Overcoming the Hydrolytic Lability of a Reaction Intermediate in

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Overcoming the hydrolytic lability of a reaction intermediate in production of protein/drug conjugates: conjugation of an acyclic nucleoside phosphonate to a model carrier protein Shengsheng Xu, and Igor A. Kaltashov Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00410 • Publication Date (Web): 17 Jul 2017 Downloaded from http://pubs.acs.org on July 18, 2017

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Molecular Pharmaceutics

Overcoming the hydrolytic lability of a reaction intermediate in production of protein/drug conjugates: conjugation of an acyclic nucleoside phosphonate to a model carrier protein Shengsheng Xu and Igor A. Kaltashov Department of Chemistry, University of Massachusetts-Amherst, Amherst, MA, USA

address correspondence to:

Igor A. Kaltashov Department of Chemistry University of Massachusetts-Amherst 240 Thatcher Way Life Science Laboratories N369 Amherst, MA 01003 Tel: (413) 545-1460 Email: [email protected]

ACS Paragon Plus Environment


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ABSTRACT Acquired immunodeficiency syndrome (AIDS) remains one of the most serious public health challenges and a significant cause of mortality for certain populations. Despite the large number of antiretrovirals developed in the past two decades, the inability of small molecule therapeutics to target HIV reservoirs directly creates a significant obstacle to their effective utilization. Indeed, achieving the desired therapeutic outcome in the absence of an effective means of targeted delivery must rely on dosage escalation, which frequently causes severe toxicity. This problem may be solved by conjugation of antiretroviral agents to endogenous proteins that are specifically recognized by HIV reservoirs (such as macrophages) for internalization and catabolism. However, conjugation of a large class of antiretroviral agents (acyclic nucleoside phosphonates, such as adefovir, tenofovir and cidofovir) to a protein is challenging due to rapid degradation of the activated form of the drug (e.g., adefovir phosphonoimidazolide) in an aqueous environment. A novel synthetic strategy introduced in this work overcomes the instability of the activated form of adefovir by emulating the first step of its metabolic pathway (phosphorylation), making it highly reactive towards primary amine groups of proteins, and at the same time less prone to hydrolysis by water. Efficient conjugation of the phosphorylated form of adefovir to a protein following activation with EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) and imidazole was demonstrated using a model protein. Mass spectrometry (MS) was used to identify conditions that favor formation of conjugates with minimal side products, and on-line ion exchange chromatography/MS analysis of the products revealed the presence of multiple positional isomers within the 1:1 protein/drug conjugates. Both LC/MS data and the analysis of ions produced upon top-down fragmentation of the 1:1 conjugates suggest that conjugation of phosphorylated adefovir to the protein occurs not only at primary amines, but also at hydroxyl groups. The new conjugation protocol can be used to attach adefovir and other acyclic nucleoside phosphonates to proteins recognized by the cell surface receptors specific to macrophages (such as the haptoglobin/hemoglobin complex), enabling targeted drug delivery directly to HIV reservoirs. KEYWORDS Human immunodeficiency virus (HIV), antiretroviral therapy, adefovir, protein-drug conjugate, electrospray ionization mass spectrometry (ESI MS), ion exchange chromatography, LC/MS, top-down MS/MS


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INTRODUCTION Acquired immunodeficiency syndrome (AIDS) remains one of the world’s most significant public health challenges and a significant cause of death for certain populations.1 Despite the relentless search for a cure of HIV infection, presently antiretroviral therapy (ART) remains the only feasible therapeutic modality that allows many patients to achieve and maintain near complete suppression of the virus for extended periods of time.2 However, the success of ART is undermined by the drug resistance,3 particularly in pediatric populations,4 placing a premium on the continuous expansion of the repertoire of available ART agents.5 Unfortunately, a significant fraction of novel antiretroviral drugs fail to deliver on their promise, as the inability of small molecule therapeutics to target the HIV reservoirs creates a significant obstacle to their effective utilization. Indeed, achieving a desired therapeutic effect in the absence of an effective means of targeted delivery frequently relies on dosage escalation, causing severe toxicity. For example, an acyclic nucleotide analog adefovir6 that was initially developed as anti-HIV therapeutic failed to gain approval for that indication due to significant nephrotoxicity, despite having demonstrated a clear antiretroviral benefit.7 Pharmacokinetic studies demonstrated that over 98% of unmodified adefovir molecules are excreted with urine during the 24-hour window following an intravenous injection,8 clearly preventing it from reaching anatomical and cellular viral reservoirs.9 Far from being unique to adefovir treatment, nephrotoxicity is a common problem for a number of antiretroviral agents,10-13 imposing significant limitations on available therapeutic options. The problem of dosage-dependent toxicity can be alleviated by targeting the ART agents directly to viral reservoirs,14, 15 and limiting their levels elsewhere in the organism. One particularly attractive opportunity in this regard is presented by haptoglobin, an acute phase plasma protein that irreversibly binds free hemoglobin molecules in circulation and delivers them to macrophages for catabolism.16 Internalization of the haptoglobin/hemoglobin complexes is mediated by CD163 receptors that are specific to macrophages and other cells of monocyte lineage17 that harbor HIV virus in AIDS patients. The potential of CD163 to serve as a target molecule for cell-directed therapies has been recognized several years ago.18 In fact, conjugation of a nucleoside analog ribavirin to hemoglobin had been explored for targeted delivery to CD163-expressing cells19 with the goal of overcoming the dose-dependent toxicity. While this particular nucleoside analog cannot be used in ART, a strategy can be envisioned where ART agents are conjugated to haptoglobin/hemoglobin complex to achieve targeted delivery to macrophages via CD163-mediated internalization to inhibit reverse transcription of HIV in infected immune cells during early stages of acute infection.20, 21 A particularly attractive class of agents includes nucleotide reverse transcriptase inhibitors (NtRTIs) incorporating structural features such as phosphonate groups that prevent them from being processed by nucleases.22 Targeted delivery of such NtRTIs utilizing vehicles capable of traversing physiological barriers (e.g., transferrin or anti-transferrin receptor antibodies23) can also solve the problem of poor accessibility of viral pools in the central nervous system, where cells of monocyte/macrophage lineage



