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DOI: 10.1002/cbic.201500041
Tuning Cerium(IV)-Assisted Hydrolysis of Phosphatidylcholine Liposomes under Mildly Acidic and Neutral Conditions Dominique E. Williams, Kanchan Basnet, and Kathryn B. Grant*[a] With the goal of designing a lysosomal phospholipase mimic, we optimized experimental variables to enhance CeIV-assisted hydrolysis of phosphatidylcholine (PC) liposomes. Our best result was obtained with the chelating agent bis–tris propane (BTP). Similar to the hydrolytic enzyme, CeIV-assisted hydrolysis of PC phosphate ester bonds was higher at lysosomal pH (~ 4.8) compared to pH 7.2. In the presence of BTP, the average cleavage yield at ~ pH 4.8 and 37 8C was: 67 1 %, 5.7-fold
higher than at ~ pH 7.2 and roughly equivalent to the percent of phospholipid found on the metal-accessible exo leaflet of small liposomes. No CeIV precipitation was observed. When BTP was absent, there was significant turbidity, and the amount of cleavage at ~ pH 4.8 (69 1 %) was 2.1-fold higher than the yield obtained at ~ pH 7.2. Our results show that BTP generates homogenous solutions of CeIV that hydrolyze phosphatidylcholine with enhanced selectivity for lysosomal pH.
Introduction Small-molecule, metal-based, synthetic hydrolytic agents have potential applications in diverse fields that include biotechnology, protein engineering, the study of biomolecular function and solution structure, and drug design and discovery.[1] The lanthanide ion cerium(IV) has caught the attention of scientists due to its ability to promote efficient hydrolysis of biological molecules under non-denaturing temperature and pH conditions. The CeIV metal ion and its complexes have been employed to cleave not only phosphate ester bonds but also amide linkages. Compounds hydrolyzed by CeIV include peptides and proteins,[2] DNA,[3] activated synthetic phosphate ester-containing derivatives,[3c, 4] and phospholipids.[5] Lipids play vital roles in biological systems as energy storage molecules and chemical messengers in cell signaling and regulation. Phospholipids are of particular significance as the major components of the biological membranes that surround all cells and organelles.[6] The impressive hydrolytic ability of CeIV has been attributed to a number of factors. CeIV ions can form complexes that possess: 1) high coordination numbers (up to 12); 2) flexible ligand geometries; and 3) fast ligand exchange rates that promote favorable interactions between substrate and water molecules.[1a, b, 4d, f, 5d, 7] The CeIV ion is a strong Lewis acid with high charge density and high affinity for negatively charged oxygen atoms. Upon binding to oxygen, the electron-withdrawing ability of CeIV enhances the electrophilicity of the carbon or phosphorous atoms in the substrate’s scissile ester and amide bonds.[7b] The metal ion can additionally generate local hydrox[a] Dr. D. E. Williams, K. Basnet, Prof. K. B. Grant Department of Chemistry, Georgia State University P.O. Box 3965, Atlanta, GA 30302-3965 (USA) E-mail: [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.201500041.
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ide nucleophiles under neutral to mildly acidic conditions by lowering the pKA of metal-bound water molecules from ~ 15.7 to ¢1.1.[8] One potential drawback of CeIV is the tendency of this metal ion center to form insoluble polynuclear hydroxo species in aqueous solution as the pH is raised above ~ 4.0.[4a, d] In this process, CeIV species lose hydrolytic activity by gradually acquiring a lower net positive charge that reduces Lewis acid strength.[4a, d] Given the proper ligand, complex formation can prevent undesired precipitation to provide soluble CeIV species of definite composition that actively promote substrate hydrolysis.[2b, 3a, b, d–f, 4a, c–g, 9] Kitamura and Komiyama employed a CeIV ethylenediaminetetraacetate (EDTA) complex to selectively cleave single-stranded gap and bulge sites in double-stranded DNA oligonucleotides under physiological conditions (37 8C and pH 7.0).[3d] In the absence of EDTA, the CeIV formed insoluble hydroxide gels that lost selectivity and hydrolyzed the single-stranded and double-stranded DNA forms at the same rate. Branum et al. utilized a polyaminocarboxylate dicerium(IV) complex as a regioselective hydrolase to convert supercoiled double-stranded plasmid to linearized DNA through preferential cleavage of 3’-O-P DNA phosphate ester bonds at 37 8C and pH 8.0.[3b] Our published research has recently focused on utilizing metals to facilitate the hydrolysis of phosphate ester bonds in naturally occurring unactivated phospholipids. With the goal of designing a lysosomal phospholipase mimic, we treated liposomes of l-a-phosphatidylcholine and sphingomyelin (PC and SM, respectively; 1 in Scheme 1) with twelve metal ions that are known to hydrolyze activated and unactivated amide and/or phosphate ester bonds in peptides, nucleic acids, and other compounds (CeIV, ZrIV, HfIV, CoII, CuII, EuIII, LaIII, NiII, PdII, YIII, YbIII, and ZnII).[5d, 7c] In the event that a metal ion cleaved both of the phosphate ester bonds of the phospholipid, free inor-
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Scheme 1. Metal-assisted hydrolysis of the phosphate ester bonds of a phospholipid (1) release inorganic phosphate (2), choline (3), and ROH. ROH = diacylglycerol for phosphatidylcholine and ceramide for sphingomyelin.
ganic phosphate (2) would be released (Scheme 1). In these first generation experiments, only CeIV, ZrIV, HfIV, EuIII, LaIII, PdII, and YbIII were capable of hydrolyzing SM and/or PC. In contrast to ZrIV, HfIV, EuIII, LaIII, PdII, and YbIII, for which hydrolysis levels were extremely low at neutral and at mildly acidic pH values, cerium(IV) ammonium nitrate (Ce(NH4)2(NO3)6) was found to be overwhelmingly superior, reacting with the phosphate ester bonds in PC and SM to liberate significant amounts of inorganic phosphate (2; 20 h at 60 8C). Similar to lysosomal phospholipase, we found that CeIV consistently hydrolyzed phospholipids more efficiently at lysosomal pH (~ 4.8) compared to near-neutral conditions. NMR and Fourier transform (FT) Raman spectra show that lanthanide cations coordinate to PC bilayers, primarily at free oxygen atom(s) of the polar head group phosphates.[10] This led to the proposal of a hydrolytic mechanism in which CeIV binds to a negatively charged phosphate oxygen in the lipid (activating the phosphorous atom towards nucleophilic attack) while delivering a hydroxide nucleophile to the scissile phosphate ester bond.[4a, d, 5d, 7b, 11] Examples of lysosomal phospholipases that engage in phosphate ester bond hydrolysis include: 1) acid sphingomyelinase,[12] which acts on SM to release ceramide and 2) lysosomal phospholipase C,[13] which cleaves phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine to produce diacylglycerol (Scheme 1). When these enzymes are impaired, symptomatology can result from a significant buildup of unhydrolyzed phospholipid substrate within the lysosomes of affected cells. In Niemann-Pick disease type A, an inherited lysosomal storage disorder caused by diminished activity of acid sphingomyelinase,[12b, 14] pathogenic levels of unhydrolyzed sphingomyelin accumulate within lysosomes of central nervous and reticuloendothelial system cells.[14a] In Sandhoff disease, low activity of the lysosomal enzyme hexosaminidase B results in the lysosomal accumulation of the ganglioside GM2 and of the glycosphingolipid GA2,[14b] yet its clinical manifestations are very similar to Niemann-Pick. In common to both disorders is an unexplained, significant buildup of phosphatidylcholine within the lysosomes of pulmonary reticuloendothelial cells.[14a, 15] This gives rise to the acute respiratory infections that are one of the major causes of death in Niemann-Pick and Sandhoff patients.[16] The ability of CeIV ions to selectively hydrolyze phospholipid liposomes under mildly acidic conditions pointed to the possibility of developing metal-based reagents to treat storage disease.[14a, b, 15–16] In our first-generation published experiments, hydrolysis of phosphatidylcholine liposomes by Ce(NH4)2(NO3)6 gave rise to 1.8 times more inorganic phosphate at pH 4.8 ChemBioChem 2015, 16, 1474 – 1482
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compared to pH 7.2 (20 h at 37 8C, 2 mm PC, 10 mm CeIV). Hydrolysis yields at ~ pH 4.8 and 7.2 were 18 3 % and 10 2 %, respectively.[5d, 7c] More efficient suppression of cleavage under neutral conditions would be critical for protecting cells from hydrolytic damage in the event of any leakage of a metalbased therapeutic agent from the lysosome into the cytoplasm. Moreover, the hydrolysis reactions contained undesired CeIV precipitation, and the yields of phosphate diester cleavage at 37 8C and ~ pH 4.8 were low. In the present paper, CeIV-assisted hydrolysis of phosphatidylcholine liposomes was optimized. We evaluated coordinating ligands and multiple reaction variables. Our aim was to tune and enhance the reactivity of CeIV to afford efficient, homogeneous hydrolysis of PC liposomes at 37 8C and ~ pH 4.8, with minimized cleavage under near-neutral conditions. Our best result for phosphatidylcholine liposomes was obtained in the presence of CeIV and bis–tris propane (BTP). Compared to our first-generation experiments,[5d, 7c] the yield of PC hydrolysis at ~ pH 4.8 and the ratio of pH 4.8 hydrolysis to pH 7.2 hydrolysis were significantly increased, and visible CeIV precipitation was cleared from hydrolysis reactions. When the single-chain nonionic surfactant Triton X-100 was used to convert the liposomes to mixed micelles, cleavage of phosphatidylcholine occurred in near-quantitative yields (37 8C and ~ pH 4.8).
Results and Discussion Evaluating ligands to optimize CeIV-assisted hydrolysis of PC liposomes In the first experiment for this paper, six prospective ligands were tested for hydrolysis of phosphatidylcholine liposomes against buffered CeIV(NH4)2(NO3)6 (Scheme 2). The anionic polyaminocarboxylates ethylenediaminetetraacetate (4, EDTA) and 1,3-diamino-2-hydroxypropane-N,N,N’,N’-tetraacetic acid (5, HPTA) form CeIV complexes that promote efficient doublestrand DNA cleavage (37–55 8C and pH 8.0).[3a, b] Because chelating ligands with negatively charged donor groups can decrease the Lewis acid character of lanthanide ions,[17] the amine and amino alcohol ligands 6–9 were chosen in the event that 4 and 5 were found to be detrimental to phosphate ester hydrolysis. Ott and Krmer utilized ZrCl4 and tris(hydroxymethyl)aminomethane (tris, 7) to produce higher rates of BNPP cleavage at ~ pH 4.8 compared to ZrIV alone, with a steep decline in reactivity as pH values approached ~ 7.2.[18] Finally, 1,10-phenanthroline (8) and histamine (9) were selected on the basis of literature findings that lanthanide complexes incorporating these ligands efficiently hydrolyze p-nitrophenyl-activated phosphate ester bonds.[19] Similar to our first generation, published experiments,[5d, 7c] the cleavage reactions contained 2 mm of PC in liposome form and 10 mm CeIV(NH4)2(NO3)6 and were run at ~ pH 4.8 and 7.2 for a period of 20 h. Twenty millimolar concentrations of each of the six ligands were added, and the reaction temperature was raised from 37 to 60 8C. The production of inorganic phosphate by CeIV-assisted hydrolysis of phosphate ester bonds (Scheme 1) was measured by using a commercially available
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Scheme 2. Ligands used to evaluate CeIV-assisted hydrolysis of phosphatidylcholine: ethylenediamine-tetraacetate (EDTA, 4); 1,3-diamino-2-hydroxypropane-N,N,N’,N’-tetraacetate (HPTA, 5); 1,3-bis-[tris(hydroxymethyl)methylamino]propane (BTP, 6); tris(hydroxymethyl)aminomethane (tris, 7); 1,10 phenanthroline (8); histamine (9). pKa values appear in parenthesis;[4d, 21–22] n.d. = not determined.
