G.L. Mattok and R.A. Heacock- The Chemistry of the Aminochromes Part VI: The Reaction of Adrenochrome with Glutathione

8/3/2019 G.L. Mattok and R.A. Heacock- The Chemistry of the Aminochromes Part VI: The Reaction of Adrenochrome with Gl

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THE CHEMISTRY OF THE AMINOCHROMESPART VI. THE REACTION OF ADRENOCHROME WITH GLUTATHIONE'*"3

G. L. MATTOK ND R. A. HEACOCKPsychiatric Research U ni t, Unirrersity Hosp ital, Saskato on, SaskatchewanReceived August 10, 1964

ABSTRACTTh e reaction between adrenochrome and glutathione ha s been studied. 'The main productsresult from the 1,4-addition of glutathione to th e Cg unsaturated carbonyl systetns in adreno-chrome involving the c6-C~ and the c4-c~~ouble bonds, a nd a re probably 7-S-glutathionyl-5,6-dihydroxy-N-methylindole nd 9-S-glutathionyl-2,3,6,9-tetrahydro-3,5-dihydroxy-6-0~0-N-methylindole respectively. 5,6-Dihydroxy-N-methylindoleis also formed during the inter-action of glutathione and adrenochrome. T he lnechanisrns by which these compounds areformed are discussed.

INTRODUCTIONThiols, such as glutathione, generally react with quinones by 1,4-addition across one

of th e a,p-unsaturated carbonyl systems. Much of the early work, dating back to 1888(I), was reviewed by Snell and Weissberger in 1939 (2). Th e reaction of thiols, includingglutathione, with 2-methyl-l,4-naphthoquinoneas been described by several workersas a 1,4-addition process (3, 4, 5), although it has recently been suggested tha t thisreaction proceeds by a simple nucleophilic substi tution of t he quinone ring by th e mer-captide anion (6). Most of the previously reported additions of th is type involvedp-quinones; however, there have been a number of examples of t he reported addition ofthiols, including glutathione, t o oxidized catechol derivatives (cf. 7-11) and recently theI$-addition of a thiol (l-phenyl-5-mercaptotetrazole) to several quinones, includingsome o-quinones, has been described (12, 13).

Preliminary paper chromatographic studies on the interaction of adrenochrome (I)with glutathione (free acid) in aqueous solution indicated th at 5,6-dihydroxy-N-methylin-dole (11) and two other products (non-ether extractable) were formed; the major product(111) had an R, value* of ca. 0.60 and appeared t o have retained th e indole nucleus andto contain an a-amino acid grouping (14, 15). Bouchilloux and Kodja subsequentlyreported that dopachrome solutions were decolorized by glutathione (phosphate buffer,pH = 5.5) with t he formation of 5,6-dihydroxyindole, 5,6-dihydroxyindole-2-carboxylicacid, and compounds described a s 4-S-glutathionyl-5,6-dihydroxyindolend its 2-carboxyderivative (10, 11). These latt er two compounds were also reported t o be obtained by th eenzymatic oxidation of 5,6-dihydroxyindole or 5,6-dihydroxyindole-2-carboxylic cidrespectively in the presence of g luta thione (10, 11). Th is paper reports th e results of a nextensive spectroscopic and paper chromatographic examinat ion of the products obtainedby the interaction of adrenochrome and glutathione.

E XP E R I M E NT ALiMaterialsAdrenochrome (16), N-ethylnoradrenochrome (17), N-isopropylnoradrenochrome (17), adrenochromemethyl ether (17), adrenochrome ethyl ether (17), and 5,6-dihydroxy-N-methylindole 18) were prepared'T hi s investigation was supported by grants fro m the Department of National H ealth and Welfare (Ottawa)and the Gorrernment of Saska tchew an (D epa rtm ent of Pu blic He alth).2Part V. Can. J . Chem. 41, 139 (1963).3A prelimin ary report of part of thi s invest igatio n appeared in Arch. Biochem. Biophys. 107, 362 (1964).*A ll the R , values referred to in this paper were obtained us ing 2% acetic acid in water as th e ru nni ng solrrent(see Experimen tal section for details).Canadia n Journal of Chemistry. Volume 43 (1965)

