Synthesis, characterization and some properties of dideoxynucleoside analogs of cytidine diphosphate diacylglycerol

45

~~~c~~~~c~et Bi~p~y~icaAcfa, 1165 (1992) 45-52

0 1992 Elsevier Science Publishers B.V. All rights reserved 0~5-276~/92/~05.00

BBALIP 54040

Synthesis, characterization and some properties of dideoxynucleoside analogs of cytidine diphosphate diacylglycero~ GMT.

van Wijk a, KY. Hostetler b*c,C.N.S.P. Suurmeijer

a and H. van den Bosch a

a Center for Bfomembranes and Lipid Enzymology, University of Utrecht,Utrecht (Netherlands), b &al Inc., San Diego, CA (USA) and ’ Department of Medicine, Uniuersity of California, San Diego and VA Medical Center, San Diego, CA (USA)

(Received 24 April 1992)

Key words: Nucleoside diphosphate diacylglycerol; HIV; AZT; ddC; 3’-Deoxythymidine

Phospholipid conjugates of antiretroviral nucleoside analogs have been proposed to have several advantageous features when compared to the parent drugs (Hostetler, K.Y. et al. (1990) 3. Biol. Chem. 265, 6112-611’7). Here we report on the synthesis of one such type of lipid conjugates, i.e., nucleosides diphosphate diacylglycerols. The syntheses of 3’-azido-3’-deo~th~idine diphosphate dia~~glycero1, 3’-deo~thymidine diphosphate diacylg~ycero1 and 2~,3~-dideo~c~tidine d~phosphat~ diac~lg~yccro~ (with different acyl chains) were performed starting from phosphatidic acid and the antiviral nucleoside. A high-performance liquid chromatography procedure for a single step purification of the compounds is presented. The compounds were characterized biochemically, using rat liver enzymes and chemically by phosphorus, fatty acid, ultraviolet, IR and ‘H-NMR analyses. Preliminary data on the behaviour in aqueous solution of some of the compounds are presented.

Introduction The human immunodcf~ci~ncy virus (HIV) is the etiologic agent of the acquired immunodeficiency syndrome (AIDS) [1,2]. 3’-~~do-3’-deo~hymidi~e (AZT, Zidovudine) was the first drug approved for treatment of HIV infection after it was shown that it reduced morbidity and mortality of patients with AIDS or AIDS-related complex [3]. At the moment several compounds with structural analogy to AZT, commonly referred to as (substituted) dideoxynucleosides, are in various stages of clinical testing [4]. After anaboIic conversion to their triphosphates by host cell kinases, dideo~nucleosides interfere with the reverse transcription of viral RNA into viral DNA [4,5]. ~though compounds like AZT, Z’-3’-dideo~inosine (dd1) and 2’,3’-dideoxycytidine (ddC) have been shown to be

Correspondence to: H. van den Bosch, Center for Biomembranes and Lipid Enzymology, University of Utrecht, Padualaan 8, 3584 CH Utrecht, Netherlands. Abbreviations: NuDP-DAG, nucleoside diphosphate diacylglycerol; AZTDP-DAG, 3’-azido9’-deoxythymidine diphosphate diacylglycerol; 3dTDP-DAG, 3’-deoxythymidine diphosphate diacylglycerol; ddCDP-DAG, 2’,3’-dideoxycytidine diphosphate diacylglycerol; PA, phosp~atidic acid; HIV, human immunodeficien~ virus; AIDS, acquired immunodef~cien~ syndrome; HPLC, high-~~ormance liquid chr~matography~ TLC, thin-layer chromatography.

