The Mechanism of Ether Bond Formation in 0-Alkyl Lipid Synthesis in Ehrlich Ascites Tumor UNUSUAL CLEAVAGE OF THE FATTY ACID MOIETY OF ACYL DIHYDROXYACETONE PHOSPHATE*

We have previously presented evidence for the for- mation of 1-0-alkyl dihydroxyacetone-P from acyl di-hydroxyacetone-P via the initial formation of an inter- mediate 1-0-acyl endiol of acyl dihydroxyacetone-P. This reaction involves a stereospecific exchange of the pro-R hydrogen of the acyl dihydroxyacetone-P moiety without change in configuration. The fatty acid is re- placed by a long chain fatty alcohol which retains the oxygen of the primary carbinol. In the absence of fatty alcohol, water substitutes and the product is dihydrox-yacetone-P which has also exchanged the pro-R hydrogen with a hydrogen from the medium. An absolute requirement of the proposed mechanism is that the loss of the fatty acid must proceed via an unusual cleavage of the dihydroxyacetone-P C-1 to oxygen bond instead of the usual cleavage at the fatty acid acyl to oxygen bond. In the present investigation, we have synthesized hexadecanoyl dihydroxyacetone-P containing oxygen-18 exclusively at the dihydroxyacetone-P C-1 oxygen. Using this substrate, we have shown that cleavage of hexadecanoyl dihydroxyacetone-P at the C-1 to oxygen bond is linked to 0-alkyl dihydroxyacetone-P synthesis. Inhibition of

with retention of the primary carbinol oxygen (10). The results of these studies suggested that 0-alkyl DHAP might be formed by a reaction in which fatty alcohol was substituted directly for the fatty acid of acyl DHAP. Further experiments demonstrated, however, that a mechanism more complex than a simple substitution was involved. It was fiist shown that incubation of [1,3-3Hz,1,3-'4Cz]DHAP in an 0-aklyl lipid-synthesizing system resulted in the formation of 0-alkyl glycerols which had exchanged one of the hydrogens of the C-1 of the DHAP moiety of acyl DHAP (1). It was further shown by separate treatment of the labeled substrate, DHAP, with fructose 1,6-diphosphate aldolase and glyceraldehyde 3-P isomerase that the hydrogen which was removed had the same stereospecificity as the hydrogen removed by isomerase (pro-R hydrogen) (2). In addition, the hydrogen lost during 0-alkyl lipid synthesis was replaced by another from the medium without inversion of configuration of the substituents of C-1 of the DHAP moiety (4, 5,11,12). These findings are consistent with an endiol intermediate (6). A precedent for this mechanism may be found in the action of isomerase in the reversible reaction between DHAP and glyceraldehyde 3-P which proceeds via an endiol mechanism with labilization of the pro-R hydrogen and without inversion of configuration of the hydrogens of C-1 of DHAP (13,14). The aldolase reaction, on the other hand, in which the pro-S hydrogen of C-1 of DHAP is labilized, proceeds via the initial formation of a Schiff base between substrate and enzyme (15)(16)(17)(18)(19). Such a Schiff base mechanism involving acyl DHAP could neither be demonstrated in our laboratory nor could it be demonstrated by other investigators (6,20). Furthermore, the lack of inversion of configuration and the stereospecificity of the reaction with retention of configuration militated against various kinds of direct substitution reactions. Additional studies showed that the presence of the fatty alcohol in the reaction mixture is not required for the hydrogen exchange and that, in the absence of fatty alcohol, water substitutes instead (reaction 111) with the result that the product of the reaction is DHAP which has also stereospecifically exchanged the pro-R hydro- The experimental observations detailed above led us to postulate the endiol mechanism shown in Fig. 1. In the ordinary hydrolysis of a carboxyl ester bond it is known that the carbonyl oxygen is retained by the fatty acid moiety and that the other oxygen is retained in the primary carbinol of the alcohol (21). As can be seen in reaction IV, however, an absolute requirement for the endiol mechanism is that the fatty acid which is removed from acyl DHAP retain both carboxyl oxygens. Therefore, demonstration of an unusual hydrolysis involving the release of fatty acid containing both gen (7).
Proposed mechanism of 0-alkyl dihydroxyacetone-P synthesis from acyl dihydroxyacetone-P. *, when R is alkyl, the product is 0-alkyl dihydroxyacetone-P. When R' is hydrogen, the product is dihydroxyacetone-P. carboxyl oxygens would support the postulated mechanism. The present investigation addresses this problem.

