Synthesis of covalently immobilized phospholipid analogues

Chemistry and Physics of Lipids, 37 (1985) 307-315 Elsevier Scientific Publishers Ireland Ltd.

307

SYNTHESIS OF C O V A L E N T L Y I M M O B I L I Z E D P H O S P H O L I P I D ANALOGUES

M. WURL, D. ENTERLING and H. KUNZE Department of Biochemical Pharmacology, Max-Planck-lnstitute for Experimental Medicine. Herin attn- Rein- Strasse 3. D-3400 Gi#tingen (1~R. G. )

Recci,,cd January 30lh, 1985 accepted April 29th, 1985

revision rcceivcd April 291h, 1985

Cyclic l-O-acyl-2-O-alkyl-glyccro-3-phosphotriesters and I-O-acyl-2-O-alkyl-glyccro-3bromocthylphosphate with a free acyl moiety in position 1 of the glycerol backbone were synthesized. These phospholipid intermediates were covalently bound to AH-Sepharosc via the carbodiimide method. After immobilization the corresponding phosphatidylethanolaminc analogues were obtained by acid hydrolysis of the cyclic phosphotriesters and by direct amination of the bromoethylphosphate. Thus, in a short, stepwise synthesis including minimum use of protecting groups, a variety of immobilized phospholipid analogues are available as attinity adsorbents for the purification of enzymes related lo phospholipid metabolism. Keywords: phospholipid analogues; cyclic phosphotriesters; phospholipid-Sepharose; affinity

adsorbents

Introduction F e w a t t e m p t s have b e e n m a d e to s y n t h e s i z e i m m o b i l i z e d p h o s p h o l i p i d s with the aim of p u r i f y i n g e n z y m e s involved in p h o s p h o l i p i d m e t a b o l i s m [1-7]. In o r d e r to purify for e x a m p l e the s e m i n a l p h o s p h o l i p a s e A 2 [8], that s t r o n g l y p r e f e r s p h o s p h a t i d y l e t h a n o l a m i n e as s u b s t r a t e , it is necessary to s y n t h e s i z e p h o s p h o l i p i d s with specific structural characteristics, c o m b i n i n g the m i n i m a l s t r u c t u r a l s u b s t r a t e r e q u i r e m e n t s [91 with a p p r o p r i a t e p r o p e r ties r e q u i r e d for i m m o b i l i z a t i o n of the p h o s p h o l i p i d a n a l o g u e a n d purification of the e n z y m e : (i) position 1 of the glycerol b a c k b o n e must be a c t i v a t e d for c o u p l i n g to solid m a t r i c e s ; (ii) the p h o s p h o l i p i d must be r e s i s t a n t to h y d r o l y s i s by the p h o s p h o l i p a s e a n d c o n t a m i n a t i n g e n z y m e s ; (iii) the p o l a r h e a d g r o u p s h o u l d have high affinity to the e n z y m e ; (iv) the gel m a t r i x must be r e s i s t a n t to o r g a n i c s o l v e n t s and stable u n d e r acidic and alkaline pH conditions. 0009-3084/85/$03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd. Published and Printed in Ireland

308 Since synthetic routes for the acylation and alkylation of glycerol are well established [10], recent advances in glycerophospholipid syntheses have dealt with the phosphorylation procedure [11, 12]. Reaction of diglycerides with bromoethylphosphoric acid dichloride [121 followed by direct amination 113] has been successful for the preparation of cyclic phosphotriesters, the oxazaphospholanes. Both, the bromoethylphosphoric acid esters of diglycerides and the oxazaphospholanes of substituted glycerols appeared to be suitably protected phospholipid intermediates, capable of covalent binding to AH-Sepharose. Conversion of these phospholipid precursors after immobilization to the solid phase would then result in different types of covalently coupled phospholipid analogues for the purification of phospholipases.