119


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120 CANADIAN JOURNAI, OF CHEMISTRY. VOL. 43. 1965by the methods described in the literature. Glutathione, its monosodium salt, and oxidized glutathionewere obtained from the Nutritional Biochemicals Corporation.SpectroscopyTh e spectra were recorded on either a Beckman DIG-2 or Unicatn SP-800 recording spectrophotometer.Th e reaction mixtures studied were prepared from adrenochrome (5.0 mg) and glu tathione (frec acid ormonosodium salt) (25.0 mg) dissolved in water (1.0 ml). Suitable dilutions were prepared for measurementof t he spectra. When requi red, ether extractions were carried out with peroxide-free ether (4 X 1 rnl). Inexperiment s designed to stu dy th e effects of added reagents, th e reaction mixture was prepared by t hemethod described above; ether extracte d (if necessary) and a slight excess of th e solid reagent (i.e. silveroxide, sodium hydrosulfite, sodium bisulfite, or sodium acetate) were added directly to the solution. In theexperiment s designed to find the optimu m pH for the formation of a given product the reaction mixturewas prepared a s described above, except t hat a suitable acetat e or phosphate buffer was used a s the reactionmedium in place of wate r.Paper ChrotnatograplzyThe aminochrome (10 mg) was dissolved in water (1.0 ml) and the solution was treated with a slightexcess of glu tathione (free acid or monosodium salt).* Whe n th e red color of the arninochrome had beencomplete ly discharged the reaction mixtu re was filtered and a sample (ca. 25-50 fil) of t he clear filtrateapplied to the chromatographic paper (acid-washed Whatman No. 1 paper). The chromatography wascarried o ut by the descending technique, using 2y0 acetic acid in water a s the running solvent. In all casesthe solvent was allowed t o descend abo ut 15 to 16 in. (this required approximately 2.5-3 h running time).After drying (in air, at room temperature), the developed chromatograms were examined for fluorescencein ultraviolet light and individua l chromatograms were sprayed with one of t he following chromogenicreagents:? (a) Ehrlich' s reagent, (b) cinnamaldehyde, (c) p-dimethylaminocinnamaldehyde, (d) Gibb'sreagent, (e) diazotized p-nitroaniline, Cf) ninhydrin/pyridine, and (g) ferric chloride.In some cases the aqueous solution of t he am inoc hro~ needuction products (prepared as described above,1.0 ml) was extracted, af te r filtration, with peroxide-free ether (4 X 1.0 ml). Spot s consisting of ca. 120-150~1 of the dried (NazSOd) ethereal e xtr act a nd ca. 4C-60 fi1 of the aqueous mother liquors were appl ied tothe paper separately and the chromatography carried out as described above.In t he experiments designed t o obtain relatively pure solutions of the individual products, the reactionmixture was prepared in th e manner described above and a sample (ca. 500 ~ 1 )as applied as a "streak"across the origin on acid-washed Whatman 3 MM paper (23 cm X 57 cm). After development with 2%acetic acid, the appro priate zones were located by spraying a narrow stri p, cut from the edge of the chroma-togram. The zones were cut out and eluted with water.To ascertain if any reaction occurred between 5 ,6 -d ihydroxy-N-methv l indo le (11) and glutathione (oroxidized glutathione) , an aqueous solution of (11) (10 mg in 1ml) was treated with a n excess of t he amino-acid and the reaction mixture examined chromtographically in the manner dcscribed abovc, aftcr it hadbeen allowed to stan d a t room temperature for a period of time co~np arabl eo that required for th ~: dreno-chrome-glutathionc reaction to go to completion.

