Esterase-catalyzed Hydrolysis Research Articles

Mammalian Esterase Activity: Implications for Peptide Prodrugs.

As a traceless, bioreversible modification, the esterification of carboxyl groups in peptides and proteins has the potential to increase their clinical utility. An impediment is the lack of strategies to quantify esterase-catalyzed hydrolysis rates for esters in esterified biologics. We have developed a continuous Förster resonance energy transfer (FRET) assay for esterase activity based on a peptidic substrate and a protease, Glu-C, that cleaves a glutamyl peptide bond only if the glutamyl side chain is a free acid. Using pig liver esterase (PLE) and human carboxylesterases, we validated the assay with substrates containing simple esters (e.g., ethyl) and esters designed to be released by self-immolation upon quinone methide elimination. We found that simple esters were not cleaved by esterases, likely for steric reasons. To account for the relatively low rate of quinone methide elimination, we extended the mathematics of the traditional Michaelis-Menten model to conclude with a first-order intermediate decay step. By exploring two regimes of our substrate → intermediate → product (SIP) model, we evaluated the rate constants for the PLE-catalyzed cleavage of an ester on a glutamyl side chain (kcat/KM = 1.63 × 103 M-1 s-1) and subsequent spontaneous quinone methide elimination to regenerate the unmodified peptide (kI = 0.00325 s-1; t1/2 = 3.55 min). The detection of esterase activity was also feasible in the human intestinal S9 fraction. Our assay and SIP model increase the understanding of the release kinetics of esterified biologics and facilitate the rational design of efficacious peptide prodrugs.

Thiamine analogues featuring amino-oxetanes as potent and selective inhibitors of pyruvate dehydrogenase

Pyruvate dehydrogenase complex (PDHc) is suppressed in some cancer types but overexpressed in others. To understand its contrasting oncogenic roles, there is a need for selective PDHc inhibitors. Its E1-subunit (PDH E1) is a thiamine pyrophosphate (TPP)-dependent enzyme and catalyses the first and rate-limiting step of the complex. In a recent study, we reported a series of ester-based thiamine analogues as selective TPP-competitive PDH E1 inhibitors with low nanomolar affinity. However, when the ester linker was replaced with an amide for stability reasons, the binding affinity was significantly reduced. In this study, we show that an amino-oxetane bioisostere of the amide improves the affinity and maintains stability towards esterase-catalysed hydrolysis.

Salivary esterases dramatically reduce biostability of dentin collagen treated with galloylated polyphenols.

To investigate the effects of salivary esterases on biostability of collagen treated by galloylated polyphenols. Human dentin was microtomed into 6-μm-thick films, which were demineralized and treated for 60s using solutions containing 0.6% and 2% of one of the crosslinkers: tannic acid (TAC), epigallocatechin gallate (EGCG), epigallocatechin (EGC), and N-[3-dimethylaminopropyl]-N'-ethylcarbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS), and for 1h using EDC/NHS. Half of the treated and untreated (control) films were subjected to human saliva incubation. Collagen biostability was assessed via exogenous protease biodegradation by weight loss and hydroxyproline release, and endogenous MMPs by in situ zymography. The degradation products of galloylated polyphenols (TAC and EGCG) by saliva were monitored using proton nuclear magnetic resonance (1H NMR) and gel permeation chromatography (GPC). The esterase activity of saliva induced by the crosslinkers was also assessed. Collagen films treated with TAC and EGCG exhibited significantly improved biostability (p<0.05); however, the enhanced biostability was severely reduced after saliva incubation (p<0.001). For EDC/NHS treated collagen, saliva incubation showed negligible effect on the biostability. 1H NMR studies confirmed the esterase-catalyzed hydrolysis of the galloyl. GPC measurements showed decreased molecular weight of TAC in saliva indicating its chemical degradation. Both TAC and EGCG showed much higher esterase activity than other treatment groups. The galloyl group plays important role in collagen crosslinking, inducing higher biostability. However, galloylated polyphenols crosslinked on collagen are highly susceptible to metabolism of human saliva by salivary esterase, dramatically compromising the enhanced biostability.

