IN THE accompanying paper (1) we described the isolation. Synthesis of sulfate esters of. phosphatidylglycerol (diphytanyl ether analog)

Transcription

1 Synthesis of sulfte esters of J phosphtidylglycerol (diphytnyl ether nlog) A. J. Hncockl nd M. Ktes2 Deprtment of Biochemistry, University of Ottw, Ottw, Ontrio, Cnd, K1N 6N5 Abstrct Synthesis of 1-sn-phosphtidyl-3 -sn-glycero-1 -sul- fte (phosphtidylglycero-1-sulfte) ws chieved bymonosulftion of l-sn-phosphtidyl-3 -sn-glycerol (diphytnyl ether nlog) with n equimolr mount of SO-pyridine complex t room temperture; with excess sulftion regent t 6OoC, the 1,2 -disulft ester ws obtined. The phosphtidylglycero-2-sulfte isomer ws synthesized by n unmbiguous route strting from the bcteril 2,3-di-O-phytnyl-sn-glycero. The synthetic phosphtidylglycerosulftes were chrcterized by nlyticl, chromtogrphic, opticl rottory, nd spectrl (infrred nd NMR) dt nd compred with the phosphtidylglycerosulfte isolted from Hlobcterium cutirubrum. Supplementry key words phosphosulfolipid hlophilic bcteri. 1-sn-phosphtidyl-3 -sn-glycero-1 -sulfte. l-sn- phosphtidyl-1 -sn-glycero-2 -sulfte. -sn-phosphtidyl-3 -snglycero-l,2 -disulfte. 2-0-myristoyl-3-0-benzyl-sn-glycerol sulftion of phosphtidylglycerol. 3-0-benzyl phosphtidylglycerol. NMR spectr. infrred spectr. opticl rottions. thin-lyer chromtogrphy N THE ccompnying pper (1) we described the isoltion nd chrcteriztion of new phosphosulfolipid in the extreme hlophile, Hlobcterium cutirubrum. Structurl studies (1) indicted tht this lipid ws probbly primry sulftester of sn-2,3-diphytnyl-glycero-lphosphoryl-sn-3 -glycerol (phosphtidylglycerol, diether nlog) (PG). Finl proof of this structure ws obtined by comprison (1) of the bcteril lipid with the synthetic sn-1- nd sn-2-sulfte esters of PG. We present here, in detil, the procedures for synthesis of these compounds. The synthesis of the 1,2-disulfte ester of PG is lso included for comprison. The procedures used re summrized in Schemes 1 nd 2. MATERALS AND METHODS The SOs-pyridine complex nd 2,4,6-triisopropvlbenzenesulfonyl chloride (TPS) were products of Aldrich Chemicl Go. (Milwukee, Wis.). Phosphorus ws determined by the method of Allen (2), nd sulfte by the brium chlornilte procedure of Spencer (3), s described in the ccompnying pper (1). Opticl rottions were mesured in Perkin-Elmer 141 utomtic-redout polrimeter. nfrred spectr were tken in solution with Beckmn R-O spectrophotometer. 100-MHz NMR spectr were recorded for dilute solutions in [2H]chloroform with Vrin HA-100 NMR spectrometer equipped with 31P-1H spin decoupler. Chemicl shifts re expressed s ppm(6) reltive to trimethylsilne tken s zero. This work forms prt of Ph.D. thesis submitted by A. J. Hncock to the Deprtment of Biochemistry, University of Ottw, for the Ph.D. degree. Supported by grnt A-5324 from the Ntionl Reserch Council of Cnd. Abbrevitions: TLC, thin-lyer chromtogrphy; NMR, nucler mgnetic resonnce; PG, phosphtidylglycerol; PGS, phosphtidylglycerosulfte; PG-14, 1-sn-phosphtidyl-3 -sn-glycero-1 - sulfte; PG-2-S, -sn-phosphtidyl-1 -sn-glycero-2 -sulfte; PG- 1,2-di-S, 1-sn-phosphtidyl-3 -sn-glycero-l,2 -disulfte; TPS, 2,- 4,6-triisopropylbenzenesulfonyl chloride. Present ddress: Deprtment of Biochemistry, School of Medicine, Cse Western Reserve University, Clevelnd, Ohio To whom requests for reprints should be ddressed. 430 Journl of Lipid Reserch Volume 14, 1973 EXPERMENTAL PROCEDURES SYNTHESS OF PHOSPHATDYLGLYCERO--SULFATE (PG-1-S) (Scheme 7) Bcteril 2,3-diphytnyl-sn-glycero-lphosphoryl-sn-3 -glycerol (PG) (1) This lipid ws isolted from totl polr lipids of H. cutirubrum s mixed (N, Mg, NHd) slt, s described in the ccompnying pper (1).

