Manipulation of Glutathione-Mediated Degradation of Thiol

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Manipulation of Glutathione-Mediated Degradation of Thiol-Maleimide Conjugates Haocheng Wu, Paige J. LeValley, Tianzhi Luo, April M. Kloxin, and Kristi L. Kiick Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00546 • Publication Date (Web): 04 Oct 2018 Downloaded from http://pubs.acs.org on October 5, 2018

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Bioconjugate Chemistry

Manipulation of Glutathione-Mediated Degradation of ThiolMaleimide Conjugates Haocheng Wua, Paige J. LeValley,b Tianzhi Luoa, April M. Kloxin,a,b Kristi L. Kiicka,c,* a

Department of Materials Science and Engineering, University of Delaware, Newark, DE, 19716, USA Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE, 19716, USA c Delaware Biotechnology Institute, Newark, DE, 19711, USA b

Abstract: The retro Michael-type addition and thiol exchange of thioether succinimide click linkages in response to thiol-containing environments offers a novel strategy for the design of glutathione-sensitive degradable hydrogels for controlled drug delivery. Here we characterize the kinetics and extent of the retro Michael-type addition and thiol exchange with changes in both the pKa of the thiols and the identity of N-substituents of maleimides. A series of N-substituted thioether succinimides were prepared through typical Michael-type addition. Model studies (1H NMR, HPLC) of 4-mercaptophenylacetic acid (MPA, pKa 6.6) conjugated to N-ethyl maleimide (NEM), Nphenyl maleimide (NPM) or N-aminoethyl maleimide (NAEM) and then incubated with glutathione showed half-lives of conversion from 3.1 h to 18 h, with extents of conversion from approximately 12% to 90%. The variations in the rates of exchange and hydrolytic ring opening appear to be mediated by resonance effects, electron-withdrawing capacity of the N-substituted moiety, as well as the potential for intramolecular catalytic hydrogen bonding of amine substituents with water (particularly in the case of ring opening). Further model studies of 4-mercaptohydrocinnamic acid (MPP, pKa 7.0) and N-acetyl-L-cysteine (NAC, pKa 9.5) conjugated to selected N-substituted maleimides and then incubated with glutathione showed half-lives of conversion from 3.6 h to 258 h, with extents of conversion from approximately 1% to 90%. A higher pKa of the thiol decreased the rate of the exchange reaction and limited the impact of other electronic effects of N-substituents on the extents of conversion. Additional factors affecting the conversion kinetics were studied on NEM conjugates. The kinetics of the retro Michael-type addition and exchange reaction were not hindered by thiol traps of lower pKa, but were retarded in conditions of lower pH. These studies shed light into details of thiol and maleimide design that could be used to tune the rates of degradation of drug and polymer conjugates for a variety of applications.

maleimides are most frequently utilized as they react quickly as Michael acceptors, yield no by-products and have high thiol specificity; the nitrogen atom in the ring allows facile functionalization and many such functionalized maleimides are commercially available. The applications of N-substituted maleimides include performing measurements of thiols in biological fluids,12 in vivo imaging studies,13, 14 structural and functional studies of proteins15-17 and synthesis of protein-drug conjugates,18, 19 as well as the development of bioconjugates such as artificial metalloenzymes20 and biosensors.21 Recent reports have demonstrated the self-healing ability of polymeric materials with thiol-Michael crosslinks at elevated pH or elevated temperature, introducing the utility of this chemistry toward developing coatings or elastomers.22-24 Others have utilized bromomaleimides to impart reversible modification of thiols for controlled bioconjugation formulations.25 Of additional interest in the bioconjugation field is the use of the thiol-maleimide reaction in the cross-linking of hydrogels, the fluorescent labeling of molecules and, more recently, for exploiting the capacity of the thioether succinimides bonds to undergo retro Michael-type addition reaction in thiol-rich environments.26 As reported, selected thioether succinimides can undergo retro Michael-type addition and subsequent thiol exchange reactions in the presence of other thiol compounds such as glutathione at physiological pH and temperature. Figure 1 shows the reversibility of the Michael-type addition reaction between maleimide and thiols under certain conditions as a potential controlled degradation mechanism.26 The first step is a retro

Introduction Thiol-sensitive biodegradable polymers and bioconjugates that serve as intracellularly triggered drug and gene delivery systems have attracted much attention.1-3 By introducing thiol-sensitive bonds in general, hydrogel degradation and/or drug release exploits intracellular or intratumoral variation in the concentration of naturally existing reducing agents, such as glutathione (GSH),4 to trigger release. Typically, the extracellular concentration of glutathione in plasma is approximately 2–20 μM, whereas the concentration in cytosol, mitochondria and cellular nuclei is well above that, in the range of 0.5–10 mM,5 with an elevated level of GSH in carcinoma cells in particular.6 This presence of distinct concentrations facilitates the triggered intracellular disruption of conjugates and hydrogels while offering a degree of stability in circulation.5-7 The most common motif in the design of thiolsensitive bioconjugates involves the incorporation of disulfides, which can undergo thiol-disulfide exchange under reductive conditions to achieve rapid cleavage, at time scales ranging from minutes to hours.8-9 The window over which the cleavage kinetics can be engineered is considerably limited even by altering the disulfide’s neighboring chemical substituents.10 In contrast to disulfide-based bioconjugation strategies, the thiol-maleimide Michael addition reaction has been widely implemented in biological systems, primarily due to the selectivity of the thiol-maleimide reaction in aqueous environments, the rapid kinetics associated with the reaction, and the stability of the thiol-maleimide product.11 N-substituted

