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Synthesis and characterization of a radioiodinated, photoreactive and physiologically active analogue of platelet activating factor
Chemistry and Physics of Lipids, 37 (1985) 215-226 Elsevier Scientific Publishers Ireland Ltd.
215
SYNTHESIS AND CHARACTERIZATION OF A RADIOIODINATED, PHOTOREACTIVE AND PHYSIOLOGICALLY ACTIVE ANALOGUE OF PLATELET ACTIVATING FACTOR
P. BI';TTE-BOBILLOa, A. BIENVENUEa,*, C. BROQUETb and L. MAURINa aLaboratoire de Biologie Physico-Chirnique, place E. Bataillon. 34060, Montpellier Cedes and bLaboratoire de Pharmacochimie Moldculaire, Universitd Paris VII, 2 Place Jussieu, 75005 Paris (France) Received January 7th, 1 9 8 5 accepted March 29th, 1985
revision received March 25th, 1985
The multistep synthesis of a photoreactive, radioactive and aggregating analogue of plateletactivating factor (PAF)-acether is described. The photoreactive and radioactive moiety was added at the last step; the specific radioactivity was higher than 1000 Ci/mmol. The concentration of this new analogue which causes 50% of aggregation of platelets were of the same order of magnitude as for synthetic snPAF-acether, so as for two other analogues having a bulky group at the co end of the fatty ether chain. The photoreactivity was proved by the covalent binding of the analogue to protein (BSA) after 10-min irradiation times at 300 nm. The binding was largely prevented by prior (not by later) addition of a high concentration of lyso phosphatidyl choline.
Key words: ether linked phospholipids; azido and radioiodinated analogues of platelet-activating factor; chemical synthesis; platelet aggregation dose; photoaffinity.
Introduction Platelet-activating factor (PAF-acether) is a mediator of many physiological responses, such as anaphylaxis, inflammation, platelet aggregation and hypotension [ 1]. Its structure is now well known: it is a glycerophospholipid with a choline polar head group, bound to a large hydrophobic tail ( 1 6 - 1 8 carbon atoms) by an ether bond at position 1 and to an acetyl group at position 2. It is generally recognized that only the R(sn) enantiomer is physiologically active [2]. The best means for detecting this product is the rabbit platelet aggregation test [3] which exhibits a very high efficiency with 1-hexadecyl compound: a concentration of 1 0 - 1 1 - 1 0 -~° M was sufficient to provoke a full aggregation of platelets. Many analogues of endogenous PAF-acether molecules have been synthesized by different authors after the first rehable method was published by Godfroid et al. [4].
*To whom correspondence should be sent. Abbreviations: BSA, bovine serum albumin; DMSO, dimethyl sulfoxide; HPLC, high performance liquid chromatography; PAl:, platelet-activating factor; SDS-PAGE,electrophoresis with 0.2% sodium dodecyl sulphate on polyacrylamide gel 10%; THF, tetrahydrofuran; TLC, thin-layer chromatography. 0009-3084/85/$03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
216 The substituents at position 1,2 or 3 were modified and there was a general agreement to the following rules: (a) The length of the chain at position 1 is not critical between 10 and 20 carbon atoms, on condition that it is bound to the glycerol backbone by an ether bond [5]. (b) The group at position 2 is very important: an increase in the chain length of the ester drastically decreases the activity [5]. However a short chain bound by an ether [2] or a carbamate bond [6] has an excellent activity, sometimes higher than the native compound. (c) The phosphoryl choline group also seems indispensable [7]. An aggregation dose on the order of nM raises the possibility of a receptor-mediated process and some papers have been devoted to this point. By using acetyl tritiated analogues of PAF-acether, many authors found that this molecule binds with high affinity to different cells [8-11] and to plasma membranes [12]. It is thus very likely that certain proteins in many cells and organs can bind PAFacether or lyso PAF-acether. One of the best method for studying this binding and for characterizing the putative 'receptor(s)', is the affinity labeling [13] since it can demonstrate three properties at the same time: the agonist (or antagonist) binding of the analogue, the specificity of the binding by a displacement reaction, and the molecular weight of the 'receptor(s)' if the analogue is radiolabeled. In this paper we describe the synthesis of an analogue of PAF-acether exhibiting the three structural requirements for efficient affinity labeling: (i) a high platelet aggregation effect, achieved by maintaining the structural features necessary for the reactivity and thus modifying only the very end of the long chain at position 1; (ii) a high specific radioactivity by radiolabeling with 12si and (iii) a high reactive yield achieved by introducing an aromatic azido group photoactivatable at relatively long wavelength (300 nm) [14]. The chemical formula of this compound (17a) is: 0 C H2OCH2(C H 2 ) 9 C H 2 N H - ~ ) - N
]
112s 3
HO
CHO-COCH3
I
oDI
+
CH20-P-OCH2CH2N(CHa)a I
OExperimental
17a Procedures
Materials Analytical grade chemicals products were purchased from Prolabo or Merck (France) except for 4-azido 2-hydroxy benzoic ester of N-hydroxy succinimide, which was obtained from Pierce. Serum albumin came from Sigma. Open-column chromatography was carried out with silica gel (Merck). Radioactive iodine was from Amersham (U.K.).
217 TLC was performed on silica-gel plates Si60/F254 (Merck), with synthetic PAF acether as reference. Spots were detected by molybdenum blue spray, fluorescence quenching, radioactive counting (ESI Nuclear type 257) or autoradiography (Kodak XOMAT-MA films). The photoreactor was equipped with three 50W, 300 nm lamps. SDS-PAGE was performed with a custom-built apparatus. NMR and IR spectra were recorded with a Brucker E M360 and Perkin Elmer type 298 spectrometers respectively. The spectra of all the products agree with the formulas given in the text. The phthalimido group was easy to identify by a characteristic NMR multiplet at 7.7-7.8 ppm and by three characteristic IR bands at 1770 (minor), 1715 (major) and 1620 cm -1 respectively. HPLC equipment consisted of a 6000A pump (Waters) and variable wave length detector (Pye UNICAM).
Cells and aggregation procedures Rabbit platelets were withdrawn, separated and maintained essentially as already described [15]. Aggregation of rabbit platelets was evaluated in comparison with synthetic, optically active PAF-acether from Dr. Godfroid's laboratory [16].
