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if3~,phYiafiYl, sn.glycero, l,phosphoric acid (monophytanyl ether acid"6aS ¢ffeCtedbY condensation of l'iodo-2:O-benzyl-3-O-phy" 'dl;P,nittobenZylph0~hate in anhydrous toluene followed by cataresultingphosphotriester to remove the benzyl and p-nitrobenzyl iymnybsn.glycero-l-phosphorybl:sn-glycerol (monophytanyl ether yl gl~rol)was carried out by conversion of the above phosphotriester e ii~i~bly blocked kVsoph0sphatidicacid which was condensed with zYi,sn~yceroliRemOvalOffheprotectingaromatic and t-butyl groups .triesterin ... intermediate gave the desired phytanyl ether analogue of lysoi lyso analogues were isolated as analyticany and chromatographically nhv~ieatm6t~ertieS and behavior towards acid hydrolysis are described.
~.,1.,In~odu~ion DUring biosynthetic studies of the diphytanyl ether phospholipid analogues present [ls0fextl .~mely halophilic bacteria [ 1] several unidentified intermediates, rapidly . . . . . . . ad~t4C; w e r e • detected [M. Kates, P.W. Deroo, J. Oliver, unpublished ~reofthese intermediates had mobilities on TLC similar to those of ,'iid........ andtysc, ,phospha:tidyl~ . . . . glycerol. Since the monophytanyl glycerol these !ysophospholipids might be expected as intermediates in the
2i3 di O.phytanyl-sn,glycerol etherphospholipids, we undertook micalsyn esi s 0f3~-phytanyl'sn,glycer°'l'ph°sph°ryl"I -sn-glycerol. Dete tnati0n0f~t~e ! ysicai~an d che,mical properties of:these "lyso'" analogues should ~!:iheir I as possible intermediates for biosynthesis of the diphytanyl . . . . . . . .identific~ ~;ion !
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by ~A.J. Hancock in partial fulfullment of the Ph.D. 973,We dedicate this paper to the memory of Prof. niversity of Missouri, Kansas City, Mo. 64110,
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158
M. Kates, A.J. Hancock, Monophytanyl ether lysophosp/zatides
Table 2 Acid hydrolysis of monophytanyl ether analogue of lysophosphatidic acid. Distribution of P, % Hydrolytic Agent
Time (hr)
2.5% methanolic-I1CI (anhydrous) 2 N methanolic-HCla,b 2 N aqcous-HCl b 2 N aqueous-HCI b 2 N aqueous-HCI b
Methanol-water phase
Chloroform phase
5
0
100
72 12 24 72
65
3S
86
14 9
91 100
a
----------------------
a Solution made by diluting 10 ml cone. HCI to 60 mt with methanol. b Sealed tube hydrolysis at 130°C.
of monophytanyl glycerol ether (Rf 0.3 on TLC in CHCI 3 -ether, 3: 1) plus traces of the chlorinated monoether (Rt 0.8 in CHCI 3 -ether, 3: 1) described previously [21. Slow hydrolysis of the lyso analogue was not due to its low solubility in the aqueouS acid, since it was hydrolyzed even slower in methanolic HCl, in which the free acid form was soluble (table 2). The synthesis of the monophytanyl ether analogue of lysophosphatidyl glycerol (se l Scheme I) involved the condensation of a suitably blocked monosilver salt of the monophytanyl ether analogue of lysophosphatidic acid with a protected glycerol iodohydrin derivative, followed by removal of the blockIng groups by mild procedures. This procedure is analogous to that used previously by van Deenen and coworkers for the synthesis of O-aminoacylphosphatidyl glycerols [4-6] and by Joo and Kates [7] for the synthesis of diphytanyl ether analogues of phosphatidyl glycerol and phosphatidyl glycerophosphate. For synthesis of the lysophosphatidyl glycerol analogue, anionic debenzylation of the triester (IV) with sodium iodide, followed by treatment with silver ion, was used to prepare the required mono-p-nitrobenzyl