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Cite This: Bioconjugate Chem. XXXX, XXX, XXX−XXX

Tyrosinase-Mediated Bioconjugation. A Versatile Approach to Chimeric Macromolecules Elita Montanari,†,‡ Arianna Gennari,†,§ Maria Pelliccia,† Lucio Manzi,∥ Roberto Donno,†,§ Neil J. Oldham,∥ Andrew MacDonald,⊥ and Nicola Tirelli*,†,§

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†

NorthWest Centre of Advanced Drug Delivery (NoWCADD), Division of Pharmacy and Optometry, School of Health Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, M13 9PT, Manchester, United Kingdom ‡ Department of Drug Chemistry and Technologies, “Sapienza” University of Rome, 00185 Rome, Italy § Laboratory of Polymers and Biomaterials, Fondazione Istituto Italiano di Tecnologia, 16163, Genova, Italy ∥ School of Chemistry, University of Nottingham, University Park, NG7 2RD, Nottingham, United Kingdom ⊥ Manchester Collaborative Centre for Inflammation Research, Division of Infection, Immunity & Respiratory Medicine, Faculty of Biology, Medicine and Health, The University of Manchester, M13 9PT, Manchester, United Kingdom S Supporting Information *

ABSTRACT: We present a method for tyrosine-selective and reversible bioconjugation; tyrosines are enzymatically converted into catechols and in situ “clicked” onto boronic acids. Importantly, our process selectively produces catechols and avoids quinones, thereby improving the control over the chemical identity of the products. We have conjugated boronic acidcontaining hyaluronic acid (HyA) to peptides bearing tyrosines in variable number and position; the use of tagging peptides for the provision of well exposed tyrosine residuesin our case the hemagglutinin-derived HA-tagmakes our approach applicable to virtually any protein; we have demonstrated this concept by conjugating HA-tagged ovalbumin to HyA, thereby also showing the feasibility of producing chimeric proteoglycans. A caveat of this appproach is that, although the formation of boronic esters does not affect the biological recognition of substrates (ovalbumin and HyA), the introduction of catechols may alter some of their biological properties: for example, only after tyrosinase treatment ovalbumin directly induced dendritic cell maturation, either alone or as a HyA conjugate.

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INTRODUCTION We present a technique for tyrosine-specific bioconjugation of peptides and proteins, which is based first on the tyrosinase (Tyrase)-catalyzed conversion of tyrosine residues into catechols, and second on their in situ coupling with a boronate-containing carrier structure (Figure 1A). The interest in a chemically selective tyrosine reaction derives from the combination of relatively low abundance with an often poor exposure to water; this in principle allows a certain degree of site selectivity in the bioconjugation. The best known examples of tyrosine bioconjugation have employed multicomponent Mannich reaction1 or diazonium ion coupling,2 and recently and most notably also ene-type reaction with cyclic diazodicarboxamides (best known as triazolinediones, TADs),3−5 although other techniques have been attempted, e.g., the formation of rhodium(III) complexes with boronic acid-containing reagents.6 However, with the exception of TADs, most approaches suffer from poor selectivity and/or the multicomponent nature of the reaction, which requires careful optimization of reagent ratios (e.g., Mannich reaction requires a large excess of formaldehyde). The bioconjugation approach reported here is based on the conversion of phenols to catechols catalyzed by a Tyrase, which © XXXX American Chemical Society

is inherently tyrosine-selective. There is, however, an important downside: enzymes of this class, as much as the functionally related laccases,7 further convert catechols to o-quinones (whereas catecholases only perform the second step8,9), which then undergo non-enzymatic, unspecific but very rapid polymerization. In nature, the latter processes preside over the biosynthesis of pheomelanines (biosynthesis triggered by Michaeltype addition of cysteins and glutathione) and eumelanins (by intramolecular Michael-type addition and production of dopachrome or dopachrome-like compounds);10,11 through conjugation to nucleophilic amino acidic residues, they are also supposed to be at the basis of cuticle sclerotization in insects.12 This chemistry has also been successfully used to in situ crosslink biomaterials such as gelatin13 or chitosan,14 in a fashion similar to what happens with horseradish peroxidase,15−17 with the advantage of not requiring hydrogen peroxide. Despite the general utility of this 2-step phenol-quinone conversion as a cross-linking process, there are only a few reports of its use for tyrosine-specific derivatization to yield soluble conjugates, e.g., Received: March 30, 2018 Revised: July 3, 2018 Published: July 5, 2018 A

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Figure 1. A. Schematic representation of the molecular mechanism of bioconjugation: tyrosine residues are hydroxylated by using resin-supported Tyrase; the resulting catechols are in situ coupled to HyA-APBA to yield a boronate linker. B. Structures of the four phenol-containing peptides used in this study; Leu-Enk has only one tyrosine residue in a terminal position, FLAG has it in internal position, and HA-tag presents three tyrosines, one of them at the N-terminus. C. HA-tag was also employed in an ovalbumin-bound form (HA-tag-OVA), where 8−10 HA-tag sequences are present on each OVA (supplier information; Western blot with a single band at 55 kDa, anti-HA-tag antibody); their oxidation and in situ coupling to HyA-APBA produces a boronic ester-linked bioconjugate.