Synthesis of" 1-O-(w-phthalimidoundecyl)2-O-acetyl (13)
3-glycero-phosphorylcholine (R,S)
Figure 1 shows the multistep procedure for the synthesis of compound 13. It is an application of the now classic procedures of Godfroid et al. [4] with modifications which appear in the text. The phthalimidyl-protected reactive amino group of 11-amino undecyl alcohol 3 was synthesized by condensation of the bromoalcohol 2 with potassium phthalimide in dry DMSO, for 7 h at 50 60°C (99% yield, m.p., 90°C). Mesylate 4 of the alcohol was prepared by adding methane sulfonyl chloride (4.8 ml in 40 ml dichloromethane) dropwise to 12.5 g of 3 dissolved in 80 ml diethylether, 11.2 ml triethylamine and 40 ml dichloromethane. The mixture was then refluxed for 2.5 h and poured into 400 ml water. Compound 4 was extracted with dichloromethane (3 X 100 ml), the organic phase dried on MgSO4 and concentrated under vacuum. After ethanol recrystallization, we obtained 13.4 g (86% yield) of pure 3 (m.p. 71 72°C). The condensation producing the ether 5 was performed by mixing, 4 g of 4, 1.1 ml of isopropylideneglycerol, 0.4 g of sodium hydride and 10 ml of DMSO distilled on sodium hydride, at 70°C. The reaction was followed by TLC with dichloromethane/ methanol (98:2, v/v) as the eluant and 5 appeared at Rf = 0.48. After 4 h, the reaction mixture was poured into 200 ml acidified water, and the pH was brought to 6 - 7 . Product 5 was extracted with 2 X 50 ml diethylether, the ether fraction was washed with 2 X 20 ml water, dried on MgSO4, and concentrated under vacuum. The orange oil (2.9 g) was chromatographed on 30 g silica eluted with dichloromethane, then dichloromethane with 1% methanol. We obtained a white solid (1 g, 25% yield) whose IR and NMR spectra correspond to 5. Diol 6 was produced by acidic cleavage of 5 (HC1 1 N in methanol) after refluxing for 1 h. An oily paste was obtained after evaporation and drying with a 95% yield.
218 The condensation of 6 (3 g) in pyridine (4.5 ml) was performed at 0°C with a dropwise addition of p-methoxytrityl chloride (2 g) in dry THF (3 ml). After 2 h stirring, the mixture was concentrated under vacuum, and chloroform (40 ml) was added. The organic phase was washed with sodium bicarbonate 10% (20 ml), dried on MgSO4 and concentrated. The oily paste was chromatographed on silica (50 g) with diethyl ether/petroleum ether (20:80, v/v). Product 7 was obtained (1.65 g; 32% yield) as an oily paste with the correct NMR and IR spectra. By acetylation of 7 with acetic anhydride in pyridine we obtained 8 quantitatively. The deprotection of 8 was performed according to Buchnea [17] on boric acidimpregnated silica. From 1 g of 8, 0.25 g of pure 9 was obtained (Rr = 0.19 diethyl ether/petroleum ether, 1 : i, v/v). Compound (13) was synthesized essentially according to Van Boeckel et al. 118] by the sequence of the three reactions shown in Fig. 1. From 45 mg of 9, we obtained an
0 O~c
DMSO
.~NK
;
Br'CH2( CH2 ) 9CHpOH
(!\
~--.C/NCI 2 ( CII?)9CII?OII
Ctl3502C1 Et3N / CH2CI 2 /
EtOEt
~
?
H20H
CHO\ /CII 3 CH-O
OH20
0
CH3
CH20/'~H 3
CH)
£ C \NCH (CH) CH OSO CHe •<...~-~,C / 2 2 9 2 ~. 4
Na~ / DMSO
5 I
HC1 / MeOH [
Q
©
~H2OCH2(CH2) 9CH2N~C~ CHOH
CH 0
I
C
? C~
CH2OCIt2(CH2)9CH2N~ / 0 CHOH ~
0
I
CH20H
Pyridine / THF
CH20
0
H3
Z 0 CH3% CH3%
Pyridine ?
?
c
/C
CH OCH (CH) CH N I I ]
2
2
29
2\c
~..,-.~...-
d
CHOCOCIt3
I
CH20H
0
SiO 2 / H3BO3
CH2OCH2(CH2)9CH2Nx/ / CHOCOCH3
0
219
N w \N~)
-['~)-N
\ N
6
HI) e - - -
Pyrldine /
II
CI I~-CI
THF Cl
CI
0 zC ~
I0
O
]
~'II20Cll 2 ( CE2 ) 9CII21~\ C: 0
I •
IIOC112CH2N (CH3)3 Tos-O
CItOCOCIIq.
b
!?
.",
1 /
~
}=<
/
fi
CH2OCH2(Cli2)9CIi2R\~ I
Pyridine / CH3CN
9,
]
0
CHOCOCH 3
*
CH20-P~OCH2CH2 N (CH3 )3
cu3/
\cu 3_] /
9 CHOCOCH3 0 0 * !H20-~-OCH2CH2N (CH3 )3 O 13
Fig. 1. Synthesis of 1-O-(ll-phthalimido undecyl)-2-O-aeetyl-3-glycero-phosphoryleholine (13).
oily product which was chromatographed on 10 g silica with a stepwise gradient of chloroform/methanol. Product 13 was obtained (32 mg), but was not completely pure. Thus, it was chromatographed by HPLC with a silica-gel column using a mixture of chloroform/methanol/water ( 6 5 : 3 5 : 6 , by vol.) according to Godfroid et al. 1H- and 3 IP.NM R spectroscopy of product 13 showed that it contained about 25% of the isomer 3-(acetyl) 2-(phosphorylcholine) (Fig. 3 and Table I). However, it exhibited a very high aggregation activity, since it induced a 50% aggregation of rabbit platelets with a concentration of about 5 . 10 -1° M (experiments carried out by E. Coeffier in Dr. Benveniste's laboratory).
Synthesis of{1 7) A priori, it is easy to link many probes to the NH 2 terminal at position 1 of compound 13 after removal of the phthalimidyl group. Deprotection was achieved by stirring 18 mg
~.20 O ;H ~ H ( C H ) CH N I 2 2 2 9 P \
e
]HOCOCH3
()
r
o
~
?°"o
÷
%o-i_-o%%.(% 13 0
H2[)CH2(CH2)gCH2NH2]
NH2NH?Ofl 2 / MeOH
.
!
CH20-P-C'CH2CH2N ( CH3 ) 3
13
L
/
|
14 (a,b)_J H
3 u
~ £5
0 "~H2
\c-c.~ 0
Et3N I CHCI 3 1 MeOH
0 '~~0~2(C~2)9CN2NH-~- ~ HO-~ 0 CX)CH 0 3
3
125INa
(
CH2OCH2(CH2)9CH2HBO_~ N3 [ HO CHO~
+
ii 0_~_0~2~42N(C~3)3 0
I125
|
Chol ramni esoMetabi dui mf istule T
17 a
0
~20_~_~2CN2N
KI
0
+
(CH3)3 16 (a,b)
qg. 2. Synthesis of (17a). Any manipulations were carried out in the dark to avoid the p h o t o d e s t :ion of the c o m p o u n d s (a) R = COCH 3 or (b) R = 1t.
~
-
f
-
-
-
I
] ppm
Fig. 3. 101 MHz 3 * P - N M R spectrum of (13) showing the ratio of the two isomers.
