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Synthesis of fluorescent and radiolabeled analogues of phosphatidic acid
Chemistry and Physics of Lipids, 36 (1985) 197--207 Elsevier Scientific Publishers Ireland Ltd.
197
SYNTHESIS OF FLUORESCENT AND RADIOLABELED ANALOGUES OF PHOSPHATIDIC ACID
KENNETH J. LONGMUIRa, ONA C. MARTIN b and RICHARD E. PAGANOh
aDepartraent of Physiology and Biophysics, College o]" Medicine, University o/ CaliJornia, lrvine, CA 9271 7 and bThe Carnegie Institution of Washington, Department of l:'mbryology, 115 I~. Unirersity Parkway. Baltimore, MD 21210 (U.S.A.) Received November 21st, 1983 revision received September 281h, 1984 accepted October 2nd, 1984 Procedures tbr the synthesis of tluorescent and radiolabeled analogues of phosphatidic acid arc described. The |luorophore 7-nitrobenzo-2.-oxa-l,3-diazole (NBD) was coupled to 6-aminocaproic acid and 12-aminododet~noic acid by reaction of NBD-chloride with the amin~ acids under mild alkaline conditions at room temperature. 1,2-Dioleoyl-sn-iU-t'~('Jglycerol 3-phosphate was prepared by acylation of sn-lU-~4C]glycerol 3-phosphate with oleic acid anhydridc using dimcthylaminopyridine as the ~talyst. This compound was converted to l-oleoyl-sn[U-~4(']glycerol 3-phosphate by hydrolysis with phospholipase Aa. The lysophosphatidic acid was reacylated with NBD-aminocaproyl imidazole or NBD-aminododecanoyl imidazol~ to form the t]uorescent, radiolabeed analogue of phospbatidic acid. Fluoresoent, non-radiolabeled analogues of phosphatidic acid were prepared by phospholipasc D hydrolysis of fluorescent phosphatidylcholine.
Keywords. 7-nitrobenzo-2-oxa-l,3-diazole; fatty acid; phosphatidic add; phospholipids; acylation reactions
Introduction Phosphatidic acid is an important intermediate for the biosynthesis of glycerolipids in mammalian cells. In order to follow the intracellular localization and metabolism o f phosphatidic acid, we have prepared fluorescent analogues where the sn-2 position contains NBD linked to either 6-aminocaproic acid or 12-aminododecanoic acid. These fluorescent phosphatidic acids are taken up by mammalian cells, transported to intracellular sites o f lipid biosynthesis and converted to end l~roducts of lipid metabolism [ 1 - 3 ]. NBD-labeled phosphatidic acids are most easily obtained by phospholipase D hydrolysis of commercially available NBD-labeled phosphatidylcholine [ 1 ]. However, for part of our investigations it was necessary to prepare phosphatidic acid with Abbreviations: NBD, 7-nitrobenzo-2-oxa-l,3-diazole; NBD..CI, 4-chloro-7-nitrobenzo-2-oxa-1. 3.-diazole; NMR, nuclear magnetic resonance; Pyr, pyridinium ion; TLC, thi~-layer chromatography. 0009-3084/85/$03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
198 both an NBD label on the fatty acid chain and a radiochemical label on the glycerol backbone. Although reports have appeared concerning the synthesis ofphosphatidic acids [4--6] and NBD-labeled fatty acids [7], the methods described in the literature failed to produce these particular analogues in either significant yield or acceptable purity. Specifically, the synthesis of NBD.fatty acids resulted in the accumulation of highly colored side products which were not eliminated by the recrystallization methods described in the literature. Second, attempts to acylate lysophosphatidic acid with the acid anhydrides of NBD-fatty acids were unsuccessful. Instead. modifications of the acyl imidazole procedure were required. Third, the acylation reactions produced a variety of side products, and considerable attention was required for their eventual separation from the final product. Finally, a number of questions remained regarding yields, purities, and time courses of all the reactions utilized in the preparation of these fluorescent and radiolabeled analogues. In this communication, we describe the modifications that were required to obtain these products in acceptable yield and purity. The synthetic reactions utilized are outlined in Scheme !.
NH e -(C
He)n--COOH
NID-CI
MIIO-NH
-- (ONe)=-- C OOH
11 Pyr
O I PyrO-P==O I O I
OH I
O (
1/1
OH
OH I HO-P=O I O I H=C - -
317
( R-~-IeO
H
H;eC--
I
HO-P=O t
0 I
P Msil4~lilmse
H
At
CH t
'CH l I
OH
o
0
C:O I R
C=O ! R OH !
HO-PIO I O
NIIO--NH- (CHI)n-- ~ - N ~ rN H C -I OH
•
CH t I
O I C"O I R
t HeC - -
"9I"
II
* Ib, II
. •
¢.:-<=.=~-~.~-~¢.=:?-
H C -!
CH,, !
0 O I I C=,O C,=O
| I (tHe) n R I NH !
NBD
m) 2
Scheme 1. Outline of synthes~s of NBD-labeledfatty acids and NBD-labeledphosphatidic acids.
199
Experimental Materials l-Acyl-2-(NBD-aminododecanoyl)-sn-glycero-3-phosphocholine was obtained from Avanti Polar Lipids, Inc., Birmingham, AL. Phospholipase D (from cabbage) was obtained from Boehringer-Mannheim, Indianapolis, IN. sn-Gylcerol 3-phosphate(di(monocyclohexylammonium) salt), 6-aminocaproic acid, oleic acid anhydride, NBD-CI, and phospholipase A2 (from porcine pancreas) were obtained from Sigma, St. Louis, MO. Dimethylaminopyridine, 12-aminododecanoic acid and carbonyldiimidazole were obtained from Aldrich, Milwaukee, Wl. sn-[U-~4C]Glycerol 3phosphate (20 ,aCi/,amol) was obtained from ICN, lrvine. CA. Silica gel 60 TLC plates (250 ,am thickness) were from E. Merck, Elmsford, NY. Silica gel for column chromatography (SilicAR CC-7) was obtained from Mallinckrodt, Paris, KY. Cation exchange resin AG 50W-X8 was obtained from Bio-Rad, Richmond, CA. Scintillation grade Triton X-IO0 was obtained from Research Products International, Mount Prospect, IL. Glass-distilled chloroform, methanol, acetone and ethyl acetate were obtained from Burdick and Jackson, Muskegon, M1. Solvents were dried by stirring in molecular sieves (Type 3A, MCB, Cincinnati, OH) and filtering. All other reagents were analytical grade. General methods Chloroform/methanol extraction was accomplished using modifications [8] of the procedures of Bligh and Dyer [9]. Lipids were dissolved in 5.0 ml of chloroform/ methanol/0.2 N HCI (1:2:0.8). (All solvent mixtures are indicated by volume.) if necessary, 6 N HCI was added to maintain a pH of 1-2 and methanol was added to maintain a single phase. The solution was separated into two phases with the addition of 1.5 ml of chloroform and 1.5 ml of 2 M KCI. The mixture was centrifuged, the upper phase was discarded, and the lower phase was washed twice with 5 ml of synthetic Folch upper phase (methanol/water/chloroform, 9 6 : 9 4 : 6 [10]). The solutions were centrifuged after each wash to insure phase separation. The lower phase was brought to dryness by evaporation with N2 and the residue dissolved in a suitable solvent. Ethyl acetate/acetone extraction was accomplished following modifications [ 1l of the procedures of Slayback et al. [ 11 ]. To 1 ml of an aqueous suspension of lipid was added 1.0 ml of 0.2 N HCI, 2.0 ml of acetone and 4.0 ml of ethyl acetate. The solution was centrifuged, and the upper phase of organic solvent removed and saved. The lower phase was extracted twice more by addition of 1.6 ml of acetone and 4.0 ml of ethyl acetate. The solution was centrifuged after each extraction to insure phase separation. The combined upper phases were brought to dryness by evaporation with N2 and the lipid residue dissolved in a suitable solvent. Lipids were separated by TLC on silica gel 60 plates (250 ,am thickness) in two solvent systems. Acidic solvent system: chloroform/acetone/methanol/acetic acid/ water (10 : 4 : 2 : 2 : 1). Basic solvent system: chloroform/methanol/NH4OH ( 13 : 7 : 1).
