Arginine-Specific Modification of Proteins with Polyethylene Glycol

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Biomacromolecules 2011, 12, 482–493

Arginine-Specific Modification of Proteins with Polyethylene Glycol Marc A. Gauthier† and Harm-Anton Klok* E´cole Polytechnique Fe´de´rale de Lausanne (EPFL), Institut des Mate´riaux and Institut des Sciences et Inge´nierie Chimiques, Laboratoire des Polyme`res, Baˆtiment MXD, Station 12, CH-1015 Lausanne, Switzerland Received October 24, 2010; Revised Manuscript Received November 30, 2010

In this study, the residue-selective modification of proteins with polymers at arginine residues is reported. The difficulty in modifying arginine residues lies in the fact that they are less reactive than lysine residues. Consequently, typical chemo-selective reactions which employ “kinetic” selectivity (active esters, Michael addition, etc.) cannot be used to target these residues. The chemistry exploited herein relies on “thermodynamic” selectivity to achieve selective modification of arginine residues. ω-Methoxy poly(ethylene glycol) bearing an R-oxo-aldehyde group was synthesized and used to demonstrate the selective modification of lysozyme at arginine residues. In addition, the optimization of reaction conditions for coupling as well as the stability of the formed adduct toward dilution, toward a nucleophilic buffer, and toward acidification are reported. It was concluded that this approach is a convenient, mild, selective, and catalyst-free method for protein modification.

Introduction Conjugation of a peptide/protein to a synthetic polymer is an elegant solution to current challenges in nanotechnology, biotechnology, and materials science.1-6 These hybrid systems can be very complex and difficult to prepare due to the high level of functionality and structural complexity of the protein. This becomes all the more true when advanced or specialized properties such as therapeutic or catalytic activity,7,8 specific (bio)recognition,9,10 and so on, are sought after, which require the modification of the protein at a specific site and with a precisely defined number of synthetic polymer chains. Several approaches are available for preparing well-defined conjugates of proteins and synthetic polymers.5,6,11-21 Among these, many residue-specific chemical reactions have been developed for the purpose of controlled protein modification. The attractiveness and popularity of this approach lies in its simplicity as it makes use of the protein’s/peptide’s intrinsic reactivity and does not require postmodification or the introduction of non-natural amino acid residues. A challenge when utilizing residue-specific peptide/protein modification reactions is to control the exact site of the modification because most proteins and peptides contain more than one copy of the different amino acids of which they are constituted. Knowledge of the protein’s primary and tertiary structure, however, may be used to guide the modification at the desired site and control the degree of modification. Different amino acids have different natural abundances and targeting one of the less abundant ones provides a method to control the degree of modification. Furthermore, as different amino acids have different propensities to be located in different sections of the peptide/protein, information about the peptide/protein structure can be used to guide the site of modification.22,23 * To whom correspondence should be addressed. E-mail: [email protected]. † Current address: Swiss Federal Institute of Technology Zu¨rich (ETHZ), Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, Drug Formulation and Delivery, Wolfgang-Pauli Str. 10, HCl J 396.4, 8093 Zu¨rich, Switzerland.

Residue-specific peptide/protein modification requires reaction conditions that allow to selectively modify the amino acid of interest, ideally in aqueous media, at pH 7, and room temperature, and which are not influenced by or lead to cross-reactivity with other amino acids. While for several amino acids, including, for example, lysine, cysteine, and glutamine, reaction conditions are available for residue-specific modification,11 a further expansion of this toolbox with methods that allow the selective modification of additional amino acids would be attractive and increase the possibilities to tailor the structure and properties of peptide/protein-synthetic polymer conjugates. In this contribution, a new, biologically inspired strategy that allows the residue-specific modification of arginine residues with synthetic polymers is described. As for lysine (5.91% of all residues in proteins), arginine is a relatively abundant residue in proteins (5.44%), though has a lower tendency to be located on the surface of the protein.11 Consequently, exploiting the surface accessibility of this residue would be advantageous for a more selective modification of proteins with ω-methoxy poly(ethylene glycol) (mPEG) in comparison to that achieved by modification of lysine residues. To the best of our knowledge, however, methods that allow a residue-specific conjugation of synthetic polymers to arginine residues have not been reported. The challenge in modifying these residues comes from their lower nucleophilicity than lysine residues. Consequently, typical chemo-selective reactions that employ “kinetic” selectivity (such as active esters, Michael addition, etc.) cannot be used to target these residues. The conjugation strategy reported here is inspired by the nonenzymatic glycation of proteins and relies on “thermodynamic” selectivity for achieving selective modification of arginine residues. This process, which is part of the Maillard reaction, involves the reaction between amino groups of a protein and reducing sugars or other endogeneous aldehydes or ketones to form so-called advanced glycation end-products.24 A particularly interesting glycating agent is the R-oxo-aldehyde methylglyoxal, which has a high tendency to preferentially modify arginine residues (slowly but permanently) in favor of lysine or cysteine (both rapidly, but reversibly).25 To date, smallmolecule reagents bearing 1,2-dicarbonyl or R-oxo-aldehyde

10.1021/bm101272g  2011 American Chemical Society Published on Web 12/23/2010

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groups have been used to study the three-dimensional structure,26 the function,27,28 the local polarity,29 and the susceptibility of proteins to be damaged by glycation in patients with diabetes.28,30 These reagents have also been used to restrict tryptic digestion to lysine residues.31 In this study, the feasibility of using R-oxo-aldehyde functional synthetic polymers for the arginine-specific modification of proteins is explored. First, conditions were established under which the guanidine side-chain of NR-acetyl arginine is modified rapidly with methylglyoxal, used as a model reagent. The stability/reversibility of ligated products was investigated toward hydrolysis, dilution, and nucleophilic displacement. Subsequently, the synthesis of mPEG bearing on one extremity a functional group structurally analogous to methylglyoxal is described, and the reaction of the latter with a model protein is characterized in detail, and the stability of the resulting conjugates examined.

