Hydrolysis of phosphonate esters catalyzed by 5'-nucleotide phosphodiesterase.

4-Nitrophenyl and 2-napthyl monoesters of phenylphosphonic acid have been synthesized, and an enzyme catalyzing their hydrolysis was resolved from alkaline phosphatase of a commerical calf intestinal alkaline phosphatase preparation by extensive ion-exchange chromatography, chromatography on L-phenylalanyl-Sepharose with a decreasing gradient of (NH4) 2SO4, and gel filtration. Detergent-solubilized enzyme from fresh bovine intestine was purified after (NH4)2SO4 fractionation by the same technique. The purified enzyme is homogeneous by polyacrylamide gel electrophoresis and sedimentation equilibrium centrifugation. It has a molecular weight of 108,000, contains approximately 21% carbohydrate, and has an amino acid composition considerably different from that reported from alkaline phosphatase from the same tissue. The homogeneous intestinal enzyme, an efficient catalyst of phosphonate ester hydoolysis but not of phosphate monoester hydrolysis, was identified as a 5'-nucleotide phosphodiesterase by its ability to hydrolyze 4-nitrophenyl esters of 5'-TMP but not of 3'-TMP. Also consistent with this identification was the ability of the enzyme to hydrolyze 5'-ATP to 5'-AMP and PPi, NAD+ to 5'-AMP and NMN, TpT to 5'-TMP and thymidine, pApApApA to 5'-AMP, and only the single-stranded portion of tRNA from the 3'-OH end. Snake venom 5'-nucleotide phosphodiesterase also hydrolyzes phosphonate esters, but 3'-nucleotide phosphodiesterase of spleen and cyclic 3',5'-AMP phosphodiesterase do not. Thus, types of phosphodiesterases can be conveniently distinguished by their ability to hydrolyze phosphonate esters. As substrates for 5'-nucleotide phosphodiesterases, phosphonate esters are preferable to the more conventional esters of nucleotides and bis(4-nitrophenyl) phosphate because of their superior stability and ease of synthesis. Furthermore, the rate of hydrolysis of phosphonate esters under saturating conditions is greater than that of the conventional substrates. At substrate concentrations of 1 mM the rates of hydrolysis of phosphonate esters and of nucleotide esters are comparable and both superior to that of bis(4-nitrophenyl) phosphate.     

Structural and functional comparisons of nucleotide pyrophosphatase/phosphodiesterase and alkaline phosphatase: implications for mechanism and evolution

The rapid expansion of the amount of genomic and structural data has provided many examples of enzymes with evolutionarily related active sites that catalyze different reactions. Functional comparisons of these active sites can provide insight into the origins of the enormous catalytic proficiency of enzymes and the evolutionary changes that can lead to different enzyme activities. The alkaline phosphatase (AP) superfamily is an ideal system to use in making such comparisons given the extensive data available on both nonenzymatic and enzymatic phosphoryl transfer reactions. Some superfamily members, such as AP itself, preferentially hydrolyze phosphate monoesters, whereas others, such as nucleotide pyrophosphatase/phosphodiesterase (NPP), preferentially hydrolyze phosphate diesters. We have measured rate constants for NPP-catalyzed hydrolysis of phosphate diesters and monoesters. NPP preferentially catalyzes diester hydrolysis by factors of 10(2)-10(6), depending on the identity of the diester substrate. To identify features of the NPP active site that could lead to preferential phosphate diester hydrolysis, we have determined the structure of NPP in the absence of ligands and in complexes with vanadate and AMP. Comparisons to existing structures of AP reveal bimetallo cores that are structurally indistinguishable, but there are several distinct structural features outside of the conserved bimetallo site. The structural and functional data together suggest that some of these distinct functional groups provide specific substrate binding interactions, whereas others tune the properties of the bimetallo active site itself to discriminate between phosphate diester and monoester substrates.     