2 0- /pipyrdm 0- c 0-r-0-1% 3 RO-CH H M H RO-CH2 so- () 5CQH C%-O-i-O-C% Hy-OH RO-YH Tm PYRDNE ROCH -03SO-CH -60. RO-CH HO-CH Q Q c~-o$-o - o - CH2"2 - ROCH 0 80-CH RO-CH 3 1 SCHEME Synthesis of phosphtidylglycero-1-sulfte nd phosphtidylglycero-1,2-disulfte (diphytnyl ether nlogs).!2,3-diphytnyl-sn-l-glycerophosphoryl-sn-3 '- glycero-1 '-sulfte (PG-1-S) (2) A solution of "mixed" slts of PG (1 ; 42 mg, 52 pmoles) in 5 ml of nhydrous benzene ws stirred with SO,-pyridine complex regent (9.5 mg, 60 pmoles) t room temperture for 6 hr (see Scheme 1). The rection mixture showed mjor spot on TLC (RF 0.38 in chloroform-methnol-90% cetic cid 30:4: 20 [v/v/v]) corresponding to the monosulfted product nd only trces of disulfted product ( RF 0.12). The mixture ws clered by centrifugtion, concentrted in nitrogen strem, nd chromtogrphed on preprtive silic gel H pltes in chloroform-methnol-concentrted mmonium hydroxide 65 : 35 : 5 (v/v/v). The monosulfted product ws eluted with 250 ml of chloroform-methnoldiethyl ether 1 : 1 : 1 (v/v/v) nd the elute ws evported to dryness in the presence of benzene; the residue ws dissolved in 10 mi of chloroform-methnol 1 : 1 (v/v), clered by centrifugtion, nd diluted with 4.5 ml of 0.5 N queous HCl. After brief centrifugtion of the biphsic system, the chloroform phse ws neutrlized with 0.2 N methnolic KOH, diluted with benzene, nd concentrted to smll volume (c. 0.5 ml), which ws diluted with 10 vol of cetone. After severl hours t O"C, the precipitted potssium slt ws centrifuged down, wshed with cold cetone, nd dried in vcuo to Synthesis of nlog). phosphtidylglycero-2-sulfte (diphytnyl ether give 38 mg (39 pmoles, 75% yield) of chromtogrphiclly pure compound (2) (white powder). The synthetic product ws indistinguishble on TLC from the bcteril PGS (Tble 1) nd gve nlyticl dt expected for the TABLE 1. Chromtogrphic mobilities of sulfted derivtives of phosphtidylglycerol RF Vlue in Solvent System: Compound 1" 2' 3' 4b Phosphtidylglycero-1-sulfte (bcteril) Phosphtidylglycero-1-sulfte (synthetic) (2) Phosphtidylglycero-2-sulfte (11) Phosphtidyl-3-0-benzyl-snglycero-2-sulfte (10) Phosphtidylgycerol-1,Z-disulfte (4) Phosphtidylglycerol (1) Phosphtidylglycero-1-sulfte, dimethyl ester (bcteril) Phosphtidylglycero-1-sulfte, dimethyl ester (synthetic) (3) Phosphtidylglycerol, monomethyl ester TLC on silic gel H in solvent systems: 1, chloroform-methnol-90yo cetic cid 30: 4: 20 (v/v/v); 2, chloroform-methnolconcd mmonium hydroxide 65 : 35 : 5 (v/v/v); 3, chloroformmethnol-wter 90: 10: 1 (v/v/v). Chromtogrphy on silicic cid-impregnted pper in solvent system 4: diisobutylketone-cetic cid-wter 40: 25 : 5 (v/v/v). Hncock nd Ktes Synthetic phosphtidylglycerosulftes 431