221 TABLE I tt-NMR PEAK ATTRIBUTION FOR COMPOUND (13) O li
g f e d c b 1t2C O-('112.-CIt~(Ctf2)~ CH2-CIt2-N
C~,.a C II 0
h O i It H C - O - C CH~
O
+
II
IIz(" O P - O
Cttz-CIt2-N(CHs) s
j
k
I
O-
1
m
Protons
Number
6 (ppm)
Multiplicity
a b c d e f g h i j k 1 m ha ia ja
211 + 2tt 21t 211 141t 21t 21t 21t 11t 3tt 2H 2ti 2H 9tt 1 It 3 tt 21t
7.83 and 7.71 3.76 1.51 1.23 1.67 3.42 3.54 5.14 2.07 4.00 4.28 3.68 3.36 4.41 2.05 4.2 4.3
m t m s m m m q s m m t s m s d+d
a For the isomer 3-(acetyl)-2-phosphorylcholine) of compound (13).
o f 13 w i t h 1.5 ml o f 0.2 M m e t h a n o l i c h y d r a z i n e s o l u t i o n at r o o m t e m p e r a t u r e for 2 days. S o l v e n t a n d excess h y d r a z i n e were r e m o v e d u n d e r v a c u u m . T h e resulting p r o d u c t d i s p l a y e d m a i n s p o t ( R f = 0.1) w i t h TLC, using c h l o r o f o r m / m e t h a n o l / w a t e r ( 6 5 : 3 5 : 6 , b y vol.) as t h e e l u a n t . This s p o t c o n t a i n e d p h o s p h o r u s (as revealed b y m o l y b d e n u m b l u e spray) a n d a m i n e (revealed b y n i n h y d r i n s p r a y ) groups. T h e y c o r r e s p o n d e d to a m i x t u r e o f P A F - a c e t h e r a n d lyso P A F - a c e t h e r (co-amino)-analogues, as c o u l d s u b s e q u e n t l y b e seen a f t e r d e r i v a t i z a t i o n o f t h e a m i n o g r o u p .
222 The high reactivity of the amino group with activated esters is the reason for its long standing use in derivatizing proteins. In our case, the side reaction with hydroxylic solvent was not a problem since the coupling reaction can be done in weakly hydroxylic solvent and thus without excessive activated ester hydrolysis. We benefited from a recent publication by Ji and Ji [14] which describes the probing of concanavalin A with photoreactive, iodinated, and activated ester 15. In our case, the reaction was performed by adding 30 mg of amino analogue mixture (14a and 14b), dissolved in 5 ml chloroform/ methanol (9 : 1, v/v) , to 40 mg of ester 15 and 20 /A of triethylamine. After 30 rain reaction time, TLC of the mixture with chloroform/methanol/water (65:35:6, by vol.) displayed 2 main spots comigrating with genuine PAF-acether and lyso PAF-acether respectively, and containing phosphate groups (molybdenum blue spray). No ninhydrin positive spots remained at Rf-values corresponding to 14a and 14b. The two main spots were separately recovered after a preparative TLC under the same conditions. We obtained 16a (11 rag) and 16b (5 rag). The two products again contained a low proportion of isomers witla exchanged acetyl and phosphoryl residues, as seen by JH- and 31p-NMR in the case of 16a. At this step, 16a was photoreactive, since irradiation at 300 nm of the product caused large spectrum changes in less than 5 rain. Iodination of 16a by 12Sl was performed according to Bolton and Hunter [19]. Typically, 2.1 /ag (about 3 nmol) of 16a was mixed to 20/al of Na 1251 (1 mCi) and 15/al chloroform. Ten microlitres of chloramine T (5 mg/ml in 0.25 M phosphate buffer, pH 7.5) was then added. After 10-30 s, excess metabisulfite (30 /~1 at 12 mg/ml in 0.05 M phosphate buffer pH 7.5), potassium iodide (10 /al of 20 mg/ml in the same buffer) then 30 tal chloroform were added. The radioactive product (17a) contained in the organic phase was purified and characterized as described below.
Results and Discussion
Our objective was to synthesize an analogue of PAF-acether which possesses three characteristics: a strong platelet aggregating effect, high radioactivity, and high photoreactivity. We demonstrate here that compound 17a, synthesized according to the procedure described in the preceding section, satisfies these conditions. Purification and chemical characterization The chloroform extract of the iodination reaction contained essentially two highly radioactive products as seen by TLC (Fig. 4B (1)). One of the two spots exhibited an Rf-value identical to a genuine PA*F-acether compound. It was purified in a microcolumn consisting of a Pasteur pipette with 50 mg silica gel. Figure 4A shows the radioactivity profile of the elution (0.5 ml fractions) with a step gradient of chloroform/methanol. Figure 4B shows the corresponding autoradiography of the TLC on some fractions. Fractions 10-29 were pooled. They together contained 40% of the initial radioactivity, i.e. 2 • 1 0 9 cpm. In this first step, a very highly radioactive com-
223 iB I
2OO
A
A x
.2
{2) IO0
o
(3)
d 0
10
{4) 20 fraction
30
number
{',) d
b
,~
Fig. 4. Purification steps for 17a: (A) silica-gel column chromatography purification of the reaction mixture; {B) autoradiography of a TLC plate (chloroform/methanol/water, 65:25:4, by vol. as eluant) on: (1) reaction mixture; {2-5) fractions number 3,5, 10 and 15, respectively. Discontinuous lines correspond to: (a) deposit; (b) lyso PAF-acether; (c) PAF-acether; {d) front. pound was prepared, having the same Rf-value as a genuine PAF-acether sample. It was stable in the dark at 0°C, for about 3 weeks. After this period a slow degradation occurred.
