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Phospholipid synthesis: effects of solvents and catalysts on acylation
Chemistry and Physics of Lipids, 48 (1988) 99--108 Elsevier Scientific Publishers Ireland Ltd.
99
Phospholipid synthesis: effects of solvents and catalysts on acylation D e v M a n g r o o a n d G e r h a r d E. G e r b e r Department of Biochemistry, McMaster University, Hamilton, Ontario L8N 3Z5 (Canada) (Received December llth, 1987; revised and accepted February 29th, 1988)
The peptide dinitrophenylprolylthreoninamide (DNP-Pro-Thr-NH2) was used as a model system to develop better acylation conditions for the synthesis of phospholipids using catalyst-activated anhydrides. The acylation rate was found to be inversely related to the polarity of the solvent, chloroform alone resulting in much better rates of reaction than did pyridine, dimethylformamide (DMF) or mixtures of these solvents. Anhydride activated by 4-pyrrofidinopyridine (PPY) was twice as reactive as that activated by 4-dimethylaminopyridine (DMAP). It was shown that the phosphate group of phosphatidylcholine (PC) interferes with the acylation by a process which could be reversed by means of the addition of a 200-fold excess of PPY. This reversal is not due to base catalysis by the PPY; the results suggest that a mixed anhydride may be formed with the phosphate and that this can be reversed by high catalyst concentrations to produce the reactive acylating agent. The acylation rates for lysophosphatidylcholine (lyso PC) using our optimum conditions were found to be approximately 50 times faster than the best rates reported in the literature, the reaction being complete within 5 rain even using only a slight excess of anhydride. Acyl group migration was assessed during these reactions and no increase in migration of the acyl groups could be detected due to these reaction conditions. The procedures described provide significant improvements over previous methods described for large scale, as well as highly radioactive microscale phospholipid synthesis.
Keywords: phospholipid synthesis; solvent and catalyst effects.
Introduction
The synthesis of phospholipids involves acylating glycero-3-phosphorylcholine (as the CdCI 2 complex) or 1-acyl-glycero-3-phosphorylcholine with fatty acid anhydride [1], fatty acid imidazolide [2--4], fatty acid chloride [5], fatty acidtrifluoroacetic anhydride [6] or 2-thiopyridyl fatty acid ester [7]. However, owing to the low reactivity of the secondary hydroxyl group of 1acyl-glycero-3-phosphorylcholine, long reaction times are required and the yields are often very low. A variety of vigorous conditions such as high temperature [2,5] and strong bases [3,4] and a large excess of the fatty acid derivatives have been used to carry out the reaction. Methods which avoid the use of such extreme conditions have also been published and involve the use of catalysts [8--11]. Although in some cases
high yields (70--90~0) have been reported, these could not always be reproduced as suggested by the large number of alternate published procedures. This was found especially so for small scale preparation of highly radioactive phospholipids and photoreactive phospholipid probes where only limiting amounts of valuable fatty acids were available. A systematic evaluation of the different parameters involved in phospholipid synthesis was therefore undertaken in order to determine the nature of the problems and hence to develop an approach which would allow more reproducible preparations of phospholipids in general. This systematic analysis was performed with the DNP-peptide, DNP-Pro-Thr-NH 2 as a model system. The dinitrophenyl chromophore can be monitored at 365 run; the peptide's secondary hydroxyl group is analogous to that of 1-acyl-
0009-3084/88/$03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
100 glycero-3-phosphorylcholine; however, unlike 1acyl-glycero-3-phosphorylcholine, the DNPpeptide does not contain other reactive groups. Conditions that affect the rate of acylation at the secondary hydroxyl group could thus be evaluated quantitatively without interference from other reactive groups. The reaction conditions for the acylation were optimized using this model system and an improved synthesis of phospholipids using these conditions is described.
Materials and methods Palmitic acid, N,N-carbonyldiimidazole (CDI), 1,2-dipalmitoyl-glycero-3-phosphorycholine (DPPC), dicyclohexylcarhodiimide, 4-dimethyiaminopyridine and 4-pyrrolidinopyridine were purchased from Sigma Chemical Co. [3HlPalmitic acid (30 Ci/mmol) was obtained from New England Nuclear and triethylamine from Pierce. 1-Palmitoyl-glycero-3-phosphorylcholine and palmitoyl chloride were purchased from Serdary. Reagent grade dimethylsulfoxide and dimethylformamide (DMF) were dried by the addition of 0.1 vol. of dry benzene and evaporation of 0.2 vol. under reduced pressure. These anhydrous solvents were then stored over 4-A molecular sieves and under nitrogen. All other anhydrous solvents were prepared by distillation under nitrogen followed by storage over 4-A molecular sieve and under nitrogen. Pyridine was refluxed with barium oxide for 2 h prior to distillation. Chloroform was shaken several times with 0.5 vol. of glass distilled water, dried with calcium chloride, then distilled from phosphorous pentoxide and stored under nitrogen at - 2 0 ° C in the dark. Tetrahydrofuran was distilled from calcium hydride. Carbon tetrachloride was dried by distillation from phosphorous pentoxide. Primary or secondary amine contaminants of triethylamine were removed by distillation of the tertiary base from ninhydrin under nitrogen. The pure triethylamine was stored over 4-A moleculax sieves, under nitrogen and at 4°C in the dark. The catalyst, 4-pyrrolidinopyridine, was puri-
fled by a modified procedure of Manson et ai. [11]; 4-pyrrolidinopyridine dissolved in a minimum volume of chloroform was extracted into petroleum ether. The ether extracts were combined, and after the solvent was evaporated under reduced pressure, the resulting residue was dissolved in chloroform and the extraction repeated. This process was continued until the catalyst was completely freed of all yellowish contaminants. PPY containing small quantities of these contaminants was found to cause a significant reduction in the rate of acylation and variable yields of the desired product. Pure 4-dimethylarninopyridine was obtained by recrystallization as described [8]. Palmitic acid, 1-palmitoyl-glycero-3-phosphorylclioline, 1,2-dipalmitoyl-glycero-3 -phosphorylcholine, pure 4-pyrrolidinopyridine and 4dimethylaminopyridine were freed of residual water by repeated addition of dry benzene and evaporation under reduced pressure.
Preparation of fatty acid imidazolide The fatty acid imidazolide was prepared [12] by transferring 20/~nol of the fatty acid (1 M in dry DMF) to a flamed pyrex screw cap tube containing 1.5 equiv, of CDI (0.2 M) in 15% DMF in benzene. The vessel was flushed with nitrogen and the reaction was allowed to proceed for 1 h at room temperature. The imidazolide was then diluted with dry benzene to 40 mM and transferred to another flamed pyrex tube containing 4 equivalents of dry Sephadex LH-20 having a CDI binding capacity of 1.15/zrnol per rag. The tube was flushed with nitrogen and the content was rotated for 1 h at room temperature; centrifugation removed the Sephadex LH-20 and afforded the imidazolide free of excess CDI.
Preparation of fatty acid anhydride Preparation of fatty acid anhydride was accomplished by reacting 0.5 equiv, of dicyclohexylcarbodiimide (0.2 M in dry CCI 4) with fatty acid (0.4 M in dry CCI 4) for 5 h at room temperature and under nitrogen [13]. After removal of the dicyclohexylurea precipitate
I01
by filtration, the solvent was evaporated under reduced pressure and the dried residue dissolved by adding the required volume of dry benzene to afford a 40 mM anhydride solution.
Preparation of 1,2-diacyl-glycero-3-phosphorylcholine To a siliconized pyrex screw cap tube containing an anhydrous residue of glycero-3-phosphorylcholine (as the CdCI 2 complex) (54 banol) was added pH]palmitic acid anhydride (185 hanoi, spec. act. 3.24 Ci/mol) and 40 equiv of PPY (4 M in dry ChC13). After the total volume was adjusted to 1.1 ml with dry chloroform and the reaction vessel was flushed with nitrogen, the reaction was allowed to proceed at room temperature in the dark. Upon completion of the reaction (as judged by TLC analysis) an equal volume of aqueous methanol (10070 H20 in MeOH, v/v) was added and after some time the solvent was removed under reduced pressure. The dried residue was dissolved by adding 500 ~1 CHCI3/MeOH (I:1, v/v) and then applied to a preparative TLC plate. The plate was developed with CHC13/MeOH/H20 (65:25:4, by vol.) and the products were visualized by autoradiography. 1,2-Diacyl-glycero-3-phosphorylcholine was eluted with CHCI3/MeOH/H20 (1: 2:0.8, by vol.) and further purified by gel filtration on a Sephadex LH-20 column (1.0 × 76 cm) pre-equilibrated with CHCI3/MeOH (1 : 1, v/v). Yield of 1,2-diacyl-glycero-3-phosphorylcholine exceeded 90070, based on radioactivity.