potent inhibitors of HIV both in vitro and in vivo, their use may be limited by toxic side effects 141.In addition, inhibition of HIV replication by dideo~~ucleosides in cells from the mono~te~macrophagc lineage, which have been reported to be a major reservoir of HIV infection [6,7], might not be very effective [8]. These non-proliferating cells have relatively low levels of nucleoside kinases [S,9] which are necessary to produce the active drug. We recently reported on the synthesis and biological activity of phospholipid analogs bearing a dideoxynucleoside as headgroup [lo]. Phospholipid conjugates of dideoxynucleosides might have a favourable metabolism, releasing an antiviral nucleoside mo~ophosphate directly, which bypasses nucleoside kinase activities. Especially dideo~nucleoside analogs of cytidine diphosphate diacylglycerol (CDP-DAG) could be metabolized in this manner. CDP-RAG is an obligatory intermediate in the biosynthesis of the anionic phospholipids phosphatidylinositol (PI), phosphatidylglycerol (PG) and diphosphatidylglycerol (DPG, cardiolipin) and upon these conversions cytidine 5’-monophosphate is released [ 111. Previously it has been shown that the enzymes involved in these biosynthetic conversions lack absolute specificity for the cytidine moiety. Adenosine-, arabinosylcytosine-, 2’-deo~cytidine-, guanosine-and uridine diphosphate dia~lg~ycerol can

46 all substitute to some extent for CDP-DAG as phosphatidyl donor [ 12- 141. This paper deals with the synthesis, purification and characterization of dideoxynucleoside analogs of CDPDAG, i.e., 3’-azido-3’-deoxythymidine diphosphate diacylglycerol (AZTDP-DAG), 3’-deoxythymidine diphosphate diacylglycerol (3dTDP-DAG) and 2’,3’-dideoxycytidine diphosphate diacylglycerol (ddCDP-DAG). All compounds showed anti-HIV activity in vitro (Ref. 10, unpublished results) and using rat liver subcellular fractions as enzyme sources, we demonstrated the release of the antiviral nucleoside 5’-monophosphates from their lipid precursor [ 15,161. Besides confirmation of the structure of the nucleoside diphosphate diacylglycerols, preliminary data on the physicochemical behaviour of some of the compounds is presented. Materials

and Methods

Materials Dimyristoyl, dipalmitoyland distearoylphosphatidic acids, all sodium salts, were obtained from Avanti Polar Lipids (Pelham, AL, USA). Dilauroylphosphatidic acid (sodium salt), 2’,3’-dideoxycytidine, 3’-deoxythymidine and Dowex 5OW (50 X 2-200, Hf form) were products from Sigma (St. Louis, MO, USA). 3’-Azido-3’-deoxythymidine 5’-monophosphate was a generous gift of Burroughs Wellcome (Research Triangle Park, NC, USA). Morpholine, dicyclohexylcarbodiimide (DC0 and 2-methyl-2-propanol (tertiary butylalcohol, t-BuOH) were the highest grade available from Aldrich (Milwaukee, WI, USA). Silica 60 F254 HPTLC plates, silica 60 F254 alufolien, phosphorusoxychloride, trimethylphosphate, HPLC grade solvents (Lichrosolv) and all other chemicals were obtained from Merck (Darmstadt, Germany), unless stated otherwise. Methods Analytical methods. Fatty acid content of nucleoside diphosphate diacylglycerols was measured as described by Shapiro [17]. Phosphorus was determined according to Rouser [18] and ultraviolet measurements were performed using a Hitachi 150-20 spectrophotometer. “P-NMR and ‘H-NMR spectra were recorded using a Brucker 300 MSL or a Brucker 360 spectrometer, respectively. Differential scanning calorimetry (DSC) was performed using a Perkin Elmer DSC-4 calorimeter. Infrared absorption spectra were obtained using a Perkin Elmer 1600 FT infrared spectrophotometer (KBr disc method). Melting points were determined with a Leitz melting point microscope. Experimental conditions are described in the legends of the figures or tables. Synthesis and purification. Nucleoside diphosphate diacylglycerols were synthesized by condensation of