3 ~~~ A (Synthetic) J z 0 v) 2 v) z W 0 w n v B DMETHYL ESTER PO- - s (SYNTHETC) WOO 2ooo WAVE NUMBER cm" FG. 1. nfrred spectr (CCl) of synthetic phosphtidylglycero-1-sulfte. A, dipotssium slt; B, dimethyl ester. TABLE 2. Dt Found Clcdo c, % H, % p, % s, % K, % S/P tomic rtio 1.oo 1.oo K/S+P tomic rtio oo Anlyticl dt for synthetic phosphtidylglycerosulftes PG-123, K Slt PG-2-S, K Slt PG-1,2-di-S, K Slt Found Clcdo Clcdb Found oo o oo 0 Clculted for C~~H~~OPSKZ.HZO (981.47). Clculted for C ~~H~~O~PS~K~.H~O (1099.6). TABLE 3. Opticl rottionp for potssium slts of sulfted derivtives of phosphtidylglycerol Compoundb (K Slts) 589 nm 578 nm 546 nm 436 nm 365 nm Phosphtidylglycero-1-sulfte (bcteril)c 1-sn-Phosphtidyl-3 '-sn-glycero O +2.14O +2.29' +4.14' +6.29' 1 '-sulfte (synthetic) +2.88' +2.97' +3.32" +5.53O sn-Phosphtidyl-3 '-sn-glycero- 1 ',2 '-disulfte +4.06' +4.13' +4.31" +6.96' +11.loo (1 n chloroform solution t 22 C. All compounds re derivtives of 2,3-di-O-phytnyl-sn-glycero-l '-phosphoryl-sn-3 '-glycerol. Dt for synthetic 1-sn-phosphtidyl-3 '-sn-glycero-2 '-sulfte re not vilble. Dt from previous pper (1). 432 Journl of Lipid Reserch Volume 14, 1973

4 PO- 1.2-di-S 2 0 v) E v) z 4 c c z W 0 W n PO-1.2-di-S TRMETHYL ESTER ~ ~.. ~ ~ ~ 30oo 2Ooo m OOQ K WAVE NUMBER cm" FG. 2. nfrred spectr (CCl) of synthetic phosphtidylglycero-1,2-disulfte. A, tripotssium slt; B, trimethyl ester. potssium slt of PGS (Tble 2). ts opticl rottions re given in Tble 3 nd its infrred spectrum is shown in Fig. 1A. Dimethyl ester of synthetic PG1-S (3) Synthetic PG-1-S (8 mg) ws converted to its dimethyl ester with dizomethne s described for the bcteril PGS (1). The RF of the dimethyl ester is given in Tble 1, nd its infrred nd NMR spectr re shown in Figs. 1B nd 4A, respectively. 2,3-Di-~phytnyl-sn-glycero-l-phosphoryl-3'-snglycero-l',Z '-disulfte (PG1,Z-di-S) (4) A solution of "mixed" slts of PG (1) (25 mg, 30 pmoles) in 5 ml of nhydrous benzene ws stirred with sulfur trioxide-pyridine regent (16 mg, 0.10 mmole) t 6OoC for 1 hr (see Scheme 1). The mixture ws centrifuged, the superntnt solution ws concentrted in nitrogen strem, nd the product ws chromtogrphed on preprtive TLC pltes in chloroform-methnolconcentrted mmonium hydroxide 65 : 35 : 5 (v/v/v) to remove trces of monosulfted product (RF 0.12 nd 0.33 for PG-1,Z-di-S nd PG-1-S, respectively). The disulfte ws eluted from the silic nd converted to its tripotssium slt s described for the monosulfted derivtive. The precipitte ws wshed with cold cetone nd dried in vcuo to give 18 mg (17 pmoles, 57% yield) of chromtogrphiclly purewhite powder. The tripotssium slt of PG-1,2-di-S hd the mobilities in vrious solvents given in Tble 1; its nlyticl dt, opticl rottion vlues, nd infrred spectrum re given in Tbles 2 nd 3 nd Fig. 2A, respectively. Trimethyl ester of PG1,Z-di-S (5) PG-1,2-di-S (6 mg) ws converted to its methyl ester with dizomethne s described for PG--S. The infrred spectrum of the trimethyl ester is shown in Fig. 2B nd the NMR spectrum in Fig. 4B. SYNTHESS OF PHOSPHATDYLGLYCERO-2-SULFATE (PG-2-S) (Schme 2) 2,3-Di-~phytnyl-sn-glycerophosphoric cid (6) This compound ws prepred by phosphoryltion of the nturl diphytnyl glycerol ether (2,3-di-O-phytnyl-sn-glycerol [4]) s described by Ktes et l. (5). The potssium slt hd [CY]$ = +1.75" (3.1 g/dl in chloroform); reported []$ = +1.78" (5). Anlysis: C ~~H~VO~PK~.H~O (827.32); clculted: P, 3.74 found: P, 3.80 Hncock nd Ktes Synthetic phosphtidylglycerosulftes 433