200 Unlabeled lipids were visualized with iodine. Radiolabeled lipids were visualized by autoradiography (Kodak X-Omat-AR film). Fluorescent lipids were visualized with a 365 nm transilluminator (Spectronics Corp., Westbury, NY). Phosphorus analyses were performed using the procedures of Ames and Dubin [12]. Fluorescence measuresments were made on a Spex Fluorolog spectrofluorometer with an excitation wavelength of 470 nm and an emission wavelength of 530 ran. Elemental analyses were performed by Galbraith Laboratories, Inc., Knoxville, TN. Melting point determinations were performed using a Fisher-Johns melting point apparatus. Radiolabeled samples were prepared for scintillation counting by thoroughly drying an aliquot of sample in a scintillation vial, then adding 0.17 ml of water followed by 2.0 ml of Triton-toluene scintillation fluid [I 3], To determine the positions of the fatty acids on the glycerol backbone, fluorescent phosphatidic acid products were treated with phospholipase A2 [14]. The reaction products were extracted into ethyl acetate/acetone and separated by TLC using the acidic solvent system. The relative fluorescence in the fatty acid and lysophosphatidic acid regions of the plate were determined using a Coming model 750 scanning densitometer. Except where specified, all reactions and lipid extractions were performed in 16 X 125 mm screw-cap glass culture tubes with Teflon-lined caps.
Preparation of l-acyl-2-{NBD-aminododecanoyl)-sn-glycerol 3-phosphate using phospholipase D Fluorescent, non-radiolabeled phosphatidic acid was prepared by phospholipase D hydrolysis of fluorescent phosphatidylcholine [14]. A chloroform solution ol commercially available l-acyl-2-(N BD-aminododecanoyl)-sn-glycero-3-phosphocholine (2.3 tamol) was evaporated to dryness with N2, then dried in vacuo. The phosphatidylcholine was suspended in 1.0 ml of 0.1 M sodium acetate (pH 6.0} followed by 0.2 ml of i M CaCI2, 0.8 ml of phospholipase D (20 mg/ml) and 0.5 ml of diethyl ether. The reaction was stirred vigorously on a magnetic stirrer, Progress of the reaction was followed by TLC in the acidic solvent system. After 1 h, 8 ml of chloroform/methanol (1:2) and 0.1 mI of 6 N HCI were added. The solution was separated into two phases with 2.0 ml each of chloroform and 2 M KCI. The mixture was centrifuged, and the lower phase filtered through glass wool to remove precipitated protein. The filtrate was washed twice with 5 ml of synthetic Folch upper phase. The lower phase was evaporated to dryness with N2, dissolved in chloroform/ methanol (95.:5), applied to a TLC plate of activated silica gel 60 (250 tan thickness), and developed in the acidic solvent system. The band containing the fluorescent phosphatidic acid was scraped, and the product recovered on a Soxhlet extractor for 5 h with chloroform/methanol (1:2). The yield, by phosphorus analysis, was 84%.
Synthesis of NBD-ammododecanoic acid (H ) The reactions of NBD-CI with tile amino acids were carried out using modifica-
201
tions of the procedures of Monti et al. [7]. NaHCOa (1 retool) was dissolved in 2.5 ml of water in a 25 ml Erlenmeyer flask. To this solution was added 12-aminododecanoic acid (0.25 mmol), followed by 7.5 ml of ethanol. The fatty acid was dissolved with mild heating (some NaHCO3 precipitated at this point). After cooling to room temperature, NBD-CI (0.75 retool) was added, and the reaction was stirred in the dark at room temperature. Progress of the reaction was monitored by removing 25 /al aliquots, quenching in chloroform/methanol/0.2 N HCI (1:2:0,8), and determining the fluorescence, After 4 h, the reaction was stopped with 1.0 ml of I N HCI. Ethanol and water were added to obtain 30 ml of ethanol/water (1 : 1). The products were dissolved with heating, then crystallized overnight at -20°C. The precipitate was harvested on glass fiber filter paper, washed with cold water, and dissolved in chloroform/ methanol (2:1). The solvents were evaporated and the products recrystallized in 45 ml of ethanol/water (1:2). The recrystallized products were dissolved in 20 ml of chloroform/methanol (99:1) and applied to a column of 25 g of activated silica gel. Non-t]uorescent material was eluted with 250 ml of chloroform. Elution continued with 250 ml of chloroform/methanol (99.5:0.5) followed by 250 ml of chloroform/methanol (99 : I), at which point a fluorescent band running ahead of the major product was eluted. The NBD-aminododecanoic acid was eluted with chloroform/methanol (98 : 2), and collected in five 50-ml fractions. The first fraction of NBD-aminododecanoic acid contained a minor fluorescent impurity, and was repurified by the same silica gel column chromatography procedure. All fractions containing purified NBDaminododecanoic acid were combined, the solvents evaporated, and the product dried in vacuo and weighed (yield 37%). Melting point 95- 97°C. Elemental analysis, calc. for Cl~-12~'q4Os: C, 57.13%; H, 6.93%; N, 14.80%; found: C, 57.08%; H, 6.89%; N, 14.61%. Rf = 0.52 (basic solvent system). IH-NMR in CDCI3:8.48 ppm (d, J=9 Hz, NH-CCHCHC--NO2); 6.16 ppm (d, J=9 Hz, NH-CCHCHC-NO2); 6.34 ppm (m, NH); 3.47 ppm (q, J=7 Hz, N-CH2); 2.33 ppm (t, J=7 Hz, CH2COO): 1.8-1.3 ppm (hydrocarbon chain protons).
Synthesis of NBD-aminocaproic acid (11] NaHCO3 (2 mmol) was dissolved in 5 ml of water in a 50 ml Erlenmeyer flask. 6-Aminocaproic acid (0.5 retool) and 15 ml of ethanol were added and the fatty acid was dissolved with mild heating. The solution was cooled to room temperature, and NBD-CI (1.5 mmol) was added. The reaction was stirred for 4 h at room temperature in the dark. Progress of the reaction was followed as described above. The reaction was stopped with 2.5 ml of 1 N HCI. The solvents were evaporated and the products crystallized at -20°C in 90 ml of ethanol/water ( 1 : 2). A second crystallization was carried out in 120 ml of ethanol/water (! :3). The products were dissolved in 20 ml of chloroform/methanol (98.5:1.5), and applied to a column of 30 g of activated silica gel. Non-fluorescent material and a minor fluorescent product were eluted with 250 ml of chloroform/methanol (99.5:0.5) followed by 250 ml of chloroform/methanol (99: 1). The fluorescent fatty acid was eluted
202 with 500 ml of chloroform/methanol (98:2), followed by 200 ml of chloroform/ methanol (96:4). The fractions containing the product were combined, the solvents evaporated, and the product dried in vacuo and weighed (yield 46%). Melting point 155-156°C. Elemental analysis, calc. for Ct2Ht4N4Os: C, 48.98%; H, 4.80%; N, 19.04%; found: C, 49.02%; H, 4.80%; N, 18.90%. Rf = 0.46 (basic solvent system).