nucleoside phosphomorpholidates with phosphatidic acids in anhydrous pyridine 1191. The reaction steps are described in detail below. Phosphorylation of 3’-deoxythymidine (3dT) and 2’, 3’-dideoxycytidine fddC). To a stirred solution of 2 mmol phosphorusoxychloride in 3-4 ml trimcthylphosphate on ice, 1 mmol of dry nucleoside was added stepwise [20,21]. The progress of the reaction was monitored by HPTLC or HPLC. For HPTLC analysis, an aliquot of the reaction mixture was spotted on a silica 60 F254 plate, which was developed with n-propanol/ 25% ammonia/water (20 : 20 : 3, v/v) as solvent system. The reaction was judged to be completed by the disappearance of the ultraviolet (UV> positive spot of 3dT or ddC and the appearance of a major UV- and phosphorus (Pi) positive spot of the respective 5’monophosphate. Alternatively, the reaction progress was monitored by HPLC using a Partisil IOSAX (Whatman) or MonoQ (Pharmacia) anion exchange column. Elution was performed as described [15] for the Partisil column or as presented below for the purification of the compounds (MonoQ). After completion of the reaction (usually within 2 h), the mixture was treated on ice with cold water to generate the product and the solution was neutralized with aqueous ammonia or NaOH to pH 7. This solution was applied to a Q-Sepharose fast flow anion exchange column (or MonoQ, for small amounts of product) and after washing with two bedvolumes of water the product was eluted isocratically with 0.1 M NH,HCO,. UV- and Pi-positive fractions were analyzed, pooled and repeatedly evaporated to dryness from methanol or methanol/water (1 : 1, v/v). Yields, after lyophilization, were generally > 70%. HPTLC analysis showed a single UV- and Pi-positive spot with I-propanol/25% ammonia/water (20 : 20 : 3, v/v) as developing system. AZT5’-monophosphate, R, = 0.63, 3dT-5’-monophosphate, R, = 0.61 and ddC-5’-monophosphate, R, = 0.5 1. All antiviral nucleoside monophosphates were readily dephosphorylated by rat liver mitochondrial enzyme preparations to the corresponding nucleosides [ 151. Nucleoside monophosphomorpholidates. Nucleoside 5’-monophosphate morpholidates were prepared essentially as described by others [ 19,221 with some modifications. A solution of 1 mmol nucleoside monophosphate was passed through a Dowex 50W (H’ form) cation exchange column (5 to 7 g wet weight) and the column was eluted with water until UV-absorbing material was no longer detected in the eluate. The eluatc was concentrated to approx. 10 ml and 10 ml t-BuOH and 4 mmol morpholine were added. This mixture was gently refluxed and a solution of 4 mmol dicyclohexylcarbodiimide in 15 ml t-BuOH was added from a dropping funnel over a period of 3 to 4 h. HPTLC analysis of the crude reaction mixture showed quantitative conversion of the nucleoside monophosphate

47 phosphatidic acid, This mixture was three times evaporated to dryness from pyridine under nitrogen. Finally, 30 ml of dry pyridine was added, approx. 15 ml was evaporated and the flask was tightly sealed. The progress of the reaction was checked by HPTLC analysis with chloroform/methanol/25% ammonia/water (70 : 38: 8: 2, v/v> as developing system. Maximal amounts of product were usually obtained between 4 to 7 days as judged by the appearance of a UV-and Pi-positive spot at R, between O-2 and 0.3, depending on the nucleoside. The reaction was stopped by evaporation of pyridine, followed by repeated evaporations from toluene. The residue was either directly purified or first extracted according to Bligh and Dyer [23]. Purification. The dry lyophilized crude reaction products were purified by silica gel chromatography using two different systems. Small amounts (up to 25 mg) were applied to an open silica gel column (silica 60, 230-400 mesh, Merck) and fractionated isocratitally using chloroform/methanol/25% ammonia/ water (70: 38: 8: 2. v/v) as eluent. Larger amounts were purified by means of HPLC using either a Waters FPorasil column (silica, 10 pm particles, 30 X 1.9 cm> or a home-made column (polygosyl, kieselgel 60, 5-20 ,um particles, 80 x 2 cm). Elution was performed isocratically with n-hexane/2_propanol/25% ammonia/ water (43 : 57: 3 : 7, v/v) as solvent, which allowed direct detection of the phospholipids [24]. This procedure allows purification of the liponucleotides in a single step, although care must be taken not to overload columns since this results in cross-contamination