Preparation of 1-acyl-glycero-3-phosphorylcholine 1,2-Di- pH]palmitoyl-glycero-3-phosphorylcholine (7.6 ~anol, spec. act. 1.1 Ci/mol) prepared as above, was suspended in 98 pl of 10 mM HEPES (pH 7.5) and sonicated under nitrogen for 15 min at room temperature. To this mixture, 2 bd of 1 M CaCI 2 and 100/A phospholipase A~ (1 mg/ml) were added. After 5 min, 960 pl of CHC13/McOH/H20 (1:1:0.4, by vol.) were added [14] and the chloroform phase subjected to gel filtration on Sephadex LH-20 as described above.
Reacylation of 1-acyl-glycero-3-phosphorylcholine To an anhydrous residue of l-[3H]palmitoylglycero-3-phosphorylcholine (12.9 banol, spec. act. 85 mCi/mmol) was added 100 /~1 of dry CHC13 and 1.1 equivalents of trifluoroacetic acid. After the 1-acyl-glycero-3-phosphorylcholine was solubilized as the trifluoroacetate salt, the solvent was evaporated with a stream of nitrogen. The resulting oil was further dried for 10 min under reduced pressure and dissolved by adding 129 IA dry CHCI 3. To dried palmitic acid anhydride (9.0 ~mol), 60 pl of 4 M 4-pyrrolidinopyridine was added, followed by 60/A of 100 mM 1-acyl-glycero-3-phosphorylcholine (as the trifluoroacetate salt). After the content was mixed and the vessel flushed with nitrogen, the reaction was allowed to proceed at room temperature in the dark. Ten minutes after the reaction was initiated, the reaction mixture was diluted to 320/d with 10070 H20 in methanol and applied onto a Sephadex LH-20 column (1.0 × 76 cm) pre-eqnilibrated with CHCI3/MeOH (1:1, v/v). The yield of 1,2-diacyl-glycero-3-phosphorylcholine, based on radioactivity, was greater than 90070.
Large scale synthesis: 1-palmitoyl-2-oleoyl-3-glycerophosphorylcholine To an anhydrous residue of 1-palmitoyl-glycero-3-phosphorylcholine (1.0 mmol) was added 50 ml of dry CHCI 3 and 1.1 equiv, of anhydrous trifluoroacetic acid. The solvent was removed under reduced pressure and the resulting oil was redissolved in CHC13 (10 ml) and added to a chloroform solution (10.0 ml) containing oleoyl anhydride (2.0 mmol) and PPY (2.0 mmol). The reaction was allowed to proceed to completion (judged by TLC analysis; 2--3 h) in the dark at room temperature. After removal of the solvent under reduced pressure, the residue was purified by chromatography on Sephadex LH-20 as described above. The yield of isolated 1-palmitoyl-2-oleoyl-glycero-3-phosphorylcholine typically exceeded 900/0.
1o2 Synthesis of DNP-prolylthreoninamide
Calculation of rate constant
D N P - P r o - T h r - N H 2 was prepared as described [15]. Purification o f the DNP-peptide was achieved by H P L C using isocratic elution o f a ~Bondapak ODS column with 15% methanol and 1% acetic acid. The D N P - P r o - T h r - N H 2 was rendered anhydrous by repeated addition o f dry benzene and evaporation under reduced pressure and was stored as a 2 m M solution in anhydrous chloroform under a nitrogen atmosphere.
Rate constants for the model peptide reactions were calculated according to the pseudofirst-order equation outlined below: k k
"-
app
[ANHYDRIDE] --
tkapp = [A]t
[A]o
"-
[peptide] at time t
31P-NMR spectroscopy
[A]t
T o dried palmitic acid anhydride (0--375 /anol) was added 3 4 / ~ n o l o f 1,2-diacyl-glycero3-phosphorylcholine (200 m M in dry CHCI3). The content was diluted with chloroform to 3.4 ml, and left at room temperature under nitrogen and in the dark. After 4 h, the reaction mixture was transferred to a s~P-NMR sample tube under nitrogen, and 3ZP-NMR spectral data were recorded at 100 M H z on a Bruker WM250 spectrometer in the Fourier transform mode. The sample was given a 45 ° pulse every 15 ~ and spectra were accumulated over a sweep width o f 20 KHz. The temperature o f the probe was kept constant at 25 °C by a Bruker temperature controller. Chemical shift values were recorded relative to 85% phosphoric acid (high frequency as positive).
[A] o = initial [peptide]
Results and discussion
The catalysts D M A P [8,9] and P P Y [10,11] greatly enhance the reactivity o f fatty acid anhydrides. Attempts to prepare highly radioactive photoreactive phospholipids probes using these I
100
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HPLC analysis of model peptide reaction The progress o f fatty acid acylation o f the model peptide, DNP-Pro-Thr-NH2, was routinely analyzed by H P L C , using a MilliporeWaters system equipped with two M-60 pumps, a 720 system controller, a M-730 data module, a WISP 710B automatic injector and a 440 absorbance detector. At specific times an aliquot o f the reaction was diluted into aqueous DMF (10% H20 in DMF) and applied onto a 4.6 x 150 mm ~Bondapak ODS column Pre-equilibrated with 15% methanol in 10 m M sodium acetate (pH 4.4). The column was eluted with a linear methanol gradient and the peptides detected by monitoring the effluent at 365 nm.
+< 40 e~ -J m_ ~ 20
0
2
4 TIME (hr)
6
Fig. 1. Comparison of the reactJvities of different fatty acid acylating reagents. DNP-Pro-Thr-NH, (50 nmol), in the presence of 25 ~anol of dry triethylamlne, was acylated with 5 ~mol of palmitoyl chloride (A), palmitoylanhydride (e) or palmitoyl imidazofide( I ) in 25 ~1 of dry chloroform. At the times indicated, aliquots were diluted into 10% water in DMF and analyzed by HPLC as described in Materials and methods.
103
catalysts resulted in poor yields; increasing the ratio of lysoPC to anhydride resulted in even poorer yields and the reactions proceeded much more slowly (results not shown). A systematic study of the conditions that affect the reactivity of catalyst-activated anhydride was therefore undertaken. The peptide, DNP-Pro-Thr-NH 2 was chosen as a model system and the rate of acylation monitored by HPLC. The only reactive group in the model peptide is a secondary hydroxyl group which should have a reactivity roughly comparable to that of the secondary hydroxyl group of lysoPC. The model peptide thus allowed the rapid quantitative monitoring of the reaction in the absence of complicating side reactions with other reactive groups as would be the case for lysoPC. The reactivity of commonly used acylating agents are compared in Fig. 1. As expected, the
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acid chloride is the most reactive, being considerably more reactive than the uncatalyzed anhydride, while the imidazolide is the least reactive. This is consistent with the literature which reports the need for strong base [3,4,16] or elevated temperature [2,17] for reaction with the latter reagent. It should be noted that the addition of optimal catalyst concentration to the anhydride resulted in a rate which was much faster than that of the acid chloride in this solvent, and these could therefore not be plotted on the same graph. The high reactivity of the catalyzed anhydride acylation thus makes this the reagent of choice and this was therefore used for all further studies. The effect of solvent on the rates of acylation of DNP-Pro-Thr-NH, was studied (Fig. 2) with palmitic acid anhydride activated with 0.02 equiv, of DMAP and the acylation carried out in chloroform, chloroform/pyridine (4: 1, v/v),
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Fig. 2. Effects of solvent on the rate of acylation of DNPPro-Thr-NH 2. Paimitic acid anhydride (1.25 p~nol), dried as described in Materials and methods, was dissolved in 10 ~1 of pyridine ( I ) , CHCl3/pyridine (4:1) (A), C H C I / D M F (1 : 1) (O), or CHCI 3 ( e ) . DMAP (0.025 pmol) in 5 ~1 of the same solvent was added and the reactions were initiated by adding 10 ~1 of a solution of the peptide (125 ~M) in the same solvent. At the times indicated, aliquots were diluted into 10V0 H20 in DMF and analyzed by HPLC.
o
6 0
2
~ 4
~
~_ 6
TIME (min)
Fig. 3. Effect of catalysts on the rate of acylation of DNPPro-Thr-NH 2. Dry chloroform (160 ~1) was added to 8 ~mol of dried palmitic acid anhydride. After the addition of 20 ~1 of dry CHCI 3 (O), 50 mM PPY in dry CHC13 (A) or 50 mM DMAP in dry CHCI 3 ( e ) , the reactions were initiated by adding 20 ~1 of a solution of the peptide (52.5 /AM) in dry CHCI 3. At the times indicated, aliquots were diluted into 10°/0 H20 in DMF and analyzed by HPLC.