5 SYNTHETC PO-2- S DMETHYL ESTER (SYNTHETC) 1 1 1,,, l 1 1,, 1 1, so ldoo WAVE NUMBER cm" FG. 3. nfrred spectr (CC14) of synthetic phosphtidylglycero-2-sulfte. A, dipotssium slt; B, dimethyl ester. The potssium slt ws converted to the free cid form s described elsewhere (5). 2-O"yristoyl-3-O-benzyl-sn-glycerol (7) This substnce ws prepred s described for the steroyl compound by De Hs nd vn Deenen (6) by monocyltion of 3-U-benzyl-sn-glycerol (7) ; it ws seprted from the 3-0-myristoyl isomer by preprtive TLC using sodium cette-impregnted pltes to prevent cyl migrtion. Exmintion of the product by NMR s described previously (6) showed tht it contined only trce of the 3-U-myristoyl isomer. 2,3-Di-~phytnyl-~~-glycero-l-phosphoryl-l'- (2 '-O-myristoyl-3 '-O-benzyl)-smglycerol (8) The dried free cid form of 2,3-di-O-phytnyl-snglycerophosphte (6), prepred from the potssium slt (120 mg, 0.16 mmole), ws mixed with solution of 2-0- myristoyl-3-0-benzyl-sn-glycerol (7) (125 mg, 0.32 mmole) in 10 ml of nhydrous pyridine, nd the mixture ws brought to dryness under reduced pressure. The residue ws dried in vcuo over phosphorus pentoxide for 12 hr nd then stirred with 20 ml of nhydrous pyridine nd dry TPS (194 mg, 0.64 mmole) t room temperture for 8 hr (Scheme 2). The mixture ws then cooled on ice, stirred with 1 ml of wter for 30 min, nd evported to dryness under reduced pressure. Triturtion of the residul oil with cold diethyl ether precipitted triisopropylbenzenesulfonic cid, which ws removed by centrifugtion nd wshed twice with diethyl ether. The combined etherel superntes were wshed with wter, 0.5 N hydrochloric cid, gin with wter, nd brought to dryness under reduced pressure in the presence of benzene. The residul oil on TLC in chloroform-methnolconcentrted mmonium hydroxide (80: 20: 2, V/V/V) showed mjor phosphte-positive spot with RF The crude product ws purified by preprtive TLC first in chloroform-diethyl ether 3 : 1 (v/v) (RF 0.00) ther in chloroform-methnol-concentrted mmonium hy droxide 90: 10: 1 (v/v/v) (Rp 0.30) in the sme direction The product ws eluted with chloroform-methnoldiethyl ether 1 : 1 : 1 (v/v/v), converted to the potssium slt, nd precipitted by cetone, s described for PGS (1). The oily precipitte ws wshed with cold cetont nd dried in vcuo; yield, 137 mg (0.2 mmole, 75%) 01 chromtogrphiclly pure product (8) hving []E = (4.1 g/dl in chloroform). Anlysis: C~TH~~RO~PK (1145.8); clculted: P, 2.70 found: P, Journl of Lipid Reserch Volume 14, 1973

6 ppm (8) " ppm (8 ppm (8) FG. 4. NMR spectr (expnded downfield region; sweepwidth, 250 Hz) in [*H]chloroform of: A, synthetic phosphtidylglycero-lsulfte dimethyl ester; B, phosphtidylglycero-l,z-disulfte, trimethyl ester; nd C, phosphtidylglycero-2-sulfte. Top spectr re lp-lh decoupled; bottom spectr re norml. The infrred spectrum (liquid film) of compound (8) showed bsorption for the following groups: ester crbonyl (1740 cm"), romtic (3025, 2065, 3095, 1495, 690 cm"), phytnyl (2850, 2930, 2960, 1455, [doublet] cm-l), myristoyl (CHz), (730 cm-'), bonded P=O (1240 cm-l)), C-0-Cy P-0- (1100 cm"), nd P-0-C (1055 cm-l). Hydroxyl bsorption ws bsent. 