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A spectrophotometric determination of carbanions with tetranitromethane
Analytica Chimica Acla Elscvlcr Publishing Coqpony, Printed in The Ncthcrlands
47
Atnstcrdam
4 SPECTROPHOTObIETRIC TETRANITROMETHANE*
DE-l-ERM
INATION
01;
CA 1WANIONS
WITH
Various organic reactions sucli as clectrol~l~ilic substitutions1 and nnnlogcms are kno\vn to involve carlxulion intermediates. Ihtecznzymc-catalyzed rcactions”~” tion of c;~rlxinions and eslxxiall~ measurcnients of tlie rates of dcprotonnt.ion of C-acids arc1 most commonly carried out by proccclurcs tllat irlvolve llydrogcn isotope any clcctrol~llilc \vliicli reacts selectiveI zxcl~angc or lialogen:~tion~~. In principle, wit11 carhnions may be suitxblc for determination of cat-hnion, proviclcd tllxt it I-cacts rapidly enough for the ionization of the C-acid to Ix rate-clctct_nlininlT. However, tlic value of sucli an analytical reagent clcpcncls in large mcasurc on Iiow rexlily its reaction can be followed qu;Lntitativcly. In tllc present study, tetranitrolnetll~~l~e leas been usecl as a rcngcnt for carbanions. It reacts rapidly with carlxuiions with tlie concomitant formation of an inten\vllicli proviclcs tllc basis for a convenient spccsely yellow by-product, nitroformate, tropliotometric assay.
Mutcvials Tetratlitrc,lilctll~ne, nitromctllane, nitroc~hanc, malononitrilc, and met11ylsulfone were obtained from Aldricll Chemical Company. x,r-Dinitrocthane was purchased from tllc same compny as a 30’:/; solution in ethylene chlot-idc which was removed before use in a rotary evaporator under reduced pressure. z,4-l~c~~tanedione (Eastman Organic Chemicals), acetonitrile (J. T. I3aker Cllemical Company), acetone (Merck), Dowex anion escllanger AG I-,%?, 200-400 mesh (12iorad Laboratories) :~ncl x,2-dichloroethne (Fisller Scientific Con~pany) were also used.
A Unicum SP-Soo recording spectrophotometcr ccluipped with an xutomatic cell changer and repetitive reading l)rogram xvas used for determining the rate of ~ik? cell-llolder \vas tllerniostatted at 25”. Absorbance at nitroformate production. discrete wavelengths \vas measured with a Zeiss PAIQ II spectropllotomcter. Alxorption spectra were obtained with a Cary 15 recording sl~ectropl~otometer and infr:Lrcd * This work \V;LS supportctl the Dcpnrtnwnt of I-Icalth,
by Gr;rnt-ill-hicl GAIIZclucation lrncl Wclfarc.
I 5003 from
tllc
Nation;11
Awtl.
Chim.
I nstitutcs Acla,
51
of I-lcalth
of
(1970) 47-52
14.4 .. n
01
zoo
300 WAVELENGTH
400 I nm)
f
~PPEC1’IIIOI’~IOTO~IETKIC
I)E’l-EIIMISAI’IOS
OF
CARHANIOSS
49
formate, in contrast to tctranitromethane, absorbs strongly at 350 rim5 (E= xg,,~oo; Fig. I), For determinati(~n of reaction rates, tile concentrations of C-acids were chosen according to the rates of ionization (cf Table I). Fast icmizing C-acids were used at low concentrations with an excess of tetranitromethane and the increase in ASIN was treated as a first-order reaction. Slowly ionizing compounds were added to an excess of TNThI and the data were plotted as zero-order r’cactions. Correction was made for the spontaneous production of nitroformate from tetrnnitromctllarle at each pn employed. Zero-order rates were calculated assuming rnc)nonitration, ant1 no product other than nitroforrnate ahsorlkg at 350 nm. In order to prcmote its dissolution, tetranitronletllalle was cuqh)-ccl as a 0.03 M solution in gsf<, ethanol.