RESULTSAddition of glutathione (monosodiunl salt ) to an aqueous solution of adrcnochronle

(I ) resulted in the discharge of the red color of the solu tion; the resulting pale yellowsolution showed well-defined absorption maxiina of app rox in~at ely qual intensity in theregions of 300 nlp and 350 1 1 1 ~ see Fig. I ) . When the reaction was carried out usingglutathione, as the free acid rathe r than its monosodiun~ alt, the absorbance at 300 n ~was ca. 70y0 greater than that at 350 illp (see Fig. 1). In view of the higher absorbancesin the 350 mp region of reaction mixtures obtained using the sodium sal t of glutathione.the effect of pH on the relative intensities of the main absorption peaks was investigated,Reaction mixtures containing adrenochronle ( I ) and glutathione were prepared in a seriesof aceta te and phosphate buffers in the pH range 1-7, and i t was observed tha t formationof the product (A = ca. 350 nip) (i.e. ( IV) ) occurred optilnally at pH = 4.7 (see Fig.2) ; the amount of (IV) formed fell off sharply a t pH values above and below 4.7 and didnot form a t all at p H values > 6 .

Paper chromatograms of these reaction mixtures on Whatman No. 1 paper with 2%*An analogoz~s .uperitilent was carried oz~t sing oxidized glz~tathione n place of glutathio~ze, ut the red colorof the adrenocl~ronze olution was not discharged in a comparable period o time.?The chrotizogenic reage?zts were prepared by the nzethods conznzonly described in the li terature (cf. (1.9)).


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MATTOIC AN D I-IEACOCK: CI3EMISTR Y OF TH E AMINOCI-IROMES.P.4RT VI 121

WAVELENGTH (mp ) pHFIG.1. Absorption spectra of ( a ) adrenochrome/glu ta th ione reaction mixture (broken line) and (b)adrenochrorne/glutathione (monosodium salt) reaction mixture (solid line).FIG.2. Th e effect of pH on the absorbance at 350 mp of buffered glutathione/adrenochrome reactionmixtures.

acetic acid as the developing solvent (cf. 14, 15) showed three spots (which gave bluecolors after spraying with Ehrlich's reagent) with R, values of 0.43, 0.64, and 0.85 re-spectively (see Table I). A yellow spot (R, = ca. 0.90), which appeared overnight onthe sprayed chromatograms, was probably due t o unchanged glutathione. Th e substancewith an R, of 0.85 showed a distinct yellow fluorescence in ultraviolet light; the substancewith an R, of 0.64 (i.e. (111)) did not fluoresce and the product with a n R, of 0.43 showedthe weak "blue-mauve" fluorescence in "long wavelength" ultraviolet light characteristicof (11). The absorption maxima for the products were obtained b y elution of t he approp-riate zones from a developed chromatogram of the adrenochrome-glutathione reactionmixture; these were as follows: R,, 0.64 (i.e. (111)), 303 mp and R,, 0.85 (i.e. ( IV)), 352mp. The unidentified product (111) exhibited color reactions expected for (i) an indole,(ii) a phenol, and (iii) an a-amino acid (see Tab le I). The color reactions of t he product(IV) were a little less certain, due to the contaminat ion of t he zone (Rfi 0.80-0.90) on thechromatograms with unchanged glutathione; however, it did appear t o react slowly withEhrlich's reagent, cinnamaldehyde, diazotized 9-nitroaniline, and ferric chloride (seeTable I). T he colors obtained from (IV) were similar to those given by t he adrenochrome -sodium bisulfite addition product with these reagents (20).

N-Ethylnoradrenochrome and N-isopropylnoradrenochrome behaved similarly toadrenochrome on treatment with glutathione in aqueous solution. Paper chromatographicexamination of the reaction mixtures showed tha t three products were formed in eachcase; the expected N-alkyl-5,G-dihydroxyindole nd products probably analogous to (111)and (IV). Adrenochrome methyl ether and adrenochrome ethyl ether also behaved in asimilar manner to adrenochrome on treatment with glutathione (see Table I). Theseresults suggest that the 1- and 3-positions of the adrenochrome nucleus are not materiallyinvolved in a ny of the reaction sequences. There was no reaction between adrenochromeand oxidized glutathione; consequently, the thiol group of glutathione must have beeninvolved in all these reactions. Th e possibility of secondary product formation by the


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C A N A D I A N JOURNAL OF CHEMISTRY. VOL. 43. 1965TABLE I

Paper chromatography of the products obtained from the reactionof some aminochromes with glutathioneAmi~lochromeeduced Average R, values (X100) of the major products