Identification of a potent Nrf2 displacement activator among aspirin-containing prodrugs

Aspirin is a desired leaving group in prodrugs aimed at treatment of neurodegeneration and other conditions. A library of aspirin derivatives of various scaffolds potentially activating Nrf2 has been tested in Neh2-luc reporter assay which screens for direct Nrf2 protein stabilizers working via disruption of Nrf2-Keap1 interaction. Most aspirin prodrugs had a pro-alkylating or pro-oxidant motif in the structure and, therefore, were toxic at high concentrations. However, among the active compounds, we identified a molecule resembling a well-known Nrf2 displacement activator, bis-1,4-(4-methoxybenzenesulfonamidyl) naphthalene (NMBSA). The direct comparison of the newly identified compound with NMBSA and its improved analog in the reporter assay showed no quenching with N-acetyl cysteine, thus pointing to Nrf2 stabilization mechanism without cysteine alkylation. The potency of the newly identified compound in the reporter assay was much stronger than NMBSA, despite its inhibitory action in the commercial fluorescence polarization assay was observed only in the millimolar range. Molecular docking predicted that mono-deacetylation of the novel prodrug should generate a potent displacement activator. The time-course of reporter activation with the novel prodrug had a pronounced lag-period pointing to a plausible intracellular transformation leading to an active product. Treatment of the novel prodrug with blood plasma or cell lysate demonstrated stepwise deacetylation as judge by liquid chromatography-mass spectrometry (LC-MS). Hence, the esterase-catalyzed hydrolysis of the prodrug liberates only acetyl groups from aspirin moiety and generates a potent Nrf2 activator. The discovered mechanism of prodrug activation makes the newly identified compound a promising lead for future optimization studies.

Mechanistic insight into esterase-catalyzed hydrolysis of phthalate esters (PAEs) based on integrated multi-spectroscopic analyses and docking simulation

A novel PAE-hydrolyzing esterase (named Hyd) gene was screened from the genomic library of Rhodococcus sp. 2G and was successfully expressed in heterologous E. coli, which was defined as a new family of esterolytic enzymes. The purified Hyd could efficiently degrade various PAEs, displaying high activity and stability with a broad range of pH (4–10) and temperature (20–60 °C). Interaction mechanism of Hyd with dibutyl phthalate (DBP) was investigated by integrated multi-spectroscopic and docking simulation methods. Fluorescence and UV–vis spectra revealed that DBP could quench the fluorescence of Hyd through a static quenching mechanism. The results from synchronous fluorescence and CD spectra confirmed that the DBP binding to Hyd triggered conformational and micro-environmental changes of Hyd, which were characterized by increased stretching extent and random coil, and decreased α-helix and β-sheet. Molecular docking study showed that DBP could be bound to the cavity of Hyd with hydrogen bonding and hydrophobic interaction. A novel and distinctive catalytic mechanism was proposed: two key residues Thr190 and Ser191 might catalyze the hydrolysis of DBP, instead of the conserved catalytic triad (Ser-His-Asp) reported elsewhere, which was confirmed by site-directed mutagenesis.

Cis/Trans Isomerization of Unsaturated Fatty Acids in Polysorbate 80 During Light Exposure of a Monoclonal Antibody-Containing Formulation.

