Fish and Clips: A Convenient Strategy to Identify Tyrosinase

Letter Cite This: ACS Macro Lett. 2019, 8, 724−729

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Fish and Clips: A Convenient Strategy to Identify Tyrosinase Substrates with Rapid Activation Behavior for Materials Science Applications Justus Horsch,† Patrick Wilke,† Heike Stephanowitz,‡ Eberhard Krause,‡ and Hans G. Börner*,† †

Department of Chemistry, Laboratory for Organic Synthesis of Functional Systems, Humboldt-Universität zu Berlin, Brook-Taylor-Str. 2, 12489 Berlin, Germany ‡ Leibniz Institute for Molecular Pharmacology, Robert-Rössle-Str. 10, 13125 Berlin, Germany

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S Supporting Information *

ABSTRACT: Peptides with suitable substrate properties for a specific tyrosinase are selected by combinatorial means from a one-bead-one-compound (OBOC) peptide library. The identified sequences exhibit tyrosine residues that are rapidly oxidized to 3,4-dihydroxyphenylalanine (Dopa), making the peptides interesting for enzyme-activated adhesives. The selection process of peptides involves tyrosinase oxidation of tyrosine-bearing sequences on a solid support, yielding dopaquinone residues (fish from the sequence pool), to which thiol-functional fluorescent probes attach by Michael-reaction (clip to mark). Labeled supports are isolated and sequence readout is feasible by MALDI-TOF-MS/MS to reveal peptides, while activation kinetics as well as enzyme-activated coating behavior are verifying the proper selection.

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peptide sequences with good tyrosinase substrate properties still constitutes challenges. Combinatorial chemistry offers tools to identify purpose-adapted peptide sequences from large libraries, being ideal to screen the sequential space spanned by peptides. Fluorescence-quenched one-bead-one-compound (OBOC) peptide libraries were applied to screen for enzyme substrates, for example, for protease.18,19 Enzymatic cleavage of the fluorescence quencher moiety then allowed for the identification of substrate sequences. More recently, phage display has been used to select mussel-inspired adhesive peptides from a large 12mer sequence library.13 The modified biopanning procedure combined the selection of peptides showing (i) material surface tailored adsorption and (ii) good tyrosinase substrate properties. However, a selection that focuses primarily on tyrosinase substrate properties would contribute to the understanding of sequence specificity effects for particular enzyme batches. Here, we report a combinatorial approach to screen for tyrosinase substrates, using a one-bead-one-compound peptide library (Figure 1). From the large sequence pool, resin-bound peptides with superior substrate properties are rapidly oxidized by a commercially available tyrosinase (fish). The enzymatic processing leads to dopaquinones that act as Michael acceptors

imicking aspects of the remarkable adhesive apparatus of marine mussels has been an intense field of research for the past decades.1,2 The bioinspired approach led to polymers that proved their applicability as novel adhesives,3−5 underwater glues,6,7 and effective saltwater coatings.8−10 Most of these polymer materials are instant adhesives that rely on the ready presentation of 3,4-dihydroxyphenylalanine (Dopa) as the key functionality to generate adhesion.5 Alternatively, Dopa moieties can be generated on demand from tyrosine residues by enzymatic transformation using tyrosinase.11 Thus, the specific enzyme came into focus in order to realize enzymatic synthesis of peptide-based dyes with tunable colors,12 or enzyme-activatable peptide-block-poly(ethylene glycol) (peptide-PEG) conjugates, that can constitute seawater stable antifouling coatings.10,13 Moreover, tyrosinase-activated polymerization of peptide-based macromonomers enabled access to segmented polymers that act as artificial mussel glue proteins.14 Those can tack various surfaces, exhibit strong adhesion behavior and generate coatings with stability against 4.2 mol/L hypersaline solution (Dead Sea water models). With respect to the understanding of biochemical reactivity, tyrosinase appears to be well-suited for materials science applications.6,13,15 Tyrosinase can be either genetically expressed16 as pure and highly active variant or extracted from biological sources,17 for example, mushrooms. While the latter provides the enzyme cost-effectively in large scale, high batchto-batch variations limit the applicability as the substrate specificity might alter. Thus, the de novo design of short © XXXX American Chemical Society