Synthesis of 1,2-dioleoyl-sn[U-14C]glycerol 3-phosphate (IV) Acylation of sn-[U34C] glycerol 3-phosphate (Ill) with oleic acid anhydride was carried out using modifications of the procedure of Gupta et al. [5]. Five g of cation exchange resin were added to a 30 cm X 1 cm chromatography column. The resin was cleaned by successive washes with 25 ml of 10 N NaOH, 100 ml of water, 25 ml of 6 N HCI, 100 ml of water, then converted to the pyridinium form with 100 ml of 2.5 M pyridine, followed by 100 ml of water. sn- [ U-14C] Glycerol 3-phosphate (I 25/aCi) and unlabeled sn-glycerol 3-phosphate were combined to obtain 25/~mol (5/~Ci//amol) in 5 ml of water. The solution was passed through the resin and collected in 10 ml of water. After evaporation of solvent, the residue was dissolved in methanol and the radioactivity determined. The methanol was evaporated, and the residue dried in vacuo over P2Os (recovery 95%). To the residue was added oleic acid anhydride (120 gmol, 2.5-fold excess), 0.2 ml of chloroform and dimethylaminopyridine (120 #mol). The reaction mixture was stirred at room temperature for 36 h. The reaction was monitored by TLC in the acidic solvent system. Radiolabeled lipids were visualized by autoradiography, and regions of the plate scraped and counted. At the end of the reaction period, the solvent was evaporated and ~eplaced with 0.5 ml of methanol/chloroform/water/pyridine, (2:1 : 1 : 1), and the reaction mixture stirred for an additional 24 h. The solvents were evaporated and the lipids isolated by extraction into chloroform/methanol. The phosphatidic acid was separated from fatty acid by column chromatography on 5 g of activated silica gel. The products were applied in chloroform, and the fatty acids eluted with 50 ml of chloroform/methanol (95 : 5), followed by 50 ml of chloroform/methanol (90: 10). Phosphatidic acid was eluted with 100 ml of chloroform/methanol (75 : 25), followed by 50 ml of chloroform/methanol (50 : 50). Column fractions were analyzed by TLC using the acidic solvent system and the lipids visualized by autoradiography, then with iodine. Fractions containing the phosphatidic acid were combined, the solvents evaporated, and the product stored in chloroform/methanol (2: 1) at -20°C (yield 63%).
Preparation of 1-oleoyl-sn-[U34C]glycerol 3-phosphate (V) Phosphatidic acid was hydrolyzed using phospholipase As [14]. 1,2-Dioleoyl-sn[U-t4C]glycerol 3-phosphate (IV) (12 tamol) was dried in vacuo to remove chloroform and methanol, then dissolved in 10 ml of diethyl ether. The reaction was started with the addition of 0.5 mg of phospholipase As in 1.0 ml of 20 mM CaCI2
203 and 0.1 M sodium borate (pH 7.0) and the solution stirred vigorously. The reaction was followed by removing lO-pl aliquots and separating the lipids by TLC in the acidic solvent system. At the end of the reaction period, the ether was evaporated and the lysophosphatidic acid was extracted into ethyl acetate]acetone. The solvents were evaporated, the residue dissolved in chloroform/methanol (95:5), then applied to a column of 5 g of silica gel as described above. Fatty acid and trace amounts of phosphatidic acid were eluted with 50 ml of chloroform/methanol (95:5), followed by 50 ml of chloroform/methanol (75:25). The product was eluted with 100 ml of chloroform• methanol (50:50). Column fractions were analyzed by TLC and the lipidsvisualized by autoradiography. Fractions containing the lysophosphatidic acid were combined, the solvents evaporated, and the product stored in chloroform/methanol (2 : 1) at -20°C (yield 59%).
Synthesis of l-oleoyl-2-(NBD-aminododeeanoyl)-sn-[U-x4C]glycerol 3-phosphate (Vl) Acylations of lysophosphatidic acid with fluorescent fatty acids were carried out using modifications of the acyl imidazole procedure [15]. Approximately 7 /amol of l-oleoyl-sn-[U-v~C] glycerol 3-phosphate (V) was dried in vacuo over P2Os. In a separate tube, NBD-aminododecanoic acid (20/Jmol) and carbonyldiimidazole (20/amol) were dissolved in 1.0 ml of dry chloroform. After 1 h at room temperature, this reaction mixture was transferred to the tube containing the lysophosphatidic acid, and the solvents evaporated. The reaction mixture was resuspended in 0.1 ml of dry chloroform and stirred for 48 h in an oil bath at 60°C. Progress of the reaction was followed by TLC in the acidic solvent system and the lipids visualized by (1) autoradiography, (2) fluorescence excitation on a 365 nm transilluminatot and (3) iodine staining. The products were extracted into ethyl acetate]acetone, dissolved in a small volume of chloroform/methanol (95:5), and applied to a silica gel 60 TLC plate (250 #m thickness) for preparative TLC in the acidic solvent system. The band containing the fluorescent phosphatidic acid was scraped, and the gel extracted four times with 5 ml of chloroform/methanol/0.2 N HCI (1:2:0.8). The products were further isolated by the chloroform]methanol extraction procedure. The final product was stored in dry tetrahydrofuran at -70°C (yield 38%). Treatment of a portion of the final product with phospholipase A2 revealed that 78% of the NBDaminododecanoic acid was acylated at position sn.2 and 22% at position sn-I.
Synthesis of 1-oleoyl-2-(NBD-aminocaproyl)-sn-[U-14C]glycerol 3.phosphate (VI ) A sample of l-oleoyl-sn-[U-14C]glycerol 3-phosphate (V) (30 /amol, 1.5 /aCi/ /amol) was dried in vacuo over P2Os. In a separate tube, NBD-aminocaproic acid (90 omol) and carbonyldiimidazole (90 /amol) were dissolved in 1.0 ml of dry tetrahydrofuran, and allowed to react at room temperature for 2 h. The reaction mixture was transferred to the tube containing the lysophosphatidic acid. The
204
solvent was evaporated and replaced with 0.5 ml of dry dichloromethane. The tube was capped tightly and the reaction mixture stirred for 48 h at 60-70°C in an oil bath. At the end of the reaction period, the fluorescent phosphatidic acid was extracted into ethyl acetate/acetone and purified by preparative TLC as described above (yield 22%). Phospholipase A2 treatment of the product revealed that 79% of the aminocaproic acid was acylated at position sn-2 and 21% at position sn-1.
Results and Discussion
Synthesis of NBD-amino acids The reaction of NBD-CI with the amino acids formed highly fluorescent products from non-fluorescent precursors. Figure I is a typical time course for the reaction of NBD-CI with 12-aminododecanoic acid. Identical results were obtained for the reaction of NBD-C1 with 6-aminocaproic acid. The reaction was essentially complete between 6 - 9 I1. However, reaction times this long resulted in the accumulation of colored, non-fluorescent reaction products which made purification of the fluorescent fatty acid difficult. For the preparation of purified fluorescent fatty acids, a reaction time of 4 h at room temperature was optimal.
!
!
l
I
1
I
I
o1.0
z 1,1.1
~.0.8 o _J It. w
0.6
t-
~0.4, 0.2
t
1
2
I
1
4
I
I
I
6
TIME, h Fig. 1. Appearance of fluorescence during the reaction of NBD-CI with 12-aminododecanoic acid. Reaction conditions are described in the Methods. Aliquots of the reaction mixture were removed at the indicated times and acidified in chloroform/methanol/0.2 N HCI ( 1 : 2 : 0 . 8 ) . Fluorescence measurements were made at hex = 470 nm and hem = 530 nm.
205 Two fluorescent products were obtained. The major product was the expected NBD-amino acid. A minor product was also isolated which on TLC in both solvent systems ran close to the solvent front. NMR analysis revealed that this minor component was the fatty acid ester. Separation of the small amount of ester from the fatty acid was achieved by stepwise elution of the silica gel column. Silica gel column chromatography was also necessary to ensure complete separation of the fluorescent product from the colored, non-fluorescent side products.