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(e.g., microglia, perivascular macrophages and meningeal macrophages) harboring HIV are protected by the blood brain barrier.24, 25 Conjugation of one of the members of the NtRTI family, cidofovir, to a serine/tyrosine dipeptide has been recently demonstrated,26-28 where the phosphonate group of cidofovir formed phosphonoester bond with the hydroxyl group of serine in N, N’-dimethylformamide (DMF). However, this conjugate undergoes rapid hydrolysis in aqueous environment at physiological pH with a half-life below two hours,26, 27 which may not provide an adequate time window for successful delivery of an ART agent to its target site. Furthermore, most carrier proteins are likely to undergo unfolding in organic solvent (such as DMF), causing aggregation and precipitation during the conjugation process. Therefore, it is highly desirable that the carrier protein/drug conjugation reactions take place in an aqueous environment under conditions that do not compromise the higher order structure of the protein. In this respect, a more attractive scenario would include two steps: chemical activation of the anti-viral agent in organic solvent followed by the transfer of the chemically active intermediate to an aqueous protein solution to produce a conjugate. This scheme was used successfully to produce a ribavirin/hemoglobin conjugate19 by activating ribavirin with carbonyldiimidazole (CDI) in DMF to generate a reactive phosphorimidazolide intermediate. However, our attempts to adopt this strategy to conjugate NtRTIs (such as adefovir) to proteins failed due to the extreme instability of the phosphonoimidazolide intermediate in aqueous environments compared to its phosphorimidazolide counterpart. In this work, a modified conjugation strategy is presented which overcomes the problem of the phosphonoimidazolide intermediate instability by chemically modifying adefovir prior to its activation. This initial modification emulates the first step in adefovir’s metabolic pathway (phosphorylation), allowing a phosphorimidazolide intermediate to be produced that is reactive towards primary amines on lysine side chains and at the N-terminus of the protein (and, to a lesser extent, hydroxyl groups on the protein side chains), and at the same time shows remarkable stability vis-à-vis hydrolytic attacks by water. The new conjugation protocol can be used to attach adefovir and other structurally related anti-viral agents to proteins recognized by the cell surface receptors specific to macrophages (such as a haptoglobin/hemoglobin complex), enabling targeted drug delivery directly to the cells harboring HIV viruses.


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EXPERIMENTAL SECTION Materials. Adefovir (9-(2-phosphonomethoxyethyl) adenine, PMEA) was purchased from AK Scientific, Inc. (Union City, CA). Lysozyme (Lz), 1, 1’-carbonyldiimidazole (CDI), phosphoric acid (crystalline, 99.999% metal trace basis) and imidazole were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

hydrochloride)

was

purchased

from

Pierce

Biotechnology (Rockland, IL). Triethylamine (TEA), N, N’-dimethylformamide (DMF) and anhydrous diethyl ether were purchased from Fisher Scientific (Fair Lawn, NJ). All other solvents and reagents were of analytical grade or higher. Phosphorylation of adefovir (PMEA) and preparation of lysozyme/adefovir conjugates (Lz/PMEA conjugates). Preparation of the Lz/PMEA conjugates is shown in Schemes 2-3. PMEA was converted to PMEA phosphonoimidazolide (PMEA-Im) using a modification of the previously published procedure.29 Briefly, PMEA (15 mg, 0.054 mmol) powder was placed in a 2 mL glass vial. CDI (0.082 mmol, 1.5 equiv) was dissolved in 400 μL DMF/TEA mixture (3:1, v:v). This DMF/TEA solution was stirred at room temperature for 1.5 hours, followed by addition of 36 μL of 3.1 M solution of anhydrous phosphoric acid (0.054 mmol, 1 equiv) in DMF. The phosphorylation was performed overnight at room temperature. Afterwards, the reaction solution was placed to cold diethyl ether to precipitate adefovir monophosphate (PMEAp). The cloudy diethyl ether was kept at -20 oC for 2 hours followed by centrifugation, after which the supernatant was decanted. The waxy precipitate at the bottom was re-dissolved in 100 μL distilled water and divided equally in two parts, which were mixed with 400 μL 0.2 M imidazole buffer (pH 6.8) and 400 μL 0.2 M sodium bicarbonate buffer (pH 8.7), respectively. EDC (0.027 mmol, 1 equiv) was added to both reaction buffers and imidazole (0.027 mmol, 1 equiv) was added to the 0.2 M sodium bicarbonate buffer. Both reaction solutions were stirred at room temperature for half an hour. A 50 μL aliquot of the 1 mM lysozyme stock solution was added to each of the reaction buffers (450 μL), giving a final protein concentration of 100 μM. The final drug-to-protein molar ratios in both solutions were around 540:1. Both reaction solutions were stirred at room temperature for 24 hours. Characterization of PMEA derived small molecules. 1 μL aliquots of DMF solution at different stages of reaction (including PMEA’s complete activation by CDI and PMEA’s phosphorylation) were diluted with 100 μL deionized water followed by analysis performed with a QStar-XL (AB-Sciex, Toronto, Canada) hybrid quadrupole/time-of-flight mass spectrometer to determine masses of the reaction/hydrolysis products. Characterization of Lz/PMEA conjugates. Mass measurements of Lz/PMEA conjugates were carried out with a QStar-XL (AB-Sciex, Toronto, Canada) hybrid quadrupole/time-of-flight mass spectrometer followed their purification by buffer exchange in 100 mM ammonium acetate; ions were produced by