suggesting the use of Michael-type addition18 or strain-promoted cycloaddition on the quinonic species19 (both approaches recently reviewed by van Delft20). The main reason is the presence of side processes: as shown in a study performed on the hemagglutinin-derived YPYDVPDYA tagging peptide (HA-tag, 96−106 aa of the influenza virus hemagglutinin glycoprotein), quinones can be generated via Tyrase-mediated oxidation and undergo addition of, e.g., hydrazides, but at the same time cross-linking and possibly also cleavage reactions can complicate the process and the identity of the products.18 In this study, therefore, we have focused on a way to stop the conversion at the level of catechols, which are in situ reacted with boronic acid derivatives to yield boronic esters; these rather popular linker groups21 are characterized by good stability under physiological conditions, but are easily cleaved at acidic pH or in the presence of oxidants.22 We have experience in boronic ester-based conjugates,23 which can therefore be employed to release a payload (e.g., a catechol-containing protein) in a responsive fashion.22 Therefore, the main point of this study is the possibility to fine-tune a very selective enzymatic conversion for the purpose of reversible bioconjugation. Here, the structure we used to link proteic/peptidic materials is a derivative of hyaluronic acid (HyA), which was chosen as a biocompatible, degradable carrier; additionally, HyA in principle also allows for biological targeting (predominantly through CD44).24 Interestingly, the literature offers only a

handful of reports for the covalent derivatization of HyA with proteins, e.g., with interferon α,25 or VEGF decoy receptor sFlt-,26 to yield soluble bioconjugates; this may stem from the rather unspecific techniques used to date to introduce the chemical links, which most often do not allow at the same time a good control over the sites of derivatization and the provision of environmental responsiveness (e.g., pH-activated release of the proteic payload). We have conjugated HyA to a number of peptides with isolated (L-Tyr), terminal (Leu-Enk), internal (FLAG), or both terminal and internal tyrosines (HA-tag) (Figure 1B); please note that to avoid confusion between similar acronyms, in this work we use HA as an abbreviation for hemagglutinin and HyA for hyaluronic acid. FLAG and HA-tag are among the most commonly used epitope tags,27 which are introduced on proteins to improve characterization, purification, or localization of the macromolecule of interest;28 such peptides can be easily grafted or even more commonly expressed with the protein as fusion constructs.29−31 Since tagging can be performed on virtually any protein via routine protocols, a tag-targeted strategy provides a flexible and virtually universal platform for bioconjugation. Here, we have focused our attention on HA-tag as an enzymatically activatable linker between proteins and HyA. The HA-tag sequence is rich in tyrosine residues, and highaffinity anti-HA-tag antibodies as well as grafted proteins are commercially available;32 for example, we have also employed B

DOI: 10.1021/acs.bioconjchem.8b00227 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Figure 2. A. The Tyrase-mediated oxidation when performed in the absence (top) and in the presence (bottom) of AA. B. The rates of the A270 decrease, i.e., the AA consumption rate, are reported as a function of Tyrase concentration, showing that L-Tyr and Leu-Enk consistently follow a more rapid kinetics than the other two peptides. C. Absorption spectra (230−600 nm) of the four Tyrase substrates in 10 mM PBS as a function of the exposure to free Tyrase (6 U/mL for L-Tyr and Leu-Enk; 65 U/mL for FLAG and HA-tag; [tyrosine] = 0.1 mM), in the absence of AA (top) and presence of 0.1 mM AA (bottom).

peptide; on the other hand, the AA oxidation rate did vary (more rapid for L-Tyr and Leu-Enk, and slower for FLAG and HA-tag; Figure 2B), therefore excluding that AA was a Tyrase substrate. At the same time, LC-MS analysis performed before AA complete consumption showed significant production of catechols (Figure 3A and B), therefore ensuring that AA did not interfere with the first step of enzymatic conversion. Incidentally, MS/MS analysis showed that Tyrase converted preferentially the internal tyrosine residues of HA-tag, with the N-terminal one being present as catechol only in the triple-oxidized peptide, which was the product present in the smallest amount (Figure 3B; see also Supporting Information, Figure S4 and S5). [Note: AA-mediated process of catechol-production may affect the HyA size: AA, in particular when metal ion contaminant (e.g., iron or copper), can contribute to HyA depolymerization. However, at the concentration used in our process, AA did not significantly affect the dynamic viscosity of both HyA and HyA-APBA solution for at least 3 days, which is an indication of the absence of significant depolymerization (see Figure S1).] Having established conditions for a selective (quinone-free) generation of catechols, we have then proven their availability for in situ conjugation with boronic acids. The latter react with diols to produce cyclic esters,35 which are specifically stable with catechols (equilibrium constants ≥103 M−1, vs ∼1−10 M−1 for aliphatic diols22). The resulting boronic esters are reversible, and can easily hydrolyze at mildly acidic pH36,37 or be cleaved

an HA-tag grafted onto ovalbumin (HA-tag-OVA) (Figure 1C), choosing the latter as a functional protein for its popular role as a model antigen in immunology and vaccine development.33 It is also worth pointing out that the resulting products can be seen as artificial and pH-reversible proteoglycans, due to the presence of a glycosoaminoglycan and of proteic material.

