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Structural determination of ascorbic acid 2-o-phosphate formed via acid hydrolysis of an ascorbic acid 3-O-phosphinate
STRUCTURAL DETERMINATION OF ASCORBIC ACID 2-O-PHOSPHATE FORMED VIA ACID HYDROLirSIS OF AN ASCORBIC ACID 3-O-PHOSPHINATE J. JERNOW,’ J. Bm.
E. O~lvrrro, A. Perutm~, P. Rosw and V. TOOME aputmcnt ot Chanii Rcsmrch,Hoffmsar&aRocbcInc.,Natky. NJ 07110. U.S.A. (Radud
in USA 24 July 1978)
_ c-stem,2 aid 9 (Pi
_
l-3).
BecauseoftheneedforamorestabIeformofvitaminC, recenthtaahuehasbeeninundataIwithvariuus methodsfortheproductioaofsuIfateandphosph&acid l?sters.Manyoftheseestershavebeenshowntobe bio@&Iyactiveoran&scorb&‘Inmanycases,itis diekldttomakestnKtwaIasaiRM&oftheseesterson thebasisofspectraIandchemkaIdataaIone.Thereported ascorbic acid 3GsuIfate 1, for .exampk. was recentIyreassi@icdthe%OlmIfatestruchue2basedon an x-ray sin& crysEalanaIysiL2 Since Cutolo and La&a’s that report on the synthesis ofan~ascorbicecidphosph&(chamt&&asitstriscycIohexylamnmnium saIt).’ many papers and patents have da& with the pmpamtive metMoIogy of this phosphate esteL” Yet, despite the m&ated interest in thisarea,theexactsbuctureofthiscompoundisstiUin diSpUtC.TbCOigid~tbon~WCIl~HiIlkk~and
Chitose%portedthisesterasthethepho&ate3(orits saIt4)whikothers,inchulinRSeii~contendeditwasthe 2-o-phorphate 5. we sol&t to ck&mine the exact SbVCtUlCOfthirCSter.
Sincea3-O-am&acidesterof&linitivesbucttue wasunknownforuseasrmodeIforcomparisoonstudks. we prepamd such an ester. Thus aIkyIationof the thaIIium 3-a&&m@ 10 with his-morpMinophosphinyl chbride (11)‘Oa&ded tbs 3u+sc&& 9. The structuraI ar&tunent of the product was cm5rmed by X-ray rmalysis. The same product could also be preparedfrom lland %O-iipyiide~~-~gscorbic acid (12)in the presence of pyridine, though this reaction was much slower. Identity was made 00 the basis of a comparison of the X-ray powder diflraction patterns of these two products. UnIike most ascorbic acid derivatives, compound 9 is thennaRy stable; it melts at 1700without decomposition. It is acidic, beii soluble in dilute sodium bicarbonate sohuion, and, upon acidiB&on, can be recovered unchanped. WhenanattemptwasmadetoobtaintheM%phosphatede+ative3viatheack%cataIyxedhydroIysisof9, twoproductswereobservaIbytlcanaIysis.OnIyoneof theseproductscouIdpeisohttedasthetris-cycbhexyIammonium saIt of mrcm-9~ needks (m.p. 173+ It
eluted as a sin& component on various paper chromatorpsphic systems, on a DE23 LC cohimn (ttkthyIaru monium carbonate gradient), and on a HPLC PWAXSO,‘- coIumn (ammonium suIfate Rradient).This saIt is identicaI (m.p., ‘H and “C NMR, W, amI HPIX amparisons)withthatprepamdbythedirectp&spharylationof 12withphos&msoxychIorkkrccordinptothe procedUnotCutdoradIJJ&fL’ we demonstrate now that the phosphate saItin questionisinfactthe2 6, pmumably formed --oftbcinitiany thlW&atlacidcltalyzediso formed3+hosphate3dur&thehydroIysisof9.’Ihis iflens is based 00 ccmparisons of the sa& pK, vahleandofthepHpro5kofitswspectrawiththose . 