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electrospray ionization (ESI) using a turbospray source. The conjugates were completely denatured during separation/purification on a 2.1 mm × 50 mm BioSuite (Waters, Milford, MA) C18 column (particle size of 3 μm and pore size of 100 Å) prior to MS based characterization. Fragmentation of conjugate ions was carried out by selecting a precursor ion (m/z selection window 1627.0 ± 0.4 u) followed by its collisional activation (50 eV collisional energy). Assessment of the conjugate heterogeneity (profiling of positional isomers) was carried out by introducing an online ion-exchange chromatography (IXC) step prior to ESI MS measurements using a method developed earlier in our laboratory.30, 31 Briefly, IXC (cation exchange) was performed on Agilent 1100 (Agilent, Santa Clara, CA) liquid chromatograph equipped with a 2.1 mm × 100 mm PolyCAT A (PolyLC Inc., Columbia, MD) weak cation-exchange column (particle size of 5 μm and pore size of 300 Å). Ammonium acetate at pH 7.0 was used for MS-compatible gradient elution (100 mM in mobile phase A and 1 M mobile phase B). The flow rate was maintained at 0.1 mL/min, and the linear gradient ran from 0% to 60% mobile phase B during a 60 min time interval. A Synapt G2Si (Waters, Milford, MA) hybrid quadrupole/time-of-flight mass spectrometer was used for on-line ESI MS detection.


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RESULTS AND DISCUSSIONS Preparation of lysozyme/adefovir conjugates. Our initial attempt to conjugate adefovir to a model protein (lysozyme) using the procedure designed for conjugating a chemically similar drug ribavirin to hemoglobin19 failed to generate any detectable conjugates despite the presence of seven primary amines (targets of the conjugation reaction) within the protein. MS analysis of the activated form of adefovir produced in DMF yielded a mass spectrum with a single peak at m/z 322 (Figure 1) consistent with the notion of the expected intermediate (adefovir phosphonoimidazolide, see Scheme 1) being produced at nearly 100% yield. However, the subsequent conjugation reaction must take place in the aqueous environment, and addition of even small amount of water to the DMF solution of adefovir phosphonoimidazolide (ca. 2.5 % by volume) resulted in a dramatic alteration of the appearance of the mass spectrum following a relatively short (15 min) incubation time period (blue trace in Figure 1). Although the adefovir phosphonoimidazolide ion is still present in the spectrum, the most abundant ionic signal corresponds to a species at m/z 527; a lower-abundance peak corresponding to unmodified adefovir (m/z 272) is also present. The mass of ion at m/z 527 is consistent with the dimeric form of adefovir (linked via pyrophosphonate bond). This species (structure shown in Scheme 1) is formed not only as a result of exposure to water, but also during diethyl ether precipitation (data not shown), an essential step required to remove unconsumed reagents and organic solvents from the chemically activated adefovir prior to its conjugation to a protein. The re-appearance of the signal of unmodified adefovir (m/z 272) in the mass spectrum of its activated form (adefovir phosphonoimidazolide) exposed to water is likely due to hydrolysis (Scheme 1) facilitated by formation of the transition zwitterion. Indeed, the methylene bridge (-CH2-) in the phosphonoamide moiety exerts an electron donating inductive effect on both phosphorus and nitrogen atoms, leading to their basicity increase. As a result, the proton of the hydroxyl group attached to the phosphorus atom is expected to migrate to the nearby basic nitrogen atom of the imidazole ring, forming a transition zwitterion and making imidazole a good leaving group.32 Formation of this species is expected to occur in the phosphonoimidazolide intermediate, with the imidazole group being rapidly replaced by water (or another agent capable of a nucleophilic attack.32 This behavior is in contrast to that of phosphorimidazolide, which had been demonstrated to be much more stable to hydrolytic nucleophilic attacks,33 and is likely due to the high basicity of the phosphorus–linked nitrogen atom (unlike phosphonoimidazolide).32 The hydrolyzed form (i.e. adefovir) of adefovir phosphonoimidazolide (PMEA-Im) is strongly nucleophilic and can also attack the phosphorus atom in the zwitterionic form of PMEA-Im, leading to formation of a pyrophosphonate-linked PMEA dimer (Scheme 1), following essentially the same mechanism as the one proposed for the hydrolytic attack by a water molecule. The molecular weight of this dimeric product is consistent with the mass of the most abundant ionic species in the mass spectrum of water-exposed