within this time, using chIorofo~/methanol/acetic acid/water (50 : 25 : 7 : 3, v/v) as developing system. The reaction mixture was cooled to room temperature, filtrated and extracted with diethylether. HPTLC analysis of the aqueous phase showed a major UV- and Pi-positive spot between R, 0.4 to 0.6, depending on the nucleoside. This layer was evaporated to dryness and lyophilized, yielding > 90% of the nucleoside 5’monophoshomorpholidate as the 4-morpholidine-~, ~‘-dicyclohe~l~arboxamidine salt. This product was used without further purification for the synthesis of the nucleoside diphosphate diacylglycerols. Conversion of phosphatidic acid, sodium salt to the free acid. Dilauroyl, dimyristoyl, dipalmitoyl- and di-

stearoylphosphatidic acids (all sodium salts) were converted to the free acid using the extraction procedure of Bligh and Dyer [23]. Phosphatidic acid (1 mmol) was stirred for 1 h in a homogenous mixture of 100 ml chloroform, 200 ml methanol and 100 ml 0.1 M HCI. To this solution 100 ml chloroform and 100 ml 0.1 M HCl were added and the chloroform layer was isolated. The aqueous methanol layer was extracted twice with 200 ml chloroform and the combined chloroform layers were evaporated to dryness and lyophilized (yield > 95%). The dried products were used without further manipulations for coupling with nucleoside monophosphate morpholidates. Synthesis of nucleoside diphosphate diacylglycerols. 1 mmol of nucleoside monophosphate morpholidate was dissolved in 5 ml of anhydrous pyridine and this solution was transferred to a flask containing 1 mmol of

POC13

H20

(CH,O),PO

t -i3uOH /Hz0

-

X

X

X

/

0

H*C-o-L(CH,),-Cl-l, ::

CH,-(CH,)“---C-0-CH

I

/ pyridine

o CH,-(CH,)&O-CH

F;’

H2y-0-C-(CH&-Cl-& o H&-O-+-O&-

Fig, 1, Synthesis of NuDP-DAGs starting with phosphatidic acid and the nucleoside. Compound 1-4: X = N,, Y = thymine: 3’-azido-3’deoxythymidine diphosphate diacylglycerol (AZTDP-DAG). (11, n = 10, AZTDP-DAG, dilauroyl (diC12); (21, n = 12, AZTDP-DAG, dimyristoyl (diC14); (31, ?z= 14, AZTDP-DAG, dipalmitoyl fdiC16); f4), n = 16, AZTDP-DAG, distearoyl (diC18). Compound 5 and 6, X = H, Y = t~ymi~e:3’-deo~thymidine diphosphate diacylglycerol (3dTDP-DAG); 151, n = 10, 3dTDP-DAG, dilauroyl (diC12); (61, FI= 12, 3dTDP-DAG, dimyristoyl (diC14); ~rn~und 7 and 8, X = H, Y = ~tosine:2’,3’-dideo~c~idine diph~sphat~ diacyl~lycerol ~ddC~P-DAG~ (71, n = 10, ddCDP-DAG, dilauroyl (diC12); IS), n = 12, ddC~~-BAG, dimyristoyl (diCl4).

48

of the desired products with unreacted phosphatidic acid, which is still present in the crude mixture in considerable amounts. After evaporation of the eluens the compounds were extracted according to Bligh and Dyer [23]. The chloroform layer was neutralized with methanol/l M ammonia (1: 1, v/v), evaporated to dryness and lyophilized. Yields varied between 15 to 40% of pure products as their ammonium salts as white, voluminous, hygroscopic powders. R, values on HPTLC with chloroform~metha~ol/ 25% ammonias water (70: 38: 8: 2, v/v) were: AZTDP-DAG, 0.30; SdTDP-DAG, 0.29 and ddCDP-DAG, 0.25. Biochemical analysis. Liponucleotides were dried with a gentle stream of N, and dispersed in either 50 mM Tris-HCl (pH 7.51, or 50 mM citric acid/NaOH (PI-I 5), with or without 50 mM NaF. After addition of rat liver mitochondria as enzyme source the formation of phosphatidic acid (HPTLC) and nucleoside monophosphates (HPLC) was investigated, essentially as described recently [ 1.51.