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RESULTS AND DISCUSSION The production of quinones is detrimental to a chemically defined bioconjugation; therefore, we first aimed to intercept the Tyrase catalysis after its first step (Figure 2A, top). Using four different tyrosine-containing substrates, it is apparent that the oxidation of tyrosines is always accompanied by the presence of quinones (peaks at around 300 nm in Figure 2C) and also of visible light-absorbing species identifiable as dopachrome analogous, due to their more pronounced presence when the phenol is close to a free amine (L-Tyr and Leu-Enk). Since it is known to be a melanogenesis inhibitor,34 we have hypothesized that ascorbic acid (AA) could be used as an in situ reducing agent to convert quinones back to catechols (Figure 2A, bottom). First, it was noteworthy that, for all substrates, quinone and “dopachrome” bands appeared only after the complete consumption of AA (Figure 2C, bottom; see also Supporting Information, Figure S3). If this effect was due to AA being more rapidly oxidized by Tyrase, its disappearance rate would not depend on the nature of the tyrosine-containing C

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Figure 3. A. HPLC analysis of L-Tyr (top) and Leu-Enk (bottom); the chromatograms show the pristine form (lower curves), and the effect of 20′ oxidation in the absence of AA (middle curves) and in its presence (0.1 mM; top curves); the peaks corresponding to catechols are marked with red arrows. For L-Tyr, the inset shows the peaks of L-Tyr and L-Dopa premixed in equimolar amounts. D-tryptophan (24 μM) was used as an internal standard (n = 3). B. ESI-MS spectra of unmodified, mono-, double-, and triple-oxidized HA-tag ([tyrosine] = 0.17 mM; [resin supportedTyrase] = 1128 U/mL; [AA] = 0.17 mM); time = 1 h after in-line HPLC separation of the various peaks. The chemical structures present the location of the oxidation sites obtained via MS/MS analysis; the nonoxidized tyrosine residues are in purple, the ones oxidized to catechols in red.

under oxidizing conditions,38 allowing their use, e.g., for responsive drug release.21,39 Recently, our group has employed boronated HyA (HyA-APBA) to react with mono- and polyfunctional catechols, respectively producing bioconjugates22 and nanoparticles.23 The choice of a HyA as a carrier structure is due to its most important receptor, CD44, being overexpressed in a number of cancers40 and upon inflammatory activation, and is responsible not only for HyA binding on cell surfaces (→targeting) but also for its endocytosis (→delivery).41 The enzymatically generated catechols easily conjugate to boronic acids. We have proven this point by using a competitive assay: a catechol reporter (alizarin red S, ARS) precomplexed to HyA-APBA is displaced by the catechols produced in situ via tyrosine oxidation, and this process is followed through the changes in the ARS absorption spectrum22 (Figure 4A, left); we have recently demonstrated22 that this method allows us to obtain catechol/boronate equilibrium constants more accurately than the more popular method based on ARS fluorescence22 (see Supporting Information for the calculation of catechol/HyA-APBA equilibrium constants and for the estimation of catechol content). We have used this assay to qualitatively prove that tyrosines are effectively converted to catechol/boronate complexes: ARS changed its maximum of absorbance only when Tyrase was added to the peptide/HyA-APBA mixtures, and the shifts followed a kinetics similar to that of catechol production, as assessed via HPLC for L-Tyr and Leu-Enk (Figure 4A, right). It is worth noting that this conversion can be performed with Tyrase both in solution and supported on an epoxy-functionalized resin (Immobead 150 p; see Supporting Information, Figure S2, also for the characterization of the activity of the

supported enzyme); the supported enzyme allows for an easy purification of the bioconjugate and therefore it was preferred for all ELISA or cell-based tests. It is noteworthy that, in the absence of AA, the presence of boronic acids was not sufficient to avoid the formation of further odixized species (see Supporting Information, Figure S6). We have also measured the equilibrium constants for the conjugation of oxidized L-Tyr and Leu-Enk, respectively, 3124 ± 224 and 3557 ± 594 M−1, which show these catchols to bind HyA-APBA better than ARS (Keq = 2550 ± 150 M−1). It is important to note that these experiments were performed in excess of catechols (tyrosine/boronate/ARS molar ratio: 1:0.26:1.5) because the competitive equilibrium requires a negligible concentration of free boronates, whereas in all further studies the conjugates were prepared using an excess of boronates, in order to maximize the retention of the catechols. However, these values of equilibrium constant ensure that in all cases the catechols quantitative bind to the HyA carrier. For the oxidized HA-tag, the evaluation of the binding constant is more complex, since there are three tyrosine residues that likely differ in both oxidizability and affinity to boronic acid when oxidized; here, we have assumed its average binding constant to be analogous to that of oxidized L-Tyr and Leu-Enk (3500 M−1), and used the ARS assay to estimate the tyrosinecatechol conversion (see ELISA tests). For this bioconjugation approach to have a general validity, the products of enzymatic oxidation and/or of boronic ester formation should not have a significant toxicity. Indeed, in a rather broad range of concentrations neither the oxidation nor the conjugation to HyA-APBA appeared to increase the (negligible) cytotoxicity of HA-tag, as evaluated on RAW D

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Figure 4. A. Top: scheme of the reactions involved in the ARS spectral shift assay. Center and bottom: the ARS maximum of absorption shifts to higher wavelengths when a catechol is generated (black squares), whereas no shift is recorded in the presence of the pristine peptides and of HyAABPA but without Tyrase (empty circles); the kinetics of the shift mirrors the generation of catechols followed via HPLC (red circles); tyrosine/ boronate/ARS molar ratio: 1:0.26:1.5 (to ensure negligible concentrations of free boronates); supported Tyrase = 161 U/mL. B. ELISA analysis of the recognition capacity of different products of supported Tyrase oxidation and bioconjugation. Top: recognition of HA-tag by its antibody before/ after 1 h oxidation and with/without HyA-APBA. Center: separate recognition of the HA-tag-OVA two components by antibodies for HA-tag and OVA, in the pristine form of the tagged protein and after 1 and 3 h of oxidation. Right: sandwich ELISA for the contemporaneous recognition of the HA-tag-OVA/HyA-APBA conjugate as an OVA- and HyA-bearing system. All results of direct ELISA are normalized against the respective positive controls (n = 3).

