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Tunable degradation of maleimide-thiol adducts in reducing environments.

ABSTRACT: Addition chemistries are widely used in preparing biological conjugates, and in particular, maleimide-thiol adducts have been widely employed. Here, we show that the resulting succinimide thioether formed by the Michael-type addition of thiols to N-ethylmaleimide (NEM), generally accepted as stable, undergoes retro and exchange reactions in the presence of other thiol compounds at physiological pH and temperature, offering a novel strategy for controlled release. Model studies ((1)H NMR, HPLC) of NEM conjugated to 4-mercaptophenylacetic acid (MPA), N-acetylcysteine, or 3-mercaptopropionic acid (MP) incubated with glutathione showed half-lives of conversion from 20 to 80 h, with extents of conversion from 20% to 90% for MPA and N-acetylcysteine conjugates. After ring-opening, the resultant succinimide thioether did not show retro and exchange reactions. The kinetics of the retro reactions and extent of exchange can be modulated by the Michael donor's reactivity; therefore, the degradation of maleimide-thiol adducts could be tuned for controlled release of drugs or degradation of materials at time scales different than those currently possible via disulfide-mediated release. Such approaches may find a new niche for controlled release in reducing environments relevant in chemotherapy and subcellular trafficking.

SUBMITTER: Baldwin AD

PROVIDER: S-EPMC3220410 | biostudies-literature | 2011 Oct

REPOSITORIES: biostudies-literature

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Tunable degradation of maleimide-thiol adducts in reducing environments.

Baldwin Aaron D AD Kiick Kristi L KL

Bioconjugate chemistry 20110926 10

Addition chemistries are widely used in preparing biological conjugates, and in particular, maleimide-thiol adducts have been widely employed. Here, we show that the resulting succinimide thioether formed by the Michael-type addition of thiols to N-ethylmaleimide (NEM), generally accepted as stable, undergoes retro and exchange reactions in the presence of other thiol compounds at physiological pH and temperature, offering a novel strategy for controlled release. Model studies ((1)H NMR, HPLC) o ...[more]

PMID: 21863904

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Project description:Michael-type addition reactions are widely used to polymerize biocompatible hydrogels. The thiol-maleimide modality achieves the highest macromer coupling efficiency of the reported Michael-type pairs, but the resulting hydrogel networks are heterogeneous because polymerization is faster than the individual components can be manually mixed. The reactivity of the thiol dictates the overall reaction speed, which can be slowed in organic solvents and acidic buffers. Since these modifications also reduce the biocompatibility of resulting hydrogels, we investigated a series of biocompatible buffers and crosslinkers to decelerate gelation while maintaining high cell viability. We found that lowering the polymer weight percentage (wt%), buffer concentration, and pH slowed gelation kinetics, but crosslinking with an electronegative peptide was optimal for both kinetics and cell viability. Including a high glucose medium supplement in the polymer solvent buffer improved the viability of the cells being encapsulated without impacting gelation time. Slowing the speed of polymerization resulted in more uniform hydrogels, both in terms of visual inspection and the diffusion of small molecules through the network. However, reactions that were too slow resulted in non-uniform particle dispersion due to settling, thus there is a trade-off in hydrogel network uniformity versus cell distribution in the hydrogels when using these networks in cell applications. STATEMENT OF SIGNIFICANCE:The polymer network of thiol-maleimide hydrogels assembles faster than individual components can be uniformly mixed due to their fast gelation kinetics. The lack of homogeneity can result in variable cell-based assay results, resulting in batch-to-batch variability and limiting their use in predictive screening assays. Although these hydrogels are incredibly useful in tissue engineering, this network heterogeneity is a known problem in the field. We screened a variety of possible techniques to slow down the reaction speed and improve the homogeneity of these hydrogels, without sacrificing the viability and distribution of encapsulated cells. As others have reported, an electronegative crosslinker was the most effective technique to slow the reaction, but the chemical modification required is technically challenging. Of interest to a broad community, we screened buffer type, strength, and crosslinker electronegativity to find an optimal reaction speed that allows for high cell viability and small molecule diffusion, without allowing cells to settle during gelation, allowing application of these materials to the drug screening industry and tissue engineering community.

Project description:The efficient radiosynthesis of biomolecules utilizing minute quantities of maleimide substrate is important for availability of novel peptide molecular imaging agents. We evaluated both 3-18F-fluoropropane-1-thiol and 2-(2-(2-(2-18F-fluoroethoxy)ethoxy)ethoxy)ethane-1-thiol (18F-fluoro-PEG4 thiol) as prosthetic groups for radiolabeling under physiological conditions. The precursor employed a benzoate for protection of the thiol and an arylsulfonate leaving group. The radiofluorination was fully automated on an Eckert & Ziegler synthesis system using standard Kryptofix222/K2CO3 conditions. In order to minimize the amount of biological molecule required for subsequent conjugation, the intermediates, S-(3-18F-fluoropropyl) benzothioate and 18F-fluoro-PEG4 benzothioate, were purified by HPLC. The intermediates were isolated from the HPLC in yields of 37-47% and 28-35%, respectively, and retrieved from eluate using solid phase extraction. Treatment of the benzothioates with sodium methoxide followed by acetic acid provided the free thiols. The desired maleimide substrate in acetonitrile or phosphate buffer was then added and incubated at room temperature for 15?min. The final radiolabeled bioconjugate was purified on a separate HPLC or NAP-5 column. Maleimides utilized for the coupling reaction included phenyl maleimide, an Evans Blue maleimide derivative, a dimeric RGDfK maleimide (E[c(RGDfK)]2), two aptamer maleimides, and PSMA maleimide derivative. Isolated radiochemical yields (non-decay corrected) of maleimide addition products based on starting 18F-fluoride ranged from 6 to 22% in a synthesis time of about 90?min. 18F-thiol prosthetic groups were further tested in vivo by conjugation to E[c(RGDfK)]2 maleimide in a U87MG xenograft model. PET studies demonstrated similar tumor accumulation of both prosthetic groups. 18F-fluoro-PEG4-S-E[c(RGDfK)]2 displayed a somewhat favorable pharmacokinetics compared to 18F-fluoropropyl-S-E[c(RGDfK)]2. Bone uptake was low for both indicating in vivo stability.

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Tunable degradation of maleimide-thiol adducts in reducing environments.

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Tunable degradation of maleimide-thiol adducts in reducing environments.

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