Adrenochrome 43* 6 4 t ~ 855911N-Ethylnoradrenochrome 51** 66 84 907N-Isopropylnoradrenochrorne 69 85 907Adrenochrome methyl ether 44' 55 t t 85 907Adrenochrome ethyl e ther 45' 65t 90766 t 85 928*5,6-Dihydroxy-N-methylindole 11).t7-S-Glutathionyl-5.6-dihydroxy-N-methylindole (111).SColor reactions of (111). Ehrlich's reagent, blue-violet' cinnam aldehyd e. red-pink + grey-violet; f i-dimethylaminocinnamal-dehyde, blue-green -+ grey-blue; diazotized p-nitroaniline: magenta -+violet-brown; Gibb's reagent, brown; nic;hydrin/pyridine,blue-mey (slow at room temperature).9-S-Glutatbionyl-2.3,6,9-tetrahydro-3,5-dihydroxy-6-oxo-N-methylindoleIV).IColor reactions of (IV).Ehrlich's reagent, blue; cinnamaldehyde, violet-brown; p-dimethylaminocinnamaldehyde, grey-blue;t . . . .diazotued p-nltroanlhne, yellow -+ brown; Gibb's reagent, brown; ferric chloride. grey-brown; ninhydrin/pyridine. probablyblue-grey, but thi s area is confuse d by the proximity of excess glutat hione.TExcess glutathione (and possibly oxidized glutathione)."N-Ethyl-5.6-dihydroxyindole.H5.6-Dihydroxy-N-isopropylindole.

interaction of 5,6-dihydroxy-N-methylindole11) with glutathione (or oxidized gluta-thione) was also ruled o ut , since no react ion occurred between (11) an d ei ther glutath ioneor oxidized glutathione in aqueou s solut ion, over periods of t im e comp arable with thoserequired for the adrenochrome/glutathione (or monosodium sa l t ) reac t ions to go tocomplet ion.I t was shown by paper chrom atograph y tha t e ther ext ract ion of th e adrenochrome-gluta thione reac t ion mixtures comple te ly removed the substance wi th an R, of 0.43(i.e. (11)). T h e ul traviolet spectrum of t he aqu eous m other l iquors, st i l l showed peak s a tca. 300 m p a n d 350 mp, al though th e intensi ty of th e 300 mp peak was much reduced,because of t he rem oval of (11) which also absorbs in th is region. T h e react ion mixture,after removal of (11) by eth er extract ion, w as ini tial ly yel low, bu t on th e add it ion ofsolid sodium aceta te or on the cau tious addit ion of aqu eous sodium hy droxide th e solutionbecame red. D ilution (ca. 100 imes) of the ether-extracted react ion mixture with water,a l so led to the form at ion of a n orange-red produ ct , a f te r 2 h a t room temp era tur e ; thecolor intensified on stan ding ov ernight . Th ese color changes did not ap pear to be affectedby bubb ling oxygen throu gh t he solut ion. Th e change in color of t he solut ion was accom-panied by a modificat ion of th e absorption sp ectr um ; the inte nsi ty of the absorption a tca . 350 mp w as markedl y reduced , and event ua ll y d i sappeared ; a t t he sam e t i me t he rewas an increase in the absorbance a t 300 mp and a new peak a t 485 mp appeared. Thered color of t he modified r eactio n mix ture wa s discharg ed on add ition of excess sodiumhydroxide, with the formation of a yellow product which exhibi ted an intense yel low-green fluorescence, usually associated with adrenolut in (5,6-dihydroxy-N-methylindox yl), in ul traviolet l ight (cf. 21). Th e red p rod uct w as also decolorized by th e addit io nof sod ium hydrosulfi te or sodium bisulfite . In th e former case an eth er extrac table produ ctwas obta ined which was shown by paper ch romato graphy and spec troscopy to be ident ica lwith 5,6-dihydroxy-N-methylindole11) , and in the la t te r case the red color could berestored b y th e cautious add it ion of alkal i ; addit ion of excess alklai resulted in the dis-charge of t he red color and th e formation of a fluorescent pro duc t similar to th at describedabove . These reac tions wi th sodium bisul fi te an d a lkal i suggests th a t the red produ ct i sder ived from (IV) and tha t i t i s probably adrenochrome (I) (A 300 m p a n d 487 mp(cf. 22)). Th e ether extracted adrenochrome-glutathione react ion mixture is capable offur ther modi f ica t ion by oxidiz ing and reducing agents . After t rea tment of the e ther