Materials and Methods

Glycerol, diaminohexane, thionylchloride, bromoethanol, sebacic acid, aminopropanol, and N-methylethanolamine were products of E. Merck (Darmstadt, F.R.G.). 1-Cyclohexyl-3-(2-morpholinoethyl)-carbodiimidemetho-p-toluenesulfonate (CMC) was purchased from Sigma Chem. Co. (Miinchen, F.R.G.), Sepharose-CI 4 B from Pharmacia (Freiburg, F.R.G.) and 2,4,6-trinitrobenzenesulfonic acid from Serva (Heidelberg, F,R.G.). All solvents and reagents were of analytical grade. Rac-2-O-hexadecyl-3-O-benzylglycerol [14, 15], sebacic acid monobenzylester 116], bromoethylphosphoric acid dichloride [12] and AH-SepharoseCI 4 B [17] were prepared as previously described. The crude products were purified by column chromatography using silica gel 60 (0.07,'k~).2mm) (5-10ggel/g crude product) (E. Merck, Darmstadt, F.R.G.). Elution was performed by increasing the polarities of different solvent systems adapted to the respective products to be chromatographed. Determination of the phospholipid bound to Sepharose was carried out by measuring the amount of phosphorus according to Chen et al. [18] after ashing with Mg(NO3) 2 (E. Merck, Darmstadt, F.R.G.) according to Ames and Dubin [19]. The qualitative determination of free amino groups was achieved by the method of Cuatrecasas [20] with saturated borate, aqueous solutions of samples and 50 la,l of 2,4,6-trinitrobenzenesulfonic acid in a total volume of 1.5 ml.

Experimental

1. Rac- l-O-benzyl-2-O-hexadecyl-3-O-(w-benzyl)-sebacylglycerol Sebacic acid monobenzylester (29.2g; 0.10tool) and freshly distilled

309 thionyl chloride (17.8g: 0.15 tool) were heated under reflux until the cnd of gas production. Excess t)f thionvl chloride was removed by distillation, and the crude product (27g: 88% yield as based on the benzylcster) was uscd for the acylation of the substituted glycerol according to Eibl et al. [11] without further purification in order It) yield rac-I-O-ben×vl-2-() hexadecvl-3-O-(w-benzyl)scbacylglycerol (85%. based on 1-O-[~enzyl-2-() hexadecylglycerol), 2.

Rac- l-O-(¢o-benzyl)sehao'l-2-O-hexadecylglycerol (I) Rac-l-O-benzyl-2-O-hexadecyl-3-O-(oo-benzyl)sebacylglycerol

was debenzylated using catalytic hydrogenation [12]. The reaction was stopped when the molar ratio of hydrogen consumed to rac-l-O-benzyl-2-O-hexadecyl-3-O-(w-benzyl)sebacylg[ycerol was l : l . The benzylester bonding remains stable. The pure product was obtained on silica gel chromatography by raising the conccntration of diisopropylether ii1 #t-hexane from 5()
3. Rac-2-(l'-O-sebacyl-2'-O-hexadecylglycerol)-l,3,2-oxazaphosphane (II) and rac-2-(l'-O-sebacyl-2"-O-hexadecylglycerol)-l,3,2-(3-methyl)-oxazaphospholane (III) Phosphorylation and subsequent debenzylation were performed by the method of Eibl [11] using aminopropanol to synthesize rac-2-(l'-O-sebacyl2'- O-hexadecylglycerol)- 1,3,2-oxazaphosphane (II) and N-methylethanolamine to synthesize rac-2-(l'-O-sebacyl-2'-O-hexadecylglycerol)1,3,2-(3-methyl)-oxazaphospholane (Ill). The crude products were purified by silica gel chromatography. Elution was started with acetone/n-hexane (l:10. v/v) the polarity increased stepwise up to acetone/n-hexane (I:1, v/v). The yields of the pure products were 8.9g (II) (72%, as based on (I)), and 7.2 g (III) (58%, as based on (I)), respectively. The analytical data of (II) and (III) are summarized in Table I.