Materials
Lithium aluminum hydride and N-methyl-N'-nitro-N-nitrosoguanidine were obtained from Aldrich. Glycolic acid and oxalyl chloride were from Eastman; chloroacetic acid was obtained from Sigma. Palmitoyl chloride was obtained from Fisher; BSTFA, trimethylchlorosilane, methoxyamine hydrochloride, and anhydrous pyridine were from Pierce Chemical Co. Heptadecanoic and octadecanoic acids and OV 17, 3%, on 100/200 mesh Gas-chrom Q, were obtained from Applied Science Laboratories. Bacterial alkaline phosphatase was obtained from Millipore Corporation. Thin layer chromatography plates, 0.25 mm, silica gel (E. Merck) were obtained from Brinkmann Instruments. Oxygen-18 water (99%) was obtained from Prochem. Dowex 50-4X-H', 100-200 mesh, was obtained from Bio-Rad. Biofluor was obtained from New England Nuclear. SP2330, IWo, on loO/Z00 mesh Chromosorb W/AW was obtained from Supelco. Triton X-IO0 was obtained from Mann Research Laboratories. Diazomethane was prepared by reacting 2.5 g of N-methyl-N'-nitro-N-nitrosoguanidine in 25 ml of ethyl ether in a separatory funnel with 7.5 ml of 40% KOH. The entire reaction was carried out at 2 "C. The product was washed twice with ice-cold water and used directly. [l-'4C]Hexadecanol was prepared by treating an ethanolic solution of [l-14C]palmitic acid (New England Nuclear, 56 mCi/mmol) with a solution of lithium aluminum hydride in ethyl ether (22). The product was purified by thin layer chromatography on 0.25-mm silica gel plates using hexane/ ethyl ether/acetic acid (5050:1, v/v/v) as the developing solvent.

Methods
Synthesis of ["O]Glycolic Acid-The method was derived from the work of Eichloff (24). To a solution of 1.125 g of monochloroacetic acid in 6 ml of ["O]water, 0.4 g of metallic sodium was cautiously added at room temperature under a stream of nitrogen. The resultant pH was approximately 12. The solution was transferred to a sealed metal tube. The bottom portion of the tube containing the solution was immersed in a sand bath and kept at 1 0 0 "C for 48 h. At the end of this period, the pH was approximately 7.0. Assay of the reaction mixture for glycolic acid (25) revealed a yield of 56.9%. The remaining [l8O]H2O was recovered by distillation at reduced pressure at 60 "C and was used later for the synthesis of ['"O]hexadecanoic acid. The dried residue was dissolved in 100 ml of water and passed through a column of 36 g of Dowex 50-4X-H+, wet weight. Additional water was passed through the column until the washings were pH 6.5. The resulting solution of glycolic acid was dried at reduced pressure at 45 "C and then under high vacuum overnight. A yellow residue remained which was shaken with 200 ml of ethyl ether. Some insoluble material in the ethyl ether solution was removed by filtration. Approximately 1 mg of the product was dried under nitrogen and then under high vacuum for 10 min, and the trimethylsilyl ether derivative was prepared by adding 0.2 ml of anhydrous pyridine and 0.2 ml of BSTFA followed by heating for 1 h at 60 "C. The product was analyzed by gas-liquid chromatography and gave a major peak with a retention time coinciding with that of authentic glycolic acid-TMS. The fragmentation pattern of the mass spectrum of the ["OITMS derivative was identical with that of authentic glycolic acid TMS except for a two-atomic mass unit difference for appropriate ions. Twenty-five mg of Triton X-100 in chloroform was then added to the tube, and the solvent was again evaporated. Tris buffer, 0.1 M, pH 8.2, containing 0.25 M sucrose, 0.1 M KCI, 0.04 M NaF, and Ehrlich ascites tumor cell microsomes (1.5 mg of protein, prepared as previously described (1)) were added to give a final volume of 5 ml. It was noted that protein concentrations higher than 300 pg/ml were inhibitory. Incubation was carried out with agitation at 37 "C for 2 h. At the conclusion of the incubation, an internal standard consisting of either IO or 20 pg of heptadecanoic acid was added. The incubation was stopped by extraction according to the method of Bligh and Dyer (28) using 1 HCI instead of water. Assay of 0-aklyl lipid synthesis was carried out by counting the activity of the total lipid extract followed by thin layer chromatography of an aliquot on 0.25-mm silica gel layers using chloroform/methanol/acetic acid (90:1010, v/v/v) as the developing solvent (29). Areas corresponding to 0-alkyl DHAP and fatty alcohol were scraped into scintillation vials. One-half ml of water was added and then 10 ml of Biofluor. Each sample was dispersed by sonication and the radioactivity was measured by liquid scintillation. Corrections for quenching and self-absorption due to silica gel were made by comparing the total counts recovered by thin layer chromatography with those obtained from the total lipid extract. In later experiments 0-alkyl DHAP was assayed by the method of Davis and Hajra (20). Similar results were obtained by both methods.