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MATTOK AND HEACOCK: CHEMISTRY O F TH E AMINOCHROMES. PART VI 123extracted reaction mixture with silver oxide, the solution became deep yellow and theoriginal peaks a t ca. 300 mp and ca. 350 mp were replaced by peaks a t 285 mp* and399 mp; this suggests tha t both compounds can undergo oxidation. T he peak a t ca.350 mp (due to (IV) ) disappeared and th e 300 mp peak increased in int ensity on trea tmen tof th e original reaction mixture (ether extracted) with sodium hydrosulfite. Paper chrom-atography showed tha t reduction with this reagent resulted in the elimination of thefaster running substance (i.e. (IV)) and the formation of (11) which could be extractedwith ethe r and identified by ch romatography and spectroscopy.

DISCUSSIONOn the basis of the observed spectroscopic, paper chromatographic, and chemical

dat a, it is possible to propose likely structures for the products obtained by the actionof glutathione on adrenochrome; the formation of all the reaction products can also beexplained by reasonable mechanisms.

It is well known that 5,6-dihydroxy-N-methylindole (i.e. (11)) is usually produced, tosome extent, when adrenochrome reacts with a reducing agent (cf. 22), and i t has previ-ously been suggested that the product with an Rf alue of 0.43 (i.e. (11)) was, in fact,5,6-dihydroxy-N-methylindole;he results of the present investigat ion are in agreementwith th is suggestion. The paper chromatographic and spectroscopic properties of (11)were identical to those of pure 5,6-dihydroxy-N-methylindole. he formation of 5,6-dihydroxy-N-methylindole (11) could result from the direc t reduction of adrenochrome(I) by glutathione. I t could also result from the reduction of (I ) by (111) (or possibly i tsimmediate precursor (i.e. (VI)) ; the reduction of a quinone by the corresponding hydro-quinone containing an electron-donating group is well known (cf. 23). To t est theplausibility of t he lat ter t ype of reaction occurring, a solution of N-isopropylnoradreno-chrome was treated with (11); paper ch romatography of this reaction mixture showedthat interaction had, in fact, occurred, with the formation of 5,6-dihydroxy-N-isopropy-lindole (i.e. the dehydrated reduction product of N-isopropylnoradrenochrome). In viewof t he difficulty of detecting oxidized glutathione, in the presence of glutathione (withthe chromatographic system used in this investigation), and the uncertainty that itspresence, if found, was not merely due to autoxidation, i t is not possible to be sure if t hedirect reduction mechanism is operating to any extent in the adrenochrome-glutathionesystem. However, since 5,6-dihydroxy-N-methylindole11) is not the major product ofthe adrenochrome/glutathione reaction and the N-isopropylnoradrenochrome/5,6-dihy-droxy-N-methylindole interact ion resulted in appreciable formation of 5,6-dihydroxy-N-isopropylindole, in a time comparable to th at required for the completion of the adreno-chrome/glutathione reaction, it is most probable tha t th e majority of t he (11) presentin the glutathione/adrenochrome system resulted from the reduction of adrenochromeby (111) or (VI) which gave "leuco-adrenochrome" (VI I) which is known to dehydratespontaneously and form 5,6-dihydroxy-N-methylindole11) (cf. 24).

There are two possible ways by which glutathione could add, by a 1,4-additionmechanism, to adrenochrome. Addition could occur across the a,p-unsaturated Cs-car-bony1 system, which includes the C6-C7 double bond, to form the adduct (V); sub-sequently this product could undergo an "enolization" of th e polarized C6-carbonyl

*T he position of the 286 m p peak could not be precisely determined since end absorption due to oxidized gluta-thione i s significant below 290 mp; this was partially compensated by the addition of oxidized glutathione to thesolution i n the reference beam of the spectrophotometer.