4, Rac- l-O-sebacyl-2-O-hexadecylglycerol-S-(co-bromoethyl)pho,v)hate

(IV)

(IV) was synthesized according to the method of Diembeck and Eibl [12] using rac-l-O-(w-benzyl)-sebacylglycerol and bromoethylphosphoric acid dichloride in a molar ratio of 1:2. Subsequent debenzylation was performed as described by Eibl [11]. The crude product was purified by silica gel chomatography. Starting with CHCI3/CH30H/25% aqueous ammonia (200: 15: l, by w)l) and increasing the polarity stepwise up to (65: 15: 1, by vol). The yield was 8.0 g (IV) (61%, as based on (l)). The analytical data of (IV) are shown in Table I.

IV

III

11

I

mediate

Inter-

C~H620~, (590.90) C32H62NO~P (619.83) C32H~NO,~P (637.83) C3tH~IBrOmP (7O4.70)

Formula {mol.wt. )

92-94

58-59

43

23- 25

(°C)

m.p.

52.84

60.26

62.00

73.17

Calcd.

'!' C

53.1)1

59.70

62.27

72.85

Found

Found 10.54 10.02 9.54 8.37

Calcd.

1(I.58 10.(18 9.79 8.44

% H

2.2/)

2.25

('aDd.

% N

The molecular weights of (111) and (IV) were calcuhlted for the monohydrates.

ELEMENTAL ANALYSIS OF T H E P H O S P H O I 3 P I I ) I N T E R M E D I A T E S

TABI.E 1

2.25

2.22

Fotlnd

4.41)

4.85

4.99

('alcd.

% P

4.18

4.49

4.98

Found

11.34

('alcd.

% Br

11.27

Found

311

5. Covalent coupling of the synthetic phospholipid analogues to AHSepharose-Cl 4 B 2× 10-4mol lipid were dissolved in 10ml aqueous tetrahydrofuran (THF)/H~O (1 : 1, v/v), pH 7.0, and combined with a solution of 1.5 × 10 3 mol CMC in 10 ml T H F / H 2 0 (pH 4.5) according to the methods described by Rock and Snyder ]2] and Kramer et al. [1]. After adding this mixture Io l g AH-Sepharose-CL 4 B previously suspended in 4 ml H20, the resulting pH was 5.(I. The reaction mixture was gently stirred for 48 h at room temperature, then filtered by suction and subsequently washed with 20(} ml CH3OH, 4(10 ml NaCI (1 M) and 800ml H20. The amount of lipid bound to the Sepharose was determined by estimation of phosphorus. The efliciencies of the coupling procedures are summarized in Table II. 6. Inactivation of residual amino moieties by acetylation Unsubstituted amino groups were acetylated as described by Inman and Dintzis [21]. After washing with NaCI (0.1 M) the gel was resus{)ended in NaCI ((I. 1 M) and 1 ml acetic acid anhydride per g gel was added dropwise. The pH was then held constant with NaOH. Reaction was complete when the pH remained constant without titration. After washing with H20 and NaCI (0. l M) no amino groups could be detected. 7. Conversion of covalently bound phospholipids Conversion of the cyclic phosphotriesters to the corresponding phospholipids was achieved by acid hydrolysis according to the method of Eibl [11] with the exception of compound (II). This latter ring system could only be cleaved with hydrochloric acid (1 M) at 37°C; the reaction time was 20 h. Controls of acylated AH-Sepharose-CI 4 B were treated in parallel. The qualitative analysis of amino.groups demonstrated complete hydrolysis of the ring without cleavage of the aminoacyl bond. No phospholipid could be detected in the washings. The bromoethylester (VII) was converted to the corresponding phosphatidylethanolamine analogue by direct amination as described by Eibl and Nicksch [13]. 2 ml (VII) were washed with acetone and dried under reduced