Received: April 4, 2019 Accepted: May 24, 2019

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DOI: 10.1021/acsmacrolett.9b00244 ACS Macro Lett. 2019, 8, 724−729

Letter

ACS Macro Letters

absence of an N-terminal Tyr1 with good activation properties. Processing of the control peptides was performed by incubating the supported peptides separately with tyrosinase in the presence of the thiol-functional fluorescent probe. After 5 min incubation, the enzyme and the probe were carefully washed off and the fluorescence intensity of the beads was examined by fluorescence microscopy (Figure 2). Whereas

Figure 1. Illustration of the fish and clip screening procedure. A supported peptide library with fixed Tyr3 and variable Xaa positions (top) was processed by tyrosinase. Labeling of suitable beads takes place only for oxidized sequences by addition of a thiol-functional fluorescent probe to dopaquinones to identify rapidly activated peptides with MS/MS sequencing. Figure 2. Fluorescence microscopy readout of the OBOC library and controls after tyrosinase activation and labeling of activatable peptides with a fluorescent probe (insets optical microscopy). Tyrosinaseactivation of supported Testpep2 (a), Testpep1 (b), and Testpep2/ Testpep1 mixtures (c) showing selective labeling of Tyr bearing sequences. Representative image of Tyrosinase activated peptide library after labeling (d) (Conditions: 100 U/mL enzyme, 0.5 equiv of fluorescent probe).

and enable an effective reaction with a thiol-functional fluorescent probe (clip), marking the support to be isolated for sequence analysis. The pool of peptide sequences was spanned by an OBOC peptide library that was synthesized via split and mix protocol.20 Aminomethyl-ChemMatrix resin was the support of choice, as it allows peptide deprotection without liberating them from the support, shows little autofluorescence, swells well in aqueous medium and has no hydrophobic interactions with either the fluorescent probe or the enzyme (SI.). The general peptide structure was designed to follow XXYXXGGGM, where X refers to randomized amino acid residues and Y corresponds to Tyr3 residue at a fixed position. A set of amino acids was selected according to their occurrence in natural mussel foot proteins (mfp-1, -3, and -5), which in addition to Dopa contain high amounts of Gly, Pro, Ser, Arg, Lys, and His.21−23 To complement the set, Ala and Gln were added as they were found in biocombinatorial screenings for mussel inspired adhesives.13 Besides the fixed position of Tyr3, additional Tyr residues were allowed within the randomized domain, with the exception of the N-terminal position. In total, nine different amino acids were included in the library to cover a sequential space of 5832 different peptides. For ease of sequence readout, the peptides were C-terminally extended with Met and a (Gly)3 spacer, allowing cyanogen bromide cleavage of the peptides from the supports. The fluorescent probe used to label the tyrosinase-activated peptides comprised a rhodamine B conjugated to Cys via a short Ser-oligo (ethylene glycol)-Ser spacer for solubility enhancement (Figure 1). For labeling, the Michael-reaction of a thiol with dopaquinone was exploited, as the reaction proceeds quantitatively with the highest reaction rates of all nucleophilic functionalities available in peptides.24 Prior to the screening of the entire library, the approach was evaluated to ensure proper on-support activation and effective labeling (SI). For that purpose, two supported reference peptides Ac-YGG-GGGM (Testpep1) and Ac-FGG-GGGM (Testpep2) were synthesized, differing in the presence and

tyrosine of Testpep1 was oxidized, leading to a significant increase in fluorescence intensity, beads containing Testpep2 showed marginal fluorescence, which indicates nonspecific interactions with the fluorophore. The latter intensity is neglectable as a clear distinction of the actively labeled and nonlabeled peptide supports was feasible (Figure 2a,b). This was confirmed by activation of a mixture of Testpep1 and Testpep2 beads, resulting in both, labeled and nonlabeled beads side-by-side (Figure 2c). Hence, the process is selective and nonspecific labeling, for example, by noncovalent peptideprobe or support-probe interactions can be disregarded. Library screening took place by tyrosinase processing of the supported peptide pool for 5 min in the presence of the fluorescent probe. After careful washing, the fluorescence intensity of the beads was examined via fluorescence microscopy (Figure 2d). About 5% of the beads showed more pronounced fluorescence and differed clearly from other beads of the pool, suggesting that tyrosine residues were activated with dissimilar rates depending on the sequence environment. From the screening, 120 highly fluorescent and 24 low-fluorescent beads were selected by hand as positive hits and negative controls. The peptides were cleaved by CNBr treatment and subsequent sequence readout by MALDI-TOFMS/MS was employed.25 Peptides close to the surface of the beads are more easily accessible by the 120 kDa enzyme, which would explain that even for highly fluorescent beads a nonquantitative labeling of the peptides was observed. This is, however, an advantage for MALDI-TOF-MS/MS sequencing as the cleavage product contains a mixture of fluorescently labeled and unmodified peptides. While the fluorescently 725