For determination of the "0 content of the free fatty acid fractions, an aliquot containing 80% of the total lipid extract was dried and dissolved in 6 ml of hexane. To isolate fatty acids, the hexane solution of the lipids was extracted with 6 ml of 0.1 N sodium hydroxide in 50% methanol containing bromphenol blue indicator. The upper phase was completely removed with several hexane washes and discarded. The bottom phase was acidified to pH 3 with 1 N HCI and the fatty acids were isolated by means of three hexane extractions. The combined extracts were dried and the fatty acids were methylated with an ethereal solution of diazomethane.
Alkaline methanolysis was carried out by the method of Hajra (30).
Muss Spectrometry-These studies were performed on a Hewlett-Packard model 5982 mass spectrometer in combination with a Hewlett-Packard model 5933 data system using either gas chromatography or direct probe insertion for introduction of the sample for analysis in the electron impact mode. Quantitative measurements were carried out by selected ion monitoring of specific ions in which the area under each peak was compared to the area from a corresponding peak in the internal standard. In the mass spectrometer the electron energy was 70 eV and the source temperature was 180 "C. For gas chromatographic separation of the methoxime-TMS derivative of 0-alkyl DNA a glass column (4 feet X 4 mm, inner diameter) packed with 3% OV17 on 100/200 mesh Gas-chrom Q was used with a column oven temperature of 240 "C. For analysis of the fatty acid methyl esters a glass column (6 feet X 4 mm, inner diameter) packed with 10% SP2330 on l00/200 mesh Chromosorb W/AW was employed at a temperature of 200 "C. In both cases the injector temperature was 250 "C, the allglass jet separator and interface lines were 250 "C, and the helium flow rate was 30 ml/min.  contained essentially no "0. This indicates that the carbonyl oxygen of ['gO]hexadecanoyl DHAP did not contain ''0 and that breakage of the substrate occurred between the acyl to oxygen bond. In order to demonstrate that these results did not occur because of exchange of the carbonyl oxygens of hexadecanoic acid with the medium, additional experiments were performed. Hexadecanoic acid containing approximately 50% '*O in the carboxyl group was methylated with diazomethane, a procedure which leaves the carboxyl oxygens intact. The results are shown in Table 11. Examination of the Mechanism of 0-Alkyl Lipid Synthesis 139 " The values were obtained as described in the text.

I. Validation
'This determination was made to measure the amount of '"0 present from unknown sources in addition to the natural abundance.
' As detailed in the text, hexadecanoyl DHAP was treated by alkaline methanolysis which breaks the carboxylic ester group at the acyl bond and yields methyl hexadecanoate which retains only the carbonyl oxygen.

TABLE I1
Determination of the stability of the 1 8 0 in f 'n007hexaclecunoic acid 1. Untreated ["Olhexadecanoic acid methyl ester" 2. Same as "1" after alkaline If both carboxyl oxygens are randomly labeled with "0, the expected result after alkaline methanolysis would be that no molecules would contain two "0 atoms. The expected per cent containing one "0 atom in the present case after acyl bond cleavage by alkaline methanolysis would be as follows: 37.2/2 + 6 = 24.6 (see "I" and "3" above).
' After incubation, the fatty acids were extracted as described under "Methods" and methylated with diazomethane. Henadecanoyl DHAP in Buffer-The nonenzymatic release of hexadecanoic acid from [lXO]hexadecanoyl DHAP was measured for the purpose of correcting data obtained in experiments which utilized an 0-alkyl lipid-synthesizing system. To determine this, ['80]hexadecanoyl DHAP, 70 nmol, and an octadecanoic acid internal standard, 352 nmol, were added to 1 ml of phosphate buffer and incubated a t 37 "C for 2 h. The sample was extracted by the method of Bligh and Dyer (28). An identical sample was extracted without incubation. Another sample contained the internal standard only. The fatty acids were isolated by thin layer chromatography and methylated with diazomethane. The fatty acid methyl esters were analyzed and quantitated by selected ion monitoring GC-MS using ions at m/z 74, 76, 270, and 272. It was determined that only 0.3 nmol was recovered as [''O]hexadecanoic acid after 2 h of incubation (0.41%). The total free hexadecanoic acid present was 4.96 nmol (7.1% of total hexadecanoyl DHAP).