A nucleophilic catalysis step is involved in the hydrolysis of aryl phosphate monoesters by human CT acylphosphatase

Acylphosphatase, one of the smallest enzymes, is expressed in all organisms. It displays hydrolytic activity on acyl phosphates, nucleoside di- and triphosphates, aryl phosphate monoesters, and polynucleotides, with acyl phosphates being the most specific substrates in vitro. The mechanism of catalysis for human acylphosphatase (the organ-common type isoenzyme) was investigated using both aryl phosphate monoesters and acyl phosphates as substrates. The enzyme is able to catalyze phosphotransfer from p-nitrophenyl phosphate to glycerol (but not from benzoyl phosphate to glycerol), as well as the inorganic phosphate-H(2)18O oxygen exchange reaction in the absence of carboxylic acids or phenols. In short, our findings point to two different catalytic pathways for aryl phosphate monoesters and acyl phosphates. In particular, in the aryl phosphate monoester hydrolysis pathway, an enzyme-phosphate covalent intermediate is formed, whereas the hydrolysis of acyl phosphates seems a more simple process in which the Michaelis complex is attacked directly by a water molecule generating the reaction products. The formation of an enzyme-phosphate covalent complex is consistent with the experiments of isotope exchange and transphosphorylation from substrates to glycerol, as well as with the measurements of the Br?nsted free energy relationships using a panel of aryl phosphates with different structures. His-25 involvement in the formation of the enzyme-phosphate covalent complex during the hydrolysis of aryl phosphate monoesters finds significant confirmation in experiments performed with the H25Q mutated enzyme.     

Leaving group dependence and proton inventory studies of the phosphorylation of a cytoplasmic phosphotyrosyl protein phosphatase from bovine heart.

The kcat and Km values for the bovine heart low molecular weight phosphotyrosyl protein phosphatase catalyzed hydrolysis of 16 aryl phosphate monoesters and of five alkyl phosphate monoesters having the structure Ar(CH2)nOPO3H2 (n = 1-5) were measured at pH 5.0 and 37 degrees C. With the exception of alpha-naphthyl phosphate and 2-chlorophenyl phosphate, which are subject to steric effects, the values of kcat are effectively constant for the aryl phosphate monoesters. This is consistent with the catalysis being nucleophilic in nature, with the existence of a common covalent phosphoenzyme intermediate, and with the breakdown of this intermediate being rate-limiting. In contrast, kcat for the alkyl phosphate monoesters is much smaller and the rate-limiting step for these substrates is interpreted to be the phosphorylation of the enzyme. A single linear correlation is observed for a plot of log (kcat/Km) vs leaving group pKa for both classes of substrates at pH 5.0: log (kcat/Km) = -0.28pKa + 6.88 (n = 19, r = 0.89), indicating a uniform catalytic mechanism for the phosphorylation event. The small change in effective charge (-0.28) on the departing oxygen of the substrate is similar to that observed in the specific acid catalyzed hydrolysis of monophosphate monoanions (-0.27) and is consistent with a strong electrophilic interaction of the enzyme with this oxygen atom in the transition state. The D2O solvent isotope effect and proton inventory experiments indicate that only one proton is "in flight" in the transition state of the phosphorylation process and that this proton transfer is responsible for the reduction of effective charge on the leaving oxygen.     

Calcineurin-catalyzed reaction with phosphite and phosphate esters of tyrosine.

A convenient synthesis is reported for the preparation of the phosphite ester of tyrosine methyl ester. By use of calcineurin, at 30 degrees C, a phosphite ester was hydrolyzed with a VM value [119 nmol/(min.micrograms of E)] approximately 500 times greater than that obtained with tyrosine phosphate [0.23 nmol/(min.microgram of E)] as substrate, but with similar KM values (12 mM for Tyr-PH ME, 11 mM for Tyr-P). Acid phosphatase, on the other hand, hydrolyzed the phosphite ester with a VM and KM value lower than those obtained with tyrosyl phosphate. The temperature dependence of the kinetic parameters (KM and VM) was evaluated, and the activation parameters were obtained with both substrates. The entropy of activation associated with the enzymatic hydrolysis of tyrosine phosphate agrees with the entrophy change for the hydrolysis of the monoanion of phosphate monoesters. The energy of activation for both substrates was in agreement with the energy change for hydrolysis of the oxygen-phosphorous linkage of phosphate monoester monoanions and phosphite esters. These results are consistent with a scheme of general acid catalysis in the action of calcineurin.     