malachite green/molybdate-based colorimetric assay kit. The blue–green complex formed between malachite green and phosphomolybdate was monitored at 620 nm by using a UV– visible spectrophotometer.[20] The results of this experiment are shown in Figure 1, where absorbance at 620 nm reflected relative hydrolysis yields. The ligands EDTA (4) and HPTA (5) completely suppressed phosphatidylcholine cleavage, even at ~ pH 4.8. Although hydrolysis was detected in the presence of tris (7), 1,10-phenanthroline (8), and histamine (9), similar levels were observed under mildly acidic and near-neutral pH values. The best results were obtained with the amino alcohol ligand BTP (6). The hydrolysis quotient, calculated by dividing the average hydrolysis at ~ pH 4.8 by the average hydrolysis at ~ pH 7.2, was 2.6, showing
that free, inorganic phosphate levels were approximately 2.6fold higher under mildly acidic conditions compared to nearneutral pH. This represents an improvement over the 1.4-fold hydrolysis quotient exhibited by Ce(NH4)2(NO3)6 in the absence of ligand (Figure 1). By comparing relative absorbance values at pH 4.8 (Figure 1), ligands 4–9 were ranked from high to low hydrolysis levels as follows: tris (pKa = 8.3)[21] BTP (pKa1 = 6.8; pKa2 = 9.1)[4d] > histamine (pKa1 = 6.1; pKa2 = 9.8)[21] phenanthroline (pKa = 4.8)[21] > EDTA (pKa3 = 2.0; pKa4 = 2.6)[21] HPTA (pKa3 = 1.5; pKa4 = 2.6).[22] Although there are exceptions, the ordering reveals a general tendency for cleavage levels to decrease as a function of increasing ligand acidity. Strongly chelating polycarboxylates, similar to EDTA (4) and HPTA (5), for which almost no hydrolysis was detected, form thermodynamically stable lanthanide complexes with reduced ligand exchange rates.[22–23] It is reasonable to suggest that the persistent negative charges associated with 4 and 5 might have strongly suppressed phosphate ester bond hydrolysis by reducing the Lewis acid strength of CeIV.[17] Although similar in structure, it is interesting to note that BTP (6) and tris (7) have different effects on CeIV-assisted PC hydrolysis. The pKa of the amino group of tris is 8.3. Whereas CeIV has the ability to lower the pKa values of nearby functional groups, tris might still possess a positive charge at ~ pH 4.8 and 7.2 that reduces metal–ligand interactions. This might be related to the relatively high levels of CeIV-assisted phosphate ester bond cleavage that occur at low and high pH values. In contrast, BTP has two amino groups with respective pKa1 and pKa2 values of 6.8 and 9.1. Similar to tris, the positive charge at ~ pH 4.8 might reduce complex formation and promote hydrolysis. At pH ~ 7.2, however, there is a possibility that one of the two amino groups of BTP is predominantly neutral. This might increase ligand–metal interactions and, in turn, suppress cleavage. In the absence of chelating agents 4–9, CeIV precipitates were immediately formed upon the addition of Ce(NH4)2(NO3)6 to the ~ pH 4.8 and 7.2 hydrolysis reactions. When the ligands were present, however, the precipitation was either completely eliminated (in the case of 4 and 5), or greatly reduced (6–9). Although all six chelating agents were clearly capable of interacting with CeIV and influencing speciation, our observations and data suggested that substantial levels of complex formation might suppress phosphate ester hydrolysis. CeIV-assisted PC hydrolysis at 37 8C
Figure 1. Percent relative absorbance at 620 nm plotted as a function of pH. A total of 2 mm PC was reacted at 60 8C for 20 h in the presence of 10 mm Ce(NH4)2(NO3)6 and 20 mm ligand (4–9, Scheme 2) at pH 5.0–5.3 (&) or pH 7.0–7.4 (&). The number of trials (n) appears in parenthesis. Error bars represent the standard deviation.
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In our next set of experiments, the reaction temperature was reduced from 60 to 37 8C (normal core body temperature). A final concentration of 20 mm of BTP was added to ~ pH 4.8 and 7.2 reactions in order to selectively suppress CeIV-assisted phosphatidylcholine hydrolysis at near-neutral pH. The reaction time (20 h) and remaining reagent concentrations (2 mm PC, 10 mm Ce(NH4)2(NO3)6) were carried over from the preceding experiment. The inorganic phosphate liberated by CeIV was quantitated with a malachite green/molybdate reagent, and a choline-specific colorimetric assay based on Amplex Red
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Full Papers dye[5d, 7c] was utilized in the event that the lower reaction temperature limited hydrolysis to the choline side of phosphorous (3; Scheme 1). As desired, treatment of PC with CeIV in the presence of BTP produced minimal amounts of phospholipid cleavage under near-neutral conditions (Figure 2). At ~ pH 4.8,
reaching a maximum at 1.75 mm of CeIV, but were significantly lower at all metal ion concentrations above 2 mm. In the presence of 10 mm CeIV, reducing the amount of PC by a factor of 57, from 2 mm to 35 mm, resulted in a relatively modest approximately eightfold increase in the average yield of inorganic phosphate (6 2 %[7c] in Figure 2 to 45 4 % in Figure 3). It is conceivable that any potential benefit to reaction yield, gained by increasing the ratio of metal to phospholipid, might have been partially offset by a drop in reaction rate caused by substantially lowering the phosphatidylcholine concentration. In the presence of 35 mm PC, reducing CeIV by a factor of 5.7, from 10 to 1.75 mm, caused an additional 1.5-fold increase in the hydrolysis yield (from 45 4 to 69 1 % in Figure 3). This suggested that there could have been a larger proportion of hydrolytically active CeIV species at the lower 1.75 mm metal ion concentration.[4d, 7a]
Figure 2. Averaged hydrolysis yields plotted as a function of pH for A) malachite green detection of free inorganic phosphate and B) Amplex Red detection of free choline. A total of 2 mm PC was reacted at 37 8C for 20 h in the presence of 10 mm Ce(NH4)2(NO3)6 and 20 mm BTP at pH 4.9–5.2 (&) or pH 7.0–7.3 (&). The number of trials (n) appears in parenthesis. Error bars represent standard deviation.
Time course: CeIV-assisted PC hydrolysis at 37 8C Our next objective was to monitor the time-dependent release of inorganic phosphate by CeIV-assisted hydrolysis of PC liposomes (Figure 4 A). Optimized reagent concentrations were employed (1.75 mm CeIV, 35 mm PC) at the standard 37 8C reac-
inorganic phosphate and choline could indeed be detected and quantitated, but the resulting yields were low (6 2 and 14 1 %, respectively). Our next goal was to increase the efficiency of CeIV-assisted hydrolysis.
Optimizing CeIV concentration CeIV hydroxo aggregates with reduced hydrolytic activity have a propensity to form at high metal ion concentrations.[4d, 7a] This suggested that it might be possible to increase cleavage yields by reducing the amount of CeIV(NH4)2(NO3)6. Phosphatidylcholine hydrolysis reactions were accordingly run in the presence of 0 to 10 mm of CeIV(NH4)2(NO3)6 (20 h at 37 8C, ~ pH 4.8, no BTP). In these experiments, the concentration of PC was lowered from 2 mm to 35 mm. The release of phosphate was then monitored at 620 nm by using malachite green. As shown in Figure 3, hydrolysis levels initially increased,
Figure 3. Absorbance at 620 nm plotted as a function of CeIV concentration for malachite green-treated hydrolysis reactions. A total of 35 mm of PC was reacted at 37 8C for 20 h in the presence of 0.0 mm to 10.0 mm Ce(NH4)2(NO3)6 (1.5 mm piperazine buffer pH 4.8). Representative percent yields of inorganic phosphate are shown. The number of trials (n) appears in parenthesis. Error bars represent standard deviation.
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Figure 4. A) Averaged hydrolysis yields plotted as a function of time for malachite green detection of free, inorganic phosphate. A total of 35 mm PC was reacted at 37 8C with 1.75 mm Ce(NH4)2(NO3)6 : 1) without BTP, at ~ pH 4.8–4.9 (&) or ~ pH 7.1–7.4 (&; 3.5 mm piperazine or HEPES buffer respectively); or 2) with 3.5 mm BTP, at ~ pH 4.8–5.1 (~; in 1 mm MES buffer) or at ~ pH 7.0– 7.3 (~). The numbers of trials at each time point ranged from 2 to 8. B) Quotients obtained by dividing the average ~ pH 4.8 hydrolysis yields by the average ~ pH 7.2 hydrolysis yields for reactions without BTP (*) and with 3.5 mm BTP (*).
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Full Papers tion temperature. Similar to the preceding experiments in this paper, the metal-to-ligand ratio (M/L) of CeIV to BTP was 1:2. Results obtained at ~ pH 4.8 are discussed as follows. From 0– 20 h, the averaged approximate pseudo-first-order rate constants for CeIV-assisted PC hydrolysis at ~ pH 4.8 were 0.0610 0.0009 h¢1 and 0.0260 0.0010 h¢1 in the absence and presence of 3.5 mm of BTP, respectively (Figure S1 in the Supporting Information). Corresponding yields of inorganic phosphate at 20 h were 69 1 and 42 3 % (Figure 4 A). Although it is evident that PC liposome hydrolysis was initially faster without a ligand, a significant drop-off in the reaction rate occurred between the 20 and 30 h time points. In contrast, when BTP was present, hydrolysis levels continued to increase after 20 h. As a result, the yields of inorganic phosphate at 30 h were within experimental error (72 4 % without vs. 65 5 % with BTP; Figure 4 A). In the absence of ligand, ~ pH 4.8 hydrolysis reactions had accumulated visible CeIV precipitation, whereas those containing BTP remained clear. Reaction times beyond 30 h did not significantly improve hydrolysis yields. Next, phosphatidylcholine time course reactions at ~ pH 7.2 were considered (Figure 4 A). As mentioned, an ideal CeIVbased lysosomal phospholipase mimic should display enhanced activity at lysosomal pH (~ 4.8), accompanied by low levels of cleavage under near-neutral conditions. Figure 4 A shows that the addition of BTP to the hydrolysis reactions reduced cleavage levels up to 79 % (37 8C): at the 20 h time point, yields of inorganic phosphate were 33 5 and 7 2 % in the absence and presence of BTP, respectively (Figure 4 A). In order to better illustrate the effects of BTP, the ~ pH 4.8/ ~ pH 7.2 hydrolysis quotients obtained for the data points in Figure 4 A were plotted in Figure 4 B. In the presence of BTP, CeIV(NH4)2(NO3)6 produced an approximately linear increase in ~ pH 4.8/ ~ pH 7.2 quotients over the 30 h time course, whereas in the absence of ligand, the quotients were relatively constant. This shows that BTP-assisted hydrolysis became increasingly selective for mildly acidic conditions as a function of time. There was visible CeIV precipitation in the ~ pH 7.2 cleavage reactions without ligand, whereas those containing BTP remained clear throughout the time-course experiment.
Figure 5. Absorbance at 620 nm plotted as a function of [CeIV] to [BTP] ratio for malachite green-treated hydrolysis reactions. A total of 35 mm of PC was reacted at 37 8C for 20 h in the presence of 1.75 mm Ce(NH4)2(NO3)6 and 3.5 mm to 0.19 mm BTP at pH ~ 4.6–5.1 (&; 1 mm piperazine buffer) or pH ~ 7.0–7.4 (~; 1 mm HEPES). Representative percent yields of inorganic phosphate are shown. Three or more trials were performed for each data point. Error bars represent standard deviation.
on the phosphate levels produced under near-neutral conditions. Phosphatidylcholine cleavage at 37 8C: Summary Figure 6 summarizes the pH 4.8/pH 7.2 hydrolysis quotients and inorganic phosphate yields obtained under the different sets of experimental variables that we have evaluated thus far. In the published first-generation reaction that served as the starting point for the research reported in this paper (2 mm phosphatidylcholine, 10 mm Ce(NH4)2(NO3)6, 37 8C, 20 h), treatment of PC liposomes with CeIV generated inorganic phosphate in relatively low yield (B in Figure 6).[7c] A major goal of the work described in the present study was to optimize cleavage while increasing the ~ pH 4.8/ ~ pH 7.2 quotient. CeIV-assisted hydrolysis of phosphatidylcholine liposomes at ~ pH 4.8 and 37 8C was improved considerably by: 1) Decreasing the con-
Optimizing BTP concentration at 37 8C An M/L ratio of 1:2 was employed in all of the BTP hydrolysis reactions described up until this point. In order to further improve cleavage yields, the optimized concentrations of phosphatidylcholine (35 mm) and CeIV (1.75 mm) were reacted at M/L ratios ranging from 1:2 up to 9:1 at the standard 37 8C temperature and reaction time (20 h). The amount of CeIV was held constant, while the concentration of BTP was incrementally decreased. Selected cleavage yields were then calculated. As shown in Figure 5, the percent of hydrolysis at ~ pH 4.8 was: 42 % at the 1:2 M/L ratio (pH 4.8/pH 7.2 quotient = 6.0); 67 1 % at the 4:1 ratio (quotient = 5.7); and 54 1 % at the 5:1 ratio (quotient = 9.6). Thus, lowering the concentration of BTP from 3.5 mm (M/L = 1:2) to 0.35 mm (M/L = 5:1) markedly improved cleavage at ~ pH 4.8 without having a significant effect ChemBioChem 2015, 16, 1474 – 1482
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Figure 6. Average yields of inorganic phosphate released upon treatment of phosphatidylcholine liposomes with Ce(NH4)2(NO3)6 in the presence and absence of BTP, plotted according to reaction conditions (~ pH 4.6–5.2 and 37 8C for 20 h). A) 10 mm CeIV, 2 mm PC, 20 mm BTP; B) 10 mm CeIV, 2 mm PC; C) 1:75 mm CeIV, 35 mm PC, 3.5 mm BTP (M/L = 1:2); D) 10 mm CeIV, 35 mm PC; E) 1.75 mm CeIV, 35 mm PC, 0.35 mm BTP (M/L = 5:1); F) 1.75 mm CeIV, 35 mm PC, 0.44 mm BTP (M/L = 4:1); G) 1.75 mm CeIV, 35 mm PC. Corresponding pH 4.8/pH 7.2 hydrolysis quotients are shown above each bar. Three or more trials were performed for each set of reactions. Error bars represent standard deviation. Data corresponding to (B) were previously published;[7c] n.d. = not determined.