‘i’he rates and proclucts of the reaction of tetrnnitroIitct1ianc with nitroctlrntw were studied in or&r to delineate tfw basic features of the tetranitromcth:tnecarbanion reaction. The general fcasibifity of detertninntion of cwbanions by this reaction was esamincd by comparing the rates of ionization of a number of C-acids chtainecl by tllc prcscnt Iwoccdurc with tllose obtained by ottlet- tncthods.
?‘etrnrlitronlcth;Ine reacts rcndiiy with nitrocthne at per I I as judged from the production of nitroformnte (Fig. zA), ?‘fw pseudo first-orclcr rate of nitrofornzate production (I:ig. ~13)~ corrected for Irydrosiclc ion catatysis of ionization, is 5.S *IO-” min-1. This value is close to the rate of i~lii~ati~~n, measured hy ~rolnili~~ti~~tl or iodination” (2.2 *IO-” min-1). The correction for liyclroside ion catalysis assumes the same ratio krr,o//corr as for acutone7, i.c. kgr,c,= kl,fit 1l5.z * 10’1. Under the prcscnt _ conditions, catalysis of the ionization by cations and by anions other than ftydroxidc
0.6 . L. =: e? ‘8 0.4 _ d 4 a \
5’)
I’. (:HJCISTlZN,
J.
I?. RIORDAN
is negligil~leH. The same rate of ionization was also obtained by an indirect assay where nitroethanc was stored at pH IO and the progress of ionization was measu-ed on aliquots withdrawn at intervals by means of the tetranitromethane reaction (Fig. 3A). The increase in /IDLE, on addition of tetranitromcthane reveals a fast reaction (k~.[TNiU] limiting), superimposed on 5 much slower reaction (h limiting). Extrapolation of the indicator reaction to zero time gives the JIWJ value corresponding to tile amount of nitroethane anion present after the indicated period at pH IO. At I>H IO the rate of ~~r~)toll~Ltioll, 1~.[EIsOf], is negligibk+ and ionization may be treated as a first-order reaction (Fig, 313). Tl~c derived rate constant of 6.3 +IO-‘% min-* is in good ng;cemcnt with tile value cletcrmined in tile direct assay (Fig. 2).
Addition of tetl.:~nitromctl~ane to a solution of nitrrtctlmne, pretreated at various p15 values to allow ionization equilibrium, results in a rapid increase in ahsorbancc at 350 nm followed by a slower incrcasc (inset, Fig. 4). Extrapolation to xero tilnc indicutes the equilibrium concentration of nitroethane anion at each pH titration curve value. A plot of A.4 ~fi,, veY,~s pH (Fig:. 4) coincides with the theoxtical for a group wit11 a pI< of 5.6, the value reported for nitroethane~~.
The product of the reaction of tetranitromethanc with nitrocthane, designated a& [N02A‘j in scheme I, was idcntificcl as x,x-dinitroctllarle. Nitroetlrane (3. x0-3 M) was trcatecl with 2 1 of 0.05 M glycine buffer, lxx 10.0, for G 11rend an cc~uimolar amount of tetrallitrometllatlc was then added. Reaction was complete after 30 min at room temperature and the alxorbance at 350 nm did not incrcasc furtllcr. The bright yellow solution was purnpctl onto a Dowcx AG I-Sz anion-exchange column (zoo-400 mesh, cl~loridc form, 18 x o.S cm). Nitroformnte remained bouncl as a yellow-brown zone at the top of t11e c01u11111. TIE other reaction product migrated slrnvly as a yellow zone during the application, of the z-l sample and was easily clutcd with I M ammonium acetate, 1’~ +o. Aliquots of each Ernctiotx wcrc added to 6 N sodium ltytlrr~sictc in order to locate the product. Those fractions which gave a yellow color by this procedure
were pooled (rgo ml) and acidified with acetic acid to prr 3.0. This solution was extracted with three ro-ml portions of x,2-dichloroetllarle which was subsequently removed under reduced pressure on a rotary evaporator. The residue, a yellow oil, was identical with authentic x,x-dinitroethane on the basis of the following criteria: absorption spectrum wit11 2.,,.x = $So run and ~380 = 16,700, pK, of 5.0 as determined of by spectrophotometric titration, and infrared sj’ectra (Fig. 5). The identification r,r-clinitroetlxu~ as the reaction product of nitrocthane and tetmnitrometi~nl~e is j>erfcctly consistent with scheme r . C-acids
with
vaviom
clcctro~t-~~it/rri7a~~~~~~
sm!bitrwrts.