Hydrolysis of primary and secondary esters of glycerol by pancreatic juice

The relative rates of hydrolysis of the secondary ester in glycerol 1,3-benzylidene 2-oleate and in glycerol 1,3-dihexadecyl ether 2-oleate, and of the primary and secondary esters in triolein were determined. Both unaltered and selectively inactivated rat pancreatic juice were used as sources of enzyme. It was found that rat pancreatic juice contains an enzyme that can hydrolyze fatty acids esterified at the 2-position of a glyceride. This enzyme is not pancreatic lipase. It may be sterol ester hydrolase. Partial glycerides, as well as complete glycerides, can serve as substrates. Pancreatic lipase, if it can hydrolyze the 2-positioned fatty acids of a triglyceride, does so at a very slow rate.     

A series of crystal structures of a meta-cleavage product hydrolase from Pseudomonas fluorescens IP01 (CumD) complexed with various cleavage products

Meta-cleavage product hydrolase (MCP-hydrolase) is one of the key enzymes in the microbial degradation of aromatic compounds. MCP-hydrolase produces 2-hydroxypenta-2,4-dienoate and various organic acids, according to the C6 substituent of the substrate. Comprehensive analysis of the substrate specificity of the MCP-hydrolase from Pseudomonas fluorescens IP01 (CumD) was carried out by determining the kinetic parameters for nine substrates and crystal structures complexed with eight cleavage products. CumD preferred substrates with long non-branched C6 substituents, but did not effectively hydrolyze a substrate with a phenyl group. Superimposition of the complex structures indicated that benzoate was bound in a significantly different direction than other aliphatic cleavage products. The directions of the bound organic acids appeared to be related with the k(cat) values of the corresponding substrates. The Ile139 and Trp143 residues on helix alpha4 appeared to cause steric hindrance with the aromatic ring of the substrate, which hampers base-catalyzed attack by water.     

Characterization of alkaline phosphatase PhoK from Sphingomonas sp. BSAR-1 for phosphate monoester synthesis and hydrolysis

The biocatalytic activity of a so far underexploited alkaline phosphatase, PhoK from Sphingomonas sp. BSAR-1, was extensively studied in transphosphorylation and hydrolysis reactions. The use of high-energy phosphate donors and oligophosphates as suitable phosphate donors was evaluated, as well as the hydrolytic activity on a variety of phosphate monoesters. While substrates bearing free hydroxy group displayed only moderate reactivity as acceptors for transphosphorylation by PhoK, strong hydrolytic activity on a broad variety of phosphate monoesters under alkaline conditions was observed. Site-directed mutagenesis of selected amino acid residues in the active site provided valuable insights on their involvement in enzyme catalysis. The key residue Thr89 so far postulated to engage in enzyme phosphorylation was confirmed to be crucial for catalysis and could be replaced by serine, albeit with much lower catalytic efficiency.     

Estrogen esters as substrates for human paraoxonases

Mammalian paraoxonases (PONs 1, 2 and 3) are a highly conserved family of esterases, with uncertain physiological functions and natural substrates. Here we characterize the ability of purified recombinant human PONs to hydrolyze estrogen esters, a class of compounds previously not known to be PON substrates. PONs hydrolyzed estrogen mono- and diesters at position 3 of the steroid A-ring. Diesters were better substrates for the PONs and were very efficiently hydrolyzed, particularly by PON3. Esters at position 17 were not cleaved by the PONs unless an adjacent double bound was present. Purified human serum butyryl cholinesterase also hydrolyzed estrogen esters, however it preferably hydrolyzed the mono-esters. The ability of the PONs' to effectively hydrolyze a variety of estrogen esters provides further insight into the structure of their active sites and suggests that natural compounds with aromatic ester groups might be relevant substrates for the PONs.     

Characterization of an extracellular medium-chain-length poly(3-hydroxyalkanoate) depolymerase from Pseudomonas alcaligenes LB19