the functional properties of a cargo; in our case this is OVA, a potentially antigenic protein whose presentation by dendritic cells (DCs) activates lymphocytes,33 but only upon coactivation by and ideally codelivery of adjuvants,42,43 which are necessary for DC maturation and antigen presentation. In this preliminary study, we have compared the effectiveness of pristine, oxidized, and HyA-conjugated HA-tag and HA-tag-OVA in inducing the maturation of primary murine DCs, using the T-cell costimulatory molecules CD40, 80, 86, and MHC II as markers of the process.44,45 HA-tag caused no DC maturation in any form (pristine, oxidized, or conjugated), which ensures that per se the linker does not cause any significant DCactivation (Figure 5A). For HA-tag-OVA, whereas no maturation was observed with pristine HA-tag-OVA and HyA-APBA (either separately or in mixture), the enzymatic oxidation led to a significant upregulation of all markers, both with the protein alone and as a conjugate (Figure 5B). Importantly, this did not appear to be due to the catechol-bearing protein/protein conjugate showing a different aggregation: the count rate (= the scattering intensity) of the samples was virtually untouched by the extent of the enzymatic oxidation reaction (see Supporting Information, Figure S10). The presence of catechols on a potentially antigenic protein but not on the linker aloneappears therefore to confer also an additional activation capacity to that structure (e.g., through pattern recognition receptors such as TLRs), which is typical of adjuvants; this potential role of catechols may be at the basis of the use of melanin as an adjuvant.46 The catechol-bearing

264.7 macrophages and primary murine dendritic cells (see Supporting Information, Figure S7). Another important point is that the enzymatic conversion should not alter the biological recognition of the linked structures, although any change in that of the tagging peptide would not be critical. Indeed, due to their density in the peptide structure (3 out of 9 residues) the HA-tag tyrosines were critical for its immunological recognition: after 1 h of oxidation, they were estimated to be 69 ± 3% converted to catechols (ARS assay), probably with a predominance of multiple catechols in transformed peptides since capillary electrophoresis showed 52 ± 3% of unconverted peptide (see Supporting Information, Figure S8); correspondingly, the binding to the anti-HA-tag antibody was reduced to about a half, in both a free and boronate-bound form (Figure 4B, left), which suggests a complete lack of recognition for all transformed peptides. Similarly also HA-tag-OVA after oxidation was no longer a substrate for the anti-HA-tag antibody, whereas its proteic part was still a good substrate for anti-OVA (Figure 4B, center); this stems from the fact that OVA is left untouched by Tyrase (see Supporting Information, Figure S9). Incidentally, this also shows that this bioconjugation technique is really site selective, since Tyrase converts the well exposed HA-tag tyrosines, but none of the ten OVA tyrosines. Furthermore, in sandwich ELISA both HyA and OVA were recognized in the Hya-APBA/HA-tag-OVA conjugate (Figure 4B, right). Having demonstrated that the bioconjugation is effective, nontoxic, and does not alter the recognition of the linked biomolecules, we have then queried which effects it may have on E

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Figure 5. Analysis of CD40, 80, 86 and MHC-II (median fluorescence intensity (MFI) from flow cytometry, normalized to that of untreated cells) in primary murine DCs after 24 h of treatment with HA-tag, HA-tag-OVA, and their oxidation products (n = 3). Statistical analysis was performed as one-way ANOVA using Prism (GraphPad Software, Inc., La Jolla, CA, USA). Differences between groups were determined by a Tukey’s multiple comparisons test. Asterisks denote statistically significant differences (*P < 0.05; **P < 0.01).

pH; Supporting Information, Figure S11), an intracellular release in endosomes can be hypothesized. Interestingly, this modification appears to increase the antigenicity of the tagged protein, and its conjugation with HyA or any other receptorbinding structure may provide the means for its selective

protein and the conjugate upregulated the markers in an identical fashion, which strongly suggests that the oxidized HAtag-OVA is released and recognized by DCs; considering the pH-sensitive nature of the boronate esters (see also the release of Tyrase-oxidized tyrosine from HyA-APBA at mildly acidic F