104 pyridine and dimethylformamide/chloroform (1: 1, v/v). In chloroform, the reaction was completed within 30 min, whereas in c h l o r o f o r m / pyridine (4: 1, v/v), pyridine and D M F / c h l o r o form ( 1 : I , v/v), the reaction was only 60~/0, 10070 and 5°7o completed, respectively in this time period. It was apparent f r o m the dependence o f the initial rates o f the reactions on the polarity o f the solvents that the rates were inversely related to the polarity o f the solvent. These findings are consistent with similar reports in the literature [18,19]. Since the reactivity o f the catalyst-activated anhydride was highest in chloroform, this solvent was used for all further reactions. It has been reported [18] that PPY-activated acetic acid anhydride was at least two times more reactive than the corresponding DMAPactivated anhydride. Shown in Fig. 3 is a comparison o f the rates o f acylation o f DNP-ProThr-NH z with fatty acid anhydride activated with D M A P or P P Y in chloroform. In the absence o f either catalysts no acylation of the DNP-peptide was observed within 6 rain. However, when the fatty acid anhydride was activated with D M A P or P P Y the rate o f acylation o f D N P - P r o - T h r - N H z was significantly enhanced. Calculation o f the initial rate constants for the catalyzed and uncatalyzed reactions revealed that the catalyst-activated anhydrides were approximately 1000 times more reactive than the anhydride alone. Although the DMAP-activated anhydride was considerably more reactive than the anhydride alone, it was approximately one half as reactive as the PPYactivated anhydride; the latter was therefore used for all further studies. It has been shown using IH-NMR spectroscopy [18] that the enhanced reactivity o f the anhydride in the presence o f the catalysts, D M A P or PPY, is due to the conversion of the anhydride to a very reactive intermediate. It was demonstrated that the reaction between the catalyst and the anhydride to give the reactive intermediate is an equilibrium dependent process; this implies that the rate of acylation is dependent on the position o f this equilibrium. As shown in Fig. 4, as the molar ratio o f P P Y to
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Fig. 4. Effect of catalyst concentration on the rate of acylation of DNP-Pro-Thr-NHz. Palmitic acid anhydride (1/amol) was dissolved in chloroform and the appropriate amount of PPY in dry chloroform was added. The volume was adjusted to 80/al with chloroform and the reactions were initiated by the addition of 20 ~1 of a solution of the peptide (46.5 w'Vl) in dry CHCI3. At 30, 60 and 90 s, aliquots were diluted into 10% H20 in DMF and analyzed by HPLC. The rate constant at each ratio of PPY to anhydride was calculated as described in Materials and methods. fatty acid anhydride was increased, the rate of acylation o f the DNP-peptide also increased. A molar ratio of 10 was required to attain the maximum rate o f acylation and presumably at this point the anhydride had been completely converted to the reactive intermediate. Preliminary studies (data not shown) indicated that an increase in the concentration o f lysoPC during the acylation reaction resulted in an acylation rate which was greatly reduced rather than increased as would be expected for a bimolecular reaction. The presence o f impurities and water was excluded (data not shown) as possible explanations for this surprising result. It thus seemed that the phosphorylcholine group itself was interfering with the reaction. The acylation o f D N P - P r o - T h r - N H 2 with completely catalyst-activated anhydride (PPY to anhydride
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p h o s p h a t e g r o u p as i n d i c a t e d b y t h e f o l l o w i n g experiments. The interaction expected to occur between the p h o s p h a t e a n d t h e r e a g e n t is reversible a c y l a t i o n o f t h e p h o s p h a t e g r o u p . It s h o u l d b e p o s s i b l e to c o n v e r t such a m i x e d a n h y d r i d e b a c k to the d e s i r e d reactive i n t e r m e d i a t e b y i n c r e a s i n g t h e c o n c e n t r a t i o n o f P P Y ; t h e d e s i r e d 1-acyl-4-pyrr o l i d i n o p y r i d i n i u m i n t e r m e d i a t e s h o u l d b e regen e r a t e d since t h e b e t t e r l e a v i n g g r o u p in a m i x e d a n h y d r i d e is e x p e c t e d t o be t h e s t r o n g e r a c i d [20]. T h e a c y l a t i o n o f D N P - P r o - T h r - N H 2 was t h e r e f o r e p e r f o r m e d with e q u i m o l a r a m o u n t s o f the a n h y d r i d e a n d 1,2-diacyl-glycero-3-phosp h o r y l c h o l i n e in t h e presence o f i n c r e a s i n g a m o u n t s o f P P Y . A s s h o w n in Fig. 6, as t h e
MOLAR RATIO (DPPC/ANHYDRIDE) Fig. 5. Effect of phosphatidylcholine concentration on the rate of acylation of DNP-Pro-Thr-NH2. A solution of PPY (10 pmol in 20/~1 of dry CHCI3) was added to a solution of palmitic acid anhydride (1/~[nol in 20 ~1 of dry chloroform). The indicated amounts of DPPC were added and the total volume adjusted to 80 pl with dry chloroform. The reaction was initiated by the addition of 20 ~1 of a solution of the peptide (31.5 /~l in dry CHCI3). At 30, 90 and 150 s, aliquots were diluted into 10% H20 in DMF and analyzed by HPLC. The rate constants were calculated as described in Materials and methods and e~pressed as a percentage of the rate constant obtained in a control reaction in the absence of DPPC.
r a t i o o f 10) in t h e p r e s e n c e o f v a r y i n g a m o u n t s of 1 , 2 - d i a c y l - g i y c e r o - 3 - p h o s p h o r y l c h o l i n e was t h e r e f o r e studied; t h e use o f P C i n s t e a d o f l y s o P C p e r m i t t e d e v a l u a t i o n o f t h e effect o f the phosphorylcholine group on the rate of acylation of the DNP-peptide without the complication of t h e presence o f o t h e r reactive g r o u p s . A s i l l u s t r a t e d in Fig. 5, as t h e r a t i o o f 1,2diacyl-giycero-3-phosphorylcholine to anhydride was i n c r e a s e d , t h e r a t e o f a c y l a t i o n o f D N P P r o - T h r - N H 2 d e c r e a s e d . A t a 1,2-diacyl-glycero3 - p h o s p h o r y l c h o l i n e t o a n h y d r i d e r a t i o o f 1, o n l y 15% o f t h e o r i g i n a l r a t e was o b t a i n e d . It is t h u s clear t h a t t h e p h o s p h o r y l c h o l i n e g r o u p d o e s i n t e r f e r e with t h e a c y l a t i o n o f t h e h y d r o x y l g r o u p . This loss o f r e a c t i v i t y is s h o w n b e l o w t o be reversible a n d m o s t likely involves the
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MOLAR RATIO (X/DPPC) Fig. 6. Effect of basic catalysts on the rate of acylation of DNP-Pro-Thr-NH2 in the presence of diacylphosphatidylcholine. A solution of palmitic acid anhydride (1 /~mol in 20 /d of dry chloroform) was prepared as described in Materials and methods. The indicated amounts of PPY ( • ) or triethylamine and 10/~mol of PPY (O) were added and the volume was adjusted to 60 /d with chloroform. A solution of DPPC (1 I~mol in 20 ~1 of dry CHCI3) were added and the reaction was initiated by the addition of 20 ~1 of a solution of the peptide (31.5 pM) in dry CHCIr At 30, 60 and 90 s, aliquots were diluted into 10~/0 H20 in DMF and analyzed by HPLC. The rate constants were calculated as described in Materials and methods.