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Michael-type addition to regenerate the free maleimide 2, which can then react with other thiols in the system, generally glutathione. The equilibrium is significantly shifted to favor the thioether succinimides 1 and 3. Meanwhile, both thioether

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succinimides 1 and 3 can undergo hydrolysis and the products respectively then no longer undergo the retro Michael-type addition.

Figure 1. Mechanism for the glutathione-mediated retro Michael-type addition reaction for a thioether succinimide adduct. Our group has previously studied the kinetics of the retro Michael-type addition and exchange reactions undergone by select thioether succinimides in the presence of excess glutathione,26 highlighting the potential of employing the strategy for controlled release of drugs or degradation of materials. These early studies indicated that the kinetics and extent of the retro Michael-type addition and thiol exchange is significantly dependent on the nucleophilicity of the Michael donor; aryl thiol substituents with a lower pKa favored the retro and exchange reaction while alkyl thiol substituents with a higher pKa yielded thioether succinimides that were fully stable under the conditions tested. Highlighted in the study, the arylthioether-succinimide adducts exhibited a cleavage rate 10–100 × lower than that reported for the similar cleavage of disulfides while more rapid than that of the cleavage of certain cysteine-maleimide adducts. The conversion for reaction of the reported arylthioethersuccinimide adducts with GSH reached approximately 90% over a few days. The retro Michael-type addition reactions thus provide similar and complementary applications to the disulfide-mediated release of drugs in reducing environments,26-29 but with increased blood stability and prolonged drug delivery timescales relative to disulfide moieties.30 Degradation strategies that exploit the retro Michael-type reaction of thioether succinimides may find biomedical applications when a slower release or degradation profile is required. The competing hydrolysis reaction can also be employed

to desired effect, as the ring-opened product is stabilized against cleavage. Fontaine et al.31 studied the rates of ring-opening hydrolysis and thiol exchange of a range of N-substituted thioether succinimides formed by maleimide−thiol conjugation, suggesting conjugates made with electron-withdrawing maleimides can be purposefully hydrolyzed rapidly in vitro to ensure in vivo stability. In fact, recent reports utilized a phenyl Nsubstituted maleimide to induce rapid maleimide ring opening (complete conversion observed within 2 h) to improve the stabilization of thiol-maleimide bioconjugates.32 The research focusing on either utilizing or avoiding retro Michael-type addition and exchange reaction of thioether succinimides indicates a dependence of kinetics of the competing reactions—thiol exchange and hydrolysis—on the nature of both thiol substituents and N-substituents of maleimides. In this work, we aim to expand this understanding of the combined effects relevant in retro Michael-type addition and exchange reactions. A series of model compounds were designed to permit probing of the effect of thiol pKa, thiol traps and the strength of the electronic effects of Nsubstituted groups. The conversion of thioether succiminide was monitored in a series of 1H NMR33 and HPLC assays to characterize the rates of the relevant reactions in the exchange. This understanding will enable the design of thiol-mediated degradable materials for specific advanced applications.

reactions when the N-substituents on the maleimide moiety were varied. MPA was selected as the standard thiol moiety owing to our previously reported observation of its ability to undergo retro Michael-type addition and thiol exchange with GSH under relevant conditions. Kinetics experiments were performed via 1H NMR spectroscopy, by monitoring the intensity of the protons in the aromatic region (6.9-7.5 ppm) as a function of time after addition of GSH. The formation of the Michael-type adducts and their degradation products were easily observed in the NMR spectra. Figure 2 shows the 1H NMR analysis for the reaction of MPA with NAEM, with subsequent incubation with GSH, with Figure 2A showing the 1H NMR spectrum for MPA alone in solution. A majority of MPA was in the reduced state, as indicated by the observed main peaks at chemical shifts centered at 6.92 and 7.18 ppm. Minor peaks centered at 7.14 and 7.42 ppm indicate the small amount of oxidized MPA present. After addition of a molar equivalent of NAEM to the solution, the aromatic protons shifted downfield to peaks centered at 7.20 and 7.39 ppm (Figure 2B), indicating the formation of MPA-NAEM, along with a small amount of ring-opened product (7.15 and 7.34 ppm). The small fraction of oxidized MPA was immediately reduced when a molar equivalent of GSH was added, indicated by a shift of peaks from 7.14 and 7.42 ppm to 6.92 and 7.18 ppm (Figure 2C). After 24 h incubation at 37 OC, much more free MPA in the reduced state was liberated from the conjugate as indicated by the increased intensity of aromatic protons centered at 6.92 and 7.18 ppm (Figure 2D); the slight downfield shift of the protons of the free