Platelet aggregation tests The platelet aggregating ability of the various products isolated in the course of the synthesis was verified. By comparison with synthetic optically active PAF-acether, 13 and 16a displayed a very high activity: concentrations causing 50% of maximum platelet aggregation were ten times higher and two times lower than synthetic snPAF-acether respectively, even though they contain about 25% of the position isomer, and are racemic mixtures. More over, in this way we could evaluate the specific radioactivity of 17a. If we suppose that the agonist effect of 17a is the same as genuine PAF.acether molecules, we usually obtained specific activities higher than 1000 Ci/mmol. It must be emphasized that a bulky group (0.4-0.5 nm mean diameter) at the end of the chain at position 1 does not seem to influence the agonist effect of PAF analogues. Photoreactivity This last property of compound 1 7a was, of course, crucial for our objective. We chose to use the known binding of lipidic compounds to serum albumin to test for photore-
224 activity. Four results were needed to prove it, namely: (1) no irreversible binding without light; (2) binding after irradiation; (3) protection of the binding by previous treatment of the mixture with saturating concentrations of a non-photolysable compound; (4) no protection if the inhibitor was added after irradiation. Figure 4 shows that these 4 conditions were fullfilled, as seen by SDS-PAGE of mixtures of BSA and compound 1 7a. The chosen binding inhibitor was lysolecithin. As observed, a very high concentration of this ligand was necessary to ensure minor binding of 17a with serum albumin. This was not unexpected since it is well k n o w n that the displacement of many drugs from BSA by compounds having much higher affinity, is difficult [20] because of the many different specialized sites in serum albumin. Finally, it should be noted that the yield of the coupling photoreaction between the azido compound and serum albumin was apparently high since about 30 60~,~ of the trapped ligands were covalently bound by serum albumin (by comparison with chemically iodinated serum albumin). Many photoreactive groups, namely azide, diazirine or diazo residues were introduced into a fatty acid chain, and later linked to a phospho--
BSA
Fig. 5. Autoradiography (microdensitometric display) of BSA-(17a) mixtures. BSA (0.l rag/roll and compound 17a (300 000 cpm) were mixed and incubated for 5 min. Chromatography on a G25 column shown that about 30% of 1 7a eluted in the void volume. Irradiation by 300 mn lamps was carried out for 10 min except for lane (1). Lane (2): without further addition; lane (3): with the addition of lyso PC (1 mM) prior to irradiation; lane (4): with the addition of lyso PC (1 mM) after irradiation. Arrow shows the BSA position.
225 lipid backbone [21]. In comparison to other groups, the one used here has the unique advantage of combining photoreactivity and high radioactivity in the last step of the synthesis. It has not yet been determined whether or not the coupling yield is so high because the reactive residue is trapped in a pocket out of which it cannot move. In this case, the coupling yield with other proteins would decrease, because the correlation time of the w-end of protein-bound lipids can be very short [22,23]. Moreover, two recent papers [24,25] were devoted to the synthesis o f a 2-acylamino analogue of PAFacether as a precursor for photoaffinity markers. In addition to the fact that their aggregation effects were lower (1.5 × 10 -8 M at best) than ours, we feel that it is much more difficult to combine photoreactivity and radioactivity at position 2, in the last step of the synthesis, without a large loss in reactivity, since it is well known that large substituents at this position drastically decrease aggregative activity [5].
Extensions o f the method Owing to the high reactivity of the terminal amino group, a different derivatization of PAF-acether or lyso PAF-acether can readily be considered namely by radioactive fluorescent or spin label groups, On the other hand, a preparation of the active form of PAF acether or lysoderivatives analogues can be carried out, due to the stereospecific activity o f A2 phospholipases [26]. Finally, ether or carbamate binding at position 2 can easily be introduced in order to produce a non hydrolyzable compound. These. different synthesis are currently in progress in our laboratory. Acknowledgements We gratefully acknowledge the aggregation test measurements performed partially in the laboratorics of" Dr. Benveniste (INSERM U200, Clamart, France) and Dr. Vargaftig (lnstitut Pasteur, Paris, France). The phosphorylation step of the synthesis was aided by C. Aymard and B. Rayner in Professor hnbach's laboratory (USTL, Montpellier, France).
References 1 B.B. Vargaftig and J. Benveniste, Trends Pharmacol. Sci. (1983) 341-343. 2 R.L. Wykle, C.H. Miller, J.C. Lewis, J.D. Schmitt, J.A. Smith, J.R. Surles, C. Piantadosi and J.T. O'Flaherty Biochem. Biophys. Res. Commun., 100 (1981) 1651 1658. 3 J. Benveniste, J.P. Le Couedic, J. Polonsky and M. Tencd, Nature, 269 (1977) 170-171. 4 J.J. Godfroid, F. Heymans, E. Michel, C. Redevilh, E. Steiner and J. Benveniste, FEBS Lett., 116 (1980) 161-164. 5 M. Tenc~, ~i. Coeffier, F. Heymans, J. Polonsky, J.J. Godfroid and J. Benveniste, Biochimie, 63 (1981) 723-727. 6 P. Hadvary J.M. Cassal, G. Hirth, R. Barner and H.R. Baumgartner, in J. Benveniste and B. Arnoud (Eds.), PAF Acether & Structurally Related Ether Lipids, Elsevier, 198 p. 57-60. 7 P. Hadvary and H.R. Baumgartner, Thromb. Res., 30 (1983) 143 156. 8 I..H. Valone, E. Coles, V.R. Reinhold and E.J. Goetzl, J. Immunol., 129 (1982) 1637-1641. 9 C.M. Chesney, D.D. Pifer and K.M. Huch, Thromb. Haemostasis, 50 (1983) 14-17.
226 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
F.H. Valone and E.J. Goetzl, Immunology, 48 (1983) 141-149. D.P. Tuffin, P.J. Wade, D.O. Lunt and K.G. Mc CuUagh, J. Pharmacol., (suppl 1) 14 (1983) 67. S.B. Hwang, C.S.C. Lee, M.J. Cheah and T.Y. Shen, Biochemistry, 22 (1983) 4756-4763. H. Bailey and J.R. Knowles, in: W.B.P. Jakoby and M. Wilchek (Ed.), Methods in Enzymology, Vol. XLVI, Academic Press, 1977, pp. 6 9 - 1 1 4 . T.H. Ji and I. Ji, Anal. Biochem., 121 (1982) 286-289. G. Camussi, J.M. Mencia-Huerta and J. Benveniste, Immunology, 33 (1977) 5 2 3 - 5 3 4 . J.M. Mencia-Huerta and J. Benveniste, Eur. J. Immunol., 9 (1979) 4 0 9 - 4 1 5 . D. Buchnea, Lipids, 9 (1974) 5 5 - 5 7 . C.A.A. Van Boeckel, G.A. Vand der Marel, P. Wester Duin and 3.H. Van Boom, Synthesis, (1982) 399-402. A.E. Bolton and W.M. Hunter, Bioc'hem. J., 133 (1973) 5 2 9 - 5 3 9 . A.A. Spector and J.E. Fletcher, Nutrition & Drug Interrelations, Academic Press, New York, 1978. R.J. Robson, R. Radhakrishnan, A.H. Ross, Y. Tak Agaki and N.G. Khorana, in: P.C. Jost and O.H. Griffith (Ed.), Lipid-Protein Interactions, Vol. 2, J. Wiley, New York, 1982, pp. 149 175. J. Davoust, M. Seigneuret, P. Herv6 and P.F. Devaux, Biochemistry, 22 (1983) 3137-3145. J. Davoust, M. Seigneuret, P. Hervd and P.F. Devaux, Biochemistry, 22 (1983) 3146 3151. N.S. Chandrakumar and J. Hajdu, J. Org. Chem., 48 (1983) 1197-- 1202. M.M. Ponpipom and R.L. Bugianesi, Chem. Phys. Lipids, 35 (1984) 2 9 - 3 8 . M. Tenc6, E. Coeffier, J. Polonsky and J. Benveniste, Biochim. Biophys. Acta, 755 (1983) 526 530.