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PMEA-Im (m/z 527 in Figure 1), lending further credibility to the proposed mechanism. The anomalous basicity of the nitrogen atom can be attenuated via a variety of ways, and perhaps the most straightforward approach is to modify α-carbon of phosphorus in PMEA with an electron withdrawing group, such as trifluoromethyl.32 However, the structural change caused by this particular modification is likely to interfere with the drug’s ability to interact with the HIV’s reverse transcriptase, making this approach unacceptable. An alternative strategy adopted in this work relies on extending the terminal phosphonate group of adefovir by converting it to adefovir monophosphate (PMEAp, see Scheme 2), which endows the molecule with a functional group that can be readily exploited vis-à-vis conjugation to a protein without raised a specter of deactivation and/or forming non-productive intermediates. Indeed, it had been reported that nucleoside diphosphorimidazolide is less prone to hydrolysis than nucleoside monophosphorimidazolide, at least in the case of adenosine 5’-diphosphorimidazolide

and

7-methylguanosine

5’-diphosphorimidazolide

and

their

monophosphorimidazolide counterparts.33 In order to determine if phosphorylation of adefovir will make the activated form of this drug less susceptible to nucleophilic attack, we first converted PMEA to the monophosphate form (an analog of deoxyadenosine-5’-diphosphate (dADP)) to enhance the stability of the reaction intermediate. The activated form of PMEAp (phosphorimidazolide) can be readily produced in aqueous solution using EDC (Scheme 3). This strategy not only stabilizes the reaction intermediate in aqueous environment without introducing significant alteration to PMEA structure, but is also expected to facilitate the conversion of PMEA to its active metabolite (PMEApp) in vivo by eliminating the need for one of the two obligatory phosphorylation steps. Conversion of PMEA to PMEAp prior to its activation (to an imidazolide form) should also protect it against a hydrolytic attack not only prior to, but also during the conjugation to a protein. Indeed, phosphorylation of PMEA to PMEAp (an analog of dADP)33 reduces the basicity of nitrogen in phosphoramide moiety because the introduced oxygen atom at the α-position of terminal phosphorus inductively withdraws electrons from the nitrogen atom.34 This inhibits the formation of a transition zwitterion and stabilizes the intermediate. PMEA phosphorylation was carried out by adding anhydrous phosphoric acid DMF solution in PMEA-Im. The ESI mass spectrum of reaction mixture acquired in the negative ion mode (black trace in Figure 1) indicates that PMEAp (m/z=352) is the major product, which coexists with a hydrolytic product (m/z 272) and a diphosphorylated product (at m/z=432). To transfer PMEAp from DMF to water, this compound was directly precipitated in cold diethyl ether. PMEAp extracted from DMF was re-dissolved in imidazole-HCl aqueous buffer (see Materials and Methods for more detail), and activated by EDC in the presence of the target protein. The activation proceeds via an O-acylisourea intermediate, which is known to be rapidly attacked by imidazole groups,35 a trait that was exploited in our work to conjugate PMEA to a protein (Scheme 3). The “on-demand” formation of PMEAp-Im in the presence of the protein greatly reduces non-productive reaction channels


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(e.g., hydrolysis of this intermediate by water molecules), while allowing the nucleophilic attacks by the primary amines (lysine side chains) to take place, leading to the conjugate formation. While the unconsumed EDC is usually quenched prior to mixing with protein solutions with 2-mercaptoethanol, we chose to avoid this step, as the presence of this reagent in the protein solution would almost certainly induce reduction and/or rearrangement (scrambling) of disulfide bonds. The downside of keeping EDC in solution is the occurrence of side reactions, although they can be suppressed by selecting appropriate reaction conditions (vide infra). Characterization of lysozyme/adefovir conjugates. ESI mass spectra of Lz/PMEA conjugates prepared at pH 6.8 and pH 8.7 are shown in Figure 2. The ionic species present in each mass spectrum display narrow charge state distributions, and the average extent of multiple charging is consistent with compact (natively oxidized) conformations in solution.36 Both spectra contain signals of intact (unmodified) Lz and a 1:1 conjugate (calculated mass shift 335.2 Da). In addition, the mass spectrum of the conjugate produced under mildly basic (pH 8.7) conditions contains ionic signals corresponding to conjugates with 2:1 stoichiometry, as well as a range of other products. The mass analysis of these modified Lz species reveals two types of chemical modification (addition of 71.1 and 128.2 Da, consistent with reactions of residual EDC with the protein molecule37), as well as their combinations with each other and with Lz/PMEA conjugates. Specifically, modification of carboxylic groups of aspartic and/or glutamic acid residues is likely to give rise to unstable O-acylisourea intermediates, which are then converted to stable N-acylurea products via the intramolecular O-to-N acyl rearrangement in the presence of excess EDC38 (see Scheme S1 in Supplementary Material). Since the two nitrogen atoms in O-acylisourea intermediate are linked with ethyl and 3-dimethylaminopropyl groups respectively, the rearrangement may lead to one of the two possible N-acylurea products, depending on which nitrogen atom the acyl group migrates to. The secondary amides in N-acylurea byproducts undergo base-catalyzed hydrolysis and produce N-acyl-N-ethyl-carbamic acid and N-acyl-N-3-dimethylaminopropyl-carbamic acid derivatives corresponding to the mass additions of 71.1 Da and 128.2 Da (see Scheme S1 in Supplementary Material). No dead-end products are observed in the mass spectrum of Lz/PMEA conjugates produced at pH 6.8, indicating that the unstable O-acylisourea intermediates undergo quick hydrolysis prior to acyl rearrangement at neutral pH. The relatively low yield of conjugation at pH 6.8 is likely due to the significantly higher stability of the reaction intermediate PMEAp-Im, as PMEA’s phosphorylation prevents formation of a zwitterionic transition state. As a result, PMEAp-Im undergoes slower hydrolysis and requires a stronger nucleophile to attack it. Primary amines (e.g., ε-amines of lysine side chains) can act as nucleophiles only if they are deprotonated. While increasing the solution pH might seem an easy solution to this problem, it comes at a price of a dramatic rise of the abundance of dead-end and mixed products. Therefore, even though both the yield of the conjugation reaction and the average drug