Results and discussion Synthesis of Thioether Succinimide Conjugates The thioether succinimide conjugates were successfully prepared through a typical Michael-type addition reaction with the base-initiated mechanism; a catalytic amount of weak base TEA was used to deprotonate available thiol. The resulting thiolate anion, a strong nucleophile, attacks the π-bond of maleimide, resulting in a strongly basic enolate intermediate. This intermediate deprotonates an additional equivalent of thiol, giving the desired addition product as well as another equivalent of thiolate that can perpetuate the catalytic cycle.34 Three different thiols (MPA (pKa 6.6), MPP ( pKa 7.0) and NAC (pKa 9.5)) were selected as Michael donors, and three different maleimides (NEM, NPM and NAEM, each with different electronic effects of the Nsubstituents) were selected as Michael acceptors. The resulting nine different thioether succinimide conjugate products were synthesized as confirmed via 1H NMR (Figure S1-S9), with a yield of approximately 90% for all reactions. 1

H NMR Analysis of MPA-Maleimide Retro Reactions

After obtaining the series of thioether succinimide conjugates, we sought to validate that retro Michael-type additions were a significant reaction for select thioether succinimides in reducing environments and to determine the difference in thiol exchange


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MPA in Figure 2D likely results from changes in the pH of the solution upon the ring opening that occurs after the addition of NAEM After 5 d of incubation (Figure 2E), oxidized MPA (7.14 and 7.42 ppm) and GS-MPA (7.18 and 7.45 ppm) started to form, and after 10 d of incubation, more GS-MPA was accumulated (Figure 2F). With more and more GSH being consumed for the retro reaction and MPA reduction, the free MPA liberated from

the adduct via retro reaction was again converted to its oxidized form and GS-MPA. These results are comparable to those in the kinetics studies previously reported by our group.26 The 1H NMR spectra for MPA-NEM and MAP-NPM are shown in Figure S10 and Figure S11, respectively. The results show a very similar process of conjugate formation, retro reaction and hydrolysis for MPA-NEM and MPA-NPM.

Figure 2. 1H NMR of aromatic protons of (A) MPA (17.8 mM) in 0.2 M phosphate buffer in 10% D2O (pH = 7.4), (B) after addition of NAEM (17.8 mM) to MPA, (C) immediately after addition of GSH (17.8 mM) to (MPA+NAEM), and after incubation of (C) at 37 OC for (D) 24 h, (E) 5 d and (F) 10 d. Evident in (D) is free MPA (at a slightly more downfield position versus that in (A) owing to changes in solution pH upon ring opening (Figure S12)) and in (F) are large amounts of ring-opened MPA-NAEM (7.15, 7.34 ppm) and GS-MPA mixed disulfide (7.45, 7.18 ppm). The fate of the thioether succinimide conjugate 1 can be simplified to the combination of hydrolysis (1→1RO) and retro and exchange reaction (1→3), followed by the ring-opening of 3 (3→3RO). Pseudo first-order rate constants, k1 can be defined for the ring-opening of 1, and since there is a large excess of the thiol trap (10 mM GSH or 5 mM GSSG), k2 can be defined as the pseudo first-order rate constant for the retro and exchange reaction of 1. HPLC and LC/MS were used for quantification and identification in the study of reaction kinetics. These maleimides with different N-substituents (ethyl, phenyl, aminoethyl, respectively) were selected to determine how the rate of exchange might vary with the electronic effects of the N-substituents. A 0.1

HPLC Evaluation of Reaction Kinetics Figure 3 illustrates the complete set of reactions for an initial thioether succinimide conjugate 1 in the presence of excess GSH, showing ring-opening of the thioether succinimides as well as the exchange with GSH. Since the exchange reaction is dependent on the concentration of GSH in the system, excess GSH was used to ensure that kinetic data for the exchange and hydrolysis reactions could be modeled using pseudo first order reaction kinetics.26, 28 Throughout our experiments (see below), no measurable amount of free maleimide 2 was detected, confirming that equilibrium favors the thioether succinimide under these experimental conditions and that any liberated maleimide from the retro Michael-type addition is rapidly consumed by reaction with GSH.


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mM solution of 1 was incubated in 10 mM GSSG in phosphate buffer at pH 7.4 and 37 OC. The ring-opening rates of the thioether succinimide were determined from solution 1 without addition of GSSG. Reaction kinetics for the formation of 3 were determined by monitoring the changes of the peak areas in the HPLC traces, and the identity of the chemical compounds in each

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fraction was confirmed by LC/MS. Representative data for a typical set of HPLC traces monitoring the incubation of 1 (for MPA-NEM, MPA-NPM, and MPA-NAEM) are shown in Figures S13-S15. It is clear in the data that the initial single peak of 1 decreased in intensity over time via its conversion to 1RO (ringopened 1) and to 3, with subsequent conversion of 3 to 3RO.