106 ratio of the catalyst to 1,2-diacyl-giycero-3phosphorylcholine was increased, the rate of acylation of the DNP-peptide also increased. At a molar ratio of 180, the original rate of acylation was restored. These results are consistent with the reversible formation of a mixed anhydride since high concentrations of the catalyst restored the original rate observed in the absence of 1,2-diacyl-giycero-3-phosphorylcholine. It is thus clear that a small catalytic amount of catalyst is insufficient and that a large excess of catalyst is required to obtain the maximal rate; even the addition of 1 equiv, of catalyst used by others for phospholipid synthesis [8] results in less than 2% of the maximal acylation rate. In order to determine the extent to which the
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(PPY/TBA-CI) Fig. 7. Effect of increasing amounts of PPY on the rate of acylation of DNP-Pro-Thr-NH2 with palmitic acid anhydride in the presence of tetrabutylammonium chloride. To a reaction tube containing palmitic acid anhydride (1 tanol) tetrabutylnmmonium chloride (1 equiv, of 0.1 M in CHCI 3) and varying amounts of PPY (0.25 M in CHCI~) were added. The volume was adjusted to 80 ~1 with dry chloroform and the reaction was initiated by the addition of 20 ~1 of a solution of the peptide (31.5 ~ in dry CHCI 3. At 30, 60 and 120 s, aliquots were diluted into 10% H20 in DMF and analyzed by HPLC. The rate constants were calculated as described in Materials and methods.
restoration in the rate of acylation of the model peptide is due to general base catalysis, by the large amounts (200-fold excess) of catalyst, the acylation of DNP-Pro-Thr-NH 2 was performed using completely catalyst-activated anhydride, in the presence of equimolar amounts of 1,2-diacyl-giycero-3-phosphorylcholine, and varying amounts of triethylamine. As shown in Fig. 6, essentially no change in the rate constant was observed as the ratio of triethylamine to 1,2diacyl-giycero-3-phosphorylcholine was increased to 200. These results exclude base catalysis as a possible explanation for the observed restoration of the acylation rate. In view of our finding that the reactivity of the catalyzed reaction is reduced by an increase in the polarity of the solvent, the effect of the quaternary ammonium group of phosphatidylcholine on the polarity of the solvent was evaluated. The effect of the addition of 1 equiv. of tetrabutylammonium chloride on the reaction rate with the model peptide was determined. This resulted in a reduction in the rate of reaction (Fig. 7); however, this reaction exhibited optimal activity at a PPY/salt ratio of 10; addition of higher concentrations of PPY failed to further increase the rate of reaction. Thus, although the increase in polarity due to the addition of a quaternary ammonium salt does result in some reduction in the rate of reaction, this cannot be reversed by the addition of high concentrations of catalyst. These results are suggestive of the formation of a less reactive mixed anhydride with the phosphate of phosphatidylcholine and that this can be reversed by the addition of high concentrations of catalyst. Attempts to obtain direct evidence for such a mixed anhydride using 31p. NMR failed (data not shown). The chemical shift observed for the phosphate was very sensitive to the presence of the organic bases added in the reaction; further, no reaction with the anhydride was observed in the absence of these bases. Although the results obtained failed to demonstrate the putative mixed anhydride, the results presented in Figs. 5--7 would nonetheless be best explained by its reversible formation. The acylation conditions derived from the
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6 30
T I M E (mln)
Fig. 8. The effect of catalyst concentration on the rate of acylation of lysophosphatidylcholine. Solutions of [3H]palmitic acid anhydride(1.88/~mol, specific activity,2.3 Ci/mol) containing0 (O), 2.5 (11), 25 (A) or 50 (o) tanol of PPY was used to acylatelysophosphatidylcholine(1.25 gmol) prepared as described in Materials and methods as the trifluoroacetate salt in a final volume of 25 ~ of chloroform. At the times indicated, aliquots were diluted into 10e/ewater in methanoland analyzedby TLC usingCHCI~/MEOH/H20 (65:25:4, by vol.) and the radioactivity visualized and quantitated as describedin Materialsand methods.
model peptide work were used to acylate l-palmitoyl-glycero-3-phosphorylcholine with [3H]palmitic acid anhydride activated with different amounts of PPY. The progress of each reaction was monitored by TLC, the radioactive products visualized by fluorography, and the amount of 1,2-diacyi-glycero-3-phosphorylcholine product determined by assaying the radioactivity in each spot. As illustrated in Fig. 8, as the PPY to 1acyl-glycero~3-phosphorylcholine ratio was increased, the time required to obtain 100% theoretical yield of 1,2-diacyl-glycero-3-phosphorylcholine decreased. Under acylation conditions which had been optimal for the model peptides, the maximum rate of acylation of 1-acyl-glycero3-phosphorylcholine was also observed; a 100o70 theoretical yield of 1,2-diacyl-glycero-3-phosphorylcholine was obtained in 5 rain. The use of high concentrations of catalyst permits rapid completion of reactions in microscale syntheses. In larger scale syntheses of phospholipids, the use of such a large amount of catalyst may be impractical; however, reducing
the concentration of catalyst results in a reduction in the rate which can be only partly compensated for by increasing the reaction time; this is illustrated by the fact that acyiation using 1.5 equiv, of fatty acid anhydride in the presence of low catalyst concentration (2 equiv.) resulted in the reaction becoming very slow at 50O70 yield (c.f. Fig. 8, m). This is presumably due to low concentrations of acylating agent resulting from competing acylation of the phosphate and can be overcome by using at least 2 equiv, of anhydride when using suboptimal catalyst concentrations as recommended in the Methods for large scale synthesis; thus the latter procedure results in essentially quantitative yield in 2--3 h using 2 equiv, of each of the catalyst and anhydride. It has recently been shown [21] that 1-acylglycero-3-phosphorylcholine exposed to the alkaline pH under which the phospholipase A 2 digestion is performed resulted in an equilibrium mixture containing 90o70 of the 1-acyl and 10% of the 2-acyl isomer and we have confirmed this for our samples of lysoPC. Analysis of phospholipids synthesized by our procedure by phospholipase A 2 treatment (data not shown) was consistent with our lysoPC containing 10% of the 2-acyl isomer. Acylation of lysoPC using either low or high concentrations (1 or 200 equiv. respectively) of catalyst resulted in 10% acylation at the 1 position and 90o70 at the 2 position. This is as expected for the equilibrium mixture of the lysoPC and consequently our interpretation is that we do not get acyl migration due to our acylation conditions; this is consistent with the low rate constant obtained by the above authors for acyl migration in chloroform, and the high rate constant we report for the acylation under our conditions. The positional purity of the product is thus limited by the purity of the lysoPC starting material.
Acknowledgements This investigation was supported by grants to G.E. Gerber by the Medical Research Council of Canada.
108
References 1 C. Robles and D. Van Den Berg (1969) Biochim. Biophys. Acta 187, 57.0---526. 2 W.F. Boss, C.J. Kelley and F.R. Landsberger (1975) Anal. Biochem. 64, 289--292. 3 T.G. Warner and A.A. Benson (1977) J. Lipid Res. 18, 548--552. 4 A. Hermetter and F. Paltauf (1981) Chem. Phys. Lipids 28, 111--115. 5 E. Baer and D. Buchane (1959) Can. J. Biochem. Physio. 37, 953--959. 6 E.L. Pugh and M. Kates (1975) J. Lipid. Res. 16, 392 --394. 7 A.W. Nicholas, L.G. Khouri, J.C. Ellington and N.A. Porter (1983) Lipids 18, 434--438. 8 C.M. Gupta, R. Radhakrishnan and H.G. Khorana (1977) Proc. Natl. Acad. Sci. 74, 4315--4319. 9 K.M. Patel, J.D. Morrisett and J.T. Sparrow (1977) J. Lipid Res. 20, 674--677. 10 B. Perly, E.J. Duforc and H.C. Jaffell (1983) J. Labelled Compounds Radiopharmaceuticals 21, 1--13.