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diluted to a final concentration of 1 μM and injected into loadtrapping mode on a C18 Pepmap300 precolumn and separated using a capillary Thermo Scientific C18 Pepmap300 column (5 μm, 150 × 0.075 mm, 300 Å). Products were eluted using mixtures of mobile phase A (95:5 water:acetonitrile + 0.1% formic acid) and B (5:95 water:acetonitrile + 0.1% formic acid) at a flow rate of 300 nL/min. A 35 min linear gradient from 0% to 55% B, followed by 5 min at 90% B and 15 min column re-equilibration, was employed. The column was connected to a Picotip emitter (New Objective, SS Scientific, Eastbourne, UK) to which a 1.7 kV voltage was applied using the standard Thermo nanoESI source. The mass spectrometer was operated in positive ion mode with the voltage and temperature of the capillary set, respectively, at 35.0 V and 275 °C and the tube lens value held at 100.0 V. MS/MS experiments were performed in order to identify the location of the modification. Precursors were isolated in the linear ion trap within an 8 Th window and fragmented by in-trap collision-induced dissociation (CID) at a nominal energy of 35.0. Tyrase-Catalyzed Reactions. All solutions were prepared in 10 mM PBS, pH = 7.4. The immobead 150p resin particles were always dispersed in PBS for 1 h prior to use. Tyr ase -Catalyzed Oxidation of L -Tyr and Leu-Enk. Oxidation of L-Tyr and Leu-enk with Free Tyrase. 50 μL of a 90 μg/mL (0.5 mM, corresponding to a dose of 0.025 μmol) L-Tyr solution or of a 278 μg/mL (corresponding to 0.5 mM of L-Tyr residues, i.e., to a 0.025 μmol concentration of phenols) Leu-enk solution were added to 50 μL of 88 μg/mL AA (0.5 mM corresponding to 1:1 mol/mol, L-Tyr: AA) and 100 μL PBS. 50 μL of solutions of Tyrase at concentrations ranging from 5 to 40 μg/mL (from 3 to 22 U/mL) were then added, recording UV−vis spectra as a function of time (230 to 600 nm; 0 to 60 min). Mixtures without AA were used as negative controls. Oxidation of L-Tyr or Leu-enk with resin supported-Tyrase. 403 U of Tyrase solubilized in 0.58 mL of PBS was added to 50 mg of resin. The reaction was kept under constant shaking (300 rpm) at 37 °C, for 19 h in a Thermomixer Comfort (Eppendorf). The resin was filtered with VWR qualitative filter paper 413 (particle size 5−13 μm) to separate it from any unreacted enzyme, and washed ten times with 1 mL aliquots of PBS. 500 μL of 90 μg/mL L-Tyr or 278 μg/mL Leu-enk solutions in 10 mM PBS, pH = 7.4, was added to 2 mL of PBS or 500 μL of AA (0.5 mM) and 1.5 mL of PBS. The mixtures were then added to the resin supported-Tyrase and kept under constant shaking for 30 min. Every 5 min, 100 μL of the reaction mixtures was sampled and analyzed via RP-HPLC. As controls, the same mixtures were prepared without supported Tyrase. Tyrase-Catalyzed Oxidation of FLAG Peptide (free Tyrase). 50 μL of 506 μg/mL of FLAG peptide solution in PBS (0.5 mM, corresponding to 0.025 μmol) was added to 50 μL of 88 μg/mL AA (0.5 mM corresponding to 1:1 mol/mol, L-Tyr:AA) and 100 μL of 10 mM PBS, pH = 7.4. Then, 50 μL aliquots of solutions of Tyrase at concentrations ranging from 15 to 120 μg/mL in 10 mM PBS, pH = 7.4 (corresponding to 8 to 65 U/mL) were added. UV−vis spectra were recorded from 230 to 600 nm at different time points (from 0 to 80 min). As controls, the mixtures were also prepared without AA. Tyrase-Catalyzed Oxidation of HA-Tag and Its In Situ Conjugation to HyA-APBA. All solutions were prepared in 10 mM PBS, pH = 7.4, except HA-tag that was used in deionized water.

targeting to CD44-expressing cells such as DCs. Importantly, this method also has an intrinsic limit: the protein cargo should maintain its activity, and this can happen because either its entire structure or at least its active site is untouched; for example, OVA is a poor substrate for Tyrase; enzymes with a broader activity such as laccases are potentially more dangerous.

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CONCLUSION We have therefore developed an efficient, one-pot, and mild method for selective and reversible bioconjugation; we have implemented it under conditions that employ easily removable, supported enzymes, and avoid the production of quinones, and applied it to the use of HA-tag as a generally applicable tag/ linker for proteins. Interestingly, this modification appears to increase the antigenicity of the tagged protein, and its conjugation with HyA or any other receptor-binding structure may provide the means for its selective targeting to CD44-expressing cells such as DCs.

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EXPERIMENTAL PROCEDURES The list of all reagents and the description of the synthetic procedures for HyA-APBA are reported in the Supporting Information, additional materials and methods. If not otherwise stated, all solutions were prepared in 0.1 M PBS obtained by dissolving sodium dihydrogen orthophosphate dihydrate, disodium hydrogen orthophosphate dihydrate, and sodium chloride, supplied by BDH (U.K.), in concentrations, respectively, of 2.3 g/L, 11.8 g/L, and 9 g/L in water purified in a Milli-Q system (Millipore, U.K.). Physico-Chemical Characterization. High Performance Liquid Chromatography (HPLC). These analyses were performed with an Agilent 1100 series Quaternary Pump Instrument (Waldbronn, Germany), equipped with a temperature controlled autosampler (G1313−87305) and vacuum degasser pump (G1322−67300). Agilent ChemStation software was used for data processing. Samples (50 μL) were injected into an Eclipse XDB-C18, 5 μm, 4.6 × 150 mm column (USA), and detected using a Jasco UV-1575 detector operating at 270 nm. L-Tyrosine (L-Tyr) and L-Dopa products were analyzed using a mobile phase consisting of H2O:AcCN:HCOOH (96:4:0.4 v/v) at a flow rate of 1 mL/min for 10 min. L-Tyr and L-Dopa calibration curves were built at the concentration range of 0.25− 0.00781 mM in PBS (10 mM, pH = 7.4). Leu-enk and its reaction products were analyzed using a 8:2 to 0:10 v/v H2O:AcCN gradient. A Leu-enk calibration curve was built using 0.17− 0.0104 mM (94.4−5.8 μg/mL) Leu-enk in 10 mM PBS at a flow rate of 1 mL/min. UV−vis Spectroscopy. All experiments were performed using a BioTek Synergy 2 multimode microplate reader. For the kinetics of Tyrase-catalyzed oxidation of L-Tyr, the absorbance at 475 nm was recorded between 30 s and 1 h after mixing the reagent solutions, subtracting the scattering component of the buffer solution using appropriate blanks. For the evaluation of the products obtained after the enzymatic reaction between L-Tyr, Leu-enk, HA-tag, or FLAG and Tyrase, the absorbance spectra were recorded in the range of 230−600 nm at 25 °C, in 10 mM PBS at pH = 7.4. HPLC Coupled to Mass Spectrometry (LC-MS) and LC-MS/ MS Analysis. This analysis was performed with Dionex U3000 nano-HPLC (Dionex, Camberley, UK) coupled to a Thermo LTQ FT-Ultra mass spectrometer (ThermoFisher Scientific, Runcorn, UK). HA-tag and oxidized HA-tag mixtures were G