9. oftheknown2-O-suIfate2andIopbosphmate Theanionfmma2~7bcarsaIinearconju@ed chromaphore,whiIethatof3-O-esterEbcarsacrossconjugated chromaphore. A distinction between these isomersmi&tbcobservedintheiipILvaIuesandW spectrawhereasweexpectbothisomerstoshowi!4lthochKunic shifts (A, shifts toward Ioqter waveleagtb) accompanyipgtheionixatkmduetoapHincrease,we WOuIdexpect deIInite diffetences intheirabsorptioniu tensity. The 2Gderivative shotrId exhibit a hyperchromic shift and the 3&krivative shuuki display a hypochromic shift. Ascorbii acid (13) displays two acidic protons of PK. values 4.25 and 11.79 for the 3- and 2-hydroxyls, respectivdy.” SuIfation of the 2-OH as in 2t edmces the acidityofthe3-OHby1unittopX.of3.1l.Ontheother haod, a phosphinyi substitution at the 3-Gposition as in 9resuItsinasignificantdropinthep~vaIueofthe 2-OH group to 6.22.” A pII. vahre of 3.49 for the ascotbicacidpbosphoteinquestionisobviousIycompat&k with a 2-O-phosphateester structure;i.e. 6 (TabIe 1)” In addition to the evidence drawn from the pK, value comparison, the pH p&Be of the UV spectra of tbc 2-O-suIfate2invaryinRridkml!dia(Pii1)cIoseIy
soIvent.H6anver,thepHpro9koftheUVspectraof tlx~ester9isdilfenM(Pii3).AIt@@ itaIsoshowsabathochromicshiftwithincr&nRbasicity,itisoccomprniedbyahypo&omk&xt.Itis
I483
2 -P~orphot@ I 2
0.26 2.48
s
2.79
4
S.08
5
1).so
0
7.06 - IL.84
&
LopI, p24000mt
6
CURVCS
t 0.8 -
pH I. 28
2
3. IS
s
s. 55
0.7 -
0.6 -
0.5 -
0.4-
0.3
0.2
0. I
0 200
220
200
t
350
6
1: A,-K,%~KOS&,Ro-R-H 2: R,=KOSCb,Fb-K,R,=R=H 2: R,=R-R=H,R=H,Kh 4: Rt = C.HoN&* P = (CH~,NH.+)PO.fb-R-H 8: R,-fi8Pcb,%=R=Re=H 2: Rt = (C&rNHa+)J%RI-WoNHa’. &-R.-H
11 T~I.Acidityofaacwbicridcwtar” Pra &errs
&uorbic
acid 33
2-o-h&.*
2-03
3cLat
11.79
4.2s 3.11
3
2-O-Pknphato 3-0-9hoophiMte
$
3.40 3
6.22
Sttwctdde~daaunb4cwib
c x Io-3 3-PhoxphIxem& CUIMS
10
PM
i
1.
2
5.82
2s
6.06
9
6.46 7.07
8 /
12.7s
148s
14%
J.JmnIwUd
‘Pa a mcont surveywe B. N. To&art, M. Dowdq. R W. Cuboa.Y.K.KhbtdEM.BakAm.N.Y.Acd&L
BdL 19,341(1911): UKIpreviom pIoen m ti swim. 3. F. HiaLloy, U.&s PaL 3$37&549to Mmck md co., Inc. (l9nE wtoae scibdll Co., Id.. Japm Par. 038179(Jlpm 1973). ‘H.T8kdk~.~.~om~n.~.Imdrad~.~kkk * cm&J u, 29 (19jl). ‘Cc.H. Lee, P. Seib. R C. Homey. Y. T. w md C. W. Iwoe. cad Cbm # 456 (1975). ‘D. L Hm+. Ik~ducm.Suc3~ dauibm GzEzz?m d J. II. Tmbull. IMd 1963(1958). “RP.&flradRR~s,~.~ySor.,~ (1961): 0. Ncbbim md R M. Pidi, AC& VJrmkd. 13, X9 (1~.SeedmLJ.H1ylermdJ.RPlarmer,QCIPrLR&w 14ez92(l%OIfatbc&ditksoftctroaicridMdo-bYdroxv_ _ . :
‘%i??iiif*M. Vo@ ml K. Ambmm, L#uozMowkmStaRta ckdachu sdnm tu WdJsdru LdJmt. D. 513. But_. _
tcrwauu, -kJom (l%l). NotetbmtHi~oki(l)exlStarpK.vdueof73 OH
E. O~lvrrro, A. Perutm~, P. Rosw and V. TOOME aputmcnt ot Chanii Rcsmrch,Hoffmsar&aRocbcInc.,Natky. NJ 07110. U.S.A. (Radud
in USA 24 July 1978)
_ c-stem,2 aid 9 (Pi
_
l-3).