Light exposure of a monoclonal antibody formulation containing polysorbate 80 (PS80) leads to cis/trans isomerization of monounsaturated and polyunsaturated fatty acids. This cis/trans isomerization was monitored by positive electrospray ionization mass spectrometry of intact PS80 components as well as by negative ion electrospray ionization mass spectrometry analysis of free fatty acids generated via esterase-catalyzed hydrolysis. The light-induced cis/trans isomerization of unsaturated fatty acids in PS80 required the presence of the monoclonal antibody, or, at a minimum (for mechanistic studies), a combination of N-acetyltryptophan amide and glutathione disulfide, suggesting the involvement of thiyl radicals generated by photoinduced electron transfer from Trp to the disulfide. Product analysis confirmed the conversion of PS80-bound oleic acid to elaidic acid; furthermore, together with linoleic acid, we detected conjugated linoleic acids in PS80, which underwent light-induced cis/trans isomerization.

Improving the enantioselectivity of an esterase toward (S)-ketoprofen ethyl ester through protein engineering

• Biochemical characteristics of Est25 were studied in various temperature, solvent and pH. • Mutated esterase (L255W) based on the modeled esterase structure has higher selectivity to (S)-ketoprofen ethyl ester than wild-type. • The addition of methanol or ethanol (as a solvent) led to drastic increases in the enantioselectivity of each enzyme. Esterases, enzymes that hydrolyze ester bonds, have been utilized to synthesize optically pure compounds like (S)-ketoprofen, which can be produced by the esterase-catalyzed hydrolysis of rac -ketoprofen ethyl ester. We previously reported a novel esterase, Est25, which was expressed in active form in Escherichia coli , and showed high hydrolyzing activities for various substrates. However, the enzyme did not show any preference for either of rac -ketoprofen ethyl ester. In this study, to improve the enantioselectivity of Est25, we used a protein engineering approach that involves building a homology model of Est25, library generation based on the model, screening for esterase activity, and screening for specificity toward (S)-ketoprofen ethyl ester. One variant, L255W was isolated showing significantly higher enantioselectivity than the wild-type enzyme. Enantioselectivity was further improved by adding one of the polar organic solvents, namely, ethanol and methanol. However, the mutant enzyme showed lower thermostability, higher sensitivity to pH, and lower resistance to polar solvents than Est25. The L255W mutation improves substrate specificity, but, at the same time, sacrifices structural stability. The approach used in this study is precise enough to engineer the substrate specificity of esterase, and general enough to be adapted to engineer other kinds of enzymes with a minimum amount of modifications.

Enzyme-Catalyzed Conversion of Chemical Structures on the Surface of Gold Nanorods

For developing metal nanoparticles of which surface chemical structures could be altered by enzymes in the cells, functional linkers caged by coumaric acids have been synthesized. Synthesized gold nanorod (GNR) conjugates possessing coumaric acid precursors underwent (porcine liver) esterase-catalyzed hydrolysis on their surface to afford GNRs coated with amino-functionalized polyethylene glycol and fluorescent coumarins as reporter molecules for monitoring the conversion. The chemical structural conversion on the GNR surfaces was successfully observed inside cells by fluorescence microscopy when GNR conjugates were incubated with tumor cells.

Degradable cationic shell cross-linked knedel-like nanoparticles: synthesis, degradation, nucleic acid binding, and in vitro evaluation.

In this work, degradable cationic shell cross-linked knedel-like (deg-cSCK) nanoparticles were developed as an alternative platform to replace similar nondegradable cSCK nanoparticles that have been utilized for nucleic acids delivery. An amphiphilic diblock copolymer poly(acrylamidoethylamine)(90)-block-poly(DL-lactide)(40) (PAEA(90)-b-PDLLA(40)) was synthesized, self-assembled in aqueous solution, and shell cross-linked using a hydrolyzable cross-linker to afford deg-cSCKs with an average core diameter of 45 ± 7 nm. These nanoparticles were fluorescently labeled for in vitro tracking. The enzymatic- and hydrolytic-degradability, siRNA binding affinity, cell uptake and cytotoxicity of the deg-cSCKs were evaluated. Esterase-catalyzed hydrolysis of the nanoparticles resulted in the degradation of ca. 24% of the PDLLA core into lactic acid within 5 d, as opposed to only ca. 9% degradation from aqueous solutions of the deg-cSCK nanoparticles in the absence of enzyme. Cellular uptake of deg-cSCKs was efficient, while exhibiting low cytotoxicity with LD50 values of ca. 90 and 30 μg/mL in RAW 264.7 mouse macrophages and MLE 12 cell lines, respectively, ca. 5- to 6-fold lower than the cytotoxicity observed for nondegradable cSCK analogs. Additionally, deg-cSCKs were able to complex siRNA at an N/P ratio as low as 2, and were efficiently able to facilitate cellular uptake of the complexed nucleic acids.