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loading are noticeably lower when the reaction is carried out at neutral pH, these conditions are still preferable to mildly basic pH, as the products are much less heterogeneous. Although the mass spectrum of the Lz/PMEA conjugates produced at neutral pH reveals only a single conjugate (in addition to the intact unmodified protein), the sample may contain several isobaric species that differ from each other by the location of the conjugation site. In order to evaluate micro-heterogeneity of this sample with respect to possible presence of several positional isomers, it was analyzed by ion exchange chromatography (IXC) with on-line detection by ESI MS. This technique was recently developed in our laboratory30 and successfully applied for characterization of samples as complex as PEGylated glycoproteins subjected to stress conditions.31 The chromatogram of the products of Lz/PMEA conjugation show a series of peaks, and the on-line MS detection provides evidence that nearly all species eluting before 55 minutes have masses consistent with 1:1 conjugation stoichiometry, which only a minor fraction of species representing 2:1 conjugates (Figure 3). Integration of the intensities of the Lz/PMEA conjugates’ peaks in UV absorption chromatogram (not shown) suggests that the yield of the conjugation reaction is 23%, which is reasonably close to the 27% yield obtained by integrating ion peaks in the mass spectrum of the reaction mixture (Figure 2). PMEAp conjugation to the protein at the lysine side chain not only eliminates the positive charges on the ε-amine group of this residue, but also introduces negative charges (phosphonate and phosphate groups of PMEAp). Although the total number of charges is the same for all isomers, the unique position of the PMEAp group on the surface of the protein will have a profound effect on the charge density surface distribution. The surface charge density pattern is an important determinant of the protein retention on the ion exchange resin;39 therefore, it should not be surprising that IXC allow a number of isomeric conjugates to be resolved (Figure 3). The most abundant 1:1 conjugate elutes at 49 min. and likely represents the product of conjugation at the N-terminus of the protein (the pKa of the α-amine is slightly lower than that of ε-amines, making it a more favorable site for the conjugation reaction compared to the lysine side chains). Six lower-intensity peaks are also observed in the extracted ion chromatogram for the 1:1 conjugate with ionic abundance ranging from 5% to 25% of the base peak. In addition, a few minor peaks (abundance < 5% of the base peak) are present in the chromatogram of the 1:1 conjugate. This observation is surprising, as the number of chromatographically resolved isobaric 1:1 conjugates exceeds the total number of amines (six ε-amines at lysine side chains and one α-amine at the N-terminus of the protein). It might be tempting to attribute this mismatch to the presence of conjugates where the integrity of the protein higher order structure had been affected by the chemical modification (leading to an altered surface charge pattern and, as a consequence, a change in the retention time). Such large-scale conformational changes result in changes in the appearance of the charge state distributions of protein ions in ESI mass spectra;36 however, the on-line ESI detection provides no evidence of alteration of the charge state distributions of ionic species representing 1:1