2,3-Di-O-phytnyl-snglycero-l-phosphoryl-l - (3'-@benzyl)-sn-glycerol (3-o-benzyl-PG) (9) The 2-0-myristoyl derivtive (8) ws decylted by modifiction of the procedure described elsewhere (8), s follows: solution of (8) (potssium slt; 137 mg, 0.12 mmole) in 2.0 ml of chloroform-methnol 2:3 ws treted with 2.0 ml of 0.2 N methnolic NOH t 40 C. Decyltion ws complete fter 40 min (RF vlues of [8] nd decylted product [9] were 0.70 nd 0.48, respectively, in chloroform-methnol-concentrted mmonium hydroxide 80 : 20: 2 [v/v/v]) ; 3.2 ml of chloroform, 0.8 ml of methnol, nd 3.6 ml of 0.5 N queous HC were then dded successively to the cooled rection mixture. The biphsic system ws briefly centrifuged, nd the chloroform phse ws neutrlized with 0.2 N methnolic KOH nd concentrted to smll volume. The residul oil ws diluted with cetone, nd the precipitted phospholipid product (9) ws centrifuged down, wshed with cold cetone to remove trces of methyl myristte, nd dried in vcuo; yield, 89 mg of oily product (0.095 mmole, 79%). An nlyticl smple ws obtined by preprtive TLC in chloroform-methnolconcentrted mmonium hydroxide 80 : 20 : 2 (v/v/v), followed by reconversion to the potssium slt s described bove. t hd [CY:] = +l.o" (2.43 g/dl in chloroform). Anlysis: CS~H~~~O~PK (935.41); clculted: C, 68.05; H, 10.78; P, 3.31 found: Cy 67.85; H, 10.21; P, 3.43 The infrred spectrum closely resembled tht of the myristoyl derivtive (8) except for the bsence of crbonyl nd (CH2), bsorption (1740 nd 730 cm-l, respectively) nd the presence of hydroxyl bsorption (3280 cm", brod). Hncock nd Ktes Synthetic phosphtidylglyccrosulftcs 435

7 2,3-Di-~phytnyl-sn-glycer~l-phosphoryl-l'- sn-glycero-3 '-0-benzyl-2 '-sulfte (10) A solution of compound (9) (potssium slt; 85 mg, mmole) in 15 ml of nhydrous benzene ws stirred t 60 C for 3 hr with SO3-pyridine complex regent (30 mg, 0.19 mmole). The cooled mixture ws clered by centrifugtion, the residue ws wshed with benzene, nd the combined superntnt solutions were concentrted to smll volume under reduced pressure. The crude product ws purified by preprtive TLC in chloroform-methnol-concentrted mmonium hydroxide 80 : 20 : 2 (v/v/v) (Rp 0.18), nd the desired product ws eluted from the silic nd converted to the potssium slt form s described for compound (9); yield of chromtogrphiclly pure compound (10) (white powder) ws 75 mg (0.071 mmole, 75%); CY]^ = "2.38" (2.10 g/dl in chloroform). Anlysis: C~~H~~O~OPSKS-HZO (1055.6); clculted: C, 60.30; H, 9.64; P, 2.93; S, 3.04; S/P tomic rtio, 1.OO found: C, 60.91; H, 9.24; P, 2.95; S, 3.23; S/P tomic rtio, 1.06 The infrred spectrum resembled tht of compound (9) except for the reltively more intense bsorption bnd centered t 1240 cm" (P=O, nd S=O symmetric stretch) nd the sulfte bnds t 1045 cm" (shoulder, symmetric stretch S=O), 940 cm-l (C-0-S), nd 840 cm" (C-0-S, wek) ; hydroxyl bsorption ws bsent. Dimethyl ester of 3-C)-benzyl-PG-2-sulfte The dimethyl ester ws prepred s described for PG-1-S. The infrred spectrum showed sulfte bsorption t 1405 nd 1195 cm" nd t 935 cm" (intense, C- 0-S, secondry sulfte). Hydroxyl bsorption ws bsent. NMR ssignments: phytnyl (78 H), ; P"OcH3, doublet 3.72, (which collpsed to singlet on 31P-1H spin decoupling) ; S-OCH3, singlet 3.946; "OCH~C~HS, 4.57; "OCH&&5, ,3-Di-~phytnyl-s~-glycero-l-phosphoryl-l '-snglycero-2'-sulfte (PG2-S) 