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Full Papers centration of phospholipid (B vs. D in Figure 6); 2) Decreasing the concentration of Ce(NH4)2(NO3)6 (D vs. G); and 3) Decreasing the concentration of BTP (C vs. E and F). Our best results were obtained with a 4:1 metal to BTP ratio in the presence of 35 mm phosphatidylcholine and 1.75 mm Ce(NH4)2(NO3)6 (20 h at 37 8C). Compared to the first generation experiments,[5d] , [7c] utilizing the optimized hydrolysis conditions raised the pH 4.8/ pH 7.2 hydrolysis quotient from 1.8 to 5.7. This was done by increasing the cleavage yield at ~ pH 4.8 from 18 to 67 1 %, without significantly changing hydrolysis at ~ pH 7.2 (10 2 % initial averaged yield vs. 12 1 % for the optimized conditions; A vs. B in Figure 7).
Figure 7. A comparison of A) first-generation reactions to optimized hydrolysis reactions in the B) absence and C) presence of 1.6 mol equiv of Triton X100. Average yields of inorganic phosphate released upon treatment of PC with Ce(NH4)2(NO3)6 for 20 h at 37 8C are plotted for the reagent concentrations: A) 10 mm CeIV, 2 mm PC; B) and C) 1.75 mm CeIV, 35 mm PC, 0.44 mm BTP at pH 4.8–5.0 (&) or pH 7.1–7.3 (&). Corresponding pH 4.8/pH 7.2 hydrolysis quotients are underlined. Three or more trials were performed for each reaction. Error bars represent standard deviation. Data corresponding to (A) were previously published.[7c] Data corresponding to (B) were taken from Figures 5 and 6.
The addition of BTP significantly increased pH 4.8/pH 7.2 quotients by selectively reducing levels of CeIV(NH4)2(NO3)6assisted phosphatidylcholine hydrolysis under near-neutral pH conditions (20 h at 37 8C; B vs. A, G vs. C, G vs. E and F; Figure 6). At ~ pH 4.8, inorganic phosphate yields in the absence and presence of 0.44 mm BTP (4:1 m:L) were within experimental error, 69 and 67 1 % respectively (20 h at 37 8C; G vs. F in Figure 6), whereas corresponding ~ pH 7.2 hydrolysis yields were decreased by BTP from 33 5 to 12 1 % (Figures 4 and 5). Under optimal conditions, BTP suppressed PC hydrolysis at ~ pH 7.2 without significantly altering cleavage levels at mildly acidic pH. A second major goal of our research was to reduce CeIV precipitation. Optimized BTP reactions were free of turbidity, irrespective of pH (C, E, and F in Figure 6). When BTP was omitted, CeIV precipitation was observed (G). Thus, the major effect of BTP was to generate homogenous solutions of CeIV that hydrolyzed phosphatidylcholine liposomes with enhanced selectivity for lysosomal pH.
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Membrane permeability and dynamics Thus far in the present publication, the highest inorganic phosphate yield obtained after treating phosphatidylcholine liposomes with CeIV was 72 4 % (30 h at 37 8C; Figure 4). This upper hydrolysis limit approaches the ~ 68 % average distribution of total phospholipid on the metal-accessible exo leaflet of small PC liposomes[24] and suggests that CeIV might preferentially cleave phosphatidylcholine distributed on the external exo liposomal leaflet. Liposomes are unilamellar, lipid bilayer structures that contain a hollow core. In general, most metal ions are unable to freely permeate across biological membranes.[25, 26] Compared to the endo leaflet, the polar head groups of phosphatidylcholine molecules in the exo leaflet are therefore expected to be considerably more likely to interact with metal ions. In addition to membrane permeability, factors relating to membrane dynamics favor preferential hydrolysis of exo liposomal phospholipids. Due to low levels of electrostatic repulsion, the rates of transverse lipid diffusion (flip-flop) between the exo and endo bilayer surfaces are extremely slow in the case of PC and other neutral and/or zwitterionic phospholipids.[27] Moreover, CeIV-assisted hydrolysis of PC generates neutral diacylglycerol,[5d] which has been shown to have little if any effect on the transverse diffusion rates of adjacent lipids in the bilayer.[27] The membrane permeability factors described above were taken into consideration in our previously published l-a-phosphatidylcholine study.[5d] Triton X-100 is a single-chain, nonionic surfactant that is used to convert PC liposomes to micelles, a lipid packing arrangement in which all of the polar head groups face the external aqueous phase.[28] When we added 1.6 mol equiv of this surfactant to our PC liposome preparations, phosphatidylcholine hydrolysis yields at ~ pH 4.8 and 60 8C were increased from 41 5 to 79 7 %.[5d] In the present work, PC liposomes were pretreated with 1.6 mol equiv of Triton X-100. Turbidity measurements at a surfactant/phosphatidylcholine molar mixing ratio of 1.6 revealed a decrease in light absorbance consistent with the conversion of the liposomes to mixed micelles (Figure S2).[28] When the resulting mixed-micellar preparation was treated with CeIV at our optimized set of reagent concentrations (35 mm PC, 1.75 mm CeIV, 0.44 mm BTP), the averaged yield of PC hydrolysis at ~ pH 4.8 was 98 2 %, and the pH 4.8/pH 7.2 hydrolysis quotient was 6.0 (20 h at 37 8C; C in Figure 7).
1
H NMR analyses
Solution interactions between BTP and CeIV were first studied by Maldonado and Yatsimirsky. With [D6]DMSO as the solvent, the 1H NMR spectrum of free BTP was compared to the spectrum of a pre-synthesized 1:2 M/L CeIV complex of the formula [Ce(BTP)2(NO3)4]·2 H2O.[4d] Complex formation with CeIV caused the 1H NMR signals associated with the -CH2CH2CH2-, -CH2NH-, and -CH2OH methylene protons of BTP to undergo significant downfield shifts (Table 1). However, when Maldonado and Yatsimirsky diluted the [D6]DMSO solutions with D2O (10 %, v/v),
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Full Papers Table 1. CeIV-induced changes in the chemical shift values of bis–tris propane (BTP) methylene protons (Dd)[a]
pD [b]
2.3 5.1–5.2[b] 7.6–7.7[b] 9.6[b] DMSO[d]
-CH2CH2CH2¢0.003 ¢0.003 + 0.023 + 0.035 + 0.540
Dd (ppm) -CH2NH¢0.004 ¢0.004 + 0.018 + 0.051 + 0.470
CH2OH ¢0.002 ¢0.003 + 0.007 + 0.017 + 0.240
BTP protonation BTPH BTPH BTPH BTP –
2+ 2 2+ 2 +
n[c] 2 2 2 1 –
[a] Dd = dcomplex¢dBTP, where complex = [Ce(BTP)2(NO3)4]·2 H2O and BTP = free bis–tris propane. [b] NMR spectra are in D2O (Figures S3 and S4). [c] n = number of trials. [d] Published data.[4d]
the spectra of BTP and [Ce(BTP)2(NO3)4]·2H2O were superimposable, suggesting that the complex had dissociated. Our goal was to utilize 1H NMR in order to investigate the interactions between BTP and CeIV as a function of pD. We recorded proton NMR spectra of BTP and [Ce(BTP)2(NO3)4]·2 H2O[4d] in D2O (100 %, v/v) after carefully adjusting the solution pD with DCl and NaOD. When additional BTP ligand was added to NMR samples containing [Ce(BTP)2(NO3)4]·2 H2O, new peaks corresponding to free BTP were not observed in any of the spectra (data not shown). This suggested that CeIV was kinetically labile and that the BTP resonances in the [Ce(BTP)2(NO3)4]·2 H2O NMR spectra were averaged values arising from free and metal bound nitrogen and oxygen donor atoms (Figures S3 and S4). The CeIV-induced chemical shift changes (dcomplex¢dBTP) observed for the -CH2CH2CH2-, -CH2NH-, and -CH2OH methylene protons of BTP are shown in Table 1. At pD values ranging from 2.3 to 5.2, the [Ce(BTP)2(NO3)4]·2H2O NMR spectra in D2O showed no evidence of complex formation. This is consistent with Maldonado and Yatsimirsky’s NMR data for [Ce(BTP)2(NO3)4]·2 H2O in D2O/ [D6]DMSO (1:9, v/v).[4d] According to the pKa values of BTP (pKa1 = 6.8; pKa2 = 9.1),[4d] it is possible that the BTPH22 + form of the ligand exists at ~ pH 4.8. With both nitrogen donor atoms protonated, BTP would not be expected to undergo significant complex formation. Notwithstanding, the addition of BTP to phosphatidylcholine reactions prevented visible CeIV precipitation over the entire pD range studied. This suggests that the metal and ligand were able to interact to some degree. When the pD was increased from 5.2 to 7.6 and 9.6, CeIV induced small, progressively increasing downfield shifts in the positions of all of the BTP methylene proton resonances. The magnitude and pattern of the chemical shift changes suggest that -CH2NH- and -CH2OH groups of BTP interact with CeIV as a function of decreasing ligand charge.[29] Maldonado and Yatsimirsky utilized [Ce(BTP)2(NO3)4]·2 H2O to conduct potentiometric titrations with the aim of characterizing the speciation of CeIV in aqueous solution.[4d] Due to extensive precipitation of CeIV, CeIV(NH4)2(NO3)6 could not be analyzed directly. At pH values greater than 7.0, Ce4(OH)151 + was the major multinuclear CeIV hydroxo species formed from [Ce(BTP)2(NO3)4]·2 H2O. Changing the pH from 7 to 5 afforded Ce4(OH)142 + and then Ce4(OH)133 + , CeIV hydroxo species that have a higher positive charge and increased Lewis acid ChemBioChem 2015, 16, 1474 – 1482
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strength.[4d] We propose that BTP is likely to weakly associate with CeIV in aqueous solutions at ~ pH 4.8. In so doing, BTP might act as a mediator to control CeIV speciation while preventing CeIV precipitation and maintaining the activity of the metal ion center towards phosphatidylcholine hydrolysis. Upon testing different BTP concentrations at ~ pH 4.8, we found that optimum PC hydrolysis occurred when the ratio of CeIV ions to ligand was 4:1 (Figure 5). This suggests that the active species is not a stable complex between the ligand and metal but is likely to be something more dynamic. Such a conclusion is in agreement with the 1H NMR data, which show no evidence for complex formation between CeIV and BTP at pD values below 5.3 (Table 1). At ~ pH 7.2, the NMR data suggest that BTP interacts more extensively with CeIV. As a result, precipitation of CeIV is prevented, and PC hydrolysis is suppressed. BTP might bind to and directly inactivate CeIV at the higher pH value or might shift the speciation equilibria away from the formation of CeIV species with the potential for high hydrolytic activity (e.g., Ce4(OH)133 + ).