BecauseoftheneedforamorestabIeformofvitaminC, recenthtaahuehasbeeninundataIwithvariuus methodsfortheproductioaofsuIfateandphosph&acid l?sters.Manyoftheseestershavebeenshowntobe bio@&Iyactiveoran&scorb&‘Inmanycases,itis diekldttomakestnKtwaIasaiRM&oftheseesterson thebasisofspectraIandchemkaIdataaIone.Thereported ascorbic acid 3GsuIfate 1, for .exampk. was recentIyreassi@icdthe%OlmIfatestruchue2basedon an x-ray sin& crysEalanaIysiL2 Since Cutolo and La&a’s that report on the synthesis ofan~ascorbicecidphosph&(chamt&&asitstriscycIohexylamnmnium saIt).’ many papers and patents have da& with the pmpamtive metMoIogy of this phosphate esteL” Yet, despite the m&ated interest in thisarea,theexactsbuctureofthiscompoundisstiUin diSpUtC.TbCOigid~tbon~WCIl~HiIlkk~and
Chitose%portedthisesterasthethepho&ate3(orits saIt4)whikothers,inchulinRSeii~contendeditwasthe 2-o-phorphate 5. we sol&t to ck&mine the exact SbVCtUlCOfthirCSter.
Sincea3-O-am&acidesterof&linitivesbucttue wasunknownforuseasrmodeIforcomparisoonstudks. we prepamd such an ester. Thus aIkyIationof the thaIIium 3-a&&m@ 10 with his-morpMinophosphinyl chbride (11)‘Oa&ded tbs 3u+sc&& 9. The structuraI ar&tunent of the product was cm5rmed by X-ray rmalysis. The same product could also be preparedfrom lland %O-iipyiide~~-~gscorbic acid (12)in the presence of pyridine, though this reaction was much slower. Identity was made 00 the basis of a comparison of the X-ray powder diflraction patterns of these two products. UnIike most ascorbic acid derivatives, compound 9 is thennaRy stable; it melts at 1700without decomposition. It is acidic, beii soluble in dilute sodium bicarbonate sohuion, and, upon acidiB&on, can be recovered unchanped. WhenanattemptwasmadetoobtaintheM%phosphatede+ative3viatheack%cataIyxedhydroIysisof9, twoproductswereobservaIbytlcanaIysis.OnIyoneof theseproductscouIdpeisohttedasthetris-cycbhexyIammonium saIt of mrcm-9~ needks (m.p. 173+ It
eluted as a sin& component on various paper chromatorpsphic systems, on a DE23 LC cohimn (ttkthyIaru monium carbonate gradient), and on a HPLC PWAXSO,‘- coIumn (ammonium suIfate Rradient).This saIt is identicaI (m.p., ‘H and “C NMR, W, amI HPIX amparisons)withthatprepamdbythedirectp&spharylationof 12withphos&msoxychIorkkrccordinptothe procedUnotCutdoradIJJ&fL’ we demonstrate now that the phosphate saItin questionisinfactthe2 6, pmumably formed --oftbcinitiany thlW&atlacidcltalyzediso formed3+hosphate3dur&thehydroIysisof9.’Ihis iflens is based 00 ccmparisons of the sa& pK, vahleandofthepHpro5kofitswspectrawiththose . 