Experimental Section Materials. NR-Acetyl arginine, NR-acetyl lysine, Nε-acetyl lysine, NR-acetyl cysteine, ω-methoxy-poly(ethylene glycol) (mPEG; 5.000 g · mol-1), lithium diisopropylamide (LDA; 1.8 M in tetrahydrofuran/ heptane/ethylbenzene), cyclohexylamine, calcium chloride, N-bromosuccinimide, triphenylphosphine (3 mmol · g-1 on polystyrene support), chicken egg white lysozyme (HEWL), sodium hydroxide, fuming hydrochloric acid, methylglyoxal 1,1-dimethyl acetal, Amberlite IR120 (H+ form), hydroxylammonium choride, sodium tetraborate, disodium hydrogen phosphate, 9,10-phenanthrenequinone (PQ), trifluoroacetic acid (TFA, HPLC grade), trifluoroacetic anhydride (TFAA) and heptafluorobutyric acid (HFBA, HPLC grade) were obtained from Aldrich (Buchs, Switzerland) and used as received. All solvents were from Aldrich and used as received, except for tetrahydrofuran (THF) (inhibitor free), which was dried through alumina using a solvent purification system. Methods. 1H and 13C NMR spectra were recorded using a Bruker AVANCE-400 spectrometer operating at 400 MHz for protons and chemical shifts referenced to the appropriate deuterated solvent. Matrixassisted laser desorption/ionization time-of-flight mass spectra (MALDITOF MS) were obtained with an Axima-CFRTM plus mass spectrometer using dithranol saturated with sodium trifluoroacetate as matrix for mPEG samples and either sinapic acid or R-cyano-4-hydroxycinnamic acid for HEWL and HEWL-mPEG conjugates. Size-exclusion chromatography in N,N-dimethylformamide (DMF) was performed on a Waters Alliance GPCV 2000 system equipped with refractive index and differential viscometer detectors. Separation was carried out at 60 °C with TSK-Gel Alpha 2500 + 3000 + 4000 columns using DMF + 0.5 g · L-1 LiCl as eluent and a flow rate of 0.6 mL · min-1. Molecular weights were determined relative to narrow polydispersity poly(methyl methacrylate) standards. Aqueous size-exclusion chromatography was performed using a Viscotek model TDA 300 system equipped with Shodex OHpak SB-803 HQ and SB-804 HQ columns as well as a HB-G guard column. The eluant was water +0.1 M NaHCO3 at a flow rate of 0.5 mL · min-1 at 25 °C. Molecular weights are given relative to narrow polydispersity PEG/PEO standards. Analytical high-pressure liquid chromatography (HPLC) was performed using a Jasco PU-2089 quaternary gradient pump, an AS-2055 autosampler, a CO-2060 column oven (set to 30 °C), and a UV-2075 intelligent UV-vis detector set to 214 nm. Liquid chromatography coupled to mass spectrometry (LCMS) was performed on a Waters AutoPurification system equipped with a 2525 binary gradient module, a column fluidics organizer, a 2767 sample manager, a 2996 PDA detector, and a Waters single quadrupole ZQ mass detector (scanning window m/z 100-1000 in 2 s). Preparative high-pressure liquid chromatography (Prep-HPLC) was performed using a Waters 600 automated gradient controller pump module connected to a Waters preparative degasser system. The elution of the analytes was monitored by using a Waters 2487 dual λ absorbance detector (set