DOI: 10.1021/acs.bioconjchem.8b00227 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry Oxidation of HA-tag with Free Tyrase. 50 μL of 184.3 μg/mL of HA-tag (0.5 mM of L-Tyr, corresponding to 0.025 μmol) was added to 50 μL of 88.06 μg/mL AA (0.5 mM corresponding to a 1:1 L-Tyr/AA molar ratio) and 100 μL of PBS. Then, 50 μL aliquots of solutions of Tyrase at concentrations ranging from 15 to 120 μg/mL (from 8 to 65 U/mL) were then added, recording UV−vis spectra as a function of time (230 to 600 nm; 0 to 60 min). Mixtures without AA were used as negative controls. Oxidation of HA-tag with Resin-Supported Tyrase. 1692 U of Tyrase solubilized in 0.58 mL of PBS was added to 100 mg of resin. The reaction was kept under constant shaking (300 rpm) at 37 °C, for 19 h in a Thermomixer Comfort (Eppendorf). The resin was filtered with VWR qualitative filter paper 413 (particle size 5−13 μm) to separate it from any unreacted enzyme, and washed ten times with 1 mL aliquots of PBS. At this point, 500 μL of PBS was added to 500 μL of 185 μg/mL HA-tag and 500 μL of 88 μg/mL AA. The resulting solution was then added to the resin supported-Tyrase and kept under constant shaking for 1 h at room temperature. At this point, 40 μL of 5 mM AA was added to avoid catechol oxidation (essential for mass spectrometry analysis). Mixtures were filtered using Millex-GV 13 mm (PVDF, 0.22 μm) filters, diluted to a final concentration of 1 μM and injected into RP-HPLC coupled to MS. HA-tag (no Tyrase) was used as a negative control. In Situ Conjugation to HyA-APBA. 500 μL of 5 mg/mL HyA-APBA (corresponding to 0.065 μmol of APBA, df = 10 mol %) was added to 500 μL of 185 μg/mL HA-tag (0.5 mM of tyrosines: 0.025 μmol) and mixed for 10 min at room temperature. Then, 500 μL of 88 μg/mL AA solution (0.5 mM) was added. The mixtures were then added to 50 mg resinsupported-Tyrase (prepared as described above) and samples were shaken for 1 h at room temperature. 600 μL of the HAtag-catechol/HyA-APBA solution and of controls prepared w/o Tyrase and/or HyA-APBA were added to 60 μL of 50 μg/mL D-tryptophan (internal standard), 40 μL of 5 mM AA, and then filtered using Millex-GV 13 mm (PVDF, 0.22 μm) filters. The solutions were then subjected to capillary electrophoresis analysis (detection at 214 nm). The calibration curves for 0.083− 0.010 mM HA-tag, and 1.6−0.2 mg/mL HyA and HyA-APBA are shown in Figure S3. Oxidation of HA-tag-Ovalbumin via Resin SupportedTyrase and Its In Situ Conjugation to HyA-APBA. 6768 U of Tyrase solubilized in 0.58 mL of PBS (10 mM, pH = 7.4) were added to 150 mg of resin. The reaction was kept under costant shaking (300 rpm) at 37 °C, for 19 h in a Thermomixer Comfort (Eppendorf). Any unreacted enzyme was removed by filtering the resin with VWR qualitative filter paper, 413 (size 150 mm, particle size 5−13 μm) and washing it ten times with 1 mL portions of PBS. At the same time, 250 μL of 120 μg/mL AA in 20 mM PBS, pH = 7.4 was mixed with 500 μL of a 30 μg/mL HA-tag-ovalbumin (HA-tag-OVA) solution in deionized water, and the resulting solution was added to resin supported-Tyrase and shaken at room temperature for 1 and 3 h (different degrees of oxidation). HA-tag-OVA was oxidized also in the presence of 250 μL of 6.68 mg/mL HyA-APBA. As control, HA-tag-OVA without Tyrase was also prepared. All reactions were sampled at different time points and analyzed by DLS (see Figure S10). Analysis via ELISA. Oxidized HA-tag and HA-tag/HyAAPBA Conjugates. Anti-HA-tag. 50 μL of starting or oxidized HA-tag (with or without HyA-APBA) and their controls were diluted in the range concentration 600−0.006 ng/mL (dilution