Modifying Methoxycarbonyl Etomidate Inter-Ester Spacer Optimizes In Vitro Metabolic Stability and In Vivo Hypnotic Potency and Duration of Action

Methoxycarbonyl etomidate is the prototypical very rapidly metabolized etomidate analog. Initial studies suggest that it may be too short acting for many clinical uses. We hypothesized that its duration of action could be lengthened and clinical utility broadened by incorporating specific aliphatic groups into the molecule to sterically protect its ester moiety from esterase-catalyzed hydrolysis. To test this hypothesis, we developed a series of methoxycarbonyl etomidate analogs (spacer-linked etomidate esters) containing various aliphatic-protecting groups and spacer lengths. Spacer-linked etomidate esters were synthesized and their hypnotic potencies and durations of action following bolus administration were measured in rats using a loss-of-righting reflexes assay. Octanol:water partition coefficients and metabolic half-lives in pooled rat blood were determined chromatographically. All spacer-linked etomidate esters produced hypnosis rapidly and in a dose-dependent manner. ED50s for loss of righting reflexes ranged from 0.69 ± 0.04 mg/kg for cyclopropyl-methoxycarbonyl metomidate to 11.1 ± 0.8 mg/kg for methoxycarbonyl metomidate. The slope of a plot of the duration of loss of righting reflexes versus the logarithm of the dose ranged 12-fold among spacer-linked etomidate esters, implying widely varying brain clearance rates. The in vitro metabolic half-lives of these compounds in rat blood varied by more than two orders of magnitude and were diastereometrically selective. We created 13 new analogs of methoxycarbonyl etomidate and identified two that have significantly higher potency and potentially address the too-brief duration of action for methoxycarbonyl etomidate. This work may provide a blueprint for optimizing the pharmacological properties of other soft drugs.

Metabolic Activation of Prasugrel: Nature of the Two Competitive Pathways Resulting in the Opening of Its Thiophene Ring

The mechanism generally admitted for the bioactivation of the antithrombotic prodrug, prasugrel, 1c, is its two-step enzymatic conversion into a biologically active thiol metabolite. The first step is an esterase-catalyzed hydrolysis of its acetate function leading to a thiolactone metabolite 2c. The second step was described as a cytochrome P450 (P450)-dependent oxidative opening of the thiolactone ring of 2c, with intermediate formation of a reactive sulfenic acid metabolite that is eventually reduced to the corresponding active thiol 3c. This article describes a detailed study of the metabolism of 1c by human liver microsomes and human sera, with an analysis by HPLC-MS under conditions allowing a complete separation of the thiol metabolite isomers, after derivatization with 3'-methoxy phenacyl bromide. It shows that there are two competing metabolic pathways for the opening of the 2c thiolactone ring. The major one, which was previously described, results from a P450- and NADPH-dependent redox bioactivation of 2c and leads to 3c, two previously reported thiol diastereomers bearing an exocyclic double bond. It occurs with NADPH-supplemented human liver microsomes but not with human sera. The second one results from a hydrolysis of 2c and leads to an isomer of 3c, 3c endo, in which the double bond has migrated from an exocyclic to an endocyclic position in the piperidine ring. It occurs both with human liver microsomes and human sera, and does not require NADPH. However, it requires Ca(2+) and is inhibited by paraoxon, which suggests that it is catalyzed by a thioesterase such as PON-1. Chemical experiments have confirmed that hydrolytic opening of thiolactone 2c exclusively leads to derivatives of the endo thiol isomer 3c endo.