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conjugates across the entire chromatogram. Therefore, it seems unlikely that conjugation triggers loss of the protein native structure. A plausible explanation for the large number of the observed isobaric species could be the occurrence of protein deamidation during the conjugation process. Deamidation is a common non-enzymatic post-translational modification (PTM), which frequently (and inadvertently) occurs during a variety of protein handling steps,40, 41 making it a serious nuisance in biopharmaceutical analysis. Asparagines followed by either a glycine or serine residue are usually prone to deamidation,42 and lysozyme contains two such asparagine residues (N59S60 and N103G104). Deamidation results in a mass increase of only 1 Da, which is nearly impossible to detect when the protein mass is large; however, in our case a one-mass unit shift in isotopic distributions of the Lz/PMEA conjugates should be readily detected because of the relatively low mass of the latter. No such shift was detected for either of the species eluting prior to 55 min. in the chromatogram shown in Figure 3, suggesting that deamidation does not take place under the conditions chosen to produce the conjugates (pH 6.8). In order to understand what contributes to the unexpectedly large number of isomeric 1:1 Lz/PMEA conjugates, collisionally activated dissociation (CAD) of the mass-selected Lz/PMEA ions was carried out and the mass spectra of the resulting fragments (Figure 4) analyzed. CAD of the Lz/PMEA ion at +9 charge state (m/z 1627.6) gives rise to a relatively small number of fragment ions. Singly charged fragment ions are observed in the low-m/z region of the mass spectrum (m/z 274 and 354) and the complementary fragments at high m/z region (m/z 1597.3, 1786.8 and 1796.8). The most abundant of the two low-mass fragments (m/z 274) corresponds to a (PMEA+H)+, produced by the cleavage of P-O bond in the phosphate group of the conjugate. The complementary ion is represented in the CAD mass spectrum with a high-intensity peak at m/z 1796.8 (charge state +8), and a fragment ion with the same mass but different charge state (+9) is observed at m/z 1597.3 (corresponding to the neutral loss of PMEA from the precursor ion). The presence of these fragment ions in the CAD mass spectrum of the Lz/PMEA conjugates is not surprising, since the phosphoester bonds are known to be labile in the gas phase and undergo dissociation prior to the cleavage of the polypeptide amide bonds.43 What is surprising, however, is the presence of the second peak in the low-m/z region of the CAD mass spectrum (a singly charged species at m/z 354). This mass corresponds to a (PMEAp+H)+ ion, which can only be produced upon fragmentation of the Lz/PMEA conjugates if PMEAp is attached to the protein via hydroxyl-bearing side chain, such as serine or threonine, as opposed to a “conventional” linkage via the amino group of lysine side chains or the polypeptide N-terminus. Phosphoserine peptides are known to undergo phosphate cleavages upon collisional activation to produce dehydroalanine via β-elimination43 and gas phase fragmentation of serine linked PMEAp probably follows the same mechanism, producing a singly charged PMEAp. The existence of the Lz/PMEA conjugates where the pro-drug is linked to the polypeptide chain via side chains with hydroxyl-group not only explains the appearance of the CAD spectrum (Figure 4), but also



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accounts for the anomalously high number of chromatographic peaks in the XIC for the 1:1 conjugate, which exceeds the number of primary amine groups in the protein (Figure 3). In retrospect, conjugation at Ser (and possibly Thr) residues using the amine-targeting chemistry presented in this work may not be that surprising, because their side chains become nucleophilic upon deprotonation which is induced by surrounding basic residues (e.g., lysine and arginine) and is probably solution pH independent, while neutral pH limits the fraction of primary amine that can participate in the attack of activated form of PMEAp. From the practical standpoint, the ability of hydroxyl groups to compete with primary amines as conjugation sites may actually prove beneficial vis-à-vis designing affective means of targeted delivery to macrophages, as no Ser and Thr residues of haptoglobin are known to participate in binding to either hemoglobin or CD163 (the cell-surface receptor that enables internalization of the Hp/Hb complexes by macrophages) via either hydrogen bonding or Van der Waals interaction, while five Lys residues (K262, K291, K297, K345 and K379) are known to participate in such interactions with hemoglobin.44-46 The significant degree of structural heterogeneity of the 1:1 protein/PMEA conjugates produced in this work using a model protein provides important lessons vis-à-vis production of adefovir conjugates with real carrier proteins (such as hemoglobin or haptoglobin). Indeed, a very large fraction of hemoglobin (αβ dimer) and haptoglobin surface (14% and 13%, respectively) are involved in the binding interface when the hemoglobin/haptoglobin complex is formed, and these regions incorporate six amino acid residues bearing primary amines (K291, K297, K345 and K379 on Hp, and V1 and K99 on Hb-α), together accounting for 11% of all surface primary amines in the entire Hp/Hb complex. In addition, several Ser and Thr residues located in the vicinity of the binding interface may also participate in PMEAp conjugation, although in the absence of human Hp crystal structure it is difficult to judge the extent of their side chain solvent accessibility and the local electrostatic environment (the two most important factors influencing their reactivity towards PMEAp-Im). Since neither hemoglobin nor haptoglobin alone can effectively interact with the CD163 receptor (a critical step in internalization of these proteins by macrophages),18 it appears that covalent attachment of PMEAp to any of these side chains will have a strong negative impact on the effectiveness of targeted delivery by compromising the integrity/stability of the hemoglobin/haptoglobin complex. In the absence of the effective chemical means to direct the conjugation reaction away from these critical sites, it would seem prudent to produce the conjugates using the hemoglobin/haptoglobin complex as a starting material, instead of either hemoglobin or haptoglobin alone. Formation of a stable complex prior to the conjugation procedure will shield the critical Lys, Ser and Thr side chains from the PMEAp-Im. In the absence of such “natural” protection during the conjugation step, due care should be exercised to ensure the ability of the conjugates to associate with the physiological partner thereby enabling the receptor recognition and conjugate internalization by the targeted cells. CONCLUSIONS