DOI: 10.1021/acsmacrolett.9b00244 ACS Macro Lett. 2019, 8, 724−729

Letter

ACS Macro Letters

screening conditions of pH 7, the tyrosinase with a pI of 4.7−5.0 would be negatively charged, which would even promote positively charged Lys and Arg residues. Therefore, the suppression of these amino acids, indicates that electrostatic interactions do not dominate the selectivity in the combinatorial screening. The sequence analysis of the 19 peptides from lowfluorescent beads verifies a proper selection process, as substantial differences between potential nonsubstrate and substrate sequences were evident. Although the results must be considered with caution due to the number of sequences in this pool, first trends are provided. Most abundant amino acids are Ala, followed by Pro and Lys. The nonsubstrate sequences exhibit, in contrast to the potential substrates, highly discriminated occurrence of Ser, Asn, and Gly (Figure 3b). In general, higher amounts of nonpolar amino acids and the presence of Lys lead to poor activation behavior due to reduced solubility or compatibility. From the set of 118 identified unique sequences, eight peptides as good tyrosinase substrates (Pep1−8) and three nonsubstrate controls (Pep9−11) were chosen to investigate the oxidation reaction kinetics with the particular tyrosinase. The sequence selection was based on frequently occurring amino acid motifs (e.g., Asn-Gly-Tyr-Xaa4-Xaa5, Ser-Tyr-TyrXaa4basic-Xaa5, Xaa1-Xaa2-Tyr-Gly-Ser, Xaa1-Xaa2-Tyr-AlaSer) and the enrichment of amino acids that populate certain positions, as revealed by the statistical analysis. A UV/vis spectroscopy assay was used to follow the enzymatic oxidation of the tyrosine residues to dopaquinones at 305 nm (SI, Figure S16). Figure 4 summarizes the initial

labeled peptides in most cases gave the mass signals with highest intensity in the MS spectra, the fragment ions of the fluorescent dye superimpose the peptide fragments, making sequencing difficult. Nonetheless, the molecule peak of labeled peptides was used as an internal standard to identify the nonlabeled peptide signal with a mass difference of −963.4 Da corresponding to the oxidation of the tyrosine residue and the labeling with a fluorescent probe (Figure 3c). This procedure enabled the identification of 87% of the sequences from highly fluorescent beads and 83% of the sequences from lowfluorescent beads, respectively.

Figure 3. Statistical evaluation of the 99 tyrosinase substrate and 19 nonsubstrate sequences as determined by the fish and clips screening process. (a) Sequence logo, giving the frequency plot over all sequences from beads with high fluorescence intensity (substrates) and (b) amino acid occurrence frequency in the substrate and nonsubstrate sequence pools with respect to the initial library. (c) Representative MALDI-TOF-MS spectrum of the Pep4 sequence from a labeled library bead as cleaved from the support for sequencing.

Figure 4. Tyrosinase substrate properties of Pep1−11 showing max activation rates from UV/vis kinetics (conditions: 305 nm, 0.25 mM peptides, 100 U/mL enzyme, pH 6.5).