215
SYNTHESIS AND CHARACTERIZATION OF A RADIOIODINATED, PHOTOREACTIVE AND PHYSIOLOGICALLY ACTIVE ANALOGUE OF PLATELET ACTIVATING FACTOR
P. BI';TTE-BOBILLOa, A. BIENVENUEa,*, C. BROQUETb and L. MAURINa aLaboratoire de Biologie Physico-Chirnique, place E. Bataillon. 34060, Montpellier Cedes and bLaboratoire de Pharmacochimie Moldculaire, Universitd Paris VII, 2 Place Jussieu, 75005 Paris (France) Received January 7th, 1 9 8 5 accepted March 29th, 1985
revision received March 25th, 1985
The multistep synthesis of a photoreactive, radioactive and aggregating analogue of plateletactivating factor (PAF)-acether is described. The photoreactive and radioactive moiety was added at the last step; the specific radioactivity was higher than 1000 Ci/mmol. The concentration of this new analogue which causes 50% of aggregation of platelets were of the same order of magnitude as for synthetic snPAF-acether, so as for two other analogues having a bulky group at the co end of the fatty ether chain. The photoreactivity was proved by the covalent binding of the analogue to protein (BSA) after 10-min irradiation times at 300 nm. The binding was largely prevented by prior (not by later) addition of a high concentration of lyso phosphatidyl choline.
Key words: ether linked phospholipids; azido and radioiodinated analogues of platelet-activating factor; chemical synthesis; platelet aggregation dose; photoaffinity.
Introduction Platelet-activating factor (PAF-acether) is a mediator of many physiological responses, such as anaphylaxis, inflammation, platelet aggregation and hypotension [ 1]. Its structure is now well known: it is a glycerophospholipid with a choline polar head group, bound to a large hydrophobic tail ( 1 6 - 1 8 carbon atoms) by an ether bond at position 1 and to an acetyl group at position 2. It is generally recognized that only the R(sn) enantiomer is physiologically active [2]. The best means for detecting this product is the rabbit platelet aggregation test [3] which exhibits a very high efficiency with 1-hexadecyl compound: a concentration of 1 0 - 1 1 - 1 0 -~° M was sufficient to provoke a full aggregation of platelets. Many analogues of endogenous PAF-acether molecules have been synthesized by different authors after the first rehable method was published by Godfroid et al. [4].
*To whom correspondence should be sent. Abbreviations: BSA, bovine serum albumin; DMSO, dimethyl sulfoxide; HPLC, high performance liquid chromatography; PAl:, platelet-activating factor; SDS-PAGE,electrophoresis with 0.2% sodium dodecyl sulphate on polyacrylamide gel 10%; THF, tetrahydrofuran; TLC, thin-layer chromatography. 0009-3084/85/$03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland
216 The substituents at position 1,2 or 3 were modified and there was a general agreement to the following rules: (a) The length of the chain at position 1 is not critical between 10 and 20 carbon atoms, on condition that it is bound to the glycerol backbone by an ether bond [5]. (b) The group at position 2 is very important: an increase in the chain length of the ester drastically decreases the activity [5]. However a short chain bound by an ether [2] or a carbamate bond [6] has an excellent activity, sometimes higher than the native compound. (c) The phosphoryl choline group also seems indispensable [7]. An aggregation dose on the order of nM raises the possibility of a receptor-mediated process and some papers have been devoted to this point. By using acetyl tritiated analogues of PAF-acether, many authors found that this molecule binds with high affinity to different cells [8-11] and to plasma membranes [12]. It is thus very likely that certain proteins in many cells and organs can bind PAFacether or lyso PAF-acether. One of the best method for studying this binding and for characterizing the putative 'receptor(s)', is the affinity labeling [13] since it can demonstrate three properties at the same time: the agonist (or antagonist) binding of the analogue, the specificity of the binding by a displacement reaction, and the molecular weight of the 'receptor(s)' if the analogue is radiolabeled. In this paper we describe the synthesis of an analogue of PAF-acether exhibiting the three structural requirements for efficient affinity labeling: (i) a high platelet aggregation effect, achieved by maintaining the structural features necessary for the reactivity and thus modifying only the very end of the long chain at position 1; (ii) a high specific radioactivity by radiolabeling with 12si and (iii) a high reactive yield achieved by introducing an aromatic azido group photoactivatable at relatively long wavelength (300 nm) [14]. The chemical formula of this compound (17a) is: 0 C H2OCH2(C H 2 ) 9 C H 2 N H - ~ ) - N
]
112s 3
HO
CHO-COCH3
I
oDI
+
CH20-P-OCH2CH2N(CHa)a I
OExperimental
17a Procedures
Materials Analytical grade chemicals products were purchased from Prolabo or Merck (France) except for 4-azido 2-hydroxy benzoic ester of N-hydroxy succinimide, which was obtained from Pierce. Serum albumin came from Sigma. Open-column chromatography was carried out with silica gel (Merck). Radioactive iodine was from Amersham (U.K.).
217 TLC was performed on silica-gel plates Si60/F254 (Merck), with synthetic PAF acether as reference. Spots were detected by molybdenum blue spray, fluorescence quenching, radioactive counting (ESI Nuclear type 257) or autoradiography (Kodak XOMAT-MA films). The photoreactor was equipped with three 50W, 300 nm lamps. SDS-PAGE was performed with a custom-built apparatus. NMR and IR spectra were recorded with a Brucker E M360 and Perkin Elmer type 298 spectrometers respectively. The spectra of all the products agree with the formulas given in the text. The phthalimido group was easy to identify by a characteristic NMR multiplet at 7.7-7.8 ppm and by three characteristic IR bands at 1770 (minor), 1715 (major) and 1620 cm -1 respectively. HPLC equipment consisted of a 6000A pump (Waters) and variable wave length detector (Pye UNICAM).
Cells and aggregation procedures Rabbit platelets were withdrawn, separated and maintained essentially as already described [15]. Aggregation of rabbit platelets was evaluated in comparison with synthetic, optically active PAF-acether from Dr. Godfroid's laboratory [16].