Conclusion The ultimate goal of the research described in this report was to lay the groundwork for the development of an efficient lysosomal phospholipase mimic based on CeIV. Towards this end, multiple ligands and experimental variables were tested in order to optimize the hydrolysis of phosphate ester bonds in phosphatidylcholine liposomes. The ideal hydrolytic agent would exhibit maximal activity at lysosomal pH (~ 4.8), accompanied by low levels of cleavage under near-neutral conditions. Although polyaminocarboxylate ligands were found to be detrimental, our best results were obtained with the amino alcohol bis–tris propane. Under optimized reaction conditions, BTP suppressed the hydrolysis of phosphatidylcholine liposomes at ~ pH 7.2 and eliminated CeIV precipitation. A comparison to first-generation experiments[5d, 7c] showed that the percent cleavage of PC liposomes at ~ pH 4.8 increased from 18 3 to 67 1 % and that the pH 4.8/pH 7.2 hydrolysis quotient increased from 1.8 to 5.7 (20 h at 37 8C; A vs. B in Figure 7). The single-chain nonionic surfactant Triton X-100, which converts liposomes to metal-accessible mixed micelles (Figure S2),[28] increased the yield of CeIV-assisted phosphate ester bond hydrolysis to 98 2 % (20 h at 37 8C and ~ pH 4.8; C in Figure 7). Our future research will therefore focus on the development of ligands that facilitate the transport of CeIV across phospholipid bilayers. This will enable CeIV to efficiently hydrolyze phosphate ester bonds located on the metal-inaccessible endo leaflet of PC liposomes. To the best of our knowledge, this study presents the first example of near-quantitative, pH-selective hydrolysis of a naturally occurring phospholipid by a metal ion center under physiological conditions of temperature and pH. Cerium complexes display low to moderate cellular toxicity and have had therapeutic applications as anti-emetic agents and in topical creams to treat full-thickness burns.[30] In principle, encapsulation of CeIV into a delivery vehicle such as a hydrolytically inert, lysosomotropic “carrier liposome” would promote the transport of
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Experimental Section General: Deionized distilled water was used in the preparation of all stock solutions. Chemicals were of the highest available purity and were utilized without further purification. l-a-Phosphatidylcholine from chicken egg (catalogue number 840051P) was obtained from Avanti Polar Lipids. Choline chloride, Ce(NH4)2(NO3)6, 1,3-diamino-2-hydroxypropane-N,N,N’,N’-tetraacetic acid (HPTA), 1,10phenanthroline, piperazine, and tris(hydroxymethyl)aminomethane (tris) were purchased from Sigma–Aldrich. DMSO, 1,3-bis[tris(hydroxymethyl)methylamino]propane (bis–tris propane, BTP), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), histamine, 4morpholineethanesulfonic acid (MES), 4-morpholinepropanesulfonic acid (MOPS), anhydrous tert-butanol ( 99.5 %), and Triton X-100 were from Sigma. EDTA was obtained from Fisher Scientific. The NMR reagents deuterium oxide (99 atom % D), sodium deuteroxide (30 wt % in D2O, 99 atom % D), [D6]DMSO (99 atom % D), and deuterium chloride (35 wt % in D2O, 99 atom % D) were acquired from Sigma–Aldrich. Known compound [Ce(BTP)2(NO3)4]·2 H2O was prepared by using a previously published procedure[4d] (Figures S5 to S7). Malachite green phosphate (catalogue number POMG-25H) and Amplex Red Sphingomyelinase Assay Kits (catalogue number A12220) were purchased from BioAssay Systems and Invitrogen, respectively. IR spectra were recorded on a PerkinElmer Spectrum 100 FT-IR spectrometer coupled with an attenuated total reflection (ATR) sampling accessory. NMR spectra were acquired on a Bruker Avance 400 MHz NMR spectrometer. Colorimetric assays were conducted on a UV-1601 Shimadzu spectrophotometer. Preparation of liposomes: l-a-Phosphatidylcholine (2–4 mg or 16–19 mg) was dissolved in chloroform (1 mL) in a round bottom flask. The chloroform solution was concentrated in vacuo overnight. To the resulting dried phospholipid film was added preheated ddH2O at 65 8C to a final concentration of 120 mm (2 mm lipid hydrolysis reactions) or 2.06 mm (35 mm lipid hydrolysis reactions). The aqueous solution was sonicated for 20 min at 65 8C, which is above the range of gel-to-fluid transition temperature (Tm) values reported for PC (41–55 8C).[31] Mixed micelles of PC and Triton X-100 were prepared from phosphatidylcholine liposomes and then detected by measuring solution turbidity as previously described (Figure S2).[5d] Lipid hydrolysis: The following additional stock solutions were prepared prior to the hydrolysis reactions: Ce(NH4)2(NO3)6 (10, 17.5, or 11.7 mm in ddH2O); buffers (MES, piperazine, or HEPES; 200, 23.3, or 10 mm in ddH2O), ligands (tris, BTP, and histamine; 200 mm in ddH2O); HPTA (50 mm in ddH2O at pH 7); EDTA (200 mm in ddH2O at pH 8); 1,10-phenanthroline (200 mm in neat DMSO). The solutions were used to prepare standard reactions consisting of either: 1) 10 mm Ce(NH4)2(NO3)6, ligand or buffer (piperazine or HEPES; 20 mm), and phosphatidylcholine (2 mm); or 2) Ce(NH4)2(NO3)6 (1.75 mm), bis–tris propane (BTP; 3.5 mm), buffer (MES, piperazine, or HEPES; 1–3.5 mm), and phosphatidylcholine (35 mm). For the 2 mm lipid reactions, the metal complexes were allowed to form in situ by adding equal volumes of an aqueous solution of Ce(NH4)2(NO3)6 (100 mm, 300 mL) to a solution of ligand (200 mm, 300 mL). For the 35 mm lipid reactions, the following solutions were combined: BTP (35 mm, 200 mL), MES buffer or equivalent volume of water (10 mm, 200 mL), and aqueous solution of ChemBioChem 2015, 16, 1474 – 1482
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Ce(NH4)2(NO3)6 (17.5 mm, 200 mL). For use in metal-free control reactions, separate sets of solutions were prepared in which the Ce(NH4)2(NO3)6 was replaced by equivalent volumes of ddH2O. The resulting metal-ligand-buffer solutions were equilibrated at room temperature for 60 min. Concentrated or dilute HCl (0–0.5 mL) and/ or 50 % NaOH (w/v) (0–0.5 mL) were added to adjust the pH to ~ 4.8 or ~ 7.2. Hydrolysis reactions were then prepared by adding reagents in the following order: ddH2O, PC liposome preparation, and pH-adjusted metal-ligand-buffer solution (1000 mL total volume). To initiate hydrolysis, one half of each solution was treated at 60 or 37 8C. The second half was kept at 4 8C for the duration of the reaction (2–40 h). The average pH of each hydrolysis reaction was calculated by averaging pre- and post-reaction pH measurements. In CeIV titration experiments, phosphatidylcholine (35 mm), piperazine buffer (1.5 mm), and Ce(NH4)2(NO3)6 (0–10.0 mm) were treated at 37 8C and ~ pH 4.8 for 20 h. In time-course experiments, phosphatidylcholine, buffer and/or BTP (35 mm), and Ce(NH4)2(NO3)6 (1.75 mm) were treated at 37 8C and ~ pH 4.8 (in 3.5 mm piperazine or 3.5 mm BTP and 1 mm MES buffer) or at ~ pH 7.2 (in 3.5 mm HEPES or 3.5 mm BTP) for 2, 4, 5, 11, 13, 15, 17, 20, and 30 h time intervals. In BTP titration experiments, phosphatidylcholine (35 mm) was treated at ~ pH 4.8 (in 1 mm piperazine) or at ~ pH 7.2 (in 1 mm HEPES) for 20 h in the presence of Ce(NH4)2(NO3)6 (1.75 mm) and increasing concentrations of BTP (0.19–3.5 mm). The lipid hydrolysis reactions containing [Ce(BTP)2(NO3)4]·2 H2O (1.75 mm)[4d] and phosphatidylcholine (35 mm) were treated at ~ pH 4.8 (in 1 mm MES buffer) or ~ pH 7.2 (in 1 mm MOPS buffer) for 20 h at 37 8C. The average pH values of the hydrolysis reactions were calculated from pre- and post-reaction measurements. Colorimetric detection of inorganic phosphate: The 2 mm lipid hydrolysis reactions at 37 and 60 8C were respectively diluted by factors of 15 and 30 with ddH2O (300 mL final volume). Malachite green/molybdate reagent (200 mL) was then added. For the 35 mm lipid hydrolysis and metal-free control reactions, malachite green/ molybdate reagent (100 mL) was combined with the diluted reaction (200 mL) or with undiluted reaction (400 mL). The resulting solutions were incubated for 30 min at room temperature. A UV-1601 Shimadzu spectrophotometer was then utilized to detect malachite green phosphomolybdate product at 620 nm against a ddH2O blank. In order to correct for pre-existing background levels of free, inorganic phosphate in laboratory solutions, absorbance after 0 h of heat treatment (t = 0) was subtracted from reaction absorbance. The absorbance differences of the negative, metal-free control reactions (Figure S8) were then subtracted from the absorbance differences of the corresponding lipid hydrolysis reactions in the presence of metal. Yields of inorganic phosphate were calculated by comparing the final differences to linear titration curves derived from inorganic phosphate standard solutions (0–11 or 0–35 mm) that contained appropriate concentrations of CeIV, ligand, and buffer (Figures S9–S14). Colorimetric detection of choline: Free choline released upon metal-assisted lipid hydrolysis was detected by using an Amplex Red Sphingomyelinase Assay Kit.[32] Reagents taken from the kit were used to make a reaction cocktail consisting of: 1 Õ tris buffer (2,850 mL), Amplex Red (40 mL), horseradish peroxidase (30 mL), choline oxidase (30 mL), and ddH2O (60 mL). The lipid hydrolysis reactions were diluted 12.5-fold to a total volume of 1000 mL with ddH2O. Then, a total of 40 mL of each diluted reaction was treated with reaction cocktail (80 mL) at 37 8C. After 55 min, 80 mL of the solution was added to ddH2O (420 mL). Resorufin product at 572 nm was then detected with a UV-1601 Shimadzu spectrophotometer
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Full Papers against a ddH2O blank. Absorbance associated with the background was removed by subtracting t = 0 h spectra from t = 20 h spectra. The absorbance differences obtained for metal-free control reactions (Figure S8) were then subtracted from the absorbance differences obtained for corresponding lipid hydrolysis reactions in the presence of metal. Yields of free choline could then be calculated by comparing final differences to linear titration curves plotted from 0–70 mm choline standard solutions containing CeIV, ligand, and buffer (Figure S15). 1
H NMR spectroscopy: Spectra of 10 mm [Ce(BTP)2(NO3)4]·2 H2O and of 20 mm BTP were recorded in D2O at room temperature. tert-Butanol was used as an internal standard. The pD values were adjusted to 2.3, 5.1–5.2, 7.6–7.7, and 9.6 with NaOD and DCl in D2O. The equation pD = pH + 0.4 was used to convert pH units to pD.
Acknowledgements We are grateful for financial support from the National Science Foundation (CHE-0718634, K.B.G.) and from the Georgia State University Molecular Basis of Disease Program (D.E.W.). Keywords: hydrolysis · lanthanides · liposomes · lysosomes · phosphate ester [1] a) S. J. Franklin, Curr. Opin. Chem. Biol. 2001, 5, 201 – 208; b) K. B. Grant, M. Kassai, Curr. Org. Chem. 2006, 10, 1035 – 1049; c) T. Y. Lee, J. H. Suh, Chem. Soc. Rev. 2009, 38, 1949 – 1957; d) H. Katada, M. Komiyama, Curr. Gene Ther. 2011, 11, 38 – 45; e) F. Mancin, P. Scrimin, P. Tecilla, Chem. Commun. 2012, 48, 5545 – 5559; f) C. Wende, C. Ludtke, N. Kulak, Eur. J. Inorg. Chem. 2014, 2597 – 2612; g) T. Zhang, M. Ozbil, A. Barman, T. J. Paul, R. P. Bora, R. Prabhakar, Acc. Chem. Res. 2015, 48, 192 – 200. [2] a) T. Takarada, M. Yashiro, M. Komiyama, Chem. Eur. J. 2000, 6, 3906 – 3913; b) K. Stroobants, E. Moelants, H. G. Ly, P. Proost, K. Bartik, T. N. Parac-Vogt, Chem. Eur. J. 2013, 19, 2848 – 2858. [3] a) M. E. Branum, L. Que, J. Biol. Inorg. Chem. 1999, 4, 593 – 600; b) M. E. Branum, A. K. Tipton, S. R. Zhu, L. Que, J. Am. Chem. Soc. 2001, 123, 1898 – 1904; c) M. Sirish, S. J. Franklin, J. Inorg. Biochem. 2002, 91, 253 – 258; d) Y. Kitamura, M. Komiyama, Nucleic Acids Res. 2002, 30, e102; e) H. Katada, H. Seino, Y. Mizobe, J. Sumaoka, M. Komiyama, J. Biol. Inorg. Chem. 2008, 13, 249 – 255; f) J. W. Yang, Y. L. Lin, C. Dong, C. Q. Zhou, J. X. Chen, B. Wang, Z. Z. Zhou, B. Sun, W. H. Chen, Eur. J. Med. Chem. 2014, 87, 168 – 174. [4] a) K. Bracken, R. A. Moss, K. G. Ragunathan, J. Am. Chem. Soc. 1997, 119, 9323 – 9324; b) R. A. Moss, H. Morales-Rojas, Org. Lett. 1999, 1, 1791 – 1793; c) J. M. Yan, M. Atsumi, D. Q. Yuan, K. Fujita, Helv. Chim. Acta 2002, 85, 1496 – 1504; d) A. L. Maldonado, A. K. Yatsimirsky, Org. Biomol. Chem. 2005, 3, 2859 – 2867; e) H. Matsumiya, H. Nakamura, M. Hiraide, Anal. Bioanal. Chem. 2009, 394, 1471 – 1476; f) R. Bonomi, P. Scrimin, F. Mancin, Org. Biomol. Chem. 2010, 8, 2622 – 2626; g) M. Zhao, C. Zhao, X. Q. Jiang, L. N. Ji, Z. W. Mao, Dalton Trans. 2012, 41, 4469 – 4476. [5] a) K. Matsumura, M. Komiyama, J. Inorg. Biochem. 1994, 55, 153 – 156; b) H. X. Liu, J. Hu, X. T. Liu, R. C. Li, K. Wang, Chin. Sci. Bull. 2001, 46, 401 – 403; c) S. S. Cepeda, K. B. Grant, New J. Chem. 2008, 32, 388 – 391; d) M. Kassai, R. Teopipithaporn, K. B. Grant, J. Inorg. Biochem. 2011, 105, 215 – 223. [6] a) Y. A. Hannun, R. M. Bell, Science 1989, 243, 500 – 507; b) E. A. Dennis, S. G. Rhee, M. M. Billah, Y. A. Hannun, FASEB J. 1991, 5, 2068 – 2077; c) P. Niemel, M. T. Hyvonen, I. Vattulainen, Biophys. J. 2004, 87, 2976 – 2989.