have superior antiviral action when compared to the parent drugs [lo]. The anti-HIV analogs of cytidine diphosphate diacylglycerol, AZTDP-DAG, ddCDPDAG and 3dTDP-DAG, were synthesized essentially as described by Agranoff and Suomi [19], with several modifications in order to obtain the pure compounds. The yields of the actual condensation reaction between phosphatidic acid and nucleoside monophosphate morpholidate is rather low (640%) ‘. Purification of nucleoside diphosphate dia~ylglycerols from crude reaction mixtures prepared by the Agranoff method is quite challenging since considerable amounts of byproducts and residual phosphatidic acid are present in the reaction mixture. Purification of NuDP-DAGs on anion exchange columns was not very successful and did not separate NuDP-DAGs from phosphatidic acid. Silica chromatography using chloroform/methanol/25% ammonia/water (70 : 38 : 8 : 2) as eluent was successful, although the liponucleotide pool was easily contaminated with PA when columns were overloaded. Phosphatidic acid and nucleoside diphosphate diacylgly~erols have very close mobilities on HPTLC with several developing systems (data not

Results and Discussion Synthesis and purification

A complete method for the synthesis of antiviral nucleoside diphosphate diacylglycerols has been described (Fig. 11, starting from phosphatidic acid and nucleoside. These compounds have been proposed to

’ At this moment nucleoside diphosphate diacylglycerols are prepared by an alternative procedure in our laboratory, which has reaction times in the order of hours and gives better yields (%I80%). This procedure will be published elsewhere.

Compound

1

Formula

a

M* a

AZTDP-DAG diC12

Ratio h FA : Pi : Nu

A“XX(nm) _ ‘.7 . c,&. 1tV’) M-‘-cm-’

865.9

2.1 :2-O: 1.0

266; 12.St

922.0

1.8:2.0:0.X

266;

978.1

1.N:Z.O: I.0

266; 12.6kO.7

192-205

1034.1

2.1:2.0:

1.0

266; 12.5+0.9

194-204

1.1

280: 11.8+0.7

n.d.

280; lost

1X5-142

AZTDP-DAG diC14

Cu~dWW’2

AZTDP-DAG diCI6

C&s,

AZTDP-DAG diCl8

C4~Hs~NsOi~Pz

ddCDP-RAG die12

C,,H,SNJO,,P~

809.9

2.oz2.0:

ddCDP-DAG diC14

C,,H7,N,%Pz

866.0

2.1:2.0:1.0

3dTDP-DAG diCI2

C,7H,,N,o,,P2

824.9

2.o:z.o:

3dTDP-DAG diC14

CUJJXNZO,JP~

881.0

1.9:2.0:1.0

W&P;!

” As free acid. ” Ratio of fatty acid (FA):phosphorus (Pi): nucleoside c In chioroform/m~thauo~ (1: I, v/vk rd., not determined.

(Nu).

I.1

I.1

11.8* 1.0

1.3

,

m.p. (“0

141-144

170-178

269;

1.9 + 0.9

n.d.

269;

7.9+ 0.8

160-165

49 shown). HPLC purification on silica columns with ythexane/2-propanol/25% ammonia/water (43 : 57 : 3 : 7, v/v) was most successful. The eluent allows direct monitoring of phospholipids at A = 206 nm. Samples up to ‘100 mg crude product could be fractionated by this procedure in 30 minutes, the nucleoside diphosphate diacylglycerol eluting as a single peak, essentially free from phosphatidic acid. Ch~r~cterizut~o~ of ~~c~e~s~~e~~phosph~te ~~u~~~g~ycero~

In Table I some analytical data of the NuDP-DAGs are presented. All compounds have essentially the correct ratio of fatty acid/phosphorus/nucIeoside which amounted to 2: 2: 1. AI1 liponucleotides readily dissolved in chloroform/methanol (1: 1, v/v) and ultraviolet data were obtained with this solvent mixture. The la and A,,, values were essentially identical to those of the free nucleosides. The melting point of the compounds increased with increasing acyl-chain length. The melting points of dilauroyl ddCDP-DAG and 3dTDPDAG were not dete~ined due to the very hygroscopic behaviour of these two compounds. Infrared analysis of ddCDP-DAG, AZTDP-DAG and 3dTDP-DAG (all dimyristoyl) showed for all three compounds the typical phosphorus vibrational region