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to 214 and 280 nm) and collected using a Waters fraction collector III. For all analytical chromatographic instruments, a Vydac 219TP54 C18 diphenyl column (4.6 × 250 mm) or a Vydac 238DE5415 monomeric C18 column (4.6 × 150 mm) were used for proteins and amino acid analysis, respectively. An Atlantis dC18 OBD 5 µm reverse phase column (Waters; 30 × 150 mm) was used for Prep-HPLC. Solvents (methanol, acetonitrile, or ultrapure H2O) contained either 15 mM TFA or 15 mM HFBA. Elution conditions are given in the appropriate sections. Procedures. R-Bromo, ω-Methoxy-poly(ethylene glycol) (2). NBromosuccinimide (2.82 g, 15.8 mmol) and PPh3 (5.28 g, 3 mmol · g-1 on polystyrene support, 15.84 mmol) were added to a 250 mL Erlenmeyer flask, which was then sealed with a rubber septum and the atmosphere replaced with N2 by a vacuum-refill cycle. The flask was cooled to -78 °C in a dry ice/acetone bath and then 120 mL of dry dichloromethane (DCM) added. The flask was agitated on an orbital agitator and the dry ice replaced regularly as required. After 6 h, mPEG (1, 3.96 g, 0.79 mmol) in 20 mL of dry DCM was added via syringe. The reaction was left to warm slowly and left under agitation for 48 h. After this period, the supernatant of the biphasic reaction mixture was recovered by centrifugation at 4 °C, then filtered to remove residual traces of polymer resin. DCM was evaporated at room temperature under vacuum on a rotary evaporator to ∼7 mL, then 2 crystallized by addition of 45 mL of acetone and cooling to -30 °C. The solid was recovered by centrifugation, redissolved in ∼5 mL DCM and crystallized in the same manner. After recovery by centrifugation, the solid was rinsed with diethyl ether, and then dried to yield 3.33 g (84% yield; 100% conversion) of a white powder. 2 was stored under argon at -30 °C until used. 1H NMR and MALDI TOF mass spectra are shown in Figure 1. Methylglyoxal 1,1-Dimethyl Acetal Cyclohexylimine. A modified protocol from Pichon et al.32 was used. Methylglyoxal 1,1-dimethyl acetal (10, 10 g, 85 mmol) was added under inert atmosphere to a flamedried 100 mL round-bottom flask containing 50 mL of anhydrous diethyl ether, cyclohexylamine (9.23 g, 93 mmol), and granular calcium chloride (0.5 g, 4.5 mmol). The contents of the flask were stirred overnight at 45 °C and then the solvent and residual methylglyoxal 1,1-dimethyl acetal removed at 70 °C in vacuo (8 mbar) on a rotary evaporator. Methylglyoxal 1,1-dimethyl acetal cyclohexylimine was isolated from the residue by distillation as a single fraction (120-125 °C/8 mbar) to yield 13.31 g (79%) of the imine as a colorless liquid, which was stored frozen as a solid at -30 °C until used. 1H and 13C NMR spectra are supplied in Figure S1 in the Supporting Information. R-(Methylglyoxal 1,1-dimethyl acetal), ω-Methoxy-poly(ethylene glycol) (3). 3 was prepared following a modified protocol from Pichon et al.32 For alkylation of 2, LDA (8.37 mL of a 1.8 M solution, 15 mmol) and 40 mL of anhydrous THF were combined in a sealed, flamedried 100 mL round-bottom flask equipped with a magnetic stirrer. The solution was left at room temperature for 30 min and then methylglyoxal 1,1-dimethyl acetal cyclohexylimine (2.73 g, 13.7 mmol), dissolved in 5 mL of anhydrous THF, was added dropwise via syringe over 10 min. The solution was left for 5 h and then 2 (1.37 g, 0.274 mmol) dissolved in 10 mL THF was added. The solution was left for 60 h after which the reaction mixture was concentrated in vacuo and the mPEG derivative precipitated in diethyl ether (repeated twice). The precipitate was collected by centrifugation and further purified by sizeexclusion chromatography (deionized water) using a Sephadex G25 column, which concurrently hydrolyzed the imine to yield 1.1 g (80% recovered yield) of a light-yellow fluffy solid after freeze-drying (3). Assigned 1H NMR and MALDI TOF mass spectra can be found in Figure 1. R-Methylglyoxal, ω-Methoxy-poly(ethylene glycol) (6-8). Several approaches were investigated. Deprotection of the acetal end group on 3 by strong-acid ion-exchange resin-catalyzed hydrolysis was accomplished by dissolving 20.9 mg of 3 (4.18 µmol) in 2 mL of H2O containing either 20.9 mg (46 µmol) or 209 mg (460 µmol) Amberlite IR-120 strong-acid resin. The tube was sealed with a rubber septum and heated at 100 °C overnight (>18 h) in complete darkness. The

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Figure 1. End-group analysis of polymers shown in Scheme 1. Full (a) and zoomed (b) MALDI-TOF mass spectra of reaction products 1, 2, 3-5, and 6-9 purified by size-exclusion chromatography and isolated by freeze-drying. (c) 1H NMR spectra of purified reaction mixtures with and without in-tube esterification with TFAA (spectra in CDCl3 except for 6-9, which was in D2O). All peak assignments given in Scheme 1. Note: (b) annotated peaks correspond to m/z (M + Na+, n ) 103 according to Scheme 1): 4591.7 (1), 4655.6 (2), 4691.8 (3), 4573.7 (4), 4591.7 (5), 4645.7 (6), 4663.7 (7), 4681.7 (8), and 4547.7 (9). Scheme 1. Synthesis of R-oxo-aldehyde Functional mPEGa

a Reaction conditions: (i) N-bromosuccinimide, PPh3, DCM; (ii) methylglyoxal 1,1-dimethyl acetal cyclohexylimine, LDA, THF, aqueous workup; (iii) Amberlite IR-120 (H+ form), H2O, 100 °C; 10 vol % TFAA added directly to NMR tube containing ∼10 mg · mL-1 sample in CDCl3. Undesired side products identified with (*).

resulting orange solution was either diluted by a factor of 2 with D2O for NMR analysis or purified by size-exclusion chromatography

(Sephadex G25, deionized water) and isolated by freeze-drying. The resulting off-white powder was then analyzed by NMR spectroscopy,

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Scheme 2. Acetal Deprotection of Methylglyoxal 1,1-Dimethylacetala

a

Three deprotection reactions examined: (i) 10 vol % H2SO4, 100 °C, 25 min; (ii) Amberlite IR-120, H2O, 100 °C, 3 h; (iii) I2, acetone, 90 °C, 1 h.