silver salt intermediate VI and the protected iodoglycerol was l-iodo-2-0-t-butyl-3-0-benzyl-sn-glycerol VII synthesized as described elsewhere [7]. Condensation of compound VI with the protected iodo compound gave the fully blocked triester (VIII) isolated in good yield. R~moval of nitrobenzyl and benzyl groups from the triester (VIII) was effected by catalytic hydrogenolysis and the butyl group was removed by treatment with anhydrous HCl in chloroform. Although removal of the aromatic protecting groups proceeded smoothly removal of the 2 -O-butyl blocking group with anhydrous CHCl 3 -HCl was accompanied by some cleavage of the phosphodiester bond. The resulting free lysophosphatidyl glycerol was converted to the monopotassium salt (X) which was freed from some contaminating monophytanyl glycerol by acetone precipitation and preparative TLC. Elemental analyses of the chromatographically pure monopotassium salt were consistent with those expected for a monohydrated salt. The potassium salt (X) was freely soluble in chloroform and benzene, sparingly soluble in Carbon tetrachloride
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silica gel H layers (0,25 mm or vely) on 20 X 20 cm glass plates. mol-30% ammonium hydroxide acetic acid (30 : 4 : 20, v/v). :id impregnated Whatman 3 MM (40 : 25 : 5, v/v) as described else-
lysophosphatidic acid (potassium salt, ca. 2 ltmoles) were hydrolysed under three different conditions as follows: (1) Samples were heated in 4 mlof 2.5% methanolic hydrogen chloride under reflux for 1,3 or 5 hr; t o each •hydr01ysate were added 4 ml of chloroform and 3.6 ml of water and the hi• : " " ~e was analysed for total P [16].(2)The hanolic,HCl (prepared by diluting 10 ml of ealedtube at 130°C for 72 hr. The cooled tuantitatively transferred to a centrifuge 1.2ml of methanol and2 ml of chloroform. I, (3) Samples were treated with 1 ml of 2N ~r 12i 24 or 72 hr. The contents of each tube '.,0 ml of methanol and 2.0 ml of chloroform. dysed for P [161.
ether analogue of &sophosphatidic acid " "-nyl glycerol) Iriphenyl 1 hr at 10°C ted as de~as purified
~Oml of
' ; ~-i.il(~ .,>:i 9 {
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163
0.27 in chlor'oi~ri(ll)as a c0iourleSs 0il~w thRf ivelyl 0.56and An: Lndbenzene,:respec analytical sample was _dieth~ Lether (3 : I,V/V), TheRRR-iso-
: •:F, :i~' :.~:i!i;RRt !:!!:,~/:~::i M:
~!:69:iiHi::~!ii:!56:RRt~::isomer:: C, 77.52; H, 11.24"
'i:~
ionitophytanyl and aromatic absorp ti0n'~NMR assignments: phytanyl (39 H) ~ 0!75-~i~:65~i:~y~rol~methyleneand methine (7.H) 3.30-3.80 8; benzylic methylene : i~(2H)4iTOi~garomatic(5 H) 7.35 6, 4, i.O,p. Toluenemlphonyl.2.0-benz y l, 3-O-phy tany l'sn'glycerol ~:. 2.0~Benzyl~3,0,phytanyl-sn'glycerol [(II); 770 mg, 1.64 mmole] was reacted :with!recrystallisedp.fiiuenesulphonyl ~chloride (620 mg, 4i0 mmole) in 10 ml anhy: ar2,~ ~nvridine at o°andlthe mixture was then stirred at 10°C for 16 hr. The reaction ' dm: to100 ml o(ice-water, and extracted with four 30-ml portions ~ture!was P0Ure . . ofpetroleUm ether (30-60°c). The combined extracts were washed wRh water, ~cecold 2 Naquous HCI and water, and dried by evaporation with benzene under reduced pressure togivethe tosyl derivative as a colourless oil (809 mg, 82% yield;Rf • 0,fi8 in chloroform). An:analytical ~mple was obtained by preparative TLC in chloroform' The pure RRR-isomer had [~]~D2 + 1.43° (c, 6.8 in chloroform).
Found: RRR.RRS-isomer: C, 72.06; H, 9.69; S, 5.71. RRR-isomer" C, 71.93; H, 9.81; S, 5,35. C37H6oO5 S(616,91)
.