3!jOO

3600

2$00

2000

1800

between 1250 and 600 cm-‘, as is shown in Fig. 2 for AZTDP-DAG and 3dTDP-DAG. The spectra of these compounds are almost superimposable, with the exception of the peak at 2110 cm -t from the azido-group of AZTDP-DAG. The following assignments could be made: AZTDP-DAG diCl4, 2110 cm-’ (-N,), 1741 cm-’ (C = Q, fatt y acid), 1704 cm-’ (C = 0, thymidine), 1246 cm-’ (P= O), 1064 cm-’ (P-O-C), 952 cm-’ (P-O-P) and 519 cm-’ (P-O-P); 3dTDP-DAG diC14, 1741 cm-i (C = 0, fatty acid), 1699 cm-’ (C = 0, thymidine), 1241 cm-’ (P = O), 1068 cm-’ (P-O-C), 955 cm-’ (P-O-P) and 519 cm-’ (P-O-P). ‘H-NMR analysis of AZTDP-DAG, ddCDP-DAG and 3dTDP-DAG confirmed the structure of the compounds and chemical shift values (6 ppm) of major structural elements are presented in Table 11 Properties of NuDP-DAGs

AZTDP-DAG, 3dTDP-DAG and ddCDP-DAG are readily incorporated into the bilayer of sonicated Iiposomes consisting of dioIeoyIphosphatidyIchoIine/ cholesterol/ diol~oylphosphatidylglycerol/liponucleotide (5 : 3 : 1: 1, moI/mol). Fractionation of sonicated Iiposomes on a MonoQ anion exchange column showed a major (> 90%) peak (at h = 206 and 254 nm), which

1600

WAVELENGHT

1400

12&l

i tiO0

800

600

(cm-l)

B

3500

3000

2500

2000

1800

1600

WAVELENGHT

1400

1200

1000

800

600

(cm-l)

Fig. 2. Infrared spectrum (KBr disc, absorption) of AZTDP-DAG, dimyristoyl (A) and 3dTDP-DAG, dimyr~stoyl CB). The spectra are almost superimposable, except for the peak at 2110 cm-’ (azido, A).

50

upon HPTLC analysis showed that all four lipids were present in this peak (data not shown). In order to get some information on the aggregation of the pure compounds in aqueous solution, a “P-NMR study was performed with the AZTDP-DAG series (compound 1-4, Fig. 1). The liponucleotides (15 pmol) were hydrated overnight (approx. 16 h) at room temperature before recording the spectra. Compounds 1, 2 and 3 gave clear opalescent solutions under these conditions, but AZTDP-DAGdiC18 had a more turbid appearance. From the ” P-NMR spectra presented in Fig. 3 it can be concluded that none of the liponucleotides forms large aggregates under these conditions. No typical bilayer or hexagonal phase spectra are observed [25] and heating (50°C) or cooling (- 20°C) did not influence the shape of the typical narrow-range signal. The spectra of compounds 1-4, which are essentially identical, might represent either some kind of micellar organization or small vesicular structures [25]. Thermally induced phase transitions of the AZTDPDAG series were determined with differential scanning calorimetry (DSC). In Fig. 4 the thermogram of AZTDP-DAGdiC14 (compound 2) is presented, from which it can be seen that a major transition occurs which is centered at 11°C. Similar experiments with dilauroyl-, dipalmitoyland distearoyl AZTDP-DAG revealed major transitions at 5, 26 and 42°C respectively. The increase in the temperature at which the

TABLE

A

AZTDP-DG

11

Proton chemical shifts ‘I for the resonances of AZTDP-DAG, DAG and ddCDP-DAG AZTDPDAG

3dTDPDAG

3dTDP-

ddCDPDAG

I 8

I 6

I 4

CHJacyl chains) LU-CH2-(acyl chains) P-CH,-(acyl chains) -CH 2-(acyl chains) CH,-(sn-1, glycerol) CH-(sn-2. glycerol) CH,-(sn-3, glycerol)

0.87 2.30 1S8

1.25 4.15, 4.36 5.23 4.04

0.88 2.31 1.61 1.27 4.43 5.25 4.25

0.88 2.30 1.60 1.26

I’ll

5.89 2.48 3.97 4.53

6.07 2.08 4.07 4.51

5.96 1.97 3.78 _

3.86

3.90

3.42

1.96 7.77

_ _

_

6.15 1.35

base CH, (thymine) H6 (thymine) H5 (cytosine) NH1 (cytosine) were recorded standard.