IZntc.s
cf
~m?vntion
nun!
rntcs
of
io9rizaticm
The general applicability of tetranitrometi~ane as a carbanion reagent is demonstrated in Table 1. Carbon acids substituted j>y nitro, sulftmyl, cym~o, or ketctnic carhonyj groups react readily wit11 tetrnnitrr>mctllnne. With all these corn-pounds, the rate of nitration, determined as in Fig. z for nitroethanc, agrees within nn order of magnitude with the reported value of the rate of ionization.
‘I‘ett’anitrnmetila~~e can be employed to determine carbanions by a convenient spectropil~)tometric method as demonstrated by detailed kinetic and analytical studies of the reaction between tctranitromethane and nitrcxthane (Figs. z-s), and by successful determination of the ionization rate constants of a number of C-acids with different ejectron-~~~itildra~~?in~ substituents (Table I). The method is based on sj~ectro~>llc)tolnetri~ measurements of the production of the colored reaction byjxoduct nitroformate (E = 14,400). En all systems wjlere tjle rate of carbanion formation is much lower than the rate of the tctranitromctjlane reaction, this indicator reaction can be employed to determine both the rnte of carbanion formation, e.g. the ionization rate of C-acids (Table I) and the absolute co?~casWztio?t of carbanion, e.g. the cquilijxium concentration as a function of j>H (Fig. 4). In systems where the generation of carbanions is too fast to be rate-limiting, ’ tctranitromethane can be employed successfully as a trapping agent for this reactive species. The rate of nitroformate production will then jw proportional to the ~ommztratio?t of carhanion, and will provide a relative measure of the equilibriuln or stcadystate concentration of this reactive species under different conditions of tile carbaniongenerating system. Such a situation (i.e. izi> >IZG (TN&I) in scheme I) will be encountered witjt rapidjy ionizing C-acids and in certain enzyme-catalyzed reactions. It has already been possibIe by this procedure to demonstrate carbanion intermediates in the reaction pntinvays of three carbon-carbon lyases, i.e. fructose I,&diphospl~ate aldolase’z and yeast aldolase from rabbit muscle~~, yeast fructose I,G-dipilospjlatc 6-phosphogluconate dehydrogenase. We would like to thank CHARLOTTE
HART for her excellent technical assistance, The constant advice and encouragement of B. L_ V~T.JZE is gratefully appreciated. One of us (Y.C.) thanks the University of Ziirich and the American-Swiss Foundation for Scientific Exchange for financial support.
j3
I’. CHRISTEN,
J. F. RIORI>AX
Tetranitrometfiane leas been employed as a reagent for determining the rates constnnts of C-acids. The intensely yellow byof innixntitm and the clissociation provides the basis for in convenient spectropl~otometric assay lwoduct, nitroformatc, at 350 nm (E= 14,4&j).
Ix t~tranitram~tl~~nc cst utilid comme rhctif pour dc%rminer les vitcsses d’ionisation et lcs constnntcs de dissociation dcs ncicles organirlucs. 1x2 nitroformiatc jaune intense clui se lot-me permet unc analyse spectropl~otomdtrique A 350 nm (t’= r4+p).
Tctrxnitromethane ist gcbraucht worclcn, um die Geschwindigkcitskc)nst~~ntcn cler Ionisation uncl die I)isso~iationskctnstanten van C-S!iuren zu bestimmen. Das iIltensiv gelbe N~l~e~~l~r~~clul~tNitr(~f~)rn~~~t crlaubt einfaclw sl~cktrnIpl~otomctriscl~e JZcstimmungen.