TABLE II BINDING EFFICIENCIES OF PHOSPHOLIPID ANALOGUES TO SEPHAROSE Analogue

~.mol lipid/g gel

% yield

II Ill IV

12.1 9.4 38.8

6.1 4.7 19.4

312

pressure. The gel was resuspended in a mixture of 2 ml 2-propanol, 2 ml CHCIa and 8 ml dimethylformamide (DMF). After addition of 8 ml aqueous ammonia (25%) the suspension was kept at 40°C for 6 h. The reaction was terminated by cooling to 0-5°C. The mixture was subsequently washed with H 2 0 , NaCI (1 M) and reswollen in NaCl (1 M) at 4°C. Qualitative analysis of amino groups showed no cleavage of the aminoacyl bond but proved the successful conversion of the bromoethylester to the ethanolamine group.

Results and Discussion

A simple method has been described for the synthesis of phospholipid intermediates which, after covalent coupling to a solid phase and conversion to the corresponding phospholipid analogues, should be useful in enzyme affinity chromatography. The reaction sequence is shown in Figs. 1-3. Coupling was achieved by the introduction of a semiprotected dicarboxylic acid into position 1 of the glycerol backbone of 2-O-alkyl-3-Obenzylglycerol using the acid-chloride-method. The chain length of the dicarboxylic acid was chosen to be 10 carbon atoms, because the total chain including the spacer arm connected with the solid phase should be comparable to palmitic acid, the naturally occurring substituent. Debenzylation of 1-O-acyl-2-O-alkyl-3-O-benzylglycerol, controlled by the molar ratio of hydrogen consumed, resulted in a quantitative and selective removal of the benzyl ether, whereas the benzyl ester remained stable. This synthesis generated (I) in an excellent yield (88% for acylchloride formation and 78%

CH2-OH I +C6Hs_CH 0 HO-CH ~ I pTs CH~-OH

+ KOH + C I - CH2- CsH5

CH2-O CH2-O I \ *KOH I \ +H~/MeOH HO-CH CH-C6H ~ +CH3SO3R" RO-CH CH-C6H ~ I / I / CH2-O i CHI-O

CH~-OH CH2-OCO-(CHz}a- COOCH~-C6H5 I I + CI OC- (CH2)B- COOCHz-C6Hs RO-CH RO- CH I I CH2-O- CH2-C6 Hs CHz-O- CHz- C6H5

CH~- OCO-(CHz}8- COOCHz-C6Hs Pd/H2 -HO-CH2-CBH5

CH2-OH I RO-CH I CH2-OH

RO-CH I CH2-OH

I

Fig. I. I-Acylation and l-alkylation of glycerol.

R= CH3-(CH2)IspTs=P-t oluenes ulfonic acid

313

I

.Poc,,

]

+POC,,

+HO (CHl)3 NH2~

T

I

+HO-ICH~12-NHCH31

CHT-OCO(CHz)8CODCH2-CBH ~

CH~-OCO-(CH2) H COOCHz-CfiH ~

I

I

RO-CH 0 0 CH;. CHT-O P~ CHz -NH-CH7/

CH2-DCO I

-HO-CHT-CBHqPd/ H~

CH2- 0C0- (CHl)FCOOH

HO CH2 C6HqPd

CH~

CH 2 OCD-(CH2)B-COOH I

RO-CH

I

O,.O_CHT\cH

CH2 0

P\NH CH2 /

I

Z

RO CH

O..O-CHz

CH 2 O-P,

I

H;

OCO(CH~%COOH 0

il

CH; O-P O-CH 2 CH? Br OH

N_CH 2 CH 3

T[

0H

CH;-O P O-CHz CH2 B~ OH

CH3

RO-CH

(CH2~8 COOCH? C~H~

d

RO CH

RO CH O/O_CH ~ CHFO-P'--~, "u '" ~"z

-HO CH2-CBHqPd/ H 2

+POC% +HO (CH,4? B~

1V

m

Fig. 2. Phosphorylation of disubsfiluted glycerol.