Synthesis of dioleoyl phosphatidic acid and lysophosphatidic acid As suggested by Gupta et al. [5], the reaction of sn-[U-t4C] glycerol 3-phosphate with oleic acid anhydride resulted in the formation of two major products when dimethylaminopyridine was used as the catalyst. These were the dioleoyl phosphatidic acid (Rf = 0.56, acidic solvent system) and a presumed dioleoyl phosphatidic acid-oleoyl anhydride (Rf = 0.86, acidic solvent system), where a third fatty acid forms an anhydride with the phosphate. After 18 h of reaction, TLC analysis showed 20% of the radioactivity as phosphatidic acid and 78% as the presumed phosphatidic acid-oleoyl anhydride. The phosphatidic acid-oleoyl anhydride was cleanly converted to phosphatidic acid by stirring for 24 h in a methanol/chloroform[water/pyridine solution [5]. TLC showed that virtually all of the radioactivity migrated as phosphatidic acid, while no radioactivity was detected as the phosphatidic acid-oleoyl anhydride. The greatest loss of product occurred during silica gel column chromatography. After extraction of the reaction products into organic solvent, 96% of the initial radioactivity was present, with virtually all of the radioactivity as phosphatidic acid. Only 63% of the initial radioactivity was recovered after column chromatography. Phospholipase A2 effectively hydrolyzed dioleoyl phosphatidic acid. For the reaction reported here, 98% of the initial phosphatidic acid was hydrolyzed to lysophosphatidic acid within 15 min. No significant conversion of lysophosphatidic acid to glycerol 3-phosphate was observed. Extraction of lysophosphatidic acid into solvent was achieved using the ethyl acetate/acetone procedure only. If chloroform/methanol extraction was used, the lysophosphatidic acid was lost into the aqueous upper phase. In contrast, phosphatidic acid was extracted either into chloroform/methanol or into ethyl acetate/ acetone with equal success. A substantial loss of lysophosphatidic acid occurred during silica gel column chromatography. After ethyl acetate/acetone extraction, 89% of the initial radioactivity was recovered, with virtually all of the radioactivity as lysophosphatidic acid. Following column chromatography, 59% of the initial radioactivity was recovered as lysophosphatidic acid.
Synthesis of 1-oleoyl-2-{NBD-aminoacyl)-sn-[U34C]glycerol 3-phosphates At first we attempted to synthesize NBD-labeled analogues of phosphatidic acid by acylation of lysophosphatidic acid with NBD-labeled fatty acid anhydrides.
206 Yields of phosphatidic acids from this procedure were negligible. As a result, all further acylations of lysophosphatidic acid were carried out using the acyl imidazole procedure [15]. Acylations with NBD-aminododecanoyl imidazole were carried out in chloroform. Acylations with NBD-aminocaproyl imidazole could be carried out only with dichloromethane as the solvent. The reactions o f l-oleoyl-sn-[U-t4C] glycerol 3-phosphate with the imidazolides of NBD-aminododecanoic and NBD-aminocaproic acid each resulted in the formation of two major products. The first was the expected fluorescent analogue of phosphatidic acid. The second product was a radiolabeled but non-fluorescent compound which, in the acidic solvent system, had an Rf-value slightly less than the fluorescent phosphatidic acids. Because this compound was cleanly separated from the fluorescent product at the end of the reaction, no attempt was made to further characterize it. However, Lapidot et al. [4] have reported that some acylation reactions result in the formation of significant quantities of cyclic lysophosphatidic acid. This compound, in various TLC solvent systems, migrates with an Rf-value slightly less than the corresponding phosphatidic acid. Table 1 lists the time course of the distribution of products obtained during the acylation of l-oleoyl-sn-[U-14C]glycerol 3-phosphate with NBD-aminododecanoyl imidazole. The reaction was essentially complete after 48 h, as indicated by the small percentage of starting material that remained. In addition to the fluorescent product and the presumed cyclic lysophosphatidic acid, some fluorescent phospha-
TABLE I TIME COURSE OF REACTION OF I-OLEOYL-~n-IU-14C]GLYCEROL3-PHOSPHATE WiTH NBD-AMINODODECANOYL IMIDAZOLE I-Oleoyi-sn-lU-14Clglycerol 3-phosphate was reacted with a 3-fold excess of NBD-aminododecanoyl imidazole for 48 h in an oil bath at 60°C. The reaction mixture was analyzed by TLC in the acidic solvent system. Lipids were visualized by auloradiography then counted. Lipid (ROa
3h (%)
24 h (%)
48 h ~%)
1-Oleoyl-sn-IU-vlC]glycerol 3-phosphate (0.19) i -Oleoyl-2-(NBD-aminodode~ noyl)sn-[ U-t4CIglycerol 3-phosphate (0.45) 1-Oleoyl-sn-[U-14C]:glycerol 2,3-cyclic phosphate (0.35) b Phosphatidic acid acyl anhydride (0.62) c
66
18
6
25
52
53
8
23
29
2
8
12
aRf-value determined by TLC in the acidic solvent system. bTentative identification of cyclic lysophosphatidic acid is based upon the work of Lapidot et al. {4 I. CTentative identification of phosphatidic acid-acyl anhydride is based upon the work of Gupta ct~.
[Sl.
207
tidic acid-acyl anhydride was also detected. The Rt-values for the various compounds produced during the acylation of 1-oleoyl-sn-[U-t4C]glycerol 3-phosphate with NBD-aminododecanoyl imidazole are listed in Table I. The Revalue of 1-oleoyl-2(NBD-aminocaproyl)-sn-[U-t4C] glycerol 3-phosphate was 0.39 in the acidic solvent system. Separation of the fluorescent product from the presumed cyclic lysophosphatidic acid was accomplished by preparative TLC on silica gel 60, followed by extraction of the gel with the chloroform/methanol extraction procedure. In our experience, lysophosphatidic acids are lost into the upper phase during this extraction procedure. TLC analysis after extraction showed a single spot corresponding to the fluorescent, radiolabeled analogue of phosphatidic acid. Non-fluorescent, radiolabeled side products were not detected after these purification procedures were performed. The principal shortcoming of the procedures described here was that about 20% of the final product contained NBD-fatty acid at position sn-1 rather than at the desired sn-2 position. This was likely due to the rather vigorous acylation procedures required to obtain NBD-labeled phosphatidic acids in acceptable yields. Further research is needed to find milder reaction conditions which can acylate glycerophosphates with the unusual fatty acids that are becoming increasingly useful for contemporary biochemical research.
Acknowledgements We wish to thank Aiisen Sykes and Christine Resele-Tiden for their technical assistance. This work was supported by USPHS grant GM-22942 (R.E.P.), USPHS grant HL-28619 (K.J.L.), and a USPHS Pulmonary Faculty Training Award HL00456 (K.J.L.).