Figure 3. Chemical reaction pathways for the complete set of retro and ring-opening reactions of thioether succinimide conjugates in solution with glutathione. Inset is the simplified reaction scheme used to calculate the reaction rate constants for the ring-opening (k1) and the retro (k2) reactions. There were no observable levels of 2 in solution so the accumulation of 3 was utilized to calculate the rate of the retro reaction as depicted in the simplified reaction scheme. Fractional concentrations of 1, 1RO, 3, 3RO (converted to the initial thioether succinimide linkage of 1) measured by HPLC were plotted as a function of time for MPA-NEM (Figure 4A), MPA-NPM (Figure 4B) and MPA-NAEM (Figure 4C). Pseudo first-order rate equations26 were employed to fit the data (R2 > 0.98) and rate constants were then obtained. The values of ringopening rate constant k1 and retro reaction rate constant k2 (with their corresponding half-lives) are shown in Table 1. By input of rate constants into the equations, curves were constructed to plot the fractional concentration of each compound as a function of time (Figure 4). The fits show perfect agreement with data points for all compounds. We note that only the sum of the fractional concentration of 3 and 3RO are shown in Figure 4C, as the corresponding peaks for each were not separately observable in the HPLC experiment. The fractional concentration of 1 decreased as a function of time and was eventually all consumed. The fractional concentration of 3 showed an initial increase and subsequent decrease, depending on the extent of retro reaction and hydrolysis. When all reactions reach equilibrium, the end products 1RO and 3RO were completely stable in solution. The final fractional concentrations of 1RO (red plateau in Figure 4) and 3RO (green/purple plateau in Figure 4) were directly related to k1 and k2: [3RO]f/[1RO]f = k2/k1, in which the ratio indicates whether the retro and exchange reaction or the ring-opening of 1 predominates in solution.

In the reactions of MPA-NAEM, 1RO is apparently the major product at equilibrium, with a rate constant k1 of 0.9828 h-1 and respective half-life of 0.7 h. In comparison, the ring-opening of MPA-NEM is three orders of magnitude slower, with an apparent rate constant k1 of 0.0045 h-1 and respective half-life of 154 h. The ring-opening of MPA-NPM, in contrast, is only one order of magnitude slower than that of MPA-NAEM, with an apparent rate constant k1 of 0.1638 h-1 and a half-life of 4.2h The dramatic differences in the ring-opening rates of the three thioether succinimides are most likely attributable to a combination of inductive and hydrogen bonding effects of the N-substituents of the maleimide moieties. The protonated aminoethyl groups at physiological pH, capable of hydrogen bonding with water, would be expected to promote hydrolysis, which facilitates rapid ring opening of MPA-NAEM. The alkyl N-substituted groups such as the ethyl group are not capable of such intramolecular catalytic effects, and in addition are weak electron-donating groups, which retards the ring-opening of MPA-NEM. The ring-opening of MPA-NPM is only 6-fold faster than that of MPA-NEM, likely owing to the weak electron-withdrawing effect of the phenyl groups. Our previous report has shown similar pseudo first-order rate constants k1 for the ring-opening of NEM conjugates (NEM conjugated to MPA, NAC and 3-mercaptopropionic acid), which range from 0.0032 h-1 to 0.0044 h-1.26


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Kanaoka and coworkers have reported that the hydrolysis halflife of NPM (1.1 min) determined at pH 7.0 and 30 OC is more than one order of magnitude smaller than that of NEM (25 min), suggesting that electronic factors introduced by aromatic substitution on the imide nitrogen is reflected in the accelerated rate of hydrolysis.35 Lyon et al.36 reported the hydrolytic enhancement of the ring opening of an NAEM by a primary amine closely positioned to the succinimide, and proposed a mechanism involving intramolecular catalysis of the hydrolysis by the hydrogen bonding of the amine with water. Fontaine et al.31 have shown recently the ring-opening of a series of thioether succinimides N-substituted by protonated amines incubated at pH 7.4 and 25 OC, with half-lives ranging from 0.41 h (NAC-NAEM) to several hours. In this report the electron withdrawing capability of the substituent was attributed as the primary cause of differences in hydrolytic ring opening, However, the reported rates of ring opening for thioether succinimides with aminefunctionalized N-substituents were approximately 4-fold greater than those observed for conjugates with quaternary ammonium N-

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substituents, also suggesting the additional effect of intramolecular hydrogen bonding of the amine with water to catalyze hydrolysis. To confirm the potential origin of the substituent effects for the compounds investigated here, we prepared and characterized an MPA conjugate with N-[2-(trimethylammonium)ethyl]maleimide (MPA-NMAEM) and compared its rate of ring opening with that of the other MPA conjugates. The most rapid rate of hydrolytic ring opening was observed for the MPA-NAEM conjugate, which was approximately 10-fold greater than that for the MPANMAEM (Figure S17), consistent with the role of intramolecular hydrogen-bonding in the hydrolytic events for the MPA-NAEM as described in the previous reports of Lyon et al. The MPANMAEM showed similar rates of ring opening as the MPA-NPM, which were 20-40 fold greater than those observed for the MPANEM. This trend is likely attributable to the electron withdrawing character of the NPM and NMAEM substituents in contrast to the electron donating character of the NEM, consistent with the reports of Fontaine et al.