,,0 C~C.NH,V~NH ~

( ~ CNB, Hm ICH~I~-NH~

+lg

c0,cH,,.-c00., O c0,H" ~CH~-0.~

,.c0

HC 0B

0

CH~.\CH~-NH / P-O CHI

HC OR

CH21 N / P 0 CH 2 CH~

I H(t)/H20

I H~/H20

(~ (~) H N-CH?-CH-CH

Of, HC-Ofll

0 P 0 CH 2

CO-NH'~NH

VTI

OH

O CO'NH'~NH ÷NH3 aq

HC OR

I Br CH~ CH, 0 P 0 CH

INH2-CH2-CH~-O-P-O-CH2

'Vil

CO iCHI B COOCH, ,, 0

O. HC-OR

(~)

CO mCH~'8COOCH~

® 0 HC OR NH 3 CH 2 CH~ 0 P O CH~ 0

Fig. 3. Coupling to AH-Sepharose-C1 4 B and conversion to the corresponding phospholipids.

314 for acylation followed by debenzylation). The analytical data are shown in Table I.

Phosphorylution by the method of Eibl using propanolamine or Nmethyl-ethanolamine resulted in the formation of either a six-membeced (I1) o r a substituted five-membered (III) cyclic intermediate, respectively. The oxazaphosphane (II) and oxazaphospholane (III) are phosphotriesters, in which amino groups arc efficiently protected by internal cyclization. After debenzylation tile products were obtained in good yields (58% (111): 72% (II)). Using bromoethylphosphoric acid dichloride as phosphorylating agent according to the well established method of Hirt and Berchthold [22], the synthesis yielded 61% of the phosphodiester (IV) after removing the benzylesler protecting group. Table I summarizes the analytical data of these phospholipid intermediates, that are suitable for coupling to a solid phase. ('ovalent coupling of the phospholipid intermediates was performed by the carbodiimide method using the procedure described by Kramer et al. [1], with T H F / H 2 0 mixtures as solvents and the water-soluble carbodiimidc CMC in a 75-fold molar excess. The pH was adjusted to 5.0 which proved to bc optimal for amidation [1 l. Table I1 summarizes the efliciencies of lhc immobilization reactions. As can be seen from Table I1 all phospholipid intermediates exhibit low binding efliciencies, especially in the case of the cyclic phosphotriesters. In comparison, commercially available 1-O-(w-carboxy-undecyl)-2-O-hexadecyl-3-glycerol-phosphorylcholine (Berchthold, Bern, Switzerland), could be bound to AH-Sepharose-CI 4 B in a yield of I0()% (D. Enterling, M. Wurl, and H. Kunze, in prep,). It would be of interest to determine, whether cyclic structure, polarity or extension of the phospholipid head group influences the coupling reaction. In order to get altinity gels, that do not destroy chromatographic conditions by unspecific ionic interactions, the unsubstituted amino groups of AH-Sepharose-C1 4 B were inactivated by acetylation. The acidic pH of the acylation procedure did not affect the cyclic phosphotriesters. Conversion of the cyclic intermediates to the corresponding phospholipid analogues occurred at pH 2.0 in case of the 5-membered (III) and at pH 0.8 for the more stable six-membered (II) ring system. In addition the oxazaphosphane (II) intermediate had to he heated to 37°C for 20 h. Neither the acylation and conversion procedures nor tile washing solutions had any effect on the gel or the covalently bound phospholipids. This was controlled by qualitative as well as quantitative determinations of amino groups and phosphorus. The bromoethylphosphoric acid ester (VII) was converted to the corresponding phosphatidylethanolamine (VIII) by direct ]ruination. This reaction proved to be quantitative, since no bromine could be detected by elemental analysis. These synthetic routes are useful for the immobilization of a variety of

315

phospholipid anah~gues with difl'erent polar head gr¢mps which can be used for ~he purilicati¢m of ph¢~spholipases A= [23, 24l

Acknowledgemenl W c arc L'ra[cful to Pr¢~f. l)r. H. EIhl for cri|icnl rcmarks.