I .84 1.35 _

I 0

I -2

I -4

I

-6

in CDCI, /MeOD

Fig. 3. “P-NMR spectra -diC14, -diC16 and -diClS. hydrated overnight with 1 Hepes (pH 7.4).

(121.4 MHz) of AZTDP-DAG diC12, Samples (15 Fmol) were lyophilized and ml 100 mM NaCI, 2 mM EDTA, 25 mM Spectra were recorded at 37°C.

5.24 4.08

rihose

2’H 3’H 4’H S’H

I

2

PPM

diacylglycerol

L’ Spectra internal

di Cl 2

(1: 1, v/v)

with TMS as

major transitions occur, is directly related to the increase in acyl chain length of the liponucleotides, a correlation that has been observed by others for various phospholipids [261. Combining the results obtained from both DSC and “‘P-NMR experiments, one might speculate that these liponucleotides spontaneously aggregate into small bilayer vesicles in aqueous solution, the observed phase transition representing the melting of the acyl chains. However, care must be taken for further generalization and interpretation of these data. Yang et al. [27,28] studied the biophysical properties of CDP-DAG and araCDP-DAG in aqueous solutions by light mi-

51

croscopy, ‘H-NMR, gelfiltration and turbidity measurements. The investigators showed that these structural analogs of AZTDP-DAG exhibit a rather anomalous aggregational behaviour. When CDP-DAG or araCDP-DAG were gently dispersed in aqueous buffer, they spontaneously formed large vesicles of several microns (sometimes up to 50 pm) in diameter [27,28]. After two h, the average diameter of the vesicles had decreased in size and gelfiltration revealed that the vesicles eluted in a similar manner as sonicated phosphatidylcholine vesicles. However, only after several days a thermodynamically stable aggregation of the liponucleotides was observed, which appeared to be micelles with a radius of approx. 4 nm. Ordinarily, the formation of small vesicles requires, e.g., sonication methods, and vesicles and micelles are usually not formed by a single (phosphojlipid species [25,26]. Our present work suggests that also AZTDP-DAG can aggregate spontaneously into small vesicles. This timedependent polymorphic phase behaviour of the dideoxynucleoside diphosphate diacylglycerols has not yet been studied further. Different acyl chain lengths of phospholipids also can have implications for their susceptibility to phospholipid-metabolizing enzymes [29]. In view of these observations we tested AZTDP-DAG diC12, -diC14, -diC16 and -diC18 as substrate for a mitochondrial pyrophosphatase activity from rat liver [15]. As is shown in Fig. 5, the order of conversion of AZTDP-DAG to AZTMP and phosphatidic acid increases with decreasing chain length. This phenomenon has also been observed by others [29] in studies on the influence of acyl-chain length of CDP-DAG in the biosynthesis of phosphatidylglycerol. Whether this enhanced hydrolysis rate of AZTDP-DAG with decreasing chain length

I

I

I

/

I

I

3

0

5

10

15

20

I

25

temperature ("C)

Fig. 4. DSC endotherm of AZTDP-DAG, dimyristoyl. The sample was cooled and heated several times before recording the spectrum. Sample composition: approx. 2 Fmol liponucleotide in 30 ~1 of 100 mM NaCI, 40 mM EDTA, 10 mM Pipes (pH 7.4).

0

0

200

400

AZTDP-DG

600

600

1000

(FM)

Fig. 5. Influence of the acyl chain on the release of AZTMP from AZTDP-DAG diC12 (W-m), -diC14 (O-O), -diClh (o-0). and -diC18 ( q -II). Assays were performed as described in Ref. 15 at pH 5, using rat liver mitochondria

as enzyme source.