A total number of 124 peptide samples could be successfully analyzed, revealing 99 unique sequences as potential tyrosinase substrates and 19 unique nonsubstrate control sequences. Statistical analysis of the sequences from fluorescent beads shows that in general polar residues are preferably incorporated, especially on terminal positions Xaa1 and Xaa5 (Figure 3a,b). Interestingly, Ser is the most abundant residue overall with a dominant presence in Xaa1, Xaa2 and Xaa5. Asn is also highly enriched and occurs especially in Xaa1. Furthermore, Xaa4 is frequently occupied by His. The other basic amino acids such as Lys and Arg are apparently less relevant to achieve good tyrosinase substrate properties as their occurrence is rather suppressed. This is unexpected considering the fact that cationic residues are enriched in different mfp’s22,23,26 as these residues contribute substantially to adhesion and cohesion.27,28 Coulomb interactions can regulate the accessibility of enzymes to resin bound peptides, which might affect the selection process.29 This means that under the

activation rates of Pep1−11 from kinetic measurements. All peptides, which originate from highly fluorescent beads can be activated by tyrosinase as an increase in UV absorbance was evident. However, differences in the activation behavior were evident, reflecting availability and sequence dependency as overlaying effects to define substrate properties. Pep1−4 and Pep8 show rapid activation by tyrosinase and thus constitute suitable enzyme substrates. Pep5 and Pep7 show moderate activation kinetics, whereas Pep6 is processed fairly slowly into the corresponding quinone-bearing sequence. The negative control sequences Pep10 and Pep11 show a very weak increase in absorbance and confirmed their nonsubstrate properties. Interestingly, Pep9 was the only control sequence that showed significant increase in absorbance. Nonetheless, the kinetics are slow and thus demonstrate the ability of the screening method to differentiate between substrates of varying activation rates. Overall, the activation kinetic experiments of the different 726

DOI: 10.1021/acsmacrolett.9b00244 ACS Macro Lett. 2019, 8, 724−729

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ACS Macro Letters

However, clear differences in coating stability were evident, as rinsing the surface coatings with 599 mM NaCl seawater model solution removed the nonactivated conjugate quantitatively, whereas 45% of the coating from activated (Pep2*)3PEG retained stable (Figure 5). Despite the more appropriate

peptides confirmed the trends that could be deduced from the statistical analysis. (i) Rapid activation is prevented by basic and nonpolar sequence environments of Tyr3 in form of Lys, Arg and Ala. (ii) A polar sequence environment through Ser and Asn, especially at the terminal flanking positions promotes a rapid activation. (iii) A direct neighboring Tyr2 next to Tyr3 slows down the activation kinetics, as can be seen by the moderate activation of Pep5 as well as by the slow activation of Pep6 and Pep9. With respect to frequently occurring motifs, the sequence dyad Tyr3-His4 was evidently relevant to achieve faster activation kinetics in the absence of the Tyr2 neighbor. Interestingly, YH dyads also occur in mfp-5 at positions 15− 16, 22−23, 27−28, and 61−62, where the tyrosine residues are mainly oxidized to Dopa.30 The highest maximum activation rates were observed for Pep2 and Pep4. Moreover, Pep2 shows similarities to a recently identified sequence from a biocombinatorial screening of tyrosinase activatable coatings.13 Therefore, these two peptides were chosen for further applications by investigating activation and adsorption properties. To enhance their adhesive properties, the 5mer sequences of Pep2 and Pep4 were synthesized as triple repeated 15mer peptide-PEG conjugates (HSYHG)3PEG ((Pep2)3-PEG) and (NGYSS)3-PEG ((Pep4)3-PEG). Kinetic UV/vis absorption measurements, applying the same conditions as used for Pep1−11 study, were performed to reveal the activation behavior of the conjugates (SI). (Pep4)3-PEG shows a stepwise increase of absorbance that ultimately suggests the oxidation of an average of two to three Tyr residues per sequence. This could be confirmed by MALDITOF-MS analysis (SI, Figure S20). The activated conjugate shows mass signal shifts of +32 and +48 Da compared to the nonoxidized conjugate, which correspond to two and three oxidized tyrosine residues, respectively. The absorbance curve indicated that the tyrosine residues present in (Pep4)3-PEG are oxidized with dissimilar rates, leading to a stepwise increase in absorbance, with the first step appearing to be completed after about 20 min. Probably, the tyrosine of the N-terminal pentapeptide segment is more easily accessible by tyrosinase compared to the other tyrosines. Stepwise activation kinetics have been observed for congeneric peptide sequences, which contained multiple tyrosine residues with dissimilar substrate properties.10,31 (Pep2)3-PEG shows an even smother and faster activation behavior than (Pep4)3-PEG. Within 60 min the absorbance levels off and suggests the oxidation of two out of three tyrosine residues (SI, Figure S18). MALDI-TOF-MS analysis confirmed this, by revealing a mass shift of +32 Da compared to the nonoxidized conjugate. Apparently two tyrosine residues in (Pep2)3-PEG are well processable by the enzyme with rather comparable rates, while one is more difficult to be accessed by the tyrosinase. Nonetheless, in both conjugates multiple Dopa residues can be generated by tyrosinase processing, which appears to be promising to realize multivalent adhesion. The adsorption behavior of the activated conjugates on aluminum oxide surfaces was studied by quartz crystal microbalance (QCM) employing alumina coated sensors. Interestingly, both Dopa presenting domains showed an entirely different behavior. For activated (Pep2*)3-PEG strong adsorption was observed, reaching a frequency shift of Δf = −26 Hz after buffer rinsing (SI, Figure S24). Considering the Voight model for viscoelastic films, an adsorbed mass of 580 ± 8 ng/cm2 could be estimated. It has to be noted, that the nonactivated (Pep2)3-PEG leads to a similar mass deposition.