.
.
.
.
.
• Calc,: C:,72,03;H, 9.80;S, 5.20.
. showed tosyl group absorption (1182, 1195 cm -1, o hydroxyl group absorption was apparent. NMR [,85 6; CH3-C6H4-(sharp singlet)2.42 8; methyl; ..O.CH2-C6H5 (sharp singlet) 4.57 6; C6H4CH3
OSO2_(doublet) "L76 6. 5. l.lodo-2-O-benzyb3"O'phytanyl'sn'glycer°l (III) ,tanyl-sn-glycerol (697 mg, 1.15 mmole) ' 6.0 mmole) in 15 ml of anhydrous acethen for 20 hr under reflux. After re~,centrifugation the acetone supernatant te residual oily solid was extracted centrifuged,washed with 5% aqueous
97%::eld).
' C30Hi~'(,2'[ (5 72' '
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Found
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~tes,A:J~Hancock,:Monophytanylether:&sophosphatides
1.65~.
e catalyst was rem0ve.d byeentrifugati0n and washed with two Jor0form-ethan01 (i : 1,v/v)~ The combined supernatants were rreducedpressure, and the residual oil, dissolved in 5ml chloro~thl 5ml methanol ~ d 4.5 ml lr.0NHCI. The biphasic system was ~iiii~h!~of0rm!pha~ was neultrali~d:(phenolphthalein end point) ~ic!i: ;e~aPoratedm the presence of benzene to0.6 iml, and ~faiiet0ne,~ o n e Afier~o01ing , i C e , the gelatinoUs precipitate was cenithcold acetone and reprecipitated from its chloroform solution by umes .... ofdiethyl ..... ett~er.~e, lysophosphatidic • acid analogue (V) was i.¢e~!mg (45% yield) of chromato~'aphically pure white solid .~lues). The R/~:,isomer had [~]~z + 7..69° (c, 2.0 in chloroform) ~r:C, 51.98;H, 8.86; P, 5.83.
9':::iii
Z- '
caic= :e,; aa4: ; H,8,96;P. 5.s6, i~e infrared spectrum ofthe lysophosphatidic acid analogue (potassium salt) is shown infig, 1. C.~
ether analogue of lysophosphatidyl glycerol
: :L Silver salt of l-O-(mono-pnitrobenzylphosphorylJ-2-O-benzyl,3~O-phy tanyl- snglycerol ( VI) Asolution o f the phosphotriester (IV) (270 rag, 0.33 mmole)and sodium iodide (500 mg, :3.3 mmole)in 20 ml of anhydrous acetone was heated under reflux with stirring for 6hr. The solvent was evaporated under reduced pressure to give an oily solid, which was extracted with chloroform.The extract was cooled to 0°C, centrifuged, and evaporated to adark Oil (448 rag), which was purified on a silicic acid column (20 g)prepared in hexane. Elution with chloroformremoved a dark oil, whose infrarcd spectrumwas consistent with that ofp,nitrobenzyl alcohol, after which elution i!~th: chlorof0rm-met~nol (1:1, v]v) gave the sodium salt of Vl (brown oil, 169 mg),Rf 0~33and 0.76 in chloroform,methanol (9 : 1, v/v)and chloroform-methanol~30%ammonium hydroxide (65:35 : 5, v/v), respectively. An analytical sample wa$obtained by preparative TLCin chloroform-methanol-wate'r (80 : 20: 2, v/v) ::(R~O:50): !~e pure sodium salt was eluted from the silica with chloroform-methanoi-diethyl eti~er(i: 1::1, v/v). The RRR-RRS-isomer had [a] 22 + 4.29 ° (c, 4.2 in
chloroform). FoUnd: RRR,RRS-isomer: C, 63.61; H, 8.57; N, 2.09; P, 4.40. bsorption bands for nitro (1050¢m -1), and ionized bands were absent.