size-exclusion chromatography, and MALDI-TOF mass spectrometry. Deprotection of the acetal end group on 3 by transacetalization was accomplished by dissolution of 10 mg of 3 (2 µmol) in 0.65 mL of acetone (10.2 mmol) containing 1.45 mg of I2 (5.7 µmol). As above, the tube was sealed and shielded from light and stirred at 90 °C for 1 h after which time an aliquot, diluted with an equal volume of acetoned6, was analyzed by NMR spectroscopy. The remainder of the dark solution was diluted with 2 mL water, the acetone removed in vacuo, and the polymer purified, isolated, and characterized as described above. The recovered mass of polymer following both deprotection procedures was ∼80%. Assigned 1H NMR and MALDI TOF mass spectra can be found in Figure 1. EValuation of Deprotection Conditions for Methylglyoxal 1,1-Dimethylacetal. A total of 20 µL (165 µmol) of methylglyoxal 1,1-dimethylacetal (10) was deprotected to methylglyoxal either by acid hydrolysis catalyzed by sulfuric acid (1 mL of 10 vol% H2SO4 in D2O, 1.88 mmol, 100 °C, 25 min), by acid hydrolysis catalyzed by a strong acid ionexchange resin (0.5 g (1.1 mmol) Amberlite IR-120 in 3 mL D2O, 100 °C, 3 h) or by I2-catalyzed transacetalization (either 4.1 mg (16.5 µmol) or 125 mg (496 µmol) I2 in 1 mL of acetone-d6, 90 °C, 1 h). All reactions were conducted in NMR tubes and reaction products (12-14) directly analyzed by 1H NMR spectroscopy. Model Reactions of Methylglyoxal with N-Acetyl Amino Acids. Methylglyoxal 1,1-dimethylacetal (10, 0.5 mL, 4.13 mmol) was hydrolyzed to methylglyoxal in a glass tube sealed with a septum and containing 3 mL of H2O and 0.5 g (1.1 mmol) Amberlite IR-120 (H+ form) resin. This solution was stirred in the dark at 100 °C for 3 h, cooled to room temperature, and then transferred quantitatively to a 10 mL volumetric flask containing 0.096 g Na2B4O7 · 10H2O. The pH of the solution was adjusted to 9 by incremental addition of saturated NaOH. The quantity of methylglyoxal in the sample was determined from the input of methylglyoxal 1,1-dimethylacetal (10) and from the ratio of the peaks for the methyl group of mono- or dihydrate species (13 and 14 in Scheme 2, respectively) relative to oligomer peaks as determined by 1H NMR spectroscopy. A total of 3 mL of this solution (1.24 mmol) was added to a 10 mL volumetric flask containing 0.25 mmol of the appropriate N-acetyl amino acid and the level of liquid adjusted to 10 mL with 100 mM sodium borate pH 9.0 (0.48 g of Na2B4O7 · 10H2O in 50 mL of H2O). The final concentrations of methylglyoxal and N-acetyl amino acid were 125 and 25 mM, respectively. The pH of the test solution was adjusted by incremental addition of either saturated aqueous NaOH or fuming HCl. Aliquots were taken at different time intervals and analyzed by HPLC (isocratic elution; 100% H2O + TFA (95/5% H2O/MeOH + HFBA for NR-acetyl arginine pH 7.4); 1 mL · min-1; detection at 214 nm; 20 µL injection volume). As the pH of the solution was observed to decrease with time, the latter was adjusted between each sampling as described above until no further evolution was observed (approximately 3 h). The progress of the reactions was monitored through the decrease of the area of the peak corresponding to the N-acetyl amino acid. Four acetyl-amino acids were examined: NR-acetyl arginine, NR-acetyl lysine, Nε-acetyl lysine, and NR-acetyl cysteine. An identical set of experiments was performed at pH 7.4 by replacing the buffer system from 100 mM sodium borate to 100 mM sodium phosphate. After 24 h, the identity of species present within the reaction mixture was evaluated by LC-MS. Stability of Methylglyoxal-N-acetyl Amino Acid Adducts. After incubation for specified times, the stability of the reaction products formed above was evaluated. The effects of acidification/basification, addition of hydroxylamine as nucleophilic buffer, and dilution were

investigated. For pH-dependent stability, 1 mL aliquots of the solutions described above (at 24 h) were transferred to 5 mL volumetric flasks, which were then completed with water and the pH adjusted to 3, 5, 7, 9, or 12. These solutions were analyzed by HPLC as described above (to compensate for dilution, 100 µL were injected rather than 20 µL). Stability toward nucleophiles was evaluated by transferring 1 mL aliquots of the reaction mixture at 24 h to 5 mL volumetric flasks containing hydroxylammonium chloride (to a final concentration of 100 mM) and the pH of the solutions adjusted to 7. These solutions were analyzed by HPLC as described above (to compensate for dilution, 100 µL were injected rather than 20 µL). Finally, dilution-dependent stability was tested by removing buffer salts and excess methylglyoxal from the reaction mixtures (24 h) by prep-HPLC (Figure S18, Supporting Information). Fractions containing methylglyoxal-amino acid adducts were pooled together, freeze-dried, resuspended in 10 mL H2O (i.e., 5-fold dilution relative to initial solution), the pH adjusted to 7, and analyzed by HPLC with time (to compensate for dilution, 100 µL were injected rather than 20 µL). Modification of Lysozyme with R-Oxo-aldehyde mPEG. Compounds 3-5 (7.7 mg, 1.54 µmol) were hydrolyzed to 6-9 with Amberlite IR120 resin (77 mg, 169 µmol) in 1.5 mL of H2O at 100 °C for 18 h in the dark. The solution was transferred quantitatively to a 2 mL volumetric flask containing 19 mg Na2B4O7 · 10H2O, the volume completed with H2O and the pH adjusted to 9. HEWL (1.56 mg, 0.11 µmol) was then added and the solution analyzed by HPLC (gradient from 7-95% acetonitrile in 110 min, 1 mL · min-1, 40 µL injection). In this reaction mixture, the mPEG reagent is in 14-fold molar excess relative to HEWL and 1.25-fold excess relative to arginine residues on HEWL. This reaction was repeated under the same conditions but with 17.5 mg (3.5 µmol) 3-5 and 1 mg of HEWL (i.e., 50-fold excess relative to HEWL, 4.5-fold excess relative to arginine residues on HEWL). In addition, HPLC elution conditions were changed to a gradient from 30-95% acetonitrile over 40 min. This last experiment was also performed at pH 7.4 in 100 mM sodium phosphate buffer rather than the borate buffer above. Identification of Lysozyme-mPEG Conjugates. For identification purposes, the reaction above (pH 9) was performed on a larger scale to isolate HEWL-polymer conjugates by Prep-HPLC. Compounds 3-5 (175 mg, 35 µmol) was hydrolyzed to 6-9 with Amberlite IR-120 resin (1.75 g, 3.85 mmol) in 15 mL of H2O at 100 °C for 18 h in the dark. The solution was transferred quantitatively to a 20 mL volumetric flask containing 190 mg Na2B4O7 · 10H2O, the volume completed with H2O, and the pH was adjusted to 9. HEWL (10 mg, 0.68 µmol) was then added, and analytical chromatograms were recorded to follow the reaction. After 18 h, this solution was purified by Prep-HPLC and the fractions were collected (chromatograms available in Supporting Information), freeze-dried, and either reconstituted in carbonate buffer for aqueous size-exclusion chromatography or in Laemli buffer (heated for 5 min at 100 °C) for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using a 14% acrylamide gel. The gels were developed with Coumassie Blue staining to reveal proteincontaining bands. Stability of Lysozyme-mPEG Conjugates. The stability of the conjugates formed after 24 h was evaluated by diluting the solution 2-fold with 200 mM NH2OH and adjusting the pH to 7. Chromatograms recorded over a 24 h period (80 µL injected rather than 40 µL to compensate for dilution). Following this period, the solution was acidified to pH 3 with fuming HCl and a final chromatogram recorded.