9. oftheknown2-O-suIfate2andIopbosphmate Theanionfmma2~7bcarsaIinearconju@ed chromaphore,whiIethatof3-O-esterEbcarsacrossconjugated chromaphore. A distinction between these isomersmi&tbcobservedintheiipILvaIuesandW spectrawhereasweexpectbothisomerstoshowi!4lthochKunic shifts (A, shifts toward Ioqter waveleagtb) accompanyipgtheionixatkmduetoapHincrease,we WOuIdexpect deIInite diffetences intheirabsorptioniu tensity. The 2Gderivative shotrId exhibit a hyperchromic shift and the 3&krivative shuuki display a hypochromic shift. Ascorbii acid (13) displays two acidic protons of PK. values 4.25 and 11.79 for the 3- and 2-hydroxyls, respectivdy.” SuIfation of the 2-OH as in 2t edmces the acidityofthe3-OHby1unittopX.of3.1l.Ontheother haod, a phosphinyi substitution at the 3-Gposition as in 9resuItsinasignificantdropinthep~vaIueofthe 2-OH group to 6.22.” A pII. vahre of 3.49 for the ascotbicacidpbosphoteinquestionisobviousIycompat&k with a 2-O-phosphateester structure;i.e. 6 (TabIe 1)” In addition to the evidence drawn from the pK, value comparison, the pH p&Be of the UV spectra of tbc 2-O-suIfate2invaryinRridkml!dia(Pii1)cIoseIy
soIvent.H6anver,thepHpro9koftheUVspectraof tlx~ester9isdilfenM(Pii3).AIt@@ itaIsoshowsabathochromicshiftwithincr&nRbasicity,itisoccomprniedbyahypo&omk&xt.Itis
I483
2 -P~orphot@ I 2
0.26 2.48
s
2.79
4
S.08
5
1).so
0
7.06 - IL.84
&
LopI, p24000mt
6
CURVCS
t 0.8 -
pH I. 28
2
3. IS
s
s. 55
0.7 -
0.6 -
0.5 -
0.4-
0.3
0.2
0. I
0 200
220
200
t
350
6
1: A,-K,%~KOS&,Ro-R-H 2: R,=KOSCb,Fb-K,R,=R=H 2: R,=R-R=H,R=H,Kh 4: Rt = C.HoN&* P = (CH~,NH.+)PO.fb-R-H 8: R,-fi8Pcb,%=R=Re=H 2: Rt = (C&rNHa+)J%RI-WoNHa’. &-R.-H
11 T~I.Acidityofaacwbicridcwtar” Pra &errs
&uorbic
acid 33
2-o-h&.*
2-03
3cLat
11.79
4.2s 3.11
3
2-O-Pknphato 3-0-9hoophiMte
$
3.40 3
6.22
Sttwctdde~daaunb4cwib
c x Io-3 3-PhoxphIxem& CUIMS
10
PM
i
1.
2
5.82
2s
6.06
9
6.46 7.07
8 /
12.7s
148s
14%
J.JmnIwUd
‘Pa a mcont surveywe B. N. To&art, M. Dowdq. R W. Cuboa.Y.K.KhbtdEM.BakAm.N.Y.Acd&L
BdL 19,341(1911): UKIpreviom pIoen m ti swim. 3. F. HiaLloy, U.&s PaL 3$37&549to Mmck md co., Inc. (l9nE wtoae scibdll Co., Id.. Japm Par. 038179(Jlpm 1973). ‘H.T8kdk~.~.~om~n.~.Imdrad~.~kkk * cm&J u, 29 (19jl). ‘Cc.H. Lee, P. Seib. R C. Homey. Y. T. w md C. W. Iwoe. cad Cbm # 456 (1975). ‘D. L Hm+. Ik~ducm.Suc3~ dauibm GzEzz?m d J. II. Tmbull. IMd 1963(1958). “RP.&flradRR~s,~.~ySor.,~ (1961): 0. Ncbbim md R M. Pidi, AC& VJrmkd. 13, X9 (1~.SeedmLJ.H1ylermdJ.RPlarmer,QCIPrLR&w 14ez92(l%OIfatbc&ditksoftctroaicridMdo-bYdroxv_ _ . :
‘%i??iiif*M. Vo@ ml K. Ambmm, L#uozMowkmStaRta ckdachu sdnm tu WdJsdru LdJmt. D. 513. But_. _
tcrwauu, -kJom (l%l). NotetbmtHi~oki(l)exlStarpK.vdueof73 OH