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Time (h) Figure 4. MPA-NEM, MPA-NPM, and MPA-NAEM thiol-maleimide conjugates were synthesized and subsequently dissolved in 50 mM phosphate buffer with 5 mM GSSG (pH = 7.4) at a concentration of 0.1 mM. Fractional conversion to the thioether succinimide, over time, for (A) MPA-NEM, (B) MPA-NPM and (C) MPA-NAEM. Relative concentrations were measured via HPLC and the curves constructed using the derived rate constants and integrated fractional concentration equations for 1, 1RO, 3 and 3RO.


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Table 1. Pseudo first-order rate constants and respective half-lives for retro and ring-opening reactions of thiol-maleimide conjugates. Data for conjugates dissolved in 50 mM phosphate buffer containing 5 mM GSSG (pH = 7.4) at a concentration of 0.1 mM.

* k2/k1 > 1 indicates exchange reaction dominates in the solution; k2/k1 < 1 indicates that the ring-opening reaction dominates in solution. Apart from hydrolysis, we observed that the retro and exchange reactions were also impacted by the electronic character of the Nsubstituents. In the reactions of MPA-NEM, 3RO is the major product at equilibrium indicating a high extent of exchange, with a retro reaction rate constant k2 of 0.0382 h-1 and a respective halflife of 18 h. By contrast, the rate constant k2 of MPA-NPM and MPA-NAEM are one order of magnitude larger, an expected result when considering that the retro-Michael-type addition reaction is a ߚ-elimination reaction involving proton abstraction and ߚ-thiolate release from the succinimide thioether.31 Electronwithdrawing effects of N-substituents thus might be expected to increase the acidity of the dissociable C–H bond, and thus the rate of ߚ-elimination. Also of note, the retro and exchange reaction of MPA-NPM was even 1.6-fold faster than that of MPA-NAEM, which is likely attributable to the resonance effect of the phenyl group, which could promote ߚ-elimination. MPA-NEM, lacking any of these N-substituent effects, thus shows the slowest rate of exchange (and ring-opening, see above). The conversion of the three MPA-maleimide conjugates to glutathione adducts, which also takes into account the hydrolytic ring opening that precludes exchange, is shown in Figure 5. The fraction of 3 generated from 1 is determined by the progress of two competing reactions and thus is equal to k2/(k1 + k2). The MPA-NEM supported the greatest conversion ratio of the initial adduct, reaching maximum conversion of 89.5% after approximately 110 h. The MPA-NPM exhibited the next greatest

extent of conversion (58.3%), and also the most rapid kinetics, achieving a maximum conversion within 12 h. The MPA-NAEM supported the lowest conversion ratio (owing to extensive hydrolytic ring opening) and a medium exchange rate, achieving a conversion of 12.3% after approximately 4 h, while MPANMAEM (Figure S17) showed both low conversion (owing to hydrolytic ring opening) and slow exchange. In the case of the exchange reaction, the resonance effect of the N-phenyl group may contribute to the most rapid thiol exchange with a half-life of 3.1 h for MPA-NPM. In the reports of Fontaine et al.31 the pseudo first-order rate constant k2 for the retro reactions of thioether succinimides with protonated amine or quaternary amine Nsubstituents show short half-lives which range from 12 h to 61 h. These data are consistent with our reports and confirm the rate enhancement of the retro and exchange reaction by the electronwithdrawing character of the N-substituents, although for applications this must be balanced by the rate of hydrolytic ring opening (which precludes exchange). On balance, therefore, thioether succinimides with alkyl or aromatic N-substituents would thus generally be beneficial for the design of degradable linkers in which exchange would dominate (higher k2/k1, Table 1), while those with electron withdrawing and/or hydrogen-bonding character would be desirable for applications in which stability of the linkage (via conversion to the ring-opened product, lower k2/k1) is desired.


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Figure 5. The conversion of thioether succinimides with various N-substituted groups on the maleimide moiety (MPA-NEM, MPA-NPM and MPA-NAEM) to glutathione adducts via retro Michael-type addition and exchange reaction. (Conjugates incubated at 0.1 mM in 50 mM phosphate buffer with 5 mM GSSG (pH = 7.4), EDG: electron-donating group, EWG: electron-withdrawing group, asterisk indicates the hydrogen-bonding capability of the amine-functionalized NAEM, which also affects the overall extent of conversion.) We next sought to investigate the combined effects on the retro and ring-opening reactions of thioether succinimides by altering the pKa of the thiols as well as the N-substituents of maleimides (ethyl, phenyl, and aminoethyl substituents). All other experimental conditions were identical to previous experiment unless otherwise noted. The fractional concentrations of 1, 1RO, 3, 3RO measured by HPLC (normalized to the initial thioether succinimide linkage of 1) were plotted and curves were constructed as a function of time for NAC-maleimide (Figure 6),

and MPP-maleimide (Figure S18). The values of the ring-opening rate constant k1 and the retro reaction rate constant k2 with their corresponding half-lives are shown in Table 1. The close pKa of MPA (pKa 6.6) and MPP (pKa 7.0) contributed to a similar set of kinetics data, as illustrated by comparisons of the data in Figure 4 and Figure S18, while the retro and exchange reaction for NAC (pKa 9.5) adducts (Figure 6) was apparently slower with longer half-lives up to 258 h.