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Residual Amino Groups. Compounds 6-9 were reacted with HEWL at either pH 9 or 7.4 as above. After specified times (below), 100 µL of this solution was transferred to two wells of a 96-well microplate. To one well, 50 µL of 0.01% TNBS in 1 M NaHCO3 (pH 8.5) was added while to the second only the buffer was added. This was done to account for the non-negligible absorbance of the reaction mixture at the wavelength of interest. HEWL incubated in the same conditions as above but without 6-9 was also transferred to two wells of the microplate and treated as above. After incubation for 2 h at room temperature, 50 µL of 10% SDS in water was added to each well followed by 25 µL of 1 N HCl. The absorbance of the four cells was measured at 335 nm and the % of residual amino groups given by 100 × (A335(HEWL+6-9+TNBS) - A335(HEWL+6-9))/(A335(HEWL+TNBS) - A335(HEWL)). Residual Arginine Residues. Compounds 6-9 were reacted with HEWL at pH 9 as above. After a two week period, 10 µL of this solution was diluted to 50 µL with water in an Eppendorf vial, then 150 µL of 9,10-phenanthrenequinone (PQ; 150 µM in ethanol) and 25 µL of 2 N NaOH were added. Samples containing either water or HEWL without 6-9 were used as controls. Additional controls containing the analyte solution without PQ (i.e., only an equivalent amount of ethanol added) were also prepared. The solutions were kept in the dark at 30 °C for 18 h and then diluted with an equal volume of 1.2 N HCl in a 96-well quartz microplate. The plate was left for 1 h in the dark at room temperature, and then the fluorescence intensity was measured (excitation: 312 nm; emission: 395 nm). Arginine solutions with concentrations of 0.4, 0.2, 0.1, 0.04, 0.02, and 0.01 mM (50 µL of each) were used to test linearity of fluorescence. The % of residual arginine residues is given by 100 × (Fl395(HEWL+6-9+PQ) - Fl395(HEWL+6-9) - Fl395(PQ))/(Fl395(HEWL+PQ) - Fl395(HEWL) - Fl395(PQ)).

Results and Discussion In this section, the synthesis of a protected mPEG reagent and its deprotection to reveal the R-oxo-aldehyde functional group is discussed. This will be followed by the evaluation of the stability of conjugates formed between methylglyoxal and N-acetyl amino acids, the modification of HEWL with the reactive mPEG, and the characterization of the resulting conjugates. Synthesis of r-(Methylglyoxal 1,1-dimethyl acetal), ω-Methoxy-poly(ethylene glycol) (3). The synthesis of the desired R-oxoaldehyde mPEG derivative is outlined in Scheme 1. While R,βdicarbonyl compounds can be prepared in a one-step reaction from, for example, 1,2-diols,33 epoxides,34 and other functional groups,35-37 the strategy proposed here involves alkylation of R-methoxy, ω-bromo mPEG with methylglyoxal 1,1-dimethylacetal. This results in an acetal protected derivative (3) that can be deprotected to afford the desired R-oxo-aldehyde mPEG in a final step, just prior to the conjugation reaction. Compared to the one-step reactions mentioned above, the strategy presented herein is advantageous as the highly reactive dicarbonyl group is only exposed in the final deprotection step, thereby improving functional group compatibility of the construct, increasing shelf life and making handling of the intermediates less complicated. In addition, none of the reactions mentioned above can be used for in situ generation of the R-oxo-aldehyde group (i.e., in the presence of the protein) and therefore do not present any net advantage in terms of number of reaction steps compared to the approach presented in this contribution. The synthesis of the desired R-oxo-aldehyde mPEG starts with bromination of mPEG (1) with N-bromosuccinimide and solid-supported PPh3, which proceeded to quantitative conversion to the desired halide as verified by end-group analysis of the MALDI-TOF mass spectra of 2 (Figure 1a and b). The absence of residual hydroxyl end groups was verified by 1H NMR spectroscopy following addition of 10 vol % trifluoro-