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CANADIAN JOURNAL O F CHEMISTRY. VOL. 43 . 1965

group to give 7-S-glutathionyl-3,5,6-trihydroxy-N-methyindolineVI). The 3,5,6-trihydroxyindoline structure is known to rapidly undergo an intramolecular dehydrationto give the 5,6-dihydroxyindole structure (cf. 24), consequently, (VI) would be expectedto dehydrate to give ?'-S-glutathionyl-5,6-dihydroxy-N-methylindole111). Thi s structureis qui te compatible with the observed properties of (111), i.e. the typical indole absorptionmaximum a t ca. 300 mp and the color reactions expected for an indole, catechol, and ana-amino acid. I t is also water soluble and was not extracted from its aqueous solutionsby ether as would have been expected for a molecule containing a large amino acidresidue. This structure is similar to that postulated by Bouchilloux and Kodja for oneof the products obtained from dopachrome by the action of g lutathione, except that theFrench authors suggested that the glutathione residue was attached to the 4-position ofthe indole nucleus (11). Alternatively, 1,4-addition of glutathione to the a,P-unsaturatedCh-carbonyl system, with the double bond between the C9-bridge-head carbon atom andC4would result in structure (IV), i.e. 9-S-glutathionyl-2,3,6,9-tetrahydro-3,5-dihydro6-0x0-N-methylindole. This st ructure is similar to th at proposed by Tse and Oesterlingfor the adrenochroine - sodium bisulfite addition product (25) and is compatible with theproperties of the glutathione/adrenochrome interaction product (IV) with R, = 0.85and A = 352 mp. There are a number of chemical similarities between (IV) and thebisulfite addition product (cf. 25, 26) although the gluta thione adduct would appear tobe less stable. In the first place, there is the apparent regeneration of adrenochrome(A,,,, ca. 300 and 487 mp) on treatment with mild alkali (cf. 14, 26); both productsexhibit an absorption maximum in the 350-360 mp range ; both compounds show a weakyellow fluorescence in ultraviolet light and (IV) and the adrenochrome - odium bisulfiteaddition product show similar paper chroinatographic behavior, e.g. they both have high


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MATTOK A X D I-IEACOCK: CHEMISTRY OF TI-IE AMINOCMROMES. PART VI 125R,'s in 2% a c et ic a c id a n d t h e y b o t h react r e l a t i v e ly s l o w l y w i t h i n d o l e reagents, s u c h asE h r l i c h ' s reagent a n d c i n n al n a ld e h y d e.

R e a c t i o n s b e tw e e n t h io l s a n d a l ~ l i n o c h r o m e s , u c h as t h o s e d e s c r i b e d above m a y be ofconsiderable s ignif icance in several biological processes. A possible mode of a t t a c h m e n tof m e l a n i n s to p r o t e i n s c o u l d be by a su l f ide l ink age formed by a n i n t er a c ti o n of o n e oft h e t h i o l g r o u p s i n t h e l a t t e r w i t h o n e of t h e i n do l e- 5 ,G - qu i no n e u n i t s i n t h e m e l a n i ns t r u c t u r e ( cf . 2 7 , 2 8 ) ; s u c h i n t e r a c t i o n s c o u l d also o c c u r ~ v i t h h e d i s ti n c t a l n i n o ch r o m eu n i t s w h i c h are cons idered by some workers to be p r e s e n t i n t h e m e l a n i n s t r u c t u r e (cf.29 , 30 ) . I t h a s b e e n s u g g e s t e d t h a t t h e s u lf i d e l i n k a g e o c c u r sat t h e 4 - p os i ti o n i n t h e i n d o l en u c l e u s ( 1 1, 3 1 ) , h o w e v e r , t h e p r e s e n t i n v e s t i g a ti o n w o ~ r l d u g g e s t t h a t t h e p o s s i b i li t yofs u c h l i n ka g e s o c c u r r i n g at t h e 7 - p o s i t io n of t h e i n d o le n u c le u s s h o u l d n o t be o v e r l o o k e d .