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[7] a) F. C. Baes, R. E. Mesmer, The Hydrolysis of Cations, Wiley, New York, 1976; b) M. Komiyama, N. Takeda, H. Shigekawa, Chem. Commun. 1999, 1443 – 1451; c) S. S. Cepeda, D. E. Williams, K. B. Grant, Biometals 2012, 25, 1207 – 1219. [8] G. Wulfsberg, Principles of Descriptive Inorganic Chemistry, Brooks/Cole, Monterey, 1987. [9] A. Kajimura, J. Sumaoka, M. Komiyama, Carbohydr. Res. 1998, 309, 345 – 351. [10] C. B. Yuan, D. Q. Zhao, B. Zhao, Y. J. Wu, J. H. Liu, J. Z. Ni, Langmuir 1996, 12, 5375 – 5378. [11] a) M. Komiyama, N. Takeda, Y. Takahashi, H. Uchida, T. Shiiba, T. Kodama, M. Yashiro, J. Chem. Soc. Perkin Trans. 2 1995, 269 – 274; b) R. A. Moss, W. G. Jiang, Langmuir 2000, 16, 49 – 51. [12] a) L. E. Quintern, G. Weitz, H. Nehrkorn, J. M. Tager, A. W. Schram, K. Sandhoff, Biochim. Biophys. Acta 1987, 922, 323 – 336; b) B. Henry, R. Ziobro, K. A. Becker, R. Kolesnick, E. Gulbins, Handb. Exp. Pharmacol. 2013, 215, 77 – 88. [13] Y. Matsuzawa, K. Y. Hostetler, J. Biol. Chem. 1980, 255, 646 – 652. [14] a) M. Ikegami, R. Dhami, E. H. Schuchman, Am. J. Physiol. Lung. Cell. Mol. Physiol. 2003, 284, L518 – 525; b) H. Schulze, K. Sandhoff, Cold Spring Harbor Perspect. Biol. 2011, 3, 1 – 19; c) R. M. Boustany, Nat. Rev. Neurol. 2013, 9, 583 – 598. [15] a) R. Buccoliero, L. Ginzburg, A. H. Futerman, J. Inherited Metab. Dis. 2004, 27, 641 – 648; b) R. Buccoliero, S. Palmeri, G. Ciarleglio, A. Collodoro, M. M. De Santi, A. Federico, J. Inherited Metab. Dis. 2007, 30, 983. [16] a) O. A. Minai, E. J. Sullivan, J. K. Stoller, Respir. Med. 2000, 94, 1241 – 1251; b) C. J. Hendriksz, P. C. Corry, J. E. Wraith, G. T. Besley, A. Cooper, C. D. Ferrie, J. Inherited Metab. Dis. 2004, 27, 241 – 249; c) N. Guillemot, C. Troadec, T. B. de Villemeur, A. Clement, B. Fauroux, Pediatr. Pulmonol. 2007, 42, 1207 – 1214. [17] a) R. Ott, R. Krmer, Appl. Microbiol. Biotechnol. 1999, 52, 761 – 767; b) A. Sreedhara, J. A. Cowan, J. Biol. Inorg. Chem. 2001, 6, 337 – 347. [18] R. Ott, R. Krmer, Angew. Chem. Int. Ed. 1998, 37, 1957 – 1960; Angew. Chem. 1998, 110, 2064 – 2067. [19] a) H. K. Lin, Y. H. Luo, H. Lin, Transit. Metal Chem. 2006, 31, 690 – 695; b) Y. Liu, X. G. Meng, J. M. Li, X. H. Li, Colloids Surf. A 2013, 436, 839 – 845. [20] E. B. Cogan, G. B. Birrell, O. H. Griffith, Anal. Biochem. 1999, 271, 29 – 35. [21] R. L. Lundblad, F. Macdonald, Handbook of Biochemistry and Molecular Biology, Fourth Edition, CRC, Boca Raton, 2010. [22] D. W. Powell, D. R. Ling, P. K. Tse, Inorg. Chem. 1986, 25, 585 – 586. [23] S. Saito, H. Hoshino, T. Yotsuyanagi, Inorg. Chem. 2001, 40, 3819 – 3823. [24] C. R. Kensil, E. A. Dennis, Biochemistry 1981, 20, 6079 – 6085. [25] D. W. Deamer, J. Bramhall, Chem. Phys. Lipids 1986, 40, 167 – 188. [26] a) R. A. Moss, B. D. Park, P. Scrimin, G. Ghirlanda, Chem. Commun. 1995, 1627 – 1628; b) P. Scrimin, S. Caruso, N. Paggiarin, P. Tecilla, Langmuir 2000, 16, 203 – 209. [27] a) R. A. Moss, Pure. Appl. Chem. 1994, 66, 851 – 858; b) F. X. Contreras, L. Snchez-Magraner, A. Alonso, F. M. GoÇi, FEBS Lett. 2010, 584, 1779 – 1786. [28] A. Alonso, A. Villena, F. M. GoÇi, FEBS Lett. 1981, 123, 200 – 204. [29] a) C. F. G. C. Geraldes, M. P. M. Marques, A. D. Sherry, Inorg. Chim. Acta 1998, 273, 288 – 298; b) M. Kassai, R. G. Ravi, S. J. Shealy, K. B. Grant, Inorg. Chem. 2004, 43, 6130 – 6132. [30] S. P. Fricker, Chem. Soc. Rev. 2006, 35, 524 – 533. [31] D. Marsh, Handbook of Lipid Bilayers, second ed., CRC, Boca Raton, 2013. [32] X. He, F. Chen, M. M. McGovern, E. H. Schuchman, Anal. Biochem. 2002, 306, 115 – 123.
Manuscript received: January 26, 2015 Accepted article published: May 8, 2015 Final article published: June 10, 2015
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DOI: 10.1002/cbic.201500041
Tuning Cerium(IV)-Assisted Hydrolysis of Phosphatidylcholine Liposomes under Mildly Acidic and Neutral Conditions Dominique E. Williams, Kanchan Basnet, and Kathryn B. Grant*[a] With the goal of designing a lysosomal phospholipase mimic, we optimized experimental variables to enhance CeIV-assisted hydrolysis of phosphatidylcholine (PC) liposomes. Our best result was obtained with the chelating agent bis–tris propane (BTP). Similar to the hydrolytic enzyme, CeIV-assisted hydrolysis of PC phosphate ester bonds was higher at lysosomal pH (~ 4.8) compared to pH 7.2. In the presence of BTP, the average cleavage yield at ~ pH 4.8 and 37 8C was: 67 1 %, 5.7-fold
higher than at ~ pH 7.2 and roughly equivalent to the percent of phospholipid found on the metal-accessible exo leaflet of small liposomes. No CeIV precipitation was observed. When BTP was absent, there was significant turbidity, and the amount of cleavage at ~ pH 4.8 (69 1 %) was 2.1-fold higher than the yield obtained at ~ pH 7.2. Our results show that BTP generates homogenous solutions of CeIV that hydrolyze phosphatidylcholine with enhanced selectivity for lysosomal pH.
Introduction Small-molecule, metal-based, synthetic hydrolytic agents have potential applications in diverse fields that include biotechnology, protein engineering, the study of biomolecular function and solution structure, and drug design and discovery.[1] The lanthanide ion cerium(IV) has caught the attention of scientists due to its ability to promote efficient hydrolysis of biological molecules under non-denaturing temperature and pH conditions. The CeIV metal ion and its complexes have been employed to cleave not only phosphate ester bonds but also amide linkages. Compounds hydrolyzed by CeIV include peptides and proteins,[2] DNA,[3] activated synthetic phosphate ester-containing derivatives,[3c, 4] and phospholipids.[5] Lipids play vital roles in biological systems as energy storage molecules and chemical messengers in cell signaling and regulation. Phospholipids are of particular significance as the major components of the biological membranes that surround all cells and organelles.[6] The impressive hydrolytic ability of CeIV has been attributed to a number of factors. CeIV ions can form complexes that possess: 1) high coordination numbers (up to 12); 2) flexible ligand geometries; and 3) fast ligand exchange rates that promote favorable interactions between substrate and water molecules.[1a, b, 4d, f, 5d, 7] The CeIV ion is a strong Lewis acid with high charge density and high affinity for negatively charged oxygen atoms. Upon binding to oxygen, the electron-withdrawing ability of CeIV enhances the electrophilicity of the carbon or phosphorous atoms in the substrate’s scissile ester and amide bonds.[7b] The metal ion can additionally generate local hydrox[a] Dr. D. E. Williams, K. Basnet, Prof. K. B. Grant Department of Chemistry, Georgia State University P.O. Box 3965, Atlanta, GA 30302-3965 (USA) E-mail: [email protected] Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.201500041.
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ide nucleophiles under neutral to mildly acidic conditions by lowering the pKA of metal-bound water molecules from ~ 15.7 to ¢1.1.[8] One potential drawback of CeIV is the tendency of this metal ion center to form insoluble polynuclear hydroxo species in aqueous solution as the pH is raised above ~ 4.0.[4a, d] In this process, CeIV species lose hydrolytic activity by gradually acquiring a lower net positive charge that reduces Lewis acid strength.[4a, d] Given the proper ligand, complex formation can prevent undesired precipitation to provide soluble CeIV species of definite composition that actively promote substrate hydrolysis.[2b, 3a, b, d–f, 4a, c–g, 9] Kitamura and Komiyama employed a CeIV ethylenediaminetetraacetate (EDTA) complex to selectively cleave single-stranded gap and bulge sites in double-stranded DNA oligonucleotides under physiological conditions (37 8C and pH 7.0).[3d] In the absence of EDTA, the CeIV formed insoluble hydroxide gels that lost selectivity and hydrolyzed the single-stranded and double-stranded DNA forms at the same rate. Branum et al. utilized a polyaminocarboxylate dicerium(IV) complex as a regioselective hydrolase to convert supercoiled double-stranded plasmid to linearized DNA through preferential cleavage of 3’-O-P DNA phosphate ester bonds at 37 8C and pH 8.0.[3b] Our published research has recently focused on utilizing metals to facilitate the hydrolysis of phosphate ester bonds in naturally occurring unactivated phospholipids. With the goal of designing a lysosomal phospholipase mimic, we treated liposomes of l-a-phosphatidylcholine and sphingomyelin (PC and SM, respectively; 1 in Scheme 1) with twelve metal ions that are known to hydrolyze activated and unactivated amide and/or phosphate ester bonds in peptides, nucleic acids, and other compounds (CeIV, ZrIV, HfIV, CoII, CuII, EuIII, LaIII, NiII, PdII, YIII, YbIII, and ZnII).[5d, 7c] In the event that a metal ion cleaved both of the phosphate ester bonds of the phospholipid, free inor-
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Scheme 1. Metal-assisted hydrolysis of the phosphate ester bonds of a phospholipid (1) release inorganic phosphate (2), choline (3), and ROH. ROH = diacylglycerol for phosphatidylcholine and ceramide for sphingomyelin.
ganic phosphate (2) would be released (Scheme 1). In these first generation experiments, only CeIV, ZrIV, HfIV, EuIII, LaIII, PdII, and YbIII were capable of hydrolyzing SM and/or PC. In contrast to ZrIV, HfIV, EuIII, LaIII, PdII, and YbIII, for which hydrolysis levels were extremely low at neutral and at mildly acidic pH values, cerium(IV) ammonium nitrate (Ce(NH4)2(NO3)6) was found to be overwhelmingly superior, reacting with the phosphate ester bonds in PC and SM to liberate significant amounts of inorganic phosphate (2; 20 h at 60 8C). Similar to lysosomal phospholipase, we found that CeIV consistently hydrolyzed phospholipids more efficiently at lysosomal pH (~ 4.8) compared to near-neutral conditions. NMR and Fourier transform (FT) Raman spectra show that lanthanide cations coordinate to PC bilayers, primarily at free oxygen atom(s) of the polar head group phosphates.[10] This led to the proposal of a hydrolytic mechanism in which CeIV binds to a negatively charged phosphate oxygen in the lipid (activating the phosphorous atom towards nucleophilic attack) while delivering a hydroxide nucleophile to the scissile phosphate ester bond.[4a, d, 5d, 7b, 11] Examples of lysosomal phospholipases that engage in phosphate ester bond hydrolysis include: 1) acid sphingomyelinase,[12] which acts on SM to release ceramide and 2) lysosomal phospholipase C,[13] which cleaves phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine to produce diacylglycerol (Scheme 1). When these enzymes are impaired, symptomatology can result from a significant buildup of unhydrolyzed phospholipid substrate within the lysosomes of affected cells. In Niemann-Pick disease type A, an inherited lysosomal storage disorder caused by diminished activity of acid sphingomyelinase,[12b, 14] pathogenic levels of unhydrolyzed sphingomyelin accumulate within lysosomes of central nervous and reticuloendothelial system cells.[14a] In Sandhoff disease, low activity of the lysosomal enzyme hexosaminidase B results in the lysosomal accumulation of the ganglioside GM2 and of the glycosphingolipid GA2,[14b] yet its clinical manifestations are very similar to Niemann-Pick. In common to both disorders is an unexplained, significant buildup of phosphatidylcholine within the lysosomes of pulmonary reticuloendothelial cells.[14a, 15] This gives rise to the acute respiratory infections that are one of the major causes of death in Niemann-Pick and Sandhoff patients.[16] The ability of CeIV ions to selectively hydrolyze phospholipid liposomes under mildly acidic conditions pointed to the possibility of developing metal-based reagents to treat storage disease.[14a, b, 15–16] In our first-generation published experiments, hydrolysis of phosphatidylcholine liposomes by Ce(NH4)2(NO3)6 gave rise to 1.8 times more inorganic phosphate at pH 4.8 ChemBioChem 2015, 16, 1474 – 1482
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compared to pH 7.2 (20 h at 37 8C, 2 mm PC, 10 mm CeIV). Hydrolysis yields at ~ pH 4.8 and 7.2 were 18 3 % and 10 2 %, respectively.[5d, 7c] More efficient suppression of cleavage under neutral conditions would be critical for protecting cells from hydrolytic damage in the event of any leakage of a metalbased therapeutic agent from the lysosome into the cytoplasm. Moreover, the hydrolysis reactions contained undesired CeIV precipitation, and the yields of phosphate diester cleavage at 37 8C and ~ pH 4.8 were low. In the present paper, CeIV-assisted hydrolysis of phosphatidylcholine liposomes was optimized. We evaluated coordinating ligands and multiple reaction variables. Our aim was to tune and enhance the reactivity of CeIV to afford efficient, homogeneous hydrolysis of PC liposomes at 37 8C and ~ pH 4.8, with minimized cleavage under near-neutral conditions. Our best result for phosphatidylcholine liposomes was obtained in the presence of CeIV and bis–tris propane (BTP). Compared to our first-generation experiments,[5d, 7c] the yield of PC hydrolysis at ~ pH 4.8 and the ratio of pH 4.8 hydrolysis to pH 7.2 hydrolysis were significantly increased, and visible CeIV precipitation was cleared from hydrolysis reactions. When the single-chain nonionic surfactant Triton X-100 was used to convert the liposomes to mixed micelles, cleavage of phosphatidylcholine occurred in near-quantitative yields (37 8C and ~ pH 4.8).