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if3~,phYiafiYl, sn.glycero, l,phosphoric acid (monophytanyl ether acid"6aS ¢ffeCtedbY condensation of l'iodo-2:O-benzyl-3-O-phy" 'dl;P,nittobenZylph0~hate in anhydrous toluene followed by cataresultingphosphotriester to remove the benzyl and p-nitrobenzyl iymnybsn.glycero-l-phosphorybl:sn-glycerol (monophytanyl ether yl gl~rol)was carried out by conversion of the above phosphotriester e ii~i~bly blocked kVsoph0sphatidicacid which was condensed with zYi,sn~yceroliRemOvalOffheprotectingaromatic and t-butyl groups .triesterin ... intermediate gave the desired phytanyl ether analogue of lysoi lyso analogues were isolated as analyticany and chromatographically nhv~ieatm6t~ertieS and behavior towards acid hydrolysis are described.
~.,1.,In~odu~ion DUring biosynthetic studies of the diphytanyl ether phospholipid analogues present [ls0fextl .~mely halophilic bacteria [ 1] several unidentified intermediates, rapidly . . . . . . . ad~t4C; w e r e • detected [M. Kates, P.W. Deroo, J. Oliver, unpublished ~reofthese intermediates had mobilities on TLC similar to those of ,'iid........ andtysc, ,phospha:tidyl~ . . . . glycerol. Since the monophytanyl glycerol these !ysophospholipids might be expected as intermediates in the
2i3 di O.phytanyl-sn,glycerol etherphospholipids, we undertook micalsyn esi s 0f3~-phytanyl'sn,glycer°'l'ph°sph°ryl"I -sn-glycerol. Dete tnati0n0f~t~e ! ysicai~an d che,mical properties of:these "lyso'" analogues should ~!:iheir I as possible intermediates for biosynthesis of the diphytanyl . . . . . . . .identific~ ~;ion !
"
r
by ~A.J. Hancock in partial fulfullment of the Ph.D. 973,We dedicate this paper to the memory of Prof. niversity of Missouri, Kansas City, Mo. 64110,
155
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,
•
.
.i."
_
,I.
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..
,
.
.
.
.
x
2
l ~
¸ : I,~O
1200 iO00 i
$00
(potassium salt) in cch.
158
M. Kates, A.J. Hancock, Monophytanyl ether lysophosp/zatides
Table 2 Acid hydrolysis of monophytanyl ether analogue of lysophosphatidic acid. Distribution of P, % Hydrolytic Agent
Time (hr)
2.5% methanolic-I1CI (anhydrous) 2 N methanolic-HCla,b 2 N aqcous-HCl b 2 N aqueous-HCI b 2 N aqueous-HCI b
Methanol-water phase
Chloroform phase
5
0
100
72 12 24 72
65
3S
86
14 9
91 100
a
----------------------
a Solution made by diluting 10 ml cone. HCI to 60 mt with methanol. b Sealed tube hydrolysis at 130°C.
of monophytanyl glycerol ether (Rf 0.3 on TLC in CHCI 3 -ether, 3: 1) plus traces of the chlorinated monoether (Rt 0.8 in CHCI 3 -ether, 3: 1) described previously [21. Slow hydrolysis of the lyso analogue was not due to its low solubility in the aqueouS acid, since it was hydrolyzed even slower in methanolic HCl, in which the free acid form was soluble (table 2). The synthesis of the monophytanyl ether analogue of lysophosphatidyl glycerol (se l Scheme I) involved the condensation of a suitably blocked monosilver salt of the monophytanyl ether analogue of lysophosphatidic acid with a protected glycerol iodohydrin derivative, followed by removal of the blockIng groups by mild procedures. This procedure is analogous to that used previously by van Deenen and coworkers for the synthesis of O-aminoacylphosphatidyl glycerols [4-6] and by Joo and Kates [7] for the synthesis of diphytanyl ether analogues of phosphatidyl glycerol and phosphatidyl glycerophosphate. For synthesis of the lysophosphatidyl glycerol analogue, anionic debenzylation of the triester (IV) with sodium iodide, followed by treatment with silver ion, was used to prepare the required mono-p-nitrobenzyl silver salt intermediate VI and the