11 J.T. Manson, A.V. Broccoli and C.H. Huan8 (1981) Anal. Biochem. 113, 96--101. 12 R.C. Morton, D. Mangroo and G.E. Gerber (1988) Can. J. Chem., in press. 13 Z. Sellnger and Y. Lapidot (1966) J. Lipid Res. 7, 174 --175. 14 E.G. Bligh and W.J. Dyer (1959) Can. J. Biocbem. Physiol. 37, 911--917. 15 R.C. Morton and G.E. Gerber (1988) J. Biol. Chem., in press. 16 A. Hermetter, H. Stutz and F. Paltauf (1983) Chem. Phys. Lipids 32, 145--152. 17 K.J. Longmuir, O.C. Martin and R.E. Pagano (1985) Chem. Phys. Lipids 36, 197--207. 18 G. Hofle, W. Stegfich and H. Vorbruggen (1978) Angew. Chem. 17, 569--583. 19 E. Guibe-Jampel, G. LeCorre and M. Wakselman (1979) Tetrabedron Lett. 13, 1157--1160. 20 J.M. Tedder (1955) Chem. Rev. 55,787--827. 21 A. Plunkthun and A.E. Dennis (1982) Biochemistry 21, 1743--1750.
99
Phospholipid synthesis: effects of solvents and catalysts on acylation D e v M a n g r o o a n d G e r h a r d E. G e r b e r Department of Biochemistry, McMaster University, Hamilton, Ontario L8N 3Z5 (Canada) (Received December llth, 1987; revised and accepted February 29th, 1988)
The peptide dinitrophenylprolylthreoninamide (DNP-Pro-Thr-NH2) was used as a model system to develop better acylation conditions for the synthesis of phospholipids using catalyst-activated anhydrides. The acylation rate was found to be inversely related to the polarity of the solvent, chloroform alone resulting in much better rates of reaction than did pyridine, dimethylformamide (DMF) or mixtures of these solvents. Anhydride activated by 4-pyrrofidinopyridine (PPY) was twice as reactive as that activated by 4-dimethylaminopyridine (DMAP). It was shown that the phosphate group of phosphatidylcholine (PC) interferes with the acylation by a process which could be reversed by means of the addition of a 200-fold excess of PPY. This reversal is not due to base catalysis by the PPY; the results suggest that a mixed anhydride may be formed with the phosphate and that this can be reversed by high catalyst concentrations to produce the reactive acylating agent. The acylation rates for lysophosphatidylcholine (lyso PC) using our optimum conditions were found to be approximately 50 times faster than the best rates reported in the literature, the reaction being complete within 5 rain even using only a slight excess of anhydride. Acyl group migration was assessed during these reactions and no increase in migration of the acyl groups could be detected due to these reaction conditions. The procedures described provide significant improvements over previous methods described for large scale, as well as highly radioactive microscale phospholipid synthesis.
Keywords: phospholipid synthesis; solvent and catalyst effects.
Introduction
The synthesis of phospholipids involves acylating glycero-3-phosphorylcholine (as the CdCI 2 complex) or 1-acyl-glycero-3-phosphorylcholine with fatty acid anhydride [1], fatty acid imidazolide [2--4], fatty acid chloride [5], fatty acidtrifluoroacetic anhydride [6] or 2-thiopyridyl fatty acid ester [7]. However, owing to the low reactivity of the secondary hydroxyl group of 1acyl-glycero-3-phosphorylcholine, long reaction times are required and the yields are often very low. A variety of vigorous conditions such as high temperature [2,5] and strong bases [3,4] and a large excess of the fatty acid derivatives have been used to carry out the reaction. Methods which avoid the use of such extreme conditions have also been published and involve the use of catalysts [8--11]. Although in some cases
high yields (70--90~0) have been reported, these could not always be reproduced as suggested by the large number of alternate published procedures. This was found especially so for small scale preparation of highly radioactive phospholipids and photoreactive phospholipid probes where only limiting amounts of valuable fatty acids were available. A systematic evaluation of the different parameters involved in phospholipid synthesis was therefore undertaken in order to determine the nature of the problems and hence to develop an approach which would allow more reproducible preparations of phospholipids in general. This systematic analysis was performed with the DNP-peptide, DNP-Pro-Thr-NH 2 as a model system. The dinitrophenyl chromophore can be monitored at 365 run; the peptide's secondary hydroxyl group is analogous to that of 1-acyl-
0009-3084/88/$03.50 © 1988 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
100 glycero-3-phosphorylcholine; however, unlike 1acyl-glycero-3-phosphorylcholine, the DNPpeptide does not contain other reactive groups. Conditions that affect the rate of acylation at the secondary hydroxyl group could thus be evaluated quantitatively without interference from other reactive groups. The reaction conditions for the acylation were optimized using this model system and an improved synthesis of phospholipids using these conditions is described.
Materials and methods Palmitic acid, N,N-carbonyldiimidazole (CDI), 1,2-dipalmitoyl-glycero-3-phosphorycholine (DPPC), dicyclohexylcarhodiimide, 4-dimethyiaminopyridine and 4-pyrrolidinopyridine were purchased from Sigma Chemical Co. [3HlPalmitic acid (30 Ci/mmol) was obtained from New England Nuclear and triethylamine from Pierce. 1-Palmitoyl-glycero-3-phosphorylcholine and palmitoyl chloride were purchased from Serdary. Reagent grade dimethylsulfoxide and dimethylformamide (DMF) were dried by the addition of 0.1 vol. of dry benzene and evaporation of 0.2 vol. under reduced pressure. These anhydrous solvents were then stored over 4-A molecular sieves and under nitrogen. All other anhydrous solvents were prepared by distillation under nitrogen followed by storage over 4-A molecular sieve and under nitrogen. Pyridine was refluxed with barium oxide for 2 h prior to distillation. Chloroform was shaken several times with 0.5 vol. of glass distilled water, dried with calcium chloride, then distilled from phosphorous pentoxide and stored under nitrogen at - 2 0 ° C in the dark. Tetrahydrofuran was distilled from calcium hydride. Carbon tetrachloride was dried by distillation from phosphorous pentoxide. Primary or secondary amine contaminants of triethylamine were removed by distillation of the tertiary base from ninhydrin under nitrogen. The pure triethylamine was stored over 4-A moleculax sieves, under nitrogen and at 4°C in the dark. The catalyst, 4-pyrrolidinopyridine, was puri-
fled by a modified procedure of Manson et ai. [11]; 4-pyrrolidinopyridine dissolved in a minimum volume of chloroform was extracted into petroleum ether. The ether extracts were combined, and after the solvent was evaporated under reduced pressure, the resulting residue was dissolved in chloroform and the extraction repeated. This process was continued until the catalyst was completely freed of all yellowish contaminants. PPY containing small quantities of these contaminants was found to cause a significant reduction in the rate of acylation and variable yields of the desired product. Pure 4-dimethylarninopyridine was obtained by recrystallization as described [8]. Palmitic acid, 1-palmitoyl-glycero-3-phosphorylclioline, 1,2-dipalmitoyl-glycero-3 -phosphorylcholine, pure 4-pyrrolidinopyridine and 4dimethylaminopyridine were freed of residual water by repeated addition of dry benzene and evaporation under reduced pressure.
Preparation of fatty acid imidazolide The fatty acid imidazolide was prepared [12] by transferring 20/~nol of the fatty acid (1 M in dry DMF) to a flamed pyrex screw cap tube containing 1.5 equiv, of CDI (0.2 M) in 15% DMF in benzene. The vessel was flushed with nitrogen and the reaction was allowed to proceed for 1 h at room temperature. The imidazolide was then diluted with dry benzene to 40 mM and transferred to another flamed pyrex tube containing 4 equivalents of dry Sephadex LH-20 having a CDI binding capacity of 1.15/zrnol per rag. The tube was flushed with nitrogen and the content was rotated for 1 h at room temperature; centrifugation removed the Sephadex LH-20 and afforded the imidazolide free of excess CDI.
Preparation of fatty acid anhydride Preparation of fatty acid anhydride was accomplished by reacting 0.5 equiv, of dicyclohexylcarbodiimide (0.2 M in dry CCI 4) with fatty acid (0.4 M in dry CCI 4) for 5 h at room temperature and under nitrogen [13]. After removal of the dicyclohexylurea precipitate
I01
by filtration, the solvent was evaporated under reduced pressure and the dried residue dissolved by adding the required volume of dry benzene to afford a 40 mM anhydride solution.