Results and Discussion Evaluating ligands to optimize CeIV-assisted hydrolysis of PC liposomes In the first experiment for this paper, six prospective ligands were tested for hydrolysis of phosphatidylcholine liposomes against buffered CeIV(NH4)2(NO3)6 (Scheme 2). The anionic polyaminocarboxylates ethylenediaminetetraacetate (4, EDTA) and 1,3-diamino-2-hydroxypropane-N,N,N’,N’-tetraacetic acid (5, HPTA) form CeIV complexes that promote efficient doublestrand DNA cleavage (37–55 8C and pH 8.0).[3a, b] Because chelating ligands with negatively charged donor groups can decrease the Lewis acid character of lanthanide ions,[17] the amine and amino alcohol ligands 6–9 were chosen in the event that 4 and 5 were found to be detrimental to phosphate ester hydrolysis. Ott and Krmer utilized ZrCl4 and tris(hydroxymethyl)aminomethane (tris, 7) to produce higher rates of BNPP cleavage at ~ pH 4.8 compared to ZrIV alone, with a steep decline in reactivity as pH values approached ~ 7.2.[18] Finally, 1,10-phenanthroline (8) and histamine (9) were selected on the basis of literature findings that lanthanide complexes incorporating these ligands efficiently hydrolyze p-nitrophenyl-activated phosphate ester bonds.[19] Similar to our first generation, published experiments,[5d, 7c] the cleavage reactions contained 2 mm of PC in liposome form and 10 mm CeIV(NH4)2(NO3)6 and were run at ~ pH 4.8 and 7.2 for a period of 20 h. Twenty millimolar concentrations of each of the six ligands were added, and the reaction temperature was raised from 37 to 60 8C. The production of inorganic phosphate by CeIV-assisted hydrolysis of phosphate ester bonds (Scheme 1) was measured by using a commercially available
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Scheme 2. Ligands used to evaluate CeIV-assisted hydrolysis of phosphatidylcholine: ethylenediamine-tetraacetate (EDTA, 4); 1,3-diamino-2-hydroxypropane-N,N,N’,N’-tetraacetate (HPTA, 5); 1,3-bis-[tris(hydroxymethyl)methylamino]propane (BTP, 6); tris(hydroxymethyl)aminomethane (tris, 7); 1,10 phenanthroline (8); histamine (9). pKa values appear in parenthesis;[4d, 21–22] n.d. = not determined.
malachite green/molybdate-based colorimetric assay kit. The blue–green complex formed between malachite green and phosphomolybdate was monitored at 620 nm by using a UV– visible spectrophotometer.[20] The results of this experiment are shown in Figure 1, where absorbance at 620 nm reflected relative hydrolysis yields. The ligands EDTA (4) and HPTA (5) completely suppressed phosphatidylcholine cleavage, even at ~ pH 4.8. Although hydrolysis was detected in the presence of tris (7), 1,10-phenanthroline (8), and histamine (9), similar levels were observed under mildly acidic and near-neutral pH values. The best results were obtained with the amino alcohol ligand BTP (6). The hydrolysis quotient, calculated by dividing the average hydrolysis at ~ pH 4.8 by the average hydrolysis at ~ pH 7.2, was 2.6, showing
that free, inorganic phosphate levels were approximately 2.6fold higher under mildly acidic conditions compared to nearneutral pH. This represents an improvement over the 1.4-fold hydrolysis quotient exhibited by Ce(NH4)2(NO3)6 in the absence of ligand (Figure 1). By comparing relative absorbance values at pH 4.8 (Figure 1), ligands 4–9 were ranked from high to low hydrolysis levels as follows: tris (pKa = 8.3)[21] BTP (pKa1 = 6.8; pKa2 = 9.1)[4d] > histamine (pKa1 = 6.1; pKa2 = 9.8)[21] phenanthroline (pKa = 4.8)[21] > EDTA (pKa3 = 2.0; pKa4 = 2.6)[21] HPTA (pKa3 = 1.5; pKa4 = 2.6).[22] Although there are exceptions, the ordering reveals a general tendency for cleavage levels to decrease as a function of increasing ligand acidity. Strongly chelating polycarboxylates, similar to EDTA (4) and HPTA (5), for which almost no hydrolysis was detected, form thermodynamically stable lanthanide complexes with reduced ligand exchange rates.[22–23] It is reasonable to suggest that the persistent negative charges associated with 4 and 5 might have strongly suppressed phosphate ester bond hydrolysis by reducing the Lewis acid strength of CeIV.[17] Although similar in structure, it is interesting to note that BTP (6) and tris (7) have different effects on CeIV-assisted PC hydrolysis. The pKa of the amino group of tris is 8.3. Whereas CeIV has the ability to lower the pKa values of nearby functional groups, tris might still possess a positive charge at ~ pH 4.8 and 7.2 that reduces metal–ligand interactions. This might be related to the relatively high levels of CeIV-assisted phosphate ester bond cleavage that occur at low and high pH values. In contrast, BTP has two amino groups with respective pKa1 and pKa2 values of 6.8 and 9.1. Similar to tris, the positive charge at ~ pH 4.8 might reduce complex formation and promote hydrolysis. At pH ~ 7.2, however, there is a possibility that one of the two amino groups of BTP is predominantly neutral. This might increase ligand–metal interactions and, in turn, suppress cleavage. In the absence of chelating agents 4–9, CeIV precipitates were immediately formed upon the addition of Ce(NH4)2(NO3)6 to the ~ pH 4.8 and 7.2 hydrolysis reactions. When the ligands were present, however, the precipitation was either completely eliminated (in the case of 4 and 5), or greatly reduced (6–9). Although all six chelating agents were clearly capable of interacting with CeIV and influencing speciation, our observations and data suggested that substantial levels of complex formation might suppress phosphate ester hydrolysis. CeIV-assisted PC hydrolysis at 37 8C
Figure 1. Percent relative absorbance at 620 nm plotted as a function of pH. A total of 2 mm PC was reacted at 60 8C for 20 h in the presence of 10 mm Ce(NH4)2(NO3)6 and 20 mm ligand (4–9, Scheme 2) at pH 5.0–5.3 (&) or pH 7.0–7.4 (&). The number of trials (n) appears in parenthesis. Error bars represent the standard deviation.
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In our next set of experiments, the reaction temperature was reduced from 60 to 37 8C (normal core body temperature). A final concentration of 20 mm of BTP was added to ~ pH 4.8 and 7.2 reactions in order to selectively suppress CeIV-assisted phosphatidylcholine hydrolysis at near-neutral pH. The reaction time (20 h) and remaining reagent concentrations (2 mm PC, 10 mm Ce(NH4)2(NO3)6) were carried over from the preceding experiment. The inorganic phosphate liberated by CeIV was quantitated with a malachite green/molybdate reagent, and a choline-specific colorimetric assay based on Amplex Red
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Full Papers dye[5d, 7c] was utilized in the event that the lower reaction temperature limited hydrolysis to the choline side of phosphorous (3; Scheme 1). As desired, treatment of PC with CeIV in the presence of BTP produced minimal amounts of phospholipid cleavage under near-neutral conditions (Figure 2). At ~ pH 4.8,
reaching a maximum at 1.75 mm of CeIV, but were significantly lower at all metal ion concentrations above 2 mm. In the presence of 10 mm CeIV, reducing the amount of PC by a factor of 57, from 2 mm to 35 mm, resulted in a relatively modest approximately eightfold increase in the average yield of inorganic phosphate (6 2 %[7c] in Figure 2 to 45 4 % in Figure 3). It is conceivable that any potential benefit to reaction yield, gained by increasing the ratio of metal to phospholipid, might have been partially offset by a drop in reaction rate caused by substantially lowering the phosphatidylcholine concentration. In the presence of 35 mm PC, reducing CeIV by a factor of 5.7, from 10 to 1.75 mm, caused an additional 1.5-fold increase in the hydrolysis yield (from 45 4 to 69 1 % in Figure 3). This suggested that there could have been a larger proportion of hydrolytically active CeIV species at the lower 1.75 mm metal ion concentration.[4d, 7a]
Figure 2. Averaged hydrolysis yields plotted as a function of pH for A) malachite green detection of free inorganic phosphate and B) Amplex Red detection of free choline. A total of 2 mm PC was reacted at 37 8C for 20 h in the presence of 10 mm Ce(NH4)2(NO3)6 and 20 mm BTP at pH 4.9–5.2 (&) or pH 7.0–7.3 (&). The number of trials (n) appears in parenthesis. Error bars represent standard deviation.
Time course: CeIV-assisted PC hydrolysis at 37 8C Our next objective was to monitor the time-dependent release of inorganic phosphate by CeIV-assisted hydrolysis of PC liposomes (Figure 4 A). Optimized reagent concentrations were employed (1.75 mm CeIV, 35 mm PC) at the standard 37 8C reac-
inorganic phosphate and choline could indeed be detected and quantitated, but the resulting yields were low (6 2 and 14 1 %, respectively). Our next goal was to increase the efficiency of CeIV-assisted hydrolysis.
Optimizing CeIV concentration CeIV hydroxo aggregates with reduced hydrolytic activity have a propensity to form at high metal ion concentrations.[4d, 7a] This suggested that it might be possible to increase cleavage yields by reducing the amount of CeIV(NH4)2(NO3)6. Phosphatidylcholine hydrolysis reactions were accordingly run in the presence of 0 to 10 mm of CeIV(NH4)2(NO3)6 (20 h at 37 8C, ~ pH 4.8, no BTP). In these experiments, the concentration of PC was lowered from 2 mm to 35 mm. The release of phosphate was then monitored at 620 nm by using malachite green. As shown in Figure 3, hydrolysis levels initially increased,
Figure 3. Absorbance at 620 nm plotted as a function of CeIV concentration for malachite green-treated hydrolysis reactions. A total of 35 mm of PC was reacted at 37 8C for 20 h in the presence of 0.0 mm to 10.0 mm Ce(NH4)2(NO3)6 (1.5 mm piperazine buffer pH 4.8). Representative percent yields of inorganic phosphate are shown. The number of trials (n) appears in parenthesis. Error bars represent standard deviation.
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Figure 4. A) Averaged hydrolysis yields plotted as a function of time for malachite green detection of free, inorganic phosphate. A total of 35 mm PC was reacted at 37 8C with 1.75 mm Ce(NH4)2(NO3)6 : 1) without BTP, at ~ pH 4.8–4.9 (&) or ~ pH 7.1–7.4 (&; 3.5 mm piperazine or HEPES buffer respectively); or 2) with 3.5 mm BTP, at ~ pH 4.8–5.1 (~; in 1 mm MES buffer) or at ~ pH 7.0– 7.3 (~). The numbers of trials at each time point ranged from 2 to 8. B) Quotients obtained by dividing the average ~ pH 4.8 hydrolysis yields by the average ~ pH 7.2 hydrolysis yields for reactions without BTP (*) and with 3.5 mm BTP (*).
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Full Papers tion temperature. Similar to the preceding experiments in this paper, the metal-to-ligand ratio (M/L) of CeIV to BTP was 1:2. Results obtained at ~ pH 4.8 are discussed as follows. From 0– 20 h, the averaged approximate pseudo-first-order rate constants for CeIV-assisted PC hydrolysis at ~ pH 4.8 were 0.0610 0.0009 h¢1 and 0.0260 0.0010 h¢1 in the absence and presence of 3.5 mm of BTP, respectively (Figure S1 in the Supporting Information). Corresponding yields of inorganic phosphate at 20 h were 69 1 and 42 3 % (Figure 4 A). Although it is evident that PC liposome hydrolysis was initially faster without a ligand, a significant drop-off in the reaction rate occurred between the 20 and 30 h time points. In contrast, when BTP was present, hydrolysis levels continued to increase after 20 h. As a result, the yields of inorganic phosphate at 30 h were within experimental error (72 4 % without vs. 65 5 % with BTP; Figure 4 A). In the absence of ligand, ~ pH 4.8 hydrolysis reactions had accumulated visible CeIV precipitation, whereas those containing BTP remained clear. Reaction times beyond 30 h did not significantly improve hydrolysis yields. Next, phosphatidylcholine time course reactions at ~ pH 7.2 were considered (Figure 4 A). As mentioned, an ideal CeIVbased lysosomal phospholipase mimic should display enhanced activity at lysosomal pH (~ 4.8), accompanied by low levels of cleavage under near-neutral conditions. Figure 4 A shows that the addition of BTP to the hydrolysis reactions reduced cleavage levels up to 79 % (37 8C): at the 20 h time point, yields of inorganic phosphate were 33 5 and 7 2 % in the absence and presence of BTP, respectively (Figure 4 A). In order to better illustrate the effects of BTP, the ~ pH 4.8/ ~ pH 7.2 hydrolysis quotients obtained for the data points in Figure 4 A were plotted in Figure 4 B. In the presence of BTP, CeIV(NH4)2(NO3)6 produced an approximately linear increase in ~ pH 4.8/ ~ pH 7.2 quotients over the 30 h time course, whereas in the absence of ligand, the quotients were relatively constant. This shows that BTP-assisted hydrolysis became increasingly selective for mildly acidic conditions as a function of time. There was visible CeIV precipitation in the ~ pH 7.2 cleavage reactions without ligand, whereas those containing BTP remained clear throughout the time-course experiment.