References 1 R.E. Pagano, K.J. Longmuir, O.C. Martin and D.K. Struck, J. Cell Biol., 91 (1981) 872-877. 2 R.E. Pagano, K.J. Longmuir and O.C. Martin, J. Biol. Chem., 258 (1983) 2034-2040. 3 R.E. Pagano and K.J. Longmuir, Trends Biochem. Sci., 8 (1983) 157-16 I. 4 Y. Lapidot, I. Barzilay and J. Hajdu, Chem. Phys. Lipids, 3 (1969) 125 - 134. 5 C.M. Gupta, R. Radhakrishnan and H.G. Khorana, Proc. Natl. Acad. Sci. U.S.A., 74 (1977) 4315-4319. 6 P.B. Kingsley and G.W. Feigenson, Chem. Phys. Lipids, 24 (1979) 135-147. 7 J.A. Monti, S.T. Christian,W.A. Shaw and W.H. Finley, Life Sci.,21 (1977) 345-356. 8 A.K. Hajra, E.B. Seguin and B.W. Agranoff, J. Biol. Chem., 243 0968) 1609 -1616. 9 E.G. Bligh and W.J. Dyer, Can. J. Biochem. Physiol.,37 (1959) 911-917. I0 J. Folch, M. Lees and G.H.S. Stanley, J. Biol.Chem., 226 (1957) 497-509. II J.R.B. Slayback, L.W.Y. Cheung and R.P. Geyer, Anal. Biochem., 83 (1977) 372-384. 12 B.N. Ames and D.T. Dubin, J. Biol. Chem., 235 (1960) 769--775. 13 J.E. Bleasdale, P. Wallis, P.C. MacDonald and J.M. Johnston, Biochim. Biophys. Acta, 575 0979) 135-147. 14 M. Kates, Techniques of Lipidology (Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 3, Part 2), North-Holland, Amsterdam, 1972, pp. 568-569. 15 W.F.C. Boss, C J . Kelley and F.R. Landsberger, Anal. Biochem., 64 (1975) 289 -292.
197
SYNTHESIS OF FLUORESCENT AND RADIOLABELED ANALOGUES OF PHOSPHATIDIC ACID
KENNETH J. LONGMUIRa, ONA C. MARTIN b and RICHARD E. PAGANOh
aDepartraent of Physiology and Biophysics, College o]" Medicine, University o/ CaliJornia, lrvine, CA 9271 7 and bThe Carnegie Institution of Washington, Department of l:'mbryology, 115 I~. Unirersity Parkway. Baltimore, MD 21210 (U.S.A.) Received November 21st, 1983 revision received September 281h, 1984 accepted October 2nd, 1984 Procedures tbr the synthesis of tluorescent and radiolabeled analogues of phosphatidic acid arc described. The |luorophore 7-nitrobenzo-2.-oxa-l,3-diazole (NBD) was coupled to 6-aminocaproic acid and 12-aminododet~noic acid by reaction of NBD-chloride with the amin~ acids under mild alkaline conditions at room temperature. 1,2-Dioleoyl-sn-iU-t'~('Jglycerol 3-phosphate was prepared by acylation of sn-lU-~4C]glycerol 3-phosphate with oleic acid anhydridc using dimcthylaminopyridine as the ~talyst. This compound was converted to l-oleoyl-sn[U-~4(']glycerol 3-phosphate by hydrolysis with phospholipase Aa. The lysophosphatidic acid was reacylated with NBD-aminocaproyl imidazole or NBD-aminododecanoyl imidazol~ to form the t]uorescent, radiolabeed analogue of phospbatidic acid. Fluoresoent, non-radiolabeled analogues of phosphatidic acid were prepared by phospholipasc D hydrolysis of fluorescent phosphatidylcholine.
Keywords. 7-nitrobenzo-2-oxa-l,3-diazole; fatty acid; phosphatidic add; phospholipids; acylation reactions
Introduction Phosphatidic acid is an important intermediate for the biosynthesis of glycerolipids in mammalian cells. In order to follow the intracellular localization and metabolism o f phosphatidic acid, we have prepared fluorescent analogues where the sn-2 position contains NBD linked to either 6-aminocaproic acid or 12-aminododecanoic acid. These fluorescent phosphatidic acids are taken up by mammalian cells, transported to intracellular sites o f lipid biosynthesis and converted to end l~roducts of lipid metabolism [ 1 - 3 ]. NBD-labeled phosphatidic acids are most easily obtained by phospholipase D hydrolysis of commercially available NBD-labeled phosphatidylcholine [ 1 ]. However, for part of our investigations it was necessary to prepare phosphatidic acid with Abbreviations: NBD, 7-nitrobenzo-2-oxa-l,3-diazole; NBD..CI, 4-chloro-7-nitrobenzo-2-oxa-1. 3.-diazole; NMR, nuclear magnetic resonance; Pyr, pyridinium ion; TLC, thi~-layer chromatography. 0009-3084/85/$03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
198 both an NBD label on the fatty acid chain and a radiochemical label on the glycerol backbone. Although reports have appeared concerning the synthesis ofphosphatidic acids [4--6] and NBD-labeled fatty acids [7], the methods described in the literature failed to produce these particular analogues in either significant yield or acceptable purity. Specifically, the synthesis of NBD.fatty acids resulted in the accumulation of highly colored side products which were not eliminated by the recrystallization methods described in the literature. Second, attempts to acylate lysophosphatidic acid with the acid anhydrides of NBD-fatty acids were unsuccessful. Instead. modifications of the acyl imidazole procedure were required. Third, the acylation reactions produced a variety of side products, and considerable attention was required for their eventual separation from the final product. Finally, a number of questions remained regarding yields, purities, and time courses of all the reactions utilized in the preparation of these fluorescent and radiolabeled analogues. In this communication, we describe the modifications that were required to obtain these products in acceptable yield and purity. The synthetic reactions utilized are outlined in Scheme !.
NH e -(C
He)n--COOH
NID-CI
MIIO-NH
-- (ONe)=-- C OOH
11 Pyr
O I PyrO-P==O I O I
OH I
O (
1/1
OH
OH I HO-P=O I O I H=C - -
317
( R-~-IeO
H
H;eC--
I
HO-P=O t
0 I
P Msil4~lilmse
H
At
CH t
'CH l I
OH
o
0
C:O I R
C=O ! R OH !
HO-PIO I O
NIIO--NH- (CHI)n-- ~ - N ~ rN H C -I OH
•
CH t I
O I C"O I R
t HeC - -
"9I"
II
* Ib, II
. •
¢.:-<=.=~-~.~-~¢.=:?-
H C -!
CH,, !
0 O I I C=,O C,=O
| I (tHe) n R I NH !
NBD
m) 2
Scheme 1. Outline of synthes~s of NBD-labeledfatty acids and NBD-labeledphosphatidic acids.
199
Experimental Materials l-Acyl-2-(NBD-aminododecanoyl)-sn-glycero-3-phosphocholine was obtained from Avanti Polar Lipids, Inc., Birmingham, AL. Phospholipase D (from cabbage) was obtained from Boehringer-Mannheim, Indianapolis, IN. sn-Gylcerol 3-phosphate(di(monocyclohexylammonium) salt), 6-aminocaproic acid, oleic acid anhydride, NBD-CI, and phospholipase A2 (from porcine pancreas) were obtained from Sigma, St. Louis, MO. Dimethylaminopyridine, 12-aminododecanoic acid and carbonyldiimidazole were obtained from Aldrich, Milwaukee, Wl. sn-[U-~4C]Glycerol 3phosphate (20 ,aCi/,amol) was obtained from ICN, lrvine. CA. Silica gel 60 TLC plates (250 ,am thickness) were from E. Merck, Elmsford, NY. Silica gel for column chromatography (SilicAR CC-7) was obtained from Mallinckrodt, Paris, KY. Cation exchange resin AG 50W-X8 was obtained from Bio-Rad, Richmond, CA. Scintillation grade Triton X-IO0 was obtained from Research Products International, Mount Prospect, IL. Glass-distilled chloroform, methanol, acetone and ethyl acetate were obtained from Burdick and Jackson, Muskegon, M1. Solvents were dried by stirring in molecular sieves (Type 3A, MCB, Cincinnati, OH) and filtering. All other reagents were analytical grade. General methods Chloroform/methanol extraction was accomplished using modifications [8] of the procedures of Bligh and Dyer [9]. Lipids were dissolved in 5.0 ml of chloroform/ methanol/0.2 N HCI (1:2:0.8). (All solvent mixtures are indicated by volume.) if necessary, 6 N HCI was added to maintain a pH of 1-2 and methanol was added to maintain a single phase. The solution was separated into two phases with the addition of 1.5 ml of chloroform and 1.5 ml of 2 M KCI. The mixture was centrifuged, the upper phase was discarded, and the lower phase was washed twice with 5 ml of synthetic Folch upper phase (methanol/water/chloroform, 9 6 : 9 4 : 6 [10]). The solutions were centrifuged after each wash to insure phase separation. The lower phase was brought to dryness by evaporation with N2 and the residue dissolved in a suitable solvent. Ethyl acetate/acetone extraction was accomplished following modifications [ 1l of the procedures of Slayback et al. [ 11 ]. To 1 ml of an aqueous suspension of lipid was added 1.0 ml of 0.2 N HCI, 2.0 ml of acetone and 4.0 ml of ethyl acetate. The solution was centrifuged, and the upper phase of organic solvent removed and saved. The lower phase was extracted twice more by addition of 1.6 ml of acetone and 4.0 ml of ethyl acetate. The solution was centrifuged after each extraction to insure phase separation. The combined upper phases were brought to dryness by evaporation with N2 and the lipid residue dissolved in a suitable solvent. Lipids were separated by TLC on silica gel 60 plates (250 ,am thickness) in two solvent systems. Acidic solvent system: chloroform/acetone/methanol/acetic acid/ water (10 : 4 : 2 : 2 : 1). Basic solvent system: chloroform/methanol/NH4OH ( 13 : 7 : 1).