.
47
Atnstcrdam
4 SPECTROPHOTObIETRIC TETRANITROMETHANE*
DE-l-ERM
INATION
01;
CA 1WANIONS
WITH
Various organic reactions sucli as clectrol~l~ilic substitutions1 and nnnlogcms are kno\vn to involve carlxulion intermediates. Ihtecznzymc-catalyzed rcactions”~” tion of c;~rlxinions and eslxxiall~ measurcnients of tlie rates of dcprotonnt.ion of C-acids arc1 most commonly carried out by proccclurcs tllat irlvolve llydrogcn isotope any clcctrol~llilc \vliicli reacts selectiveI zxcl~angc or lialogen:~tion~~. In principle, wit11 carhnions may be suitxblc for determination of cat-hnion, proviclcd tllxt it I-cacts rapidly enough for the ionization of the C-acid to Ix rate-clctct_nlininlT. However, tlic value of sucli an analytical reagent clcpcncls in large mcasurc on Iiow rexlily its reaction can be followed qu;Lntitativcly. In tllc present study, tetranitrolnetll~~l~e leas been usecl as a rcngcnt for carbanions. It reacts rapidly with carlxuiions with tlie concomitant formation of an inten\vllicli proviclcs tllc basis for a convenient spccsely yellow by-product, nitroformate, tropliotometric assay.
Mutcvials Tetratlitrc,lilctll~ne, nitromctllane, nitroc~hanc, malononitrilc, and met11ylsulfone were obtained from Aldricll Chemical Company. x,r-Dinitrocthane was purchased from tllc same compny as a 30’:/; solution in ethylene chlot-idc which was removed before use in a rotary evaporator under reduced pressure. z,4-l~c~~tanedione (Eastman Organic Chemicals), acetonitrile (J. T. I3aker Cllemical Company), acetone (Merck), Dowex anion escllanger AG I-,%?, 200-400 mesh (12iorad Laboratories) :~ncl x,2-dichloroethne (Fisller Scientific Con~pany) were also used.
A Unicum SP-Soo recording spectrophotometcr ccluipped with an xutomatic cell changer and repetitive reading l)rogram xvas used for determining the rate of ~ik? cell-llolder \vas tllerniostatted at 25”. Absorbance at nitroformate production. discrete wavelengths \vas measured with a Zeiss PAIQ II spectropllotomcter. Alxorption spectra were obtained with a Cary 15 recording sl~ectropl~otometer and infr:Lrcd * This work \V;LS supportctl the Dcpnrtnwnt of I-Icalth,
by Gr;rnt-ill-hicl GAIIZclucation lrncl Wclfarc.
I 5003 from
tllc
Nation;11
Awtl.
Chim.
I nstitutcs Acla,
51
of I-lcalth
of
(1970) 47-52
14.4 .. n
01
zoo
300 WAVELENGTH
400 I nm)
f
~PPEC1’IIIOI’~IOTO~IETKIC
I)E’l-EIIMISAI’IOS
OF
CARHANIOSS
49
formate, in contrast to tctranitromethane, absorbs strongly at 350 rim5 (E= xg,,~oo; Fig. I), For determinati(~n of reaction rates, tile concentrations of C-acids were chosen according to the rates of ionization (cf Table I). Fast icmizing C-acids were used at low concentrations with an excess of tetranitromethane and the increase in ASIN was treated as a first-order reaction. Slowly ionizing compounds were added to an excess of TNThI and the data were plotted as zero-order r’cactions. Correction was made for the spontaneous production of nitroformate from tetrnnitromctllarle at each pn employed. Zero-order rates were calculated assuming rnc)nonitration, ant1 no product other than nitroforrnate ahsorlkg at 350 nm. In order to prcmote its dissolution, tetranitronletllalle was cuqh)-ccl as a 0.03 M solution in gsf<, ethanol.
