Tyrosine-Triazolinedione Bioconjugation as Site-Selective Protein

Nov 29, 2017 - The electrophilic aromatic substitution (SEAr) reaction of triazolinediones (TADs) with the phenol moiety of tyrosine amino acid residu...
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Letter Cite This: ACS Macro Lett. 2017, 6, 1368−1372

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Tyrosine-Triazolinedione Bioconjugation as Site-Selective Protein Modification Starting from RAFT-Derived Polymers Stef Vandewalle,‡,† Ruben De Coen,‡,§ Bruno G. De Geest,*,§ and Filip E. Du Prez*,† †

Polymer Chemistry Research Group, Centre of Macromolecular Chemistry (CMaC), Department of Organic and Macromolecular Chemistry, Ghent University, Krijgslaan 281 S4-bis, B-9000 Ghent, Belgium § Department of Pharmaceutics, Ghent University, Ottergemsesteenweg 460, B-9000 Ghent, Belgium S Supporting Information *

ABSTRACT: The electrophilic aromatic substitution (SEAr) reaction of triazolinediones (TADs) with the phenol moiety of tyrosine amino acid residues is a potent method for the siteselective formation of polymer−protein conjugates. Herein, using poly(N,N-dimethylacrylamide) (pDMA) and bovine serum albumin (BSA) as model reagents, the performance of this tyrosine-TAD bioconjugation in aqueous solutions is explored. At first, reversible addition−fragmentation chain transfer (RAFT) polymerization with a functional urazole, a precursor for TAD, chain transfer agent is used for the synthesis of a TAD endfunctionalized pDMA. Eventually, the BSA ligation efficiency and selectivity of this polymer was evaluated in different aqueous solvent mixtures using SDS-PAGE and mass spectroscopy after trypsin digestion.

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tyrosine containing peptides were PEGylated through reaction with diazonium salt terminated poly(ethylene glycol).7 Although both reactions proceed under benign conditions, they feature slow reaction kinetics and demand distinct pH (4.5) to ensure site-selective labeling of tyrosine residues in protein modification. Recently, Barbas and co-workers documented the tyrosine ene-type reaction with cyclic diazodicarboxamides, in the form of triazolinediones (TAD’s), as a robust and fast bioconjugation method with click characteristics.8 In aqueous buffered medium and in the presence of other TAD-reactive amino acids (e.g., tryphtophane and lysine), stable tyrosine-TAD adducts are selectively formed. This was exemplified for the purpose of PEGylation of chymotrypsinogen and for the design of antibody-drug conjugates.9 Very recently, the group of Börner synthesized, in collaboration with our research group, TAD-end-capped peptides to perform chemical polymer ligations.10 Moreover, Heise and co-workers used a bifunctional TAD molecule for the efficient cross-linking of polypeptides through site-selective reaction with tyrosine residues.11 In this work, the performance of the tyrosine-TAD reaction for polymer−protein conjugation is explored in detail using an acrylamide polymer that can easily be prepared by RAFT. Poly(N,N-dimethylacrylamide) and bovine serum albumin (BSA) are used as model components in this study. As this bioconjugation is typically performed in aqueous environment,

olymer conjugation of proteins can lead to the formation of bioconjugates with improved properties in a complex biological environment.1 These polymer bioconjugates can be of benefit for various therapeutic applications. For example, introducing hydrophilic polymers (e.g., PEGylation) enhances the protein’s stability and solubility and extends their residence time in the human body.2 Furthermore, polymer materials that can adapt their morphology based on external triggers are of interest. Through ligation of such smart polymers, the bioactivity3 or immune-biological properties4 of proteins can be regulated by changes in the biological environment such as a temperature variation. A first approach to form polymer bioconjugates is “grafting-from”, where a protein macroinitiator is used to grow a polymer chain on the protein’s surface.5 A second approach that can be used to covalently link polymers to proteins is the “grafting-to” method. Here, native amino acid residues (predominantly lysines and cysteines) or unnatural amino acids, such as azidolysine, are chemoselectively modified with end-functionalized tailor-made polymers that can be synthesized using various reversible deactivation radical polymerization (RDRP) techniques.1b,6 Lysines are crucial for determining the pKa of a protein, while cysteine residues play a crucial role in 3D folding. Therefore, as an alternative to the conjugation of lysines with activated esters and cysteines with maleimide, vinylsulfone, or pyridyldisulfide chemistry,1b targeting other amino acid residues might be of great significance. In this regard, modification of tyrosines has gained considerable attention over the past years. Tyrosine modification of proteins has been performed by Mannich condensation reactions with low molecular imines, while © XXXX American Chemical Society

