01 3.ii to J.0 ani1)ercsf l o ~ c t through l t h e circuit a t a voltage of 3.8 t o 4.0, T h e tcrniwi-attire of the aiiolyte was inaintainetl at 40 t o 45’. At the eriti of eight hours the oxidation ‘I,.as col,il,~ctc, T h e rcact.ion Inixture ,rorketl ul, as in thc. case of the qiiinolitie oxidation arid copper nicociliate (0 ti g . UT tW‘ , j \vas c;Ltaiiied.
Summary I . {~iiinolinehas been oxidized electrolytically
[ ( ~ O S T R I I 3 L . T I O SFROM .THE CIIEMICAL
a t a platinum anode in ycj5’, sulfuric acid to quinalinic with a of 77cj,. 2. Nicotinic acid has been obtained by decarbo tiori of quinolinic acid in cyclohexanol nnd also by the electrolytic oxidation of 3-picoline a t a lead anode. RECEIVED J U N E 17. 1 9 4 O
~ , C I ; I , P F TOS’rARIII ,
LADORATCJRIES O F TIIS JOIISS HOPKINS ~NIVERSITY]
A Synthetic Ferroporphyrin Complex that is Passive to Oxygen1 BY ,ILSOPH 1-1. CORWIN AND J. GORDON ERDMAN’ Hemoglobin possesses remarkable chemical properties other than its ability to combine reversibly with molecular oxygen. One of these is its passivity to oxygen, a property which has not been reported heretofore in ferroporphyrIn coniplexes of known structure. Hemoglobin is a ferrous coniplex, potentially susceptible to oxidation, yet capable of combining with a i i t l transporting oxygen without being oxidized to the fcrric state. An eluciclation of the conditions under which 1x1ssivity to oxygen may be expected slioultl provide one of the specifications for the synthesis of conipounds paralleling the chemical behavior of hemoglobin. The first step in a program with this objective would be the preparation o f solutions of hemes, ‘free From reducing agents, suitable for oxidation studies. This paper reports experimental conditions under which this objective may be attained. I n the course of this work, conditions were disco\-crId under which a ferroporphyrin complex coupled with pj’ridine is passive to atmospheric oxygen. 7’1s riecessary reactions have been follmvect and the preparations analyzetl spectroscopically. Preparation of pure ferroporphyritis, or hemes, has prove(? tlil’ficult, due t o their extreme scnsitivity to oxidation. So far an absorption spectrum for o m of these substances in ;i non-coiirtlinating solwrit h i s not been reported. Fischer, ’Treibs a n d Zeile:’ prepared crystalline hemes for the first time. They report powder spectra oi’ three bands somewh:it resenibling the ferric, porphyrin chlorid~~s or hemins. I’owtlcr spcctra are coniplica tctl b? strong itiolccular intcr:ictions a t i t 1 by selective. i.cattt.riii;: of the prticles. Spectra ol)t~i,ine(lby the addition of reducing agents such :is h y i r u i n t , 1iytlr:ite or sodium hydrosulfite to solutions oi the ferric porphyrins i n alkaline ~ t q u c o ~ media is or in nitrogen bases such as pyritli ne :ire complicated b y the tendency of the iron to form coniplcxes through its two reriiaining coiird .nate ,;alencit-s. I n the presence of large
amounts of nitrogen bases the formation of the complex, or hemochromogen, is accompanied by a shift from para- to diamagnetism and the formation of a characteristic spectrum. I n aqueous solutions the spectrum is spread out into a broad hump through partial complex formation with either water or the reducing agent which usually must be present in large excess. Knowledge of the nature of the binding in the ferroporphyrins and their complexes is vital to our understanding of their action in living systems. The original purpose of the present work was to prepare a crystalline heme, to devise means f u r determining the spectrum in a non-coordinating solvent, and finally t o examine its stability w.th respect to oxidation, both with and without a coijrdinating base. These objectives were a t tained by the use of special apparatus designed to exclude atmospheric oxygen during the nianipulations. ’ llesoherne IX dimethyl ester, I , crystallized in red needles which possessed a peculiar golden lustre unlike any of the other iron compounds of CH?--COOCH,g H,C;pCH? i