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


In this work, we introduced a robust synthetic strategy to overcome the instability of adefovir phosphonoimidazolide by emulating the first step of adefovir metabolic pathway and used this strategy to demonstrate successful conjugation of this anti-viral drug with a model protein. The phosphorylation of adefovir allows the conversion of α-position functional group of the terminal phosphorus from methylene (-CH2-) to oxygen, efficiently inhibiting a formation of transition zwitterion, which frequently occurs in phosphonoimidazolide based intermediates and causes their hydrolytic instability. Mass spectrometry was used to monitor the outcomes of individual steps in the adefovir’s activation, allowing us to understand why the classical conjugation chemistry failed in this drug. Use of mass spectrometry was also critical in selecting the optimal conditions for the coupling of adefovir monophosphate to the model protein, as the conditions favoring higher conjugation yield (and higher drug-to-protein loading ratio) were shown to also lead to formation to a large amount of dead-end cross-links and mixed products. A reasonable compromise between modest conjugation yields (27%) and product purity (almost exclusively 1:1 conjugate) was found; however, the conjugate was shown to exhibit significant structural micro-heterogeneity due to distribution of the conjugation sites among 12 protein site chains. This exceeds the number of primary amines (the intended targets of the conjugation reaction), suggesting involvement of other group(s) in conjugation. Fragmentation patterns of the conjugates activated by collisions in the gas phase provide unequivocal evidence that primary alcohols (side chains of Ser and Thr) also participate in conjugation reactions. The novel synthetic strategy developed in this work can be applied to other anti-viral drugs (such as tenofovir, which exhibits a structural template similar to adefovir, including the presence of a phosphonate group, as well as other members of acyclic nucleoside phosphonate family). A work is currently underway in our laboratory to apply the new synthetic strategy to conjugate adefovir to a hemoglobin/haptoglobin complex, enabling targeted delivery of this drug directly to cellular reservoirs of HIV. Catabolic processing of the hemoglobin-haptoglobin complexes occurs inside the lysosomes. These organelles contain a number of enzymes allowing them to effectively degrade a variety of biomolecules, including proteins and nucleic acids. In particular, cells of monocyte/macrophage lineage contain a specialized acid phosphatase (tartrate-resistant acid phosphatase, sometimes also referred to as macrophage acid phosphatase or type V acid phosphatase47), which not only hydrolyzes a wide range of phosphates, but also dephosphorylates proteins.48 Perinuclear localization of the majority of lysosomes,49 as well as the presence of nucleoside-specific porters50 are likely to facilitate the transfer of the nucleoside analogs cleaved off their carriers (hemoglobin and/or haptoglobin) from lysosomes to their ultimate destination, the cell nucleus. ASSOCIATED CONTENT Supporting Information Proposed mechanism of formation of N-acyl-N-3-dimethylaminopropyl carbamic acid (NDCA) and



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Page 14 of 27

N-acyl-N-3-ethyl carbamic acid (NECA) derivatives in production of Lz/PMEA conjugates in pH 8.7 condition. AUTHOR INFORMATION Corresponding Author: Igor A. Kaltashov, Department of Chemistry, University of MassachusettsAmherst, 240 Thatcher Way, Life Science Laboratories N369, Amherst, MA 01003. Tel: (413) 545-1460; email: [email protected] Notes: The authors declare no competing financial interest. ACKNOWLEDGMENTS The authors are grateful to Dr. Khaja Muneeruddin (presently at University of Massachusetts Medical School) for help with setting up online cation exchange chromatography mass spectrometry (carried out in the Mass Spectrometry Center at UMass-Amherst), to Dr. Cedric E. Bobst (UMass-Amherst) for helpful discussions.


Page 15 of 27

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Schemes Scheme 1. Activation of PMEA with CDI in DMF (top), formation of PMEA-Im transition zwitterion (middle) and formation of dimeric form of PMEA (via pyrophosphonate bond) (bottom). Scheme 2. Phosphorylation of PMEA. Scheme 3. Activation of PMEAp with EDC in aqueous solution (top), conjugation of PMEAp with primary amine (middle) and hydroxyl (bottom) groups on protein surface. Figure legends Figure 1. Hydrolytic instability of PMEA-Im in the presence of H2O as exemplified by the ESI mass spectra (negative ion mode) of this activated form of adefovir in DMF (brown trace) and DMF/H2O mixture, 97.5:2.5 v:v (blue). See Scheme 1 for structures of species whose peaks are labeled in the mass spectra. The mass spectrum of the phosphorylated form of PMEA (PMEAp) in DMF is shown in black. Figure 2. ESI mass spectra of the products of Lz conjugation with PMEAp carried out at pH 8.7 (red trace) and 6.8 (blue trace). The inset shows a zoomed view of ions with charge state +9: the base peak at m/z 1590.4 corresponds to intact Lz; mass shift of 335.2 Da corresponds to conjugation of a single PMEAp to the protein, while additions of 71.1, 128.2 and 199.3 Da represent Lz’s N-acyl-N-3-ethyl carbamic acid derivative, N-acyl-N-3-dimethylaminopropyl carbamic acid derivative and their combination respectively. Figure 3. Total ion chromatogram (black trace) of Lz/PMEA conjugates produced at pH 6.8 and extracted ion chromatograms for the unmodified protein (blue), 1:1 conjugate (purple) and 2:1 conjugate (red) plotted with different magnification factors (the m/z values indicate the ions selected for plotting each XIC). The inset shows zoomed views of ion peaks at charge state +8 from the mass spectra averaged across the elution windows as indicated on the panel (peaks labeled with stars correspond to sodium adducts). Figure 4. Mass spectrum of fragment ions produced by collisional activation of 1:1 Lz/PMEA conjugates (m/z 1627.6). The diagrams in the inset show structure of observed fragment ions (fragments that can originate from precursors where PMEAp is conjugated either to primary amines or alcohol hydroxyls are labeled in red, while the fragments that can originate exclusively from precursors where PMEAp is conjugated to alcohol hydroxyls are labeled in blue).