Gauthier and Klok

acetic anhydride to 2 in CDCl3 (Figure 1c). As can be seen for the equivalent experiment performed on mPEG (1), residual hydroxyl groups become esterified by this reaction, which results in the appearance of a triplet at 4.45 ppm. The absence of this signal for 2 indicates that no hydroxyl groups remain following halogenation. The use of non-solid-supported PPh3 rendered purification of 2 tedious and resulted in lower recovered yields. Drying of 2 following synthesis was accomplished in vacuo, but without heating. Heating, even to a small extent (ca. >40 °C), resulted in yellowing of the sample, possibly due to dehalogenation. Next, the brominated mPEG derivative 2 was alkylated with methylglyoxal 1,1-dimethylacetal cyclohexylimine using LDA. For this reaction, the lithium enamide of methylglyoxal 1,1dimethyl acetal was used because of self-condensation side reactions, which occur when the corresponding lithium enolate is used.32 In addition, the alkylation of isopropyl and cyclohexyl iminoacetals of methylglyoxal 1,1-dimethyl acetal mediated by lithium diisopropylamide has previously been shown to proceed to high conversion in the presence of either stoichiometric amounts or excess halide.32,38,39 After about 48 h, no further evolution of the degree of conversion to the alkylated product 3 was observed. Typical conversion to the desired end group was in the 40-50% range as determined by integrating peaks d, e, f, and g relative to the internal standard b (ω-methoxy group on the polymer; Figure 1c). The conversion value given above is given as a range calculated from the most intense and least intense of these peaks. It was noticed that prolonged storage of 3 in D2O resulted in the total disappearance of peaks d, e, and f over a period of several hours due to H-D exchange with the solvent due to their relative acidity; e was particularly affected, possibly due to enolization. In general, conversion to the desired compound 3 was always around 40%. The incomplete conversion to the alkylated product 3 is the result of both competitive hydrolysis and 1,2-dehydrobromination reactions of 2, both of these side reactions being directly evidenced by MALDI-TOF mass spectrometry (peaks 4 and 5 in Figure 1b). 1 H NMR spectroscopy of the reaction mixture in CDCl3 revealed ∼23% 1,2-dehydrobromination product (4), as determined by integrating peaks h and i relative to b. Upon addition of 10 vol % trifluoroacetic anhydride to the NMR tube, peaks h and i disappear, owing to acid-mediated deprotection of the vinyl ether end group concurrent with the appearance of peak c, accounting for 60% of end groups. Thus, the hydrolysis byproduct accounts for about 27-37% of the reaction mixture prior to trifluoroacetic anhydride modification. To improve the conversion of 2 to 3, it was attempted to eliminate hydrolysis side reactions by extensively drying 2 prior to reaction. Various chemical and physical drying methods were employed, however, these often resulted in dehalogenation (yellowing of 2) and lower conversion to 3. As another alternative, it was rationalized that by decreasing the reactivity of the halogen leaving group, a more aggressive drying protocol could be employed without dehalogenation. In addition, it was anticipated that 1,2-dehydrohalogenation induced by the sterically hindered lithium enamide base would also be reduced by this modification. To this effect, R-chloro, ω-methoxy poly(ethylene glycol) was prepared (Supporting Information) by chlorination of 1 with thionyl chloride and pyridine, as described by Zalipsky.40 The improved stability of this end group permitted azeotropic drying with toluene followed by vacuum drying in the molten state (>60 °C at 0.1 mbar overnight) without dehalogenation, as evidenced by MALDI-TOF mass spectrometry and 1H NMR spectroscopy. However, alkylation

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of this compound in the manner described above resulted in similar reaction mixtures (both in terms of the nature and relative proportion of side products) to those obtained with 2 (Supporting Information, Figure S2). In any event, as all the side products of the alkylation reaction can be converted to R-hydroxy, ω-methoxy poly(ethylene glycol) (i.e., mPEG, 1) by hydrolysis and because these are not reactive toward the side chains of proteins under the conditions examined, these are inconsequential. A minor amount of dimer of 3 was observed by sizeexclusion chromatography, possibly due to aldol condensation of the end group. Acetal Deprotection to r-(Methyl glyoxal), ω-Methoxypoly(ethylene glycol) (6-8). Deprotection of the acetal end group on 3 to reveal the R-oxo-aldehyde group may be accomplished in a number of manners. An analogous reaction, the deprotection of methylglyoxal 1,1-dimethylacetal via acid hydrolysis, has received much attention in the past given that this is the main route for preparing high quality methylglyoxal (vide infra).41,42 In general, the deprotection of ketoacetals such as methylglyoxal 1,1-dimethylacetal and the structurally analogous group on 3 is complicated by the high tendency of R-oxo-aldehydes to selfreact in the form of hydration, dimerization, ring closure, and aldol condensation reactions.43 These reactions are often catalyzed by the aqueous acidic conditions used for hydrolysis,35 a problem that is generally overcome in the case of methylglyoxal by distillation to isolate the monomeric species from oligomers. The low recovered yield of monomeric methylglyoxal reported in the literature, on the order of about 50-60% from methylglyoxal 1,1-dimethylacetal,41,42 is likely to result from these side reactions. In the context of the hydrolysis of 3, however, such oligomerization is problematic owing to the difficulty of isolating the monomeric 6-8 from oligomeric 6-8. Therefore, before attempting the deprotection of 3, the reaction conditions for the acetal deprotection of methylglyoxal1,1-dimethylacetal (10), a structurally relevant model compound, were investigated. The aim of these experiments was to identify reaction conditions that promote direct formation of monomeric R-oxo-aldehydes with minimal oligomerization (Scheme 2). Three different reaction conditions were investigated: (i) hydrolysis catalyzed by sulfuric acid (10 vol % H2SO4, 100 °C, 25 min),41,44 (ii) hydrolysis catalyzed by a strong-acid ionexchange resin (Amberlite IR-120, 100 °C, 3 h),42 and (iii) I2catalyzed transacetalization (acetone, 90 °C, 1 h).45 Figure 2 shows the 1H NMR spectra of methylglyoxal 1,1-dimethylacetal (10) in equilibrium with its monohydrated form 11 in D2O (Figure 2a) as well as the reaction mixtures following deprotection, without any purification (Figure 2b-d). Peak assignments are given in Scheme 2 for the produced methylglyoxal 12 in equilibrium with its hydrated forms 13 and 14. The extent of acetal deprotection was monitored by comparison of the integrals of the acetal group relative to those of its hydrolyzed products (i.e., methanol and others), which appear shifted upfield. Marked differences were observed between the products obtained by the different deprotection reactions. Following sulfuric acid-catalyzed deprotection, a relatively complicated 1 H NMR spectrum was observed. The peaks attributed to the mono- (13) and dihydrate (14) of methylglyoxal are broad and shifted compared to previously published values of hydrated monomeric methylglyoxal and various small sharper peaks in the 1.2-1.7 ppm region were observed, which is in accordance with the production of oligomeric species according to the interpretation of Nemet et al.42 A small amount of unhydrated methylglyoxal (12) was observed at 8.2 ppm. In contrast, the use of the ion-exchange resin produced sharp monomeric peaks