Figure 5. Absorbance at 620 nm plotted as a function of [CeIV] to [BTP] ratio for malachite green-treated hydrolysis reactions. A total of 35 mm of PC was reacted at 37 8C for 20 h in the presence of 1.75 mm Ce(NH4)2(NO3)6 and 3.5 mm to 0.19 mm BTP at pH ~ 4.6–5.1 (&; 1 mm piperazine buffer) or pH ~ 7.0–7.4 (~; 1 mm HEPES). Representative percent yields of inorganic phosphate are shown. Three or more trials were performed for each data point. Error bars represent standard deviation.
on the phosphate levels produced under near-neutral conditions. Phosphatidylcholine cleavage at 37 8C: Summary Figure 6 summarizes the pH 4.8/pH 7.2 hydrolysis quotients and inorganic phosphate yields obtained under the different sets of experimental variables that we have evaluated thus far. In the published first-generation reaction that served as the starting point for the research reported in this paper (2 mm phosphatidylcholine, 10 mm Ce(NH4)2(NO3)6, 37 8C, 20 h), treatment of PC liposomes with CeIV generated inorganic phosphate in relatively low yield (B in Figure 6).[7c] A major goal of the work described in the present study was to optimize cleavage while increasing the ~ pH 4.8/ ~ pH 7.2 quotient. CeIV-assisted hydrolysis of phosphatidylcholine liposomes at ~ pH 4.8 and 37 8C was improved considerably by: 1) Decreasing the con-
Optimizing BTP concentration at 37 8C An M/L ratio of 1:2 was employed in all of the BTP hydrolysis reactions described up until this point. In order to further improve cleavage yields, the optimized concentrations of phosphatidylcholine (35 mm) and CeIV (1.75 mm) were reacted at M/L ratios ranging from 1:2 up to 9:1 at the standard 37 8C temperature and reaction time (20 h). The amount of CeIV was held constant, while the concentration of BTP was incrementally decreased. Selected cleavage yields were then calculated. As shown in Figure 5, the percent of hydrolysis at ~ pH 4.8 was: 42 % at the 1:2 M/L ratio (pH 4.8/pH 7.2 quotient = 6.0); 67 1 % at the 4:1 ratio (quotient = 5.7); and 54 1 % at the 5:1 ratio (quotient = 9.6). Thus, lowering the concentration of BTP from 3.5 mm (M/L = 1:2) to 0.35 mm (M/L = 5:1) markedly improved cleavage at ~ pH 4.8 without having a significant effect ChemBioChem 2015, 16, 1474 – 1482
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Figure 6. Average yields of inorganic phosphate released upon treatment of phosphatidylcholine liposomes with Ce(NH4)2(NO3)6 in the presence and absence of BTP, plotted according to reaction conditions (~ pH 4.6–5.2 and 37 8C for 20 h). A) 10 mm CeIV, 2 mm PC, 20 mm BTP; B) 10 mm CeIV, 2 mm PC; C) 1:75 mm CeIV, 35 mm PC, 3.5 mm BTP (M/L = 1:2); D) 10 mm CeIV, 35 mm PC; E) 1.75 mm CeIV, 35 mm PC, 0.35 mm BTP (M/L = 5:1); F) 1.75 mm CeIV, 35 mm PC, 0.44 mm BTP (M/L = 4:1); G) 1.75 mm CeIV, 35 mm PC. Corresponding pH 4.8/pH 7.2 hydrolysis quotients are shown above each bar. Three or more trials were performed for each set of reactions. Error bars represent standard deviation. Data corresponding to (B) were previously published;[7c] n.d. = not determined.
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Full Papers centration of phospholipid (B vs. D in Figure 6); 2) Decreasing the concentration of Ce(NH4)2(NO3)6 (D vs. G); and 3) Decreasing the concentration of BTP (C vs. E and F). Our best results were obtained with a 4:1 metal to BTP ratio in the presence of 35 mm phosphatidylcholine and 1.75 mm Ce(NH4)2(NO3)6 (20 h at 37 8C). Compared to the first generation experiments,[5d] , [7c] utilizing the optimized hydrolysis conditions raised the pH 4.8/ pH 7.2 hydrolysis quotient from 1.8 to 5.7. This was done by increasing the cleavage yield at ~ pH 4.8 from 18 to 67 1 %, without significantly changing hydrolysis at ~ pH 7.2 (10 2 % initial averaged yield vs. 12 1 % for the optimized conditions; A vs. B in Figure 7).
Figure 7. A comparison of A) first-generation reactions to optimized hydrolysis reactions in the B) absence and C) presence of 1.6 mol equiv of Triton X100. Average yields of inorganic phosphate released upon treatment of PC with Ce(NH4)2(NO3)6 for 20 h at 37 8C are plotted for the reagent concentrations: A) 10 mm CeIV, 2 mm PC; B) and C) 1.75 mm CeIV, 35 mm PC, 0.44 mm BTP at pH 4.8–5.0 (&) or pH 7.1–7.3 (&). Corresponding pH 4.8/pH 7.2 hydrolysis quotients are underlined. Three or more trials were performed for each reaction. Error bars represent standard deviation. Data corresponding to (A) were previously published.[7c] Data corresponding to (B) were taken from Figures 5 and 6.
The addition of BTP significantly increased pH 4.8/pH 7.2 quotients by selectively reducing levels of CeIV(NH4)2(NO3)6assisted phosphatidylcholine hydrolysis under near-neutral pH conditions (20 h at 37 8C; B vs. A, G vs. C, G vs. E and F; Figure 6). At ~ pH 4.8, inorganic phosphate yields in the absence and presence of 0.44 mm BTP (4:1 m:L) were within experimental error, 69 and 67 1 % respectively (20 h at 37 8C; G vs. F in Figure 6), whereas corresponding ~ pH 7.2 hydrolysis yields were decreased by BTP from 33 5 to 12 1 % (Figures 4 and 5). Under optimal conditions, BTP suppressed PC hydrolysis at ~ pH 7.2 without significantly altering cleavage levels at mildly acidic pH. A second major goal of our research was to reduce CeIV precipitation. Optimized BTP reactions were free of turbidity, irrespective of pH (C, E, and F in Figure 6). When BTP was omitted, CeIV precipitation was observed (G). Thus, the major effect of BTP was to generate homogenous solutions of CeIV that hydrolyzed phosphatidylcholine liposomes with enhanced selectivity for lysosomal pH.
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Membrane permeability and dynamics Thus far in the present publication, the highest inorganic phosphate yield obtained after treating phosphatidylcholine liposomes with CeIV was 72 4 % (30 h at 37 8C; Figure 4). This upper hydrolysis limit approaches the ~ 68 % average distribution of total phospholipid on the metal-accessible exo leaflet of small PC liposomes[24] and suggests that CeIV might preferentially cleave phosphatidylcholine distributed on the external exo liposomal leaflet. Liposomes are unilamellar, lipid bilayer structures that contain a hollow core. In general, most metal ions are unable to freely permeate across biological membranes.[25, 26] Compared to the endo leaflet, the polar head groups of phosphatidylcholine molecules in the exo leaflet are therefore expected to be considerably more likely to interact with metal ions. In addition to membrane permeability, factors relating to membrane dynamics favor preferential hydrolysis of exo liposomal phospholipids. Due to low levels of electrostatic repulsion, the rates of transverse lipid diffusion (flip-flop) between the exo and endo bilayer surfaces are extremely slow in the case of PC and other neutral and/or zwitterionic phospholipids.[27] Moreover, CeIV-assisted hydrolysis of PC generates neutral diacylglycerol,[5d] which has been shown to have little if any effect on the transverse diffusion rates of adjacent lipids in the bilayer.[27] The membrane permeability factors described above were taken into consideration in our previously published l-a-phosphatidylcholine study.[5d] Triton X-100 is a single-chain, nonionic surfactant that is used to convert PC liposomes to micelles, a lipid packing arrangement in which all of the polar head groups face the external aqueous phase.[28] When we added 1.6 mol equiv of this surfactant to our PC liposome preparations, phosphatidylcholine hydrolysis yields at ~ pH 4.8 and 60 8C were increased from 41 5 to 79 7 %.[5d] In the present work, PC liposomes were pretreated with 1.6 mol equiv of Triton X-100. Turbidity measurements at a surfactant/phosphatidylcholine molar mixing ratio of 1.6 revealed a decrease in light absorbance consistent with the conversion of the liposomes to mixed micelles (Figure S2).[28] When the resulting mixed-micellar preparation was treated with CeIV at our optimized set of reagent concentrations (35 mm PC, 1.75 mm CeIV, 0.44 mm BTP), the averaged yield of PC hydrolysis at ~ pH 4.8 was 98 2 %, and the pH 4.8/pH 7.2 hydrolysis quotient was 6.0 (20 h at 37 8C; C in Figure 7).
1
H NMR analyses
Solution interactions between BTP and CeIV were first studied by Maldonado and Yatsimirsky. With [D6]DMSO as the solvent, the 1H NMR spectrum of free BTP was compared to the spectrum of a pre-synthesized 1:2 M/L CeIV complex of the formula [Ce(BTP)2(NO3)4]·2 H2O.[4d] Complex formation with CeIV caused the 1H NMR signals associated with the -CH2CH2CH2-, -CH2NH-, and -CH2OH methylene protons of BTP to undergo significant downfield shifts (Table 1). However, when Maldonado and Yatsimirsky diluted the [D6]DMSO solutions with D2O (10 %, v/v),
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pD [b]
2.3 5.1–5.2[b] 7.6–7.7[b] 9.6[b] DMSO[d]
-CH2CH2CH2¢0.003 ¢0.003 + 0.023 + 0.035 + 0.540
Dd (ppm) -CH2NH¢0.004 ¢0.004 + 0.018 + 0.051 + 0.470
CH2OH ¢0.002 ¢0.003 + 0.007 + 0.017 + 0.240
BTP protonation BTPH BTPH BTPH BTP –
2+ 2 2+ 2 +
n[c] 2 2 2 1 –
[a] Dd = dcomplex¢dBTP, where complex = [Ce(BTP)2(NO3)4]·2 H2O and BTP = free bis–tris propane. [b] NMR spectra are in D2O (Figures S3 and S4). [c] n = number of trials. [d] Published data.[4d]
the spectra of BTP and [Ce(BTP)2(NO3)4]·2H2O were superimposable, suggesting that the complex had dissociated. Our goal was to utilize 1H NMR in order to investigate the interactions between BTP and CeIV as a function of pD. We recorded proton NMR spectra of BTP and [Ce(BTP)2(NO3)4]·2 H2O[4d] in D2O (100 %, v/v) after carefully adjusting the solution pD with DCl and NaOD. When additional BTP ligand was added to NMR samples containing [Ce(BTP)2(NO3)4]·2 H2O, new peaks corresponding to free BTP were not observed in any of the spectra (data not shown). This suggested that CeIV was kinetically labile and that the BTP resonances in the [Ce(BTP)2(NO3)4]·2 H2O NMR spectra were averaged values arising from free and metal bound nitrogen and oxygen donor atoms (Figures S3 and S4). The CeIV-induced chemical shift changes (dcomplex¢dBTP) observed for the -CH2CH2CH2-, -CH2NH-, and -CH2OH methylene protons of BTP are shown in Table 1. At pD values ranging from 2.3 to 5.2, the [Ce(BTP)2(NO3)4]·2H2O NMR spectra in D2O showed no evidence of complex formation. This is consistent with Maldonado and Yatsimirsky’s NMR data for [Ce(BTP)2(NO3)4]·2 H2O in D2O/ [D6]DMSO (1:9, v/v).[4d] According to the pKa values of BTP (pKa1 = 6.8; pKa2 = 9.1),[4d] it is possible that the BTPH22 + form of the ligand exists at ~ pH 4.8. With both nitrogen donor atoms protonated, BTP would not be expected to undergo significant complex formation. Notwithstanding, the addition of BTP to phosphatidylcholine reactions prevented visible CeIV precipitation over the entire pD range studied. This suggests that the metal and ligand were able to interact to some degree. When the pD was increased from 5.2 to 7.6 and 9.6, CeIV induced small, progressively increasing downfield shifts in the positions of all of the BTP methylene proton resonances. The magnitude and pattern of the chemical shift changes suggest that -CH2NH- and -CH2OH groups of BTP interact with CeIV as a function of decreasing ligand charge.[29] Maldonado and Yatsimirsky utilized [Ce(BTP)2(NO3)4]·2 H2O to conduct potentiometric titrations with the aim of characterizing the speciation of CeIV in aqueous solution.[4d] Due to extensive precipitation of CeIV, CeIV(NH4)2(NO3)6 could not be analyzed directly. At pH values greater than 7.0, Ce4(OH)151 + was the major multinuclear CeIV hydroxo species formed from [Ce(BTP)2(NO3)4]·2 H2O. Changing the pH from 7 to 5 afforded Ce4(OH)142 + and then Ce4(OH)133 + , CeIV hydroxo species that have a higher positive charge and increased Lewis acid ChemBioChem 2015, 16, 1474 – 1482
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strength.[4d] We propose that BTP is likely to weakly associate with CeIV in aqueous solutions at ~ pH 4.8. In so doing, BTP might act as a mediator to control CeIV speciation while preventing CeIV precipitation and maintaining the activity of the metal ion center towards phosphatidylcholine hydrolysis. Upon testing different BTP concentrations at ~ pH 4.8, we found that optimum PC hydrolysis occurred when the ratio of CeIV ions to ligand was 4:1 (Figure 5). This suggests that the active species is not a stable complex between the ligand and metal but is likely to be something more dynamic. Such a conclusion is in agreement with the 1H NMR data, which show no evidence for complex formation between CeIV and BTP at pD values below 5.3 (Table 1). At ~ pH 7.2, the NMR data suggest that BTP interacts more extensively with CeIV. As a result, precipitation of CeIV is prevented, and PC hydrolysis is suppressed. BTP might bind to and directly inactivate CeIV at the higher pH value or might shift the speciation equilibria away from the formation of CeIV species with the potential for high hydrolytic activity (e.g., Ce4(OH)133 + ).