3-0-Benzyl phosphtidylglycerosulfte (10) (potssium slt; 40 mg, 0.38 mmole) ws hydrogenolyzed with plldium-chrcol ctlyst (4) in 6 ml of chloroformmethnol 1 : 1 (v/v) t room temperture nd pressure. After 30 min, the ctlyst ws removed by centrifugtion nd wshed with 4 ml of chloroform-methnol 1 : 1 (v/v); the combined superntnt solutions were immeditely diluted with 4.5 ml of 0.1 N queous HCl, nd the biphsic system ws briefly centrifuged. The chloroform phse ws neutrlized with 0.2 N methnolic KOH, nd the potssium slt of the PG-2-S (11) ws isolted [CY]: s described for compound (10). Yield, 35 mg (94y0); = -6.08" (1.65 g/dl in chloroform). Anlyticl dt for PG-2-S potssium slt re given in Tble 2. The PG-2-S migrted s single spot in three solvent systems (see Tble 1) ; in ech system the PG-2-S hd slightly higher mobilities thn the bcteril PG-1-S. The infrred spectrum of PG-2-S potssium slt is shown in Fig. 3A. Dimethyl ester of PG-2-S (12) The ester ws prepred s described for the PG-1-S. The infrred nd NMR spectr re shown in Figs. 3B nd 4C, respectively. Bcteril PGS The "nturl" form of PGS ws isolted from the lipids of H. cutirubrum s described in the ccompnying pper (1). RESULTS AND DSCUSSON The synthesis of the PG-1-sulfte ws crried out by direct sulftion of the pure bcteril phosphtidylglycerol (1) (diphytnyl ether nlog) (Scheme l), using n equimolr mount of SO3-pyridine complex t room temperture. Under these conditions, monosulftion occurred lmost exclusively ( ) ; the resulting PGS (2) ws isolted s the dipotssium slt in good yield (c. 75%) in chromtogrphiclly (Tble 1) nd nlyticlly (Tble 2) pure stte. The infrred spectrum of the dipotssium slt, s well s of the dimethyl ester (3) (Fig. l), showed bsorption t 980 cm" (shoulder), indictive of primry sulfte, nd only wek bsorption t 935 cm", ttributble to secondry sulfte (9). The mjor S-OCH signl t in the NMR spectrum of the methyl ester (Fig. 4A) could therefore be ssigned to primry S-OCH group nd the minor S-OCH, signl t to secondry sulfte group. t ppered then tht monosulftion of PG occurred predominntly t position 1 nd only to limited extent (c. 8%) t position 2. Sulftion of PG t 6OoC with n excess of SOs-pyridine complex (Scheme 1) gve the chromtogrphiclly (Tble 1) nd nlyticlly (Tble 2) pure PG-1,2- disulfte (4). ts infrred spectrum (Fig. 2) showed both primry nd secondry sulfte C-0"s bnds (995 nd 940 cm", respectively), nd its NMR spectrum (Fig. 4B) hd two well-resolved S-OCH3 signls of equl intensity t ppm. These dt indicted tht discrete S-OCH, signls (differing by 3 Hz) in the NMR spectrum re in fct given by primry nd secondry sulfte esters,.the ssignments for these groups being most likely the upfield nd downfield signls, respectively. 436 Journl of Lipid Reserch Volume 14, 1973