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Figure 2. 1H NMR spectra of the reaction mixtures resulting from model acetal deprotection reactions: (a) methylglyoxal 1,1-dimethylacetal in D2O, (b) H2SO4-catalyzed hydrolysis in D2O, (c) ionexchange resin-catalyzed hydrolysis in D2O, and (d) I2-catalyzed transacetalization in acetone-d6. Peak assignments given in Scheme 2.

of the mono- (13) and dihydrates (14) of methylglyoxal in addition to small multiplets at 2.27 and 1.34 ppm, likely corresponding to methyl protons on oligomeric species. Integration of these peaks relative to the sharp monomeric peaks x, z, and ab indicated that oligomerization accounted for about 24% of the sample, a value that is consistent with the recovered yields for this compound prepared by this method after distillation of the reaction mixture (above). Finally, acetal deprotection was also attempted via transacetalization with acetone, a competitive ketone solvent, catalyzed by 10 mol % I2 relative to the acetal. Under these conditions partial deprotection of the acetal to a hemiacetal was observed by the appearance of a singlet at 4.72 ppm (Figure S3 in Supporting Information). Increasing the content of I2 to 3 mol equiv relative to acetal, however, afforded complete deprotection to the unhydrated (12, 8.2 ppm) and monohydrate methylglyoxal (13), as observed in Figure 2d. Small oligomer peaks in the 1-2 ppm region were also observed which accounted for about 25% of the sample. The extent of oligomerization was comparable to that observed by hydrolysis using the ion-exchange resin and therefore also constitutes a viable means for producing methylglyoxal from its protected acetal form, despite ulterior purification steps for the removal of iodine. Next, the conditions used for the ion-exchange resin-catalyzed hydrolysis of methylglyoxal 1,1-dimethylacetal (10) were applied to the hydrolysis of the acetal on 3. The deprotection was much slower (ca. 14% after 1 h at 100 °C), and near complete deprotection was only achieved after stirring for 18 h at this temperature, as monitored by the disappearance of the acetal peak g (3.38 ppm) in the 1H NMR spectrum of aliquots taken at specified times. In an attempt to increase the rate of the deprotection, the amount of acid catalyst was increased by a factor of 10 (i.e., to 10 mg Amberlite IR-120 per mg 3-5), but this did not significantly affect the rate of hydrolysis. Hydrolysis was, however, complete after the 18 h period. In contrast, I2catalyzed deprotection of the acetal (3 mol equiv relative to acetal) was much more rapid, being complete after 1 h at 90 °C in acetone. Representative spectra of the unpurified and purified reaction mixtures obtained by both deprotection methods are shown in Figure 3. The nature of the species present in the reaction mixtures was determined based on chemical shift

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Figure 3. 1H NMR spectra of (a) 3, (b) deprotection products of 3 by acid hydrolysis crude (18 h at 100 °C), (c) acid hydrolysis purified, (d) transacetalization crude (1 h at 90 °C), and (e) transacetalization purified. Spectra (a) taken in CDCl3, (b, c, and e) in D2O, and (d) in acetone-d6. Peak assignments given in Scheme 1. Unidentified impurities or intermediates are marked with a star (*).