An extracellular medium-chain-length poly(3-hydroxyalkanoate) (MCL-PHA) depolymerase from an isolate, Pseudomonas alcaligenes LB19, was purified to electrophoretic homogeneity by hydrophobic interaction chromatography using Octyl-Sepharose CL-4B and gel permeation chromatography using Sephadex G-150. The molecular mass of the enzyme, which consisted of a single polypeptide chain, was approximately 27.6 kDa. The pI value of the enzyme was estimated to be 5.7, and its maximum activity was observed at pH 9.0 and 45 degreesC. The enzyme was significantly inactivated by EDTA and phenylmethylsulfonyl fluoride (PMSF) but insensitive to dithiothreitol. It was also markedly inhibited by 0.1% Tween 80 and 0.05% Triton X-100. The purified enzyme could hydrolyze various types of bacterial aliphatic and aromatic MCL-PHAs but not poly(3-hydroxybutyrate), polycaprolactone, and poly(L-lactide). Biodegradation rates of the aromatic MCL-PHAs were significantly lower than those of the aliphatic MCL-PHAs, regardless of the compositions and types of aromatic substituents. It was able to hydrolyze medium-chain-length p-nitrophenylalkanoates more efficiently than the shorter-chain forms. The main hydrolysis products of poly(3-hydroxynonanoate) were identified as monomer units. The results demonstrated in this study suggest that the MCL-PHA depolymerase from P. alcaligenes LB19 is a distinct enzyme, which are different from those of other MCL-PHA degrading bacteria in its quaternary structure, pI value, sensitivity to EDTA and PMSF, and hydrolysis products of MCL-PHA.     

LDH and structural analogues of pyruvate (author's transl)]

Pyruvate related compounds have been tested for their active or inhibitory properties on LDH. Special structure features were needed for compounds to be bound to the enzyme active site. Pyruvate and the LDH molecule were bound by the interaction of the carboxyl group or its ester derivative, with the enzyme. Ethyl pyruvate seemed to behave as a substrate of the enzyme whereas acetophenone, ethyl acetoacetate and beta-oxoglutarate did not act as substrates or inhibitors of LDH. Phenyl pyruvate, alpha-oxoglutarate and L-mandelate are not substrates but inhibitors of the LDH. It seems that a structure having a carbonyl group in the alpha-position to the carboxyl is required for the binding of a compound to the LDH molecule. Glyoxylate alpha-oxobutyrate and alpha-oxovalerianate appear to be worse LDH substrates than pyruvate itself. This seems to suggest that the shortening or the extension of the pyruvate aliphatic chain induces a decrease of the affinity of the enzyme towards their substrate homologues.     

Inhibition of class D beta-lactamases by diaroyl phosphates

The production of beta-lactamases is an important component of bacterial resistance to beta-lactam antibiotics. These enzymes catalyze the hydrolytic destruction of beta-lactams. The class D serine beta-lactamases have, in recent years, been expanding in sequence space and substrate spectrum under the challenge of currently dispensed beta-lactams. Further, the beta-lactamase inhibitors now employed in medicine are not generally effective against class D enzymes. In this paper, we show that diaroyl phosphates are very effective inhibitory substrates of these enzymes. Reaction of the OXA-1 beta-lactamase, a typical class D enzyme, with diaroyl phosphates involves acylation of the active site with departure of an aroyl phosphate leaving group. The interaction of the latter with polar active-site residues is most likely responsible for the general reactivity of these molecules with the enzyme. The rate of acylation of the OXA-1 beta-lactamase by diaroyl phosphates is not greatly affected by the electronic effects of substituents, probably because of compensation phenomena, but is greatly enhanced by hydrophobic substituents; the second-order rate constant for acylation of the OXA-1 beta-lactamase by bis(4-phenylbenzoyl) phosphate, for example, is 1.1 x 10(7) s(-)(1) M(-)(1). This acylation reactivity correlates with the hydrophobic nature of the beta-lactam side-chain binding site of class D beta-lactamases. Deacylation of the enzyme is slow, e.g., 1.24 x 10(-)(3) s(-)(1) for the above-mentioned phosphate and directly influenced by the electronic effects of substituents. The effective steady-state inhibition constants, K(i), are nanomolar, e.g., 0.11 nM for the above-mentioned phosphate. The diaroyl phosphates, which have now been shown to be inhibitory substrates of all serine beta-lactamases, represent an intriguing new platform for the design of beta-lactamase inhibitors.     