Figure 6. NAC-NEM, NAC-NPM, and NAC-NAEM thiol-maleimide conjugates were synthesized and subsequently dissolved in 50 mM phosphate buffer with 5 mM GSSG (pH = 7.4) at a concentration of 0.1 mM. Fractional conversion to the thioether succinimide, over time, for (A) NAC-NEM, (B) NAC-NPM and (C) NAC-NAEM. Relative concentrations were measured via HPLC and the curves constructed using the derived rate constants and integrated fractional concentration equations for 1, 1RO, 3 and 3RO.


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A summary of the pseudo first-order rate constants and halflives for selected thioether succinimides are shown in Figure 7. In each diagram, the three groups of thiol adducts for comparison were fixed based on the identities of the thiols, which were MPA (pKa 6.6), MPP (pKa 7.0) and NAC (pKa 9.5); the thiol trap GSH has the highest pKa of 9.65 in solution. For each type of thiol adduct, the maleimides employed are indicated by different color bars in the bar graph (NEM, NPM and NAEM represented by navy, green and gray bars, respectively). Figure 7A shows the value of the ring-opening rate constant k1 for the thioether succinimide adducts, illustrating that for a given thiol, an increase in the electron-withdrawing capacity of the maleimide Nsubstituents increases the rate of ring-opening, with a dramatic additional effect for the amine-containing N-substituents where intramolecular catalytic hydrogen bonding is possible. In contrast, when the maleimide moiety is fixed (e.g., compare rates for bars of the same color), the difference in solubility of three thiols: S(NAC) > S(MPA) > S(MPP), is suggested to have a slight impact on the rate of ring-opening; an improved solubility of adducts would be expected to facilitate hydrolysis. Thus, the value of k1 is suggested to depend on the inductive and hydrogen-bonding effects of the N-substituents and is affected slightly by the adducts’ solubility. Figure 7B shows the value of k2 for the thioether succinimide adducts. Of the thiol adducts, those with phenyl groups showed the most rapid exchange reaction likely due to resonance effects, followed by the electron-withdrawing and hydrogen-bonding aminoethyl group. N-substituents that lack either of these features

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show the slowest rate of exchange. When the maleimide moiety is fixed, the rate of exchange of the initial adduct is clearly impacted by the thiol pKa, with higher pKa decreasing the rate (NAC > MPP > MPA). The value of k2 is thus indicated to be significantly affected by both the electronic effects of the N-substituents and the pKa of the respective thiols. Figure 7C summarizes the value of k2/(k1 + k2) for the various conjugates, which also represents the conversion ratio of initial adduct to glutathione adduct. Comparison of Figures 7B and 7C illustrates that since the pKa of the thiols has a greater effect on the value of k2 than it does on the value of k1, the pKa thus is a key determinant in setting the tunable range of the value of k2/(k1 + k2); the lower pKa values (MPA, MPP) yield the wider the tunable range. The electronic effects of N-substituents had significant impact on the values of both k1 and k2, and thus differentiated the value of k2/(k1 + k2) for a given thiol. Figure 7D shows the half-lives for the ring-opening reaction, which range from 0.7 h (NAC-NAEM) to 198 h (MPPNEM). Figure 7E shows the half-lives for the exchange reaction, which show a smaller range from 3.1 h (MPA-NPM) to 258 h (NAC-NEM). Fontaine et al.31 have shown comparable results that, for a series of thioether succinimides with amine substituents, the rates of the retro-Michael reactions span only 30fold, whereas ring-opening reactions span over 600-fold, which also indicates that the retro reaction is less sensitive than the hydrolysis of thioether succinimide to the effects of Nsubstituents.

Figure 7. Pseudo first-order rate constants and half-lives for selected succinimide thioether adducts (0.1 mM) incubated in phosphate buffer containing 5 mM GSSG (pH = 7.4). (A) Ring-opening rate constant k1, (B) retro and exchange reaction rate constant k2, (C) conversion ratio of initial adduct to glutathione adduct: k2/(k1 + k2), (D) half-lives for k1, (E) half-lives for k2. The degradation kinetics of 0.1 mM thiol-NEM adducts in the presence of 5 mM GSSG or 10 mM NDC were studied to help understand the relative impact of thiol moieties and thiol traps in the retro and exchange reactions (Table 2). The kinetics profiles (Figure S19) are similar, as suggested by the comparison of the curves of 1 and 1RO for the same thiol-NEM adduct. The values of k1 and k2 are also on the same order of magnitude. The difference

is that 3 (NDC-NEM) generated in the presence of NDC ringopened faster than 3 (GS-NEM) generated in the presence of GSSG. Degradation profiles of NAC-NEM in the presence of NDC (Figure S19F) indicates that pKa of the thiol trap does not affect the occurrence of the retro and exchange reaction, even though the pKa of NDC (7.8) is much lower than that of NAC (9.5). These results are also consistent with the retro-Michael