8 To estblish the ssignments for primry nd secondry monosulfte S-OCH, signls in the NMR spectrum unequivocbly, the synthesis of the phosphtidylglycero- 2-sulfte ws crried out by the unmbiguous route shown in Scheme 2. n this procedure, bcteril 2,3-di- 0-phytnyl-m-glycerol ether ws converted (5) to diphytnyl ether phosphtidic cid (6), which ws redily condensed in the presence of TPS in dry pyridine (10) with 2-0-myristoyl-3-0-benzyl-sn-glycerol (7) to give the cyl PG benzyl ether (8). The ltter ws decylted in wrm methnolic NOH, nd the benzylted product (9) wsulfted with SO-pyridine complex in dry benzene t 60 C. Debenzyltion of the resulting compound (10) ws redily chieved by hydrogenolysis over plldium-chrcol ctlyst, nd the synthetic PG-2-S3 (11) ws isolted s its potssium slt or converted to its methyl ester (12). The chromtogrphiclly (Tble 1) nd nlyticlly (Tble 2) pure product obtined showed only the secondry sulfte bsorption bnd (935 cm-l) in the infrred (Fig. 3) nd gve single S- OCH3 NMR signl t ppm (Fig. 4C). The upfield S-OCH3 signl ( ppm, Fig. 4) my be ssigned unmbiguously to the primry sulfte ester group, nd the synthetic monosulfted PG (2) must therefore be the PG--S isomer. Since only the synthetic PG-1-S isomer ws found to be identicl with the PGS isolted from H. cutirubrum (Tbles 1-3 nd Figs. 1 nd 4; see Ref. 1) the nturl compound must hve the structure l-sn-phosphtidyl-3 '-sn-glycero-1'-sulfte (diphytnyl ether nlog) (see Fig. 4, Ref. l). This compound hs the configurtion sn-l-phosphtidyl-1'- sn-glycero-2 "sulfte. The sn-1-phosphtidyl-3 '-sn-glycero-2'-sulfte isomer would hve been more pproprite, but its synthesis required the 1-0-benzyl-sn-glycerol (11) s strting mteril, which ws less redily ccessible thn the 3-0-benzyl isomer (7) used here. NMR nd R spectrl ssignments for the secondry sulfte re, however, independent of the configurtion of the sulfted glycerol moiety. The uthors re indebted to Mr. F. McClusky for the NMR spectr, nd to Mrs. E. Szbo nd Mr. G. Ben-Tchvtchvdze for the figures. Elementl nlyses were performed by A. Bernhrdt, Mikronlytisches Lbortorium, Elbch uber Engelskirchen, West Germny. The support of this work by grnt (A-5324) from Ntionl Reserch Council of Cnd is grtefully cknowledged. Mnuscript received 24 October 7972; ccepted 27 Februry REFERENCES 1. Hncock, A. J., nd M. Ktes Structure determintion of the phosphtidylglycerosulfte (diether nlog) from Hlobcterium cutirubrum. J. Lipid Res. 14: Allen, R. J. L The estimtion of phosphorus. Biochem. J. 34: Spencer, B The ultrmicro determintion of inorgnic sulfte. Biochem. J. 75: Joo, C. N., T. Shier, nd M. Ktes Chrcteriztion nd synthesis of mono- nd diphytnyl ethers of glycerol. J. Lipid Res. 9: Ktes, M., C. E. Prk, B. Plmet, nd C. N. Joo Synthesis of diphytnyl ether nlogues of phosphtidic cid nd cytidine diphosphte diglyceride. Cn. J. Biochem. 49: De Hs, G. H., nd L. L. M. vn Deenen Structurl identifiction of isomeric lysolecithins. Biochim. Biophys. Act. 106: Ktes, M., T. H. Chn, nd N. Z. Stncev Aliphtic diether nlogs of glyceride-derived lipids.. Synthesis of D-,p-dilkyl glyceryl ethers. Biochemishy. 2: Mrshll, M. O., nd M. Ktes Biosynthesis of phosphtidylglycerol by cell-free preprtions from spinch leves. Biochim. Biophys. Act. 260: Hines, T. H The chemistry of the sulfolipids. Progr. Chem. Fts 0therLipid.c. ll(3): Anej, R., nd A.P. Dvies The synthesis of spin-lbelled glycero-phospholipid. Chern. Phys. Lipids. 4: Gigg, J., nd R. Gigg Benzyl-L-glycerol nd D-(glycerol1,2trbonte). J. Chem. Soc. C Hncock nd Kks Synthetic phophtidylglyceroeulfrrtee 437