The Tetrahymena intervening sequence ribonucleic acid enzyme is a phosphotransferase and an acid phosphatase

A shortened form of the Tetrahymena intervening sequence (IVS) RNA acts as an enzyme, catalyzing nucleotidyl transfer and hydrolysis reactions with oligo(cytidylic acid) substrates [Zaug, A. J., & Cech, T. R. (1986) Science (Washington, D.C.) 231, 470-475]. These reactions involve phosphodiester substrates. We now show that the same enzyme has activity toward phosphate monoesters. The 3'-phosphate of C5p or C6p is transferred to the 3'-terminal guanosine of the enzyme. The pH dependence of the reaction (optimum at pH 5) indicates that the enzyme has activity toward the dianion and much greater activity toward the monoanion form of the 3'-phosphate of the substrate. Phosphorylation of the enzyme is reversible by C5-OH and other oligo(pyrimidines) such as UCU-OH. Thus, the RNA enzyme acts as a phosphotransferase, transferring the 3'-terminal phosphate of C5p to UCU-OH with multiple turnover. At pH 4 and 5, the phosphoenzyme undergoes slow hydrolysis to yield inorganic phosphate. Thus, the enzyme has acid phosphatase activity. The RNA enzyme dephosphorylates oligonucleotide substrates with high sequence specificity, which distinguishes it from known protein enzymes.     

Hydrolysis of fully esterified alcohols containing from one to eight hydroxyl groups by the lipolytic enzymes of rat pancreatic juice

The enzymatic hydrolysis in vitro of the esters of methanol, ethylene glycol, glycerol, erythritol, pentaerythritol, adonitol, sorbitol, and sucrose in which all alcohol groups were esterified with oleic acid was studied. Various preparations of rat pancreatic juice, including pure lipase, were used as the sources of enzymes. Lipase (EC 3.1.1.3) did not hydrolyze compounds that contained more than three ester groups. Compounds containing four and five ester groups were hydrolyzed by certain preparations of pancreatic juice; this activity is attributed to the enzyme, nonspecific lipase. This enzyme also hydrolyzed esters of primary alcohols. The compounds containing six (sorbitol) and eight (sucrose) ester groups were not hydrolyzed.     

Studies on the substrate specificity of a carboxyl ester hydrolase from human pancreatic juice. I. Action on carboxyl esters,glycerides and phospholipids

Purified carboxyl ester hydrolase (carboxylic-ester hydrolase, EC 3.1.1.1) from human pancreatic juice was found to hydrolyze triacetin, methyl butyrate and glycerides solubilized by bile salts. It has no activity on substrate presented as emulsion or monomolecular films.The human enzyme was found to deacylate phospholipids and lysophospholipids at different rates. The hydrolysis of short-chain phospholipids and lysophospholipids at different rates. The hydrolysis of short-chain phosphatidylcholines was dependent of substrate solubility and dioctanoyl phosphatidylcholine was deacylated with the highest rate. Long-chain phosphatidylcholines and lysophosphatidylcholines present in microsomal membranes were deacylated with very low rates, only lysophosphatidylcholine deacylation was faster. Evidence is presented that human carboxyl ester hydrolase is the lyophosphatidylcholine-hydrolyzing enzyme corresponding to bovine lysophospholipase.Bile salts play an important part on the activity of human carboxyl ester hydrolase, in addition to the role of detergent that they have on insoluble substrates.     

Synthesis and beta-lactamase reactivity of alpha-substituted phenaceturates

Beta-lactams with 6alpha (penicillins) or 7alpha (cephalosporins) substituents are often beta-lactamase inhibitors. This paper assesses the effect of such substituents on acyclic beta-lactamase substrates. Thus, a series of m-carboxyphenyl phenaceturates, substituted at the glycyl alpha-carbon by -OMe, -CH(2)OH, -CO(2)(-), and -CH(2)NH(3)(+), have been prepared, and tested for their reactivity against serine beta-lactamases. The latter two are novel substituents in beta-lactamase substrates. The methoxy and hydroxymethyl compounds were found to be poor to moderately good substrates, depending on the enzyme. The aminomethyl compound gave rise to a transiently stable (t(1/2)=4.6s) complex on its reaction with a class C beta-lactamase. The reactivity of the compounds against three low molecular weight DD-peptidases was also tested. Again, the methoxy and hydroxymethyl compounds proved to be quite good substrates with no sign of inhibitory complexes. The DD-peptidases reacted with one enantiomer (the compounds were prepared as racemates), presumably the D compound. The class C beta-lactamase reacted with both D and L enantiomers although it preferred the latter. The structural bases of these stereo-preferences were explored by reference to the crystal structure of the enzyme by molecular modeling studies. The aminomethyl compound was unreactive with the DD-peptidases, whereas the carboxy compound did not react with any of the above-mentioned enzymes. The inhibitory effects of the -OMe and -CH(2)OH substituents in beta-lactams apparently require a combination of the substituent and the pendant leaving group of the beta-lactam at the acyl-enzyme stage.     