A kinetic method for the determination of plasma protein binding of compounds unstable in plasma: Specific application to enalapril

Traditional methods for the determination of plasma protein binding (PPB), such as equilibrium dialysis and ultrafiltration, normally operate on a timescale ranging from tens of minutes to several hours and are not suitable for measuring compounds that have significant chemical degradation on this timescale. One such compound is enalapril. Although stable in human plasma enalapril is subject to rapid esterase-catalyzed hydrolysis in rat plasma. A method has been developed which allows the extent of rat PPB of enalapril to be determined from initial rates kinetics of the adsorption of the unstable compound to dextran coated charcoal (DCC). The method has been applied to stable compounds, and the results are consistent with those from traditional equilibrium dialysis experiments. The experimental method is simple to run, requires no specialized equipment, and can potentially be applied to other compounds that show instability in plasma where traditional experimental techniques are unsuitable.

Liposomal formulation of α-tocopheryl maleamide: In vitro and in vivo toxicological profile and anticancer effect against spontaneous breast carcinomas in mice

The vitamin E analogue α-tocopheryl succinate (α-TOS) is an efficient anti-cancer drug. Improved efficacy was achieved through the synthesis of α-tocopheryl maleamide (α-TAM), an esterase-resistant analogue of α-tocopheryl maleate. In vitro tests demonstrated significantly higher cytotoxicity of α-TAM towards cancer cells (MCF-7, B16F10) compared to α-TOS and other analogues prone to esterase-catalyzed hydrolysis. However, in vitro models demonstrated that α-TAM was cytotoxic to non-malignant cells ( e.g. lymphocytes and bone marrow progenitors). Thus we developed lyophilized liposomal formulations of both α-TOS and α-TAM to solve the problem with cytotoxicity of free α-TAM (neurotoxicity and anaphylaxis), as well as the low solubility of both drugs. Remarkably, neither acute toxicity nor immunotoxicity implicated by in vitro tests was detected in vivo after application of liposomal α-TAM, which significantly reduced the growth of cancer cells in hollow fiber implants. Moreover, liposomal formulation of α-TAM and α-TOS each prevented the growth of tumours in transgenic FVB/N c-neu mice bearing spontaneous breast carcinomas. Liposomal formulation of α-TAM demonstrated anti-cancer activity at levels 10-fold lower than those of α-TOS. Thus, the liposomal formulation of α-TAM preserved its strong anti-cancer efficacy while eliminating the in vivo toxicity found of the free drug applied in DMSO. Liposome-based targeted delivery systems for analogues of vitamin E are of interest for further development of efficient and safe drug formulations for clinical trials.

Enhancement of Electrochemical Signal on Gold Electrodes by Polyvalent Esterase−Dendrimer Clusters

5'-Maleimide-oligodeoxynucleotide was conjugated with single sulfhydryl group of cystamine core poly(amidoamine) dendrimers of different generations. Amino groups on the dendrimer moiety were modified with maleimide and coupled to the cysteine 118 of esterase 2 from Alicyclobacillus acidocaldarius in a site-specific manner. Polyvalent esterase-dendrimer-oligodeoxynucleotide clusters were hybridized to capture oligodeoxynucleotides immobilized on a gold electrode. The amperometric signal of p-aminophenol was detected following the esterase-catalyzed hydrolysis of p-aminophenylbutyrate. The multiple anchoring of the esterase reporter via generation 3-and generation 5-derived clusters exhibited 10- and 100-fold signal enhancement, respectively, as compared to monovalent esterase-oligonucleotide conjugate. The polyvalent and monovalent reporters were comparable in their abilities regarding mismatch discrimination.