‘i’he rates and proclucts of the reaction of tetrnnitroIitct1ianc with nitroctlrntw were studied in or&r to delineate tfw basic features of the tetranitromcth:tnecarbanion reaction. The general fcasibifity of detertninntion of cwbanions by this reaction was esamincd by comparing the rates of ionization of a number of C-acids chtainecl by tllc prcscnt Iwoccdurc with tllose obtained by ottlet- tncthods.
?‘etrnrlitronlcth;Ine reacts rcndiiy with nitrocthne at per I I as judged from the production of nitroformnte (Fig. zA), ?‘fw pseudo first-orclcr rate of nitrofornzate production (I:ig. ~13)~ corrected for Irydrosiclc ion catatysis of ionization, is 5.S *IO-” min-1. This value is close to the rate of i~lii~ati~~n, measured hy ~rolnili~~ti~~tl or iodination” (2.2 *IO-” min-1). The correction for liyclroside ion catalysis assumes the same ratio krr,o//corr as for acutone7, i.c. kgr,c,= kl,fit 1l5.z * 10’1. Under the prcscnt _ conditions, catalysis of the ionization by cations and by anions other than ftydroxidc
0.6 . L. =: e? ‘8 0.4 _ d 4 a \
5’)
I’. (:HJCISTlZN,
J.
I?. RIORDAN
is negligil~leH. The same rate of ionization was also obtained by an indirect assay where nitroethanc was stored at pH IO and the progress of ionization was measu-ed on aliquots withdrawn at intervals by means of the tetranitromethane reaction (Fig. 3A). The increase in /IDLE, on addition of tetranitromcthane reveals a fast reaction (k~.[TNiU] limiting), superimposed on 5 much slower reaction (h limiting). Extrapolation of the indicator reaction to zero time gives the JIWJ value corresponding to tile amount of nitroethane anion present after the indicated period at pH IO. At I>H IO the rate of ~~r~)toll~Ltioll, 1~.[EIsOf], is negligibk+ and ionization may be treated as a first-order reaction (Fig, 313). Tl~c derived rate constant of 6.3 +IO-‘% min-* is in good ng;cemcnt with tile value cletcrmined in tile direct assay (Fig. 2).
Addition of tetl.:~nitromctl~ane to a solution of nitrrtctlmne, pretreated at various p15 values to allow ionization equilibrium, results in a rapid increase in ahsorbancc at 350 nm followed by a slower incrcasc (inset, Fig. 4). Extrapolation to xero tilnc indicutes the equilibrium concentration of nitroethane anion at each pH titration curve value. A plot of A.4 ~fi,, veY,~s pH (Fig:. 4) coincides with the theoxtical for a group wit11 a pI< of 5.6, the value reported for nitroethane~~.
The product of the reaction of tetranitromethanc with nitrocthane, designated a& [N02A‘j in scheme I, was idcntificcl as x,x-dinitroctllarle. Nitroetlrane (3. x0-3 M) was trcatecl with 2 1 of 0.05 M glycine buffer, lxx 10.0, for G 11rend an cc~uimolar amount of tetrallitrometllatlc was then added. Reaction was complete after 30 min at room temperature and the alxorbance at 350 nm did not incrcasc furtllcr. The bright yellow solution was purnpctl onto a Dowcx AG I-Sz anion-exchange column (zoo-400 mesh, cl~loridc form, 18 x o.S cm). Nitroformnte remained bouncl as a yellow-brown zone at the top of t11e c01u11111. TIE other reaction product migrated slrnvly as a yellow zone during the application, of the z-l sample and was easily clutcd with I M ammonium acetate, 1’~ +o. Aliquots of each Ernctiotx wcrc added to 6 N sodium ltytlrr~sictc in order to locate the product. Those fractions which gave a yellow color by this procedure
were pooled (rgo ml) and acidified with acetic acid to prr 3.0. This solution was extracted with three ro-ml portions of x,2-dichloroetllarle which was subsequently removed under reduced pressure on a rotary evaporator. The residue, a yellow oil, was identical with authentic x,x-dinitroethane on the basis of the following criteria: absorption spectrum wit11 2.,,.x = $So run and ~380 = 16,700, pK, of 5.0 as determined of by spectrophotometric titration, and infrared sj’ectra (Fig. 5). The identification r,r-clinitroetlxu~ as the reaction product of nitrocthane and tetmnitrometi~nl~e is j>erfcctly consistent with scheme r . C-acids
with
vaviom
clcctro~t-~~it/rri7a~~~~~~
sm!bitrwrts.