factor 1:10) with 10 mM PBS (pH = 7.4) and then tested through ELISA immunoassay. Briefly, samples (50 μL) were coated onto PVC microtiter plate (F96 maxisorp nunc-immuno plate, Thermo Scientific) overnight at 4 °C. After removal of excess samples, wells were washed twice with washing buffer (PBS containing 0.05% v/v Tween-20) and blocked with 200 μL of blocking buffer (1% w/v BSA in 10 mM PBS at pH = 7.4) for 1 h at room temperature. Plates were washed twice, followed by the addition of primary antibody solution (anti-rabbit anti-HA-tag, Sigma H6908, 0.25 μg/mL in blocking buffer, 100 μL/well) for 2 h at room temperature. After 5 washes, HRP-conjugated goat antirabbit IgG (Sigma, A0545, diluted 1:30.000 in blocking buffer, 100 μL/well) were added for 1 h at room temperature. Plates were washed 5 times and the reaction was visualized by the addition of 100 μL of the chromogenic substrate for 10 min in the dark. The reaction was stopped with 50 μL of 2 M H2SO4 and the absorbance was measured at 450 nm using microplate reader. The HA-tag calibration used concentrations in the range of 0.006−6 ng/mL. Oxidized HA-tag-OVA and HA-tag-OVA/HyA-APBA Conjugates. Direct ELISA (anti-HA-tag). The HA-tag-OVA was dissolved in PBS at concentrations in the range of 0.015 to 15 μg/mL; oxidized HA-tag-OVA after 1 or 3 h reaction, with or without HyA-APBA, and their controls were also tested in the same concentration range. Samples (50 μL) were added to PVC microtiter plate (F96 Maxisorp Nunc-immuno plate, Thermo Scientific) overnight at 4 °C. After coating the plates, the excess liquid was removed, the wells were washed twice with washing buffer (PBS containing 0.05% v/v Tween-20) and blocked with 200 μL of blocking buffer (1% w/v BSA in 10 mM PBS at pH = 7.4) for 1 h at room temperature. Plates were washed twice, followed by the addition of primary antibody solution (anti-rabbit anti-HA-tag, sigma H6908, 0.25 μg/mL in blocking buffer, 100 μL/well) for 2 h at room temperature. After five washes, HRP-conjugated goat anti-rabbit IgG (sigma, A0545, diluted 1:30 000 in blocking buffer, 100 μL/well) were added for 1 h at room temperature. Plates were washed five times, then 100 μL of chromogenic substrate were added for 10 min in the dark. The reaction was stopped with 50 μL of 2 M H2SO4 and the absorbance was measured at 450 nm with microplate reader (n = 3). Direct ELISA (Anti-Ovalbumin) Analysis. Ovalbumin (OVA) and HA-tag-OVA solutions were prepared in PBS at concentrations in the range of 0.015 to 15 μg/mL; solutions with similar concentrations of HA-tag-OVA oxidized for 1 or 3 h with or without HyA-APBA and their controls were also analyzed. Wells of microtiter plates were coated by adding 50 μL of these samples (4 °C, overnight). The coating solutions were removed and 200 μL of blocking buffer were added for 1 h at room temperature. Primary antibody solution (anti-rabbit anti-OVA, Millipore AB1225; 1:3000 in blocking buffer, 100 μL/well), was added for 2 h at room temperature. Then, HRP-conjugated goat anti-rabbit IgG (1:30.000 in blocking buffer, 100 μL/well) was added for 1 h at room temperature, followed by the addition of substrate solution (100 μL for 10 min in the dark). The reaction was stopped with 50 μL of 2 M H2SO4 and the absorbance was read at 450 nm with microplate reader. Plates were washed 2−5 times with washing buffer after each step (n = 3). Sandwich ELISA Analysis. Anti-sheep anti-HyA antibody was diluted 1:5000 in PBS and coated (50 μL) onto the wells of the microplate (4 °C overnight). Coating solutions were H

DOI: 10.1021/acs.bioconjchem.8b00227 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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DC Surface Marker Analysis through Flow Cytometry. After 24 h of treatment, cells were harvested and stained with Zombie Aqua dye (Biolegend) or Live cell/Dead cell discrimination by following manufacturer instructions. Cells were washed with 2 mL of 1% FBS-PBS, treated with 1% FBS-PBS (blocking buffer) for 30 min on ice and then surface stained with a cocktail of anti-mouse mAb (at appropriate dilutions in 100 μL of blocking buffer), consisting of CD11cAPC/eFluor780 (eBioscience, cat no. 47−0114−82, dilution 1:200), MHCIIe450 (MHC class II (I-A/I-E) antibody, eBioscience, cat no. 48−5321−82, dilution 1:2000), CD80-APC (Biolegend, cat no. 104714, dilution 1:800), CD40-PE (Biolegend, cat no. 124610, dilution 1:200), CD86-e488 (Biolegend, cat no. 105018, dilution 1:200). Cells were stained with specific antibodies or appropriate isotype controls for 20 min on ice and protected from light. Cells were washed with blocking buffer, centrifuged, resuspended in cold PBS, and immediately analyzed with BD LSRFortessa flow cytometer (BDBioscience, San Jose, CA, USA). Dead cells were excluded from the analysis by by using gates based on forward and sideward light scattering and on Zombie Acqua negative cells (live cells were above 90%) . Data were collected for 10 000 living cells/sample. Cells were 95% CD11c+. Data analysis was performed with FlowJo software (TreeStar, Inc., Ashland, OR) by following the gating strategy described above. Percentage of positive cells and Median Fluorescence Intensity (MFI) values were determined for each cell marker surface. Statistical Analysis. One-way ANOVA analysis was performed in Prism (GraphPad Software, Inc., La Jolla, CA, USA). Differences between groups were determined by a Tukey’s multiple comparison test. Asterisks denote statistically significant differences (*P < 0.05; **P < 0.01).