T h e f o r i ll a t io n of a m i n oc h r o me - t h io l a d d i t i o n p r o d u c t s s u c h as t h e co n l p o u n d ( I V )o f fe r s t h e i n t r i g u i n g p o s s i b i li t y t h a t some t h io l s cou ld conce ivab ly ac t as " a m i n o c h r o m e -car ri e rs" , wh ich cou ld read i ly regenerate t h e a m i n o c h r o m e u n d e r t h e a p p r o p r i a t e con-d i t i o n s . H a d l e r et al . h a v e recently d e s cr i b ed t h e s o m e w h a t a n a l o g o u s c o n j u g a t i o n ofc y s t e i n e d u r i n g i t s o x i d a t i o n by 2,G-dichlorophenolindophenol,nd s u g g e s t e d that reac-t i o n s of t h i s type m a y b e of biological s ignif icance (32, 33).

ACKNOWLEDGMENTST h e a s s i st a n ce of Mrs . B. D. Scott a n d RlIrs. D. L. Wilson w i t h some of t h e p a p e r

c h r o m a t o g r a p h i c w o r k i s g r at e f u l l y acknowledged.REFERENCES

1. J. BONGARTZ. er. 21, 483 (1888).2. J. M . SNEL L nd A. \.\IEISSBERGER.. Am. Chem. SOC. 1, 450 (1939).3. L. F. FIESER nd &I. FIESER. Organic chemistry. D. C. Heath and Co., Boston, Mass. 1944. p. 738.4. L. F. FIESER nd R. B. T URN ER. . Am. Chem. Soc. 69, 2335 (1947).5. E. FRIED.\IAN, . H. ~ I A R R I A N ,nd I. SIMON-REUSS. rit. J. Pharmacol. 3, 335 (1948).6. W. J . X r c s ~ a s o x , . FALCONE,nd G. STRAUSS.Biochemistry, 2, 537 (1963).7. H. S. ~ I A S O N . Nature, 175, 771 (1955).8. E. A. H . ROBERTS.Chem. Ind. London, 955 (1959).9. A. KODTAnd S. BOUCHILLOUX.omot. Rend. Soc. Biol. 154. 737 (1960).~ ~,10. S. B O U ~ H I L L O U Xand A. KODJA. ~ o m p t . k n d .51, 1920 (1960);11. S. BOUCHILLOUXnd A. KODJA. Bull. Soc. Chim. Biol. 42, 69 (1960).12. R. F. PORTER,W. W. REES, E. FRAUENGLAS S,. S. WILGUS, . H . NAWN, . P. CHIESA, nd J. W.GATES. J. Org. Chem . 29, 588 (1964).13. H. S. WILGUS, . FRAUENGLASS,. T. JONES,R. F. PORTER,nd J. W. GATES. J. Org. Chem. 29, 594(1964).14. R. k . H ~ A C O C Kand B. D. LAIDLAW.Chem. Ind. London, 1510 (1958).15. R. A. HEACOCKnd B. D. SCOTT. Can. J. Biochem. Physiol. 37, 1087 (1959).16. R. A. HEACOCK,. N ~ ~ ~ ~ ~ ~ ~nd A. N. PAYZA. Can. J . Chem. 36, 853 (1958).17. R. A. HEACOCKnd B. D. SCOTT. Can. J. Chem. 38, 516 (1960).18. G. L. MATTOKnd R. A. HEACOCK.Can. J. Chem. 42, 484 (1964).19. R. A. HEACOCK, . E. MAHON,nd B. D. SCOTT. Can. J. Chem. 39, 231 (1961).20. R. A. HEACOCKnd B. D. SCOTT. Unpublished results.21. R. A . HEACOCKnd G. L. MATTOK.Can. J. Chem. 41, 139 (1963).22. R. A. HEACOCK. hem. Rev. 59, 181 (1959).23. R. CECIL nd J. R. M CPHEE. Advan. Protein Chem. 14, 255 (1959).24. J. HARLEY-MASON.. Chem. Soc. 1276 (1950).25. R. L. TSE and M. 1. OESTERLING. lin. Chim. Acta. 8. 393 (1963)., - - ~ -~ ,

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