is a true enzymatic preference is not known. It might well be that the organization of the liponucleotides in aqueous solution and their subsequent association with mitochondria is responsible for this effect. In itself, the conversion of NuDP-DAG to the antiviral nucleoside monophosphates and phosphatidic acids which has been proven for all eight liponucleotides indicated in Table I (Ref. 15, unpublished results) confirms the structure of these compounds. Conclusion

A complete method for the synthesis and purification of dideoxynucleoside diphosphate diacylglycerols is described. The compounds are of potential importance in treating the monocyte/macrophage reservoir of HIV infection [lo]. Cleavage of the pyrophosphate bridge of the liponucleotides with rat liver mitochondrial enzyme preparations results in the formation of phosphatidic acid and the nucleoside monophosphates, confirming the structure of the compounds. The correctness of the structure of the compounds was further determined by ‘H-NMR-, IR-, phosphorus-, ultraviolet- and fatty acid analyses. 31P-NMR spectra of 15 mM preparations of the AZT series showed essentially no difference and the shape of the spectra suggests that these lipids, in pure form, are not assembled into large bilayer structures, although more detailed studies will have to be performed to elucidate the biophysical properties of this type of compounds [27,28]. On the other hand, AZTDP-DAG, ddCDP-DAG and 3dTDPDAG (all dimyristoyl) are readily incorporated into liposomes (10% liponucleotide of total lipid). Preliminary experiments using [ 3H]3dTDP-DAG diC14 incorporated into sonicated vesicles indicated that the compound is rather rapidly (t ,,* is 20 to 30 min) transferred between artificial membranes and that there is essentially no flip-flop (data not shown). Recently we started studies on the liposomal behaviour of 3dTDP-

52 DAG, making use of pyrene-labeled analogs of 3dTDP-DAG, which wil1 allow more accurate and continuous monitoring of these types of compounds in artificial membrane systems [30]. Acknowledgements This research was supported in part by Vical Inc., San Diego, CA, USA. Dr. KY, Hostetler is supported by NIH grant GM 24979 and by the Research Center for AIDS and HIV infection of the San Diego VA Medical Center, San Diego, CA, USA. Mr. W.S.M. Geurts van Kessei is gratefully acknowledged for his advices on the HPLC purification of the iiponucleotides. We would like to thank Dr. J.A. Killian and Dr. K. Nicolay for discussing “‘P-NMR results. Mr. E.Th.G. Lutz is gratefully acknowledged for recording and discussing FT-IR spectra. References errs-S~noussi, F., Chermann, J.C., Rey, R., Rey, F., Nugeyre, MT., Chamaret, S., Gruest, J., Dauguet, C.. Axler-Blin, C., Vezinet-Brun, F., Rouzioux, C., Rozenbaum, W. and Montagnier. L. (1983) Science 220, 868-871. Gallo, RX., Salahuddin, S.Z., Popovic, M., Shearer, G., Kaplan, M., Hayes, B., Palker, T., Redfield, R., Oleske, J., Safai, B., Whites, G., Foster, P. and Markham, P. (1984) Science 224, 500-503. Fischl, M.A.. Richman, D.D., Grieco, M.H., Cottlieb, M.S., Volherding, P.A.. Ldskin, O.L., Leedom, J.M., Groopman, J.E.. Mildvan, D., Schooley, R.T., Jackson, G.G., Durack, D.T. and King, D. (19871 N. Engl. J. Med. 317, 1X5-191. Yarchoan, R., Mitsuya, H., Meyers, C.E. and Broder, S. (1989) N. En& J. Med. 321, 727-738. Furman, P.A., Fyfe, J.A., St.Clair, M.H., Wcinhold, K., Rideout, J.L., Freeman, G.A., Lehrman, S.N., Bolognesi, D.P., Brader, S., Mitsuya, H. and Barry, D.W. (1986) Proc. Nat]. Acad. Sci. USA 83, 8333-8337. Gendelman, H.E., Orenstein, J.M., Baca, L.M., Weiser, B., Burger, H., Kalter, D.C. and Meltzer, MS. (198Y) AIDS 3, 475-4’)s.

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