Referen ces I R M . Kramcr. C. W(ithrich. ('. l~llier. P R . Allcgrini and P. Zahler, l~i,~chim, l~i~ph~. Acta, 5()7 (lt)7g) 3gl 3t)4. 2 ('.(). R~ck and t:. Snyttcr, J. Biol. ('hem., 2e,(I (It)75)~5~4 65~¢'~. 3 ll.M. Vcrheii, A..I. Sl()II'~()~)m allt[ (;.H. tic ['laits, Re',. Pllysh)l., t)l (l()Sl)OI 2()3. 4 R. Berchlh~dd, ( ' h e m . Phys. l.ipids, 2g (19Nl) 55 -6(l. 5 R.J. Apitz-Castro, M.A. Max, M.R. Cruz and M.K. Jain, Biochem. Biophys. Rex. Commun., ~)1 (It)7t)) { ~ 7 I . ¢'~ %. Tahir and R ( ' . Hitler, Anal. Bi()chem., 135 (l~;g3) 332 334. 7 A.J. Aarsman, F. Neys and H. Van den Bosch, Biochim. Biophys. Acta, 792 (1984) 36_'~366. g H. Kunze, N. Nahas, J.R. Traynor and M. Wurl, Biochim. Biophys. Acta, 441 (1976} 93-102. t] A..l. Sl{)lh~l(tln allltI P.P.M. B(mxen, ( ' h e m . Phys. I.ipitls, 5 (It)7(t)3(11 3~),X. Ill It. Eihl, Proc. Nail. Acltd. Sci. U.S.A., 75 (1~78)41174~4II77. II H. Eibl, ( ' h c m . Phys. l.ipids, 2(~ (19811) 4II5-42t). 12 W. l)iembcck amd g . Eibl, ( ' h e m . Phys. l.ipids, 24 (lt)7t)) 237 244. 13 H. EIhl ~tlltt /\. Nicksch, ('hem. Phys. l.ipids, 22 (D)78) I S. 14 W..I. Baum~mn and |I.K. Mallg~tld, J. ()rg. Chem., 2~) (lt)(~4)3055 3057. 15 W..I. Baumamn and tt.K. Mangold, ,I. ()rg. ( ' h c m . , 3t (196(~) 4t)8 500. I¢'~ Y. lwakura and K. Wno. ( ' h e m . S(~c..lap. (.1. Pure (~hcm. N(~c.), 78 (1957) 15tl7 1511. 17 L.l. Barsukov, C.W. Dam, L.D. Bergelson, G.I. Muzija and K.W.A. Wirtz, Biochim. Biophys. Acta, 513 (It)7S} lt)bl-2It4. IbI P.S. Chen, "I'.Y. Torihara ~llld U. Warner, Anal. ( ' h e m . , 28 (195G) 175¢',- 175S. I t) B.N. A m e s and D.T. | ) u b i n , J. Biol. Chem., 235 (1961]) 76 t) 775. 211 P. ('uatrccasas, J. Biol. Chem., 245 (1970) 3(15()-3065. 21 .I.K. lnman and ll.M. l)inlzix, Biochemistry, b¢ (It)6tl)41)74MII,R2. 22 R. Hirt alnd R. Berchthold, Pharm. Acla Heir., 33 (195g) 349-35~'~. 23 M. Wurl and H. Kunze, t t o p p e - S e y l e r ' s Z. Physical. ('hem., 3~'~1 (1()£II) 13f,0. 24 M. Wurl and H. Kunze, Biochim. Biophys. Acta, 8,34 (1985) 411-418.