IZntc.s
cf
~m?vntion
nun!
rntcs
of
io9rizaticm
The general applicability of tetranitrometi~ane as a carbanion reagent is demonstrated in Table 1. Carbon acids substituted j>y nitro, sulftmyl, cym~o, or ketctnic carhonyj groups react readily wit11 tetrnnitrr>mctllnne. With all these corn-pounds, the rate of nitration, determined as in Fig. z for nitroethanc, agrees within nn order of magnitude with the reported value of the rate of ionization.
‘I‘ett’anitrnmetila~~e can be employed to determine carbanions by a convenient spectropil~)tometric method as demonstrated by detailed kinetic and analytical studies of the reaction between tctranitromethane and nitrcxthane (Figs. z-s), and by successful determination of the ionization rate constants of a number of C-acids with different ejectron-~~~itildra~~?in~ substituents (Table I). The method is based on sj~ectro~>llc)tolnetri~ measurements of the production of the colored reaction byjxoduct nitroformate (E = 14,400). En all systems wjlere tjle rate of carbanion formation is much lower than the rate of the tctranitromctjlane reaction, this indicator reaction can be employed to determine both the rnte of carbanion formation, e.g. the ionization rate of C-acids (Table I) and the absolute co?~casWztio?t of carbanion, e.g. the cquilijxium concentration as a function of j>H (Fig. 4). In systems where the generation of carbanions is too fast to be rate-limiting, ’ tctranitromethane can be employed successfully as a trapping agent for this reactive species. The rate of nitroformate production will then jw proportional to the ~ommztratio?t of carhanion, and will provide a relative measure of the equilibriuln or stcadystate concentration of this reactive species under different conditions of tile carbaniongenerating system. Such a situation (i.e. izi> >IZG (TN&I) in scheme I) will be encountered witjt rapidjy ionizing C-acids and in certain enzyme-catalyzed reactions. It has already been possibIe by this procedure to demonstrate carbanion intermediates in the reaction pntinvays of three carbon-carbon lyases, i.e. fructose I,&diphospl~ate aldolase’z and yeast aldolase from rabbit muscle~~, yeast fructose I,G-dipilospjlatc 6-phosphogluconate dehydrogenase. We would like to thank CHARLOTTE
HART for her excellent technical assistance, The constant advice and encouragement of B. L_ V~T.JZE is gratefully appreciated. One of us (Y.C.) thanks the University of Ziirich and the American-Swiss Foundation for Scientific Exchange for financial support.
j3
I’. CHRISTEN,
J. F. RIORI>AX
Tetranitrometfiane leas been employed as a reagent for determining the rates constnnts of C-acids. The intensely yellow byof innixntitm and the clissociation provides the basis for in convenient spectropl~otometric assay lwoduct, nitroformatc, at 350 nm (E= 14,4&j).
Ix t~tranitram~tl~~nc cst utilid comme rhctif pour dc%rminer les vitcsses d’ionisation et lcs constnntcs de dissociation dcs ncicles organirlucs. 1x2 nitroformiatc jaune intense clui se lot-me permet unc analyse spectropl~otomdtrique A 350 nm (t’= r4+p).
Tctrxnitromethane ist gcbraucht worclcn, um die Geschwindigkcitskc)nst~~ntcn cler Ionisation uncl die I)isso~iationskctnstanten van C-S!iuren zu bestimmen. Das iIltensiv gelbe N~l~e~~l~r~~clul~tNitr(~f~)rn~~~t crlaubt einfaclw sl~cktrnIpl~otomctriscl~e JZcstimmungen.
.