Figure 5. Comparison of areal mass densities of a nonactivated reference of (Pep2)3-PEG, activated (Pep2*)3-PEG and activated (Pep4*)3-PEG after incubation (blue), rinsing with buffer (green) and 599 mM NaCl solution (red).

oxidation behavior of (Pep4*)3-PEG, the activated conjugate leads to a minor frequency shift of Δf = −9 Hz and the generated coatings are not stable during buffer rinsing (SI, Figure S27). Reference measurements of nonactivated conjugates confirmed the poor binding capability of (Pep4)3PEG exhibiting neglectable surface adsorption (SI, Figure S26). Obviously, the screening procedure selected peptides with suitable tyrosinase substrate properties, but not necessarily robust coating systems were found. This could be expected as no selection pressure has been implemented to the screening to drive the selection toward strong adhesive peptides. Although not perfect, these results demonstrate the enhanced stability of Dopa-containing coatings. According to previous results on alumina,14 Dopa seems to be the key to strong adhesion, but relies on the sequence environment to initialize and consolidate surface contacts. Moreover, cationic residues seem to play a significant role in the adsorption process, although generally discriminated during a search for tyrosinase substrates. In conclusion, a screening procedure to identify peptides with excellent substrate properties for a specific tyrosinase was demonstrated. By incubating a large OBOC peptide library with tyrosinase, only peptides with enzyme substrate properties were oxidized to generate dopaquinone residues. Thiolfunctional fluorescent probes selectively reacted with them, to label the beads for sequence readout by MS/MS analysis. The set of 118 peptides showed, in addition to the central Tyr residue, a common enrichment of Ser, Asn, and Gly, whereas Lys and Arg were generally discriminated. Apparently, a polar and conformational flexible sequence environment of the Tyr residue promotes the enzymatic oxidation. UV/vis kinetic activation assays proved that peptides selected as enzyme substrates could be effectively oxidized and differences in activation rates indicated sequence environment effects. The most rapidly activatable sequences were conjugated as three repeats to PEG blocks to yield (HSYHG)3-PEG and (NGYSS)3-PEG conjugates. Adsorption properties of corresponding conjugates and coating stabilities were evaluated. QCM analysis revealed that the activated (HSY*HG)3-PEG conjugate adhered to alumina, forming a coating that showed a certain degree of stability against rinsing with seawater model 727