protected iodoglycerol was l-iodo-2-0-t-butyl-3-0-benzyl-sn-glycerol VII synthesized as described elsewhere [7]. Condensation of compound VI with the protected iodo compound gave the fully blocked triester (VIII) isolated in good yield. R~moval of nitrobenzyl and benzyl groups from the triester (VIII) was effected by catalytic hydrogenolysis and the butyl group was removed by treatment with anhydrous HCl in chloroform. Although removal of the aromatic protecting groups proceeded smoothly removal of the 2 -O-butyl blocking group with anhydrous CHCl 3 -HCl was accompanied by some cleavage of the phosphodiester bond. The resulting free lysophosphatidyl glycerol was converted to the monopotassium salt (X) which was freed from some contaminating monophytanyl glycerol by acetone precipitation and preparative TLC. Elemental analyses of the chromatographically pure monopotassium salt were consistent with those expected for a monohydrated salt. The potassium salt (X) was freely soluble in chloroform and benzene, sparingly soluble in Carbon tetrachloride
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n, tp.trae.h]~ride solution with ~..... [ZH]" ~ , N M R spectra were . . . . . .recorded ' t 00NMR spect'rometer. Optical rotations at22°C ina Perkin-Elmer polarimeter model
silica gel H layers (0,25 mm or vely) on 20 X 20 cm glass plates. mol-30% ammonium hydroxide acetic acid (30 : 4 : 20, v/v). :id impregnated Whatman 3 MM (40 : 25 : 5, v/v) as described else-
lysophosphatidic acid (potassium salt, ca. 2 ltmoles) were hydrolysed under three different conditions as follows: (1) Samples were heated in 4 mlof 2.5% methanolic hydrogen chloride under reflux for 1,3 or 5 hr; t o each •hydr01ysate were added 4 ml of chloroform and 3.6 ml of water and the hi• : " " ~e was analysed for total P [16].(2)The hanolic,HCl (prepared by diluting 10 ml of ealedtube at 130°C for 72 hr. The cooled tuantitatively transferred to a centrifuge 1.2ml of methanol and2 ml of chloroform. I, (3) Samples were treated with 1 ml of 2N ~r 12i 24 or 72 hr. The contents of each tube '.,0 ml of methanol and 2.0 ml of chloroform. dysed for P [161.
ether analogue of &sophosphatidic acid " "-nyl glycerol) Iriphenyl 1 hr at 10°C ted as de~as purified
~Oml of
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meoek~i~:Monophytanylether~tysophosphatid s
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163
0.27 in chlor'oi~ri(ll)as a c0iourleSs 0il~w thRf ivelyl 0.56and An: Lndbenzene,:respec analytical sample was _dieth~ Lether (3 : I,V/V), TheRRR-iso-
: •:F, :i~' :.~:i!i;RRt !:!!:,~/:~::i M:
~!:69:iiHi::~!ii:!56:RRt~::isomer:: C, 77.52; H, 11.24"
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ionitophytanyl and aromatic absorp ti0n'~NMR assignments: phytanyl (39 H) ~ 0!75-~i~:65~i:~y~rol~methyleneand methine (7.H) 3.30-3.80 8; benzylic methylene : i~(2H)4iTOi~garomatic(5 H) 7.35 6, 4, i.O,p. Toluenemlphonyl.2.0-benz y l, 3-O-phy tany l'sn'glycerol ~:. 2.0~Benzyl~3,0,phytanyl-sn'glycerol [(II); 770 mg, 1.64 mmole] was reacted :with!recrystallisedp.fiiuenesulphonyl ~chloride (620 mg, 4i0 mmole) in 10 ml anhy: ar2,~ ~nvridine at o°andlthe mixture was then stirred at 10°C for 16 hr. The reaction ' dm: to100 ml o(ice-water, and extracted with four 30-ml portions ~ture!was P0Ure . . ofpetroleUm ether (30-60°c). The combined extracts were washed wRh water, ~cecold 2 Naquous HCI and water, and dried by evaporation with benzene under reduced pressure togivethe tosyl derivative as a colourless oil (809 mg, 82% yield;Rf • 0,fi8 in chloroform). An:analytical ~mple was obtained by preparative TLC in chloroform' The pure RRR-isomer had [~]~D2 + 1.43° (c, 6.8 in chloroform).