Received: October 6, 2017 Accepted: November 27, 2017

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DOI: 10.1021/acsmacrolett.7b00795 ACS Macro Lett. 2017, 6, 1368−1372

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ACS Macro Letters

kDa, Đ = 1.16, Figure S5), which was further used for tyrosine bioconjugation. In a first attempt, TAD-pDMA was conjugated to BSA by dissolving both TAD-pDMA and BSA in PBS. SDS-PAGE analysis and optical density integration indicated only moderate conjugation efficiency. When performing a blank control experiment, that is, in the absence of BSA, we noticed that the dark red colored TAD-pDMA solution rapidly (seconds) changed into faint yellowish upon addition to PBS, which suggests hydrolytic instability of the TAD moiety in aqueous medium. Therefore, in a second series of conjugation experiments, we altered the conjugation conditions and dissolved the TAD-pDMA first in DMSO or ACN and added it to a BSA solution in PBS in 28% v/v. As shown in Figure 1 and Table 1, these conditions yielded much higher conjugation efficiency up to 85%, with ACN outperforming DMSO as conjugation medium.

competitive hydrolysis of TAD groups occurs (Scheme S1).9c,12 For this, the hydrolytic stability of TAD was quantified in different aqueous solvent mixtures and was related to the associated tyrosine-TAD ligation efficiencies. Scheme 1. (a) General Synthesis of TAD-pDMA Using RAFT Chemistry; (b) Tyrosine Modification of BSA with TAD-pDMA

Initially, a poly(N,N-dimethylacrylamide) homopolymer (pDMA) containing a clickable TAD end group was synthesized using an optimized literature procedure.13 Starting from a urazole containing chain transfer agent (Scheme 1a, UrTTC), reversible addition−fragmentation chain transfer (RAFT) polymerization was used to synthesize well-defined pDMA with moderate molecular weight (DP30, Mn = 3.0 kDa, Đ = 1.2; Figure S1). Prior to oxidizing the urazole moiety into a TAD moiety, the pDMA was thoroughly dried over crushed molecular sieves in dry dichloromethane. Subsequent heterogeneous oxidation of the urazole end groups with a tetrameric complex of 1,4-diazabicyclo[2.2.2]octane and bromine (DABCO-Br) under inert atmosphere for 3 h afforded the formation of a reactive TAD end-functionalized pDMA. The generated TAD-pDMA was immediately purified by filtration of the molecular sieves and oxidant, yielding a clear red solution, characteristic for the TAD chromophore. This color change from yellow to red was a first visual indication for successful oxidation of the urazole polymer end groups. In order to quantify this oxidation more in detail, the reactive TAD end groups were trapped in an (ultra)fast Diels−Alder reaction (kp ∼ 200 mol·L−1 s−1) with a slight excess of E,E-2,4-hexadien-1-ol (HDEO).14 After purification, the modified HDEO-pDMA was analyzed with SEC, 1H NMR and MALDI-TOF. SEC analysis showed unimodal SEC traces with narrow dispersity for the oxidized polymer (Figure S1). Complete oxidation was confirmed by 1H NMR spectroscopy in DMSO-d6 (Figure S2), showing the disappearance of the distinctive urazole singlet at 10 ppm and the presence of new signals associated with the introduced HDEO end group (3−6 ppm) in the spectrum. Furthermore, MALDI-TOF analysis of the pDMA indicates quantitative oxidation. In the spectrum of the urazole-pDMA, the main distribution corresponds to polymer fragments ionized with one sodium, while the secondary distribution is attributed to single charged urazole-pDMA fragments with an additional sodium atom associated with a deprotonated urazole end group. After oxidation, the main distribution shifts to m/z values corresponding to HDEO-pDMA fragments, while the secondary distribution, typical for the acidic urazole moieties, disappeared completely (Figure S3). Additional evidence is provided by the similarity in the theoretical and experimental isotope distribution (Figure S4). This successful oxidation procedure was subsequently applied for the synthesis of TAD-pDMA (DP37, Mn = 4

Figure 1. SDS-PAGE analysis of BSA (lanes 1 and 2 (duplicate)) and BSA:TAD-pDMA conjugates in a 1:20 (triplicates in lanes 3−5) and 1:50 (triplicates in lanes 6−8) protein to polymer ratio. Lane 9 corresponds to TAD-pDMA in the absence of protein. Polymer conjugation was performed in three different solvent mixtures, as depicted in Table 1.