200 Unlabeled lipids were visualized with iodine. Radiolabeled lipids were visualized by autoradiography (Kodak X-Omat-AR film). Fluorescent lipids were visualized with a 365 nm transilluminator (Spectronics Corp., Westbury, NY). Phosphorus analyses were performed using the procedures of Ames and Dubin [12]. Fluorescence measuresments were made on a Spex Fluorolog spectrofluorometer with an excitation wavelength of 470 nm and an emission wavelength of 530 ran. Elemental analyses were performed by Galbraith Laboratories, Inc., Knoxville, TN. Melting point determinations were performed using a Fisher-Johns melting point apparatus. Radiolabeled samples were prepared for scintillation counting by thoroughly drying an aliquot of sample in a scintillation vial, then adding 0.17 ml of water followed by 2.0 ml of Triton-toluene scintillation fluid [I 3], To determine the positions of the fatty acids on the glycerol backbone, fluorescent phosphatidic acid products were treated with phospholipase A2 [14]. The reaction products were extracted into ethyl acetate/acetone and separated by TLC using the acidic solvent system. The relative fluorescence in the fatty acid and lysophosphatidic acid regions of the plate were determined using a Coming model 750 scanning densitometer. Except where specified, all reactions and lipid extractions were performed in 16 X 125 mm screw-cap glass culture tubes with Teflon-lined caps.
Preparation of l-acyl-2-{NBD-aminododecanoyl)-sn-glycerol 3-phosphate using phospholipase D Fluorescent, non-radiolabeled phosphatidic acid was prepared by phospholipase D hydrolysis of fluorescent phosphatidylcholine [14]. A chloroform solution ol commercially available l-acyl-2-(N BD-aminododecanoyl)-sn-glycero-3-phosphocholine (2.3 tamol) was evaporated to dryness with N2, then dried in vacuo. The phosphatidylcholine was suspended in 1.0 ml of 0.1 M sodium acetate (pH 6.0} followed by 0.2 ml of i M CaCI2, 0.8 ml of phospholipase D (20 mg/ml) and 0.5 ml of diethyl ether. The reaction was stirred vigorously on a magnetic stirrer, Progress of the reaction was followed by TLC in the acidic solvent system. After 1 h, 8 ml of chloroform/methanol (1:2) and 0.1 mI of 6 N HCI were added. The solution was separated into two phases with 2.0 ml each of chloroform and 2 M KCI. The mixture was centrifuged, and the lower phase filtered through glass wool to remove precipitated protein. The filtrate was washed twice with 5 ml of synthetic Folch upper phase. The lower phase was evaporated to dryness with N2, dissolved in chloroform/ methanol (95.:5), applied to a TLC plate of activated silica gel 60 (250 tan thickness), and developed in the acidic solvent system. The band containing the fluorescent phosphatidic acid was scraped, and the product recovered on a Soxhlet extractor for 5 h with chloroform/methanol (1:2). The yield, by phosphorus analysis, was 84%.
Synthesis of NBD-ammododecanoic acid (H ) The reactions of NBD-CI with tile amino acids were carried out using modifica-
201
tions of the procedures of Monti et al. [7]. NaHCOa (1 retool) was dissolved in 2.5 ml of water in a 25 ml Erlenmeyer flask. To this solution was added 12-aminododecanoic acid (0.25 mmol), followed by 7.5 ml of ethanol. The fatty acid was dissolved with mild heating (some NaHCO3 precipitated at this point). After cooling to room temperature, NBD-CI (0.75 retool) was added, and the reaction was stirred in the dark at room temperature. Progress of the reaction was monitored by removing 25 /al aliquots, quenching in chloroform/methanol/0.2 N HCI (1:2:0,8), and determining the fluorescence, After 4 h, the reaction was stopped with 1.0 ml of I N HCI. Ethanol and water were added to obtain 30 ml of ethanol/water (1 : 1). The products were dissolved with heating, then crystallized overnight at -20°C. The precipitate was harvested on glass fiber filter paper, washed with cold water, and dissolved in chloroform/ methanol (2:1). The solvents were evaporated and the products recrystallized in 45 ml of ethanol/water (1:2). The recrystallized products were dissolved in 20 ml of chloroform/methanol (99:1) and applied to a column of 25 g of activated silica gel. Non-t]uorescent material was eluted with 250 ml of chloroform. Elution continued with 250 ml of chloroform/methanol (99.5:0.5) followed by 250 ml of chloroform/methanol (99 : I), at which point a fluorescent band running ahead of the major product was eluted. The NBD-aminododecanoic acid was eluted with chloroform/methanol (98 : 2), and collected in five 50-ml fractions. The first fraction of NBD-aminododecanoic acid contained a minor fluorescent impurity, and was repurified by the same silica gel column chromatography procedure. All fractions containing purified NBDaminododecanoic acid were combined, the solvents evaporated, and the product dried in vacuo and weighed (yield 37%). Melting point 95- 97°C. Elemental analysis, calc. for Cl~-12~'q4Os: C, 57.13%; H, 6.93%; N, 14.80%; found: C, 57.08%; H, 6.89%; N, 14.61%. Rf = 0.52 (basic solvent system). IH-NMR in CDCI3:8.48 ppm (d, J=9 Hz, NH-CCHCHC--NO2); 6.16 ppm (d, J=9 Hz, NH-CCHCHC-NO2); 6.34 ppm (m, NH); 3.47 ppm (q, J=7 Hz, N-CH2); 2.33 ppm (t, J=7 Hz, CH2COO): 1.8-1.3 ppm (hydrocarbon chain protons).
Synthesis of NBD-aminocaproic acid (11] NaHCO3 (2 mmol) was dissolved in 5 ml of water in a 50 ml Erlenmeyer flask. 6-Aminocaproic acid (0.5 retool) and 15 ml of ethanol were added and the fatty acid was dissolved with mild heating. The solution was cooled to room temperature, and NBD-CI (1.5 mmol) was added. The reaction was stirred for 4 h at room temperature in the dark. Progress of the reaction was followed as described above. The reaction was stopped with 2.5 ml of 1 N HCI. The solvents were evaporated and the products crystallized at -20°C in 90 ml of ethanol/water ( 1 : 2). A second crystallization was carried out in 120 ml of ethanol/water (! :3). The products were dissolved in 20 ml of chloroform/methanol (98.5:1.5), and applied to a column of 30 g of activated silica gel. Non-fluorescent material and a minor fluorescent product were eluted with 250 ml of chloroform/methanol (99.5:0.5) followed by 250 ml of chloroform/methanol (99: 1). The fluorescent fatty acid was eluted
202 with 500 ml of chloroform/methanol (98:2), followed by 200 ml of chloroform/ methanol (96:4). The fractions containing the product were combined, the solvents evaporated, and the product dried in vacuo and weighed (yield 46%). Melting point 155-156°C. Elemental analysis, calc. for Ct2Ht4N4Os: C, 48.98%; H, 4.80%; N, 19.04%; found: C, 49.02%; H, 4.80%; N, 18.90%. Rf = 0.46 (basic solvent system).