Conclusion The ultimate goal of the research described in this report was to lay the groundwork for the development of an efficient lysosomal phospholipase mimic based on CeIV. Towards this end, multiple ligands and experimental variables were tested in order to optimize the hydrolysis of phosphate ester bonds in phosphatidylcholine liposomes. The ideal hydrolytic agent would exhibit maximal activity at lysosomal pH (~ 4.8), accompanied by low levels of cleavage under near-neutral conditions. Although polyaminocarboxylate ligands were found to be detrimental, our best results were obtained with the amino alcohol bis–tris propane. Under optimized reaction conditions, BTP suppressed the hydrolysis of phosphatidylcholine liposomes at ~ pH 7.2 and eliminated CeIV precipitation. A comparison to first-generation experiments[5d, 7c] showed that the percent cleavage of PC liposomes at ~ pH 4.8 increased from 18 3 to 67 1 % and that the pH 4.8/pH 7.2 hydrolysis quotient increased from 1.8 to 5.7 (20 h at 37 8C; A vs. B in Figure 7). The single-chain nonionic surfactant Triton X-100, which converts liposomes to metal-accessible mixed micelles (Figure S2),[28] increased the yield of CeIV-assisted phosphate ester bond hydrolysis to 98 2 % (20 h at 37 8C and ~ pH 4.8; C in Figure 7). Our future research will therefore focus on the development of ligands that facilitate the transport of CeIV across phospholipid bilayers. This will enable CeIV to efficiently hydrolyze phosphate ester bonds located on the metal-inaccessible endo leaflet of PC liposomes. To the best of our knowledge, this study presents the first example of near-quantitative, pH-selective hydrolysis of a naturally occurring phospholipid by a metal ion center under physiological conditions of temperature and pH. Cerium complexes display low to moderate cellular toxicity and have had therapeutic applications as anti-emetic agents and in topical creams to treat full-thickness burns.[30] In principle, encapsulation of CeIV into a delivery vehicle such as a hydrolytically inert, lysosomotropic “carrier liposome” would promote the transport of
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Full Papers the metal ion center into lysosomes while minimizing undesired interactions with molecules in the rest of the cell. It is therefore conceivable that reagents based on cerium might one day be used as hydrolytic agents for in vivo applications.
Experimental Section General: Deionized distilled water was used in the preparation of all stock solutions. Chemicals were of the highest available purity and were utilized without further purification. l-a-Phosphatidylcholine from chicken egg (catalogue number 840051P) was obtained from Avanti Polar Lipids. Choline chloride, Ce(NH4)2(NO3)6, 1,3-diamino-2-hydroxypropane-N,N,N’,N’-tetraacetic acid (HPTA), 1,10phenanthroline, piperazine, and tris(hydroxymethyl)aminomethane (tris) were purchased from Sigma–Aldrich. DMSO, 1,3-bis[tris(hydroxymethyl)methylamino]propane (bis–tris propane, BTP), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), histamine, 4morpholineethanesulfonic acid (MES), 4-morpholinepropanesulfonic acid (MOPS), anhydrous tert-butanol ( 99.5 %), and Triton X-100 were from Sigma. EDTA was obtained from Fisher Scientific. The NMR reagents deuterium oxide (99 atom % D), sodium deuteroxide (30 wt % in D2O, 99 atom % D), [D6]DMSO (99 atom % D), and deuterium chloride (35 wt % in D2O, 99 atom % D) were acquired from Sigma–Aldrich. Known compound [Ce(BTP)2(NO3)4]·2 H2O was prepared by using a previously published procedure[4d] (Figures S5 to S7). Malachite green phosphate (catalogue number POMG-25H) and Amplex Red Sphingomyelinase Assay Kits (catalogue number A12220) were purchased from BioAssay Systems and Invitrogen, respectively. IR spectra were recorded on a PerkinElmer Spectrum 100 FT-IR spectrometer coupled with an attenuated total reflection (ATR) sampling accessory. NMR spectra were acquired on a Bruker Avance 400 MHz NMR spectrometer. Colorimetric assays were conducted on a UV-1601 Shimadzu spectrophotometer. Preparation of liposomes: l-a-Phosphatidylcholine (2–4 mg or 16–19 mg) was dissolved in chloroform (1 mL) in a round bottom flask. The chloroform solution was concentrated in vacuo overnight. To the resulting dried phospholipid film was added preheated ddH2O at 65 8C to a final concentration of 120 mm (2 mm lipid hydrolysis reactions) or 2.06 mm (35 mm lipid hydrolysis reactions). The aqueous solution was sonicated for 20 min at 65 8C, which is above the range of gel-to-fluid transition temperature (Tm) values reported for PC (41–55 8C).[31] Mixed micelles of PC and Triton X-100 were prepared from phosphatidylcholine liposomes and then detected by measuring solution turbidity as previously described (Figure S2).[5d] Lipid hydrolysis: The following additional stock solutions were prepared prior to the hydrolysis reactions: Ce(NH4)2(NO3)6 (10, 17.5, or 11.7 mm in ddH2O); buffers (MES, piperazine, or HEPES; 200, 23.3, or 10 mm in ddH2O), ligands (tris, BTP, and histamine; 200 mm in ddH2O); HPTA (50 mm in ddH2O at pH 7); EDTA (200 mm in ddH2O at pH 8); 1,10-phenanthroline (200 mm in neat DMSO). The solutions were used to prepare standard reactions consisting of either: 1) 10 mm Ce(NH4)2(NO3)6, ligand or buffer (piperazine or HEPES; 20 mm), and phosphatidylcholine (2 mm); or 2) Ce(NH4)2(NO3)6 (1.75 mm), bis–tris propane (BTP; 3.5 mm), buffer (MES, piperazine, or HEPES; 1–3.5 mm), and phosphatidylcholine (35 mm). For the 2 mm lipid reactions, the metal complexes were allowed to form in situ by adding equal volumes of an aqueous solution of Ce(NH4)2(NO3)6 (100 mm, 300 mL) to a solution of ligand (200 mm, 300 mL). For the 35 mm lipid reactions, the following solutions were combined: BTP (35 mm, 200 mL), MES buffer or equivalent volume of water (10 mm, 200 mL), and aqueous solution of ChemBioChem 2015, 16, 1474 – 1482
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Ce(NH4)2(NO3)6 (17.5 mm, 200 mL). For use in metal-free control reactions, separate sets of solutions were prepared in which the Ce(NH4)2(NO3)6 was replaced by equivalent volumes of ddH2O. The resulting metal-ligand-buffer solutions were equilibrated at room temperature for 60 min. Concentrated or dilute HCl (0–0.5 mL) and/ or 50 % NaOH (w/v) (0–0.5 mL) were added to adjust the pH to ~ 4.8 or ~ 7.2. Hydrolysis reactions were then prepared by adding reagents in the following order: ddH2O, PC liposome preparation, and pH-adjusted metal-ligand-buffer solution (1000 mL total volume). To initiate hydrolysis, one half of each solution was treated at 60 or 37 8C. The second half was kept at 4 8C for the duration of the reaction (2–40 h). The average pH of each hydrolysis reaction was calculated by averaging pre- and post-reaction pH measurements. In CeIV titration experiments, phosphatidylcholine (35 mm), piperazine buffer (1.5 mm), and Ce(NH4)2(NO3)6 (0–10.0 mm) were treated at 37 8C and ~ pH 4.8 for 20 h. In time-course experiments, phosphatidylcholine, buffer and/or BTP (35 mm), and Ce(NH4)2(NO3)6 (1.75 mm) were treated at 37 8C and ~ pH 4.8 (in 3.5 mm piperazine or 3.5 mm BTP and 1 mm MES buffer) or at ~ pH 7.2 (in 3.5 mm HEPES or 3.5 mm BTP) for 2, 4, 5, 11, 13, 15, 17, 20, and 30 h time intervals. In BTP titration experiments, phosphatidylcholine (35 mm) was treated at ~ pH 4.8 (in 1 mm piperazine) or at ~ pH 7.2 (in 1 mm HEPES) for 20 h in the presence of Ce(NH4)2(NO3)6 (1.75 mm) and increasing concentrations of BTP (0.19–3.5 mm). The lipid hydrolysis reactions containing [Ce(BTP)2(NO3)4]·2 H2O (1.75 mm)[4d] and phosphatidylcholine (35 mm) were treated at ~ pH 4.8 (in 1 mm MES buffer) or ~ pH 7.2 (in 1 mm MOPS buffer) for 20 h at 37 8C. The average pH values of the hydrolysis reactions were calculated from pre- and post-reaction measurements. Colorimetric detection of inorganic phosphate: The 2 mm lipid hydrolysis reactions at 37 and 60 8C were respectively diluted by factors of 15 and 30 with ddH2O (300 mL final volume). Malachite green/molybdate reagent (200 mL) was then added. For the 35 mm lipid hydrolysis and metal-free control reactions, malachite green/ molybdate reagent (100 mL) was combined with the diluted reaction (200 mL) or with undiluted reaction (400 mL). The resulting solutions were incubated for 30 min at room temperature. A UV-1601 Shimadzu spectrophotometer was then utilized to detect malachite green phosphomolybdate product at 620 nm against a ddH2O blank. In order to correct for pre-existing background levels of free, inorganic phosphate in laboratory solutions, absorbance after 0 h of heat treatment (t = 0) was subtracted from reaction absorbance. The absorbance differences of the negative, metal-free control reactions (Figure S8) were then subtracted from the absorbance differences of the corresponding lipid hydrolysis reactions in the presence of metal. Yields of inorganic phosphate were calculated by comparing the final differences to linear titration curves derived from inorganic phosphate standard solutions (0–11 or 0–35 mm) that contained appropriate concentrations of CeIV, ligand, and buffer (Figures S9–S14). Colorimetric detection of choline: Free choline released upon metal-assisted lipid hydrolysis was detected by using an Amplex Red Sphingomyelinase Assay Kit.[32] Reagents taken from the kit were used to make a reaction cocktail consisting of: 1 Õ tris buffer (2,850 mL), Amplex Red (40 mL), horseradish peroxidase (30 mL), choline oxidase (30 mL), and ddH2O (60 mL). The lipid hydrolysis reactions were diluted 12.5-fold to a total volume of 1000 mL with ddH2O. Then, a total of 40 mL of each diluted reaction was treated with reaction cocktail (80 mL) at 37 8C. After 55 min, 80 mL of the solution was added to ddH2O (420 mL). Resorufin product at 572 nm was then detected with a UV-1601 Shimadzu spectrophotometer
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Full Papers against a ddH2O blank. Absorbance associated with the background was removed by subtracting t = 0 h spectra from t = 20 h spectra. The absorbance differences obtained for metal-free control reactions (Figure S8) were then subtracted from the absorbance differences obtained for corresponding lipid hydrolysis reactions in the presence of metal. Yields of free choline could then be calculated by comparing final differences to linear titration curves plotted from 0–70 mm choline standard solutions containing CeIV, ligand, and buffer (Figure S15). 1
H NMR spectroscopy: Spectra of 10 mm [Ce(BTP)2(NO3)4]·2 H2O and of 20 mm BTP were recorded in D2O at room temperature. tert-Butanol was used as an internal standard. The pD values were adjusted to 2.3, 5.1–5.2, 7.6–7.7, and 9.6 with NaOD and DCl in D2O. The equation pD = pH + 0.4 was used to convert pH units to pD.
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Manuscript received: January 26, 2015 Accepted article published: May 8, 2015 Final article published: June 10, 2015
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