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reaction mechanism: an increase in the leaving-group ability of the thiolate ions determines the occurrence of the retro reaction, which is correlated to the acidity (pKa) of the thiol moiety; a stoichiometric excess of a thiol trap drives the equilibrium toward thiol exchange. In our previous report, we have demonstrated that the kinetics of the retro-Michael addition and thiol exchange was independent of the use of GSSG or GSH as a thiol trap.26 Baker and coworkers have described the bromination of maleimides for reversible conjugation of thiols as a bioconjugate technique.25, 37

The cysteine-bromomaleimide conjugate was found to be reversible by incubation with excess thiols such as 2mercaptoethanol or GSH with complete conversion within 4 h.37 The results are consistent with our findings that the kinetics of the retro and exchange reaction is independent of the identity of the thiol trap. It also indicates that the reduced reactivity of maleimides compared with bromomaleimdies correlates with the rate of the retro reaction.

Table 2. Pseudo first-order rate constants and respective half-lives for retro and ring-opening reactions of thiol-NEM conjugates in 5 mM GSSG and 10 mM NDC thiol traps.

* k2/k1 > 1 indicates exchange reaction dominates in the solution; k2/k1 < 1 indicates ring-opening reaction dominates in the solution. Additional factors such as the pH of the solvent may further affect the kinetics of the reaction in solution reactions. Here the influence of lower pH on the retro reaction kinetics was studied (Table 3). Figure S20 clearly shows slower retro and ring-opening reaction kinetics for MPA-NEM incubated in solutions of lower pH. The value of k1 and k2 for MPA-NEM is one order of magnitude smaller at pH 6.0 and two orders of magnitude smaller at pH 5.0, compared to that at physiological pH. We noticed that k2/k1 remained unchanged with the reduction of solution pH to 6.0, which indicates the conversion ratio from initial adduct to glutathione adduct remained high with the added potential advantage that a longer release or degradation period can be

achieved at lower pH. These properties of the retro and thiol exchange reactions suggest their potential use in the acidic microenvironment (pH 6.5–6.9) of carcinoma cells.38 Specifically, the glutathione-sensitive thioether succinimide linkages may be employed in either the formation of hydrogels or the coupling of anticancer drugs to synthetic polymers, for the purpose of developing biodegradable polymer systems for controlled release of anticancer drugs.30, 39, 40 A longer release timescale could improve the efficacy and reduce the side effects of the drugs, as therapeutic levels of the desired anticancer agents might be maintained locally for prolonged periods while reducing systemic side effects.22, 23, 28, 32, 41, 42

Table 3. Pseudo first-order rate constants and respective half-lives for retro and ring-opening reactions of MPA-NEM incubated at different pH. Data for conjugates dissolved in 50 mM phosphate buffer containing 5 mM GSSG at a concentration of 0.1 mM

* k2/k1 > 1 indicates exchange reaction dominates in the solution; k2/k1 < 1 indicates ring-opening reaction dominates in the solution.

character and hydrogen bonding, in contrast, show slow rates of both hydrolysis and exchange. Interestingly, a low pKa of the thiols enhances this influence of the thioether succinimide Nsubstituents on the rate enhancements of the retro and exchange reactions, yielding a substantially wider tunable range of exchange kinetics. Thiols with higher pKa values show more minimal influence, on the extent of conversion, of the effects of the N-substituents. Our studies also illustrate that the occurrence and kinetics of the retro and exchange reaction are not affected by the pKa of a thiol trap, but can be retarded at lower solution pH.

Conclusions We have shown that the kinetics of the GSH-mediated retro Michael-type addition can be manipulated by modifying the Nsubstituents of the Michael acceptor along with the pKa values of the Michael donor. Thioether succinimides with N-substituents with hydrogen-bonding capability or electron-withdrawing groups favor a high level of hydrolytic ring opening and enhancement of the exchange reaction, respectively, while the resonance effect of N-phenyl groups in particular is suggested to contribute to faster thiol exchange. N-substituents that lack electron-withdrawing


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Our findings altogether thus suggest the joint impact of both Nsubstituents and identity of thiols for manipulating the kinetics of the retro Michael-type addition of maleimides and thiols: (i) the conversion ratio k2/(k1 + k2) must be significantly high to favor conversion; and (ii) the half-lives of kexchange should match the need of the specific application. In the case of tumor-targeted drug delivery, a prompt destabilization of drug carriers inside cells and rapid release (within hours) of the loaded therapeutics is preferred to enhance drug efficacy, overcome multi-drug resistance (MDR), and minimize drug and carrier-associated side effects;43 while for localized chemotherapy such as a site-specific delivery of doxorubicin (DOX) from hydrogels, a sustained and tunable release (over weeks) may be preferred for a long-term control of cytotoxicity of cancer cells in vivo.44 By imparting tunable glutathione-sensitive linkages into micelles, vessels, tethered drugs or other bioconjugates our reported observations should be widely applicable for tailoring conjugates for specific applications such as sensing,45 tissue engineering46 and drug delivery.47