Synthesis of 1,2-dioleoyl-sn[U-14C]glycerol 3-phosphate (IV) Acylation of sn-[U34C] glycerol 3-phosphate (Ill) with oleic acid anhydride was carried out using modifications of the procedure of Gupta et al. [5]. Five g of cation exchange resin were added to a 30 cm X 1 cm chromatography column. The resin was cleaned by successive washes with 25 ml of 10 N NaOH, 100 ml of water, 25 ml of 6 N HCI, 100 ml of water, then converted to the pyridinium form with 100 ml of 2.5 M pyridine, followed by 100 ml of water. sn- [ U-14C] Glycerol 3-phosphate (I 25/aCi) and unlabeled sn-glycerol 3-phosphate were combined to obtain 25/~mol (5/~Ci//amol) in 5 ml of water. The solution was passed through the resin and collected in 10 ml of water. After evaporation of solvent, the residue was dissolved in methanol and the radioactivity determined. The methanol was evaporated, and the residue dried in vacuo over P2Os (recovery 95%). To the residue was added oleic acid anhydride (120 gmol, 2.5-fold excess), 0.2 ml of chloroform and dimethylaminopyridine (120 #mol). The reaction mixture was stirred at room temperature for 36 h. The reaction was monitored by TLC in the acidic solvent system. Radiolabeled lipids were visualized by autoradiography, and regions of the plate scraped and counted. At the end of the reaction period, the solvent was evaporated and ~eplaced with 0.5 ml of methanol/chloroform/water/pyridine, (2:1 : 1 : 1), and the reaction mixture stirred for an additional 24 h. The solvents were evaporated and the lipids isolated by extraction into chloroform/methanol. The phosphatidic acid was separated from fatty acid by column chromatography on 5 g of activated silica gel. The products were applied in chloroform, and the fatty acids eluted with 50 ml of chloroform/methanol (95 : 5), followed by 50 ml of chloroform/methanol (90: 10). Phosphatidic acid was eluted with 100 ml of chloroform/methanol (75 : 25), followed by 50 ml of chloroform/methanol (50 : 50). Column fractions were analyzed by TLC using the acidic solvent system and the lipids visualized by autoradiography, then with iodine. Fractions containing the phosphatidic acid were combined, the solvents evaporated, and the product stored in chloroform/methanol (2: 1) at -20°C (yield 63%).
Preparation of 1-oleoyl-sn-[U34C]glycerol 3-phosphate (V) Phosphatidic acid was hydrolyzed using phospholipase As [14]. 1,2-Dioleoyl-sn[U-t4C]glycerol 3-phosphate (IV) (12 tamol) was dried in vacuo to remove chloroform and methanol, then dissolved in 10 ml of diethyl ether. The reaction was started with the addition of 0.5 mg of phospholipase As in 1.0 ml of 20 mM CaCI2
203 and 0.1 M sodium borate (pH 7.0) and the solution stirred vigorously. The reaction was followed by removing lO-pl aliquots and separating the lipids by TLC in the acidic solvent system. At the end of the reaction period, the ether was evaporated and the lysophosphatidic acid was extracted into ethyl acetate]acetone. The solvents were evaporated, the residue dissolved in chloroform/methanol (95:5), then applied to a column of 5 g of silica gel as described above. Fatty acid and trace amounts of phosphatidic acid were eluted with 50 ml of chloroform/methanol (95:5), followed by 50 ml of chloroform/methanol (75:25). The product was eluted with 100 ml of chloroform• methanol (50:50). Column fractions were analyzed by TLC and the lipidsvisualized by autoradiography. Fractions containing the lysophosphatidic acid were combined, the solvents evaporated, and the product stored in chloroform/methanol (2 : 1) at -20°C (yield 59%).
Synthesis of l-oleoyl-2-(NBD-aminododeeanoyl)-sn-[U-x4C]glycerol 3-phosphate (Vl) Acylations of lysophosphatidic acid with fluorescent fatty acids were carried out using modifications of the acyl imidazole procedure [15]. Approximately 7 /amol of l-oleoyl-sn-[U-v~C] glycerol 3-phosphate (V) was dried in vacuo over P2Os. In a separate tube, NBD-aminododecanoic acid (20/Jmol) and carbonyldiimidazole (20/amol) were dissolved in 1.0 ml of dry chloroform. After 1 h at room temperature, this reaction mixture was transferred to the tube containing the lysophosphatidic acid, and the solvents evaporated. The reaction mixture was resuspended in 0.1 ml of dry chloroform and stirred for 48 h in an oil bath at 60°C. Progress of the reaction was followed by TLC in the acidic solvent system and the lipids visualized by (1) autoradiography, (2) fluorescence excitation on a 365 nm transilluminatot and (3) iodine staining. The products were extracted into ethyl acetate]acetone, dissolved in a small volume of chloroform/methanol (95:5), and applied to a silica gel 60 TLC plate (250 #m thickness) for preparative TLC in the acidic solvent system. The band containing the fluorescent phosphatidic acid was scraped, and the gel extracted four times with 5 ml of chloroform/methanol/0.2 N HCI (1:2:0.8). The products were further isolated by the chloroform]methanol extraction procedure. The final product was stored in dry tetrahydrofuran at -70°C (yield 38%). Treatment of a portion of the final product with phospholipase A2 revealed that 78% of the NBDaminododecanoic acid was acylated at position sn.2 and 22% at position sn-I.
Synthesis of 1-oleoyl-2-(NBD-aminocaproyl)-sn-[U-14C]glycerol 3.phosphate (VI ) A sample of l-oleoyl-sn-[U-14C]glycerol 3-phosphate (V) (30 /amol, 1.5 /aCi/ /amol) was dried in vacuo over P2Os. In a separate tube, NBD-aminocaproic acid (90 omol) and carbonyldiimidazole (90 /amol) were dissolved in 1.0 ml of dry tetrahydrofuran, and allowed to react at room temperature for 2 h. The reaction mixture was transferred to the tube containing the lysophosphatidic acid. The
204
solvent was evaporated and replaced with 0.5 ml of dry dichloromethane. The tube was capped tightly and the reaction mixture stirred for 48 h at 60-70°C in an oil bath. At the end of the reaction period, the fluorescent phosphatidic acid was extracted into ethyl acetate/acetone and purified by preparative TLC as described above (yield 22%). Phospholipase A2 treatment of the product revealed that 79% of the aminocaproic acid was acylated at position sn-2 and 21% at position sn-1.
Results and Discussion
Synthesis of NBD-amino acids The reaction of NBD-CI with the amino acids formed highly fluorescent products from non-fluorescent precursors. Figure I is a typical time course for the reaction of NBD-CI with 12-aminododecanoic acid. Identical results were obtained for the reaction of NBD-C1 with 6-aminocaproic acid. The reaction was essentially complete between 6 - 9 I1. However, reaction times this long resulted in the accumulation of colored, non-fluorescent reaction products which made purification of the fluorescent fatty acid difficult. For the preparation of purified fluorescent fatty acids, a reaction time of 4 h at room temperature was optimal.
!