removed and the plates washed; wells were blocked with blocking buffer for 1 h at room temperature. 100 μL of starting or oxidized HA-tag-OVA (1 or 3 h), with or without HyAAPBA, and their controls were added for 2 h at room temperature. Wells were washed five times, followed by the addition of primary antibody solution (anti-rabbit anti-OVA, 1:3000 in blocking buffer, 100 μL/well), for 1 h at room temperature. Plates were washed five times and then HRP-conjugated goat anti-rabbit IgG (1:30 000 in blocking buffer, 100 μL/well) was added for 1 h at room temperature. After five washes, 100 μL of TMB substrate were added for 10 min in the dark, followed by the addition of 2 M H2SO4. The absorbance at 450 nm was measured with microplate reader (n = 3). ARS Assay for the Calculation of Catechol/HyA-APBA Equilibrium Constants and for the Estimation of Catechol Content. Experimental Section. All solutions were prepared in 10 mM PBS, pH = 7.4. Alizarin Red S (ARS) was used as a primary binder for HyAAPBA, employing its absorbance shifts upon the presence of competing catechols in a competitive assay. 500 μL of 0.5 mg/mL HyA-APBA in PBS (df = 10 mol %; 0.065 μmol of boronates) were added to 500 μL of 0.5 mM L-Tyr or Leu-enk or 0.167 mM HA-tag solutions in PBS (all correspond to 0.5 mM of phenols; 0.25 μmol) and mixed for 10 min at room temperature. Then, 500 μL of 0.5 mM AA, 500 μL of PBS, and 500 μL of 0.75 mM ARS were added (corresponding 0.375 μmol). The mixtures were then added to 100 mg of resin-supported Tyrase, prepared as described above, and shaken for 30 min (L-Tyr and Leuenk) or 1 h (HA-tag). At specific time points, 250 μL aliquots were sampled and 400−700 nm absorption spectra recorded (for HPLC analysis, see SI Section 1.5). Control samples without Tyrase were also prepared. Additional details on the method used to calculate binding constants can be found in the Supporting Information. Experiments on Primary Dendritic Cells. Isolation and Generation of Murine Dendritic Cells. Bone marrow-derived dendritic cells (BMDCs) were generated as previously described.47 In brief, bone marrow was isolated from femurs of a C57BL/6 mouse (male, 6−8 weeks old) maintained in the animal facilities of the University of Manchester (Manchester, UK). 2 × 105/mL cells were seeded into bacteriological Petri dishes (Fisher), in 10 mL DC medium consisting of RPMI 1640 (Sigma) that was supplemented with 10% (v/v) heatinactivated FCS (HyClone), 2 mM L-glutamine (Life Technologies, Gaithersburg, MD), 100 U/mL penicillin/streptomycin (Life Technologies), and 20 ng/mL (day 0, 3, 6) or 5 ng/mL (day 10) recombinant human granulocyte macrophage colonystimulating factor GM-CSF (Peprotech, Rocky Hill, NJ). DCs were incubated for 10 days at 37 °C in an atmosphere of 5% CO2. On day 10, immature DCs (iDCs) were harvested and seeded at 1 × 106 per well (48 well-plate) in 125 μL of 2× RPMI 1640 medium, containing 20% FCS, 4 mM L-glutamine, 200 U/mL penicillin/streptomycin, and 10 ng/mL GM-CSF and incubated with 125 μL of pristine or oxidized HA-tag or HA-tag-OVA with or without HyA-APBA and their controls. Two different HA-tag or HA-tag-OVA concentrations (1.5 and 3, and 3.74 and 7.5 μg/mL, respectively) were tested. As positive control (DC activation), cells were treated with 2 μg/mL LPS in 125 μL PBS (for a final LPS concentration of 1 μg/mL); on the contrary, the negative control was treated with 125 μL of PBS. Samples were incubated for 24 h at 37 °C (n = 3).

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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/acs.bioconjchem.8b00227.

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Additional materials and methods (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Nicola Tirelli: 0000-0002-4879-3949 Author Contributions

All authors have given approval to the final version of the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge financial support from the EU FP7 project UniVax (grant number 601738). The authors are endebted to Dr. James Crooks for the assistance with the isolation and generation of bone marrow-derived dendritic cells; they also want to thank Dr. Chiara Di Meo, Dr. Pietro Matricardi, Prof. Tommasina Coviello, and Prof. Franco Alhaique for their collaboration, and the “Sapienza” University of Rome for the PhD studentship to Dr. Montanari, which allowed to initiate the work. I

DOI: 10.1021/acs.bioconjchem.8b00227 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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ABBREVIATIONS Tyrase,Tyrosinase; HyA,hyaluronic acid; APBA,aminophenilboronic acid; L-Tyr,L-tyrosine; Leu-Enk,leu-enkephalin; HAtag,hemagglutinin peptide; OVA,ovalbumin; AA,ascorbic acid; ARS,alizarin red S; LC-MS,liquid-chromatography−mass spectrometry; DCs,dendritic cells

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