considerations as well as by end-group analysis of 6-9 produced by acid-catalyzed hydrolysis submitted to MALDI TOF mass spectrometry (Figure 1a and b). Ion-exchange resin-catalyzed hydrolysis of the acetal groups resulted in complete hydrolysis of the acetal and few artifact peaks. Peaks o, n, and m, corresponding to the monohydrate (7) were easily observed in the 1H NMR spectrum of the crude reaction mixture, in addition to a triplet at 2.45 ppm which may correspond to q of the dihydrate species (8) based on chemical shift and multiplicity considerations. Purification of 6-9 by size-exclusion chromatography followed by isolation by freeze-drying produced a considerably cleaner spectrum in which could be observed the aldehyde peak of the unhydrated species (6) at 9.1-9.2 ppm and an increase of a broad multiplet at 1.70 ppm, which is believed to be the result of oligomerization of the R-oxoaldehyde group. The assignment of this peak to an oligomeric species was based on chemical shift considerations, on the experiments performed with methylglyoxal 1,1-dimethylacetal (Figure 2), and by the known existence of dimeric and trimetric species evidenced by analytical size-exclusion chromatography (Figure 4). Deprotection of the acetal group by I2-catalyzed transacetalization resulted in the production of the unhydrated R-oxo-aldehyde 6 along with the monohydrate 7. Purification and isolation of 6-9, however, produced a 1H NMR spectrum very similar to that obtained by acid-catalyzed hydrolysis with the monohydrate 7 being the major present species and oligomers being observed. The relative proportion of 6-8 within 6-9, which was 12-33% of total end groups, was comparable by both hydrolysis and transacetalization, indicating that both methods are applicable for end-group deprotection. Less than ∼3% of the anhydrate 6 was observed following deprotection by either method. Despite the observation that methylglyoxal oligomerized upon freeze-drying, no change in the intensity of these peaks was observed following a second identical purification/isolation procedure by size-exclusion chromatography and freeze-drying. As a result, both methods seem equivalent in terms of efficacy of deprotection of the end groups of 3, though as the ion-exchange resin catalyst is easily removed by filtration following the reaction, this deprotection strategy was selected for all the following experiments.

Gauthier and Klok

Figure 4. Size-exclusion chromatography in DMF of mPEG samples with different end groups. Chromatograms recorded following acid hydrolysis (18 h at 100 °C) and transacetalization after 1 h at 90 °C.

Model Reactions of Methylglyoxal with Nr- or Nε-Acetyl Amino Acids. Prior to exploring the possibility of using the R-oxo-aldehyde mPEG derivatives 6-8 to modify the arginine residues in proteins, the reaction of methylglyoxal with selected model amino acids was investigated. The specific aim of these experiments was to study the pH and dilution stability of the formed products. The stability of adducts of NR-acetyl amino acids with 1,2-cyclohexanedione and p-substituted phenylglyoxal has been evaluated in past research31,46 and provides a starting point for the conditions examined herein. The stability of methylglyoxal-amino acid adducts was studied rather than adducts of 6-9 because this model system provides direct and simple evaluation of formed species by mass spectrometry. The results gathered with methylglyoxal will be used to interpret the stability of HEWL-polymer conjugates prepared with 6-9 in the following sections and tested under similar conditions. After following the reaction of methylglyoxal (125 mM, produced by ion-exchange hydrolysis of methylglyoxal 1,1dimethyl acetal (10)) with N-acetyl amino acids (25 mM) in either borate buffer (pH 9) or phosphate buffer (pH 7.4), the stability of the adducts toward hydrolysis between pH 3-12, toward the nucleophilic buffer hydroxylamine (at pH 7), and toward dilution (to displace potential equilibrium species) was investigated. Hydroxylamine was chosen both to quench excess R-oxo-aldehyde reagent and to cleave any reaction products that may have formed between the latter and lysine residues. Reaction kinetics were monitored by HPLC through a decrease (or increase) of the intensity of the original N-acetyl amino acid signal, which was distinct from all other peaks. Identification of formed compounds after 2 weeks was determined by LCMS (direct ESI was not possible because the buffers suppressed ionization). All chromatograms and mass spectra are available as Supporting Information (Figures S4-14). Lo et al.25 have already evaluated the kinetics of the reaction of NR-acetyl arginine, NR-acetyl lysine, and NR-acetyl cysteine with an equimolar amount of methylglyoxal (20 mM) in 200 mM phosphate buffer solution pH 7.4 by 1H NMR spectroscopy. Similar to these authors’ findings, rapid and quantitative disappearance of NR-acetyl cysteine was observed at both pH 9 and 7.4, even before the first chromatogram could be recorded (Figures S13 and S14). The formation of a hemithioacetal (22 in Scheme 3) between the amino acid derivative and methylglyoxal was confirmed by mass spectrometry (Figure S5). In a

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Scheme 3. Compounds Formed after Reaction of NR-Acetyl Arginine and NR-Acetyl Cysteine with 5 Mol Equiv Methylglyoxal

recent study, glyoxal and methylglyoxal were shown to react with NR-acetyl cysteine and cysteine-containing peptides, indicating that reduced cysteine residues on proteins may be major targets for dicarbonyl modifications.47 Hydrolysis of 22 by acidification of the reaction mixture to pH 3 was found to be possible (Figure S4). Immediately following acidification to this pH, recovery of 71% (from reaction at pH 9) and 43% (from reaction at pH 7.4) of the initial NR-acetyl cysteine was observed concurrent with the appearance of a new peak corresponding to its disulfide. With time, the intensity of the peak of the reduced amino acid derivative decreased due to disulfide formation (aside from acidification, no precautions were taken to exclude oxygen from the reaction mixture). Also, removal of excess methylglyoxal by Prep-HPLC and resuspension of NR-acetyl cysteine-methylglyoxal adducts in water (pH 7) was found to lead to regeneration of 71% (from reaction at pH 9) and 77% (from reaction at pH 7.4) of the original amino acid derivative (Figure S20). Quantification of the non-negligible amounts of disulfide also observed during this process was not performed. Hydroxylamine was found not to displace the hemithioacetal to recover the amino acid derivative. Rather a marked increase of absorbance of the peaks observed by HPLC was observed, likely due to formation of oxime derivatives of 22 (Figure S15). This was confirmed by 1H NMR spectroscopy of the reaction mixture containing hydroxylamine (Figure S16). At both pH values examined, NR- and Nε-acetyl lysine were not significantly modified by methylglyoxal (