Studies on the porcine liver esterase-catalyzed hydrolysis of pentaacetyl catechin and epicatechin: Application to the synthesis of novel dimers and trimers

Porcine liver esterase-catalyzed hydrolysis of 3,5,7,3′,4′-pentaacetylated catechin was studied. The selectivity of the enzyme in hydrolyzing the acetate moiety is time dependent. Careful control of the duration of hydrolysis makes it possible to isolate the differentially protected catechins. Similar result was also obtained in the epicatechin series. These results are important for elaboration of epicatechin or catechin into different derivatives with defined regiochemistry. These include novel dimeric and trimeric architectures.

Development of an optical fibre reflectance sensor for p-aminophenol detection based on immobilised bis-8-hydroxyquinoline

2,2′-(1,4-Phenylenedivinylene)bis-8-hydroxyquinoline (PBHQ), a highly sensitive reagent used for the colorimetric determination of p-aminophenol (PAP), was successfully immobilised on XAD-7 and coupled with optical fibres to investigate a sensor-based approach for determining p-aminophenol. The solid-state sensor is based on the reaction of PAP with PBHQ in presence of an oxidant to produce an indophenol dye. The reflectance measurements were carried out at a wavelength of 647 nm since it yielded the largest divergence different in reflectance spectra before and after reaction with the analyte. The linear dynamic range of PAP was found within the concentration range of 0.1–2.18 mg l −1 with its LOD of 0.02 mg l −1. The sensor response from different probes ( n = 7) gave a R.S.D. of 4.4% at 1.09 mg l −1 PAP concentration. The response time of the optical one-shot sensor was 5 min for a stable solution. As this PAP sensor is irreversible, a fresh sensor has to be used for each measurement. All the experimental parameters were optimized for the determination of PAP. Using the optical sensing probe, PAP in pharmaceutical wastewater and paracetamol was determined. The effect of potential interferences such as inorganic and organic compounds was also evaluated. Potential on-site determination of PAP with such sensors can indirectly aid detection of organo-phosphorus nerve agents and pesticides in the field by inhibition of acetylcholine esterase-catalyzed hydrolysis of p-aminophenyl acetate to p-aminophenol.

Antiatherosclerotic properties of 4-(2,6-dimethylheptyl)-and 4-(2,6,10-trimethylundecyl)benzoic acids and their amides

Two amides with polyol-rich moieties and two other analogs with N-(β-aminoethyl)amide groups, all derived from 4-(2,6-dimethylheptyl)benzoic acid (I), and two polyol amides derived from 4-(2,6,10)-trimetylundecyl) benzoic acid (II) were synthesized and tested for antiatherosclerotic activity in vitro. None of the amides prevented excessive accumulation of cholesterol in the culture of smooth muscle cells (SMCs) raised from human aorta sclerotic plaque, although polyol amides from acid I inhibited protein synthesis in this cell culture. In human liver homogenates (HLHs) all amides derived from acid I accelerated the human cholesterol esterase-catalyzed hydrolysis of cholesteryl palmitate. Acid I displayed antiatherosclerotic effects in both SMC and HLH assays, while acid II and its amides were ineffective in either of these test systems.

Lipophilicity Plays a Major Role in Modulating the Inhibition of Monoamine Oxidase B by 7‐Substituted Coumarins