Synthesis of" 1-O-(w-phthalimidoundecyl)2-O-acetyl (13)
3-glycero-phosphorylcholine (R,S)
Figure 1 shows the multistep procedure for the synthesis of compound 13. It is an application of the now classic procedures of Godfroid et al. [4] with modifications which appear in the text. The phthalimidyl-protected reactive amino group of 11-amino undecyl alcohol 3 was synthesized by condensation of the bromoalcohol 2 with potassium phthalimide in dry DMSO, for 7 h at 50 60°C (99% yield, m.p., 90°C). Mesylate 4 of the alcohol was prepared by adding methane sulfonyl chloride (4.8 ml in 40 ml dichloromethane) dropwise to 12.5 g of 3 dissolved in 80 ml diethylether, 11.2 ml triethylamine and 40 ml dichloromethane. The mixture was then refluxed for 2.5 h and poured into 400 ml water. Compound 4 was extracted with dichloromethane (3 X 100 ml), the organic phase dried on MgSO4 and concentrated under vacuum. After ethanol recrystallization, we obtained 13.4 g (86% yield) of pure 3 (m.p. 71 72°C). The condensation producing the ether 5 was performed by mixing, 4 g of 4, 1.1 ml of isopropylideneglycerol, 0.4 g of sodium hydride and 10 ml of DMSO distilled on sodium hydride, at 70°C. The reaction was followed by TLC with dichloromethane/ methanol (98:2, v/v) as the eluant and 5 appeared at Rf = 0.48. After 4 h, the reaction mixture was poured into 200 ml acidified water, and the pH was brought to 6 - 7 . Product 5 was extracted with 2 X 50 ml diethylether, the ether fraction was washed with 2 X 20 ml water, dried on MgSO4, and concentrated under vacuum. The orange oil (2.9 g) was chromatographed on 30 g silica eluted with dichloromethane, then dichloromethane with 1% methanol. We obtained a white solid (1 g, 25% yield) whose IR and NMR spectra correspond to 5. Diol 6 was produced by acidic cleavage of 5 (HC1 1 N in methanol) after refluxing for 1 h. An oily paste was obtained after evaporation and drying with a 95% yield.
218 The condensation of 6 (3 g) in pyridine (4.5 ml) was performed at 0°C with a dropwise addition of p-methoxytrityl chloride (2 g) in dry THF (3 ml). After 2 h stirring, the mixture was concentrated under vacuum, and chloroform (40 ml) was added. The organic phase was washed with sodium bicarbonate 10% (20 ml), dried on MgSO4 and concentrated. The oily paste was chromatographed on silica (50 g) with diethyl ether/petroleum ether (20:80, v/v). Product 7 was obtained (1.65 g; 32% yield) as an oily paste with the correct NMR and IR spectra. By acetylation of 7 with acetic anhydride in pyridine we obtained 8 quantitatively. The deprotection of 8 was performed according to Buchnea [17] on boric acidimpregnated silica. From 1 g of 8, 0.25 g of pure 9 was obtained (Rr = 0.19 diethyl ether/petroleum ether, 1 : i, v/v). Compound (13) was synthesized essentially according to Van Boeckel et al. 118] by the sequence of the three reactions shown in Fig. 1. From 45 mg of 9, we obtained an
0 O~c
DMSO
.~NK
;
Br'CH2( CH2 ) 9CHpOH
(!\
~--.C/NCI 2 ( CII?)9CII?OII
Ctl3502C1 Et3N / CH2CI 2 /
EtOEt
~
?
H20H
CHO\ /CII 3 CH-O
OH20
0
CH3
CH20/'~H 3
CH)
£ C \NCH (CH) CH OSO CHe •<...~-~,C / 2 2 9 2 ~. 4
Na~ / DMSO
5 I
HC1 / MeOH [
Q
©
~H2OCH2(CH2) 9CH2N~C~ CHOH
CH 0
I
C
? C~
CH2OCIt2(CH2)9CH2N~ / 0 CHOH ~
0
I
CH20H
Pyridine / THF
CH20
0
H3
Z 0 CH3% CH3%
Pyridine ?
?
c
/C
CH OCH (CH) CH N I I ]
2
2
29
2\c
~..,-.~...-
d
CHOCOCIt3
I
CH20H
0
SiO 2 / H3BO3
CH2OCH2(CH2)9CH2Nx/ / CHOCOCH3
0
219
N w \N~)
-['~)-N
\ N
6
HI) e - - -
Pyrldine /
II
CI I~-CI
THF Cl
CI
0 zC ~
I0
O
]
~'II20Cll 2 ( CE2 ) 9CII21~\ C: 0
I •
IIOC112CH2N (CH3)3 Tos-O
CItOCOCIIq.
b
!?
.",
1 /
~
}=<
/
fi
CH2OCH2(Cli2)9CIi2R\~ I
Pyridine / CH3CN
9,
]
0
CHOCOCH 3
*
CH20-P~OCH2CH2 N (CH3 )3
cu3/
\cu 3_] /
9 CHOCOCH3 0 0 * !H20-~-OCH2CH2N (CH3 )3 O 13
Fig. 1. Synthesis of 1-O-(ll-phthalimido undecyl)-2-O-aeetyl-3-glycero-phosphoryleholine (13).
oily product which was chromatographed on 10 g silica with a stepwise gradient of chloroform/methanol. Product 13 was obtained (32 mg), but was not completely pure. Thus, it was chromatographed by HPLC with a silica-gel column using a mixture of chloroform/methanol/water ( 6 5 : 3 5 : 6 , by vol.) according to Godfroid et al. 1H- and 3 IP.NM R spectroscopy of product 13 showed that it contained about 25% of the isomer 3-(acetyl) 2-(phosphorylcholine) (Fig. 3 and Table I). However, it exhibited a very high aggregation activity, since it induced a 50% aggregation of rabbit platelets with a concentration of about 5 . 10 -1° M (experiments carried out by E. Coeffier in Dr. Benveniste's laboratory).
Synthesis of{1 7) A priori, it is easy to link many probes to the NH 2 terminal at position 1 of compound 13 after removal of the phthalimidyl group. Deprotection was achieved by stirring 18 mg
~.20 O ;H ~ H ( C H ) CH N I 2 2 2 9 P \
e
]HOCOCH3
()
r
o
~
?°"o
÷
%o-i_-o%%.(% 13 0
H2[)CH2(CH2)gCH2NH2]
NH2NH?Ofl 2 / MeOH
.
!
CH20-P-C'CH2CH2N ( CH3 ) 3
13
L
/
|
14 (a,b)_J H
3 u
~ £5
0 "~H2
\c-c.~ 0
Et3N I CHCI 3 1 MeOH
0 '~~0~2(C~2)9CN2NH-~- ~ HO-~ 0 CX)CH 0 3
3
125INa
(
CH2OCH2(CH2)9CH2HBO_~ N3 [ HO CHO~
+
ii 0_~_0~2~42N(C~3)3 0
I125
|
Chol ramni esoMetabi dui mf istule T
17 a
0
~20_~_~2CN2N
KI
0
+
(CH3)3 16 (a,b)
qg. 2. Synthesis of (17a). Any manipulations were carried out in the dark to avoid the p h o t o d e s t :ion of the c o m p o u n d s (a) R = COCH 3 or (b) R = 1t.
~
-
f
-
-
-
I
] ppm
Fig. 3. 101 MHz 3 * P - N M R spectrum of (13) showing the ratio of the two isomers.