Preparation of 1,2-diacyl-glycero-3-phosphorylcholine To a siliconized pyrex screw cap tube containing an anhydrous residue of glycero-3-phosphorylcholine (as the CdCI 2 complex) (54 banol) was added pH]palmitic acid anhydride (185 hanoi, spec. act. 3.24 Ci/mol) and 40 equiv of PPY (4 M in dry ChC13). After the total volume was adjusted to 1.1 ml with dry chloroform and the reaction vessel was flushed with nitrogen, the reaction was allowed to proceed at room temperature in the dark. Upon completion of the reaction (as judged by TLC analysis) an equal volume of aqueous methanol (10070 H20 in MeOH, v/v) was added and after some time the solvent was removed under reduced pressure. The dried residue was dissolved by adding 500 ~1 CHCI3/MeOH (I:1, v/v) and then applied to a preparative TLC plate. The plate was developed with CHC13/MeOH/H20 (65:25:4, by vol.) and the products were visualized by autoradiography. 1,2-Diacyl-glycero-3-phosphorylcholine was eluted with CHCI3/MeOH/H20 (1: 2:0.8, by vol.) and further purified by gel filtration on a Sephadex LH-20 column (1.0 × 76 cm) pre-equilibrated with CHCI3/MeOH (1 : 1, v/v). Yield of 1,2-diacyl-glycero-3-phosphorylcholine exceeded 90070, based on radioactivity.
Preparation of 1-acyl-glycero-3-phosphorylcholine 1,2-Di- pH]palmitoyl-glycero-3-phosphorylcholine (7.6 ~anol, spec. act. 1.1 Ci/mol) prepared as above, was suspended in 98 pl of 10 mM HEPES (pH 7.5) and sonicated under nitrogen for 15 min at room temperature. To this mixture, 2 bd of 1 M CaCI 2 and 100/A phospholipase A~ (1 mg/ml) were added. After 5 min, 960 pl of CHC13/McOH/H20 (1:1:0.4, by vol.) were added [14] and the chloroform phase subjected to gel filtration on Sephadex LH-20 as described above.
Reacylation of 1-acyl-glycero-3-phosphorylcholine To an anhydrous residue of l-[3H]palmitoylglycero-3-phosphorylcholine (12.9 banol, spec. act. 85 mCi/mmol) was added 100 /~1 of dry CHC13 and 1.1 equivalents of trifluoroacetic acid. After the 1-acyl-glycero-3-phosphorylcholine was solubilized as the trifluoroacetate salt, the solvent was evaporated with a stream of nitrogen. The resulting oil was further dried for 10 min under reduced pressure and dissolved by adding 129 IA dry CHCI 3. To dried palmitic acid anhydride (9.0 ~mol), 60 pl of 4 M 4-pyrrolidinopyridine was added, followed by 60/A of 100 mM 1-acyl-glycero-3-phosphorylcholine (as the trifluoroacetate salt). After the content was mixed and the vessel flushed with nitrogen, the reaction was allowed to proceed at room temperature in the dark. Ten minutes after the reaction was initiated, the reaction mixture was diluted to 320/d with 10070 H20 in methanol and applied onto a Sephadex LH-20 column (1.0 × 76 cm) pre-eqnilibrated with CHCI3/MeOH (1:1, v/v). The yield of 1,2-diacyl-glycero-3-phosphorylcholine, based on radioactivity, was greater than 90070.
Large scale synthesis: 1-palmitoyl-2-oleoyl-3-glycerophosphorylcholine To an anhydrous residue of 1-palmitoyl-glycero-3-phosphorylcholine (1.0 mmol) was added 50 ml of dry CHCI 3 and 1.1 equiv, of anhydrous trifluoroacetic acid. The solvent was removed under reduced pressure and the resulting oil was redissolved in CHC13 (10 ml) and added to a chloroform solution (10.0 ml) containing oleoyl anhydride (2.0 mmol) and PPY (2.0 mmol). The reaction was allowed to proceed to completion (judged by TLC analysis; 2--3 h) in the dark at room temperature. After removal of the solvent under reduced pressure, the residue was purified by chromatography on Sephadex LH-20 as described above. The yield of isolated 1-palmitoyl-2-oleoyl-glycero-3-phosphorylcholine typically exceeded 900/0.
1o2 Synthesis of DNP-prolylthreoninamide
Calculation of rate constant
D N P - P r o - T h r - N H 2 was prepared as described [15]. Purification o f the DNP-peptide was achieved by H P L C using isocratic elution o f a ~Bondapak ODS column with 15% methanol and 1% acetic acid. The D N P - P r o - T h r - N H 2 was rendered anhydrous by repeated addition o f dry benzene and evaporation under reduced pressure and was stored as a 2 m M solution in anhydrous chloroform under a nitrogen atmosphere.
Rate constants for the model peptide reactions were calculated according to the pseudofirst-order equation outlined below: k k
"-
app
[ANHYDRIDE] --
tkapp = [A]t
[A]o
"-
[peptide] at time t
31P-NMR spectroscopy
[A]t
T o dried palmitic acid anhydride (0--375 /anol) was added 3 4 / ~ n o l o f 1,2-diacyl-glycero3-phosphorylcholine (200 m M in dry CHCI3). The content was diluted with chloroform to 3.4 ml, and left at room temperature under nitrogen and in the dark. After 4 h, the reaction mixture was transferred to a s~P-NMR sample tube under nitrogen, and 3ZP-NMR spectral data were recorded at 100 M H z on a Bruker WM250 spectrometer in the Fourier transform mode. The sample was given a 45 ° pulse every 15 ~ and spectra were accumulated over a sweep width o f 20 KHz. The temperature o f the probe was kept constant at 25 °C by a Bruker temperature controller. Chemical shift values were recorded relative to 85% phosphoric acid (high frequency as positive).
[A] o = initial [peptide]
Results and discussion
The catalysts D M A P [8,9] and P P Y [10,11] greatly enhance the reactivity o f fatty acid anhydrides. Attempts to prepare highly radioactive photoreactive phospholipids probes using these I
100
I
~
I
I
~-
I
I
.L
80 t~
i +o
HPLC analysis of model peptide reaction The progress o f fatty acid acylation o f the model peptide, DNP-Pro-Thr-NH2, was routinely analyzed by H P L C , using a MilliporeWaters system equipped with two M-60 pumps, a 720 system controller, a M-730 data module, a WISP 710B automatic injector and a 440 absorbance detector. At specific times an aliquot o f the reaction was diluted into aqueous DMF (10% H20 in DMF) and applied onto a 4.6 x 150 mm ~Bondapak ODS column Pre-equilibrated with 15% methanol in 10 m M sodium acetate (pH 4.4). The column was eluted with a linear methanol gradient and the peptides detected by monitoring the effluent at 365 nm.
+< 40 e~ -J m_ ~ 20
0
2
4 TIME (hr)
6
Fig. 1. Comparison of the reactJvities of different fatty acid acylating reagents. DNP-Pro-Thr-NH, (50 nmol), in the presence of 25 ~anol of dry triethylamlne, was acylated with 5 ~mol of palmitoyl chloride (A), palmitoylanhydride (e) or palmitoyl imidazofide( I ) in 25 ~1 of dry chloroform. At the times indicated, aliquots were diluted into 10% water in DMF and analyzed by HPLC as described in Materials and methods.
103
catalysts resulted in poor yields; increasing the ratio of lysoPC to anhydride resulted in even poorer yields and the reactions proceeded much more slowly (results not shown). A systematic study of the conditions that affect the reactivity of catalyst-activated anhydride was therefore undertaken. The peptide, DNP-Pro-Thr-NH 2 was chosen as a model system and the rate of acylation monitored by HPLC. The only reactive group in the model peptide is a secondary hydroxyl group which should have a reactivity roughly comparable to that of the secondary hydroxyl group of lysoPC. The model peptide thus allowed the rapid quantitative monitoring of the reaction in the absence of complicating side reactions with other reactive groups as would be the case for lysoPC. The reactivity of commonly used acylating agents are compared in Fig. 1. As expected, the
I
-~
I
I
I
I
I
I
100
100
8O
-~ 8 o .2
.2 o
acid chloride is the most reactive, being considerably more reactive than the uncatalyzed anhydride, while the imidazolide is the least reactive. This is consistent with the literature which reports the need for strong base [3,4,16] or elevated temperature [2,17] for reaction with the latter reagent. It should be noted that the addition of optimal catalyst concentration to the anhydride resulted in a rate which was much faster than that of the acid chloride in this solvent, and these could therefore not be plotted on the same graph. The high reactivity of the catalyzed anhydride acylation thus makes this the reagent of choice and this was therefore used for all further studies. The effect of solvent on the rates of acylation of DNP-Pro-Thr-NH, was studied (Fig. 2) with palmitic acid anhydride activated with 0.02 equiv, of DMAP and the acylation carried out in chloroform, chloroform/pyridine (4: 1, v/v),
60
o
60
"" 40 ¢3 ..I
'-" _1
40
>.