A series of coumarin derivatives (1-22), bearing at the 7-position ether, ketone, ester, carbamate, or amide functions of varying size and lipophilicity, were synthesized and investigated for their in vitro monoamine oxidase-A and -B (MAO-A and -B) inhibitory activities. Most of the compounds acted preferentially as MAO-B inhibitors, with IC(50) values in the micromolar to low-nanomolar range. A structure-activity-relationship (SAR) study highlighted lipophilicity as an important property modulating the MAO-B inhibition potency of 7-substituted coumarins, as shown by a linear correlation (n=20, r(2)=0.72) between pIC(50) and calculated log P values. The stability of ester-containing coumarin derivatives in rat plasma provided information on factors that either favor (lipophilicity) or decrease (steric hindrance) esterase-catalyzed hydrolysis. Two compounds (14 and 22) were selected to investigate how lipophilicity and enzymatic stability may affect in vivo MAO activities, as assayed ex vivo in rat. The most-potent and -selective MAO-B inhibitor 22 (=7-[(3,4-difluorobenzyl)oxy]-3,4-dimethyl-1-benzopyran-2(2H)-one) within the examined series significantly inhibited (>60%) ex vivo rat-liver and striatal MAO-B activities 1 h after intraperitoneal administration of high doses (100 and 300 mumol kg(-1)), revealing its ability to cross the blood-brain barrier. At the same doses, liver and striatum MAO-A was less inhibited in vivo, somehow reflecting MAO-B selectivity, as assessed in vitro. In contrast, the metabolically less stable derivative 14, bearing an isopropyl ester in the lateral chain, had a weak effect on hepatic MAO-B activity in vivo, and none on striatal MAO-B, but, surprisingly, displayed inhibitory effects on MAO-A in both peripheral and brain tissues.

Esterase-Activated Two-Fluorophore System for Ratiometric Sensing of Biological Zinc(II)

Intracellular ester hydrolysis by cytosolic esterases is a common strategy used to trap fluorescent sensors within the cell. We have prepared analogues of Zinpyr-1 (ZP1), an intensity-based fluorescent sensor for Zn2+, that are linked via an amido-ester or diester moiety to a calibrating fluorophore, coumarin 343. These compounds, designated Coumazin-1 and -2, are nonpolar and are quenched by intramolecular interactions between the two fluorophores. Esterase-catalyzed hydrolysis generates a Zn2+-sensitive ZP1-like fluorophore and a Zn2+-insensitive coumarin as a calibrating fluorophore. Upon excitation of the fluorophores, coumarin 343 emission relays information concerning sensor concentration whereas ZP1 emission indicates the relative concentration of Zn2+-bound sensor. This approach enables intracellular monitoring of total sensor concentration and provides a ratiometric system for sensing biological zinc ion.

Prodrug modification increases potassium tricyclo[5.2.1.0(2,6)]-decan-8-yl dithiocarbonate (D609) chemical stability and cytotoxicity against U937 leukemia cells.

Potassium tricyclo[5.2.1.0(2,6)]-decan-8-yl dithiocarbonate (D609) is a selective antitumor agent, potent antioxidant, and cytoprotectant. It has the potential to be developed as a unique chemotherapeutic agent that may provide dual therapeutic benefits against cancer, e.g., enhancing tumor cell death while protecting normal tissues from damage. However, D609 contains a dithiocarbonate (xanthate) group [O-C(=S)S(-)/O-C(=S)SH], which is chemically unstable, being readily oxidized to form a disulfide bond with subsequent loss of all biological activities. Therefore, we developed the synthesis of a series of S-(alkoxyacyl) D609 prodrugs by connecting the xanthate group of D609 to an ester via a self-immolative methyleneoxyl group. These S-(alkoxylacyl)-D609 prodrugs are designed to release D609 in two steps: esterase-catalyzed hydrolysis of the acyl ester bond followed by conversion of the resulting hydroxymethyl D609 to formaldehyde and D609. Three S-(alkoxyacyl) D609 prodrugs were synthesized by varying the steric bulkiness of the acyl group. These prodrugs are stable to ambient conditions, but readily hydrolyzed by esterases to liberate D609 in a controlled manner. More importantly, the lead prodrug methyleneoxybutyryl D609 is biologically more effective than D609 in inhibiting sphingomyelin synthase, thereby increasing the level of ceramide and inducing apoptosis in U937 leukemia cells. The prodrug has a significantly lower LD(50) value than that of D609 (56.6 versus 117 microM) against U937 cells. These findings demonstrate that prodrug modification of the xanthate moiety with an alkoxyacyl group can improve D609 oxidative stability and enhance its antitumor activity.