DOI: 10.1021/acsmacrolett.9b00244 ACS Macro Lett. 2019, 8, 724−729

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(12) Lampel, A.; McPhee, S. A.; Park, H.-A.; Scott, G. G.; Humagain, S.; Hekstra, D. R.; Yoo, B.; Frederix, P. W. J. M.; Li, T.D.; Abzalimov, R. R.; Greenbaum, S. G.; Tuttle, T.; Hu, C.; Bettinger, C. J.; Ulijn, R. V. Polymeric peptide pigments with sequence-encoded properties. Science 2017, 356 (6342), 1064−1068. (13) Wilke, P.; Helfricht, N.; Mark, A.; Papastavrou, G.; Faivre, D.; Börner, H. G. A Direct Biocombinatorial Strategy toward Next Generation, Mussel-Glue Inspired Saltwater Adhesives. J. Am. Chem. Soc. 2014, 136 (36), 12667−12674. (14) Horsch, J.; Wilke, P.; Pretzler, M.; Seuss, M.; Melnyk, I.; Remmler, D.; Fery, A.; Rompel, A.; Börner, H. G. Polymerizing Like Mussels Do: Toward Synthetic Mussel Foot Proteins and Resistant Glues. Angew. Chem., Int. Ed. 2018, 57 (48), 15728−15732. (15) Wei, W.; Petrone, L.; Tan, Y.; Cai, H.; Israelachvili, J. N.; Miserez, A.; Waite, J. H. An Underwater Surface-Drying Peptide Inspired by a Mussel Adhesive Protein. Adv. Funct. Mater. 2016, 26 (20), 3496−3507. (16) Pretzler, M.; Bijelic, A.; Rompel, A. Heterologous expression and characterization of functional mushroom tyrosinase (AbPPO4). Sci. Rep. 2017, 7 (1), 1810. (17) Papa, G.; Pessione, E.; Leone, V.; Giunta, C. Agaricus bisporus tyrosinaseI. Progress made in preparative methods. Int. J. Biochem. 1994, 26 (2), 215−221. (18) Meldal, M.; Svendsen, I.; Breddam, K.; Auzanneau, F. I. Portion-mixing peptide libraries of quenched fluorogenic substrates for complete subsite mapping of endoprotease specificity. Proc. Natl. Acad. Sci. U. S. A. 1994, 91 (8), 3314−3318. (19) St. Hilaire, P. M.; Willert, M.; Juliano, M. A.; Juliano, L.; Meldal, M. Fluorescence-Quenched Solid Phase Combinatorial Libraries in the Characterization of Cysteine Protease Substrate Specificity. J. Comb. Chem. 1999, 1 (6), 509−523. (20) Lam, K. S.; Salmon, S. E.; Hersh, E. M.; Hruby, V. J.; Kazmierski, W. M.; Knapp, R. J. A new type of synthetic peptide library for identifiying ligand-binding activity. Nature 1991, 354, 82− 84. (21) Waite, J. H.; Qin, X. Polyphosphoprotein from the Adhesive Pads of Mytilus edulis. Biochemistry 2001, 40 (9), 2887−2893. (22) Lin, Q.; Gourdon, D.; Sun, C.; Holten-Andersen, N.; Anderson, T. H.; Waite, J. H.; Israelachvili, J. N. Adhesion mechanisms of the mussel foot proteins mfp-1 and mfp-3. Proc. Natl. Acad. Sci. U. S. A. 2007, 104 (10), 3782−3786. (23) Papov, V. V.; Diamond, T. V.; Biemann, K.; Waite, J. H. Hydroxyarginine-containing Polyphenolic Proteins in the Adhesive Plaques of the Marine Mussel Mytilus edulis. J. Biol. Chem. 1995, 270 (34), 20183−20192. (24) Tse, D. C. S.; McCreery, R. L.; Adams, R. N. Potential oxidative pathways of brain catecholamines. J. Med. Chem. 1976, 19 (1), 37−40. (25) Wieczorek, S.; Krause, E.; Hackbarth, S.; Röder, B.; Hirsch, A. K. H.; Börner, G. H. Exploiting specific interactions toward nextgeneration polymeric drug transporters. J. Am. Chem. Soc. 2013, 135, 1711−1714. (26) Hwang, D. S.; Yoo, H. J.; Jun, J. H.; Moon, W. K.; Cha, H. J. Expression of Functional Recombinant Mussel Adhesive Protein Mgfp-5 in Escherichia coli. Appl. Environm. Microbiol. 2004, 70 (6), 3352−3359. (27) Rapp, M. V.; Maier, G. P.; Dobbs, H. A.; Higdon, N. J.; Waite, J. H.; Butler, A.; Israelachvili, J. N. Defining the Catechol−Cation Synergy for Enhanced Wet Adhesion to Mineral Surfaces. J. Am. Chem. Soc. 2016, 138 (29), 9013−9016. (28) Gebbie, M. A.; Wei, W.; Schrader, A. M.; Cristiani, T. R.; Dobbs, H. A.; Idso, M.; Chmelka, B. F.; Waite, J. H.; Israelachvili, J. N. Tuning underwater adhesion with cation−π interactions. Nat. Chem. 2017, 9 (5), 473−479. (29) Basso, A.; Martin, L. D.; Gardossi, L.; Margetts, G.; Brazendale, I.; Bosma, A. Y.; Ulijn, R. V.; Flitsch, S. L. Improved biotransformations on charged PEGA supports. Chem. Commun. 2003, No. 11, 1296−1297. (30) Kan, Y.; Danner, E. W.; Israelachvili, J. N.; Chen, Y.; Waite, J. H. Boronate Complex Formation with Dopa Containing Mussel