Found: RRR.RRS-isomer: C, 72.06; H, 9.69; S, 5.71. RRR-isomer" C, 71.93; H, 9.81; S, 5,35. C37H6oO5 S(616,91)
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• Calc,: C:,72,03;H, 9.80;S, 5.20.
. showed tosyl group absorption (1182, 1195 cm -1, o hydroxyl group absorption was apparent. NMR [,85 6; CH3-C6H4-(sharp singlet)2.42 8; methyl; ..O.CH2-C6H5 (sharp singlet) 4.57 6; C6H4CH3
OSO2_(doublet) "L76 6. 5. l.lodo-2-O-benzyb3"O'phytanyl'sn'glycer°l (III) ,tanyl-sn-glycerol (697 mg, 1.15 mmole) ' 6.0 mmole) in 15 ml of anhydrous acethen for 20 hr under reflux. After re~,centrifugation the acetone supernatant te residual oily solid was extracted centrifuged,washed with 5% aqueous
97%::eld).
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(585.mg~ ~72:%yiel, a faintlyyellow oil l 0,52);. ~ e R R R , ~ mer had [0~]22 ,,,~r~4i:,
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Found
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e catalyst was rem0ve.d byeentrifugati0n and washed with two Jor0form-ethan01 (i : 1,v/v)~ The combined supernatants were rreducedpressure, and the residual oil, dissolved in 5ml chloro~thl 5ml methanol ~ d 4.5 ml lr.0NHCI. The biphasic system was ~iiii~h!~of0rm!pha~ was neultrali~d:(phenolphthalein end point) ~ic!i: ;e~aPoratedm the presence of benzene to0.6 iml, and ~faiiet0ne,~ o n e Afier~o01ing , i C e , the gelatinoUs precipitate was cenithcold acetone and reprecipitated from its chloroform solution by umes .... ofdiethyl ..... ett~er.~e, lysophosphatidic • acid analogue (V) was i.¢e~!mg (45% yield) of chromato~'aphically pure white solid .~lues). The R/~:,isomer had [~]~z + 7..69° (c, 2.0 in chloroform) ~r:C, 51.98;H, 8.86; P, 5.83.
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caic= :e,; aa4: ; H,8,96;P. 5.s6, i~e infrared spectrum ofthe lysophosphatidic acid analogue (potassium salt) is shown infig, 1. C.~
ether analogue of lysophosphatidyl glycerol
: :L Silver salt of l-O-(mono-pnitrobenzylphosphorylJ-2-O-benzyl,3~O-phy tanyl- snglycerol ( VI) Asolution o f the phosphotriester (IV) (270 rag, 0.33 mmole)and sodium iodide (500 mg, :3.3 mmole)in 20 ml of anhydrous acetone was heated under reflux with stirring for 6hr. The solvent was evaporated under reduced pressure to give an oily solid, which was extracted with chloroform.The extract was cooled to 0°C, centrifuged, and evaporated to adark Oil (448 rag), which was purified on a silicic acid column (20 g)prepared in hexane. Elution with chloroformremoved a dark oil, whose infrarcd spectrumwas consistent with that ofp,nitrobenzyl alcohol, after which elution i!~th: chlorof0rm-met~nol (1:1, v]v) gave the sodium salt of Vl (brown oil, 169 mg),Rf 0~33and 0.76 in chloroform,methanol (9 : 1, v/v)and chloroform-methanol~30%ammonium hydroxide (65:35 : 5, v/v), respectively. An analytical sample wa$obtained by preparative TLCin chloroform-methanol-wate'r (80 : 20: 2, v/v) ::(R~O:50): !~e pure sodium salt was eluted from the silica with chloroform-methanoi-diethyl eti~er(i: 1::1, v/v). The RRR-RRS-isomer had [a] 22 + 4.29 ° (c, 4.2 in
chloroform). FoUnd: RRR,RRS-isomer: C, 63.61; H, 8.57; N, 2.09; P, 4.40. bsorption bands for nitro (1050¢m -1), and ionized bands were absent.
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