>.
o
0 ,,1¢
20
0
I
I
l
i
I
I
I
20
0
10 20 T I M E (rain)
30
Fig. 2. Effects of solvent on the rate of acylation of DNPPro-Thr-NH 2. Paimitic acid anhydride (1.25 p~nol), dried as described in Materials and methods, was dissolved in 10 ~1 of pyridine ( I ) , CHCl3/pyridine (4:1) (A), C H C I / D M F (1 : 1) (O), or CHCI 3 ( e ) . DMAP (0.025 pmol) in 5 ~1 of the same solvent was added and the reactions were initiated by adding 10 ~1 of a solution of the peptide (125 ~M) in the same solvent. At the times indicated, aliquots were diluted into 10V0 H20 in DMF and analyzed by HPLC.
o
6 0
2
~ 4
~
~_ 6
TIME (min)
Fig. 3. Effect of catalysts on the rate of acylation of DNPPro-Thr-NH 2. Dry chloroform (160 ~1) was added to 8 ~mol of dried palmitic acid anhydride. After the addition of 20 ~1 of dry CHCI 3 (O), 50 mM PPY in dry CHC13 (A) or 50 mM DMAP in dry CHCI 3 ( e ) , the reactions were initiated by adding 20 ~1 of a solution of the peptide (52.5 /AM) in dry CHCI 3. At the times indicated, aliquots were diluted into 10°/0 H20 in DMF and analyzed by HPLC.
104 pyridine and dimethylformamide/chloroform (1: 1, v/v). In chloroform, the reaction was completed within 30 min, whereas in c h l o r o f o r m / pyridine (4: 1, v/v), pyridine and D M F / c h l o r o form ( 1 : I , v/v), the reaction was only 60~/0, 10070 and 5°7o completed, respectively in this time period. It was apparent f r o m the dependence o f the initial rates o f the reactions on the polarity o f the solvents that the rates were inversely related to the polarity o f the solvent. These findings are consistent with similar reports in the literature [18,19]. Since the reactivity o f the catalyst-activated anhydride was highest in chloroform, this solvent was used for all further reactions. It has been reported [18] that PPY-activated acetic acid anhydride was at least two times more reactive than the corresponding DMAPactivated anhydride. Shown in Fig. 3 is a comparison o f the rates o f acylation o f DNP-ProThr-NH z with fatty acid anhydride activated with D M A P or P P Y in chloroform. In the absence o f either catalysts no acylation of the DNP-peptide was observed within 6 rain. However, when the fatty acid anhydride was activated with D M A P or P P Y the rate o f acylation o f D N P - P r o - T h r - N H z was significantly enhanced. Calculation o f the initial rate constants for the catalyzed and uncatalyzed reactions revealed that the catalyst-activated anhydrides were approximately 1000 times more reactive than the anhydride alone. Although the DMAP-activated anhydride was considerably more reactive than the anhydride alone, it was approximately one half as reactive as the PPYactivated anhydride; the latter was therefore used for all further studies. It has been shown using IH-NMR spectroscopy [18] that the enhanced reactivity o f the anhydride in the presence o f the catalysts, D M A P or PPY, is due to the conversion of the anhydride to a very reactive intermediate. It was demonstrated that the reaction between the catalyst and the anhydride to give the reactive intermediate is an equilibrium dependent process; this implies that the rate of acylation is dependent on the position o f this equilibrium. As shown in Fig. 4, as the molar ratio o f P P Y to
I
I
I
I
I
~
I
5.0
x" I
4.0
I.-
3.0
z
z
2.0
~
1.0
0 0 w
0
1 0
I [ I I [ 4 8 12 M O L A R RATIO (PPY/ANHYDRIDE)
I
Fig. 4. Effect of catalyst concentration on the rate of acylation of DNP-Pro-Thr-NHz. Palmitic acid anhydride (1/amol) was dissolved in chloroform and the appropriate amount of PPY in dry chloroform was added. The volume was adjusted to 80/al with chloroform and the reactions were initiated by the addition of 20 ~1 of a solution of the peptide (46.5 w'Vl) in dry CHCI3. At 30, 60 and 90 s, aliquots were diluted into 10% H20 in DMF and analyzed by HPLC. The rate constant at each ratio of PPY to anhydride was calculated as described in Materials and methods. fatty acid anhydride was increased, the rate of acylation o f the DNP-peptide also increased. A molar ratio of 10 was required to attain the maximum rate o f acylation and presumably at this point the anhydride had been completely converted to the reactive intermediate. Preliminary studies (data not shown) indicated that an increase in the concentration o f lysoPC during the acylation reaction resulted in an acylation rate which was greatly reduced rather than increased as would be expected for a bimolecular reaction. The presence o f impurities and water was excluded (data not shown) as possible explanations for this surprising result. It thus seemed that the phosphorylcholine group itself was interfering with the reaction. The acylation o f D N P - P r o - T h r - N H 2 with completely catalyst-activated anhydride (PPY to anhydride
105 I
I
I
I
100 An t~ 0
80 0 0
Iz ~
z 0 ul
40
00
1
2
3
4
p h o s p h a t e g r o u p as i n d i c a t e d b y t h e f o l l o w i n g experiments. The interaction expected to occur between the p h o s p h a t e a n d t h e r e a g e n t is reversible a c y l a t i o n o f t h e p h o s p h a t e g r o u p . It s h o u l d b e p o s s i b l e to c o n v e r t such a m i x e d a n h y d r i d e b a c k to the d e s i r e d reactive i n t e r m e d i a t e b y i n c r e a s i n g t h e c o n c e n t r a t i o n o f P P Y ; t h e d e s i r e d 1-acyl-4-pyrr o l i d i n o p y r i d i n i u m i n t e r m e d i a t e s h o u l d b e regen e r a t e d since t h e b e t t e r l e a v i n g g r o u p in a m i x e d a n h y d r i d e is e x p e c t e d t o be t h e s t r o n g e r a c i d [20]. T h e a c y l a t i o n o f D N P - P r o - T h r - N H 2 was t h e r e f o r e p e r f o r m e d with e q u i m o l a r a m o u n t s o f the a n h y d r i d e a n d 1,2-diacyl-glycero-3-phosp h o r y l c h o l i n e in t h e presence o f i n c r e a s i n g a m o u n t s o f P P Y . A s s h o w n in Fig. 6, as t h e
MOLAR RATIO (DPPC/ANHYDRIDE) Fig. 5. Effect of phosphatidylcholine concentration on the rate of acylation of DNP-Pro-Thr-NH2. A solution of PPY (10 pmol in 20/~1 of dry CHCI3) was added to a solution of palmitic acid anhydride (1/~[nol in 20 ~1 of dry chloroform). The indicated amounts of DPPC were added and the total volume adjusted to 80 pl with dry chloroform. The reaction was initiated by the addition of 20 ~1 of a solution of the peptide (31.5 /~l in dry CHCI3). At 30, 90 and 150 s, aliquots were diluted into 10% H20 in DMF and analyzed by HPLC. The rate constants were calculated as described in Materials and methods and e~pressed as a percentage of the rate constant obtained in a control reaction in the absence of DPPC.