solution. This highlights the potential of the screening method to elucidate sequence−substrate relationships for specific tyrosinases and to identify rapidly activatable peptides that show interesting adsorption properties to mimic aspects of mussel adhesive proteins.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.9b00244.

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Experimental procedures and analytical data (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Eberhard Krause: 0000-0001-5019-522X Hans G. Börner: 0000-0001-9333-9780 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge Dr. Stefan Weidner (BAM Berlin) for MALDI-TOF-MS measurements. Financial support was granted by the German Research Council DFG: BO 1762/9-1.

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REFERENCES

(1) Lee, B. P.; Messersmith, P. B.; Israelachvili, J. N.; Waite, J. H. Mussel-Inspired Adhesives and Coatings. Annu. Rev. Mater. Res. 2011, 41 (1), 99−132. (2) Ahn, B. K. Perspectives on Mussel-Inspired Wet Adhesion. J. Am. Chem. Soc. 2017, 139 (30), 10166−10171. (3) Lee, H.; Lee, B. P.; Messersmith, P. B. A reversible wet/dry adhesive inspired by mussels and geckos. Nature 2007, 448 (7151), 338−341. (4) Ahn, B. K.; Das, S.; Linstadt, R.; Kaufman, Y.; MartinezRodriguez, N. R.; Mirshafian, R.; Kesselman, E.; Talmon, Y.; Lipshutz, B. H.; Israelachvili, J. N.; Waite, J. H. High-performance musselinspired adhesives of reduced complexity. Nat. Commun. 2015, 6, 8663. (5) Kord Forooshani, P.; Lee, B. P. Recent approaches in designing bioadhesive materials inspired by mussel adhesive protein. J. Polym. Sci., Part A: Polym. Chem. 2017, 55 (1), 9−33. (6) Zhong, C.; Gurry, T.; Cheng, A. A.; Downey, J.; Deng, Z.; Stultz, C. M.; Lu, T. K. Strong underwater adhesives made by self-assembling multi-protein nanofibres. Nat. Nanotechnol. 2014, 9 (10), 858−866. (7) White, J. D.; Wilker, J. J. Underwater Bonding with Charged Polymer Mimics of Marine Mussel Adhesive Proteins. Macromolecules 2011, 44 (13), 5085−5088. (8) Statz, A. R.; Meagher, R. J.; Barron, A. E.; Messersmith, P. B. New Peptidomimetic Polymers for Antifouling Surfaces. J. Am. Chem. Soc. 2005, 127 (22), 7972−7973. (9) Wei, Q.; Becherer, T.; Mutihac, R.-C.; Noeske, P.-L. M.; Paulus, F.; Haag, R.; Grunwald, I. Multivalent Anchoring and Cross-Linking of Mussel-Inspired Antifouling Surface Coatings. Biomacromolecules 2014, 15 (8), 3061−3071. (10) Wilke, P.; Börner, H. G. Mussel-Glue Derived Peptide− Polymer Conjugates to Realize Enzyme-Activated Antifouling Coatings. ACS Macro Lett. 2012, 1 (7), 871−875. (11) Sánchez-Ferrer, Á .; Neptuno Rodríguez-López, J.; GarcíaCánovas, F.; García-Carmona, F. Tyrosinase: a comprehensive review of its mechanism. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 1995, 1247 (1), 1−11. 728

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ACS Macro Letters Adhesive Protein Retards pH-Induced Oxidation and Enables Adhesion to Mica. PLoS One 2014, 9 (10), No. e108869. (31) Marumo, K.; Waite, J. H. Optimization of hydroxylation of tyrosine and tyrosine-containing peptides by mushroom tyrosinase. Biochim. Biophys. Acta, Protein Struct. Mol. Enzymol. 1986, 872 (1−2), 98−103.

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