If. Demonstration of a n Unusual Cleavage of Acyl DHAP in 0-Alkyl DHAP Synthesis A. The Effect of NADPH-Hexadecanoyl DHAP, 150 nmol, containing 85.4% of "0 at C-1 of DHAP was incubated in an 0-alkyl lipid-synthesizing system as described under "Methods." The free hexadecanoic acid present after incubation was quantitated as the methyl ester, and its "0 content was measured by GC-selected ion-monitoring MS of the ions at m/z 74 and m / z 76. These ions, as shown in Fig. 3, represent, respectively, the McLafferty rearrangement (31) of methyl hexadecanoate containing only or one ''0 atom and one l60 atom. The data were corrected for the natural abundances  Fig. 2. A, methyl hexadecanoate derived from standard hexadecanoic acid after extraction and methylation as described under "Methods"; B, methyl hexadecanoate isolated from the microsomal 0-alkyl lipidsynthesizing system using a substrate of hexadecanoyl-DHAP which had 85.46 of the oxygen a t C-I of the DHAP moiety of hexadecanoyl DHAP labeled with "0.  of "0 and 13C. As detailed below, these experiments showed that in the absence of 0-alkyl lipid synthesis the hexadecanoic acid liberated from hexadecanoyl DHAP was the product of hydrolysis at the acyl to oxygen bond and retained only the carbonyl oxygen. However, in the presence of 0-alkyl lipid synthesis, a large portion of the hexadecanoic acid which was liberated contained I8O, i.e. cleavage occurred at the DHAP C-1 to oxygen bond. The results are presented in  (Table 111, column B). In the microsomal enzyme system used in these studies, NADPH inhibits 0alkyl lipid synthesis by rapidly converting acyl DHAP to 1acyl glycerophosphate (9). When NADPH was added, the net quantities of hexadecanoic acid cleaved from hexadecanoyl DHAP, or more precisely, from its NADPH-derived product, 1-hexadecyl glycerol-3-P, were 118.2 and 104.3 nmol. At the same time both 0-alkyl lipid synthesis and the unusual cleavage at the DHAP C-1 to oxygen bond were greatly decreased (Table 111,  B. The Effect of Magnesium-In these experiments, we elected to inhibit 0-alkyl lipid synthesis by means of magnesium (11), which we hypothesized might only minimally affect lipase-catalyzed "ordinary" hydrolysis. At the same time we chose to provide additional data demonstrating the unusual hydrolysis operative in ether bond formation. Because of the presence of the carbonyl group at C-2 of the DHAP moiety, the mechanism of acyl DHAP lipolysis might conceivably have been different from that of 1-acyl glycerophosphate and might also in part have proceeded by the unusual mechanism demonstrated above. Although there exists a substantial body of information on the enzyme-catalyzed lipolysis of acyl glycerols, to our knowledge the lipolysis of acyl DHAP has not been investigated. The results are presented in Table IV. In column A-1 it can be seen that the yields of ['60]hexadecanoic acid measured in experiments 1 and 2 were greater than those of 3 and 4. Experiments 1 and 2 contained hexadecanoyl DHAP whereas 3 and 4 did not. The hexadecanoic acid obtained from experiments 3 and 4 was the free hexadecanoic acid derived from endogenous microsomal sources at the end of incubation.
The numbers in column B , which were calculated from those in column A-1, show the net quantities of [160]hexadecanoic acid derived from ['*O]hexadecanoyl DHAP. These results reveal without ambiguity that magnesium did not inhibit lipolysis of ['80]hexadecanoyl DHAP which proceeds by the usual mechanism, i.e. breakage of the acyl to oxygen bond.