221 TABLE I tt-NMR PEAK ATTRIBUTION FOR COMPOUND (13) O li
g f e d c b 1t2C O-('112.-CIt~(Ctf2)~ CH2-CIt2-N
C~,.a C II 0
h O i It H C - O - C CH~
O
+
II
IIz(" O P - O
Cttz-CIt2-N(CHs) s
j
k
I
O-
1
m
Protons
Number
6 (ppm)
Multiplicity
a b c d e f g h i j k 1 m ha ia ja
211 + 2tt 21t 211 141t 21t 21t 21t 11t 3tt 2H 2ti 2H 9tt 1 It 3 tt 21t
7.83 and 7.71 3.76 1.51 1.23 1.67 3.42 3.54 5.14 2.07 4.00 4.28 3.68 3.36 4.41 2.05 4.2 4.3
m t m s m m m q s m m t s m s d+d
a For the isomer 3-(acetyl)-2-phosphorylcholine) of compound (13).
o f 13 w i t h 1.5 ml o f 0.2 M m e t h a n o l i c h y d r a z i n e s o l u t i o n at r o o m t e m p e r a t u r e for 2 days. S o l v e n t a n d excess h y d r a z i n e were r e m o v e d u n d e r v a c u u m . T h e resulting p r o d u c t d i s p l a y e d m a i n s p o t ( R f = 0.1) w i t h TLC, using c h l o r o f o r m / m e t h a n o l / w a t e r ( 6 5 : 3 5 : 6 , b y vol.) as t h e e l u a n t . This s p o t c o n t a i n e d p h o s p h o r u s (as revealed b y m o l y b d e n u m b l u e spray) a n d a m i n e (revealed b y n i n h y d r i n s p r a y ) groups. T h e y c o r r e s p o n d e d to a m i x t u r e o f P A F - a c e t h e r a n d lyso P A F - a c e t h e r (co-amino)-analogues, as c o u l d s u b s e q u e n t l y b e seen a f t e r d e r i v a t i z a t i o n o f t h e a m i n o g r o u p .
222 The high reactivity of the amino group with activated esters is the reason for its long standing use in derivatizing proteins. In our case, the side reaction with hydroxylic solvent was not a problem since the coupling reaction can be done in weakly hydroxylic solvent and thus without excessive activated ester hydrolysis. We benefited from a recent publication by Ji and Ji [14] which describes the probing of concanavalin A with photoreactive, iodinated, and activated ester 15. In our case, the reaction was performed by adding 30 mg of amino analogue mixture (14a and 14b), dissolved in 5 ml chloroform/ methanol (9 : 1, v/v) , to 40 mg of ester 15 and 20 /A of triethylamine. After 30 rain reaction time, TLC of the mixture with chloroform/methanol/water (65:35:6, by vol.) displayed 2 main spots comigrating with genuine PAF-acether and lyso PAF-acether respectively, and containing phosphate groups (molybdenum blue spray). No ninhydrin positive spots remained at Rf-values corresponding to 14a and 14b. The two main spots were separately recovered after a preparative TLC under the same conditions. We obtained 16a (11 rag) and 16b (5 rag). The two products again contained a low proportion of isomers witla exchanged acetyl and phosphoryl residues, as seen by JH- and 31p-NMR in the case of 16a. At this step, 16a was photoreactive, since irradiation at 300 nm of the product caused large spectrum changes in less than 5 rain. Iodination of 16a by 12Sl was performed according to Bolton and Hunter [19]. Typically, 2.1 /ag (about 3 nmol) of 16a was mixed to 20/al of Na 1251 (1 mCi) and 15/al chloroform. Ten microlitres of chloramine T (5 mg/ml in 0.25 M phosphate buffer, pH 7.5) was then added. After 10-30 s, excess metabisulfite (30 /~1 at 12 mg/ml in 0.05 M phosphate buffer pH 7.5), potassium iodide (10 /al of 20 mg/ml in the same buffer) then 30 tal chloroform were added. The radioactive product (17a) contained in the organic phase was purified and characterized as described below.
Results and Discussion
Our objective was to synthesize an analogue of PAF-acether which possesses three characteristics: a strong platelet aggregating effect, high radioactivity, and high photoreactivity. We demonstrate here that compound 17a, synthesized according to the procedure described in the preceding section, satisfies these conditions. Purification and chemical characterization The chloroform extract of the iodination reaction contained essentially two highly radioactive products as seen by TLC (Fig. 4B (1)). One of the two spots exhibited an Rf-value identical to a genuine PA*F-acether compound. It was purified in a microcolumn consisting of a Pasteur pipette with 50 mg silica gel. Figure 4A shows the radioactivity profile of the elution (0.5 ml fractions) with a step gradient of chloroform/methanol. Figure 4B shows the corresponding autoradiography of the TLC on some fractions. Fractions 10-29 were pooled. They together contained 40% of the initial radioactivity, i.e. 2 • 1 0 9 cpm. In this first step, a very highly radioactive com-
223 iB I
2OO
A
A x
.2
{2) IO0
o
(3)
d 0
10
{4) 20 fraction
30
number
{',) d
b
,~
Fig. 4. Purification steps for 17a: (A) silica-gel column chromatography purification of the reaction mixture; {B) autoradiography of a TLC plate (chloroform/methanol/water, 65:25:4, by vol. as eluant) on: (1) reaction mixture; {2-5) fractions number 3,5, 10 and 15, respectively. Discontinuous lines correspond to: (a) deposit; (b) lyso PAF-acether; (c) PAF-acether; {d) front. pound was prepared, having the same Rf-value as a genuine PAF-acether sample. It was stable in the dark at 0°C, for about 3 weeks. After this period a slow degradation occurred.
Platelet aggregation tests The platelet aggregating ability of the various products isolated in the course of the synthesis was verified. By comparison with synthetic optically active PAF-acether, 13 and 16a displayed a very high activity: concentrations causing 50% of maximum platelet aggregation were ten times higher and two times lower than synthetic snPAF-acether respectively, even though they contain about 25% of the position isomer, and are racemic mixtures. More over, in this way we could evaluate the specific radioactivity of 17a. If we suppose that the agonist effect of 17a is the same as genuine PAF.acether molecules, we usually obtained specific activities higher than 1000 Ci/mmol. It must be emphasized that a bulky group (0.4-0.5 nm mean diameter) at the end of the chain at position 1 does not seem to influence the agonist effect of PAF analogues. Photoreactivity This last property of compound 1 7a was, of course, crucial for our objective. We chose to use the known binding of lipidic compounds to serum albumin to test for photore-
224 activity. Four results were needed to prove it, namely: (1) no irreversible binding without light; (2) binding after irradiation; (3) protection of the binding by previous treatment of the mixture with saturating concentrations of a non-photolysable compound; (4) no protection if the inhibitor was added after irradiation. Figure 4 shows that these 4 conditions were fullfilled, as seen by SDS-PAGE of mixtures of BSA and compound 1 7a. The chosen binding inhibitor was lysolecithin. As observed, a very high concentration of this ligand was necessary to ensure minor binding of 17a with serum albumin. This was not unexpected since it is well k n o w n that the displacement of many drugs from BSA by compounds having much higher affinity, is difficult [20] because of the many different specialized sites in serum albumin. Finally, it should be noted that the yield of the coupling photoreaction between the azido compound and serum albumin was apparently high since about 30 60~,~ of the trapped ligands were covalently bound by serum albumin (by comparison with chemically iodinated serum albumin). Many photoreactive groups, namely azide, diazirine or diazo residues were introduced into a fatty acid chain, and later linked to a phospho--
BSA
Fig. 5. Autoradiography (microdensitometric display) of BSA-(17a) mixtures. BSA (0.l rag/roll and compound 17a (300 000 cpm) were mixed and incubated for 5 min. Chromatography on a G25 column shown that about 30% of 1 7a eluted in the void volume. Irradiation by 300 mn lamps was carried out for 10 min except for lane (1). Lane (2): without further addition; lane (3): with the addition of lyso PC (1 mM) prior to irradiation; lane (4): with the addition of lyso PC (1 mM) after irradiation. Arrow shows the BSA position.