Hydrolytic and enzymatic degradation of liquid-crystalline aromatic/aliphatic copolyesters

Aromatic/aliphatic copolyesters containing hydrophilic moieties in the main chain or side chain were synthesized by bulk polycondensation of aromatic monomers without or with solubilizing substituents and aliphatic monomers. Hydrolytic and enzymatic degradation studies were carried out in vitro at 37 degrees C in pH 7.4 phosphate buffer and in Tris-HCl buffer containing proteinase K. The results indicate that liquid-crystalline aromatic/aliphatic copolyesters are degradable hydrolytically as well as enzymatically. The change in composition and morphology of the polyester films were monitored by nuclear magnetic resonance and scanning electron microscopy. The results suggested that aromatic species and aliphatic moieties could be released into aqueous solution during hydrolytic degradation of aromatic/aliphatic copolyesters with ethyleneoxy groups on the side chain. Modifying aromatic species with hydrophilic groups in aromatic/aliphatic copolyesters was an efficient method to improve degradability and biocompatibility due to improved solubility of degradation products in aqueous solution. Mechanical tests indicated that the copolyesters exhibited good mechanical properties prior to degradation, which can be of relevance for bone tissue engineering.     

Characterization of a phosphodiesterase capable of hydrolyzing EA 2192, the most toxic degradation product of the nerve agent VX

Glycerophosphodiesterase (GpdQ) from Enterobacter aerogenes is a nonspecific diesterase that enables Escherichia coli to utilize alkyl phosphodiesters, such as diethyl phosphate, as the sole phosphorus source. The catalytic properties of GpdQ were determined, and the best substrate found was bis(p-nitrophenyl) phosphate with a kcat/Km value of 6.7 x 10(3) M-1 s-1. In addition, the E. aerogenes diesterase was tested as a catalyst for the hydrolysis of a series of phosphonate monoesters which are the hydrolysis products of the highly toxic organophosphonate nerve agents sarin, soman, GF, VX, and rVX. Among the phosphonate monoesters tested, the hydrolysis product of rVX, isobutyl methyl phosphonate, was the best substrate with a kcat/Km value of 33 M-1 s-1. The ability of GpdQ to hydrolyze the phosphonate monoesters provides an alternative selection strategy in the search of enhanced variants of the bacterial phosphotriesterase (PTE) for the hydrolysis of organophosphonate nerve agents. This investigation demonstrated that the previously reported activity of GpdQ toward the hydrolysis of methyl demeton-S is due to the presence of a diester contaminant in the commercial material. Furthermore, it was shown that GpdQ is capable of hydrolyzing a close analogue of EA 2192, the most toxic and persistent degradation product of the nerve agent VX.     

Investigating the promiscuity of the chloramphenicol nitroreductase from Haemophilus influenzae towards the reduction of 4-nitrobenzene derivatives

Chloramphenicol nitroreductase (CNR), a drug-modifying enzyme from Haemophilus influenzae, has been shown to be responsible for the conversion of the nitro group into an amine in the antibiotic chloramphenicol (CAM). Since CAM structurally bears a 4-nitrobenzene moiety, we explored the substrate promiscuity of CNR by investigating its nitroreduction of 4-nitrobenzyl derivatives. We tested twenty compounds containing a nitrobenzene core, two nitropyridines, one compound with a vinylogous nitro group, and two aliphatic nitro compounds. In addition, we also synthesized twenty-eight 4-nitrobenzyl derivatives with ether, ester, and thioether substituents and assessed the relative activity of CNR in their presence. We found several of these compounds to be modified by CNR, with the enzyme activity ranging from 1 to 150% when compared to CAM. This data provides insights into two areas: (i) chemoenzymatic reduction of select compounds to avoid harsh chemicals and heavy metals routinely used in reductions of nitro groups and (ii) functional groups that would aid CAM in overcoming the activity of this enzyme.     

The synthesis of water soluble prodrugs analogs of echinocandin B.

A facile synthesis of phosphonate and phosphate ester prodrugs on the phenolic hydroxy of two echinocandin semisynthetic derivatives is reported. The water solubility and stability profiles of the ECB compounds varied with the choice of alkyl group used. In some cases, the ester prodrugs with small aliphatic side chains retained antifungal activity while enhancing water solubility.