r a t i o o f 10) in t h e p r e s e n c e o f v a r y i n g a m o u n t s of 1 , 2 - d i a c y l - g i y c e r o - 3 - p h o s p h o r y l c h o l i n e was t h e r e f o r e studied; t h e use o f P C i n s t e a d o f l y s o P C p e r m i t t e d e v a l u a t i o n o f t h e effect o f the phosphorylcholine group on the rate of acylation of the DNP-peptide without the complication of t h e presence o f o t h e r reactive g r o u p s . A s i l l u s t r a t e d in Fig. 5, as t h e r a t i o o f 1,2diacyl-giycero-3-phosphorylcholine to anhydride was i n c r e a s e d , t h e r a t e o f a c y l a t i o n o f D N P P r o - T h r - N H 2 d e c r e a s e d . A t a 1,2-diacyl-glycero3 - p h o s p h o r y l c h o l i n e t o a n h y d r i d e r a t i o o f 1, o n l y 15% o f t h e o r i g i n a l r a t e was o b t a i n e d . It is t h u s clear t h a t t h e p h o s p h o r y l c h o l i n e g r o u p d o e s i n t e r f e r e with t h e a c y l a t i o n o f t h e h y d r o x y l g r o u p . This loss o f r e a c t i v i t y is s h o w n b e l o w t o be reversible a n d m o s t likely involves the
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MOLAR RATIO (X/DPPC) Fig. 6. Effect of basic catalysts on the rate of acylation of DNP-Pro-Thr-NH2 in the presence of diacylphosphatidylcholine. A solution of palmitic acid anhydride (1 /~mol in 20 /d of dry chloroform) was prepared as described in Materials and methods. The indicated amounts of PPY ( • ) or triethylamine and 10/~mol of PPY (O) were added and the volume was adjusted to 60 /d with chloroform. A solution of DPPC (1 I~mol in 20 ~1 of dry CHCI3) were added and the reaction was initiated by the addition of 20 ~1 of a solution of the peptide (31.5 pM) in dry CHCIr At 30, 60 and 90 s, aliquots were diluted into 10~/0 H20 in DMF and analyzed by HPLC. The rate constants were calculated as described in Materials and methods.
106 ratio of the catalyst to 1,2-diacyl-giycero-3phosphorylcholine was increased, the rate of acylation of the DNP-peptide also increased. At a molar ratio of 180, the original rate of acylation was restored. These results are consistent with the reversible formation of a mixed anhydride since high concentrations of the catalyst restored the original rate observed in the absence of 1,2-diacyl-giycero-3-phosphorylcholine. It is thus clear that a small catalytic amount of catalyst is insufficient and that a large excess of catalyst is required to obtain the maximal rate; even the addition of 1 equiv, of catalyst used by others for phospholipid synthesis [8] results in less than 2% of the maximal acylation rate. In order to determine the extent to which the
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(PPY/TBA-CI) Fig. 7. Effect of increasing amounts of PPY on the rate of acylation of DNP-Pro-Thr-NH2 with palmitic acid anhydride in the presence of tetrabutylammonium chloride. To a reaction tube containing palmitic acid anhydride (1 tanol) tetrabutylnmmonium chloride (1 equiv, of 0.1 M in CHCI 3) and varying amounts of PPY (0.25 M in CHCI~) were added. The volume was adjusted to 80 ~1 with dry chloroform and the reaction was initiated by the addition of 20 ~1 of a solution of the peptide (31.5 ~ in dry CHCI 3. At 30, 60 and 120 s, aliquots were diluted into 10% H20 in DMF and analyzed by HPLC. The rate constants were calculated as described in Materials and methods.
restoration in the rate of acylation of the model peptide is due to general base catalysis, by the large amounts (200-fold excess) of catalyst, the acylation of DNP-Pro-Thr-NH 2 was performed using completely catalyst-activated anhydride, in the presence of equimolar amounts of 1,2-diacyl-giycero-3-phosphorylcholine, and varying amounts of triethylamine. As shown in Fig. 6, essentially no change in the rate constant was observed as the ratio of triethylamine to 1,2diacyl-giycero-3-phosphorylcholine was increased to 200. These results exclude base catalysis as a possible explanation for the observed restoration of the acylation rate. In view of our finding that the reactivity of the catalyzed reaction is reduced by an increase in the polarity of the solvent, the effect of the quaternary ammonium group of phosphatidylcholine on the polarity of the solvent was evaluated. The effect of the addition of 1 equiv. of tetrabutylammonium chloride on the reaction rate with the model peptide was determined. This resulted in a reduction in the rate of reaction (Fig. 7); however, this reaction exhibited optimal activity at a PPY/salt ratio of 10; addition of higher concentrations of PPY failed to further increase the rate of reaction. Thus, although the increase in polarity due to the addition of a quaternary ammonium salt does result in some reduction in the rate of reaction, this cannot be reversed by the addition of high concentrations of catalyst. These results are suggestive of the formation of a less reactive mixed anhydride with the phosphate of phosphatidylcholine and that this can be reversed by the addition of high concentrations of catalyst. Attempts to obtain direct evidence for such a mixed anhydride using 31p. NMR failed (data not shown). The chemical shift observed for the phosphate was very sensitive to the presence of the organic bases added in the reaction; further, no reaction with the anhydride was observed in the absence of these bases. Although the results obtained failed to demonstrate the putative mixed anhydride, the results presented in Figs. 5--7 would nonetheless be best explained by its reversible formation. The acylation conditions derived from the
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Fig. 8. The effect of catalyst concentration on the rate of acylation of lysophosphatidylcholine. Solutions of [3H]palmitic acid anhydride(1.88/~mol, specific activity,2.3 Ci/mol) containing0 (O), 2.5 (11), 25 (A) or 50 (o) tanol of PPY was used to acylatelysophosphatidylcholine(1.25 gmol) prepared as described in Materials and methods as the trifluoroacetate salt in a final volume of 25 ~ of chloroform. At the times indicated, aliquots were diluted into 10e/ewater in methanoland analyzedby TLC usingCHCI~/MEOH/H20 (65:25:4, by vol.) and the radioactivity visualized and quantitated as describedin Materialsand methods.
model peptide work were used to acylate l-palmitoyl-glycero-3-phosphorylcholine with [3H]palmitic acid anhydride activated with different amounts of PPY. The progress of each reaction was monitored by TLC, the radioactive products visualized by fluorography, and the amount of 1,2-diacyi-glycero-3-phosphorylcholine product determined by assaying the radioactivity in each spot. As illustrated in Fig. 8, as the PPY to 1acyl-glycero~3-phosphorylcholine ratio was increased, the time required to obtain 100% theoretical yield of 1,2-diacyl-glycero-3-phosphorylcholine decreased. Under acylation conditions which had been optimal for the model peptides, the maximum rate of acylation of 1-acyl-glycero3-phosphorylcholine was also observed; a 100o70 theoretical yield of 1,2-diacyl-glycero-3-phosphorylcholine was obtained in 5 rain. The use of high concentrations of catalyst permits rapid completion of reactions in microscale syntheses. In larger scale syntheses of phospholipids, the use of such a large amount of catalyst may be impractical; however, reducing
the concentration of catalyst results in a reduction in the rate which can be only partly compensated for by increasing the reaction time; this is illustrated by the fact that acyiation using 1.5 equiv, of fatty acid anhydride in the presence of low catalyst concentration (2 equiv.) resulted in the reaction becoming very slow at 50O70 yield (c.f. Fig. 8, m). This is presumably due to low concentrations of acylating agent resulting from competing acylation of the phosphate and can be overcome by using at least 2 equiv, of anhydride when using suboptimal catalyst concentrations as recommended in the Methods for large scale synthesis; thus the latter procedure results in essentially quantitative yield in 2--3 h using 2 equiv, of each of the catalyst and anhydride. It has recently been shown [21] that 1-acylglycero-3-phosphorylcholine exposed to the alkaline pH under which the phospholipase A 2 digestion is performed resulted in an equilibrium mixture containing 90o70 of the 1-acyl and 10% of the 2-acyl isomer and we have confirmed this for our samples of lysoPC. Analysis of phospholipids synthesized by our procedure by phospholipase A 2 treatment (data not shown) was consistent with our lysoPC containing 10% of the 2-acyl isomer. Acylation of lysoPC using either low or high concentrations (1 or 200 equiv. respectively) of catalyst resulted in 10% acylation at the 1 position and 90o70 at the 2 position. This is as expected for the equilibrium mixture of the lysoPC and consequently our interpretation is that we do not get acyl migration due to our acylation conditions; this is consistent with the low rate constant obtained by the above authors for acyl migration in chloroform, and the high rate constant we report for the acylation under our conditions. The positional purity of the product is thus limited by the purity of the lysoPC starting material.
Acknowledgements This investigation was supported by grants to G.E. Gerber by the Medical Research Council of Canada.
108
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