The release of hexadecanoic acid which retained both carboxyl oxygens was substantial and was, furthermore, consonant with the yield of 0-alkyl DHAP (column D). The release of this "0-containing species of hexadecanoic acid was almost completely inhibited by magnesium. Hence, both 0-alkyl lipid synthesis and the release of hexadecanoic acid which was cleaved at the DHAP C-1 to oxygen bond were simultaneously inhibited by magnesium. It may also be noted that the ordinary hydrolysis at the acyl to oxygen bond exceeded the unusual hydrolysis which occws at the DHAP C-1 to oxygen bond.
In column D, it can be seen that the yield of 0-alkyl DHAP was 16.1 & 1.7 nmol in the absence of magnesium. These amounts, as expected, are somewhat less than the amounts of hexadecanoic released by the unusual cleavage (column C).
In the presence of magnesium, the yield of 0-alkyl DHAP was negligible.
C. Experiments With and Without Hexadecanol-According to the mechanism which we have postulated for 0-alkyl DHAP synthesis (Fig. l)

DISCUSSION
The endiol mechanism which we have proposed has gained support elsewhere. Davis and Hajra (20) have postulated that a n electrophilic enol ester may be the intermediate which is attacked by the nucleophilic alcoholate ion. Presumably, this mechanism (20) is similar, if not identical, with the one which we proposed. These authors also suggest, and we concur, that the enol ester intermediate might be 1,2-cyclic, a possibility which is also in accord with the available data and which represents a slight variation of the basic mechanism shown in Fig. 1.
The present investigation augments previous experimental evidence supporting the endiol concept of 0-alkyl lipid ether bond synthesis. We have shown here that loss of the fatty acid of acyl DHAP in the process of 0-alkyl DHAP synthesis occurs via an unusual cleavage of the fatty acid at the DHAP C-1 to oxygen bond, i e . the C-1 oxygen of DHAP remains with the fatty acid. This loss of the DHAP C-1 oxygen is in accord with the endiol mechanism for 0-alkyl lipid bond formation which we have proposed (5,6) and is also in accord with the previously demonstrated fact that the oxygen of the fatty alcohol is retained by the product, 0-alkyl DHAP (10). In addition to this unusual cleavage, acyl DHAP also undergoes an ordinary hydrolysis at the acyl to oxygen bond in the crude microsomal 0-alkyl lipid-synthesizing system used in these studies. This ordinary hydrolysis is presumably mediated by a phospholipase A-1. When 0-alkyl DHAP synthesis is inhibited by NADPH or magnesium, only the ordinary hydrolysis occurs. Further support for the endiol mechanism derives from the demonstration in the present study that the fatty alcohol is not necessary for the unusual cleavage of the fatty acid of acyl DHAP. Neither is the fatty alcohol obligatory, as demonstrated in a previous study (6), for the DHAP C-1 p~0 -R hydrogen exchange. Davis and Hajra (32) have demonstrated that Ehrlich ascites cell microsomes can catalyze the exchange of the acyl group of acyl DHAP with free fatty acids and have postulated that this reaction is mediated by the same enzyme which is responsible for the synthesis of 0-alkyl DHAP. The role which this reaction may play in 0-alkyl DHAP synthesis has not been determined a t this time.
In earlier investigations, we have shown that the number of hydrogen atoms lost from C-1 of acyl DHAP substantially exceeds the number of molecules of 0-alkyl DHAP formed (7). The difference is accounted for by the formation of DHAP which has exchanged the C-1 pro-R hydrogen. By inference, the amount of fatty acid liberated with retention of both carboxyl oxygens should also exceed the amount of 0-alkyl DHAP formed. However, the incubation conditions used in the present investigation produced much higher yields of 0alkyl DHAP than we have obtained under previously used conditions. This may explain the closer stoichiometric equivalence of 0-alkyl DHAP production and ['80]hexadecanoic acid release seen in the present experiments.
In the proposed mechanism, the step in which the hydrogen is lost is irreversible, as evidenced by the fact that acyl DHAP incubated in [?H]H20 does not become labeled (4). Therefore, the putative endiol intermediate is left to react with either a molecule of fatty alcohol or a molecule of water (7). In the absence of fatty alcohol, as mentioned above, the endiol reacts exclusively with water, in which case the product is DHAP which has exchanged the p~0 -R hydrogen (7). Consequently, if the intermediary endiol does not accumulate, a necessary condition for the proposed mechanism is that the total amount of ['80]hexadecanoic acid liberated should be equal to the amount of p 0 -R hydrogen exchanged. Demonstrating this stoichiometric relationship, however, was beyond the scope of the present investigation and could not be addressed at the present time.