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 ACS Paragon Plus Environment

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relative abundance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 27

352

100

527 322

75

50

432

272 25

0 200

250

300

350

400

450

500

m/z


Xu and Kaltashov, Figure 1

Page 21 of 27

+9

100

+ 335.2 Da

+10

+8

50

25

1600

0

1250

1500

1750

2000

2250

+ 334.8 Da

+ 71.1 Da + 127.8 Da + 198.9 Da

75

+ 71.1 Da + 128.2 Da + 199.3 Da

relative abundance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48


1620

1640

1660

1680 m/z

m/z




4.00

1.00

3.00

2.00

+ 335 Da

+ 335 Da

58-59 min

48.5-49.5 min 34-35 min

0.75

0.50

1780 1800 1820 1840 1860 1880 m/z

m/z 1789 1.00

m/z 1831 (x 2)

0.25

total ion count, 107 cts.

total ion count, 107 cts.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

Page 22 of 27

m/z 1873 (x 20) 0.00

30

35

40

45

50

55

60

0.00

elution time, min.



Page 23 of 27

NH2

1796.8 8+

1786.8

274

N

+

354

80

N

+

O O O P O P OH OH

N

O

NH2

60

N N

OH O H O N P O P OH OH

40

N N

1796.8

NH2

1796.88+ 274+

O

NH2

1597.39+

N N

O H O N P O P OH OH

354 400

2730

OH

20 274.1

0 200

1627.6 (+9)

N

1786.8

100

8+

1597.3

relative abundance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48


600

800

N N

1780

1790

1800

m/z

O

1000 1200 1400 1600 1800

m/z




PMEA activation with CDI PMEA-Im (m/z 322)

NH2

: :

P O:

H N

+

N

N

22oC

O

N

N

CDI, TEA

O

OH

O

N

+

CO2

N

P N OH

of PMEA-IM transition zwitterion and hydrolysis N

N N O P N :: :O H

H2O

N O

N

N

N

O P HN

N

N

PMEA (m/z 272) O

N

:O:

H N

+

O

N

P O:

: :

NH2

NH2

:

OH

of PMEA dimer NH2

: :

O:

:O

P HN

:

:O:

P OH N

O

N

N N

N

N NH2

N N

:O :

O

P HN

:O:

O

HO P O

N

N

N

PMEA dimer (m/z 527)

N N

NH2 N N

N O

O

O

P O P

:O:

:

O

N

NH2

NH2

:

N

N

:

N

:

1 NH 2 2 N N3 4 N N 5 6 O 7 8 9 10 11 12 13 14 Formation 15 16 17 NH 18 2 N N19 20 N 21N 22 O 23 24 25 26 27 28 Formation 29 30 31 32 33 34 NH2 35 N N 36 37 N N 38 39 O 40 41 42 43 44 45 46 47 48

Page 24 of 27

N N

H N N

O

OH

O


Xu and Kaltashov, Scheme 1

Page 25 of 27


Monophosphorylation of PMEA

:

PMEApp (m/z 432)

NH2 N

N N

N

+ O

O O O P O P O P O: OH OH OH

H N N

: :

: :

:

: :

1 NH2 NH2 2 PMEAp N 3 N N N (m/z 352) H 4 anhydrous phosphoric acid N N N N 5 N + 22oC N 6 O O O O 7 O N P O P O: P HN 8 OH OH : O: 9 10 11 12 13 14 Diphosphorylation of PMEA 15 16 NH2 17 NH2 N N 18 N N anhydrous phosphoric acid 19 CDI, TEA N N N 20 N 22oC 22oC O O 21 O O O N 22 O P O P N P O P O: 23 OH : O: OH OH 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 ACS Paragon Plus Environment 47 48


PMEAp activation with EDC and imidazole


: :

NH2 1 NH2 2 N N N N 3 EDC, imidazole N N N 4 N 5 O O O O 6 O N O O P N P P O P OH 7 OH OH OH OH 8 9 10 11 12 13 14 PMEAp conjugation with primary amine groups from protein surface 15 16 NH2 17 NH2 NH2 18 N N N N 19 20 N N N N 21 O O O O 22 O O N P O P N P O P N 23 H OH OH OH OH 24 25 26 27 28 29 30 PMEAp conjugation with hydroxyl groups from protein surface 31 32 NH2 33 NH 2 34 N N N 35 N : O 36 N N N 37 N O O 38 O O O O N O P O P 39 P O P N OH OH 40 OH OH 41 42 43 44 45 46 ACS Paragon Plus Environment 47 48

Page 26 of 27


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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ToC graphic 80x43mm (300 x 300 DPI)


Overcoming the Hydrolytic Lability of a Reaction Intermediate in

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