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GSSG. A GSSG thiol trap rather than a GSH diluent was used in these studies for the convenience of not having to avoid thiol oxidation over long periods while yielding the same rates of hydrolysis and thiol release.26 Lower buffer concentrations were employed to permit quantitative detection by HPLC, and these lower concentrations were sufficient to maintain a constant pH throughout the experiment. The kinetics of thioether succinimide ring-opening were measured by monitoring reactions incubated without reductant. The pH values of all samples were verified and adjusted to 7.4 with 0.2 M NaOH if needed before incubation at 37 OC. 150 µL samples were collected periodically and added to 150 µL of 0.5% formic acid solution to reduce the pH and quench the retro and ring-opening reactions. Samples were stored at -20 O C until analyzed. RP-HPLC injections were conducted under the above-defined conditions and the areas of peaks were integrated to calculate conversion curves. The identities of the compounds present in each peak were determined using LC-MS. The same reactions of certain synthesized conjugates were also investigated under slightly different conditions to probe the effects of thiol traps and solution pH. Specifically, the 5 mM GSSG thiol trap was replaced by 10 mM NDC and the pH was maintained at 5.0 (50 mM acetate buffer, pH 5) and 6.0 (50 mM phosphate buffer, pH 6) respectively throughout the reactions. The kinetics model applied to the measured data was simplified to the combination of consecutive and parallel reactions (Figure 3 inset). Pseudo first-order rate constants k1 and k3 were defined for the ring-opening of 1 and 3; k2 is defined for the retro and exchange reaction of 1 to yield 3 in the presence of excess thiol. Reaction rate equations (see the SI for details) were employed to fit the data measured by HPLC, yielding k1, k2 and k3 with corresponding half-lives. Curves to plot the fractional concentrations of the compounds as a function of time were also constructed.

Experimental Section Materials N-ethylmaleimide (NEM), N-phenylmaleimide (NPM), N-(2aminoethyl)maleimide (NAEM), 4-mercaptophenylacetic acid (MPA), N-acetyl-L-cysteine (NAC), N-diethyl-cysteamine (NDC) were purchased from Sigma-Aldrich (St. Louis, MO) and used without further purification. 4-mercaptohydrocinnamic acid (MPP) was purchased from TCI America (Portland, OR, USA). All other reagents and materials including glutathione (GSH) and oxidized glutathione (GSSG) were purchased from Fisher Scientific (Pittsburgh, PA, USA) unless otherwise noted. 1H NMR spectra were acquired under standard quantitative conditions at ambient temperature on a Bruker AV400 NMR spectrometer (Billerica, MA, USA).

Associated Content

Synthesis of Thioether Succinimide Conjugates

Supporting information. Characterization data (1H NMR spectra, HPLC data), and details of the analysis of the fractional conversion of thiol-maleimide conjugates is available free of charge at http://pubs.acs.org.

The thiols including the mercapto-acids MPA and MPP, and the cysteine derivative NAC, were dissolved at a concentration of 50 mg/mL in 1 mL acetonitrile and reacted with molar equivalents of the maleimides, NEM, NPM and NAEM. A catalytic amount of triethylamine (0.01×) was added to the mixture. The reaction was stirred for 1 h at room temperature. The crude product was diluted to 5 mL with water and purified via reverse-phase HPLC (Waters Inc., Milford, MA) on a Waters Xbridge BEH130 Prep C-18 column. Purified fractions were collected and freeze–dried, with approximately 90% yield for all reactions.

Author Information Corresponding Author *Phone: +1 302 831 0201. [email protected]

1

H NMR Analysis of MPA-Maleimide Retro Michael-type Reactions 1

H NMR spectroscopy with W5 water suppression33 was used to monitor the retro reactions of MPA-maleimide conjugates. Samples of MPA were dissolved at a concentration of 3 mg/mL in 0.2 M phosphate buffer pH 7.4 with 10% D2O. High buffer concentrations were needed (relative to those employed in the HPLC experiments below) in order to maintain a constant pH throughout the experiment, owing to the high concentration of MPA necessary for NMR investigation. A molar equivalent of maleimide (NEM, NPM, NAEM) was added to each vessel and the NMR spectrum was recorded. Molar equivalents of GSH were added to the samples.26 After addition, the pH was adjusted to 7.4 with 0.2 M NaOH if necessary. Samples were incubated at 37 OC and spectra were recorded at 0 h, 24 h, 5 d and 10 d.

Fax: +1 302 831 4545. Email:

Acknowledgments This work was supported by the University of Delaware and the Delaware Bioscience Center for Advanced Technology (12A00448). This project was also supported (instrumentation) by grants from the National Institute of General Medical Sciences (5 P30 GM110758-02, 1 P30 GM103519, and P20 GM104316) at the National Institutes of Health (NIH). The contents of the manuscript are the sole responsibility of the authors and do not necessarily reflect the official views of the University of Delaware or the NIH. The authors also acknowledge Prathamesh Kharkar and Yingkai Liang for helpful discussions to initiate the project.

HPLC Evaluation of Reaction Kinetics Synthesized conjugates were dissolved at a concentration of 0.1 mM in 50 mM phosphate buffers (pH 7.4) containing 5 mM

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Manipulation of Glutathione-Mediated Degradation of Thiol

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