Table 1. Conjugation Efficiency of TAD-pDMA to BSA in a 1:20 and 1:50 Protein to Polymer Ratio in Different Solvent Mixtures (28% v/v)a protein/polymer ratio PBS PBS:DMSO PBS:ACN

1:20

1:50

38.1 ± 1.37% 22.9 ± 1.45% 65.9 ± 1.45%

32.7 ± 0.66% 68.7 ± 0.55% 84.8 ± 0.59%

a

Calculation is based on optical density integration of SDS-PAGE analysis (n = 3).

To investigate whether these observations can be linked to the hydrolytic stability of TAD in aqueous mixtures of DMSO and ACN we measured the hydrolysis kinetics of butyl-TAD as a small molecule model TAD compound. The visual color change from red to yellow allows for straightforward monitoring of the reaction kinetics by UV−vis spectroscopy by measuring the decrease of the characteristic TAD absorbance at 540 nm (Figure S6). Hydrolysis experiments 1369

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ACS Macro Letters were conducted in 1:1, 3:7 and 1:9 mixtures of H2O/DMSO, respectively, H2O:ACN. Kinetic plots, depicted in Figure 2, clearly demonstrate that increased concentrations of water strongly accelerate TAD hydrolysis. Interestingly, TAD hydrolysis in H2O:ACN mixtures is much slower than in H2O:DMSO mixtures (Table S1), which corresponds to the protein-conjugation data that also shed higher polymer− protein conjugation efficiency in PBS:ACN mixtures. This difference can possibly be explained by different solvation mechanisms for both mixtures. We suggest that in H2O:DMSO, there is a fast exchange of the hydrogen bonds between both molecules, while acetonitrile has the tendency to form ACN clusters in water.15 Hence, in a H2O:ACN solution, the TAD groups are shielded from the water in a way, resulting in slower hydrolysis. Comparable results were obtained for the hydrolysis kinetics of TAD-pDMA (Figure S7).

Figure 3. MALDI-TOF spectrum of peptide fragments after trypsin digestion of the with butyl-TAD modified BSA.

acids exist in their protonated unreactive form at the pH of the phosphate-buffered saline solution. To provide additional evidence for the site-selectivity of the tyrosine-TAD reaction, tandem mass spectroscopy (MS/MS) was performed on the predetermined modified peptide fragments (P1*−P4*) and the produced ions were compared to the associated unmodified ion fragments. Using this method, the precise sequence position on which tyrosine modification occurs can readily be identified. The MS/MS spectrum of peptide P1 (YLYEIAR) and peptide P3 (LGEYGFQNALIVR) before and after modification is given in Figure 4. In peptide P1, both tyrosine positions can be modified by TAD, as evidenced by the evolution of the b and y ion series in the MS/MS spectrum of the modified sample (Table S3). For instance, the mass of all the b ions is increased with 155.16 Da, for example, b3 with a typical mass of 440.22 Da shifts to b′3 with a mass of 595.38 Da. When the y ions are considered, it is clear that the TAD modification can occur on both tyrosine residues. The presence of ion y5, y6 and y′7 in the spectrum confirms the tyrosine modification on position 161, while the presence of y′6, y# and y′# indicates modification of the tyrosine on position 163. Most probably, the y# fragments originate from a scission reaction of the tyrosine residue next to the amino terminus of the peptide fragment during analysis. In the same way, the MS/MS spectrum for the modified P3 fragment is characterized by modified b ions (e.g., b′10) and y ions, that is, the peak for y10 is shifted upward to y′10, clearly indicating the selective reaction with the tyrosine moiety. To investigate the influence of the conjugation conditions and the modification of tyrosine amino acids with butyl-TAD on the protein structure and its bioactivity, UV−vis and circular dichroism (CD) spectroscopy have been performed on native BSA, BSA exposed to 30% v/v ACN:H2O, and on butyl-TAD modified BSA (Figure S9). From the close resemblance of the spectra it can be concluded that the solution structure of BSA is not affected by the presence of the acetonitrile cosolvent. Moreover, the modification of BSA with butyl-TAD has no significant effect on the α-helical secondary structure of BSA, as indicated by the two minima at 208 and 222 nm. Besides the determination of the secondary structure, the effect of BSA modification with butyl-TAD on its ability to exert enzymatic activity was examined by an esterase activity assay utilizing p-nitrophenyl acetate as substrate.16 In this experiment, the formation of p-nitrophenol is followed over time by UV−vis spectroscopy at 37 °C. As indicated by Figure

Figure 2. Hydrolysis kinetics of butyl-TAD in 1:1, 3:7, 1:9 mixtures of H2O:DMSO and H2O:ACN measured by UV−vis spectroscopy (lines are drawn as guide to the eye).

