarly, a salicylate-type interaction
involving the o-hydroxyl oxygen and the carbonyl group is
possible36 but not expected.37 After displacement of the
hydrated salt layer, the role of the cationic amine group is
unconfirmed; however, we strongly suspect that the cationic
groups participate in adhesion to the mica surface through
Coulomb interactions at the negatively charged alumina sites.
Over the first few molecular layers extending from an
aqueous mineral surface, paired catechol−cation functionalities
cooperate to displace hydration layers and promote robust
adhesion between the underlying surfaces. This synergy is not
unique to amine cations: guanidinium groups likewise enhance
aqueous adhesion, which provides a rationale for the high
content of Arg residues in certain Mfps. In small-molecule
adhesives, increasing the number of cationic groups per
molecule increases the affinity for negatively charged surfaces
but decreases the equilibrium adhesion energy by lowering the
density of bidentate-binding catechol groups. Although the
specific surface conformations of paired catechol−cation
siderophores and analogues await characterization in future
studies, it is apparent that the geometry and configurational
entropy significantly affect their intermolecular interactions.
Furthermore, while small changes to the separation distance
between the catechol and cation do not significantly alter the
adhesion synergy, a cutoff separation should exist beyond which
synergy is not observed. In analogy to the remarkably high Fe3+
stability constant of triscatechol siderophores compared with
monocatechol compounds,38 intramolecular adjacency of
binding functionalities contributes a significant energetic gain
upon adsorption to a wet surface.
Figure 2. (A) Structure of the siderophore analogue TLLC. (B) SFA
force−distance interactions for two mica surfaces in aqueous buffer
(150 mM KNO3 + 50 mM acetate, pH 3.3) (black circles) and in 2
μM TLLC (red circles). Open circles represent measurements during
the approach of the two surfaces, whereas solid circles represent
measurements during separation. The inset depicts the surfaces as they
interact throughout the measurement.
groups the same, thereby decreasing the density of robust
bidentate interactions per unit area across the surface. However,
the three additional Lys groups increase the electrostatic charge
density within the molecule and lower the CAC for TLLC by
an order of magnitude (Figure S10). TLLC’s adhesion
performance is an interesting alternative result to the work of
Wang et al.,29 where increasing the concentration of Dopa
groups in a cationic polymer had a negligible effect on the
polymer’s total adhesion. We ascribe these contrary results to
differences in the geometries in our respective adhesive
molecules; the excluded volume of random-coil polymers
sterically hinders high catechol surface densities, while small
molecules may assemble into more dense films.
Do cation and catechol functionalities require intramolecular
proximity to enhance adhesion? To determine whether a
mixture of two siderophore analoguesone without amine
(Tren-Cam, TC) and the other without catechol (Tren-Lys-
Bam, TLB)promotes adhesion at aqueous mineral surfaces,
we performed further SFA force−distance measurements
between mica surfaces, as shown in Figure S11. When the
ratio of TC (0.02−1 mM) and TLB (0.02−0.2 mM) was
varied, no enhanced adhesion or synergy between the
siderophore analogues was detected. At a concentration of
0.2 mM, TLB preferentially adsorbed onto the mica surface
over TC, displaced the hydration layer, and mediated modest
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.6b03453.
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DOI: 10.1021/jacs.6b03453
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
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■
Corresponding Authors
*herbert.waite@lifesci@ucsb.edu
*butler@chem.ucsb.edu
*jacob@engineering.ucsb.edu
Author Contributions
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(32) Li, Y.; Qin, M.; Li, Y.; Cao, Y.; Wang, W. Langmuir 2014, 30,
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ez, C.; Soengas, R. G. Metallomics
⊥M.V.R. and G.P.M. contributed equally.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
We are grateful for support from the Materials Research and
Science Engineering Centers Program of the National Science
Foundation under Award DMR 1121053, NSF CHE-1411942
(A.B.), and NSF GRFP (M.V.R.).
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