225 lipid backbone [21]. In comparison to other groups, the one used here has the unique advantage of combining photoreactivity and high radioactivity in the last step of the synthesis. It has not yet been determined whether or not the coupling yield is so high because the reactive residue is trapped in a pocket out of which it cannot move. In this case, the coupling yield with other proteins would decrease, because the correlation time of the w-end of protein-bound lipids can be very short [22,23]. Moreover, two recent papers [24,25] were devoted to the synthesis o f a 2-acylamino analogue of PAFacether as a precursor for photoaffinity markers. In addition to the fact that their aggregation effects were lower (1.5 × 10 -8 M at best) than ours, we feel that it is much more difficult to combine photoreactivity and radioactivity at position 2, in the last step of the synthesis, without a large loss in reactivity, since it is well known that large substituents at this position drastically decrease aggregative activity [5].
Extensions o f the method Owing to the high reactivity of the terminal amino group, a different derivatization of PAF-acether or lyso PAF-acether can readily be considered namely by radioactive fluorescent or spin label groups, On the other hand, a preparation of the active form of PAF acether or lysoderivatives analogues can be carried out, due to the stereospecific activity o f A2 phospholipases [26]. Finally, ether or carbamate binding at position 2 can easily be introduced in order to produce a non hydrolyzable compound. These. different synthesis are currently in progress in our laboratory. Acknowledgements We gratefully acknowledge the aggregation test measurements performed partially in the laboratorics of" Dr. Benveniste (INSERM U200, Clamart, France) and Dr. Vargaftig (lnstitut Pasteur, Paris, France). The phosphorylation step of the synthesis was aided by C. Aymard and B. Rayner in Professor hnbach's laboratory (USTL, Montpellier, France).
References 1 B.B. Vargaftig and J. Benveniste, Trends Pharmacol. Sci. (1983) 341-343. 2 R.L. Wykle, C.H. Miller, J.C. Lewis, J.D. Schmitt, J.A. Smith, J.R. Surles, C. Piantadosi and J.T. O'Flaherty Biochem. Biophys. Res. Commun., 100 (1981) 1651 1658. 3 J. Benveniste, J.P. Le Couedic, J. Polonsky and M. Tencd, Nature, 269 (1977) 170-171. 4 J.J. Godfroid, F. Heymans, E. Michel, C. Redevilh, E. Steiner and J. Benveniste, FEBS Lett., 116 (1980) 161-164. 5 M. Tenc~, ~i. Coeffier, F. Heymans, J. Polonsky, J.J. Godfroid and J. Benveniste, Biochimie, 63 (1981) 723-727. 6 P. Hadvary J.M. Cassal, G. Hirth, R. Barner and H.R. Baumgartner, in J. Benveniste and B. Arnoud (Eds.), PAF Acether & Structurally Related Ether Lipids, Elsevier, 198 p. 57-60. 7 P. Hadvary and H.R. Baumgartner, Thromb. Res., 30 (1983) 143 156. 8 I..H. Valone, E. Coles, V.R. Reinhold and E.J. Goetzl, J. Immunol., 129 (1982) 1637-1641. 9 C.M. Chesney, D.D. Pifer and K.M. Huch, Thromb. Haemostasis, 50 (1983) 14-17.
226 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26
F.H. Valone and E.J. Goetzl, Immunology, 48 (1983) 141-149. D.P. Tuffin, P.J. Wade, D.O. Lunt and K.G. Mc CuUagh, J. Pharmacol., (suppl 1) 14 (1983) 67. S.B. Hwang, C.S.C. Lee, M.J. Cheah and T.Y. Shen, Biochemistry, 22 (1983) 4756-4763. H. Bailey and J.R. Knowles, in: W.B.P. Jakoby and M. Wilchek (Ed.), Methods in Enzymology, Vol. XLVI, Academic Press, 1977, pp. 6 9 - 1 1 4 . T.H. Ji and I. Ji, Anal. Biochem., 121 (1982) 286-289. G. Camussi, J.M. Mencia-Huerta and J. Benveniste, Immunology, 33 (1977) 5 2 3 - 5 3 4 . J.M. Mencia-Huerta and J. Benveniste, Eur. J. Immunol., 9 (1979) 4 0 9 - 4 1 5 . D. Buchnea, Lipids, 9 (1974) 5 5 - 5 7 . C.A.A. Van Boeckel, G.A. Vand der Marel, P. Wester Duin and 3.H. Van Boom, Synthesis, (1982) 399-402. A.E. Bolton and W.M. Hunter, Bioc'hem. J., 133 (1973) 5 2 9 - 5 3 9 . A.A. Spector and J.E. Fletcher, Nutrition & Drug Interrelations, Academic Press, New York, 1978. R.J. Robson, R. Radhakrishnan, A.H. Ross, Y. Tak Agaki and N.G. Khorana, in: P.C. Jost and O.H. Griffith (Ed.), Lipid-Protein Interactions, Vol. 2, J. Wiley, New York, 1982, pp. 149 175. J. Davoust, M. Seigneuret, P. Herv6 and P.F. Devaux, Biochemistry, 22 (1983) 3137-3145. J. Davoust, M. Seigneuret, P. Hervd and P.F. Devaux, Biochemistry, 22 (1983) 3146 3151. N.S. Chandrakumar and J. Hajdu, J. Org. Chem., 48 (1983) 1197-- 1202. M.M. Ponpipom and R.L. Bugianesi, Chem. Phys. Lipids, 35 (1984) 2 9 - 3 8 . M. Tenc6, E. Coeffier, J. Polonsky and J. Benveniste, Biochim. Biophys. Acta, 755 (1983) 526 530.