From the SDS-PAGE data, it is clear that triazolinedione end functionalized polymer can be covalently attached to the proteins to a certain extent, with the conjugation efficiency strongly dependent on the TAD hydrolysis kinetics. To certify whether the triazolinedione is orthogonally reacting in an electrophilic aromatic substitution (SEAr) with the phenol functionality of tyrosine residues, BSA was reacted with butylTAD (20 equiv) as model reagent in a PBS:ACN mixture (0.28:0.72) and subsequently analyzed with mass spectroscopy. The modified BSA was subjected to a trypsin digestion wherein the mass (m/z) of the cleaved peptide fragments originating from the modified protein is compared to the m/z of the native unmodified peptide fragments (Figures 3 and S8). When comparing both mass spectra, it is clear that additional peaks appear in the spectrum of the modified protein (P1*− P4*). These intrinsic peaks are defined by a mass increase of 155.16 Da, being the molecular mass of butyl-TAD, compared to distinctive unmodified tyrosine containing peptide fragments (P1−P4), showing successful coupling of butyl-TAD to BSA. From this model experiment, it can be concluded that, under the applied reaction conditions, butyl-TAD orthogonally reacts with tyrosine residues. Indeed, only peptides containing tyrosine amino acids are shifted upward in the mass spectrum (Table S2). As expected, peptides with TAD-reactive lysine residues were not altered during modification since these amino 1370

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Figure 4. MS/MS spectra of peptide P1 (YLYEIAR) (left) and peptide P3 (LGEYGFQNALIVR) (right) before (bottom) and after (top) butylTAD modification.

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S10, the esterase activity of BSA is not influenced by the presence of ACN (i.e., 30% v/v ACN:PBS for 30 min) during protein modification. However, despite the fact that the secondary structure of the BSA is not affected by the modified tyrosine residues, the butyl-TAD modification does influence the esterase activity of BSA. Butyl-TAD modification does not render the protein inactive, but causes a decreased esterase activity of 31.5 ± 3.8% relative to native BSA (Figure S10). In summary, the polymer−protein conjugation using the efficient SEAr reaction between a TAD end-functionalized polymer, synthesized by RAFT, and tyrosine units of a BSA protein was evaluated. First, the procedure to introduce triazolinedione end groups in polyacrylamides was successfully optimized. Then, BSA was successfully and site-selectively modified at the tyrosine residues with this polymer, as evidenced by SDS-PAGE analysis and tandem mass spectroscopy. However, when performing conjugation in aqueous environments, one has to cope with hydrolysis of the triazolinedione moiety as an undesirable side-reaction. Significant reduction of this unfavorable side-reaction can be obtained by altering the combination of solvents, to ensure high coupling efficiencies. While the modification of the tyrosine amino acids with butyl-TAD does not affect the α-helical secondary structure of BSA, it causes a decrease in enzymatic activity of the protein, but does not render the protein inactive. Since standard RAFT conditions are used to prepare these polymer-BSA conjugates, we believe that this method could easily be expanded in the future for the synthesis and coupling of alternative TAD functionalized hydrophilic polymers starting from functional monomers.

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Bruno G. De Geest: 0000-0001-9826-6170 Filip E. Du Prez: 0000-0001-7727-4155 Author Contributions ‡

These authors equally contributed to the work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Isabel Vandenberghe of the laboratory for Protein Biochemistry and Biomolecular Engineering (L-ProBE) guided by Prof. Dr. B. Devreese is acknowledged for the trypsin digestion and the tandem mass spectroscopy of the BSA proteins. Carl Mensch and his promotors Prof. Dr. Christian Johannessen (University of Antwerp) and Prof. Dr. Patrick Bultinck (Ghent University) are acknowledged for the circular dichroism (CD) measurements. Bernhard De Meyer is acknowledged for his support during the GPC measurements. Frank Driessen is acknowledged for his help with the optimization of the urazole oxidation. F.D.P. and B.D.G. thank Ghent University for BOF funding (GOA-project).

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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.7b00795. Experimental procedures; synthesis of Ur-TTC RAFT CTA, polymers, procedure for BSA modification; NMR, UV−vis, circular dichroism (CD), MALDI-TOF, SEC data and tables with masses of peptide fragments (PDF).

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