!
l
I
1
I
I
o1.0
z 1,1.1
~.0.8 o _J It. w
0.6
t-
~0.4, 0.2
t
1
2
I
1
4
I
I
I
6
TIME, h Fig. 1. Appearance of fluorescence during the reaction of NBD-CI with 12-aminododecanoic acid. Reaction conditions are described in the Methods. Aliquots of the reaction mixture were removed at the indicated times and acidified in chloroform/methanol/0.2 N HCI ( 1 : 2 : 0 . 8 ) . Fluorescence measurements were made at hex = 470 nm and hem = 530 nm.
205 Two fluorescent products were obtained. The major product was the expected NBD-amino acid. A minor product was also isolated which on TLC in both solvent systems ran close to the solvent front. NMR analysis revealed that this minor component was the fatty acid ester. Separation of the small amount of ester from the fatty acid was achieved by stepwise elution of the silica gel column. Silica gel column chromatography was also necessary to ensure complete separation of the fluorescent product from the colored, non-fluorescent side products.
Synthesis of dioleoyl phosphatidic acid and lysophosphatidic acid As suggested by Gupta et al. [5], the reaction of sn-[U-t4C] glycerol 3-phosphate with oleic acid anhydride resulted in the formation of two major products when dimethylaminopyridine was used as the catalyst. These were the dioleoyl phosphatidic acid (Rf = 0.56, acidic solvent system) and a presumed dioleoyl phosphatidic acid-oleoyl anhydride (Rf = 0.86, acidic solvent system), where a third fatty acid forms an anhydride with the phosphate. After 18 h of reaction, TLC analysis showed 20% of the radioactivity as phosphatidic acid and 78% as the presumed phosphatidic acid-oleoyl anhydride. The phosphatidic acid-oleoyl anhydride was cleanly converted to phosphatidic acid by stirring for 24 h in a methanol/chloroform[water/pyridine solution [5]. TLC showed that virtually all of the radioactivity migrated as phosphatidic acid, while no radioactivity was detected as the phosphatidic acid-oleoyl anhydride. The greatest loss of product occurred during silica gel column chromatography. After extraction of the reaction products into organic solvent, 96% of the initial radioactivity was present, with virtually all of the radioactivity as phosphatidic acid. Only 63% of the initial radioactivity was recovered after column chromatography. Phospholipase A2 effectively hydrolyzed dioleoyl phosphatidic acid. For the reaction reported here, 98% of the initial phosphatidic acid was hydrolyzed to lysophosphatidic acid within 15 min. No significant conversion of lysophosphatidic acid to glycerol 3-phosphate was observed. Extraction of lysophosphatidic acid into solvent was achieved using the ethyl acetate/acetone procedure only. If chloroform/methanol extraction was used, the lysophosphatidic acid was lost into the aqueous upper phase. In contrast, phosphatidic acid was extracted either into chloroform/methanol or into ethyl acetate/ acetone with equal success. A substantial loss of lysophosphatidic acid occurred during silica gel column chromatography. After ethyl acetate/acetone extraction, 89% of the initial radioactivity was recovered, with virtually all of the radioactivity as lysophosphatidic acid. Following column chromatography, 59% of the initial radioactivity was recovered as lysophosphatidic acid.
Synthesis of 1-oleoyl-2-{NBD-aminoacyl)-sn-[U34C]glycerol 3-phosphates At first we attempted to synthesize NBD-labeled analogues of phosphatidic acid by acylation of lysophosphatidic acid with NBD-labeled fatty acid anhydrides.
206 Yields of phosphatidic acids from this procedure were negligible. As a result, all further acylations of lysophosphatidic acid were carried out using the acyl imidazole procedure [15]. Acylations with NBD-aminododecanoyl imidazole were carried out in chloroform. Acylations with NBD-aminocaproyl imidazole could be carried out only with dichloromethane as the solvent. The reactions o f l-oleoyl-sn-[U-t4C] glycerol 3-phosphate with the imidazolides of NBD-aminododecanoic and NBD-aminocaproic acid each resulted in the formation of two major products. The first was the expected fluorescent analogue of phosphatidic acid. The second product was a radiolabeled but non-fluorescent compound which, in the acidic solvent system, had an Rf-value slightly less than the fluorescent phosphatidic acids. Because this compound was cleanly separated from the fluorescent product at the end of the reaction, no attempt was made to further characterize it. However, Lapidot et al. [4] have reported that some acylation reactions result in the formation of significant quantities of cyclic lysophosphatidic acid. This compound, in various TLC solvent systems, migrates with an Rf-value slightly less than the corresponding phosphatidic acid. Table 1 lists the time course of the distribution of products obtained during the acylation of l-oleoyl-sn-[U-14C]glycerol 3-phosphate with NBD-aminododecanoyl imidazole. The reaction was essentially complete after 48 h, as indicated by the small percentage of starting material that remained. In addition to the fluorescent product and the presumed cyclic lysophosphatidic acid, some fluorescent phospha-
TABLE I TIME COURSE OF REACTION OF I-OLEOYL-~n-IU-14C]GLYCEROL3-PHOSPHATE WiTH NBD-AMINODODECANOYL IMIDAZOLE I-Oleoyi-sn-lU-14Clglycerol 3-phosphate was reacted with a 3-fold excess of NBD-aminododecanoyl imidazole for 48 h in an oil bath at 60°C. The reaction mixture was analyzed by TLC in the acidic solvent system. Lipids were visualized by auloradiography then counted. Lipid (ROa
3h (%)
24 h (%)
48 h ~%)
1-Oleoyl-sn-IU-vlC]glycerol 3-phosphate (0.19) i -Oleoyl-2-(NBD-aminodode~ noyl)sn-[ U-t4CIglycerol 3-phosphate (0.45) 1-Oleoyl-sn-[U-14C]:glycerol 2,3-cyclic phosphate (0.35) b Phosphatidic acid acyl anhydride (0.62) c
66
18
6
25
52
53
8
23
29
2
8
12
aRf-value determined by TLC in the acidic solvent system. bTentative identification of cyclic lysophosphatidic acid is based upon the work of Lapidot et al. {4 I. CTentative identification of phosphatidic acid-acyl anhydride is based upon the work of Gupta ct~.
[Sl.
207
tidic acid-acyl anhydride was also detected. The Rt-values for the various compounds produced during the acylation of 1-oleoyl-sn-[U-t4C]glycerol 3-phosphate with NBD-aminododecanoyl imidazole are listed in Table I. The Revalue of 1-oleoyl-2(NBD-aminocaproyl)-sn-[U-t4C] glycerol 3-phosphate was 0.39 in the acidic solvent system. Separation of the fluorescent product from the presumed cyclic lysophosphatidic acid was accomplished by preparative TLC on silica gel 60, followed by extraction of the gel with the chloroform/methanol extraction procedure. In our experience, lysophosphatidic acids are lost into the upper phase during this extraction procedure. TLC analysis after extraction showed a single spot corresponding to the fluorescent, radiolabeled analogue of phosphatidic acid. Non-fluorescent, radiolabeled side products were not detected after these purification procedures were performed. The principal shortcoming of the procedures described here was that about 20% of the final product contained NBD-fatty acid at position sn-1 rather than at the desired sn-2 position. This was likely due to the rather vigorous acylation procedures required to obtain NBD-labeled phosphatidic acids in acceptable yields. Further research is needed to find milder reaction conditions which can acylate glycerophosphates with the unusual fatty acids that are becoming increasingly useful for contemporary biochemical research.
Acknowledgements We wish to thank Aiisen Sykes and Christine Resele-Tiden for their technical assistance. This work was supported by USPHS grant GM-22942 (R.E.P.), USPHS grant HL-28619 (K.J.L.), and a USPHS Pulmonary Faculty Training Award HL00456 (K.J.L.).
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