H$ ‘ --.\si
I
H , C1 1 -L.,
x-FeHiC! -1
1
~;
CH )--;,CH?CH?COOCI-I:I N 1 ~7 ‘CH:,
----
H& ~ --v,\CH
H,,C-==C,Hj
(1) Stuciie; i n t h e Pyrrulc Series X V I I I , 1’:iUer X V I I , 1:rilman a n d Corwin, T i r i i JoI.Rs.\I,, 6 8 , 188.7 f I O - l i i ) . ( 2 ) Preient :i!I.; c~)l(lfiiiger suspcntlctl in the neck, 311d the flask iiiimediately y heated glycerol-hath t o a depth plunged iiit~)aii elect x.\-ithii!aiitl without the flask \ v e x 16 z t icks isere addeil t o preveii t iic acitl began to reflux within two or three minutcs. I n about fifteen minutes the hro~viisusperision changctl to a deep i~urple-redsolutioii. The misture wah reflustti for- oiic an(1 one-half hour\ though spectroscopic a s a y of aliquot.; takeii during se\-era1 runs iridicatcd t h a t the reaction was practically conil)lctc~after half an hour. Marked decoinpoiition liecaiiie cvitlent after t w o to three hours. The cooled solution was filtered through a large sintcretl glass funnel t o remove the catalyst and then poured slowly with efficient stirring i:ito 600 nil. of 30r( aiiiiiiotiitiiii acet a t e solution. After a half hour, the prcc.ipitate was ceiitrifugetl t!o\vii, cd with distilled water, aiitl tlissolved i n 140 nil. of aqueous ammonia. Twenty-foulcubic centiineters o " i , disodium tartratc was added with thorough stirring, and the inisturc allowed to stand for a half hour. The lxight red precipitate \vas ceiitrifugetl out, arid the supernatant liquid caref~illypulled off. T h e latter varied from Iirown t o the pale ainlxr-red of disodium iriewporphyrin. The precipitate \va\ redissolveti i n 140 nil. of amiiionia and treated a c ahove. 111 most cases the motlicr liquor containetl o t i l iiiesoporphyriii. If it \vas h t i l l ljrorvn the pro\ peatccl a third time. The wet residue was heated o;i the stear~i-l)athto 100" and 150 cc. of boiling 25' hydrochloric acid addcd with vigorous stirring. Fine purple iieedles of t h c inesoporphyriii dihydrochloritie began to separate almost iniinetliatcly. After cooliiig o\-ernight i n the ice110 hy suctioii antl wailied Jvith a little c acid. The cntii-e fuiiiiel and coiit vaeuuni desiccator over potassium oughly dry. The rnesnporphyrin dihydrocliloi-itle \\-as then lifted out as a thiii brittle purple microcryitalliiic cake. Concentration of the nicther liquor yicltletl :i'i atltlitioiial averax(. yicltl I . i 8 g. small ainouiit of the iiih~~lrochloritle; or 90.5:;. Since the reaction time in the aliovc prcparation was rcciuced, it was felt desirable to prove that the IJroduct \\-as not protoporphyriii IX b y coiiipariiig it \vitli a sani~)le of the latter prcparetl by a method not involving catalytic reduction. X small sample of protoporphvriii IX was therefore prepared by dehydration of heiiiatoporlihyriii which was in turn obtained by the reactioii of acetic acitlhydrogen bromide on hemin. Comparisoni !\ ere made of melting points and spectra of t h e diinethyl complex antl of t h e ~reforiiiedpyricliiir 11 antl mixed iiicrlting point,; were t a k e n T h move coiiciusivelv that the reduction in the a1)ove imijara. . tion yielded inesoporphyriii. grainy of Mesoporphyrin IX Dimethyl Ester.--Two mesoDorDhvrin dihvdrochloride was added t c i 2 liters of ahsol;te'ii~cthanol containing 10 g. of dry hydrogen chloride. T h e mixture was refluxed for one hour, filtered while hot through a large sintered glass funnel, anrl thcu poiireti slowly atid with stirring into 3 liters of 2l 2' sot!ium hi.~...___
(11) S t a r r a n d Hixon, "Organic Syntheses," 16, 77 1930j.
carbonate solution containing 1 kg. of iincly crushed ice aiid 4 g. of celite. . 4 filter \\-as preparctl by pouring 1 g . of celite, suspended in water, through an 8-ciii. 1;iiclincr funnel. The layer of celite thus deposited prevciitetl some of t h e porphyrin which floats on the surface froin cloggiiig the filter paper. The cclite suspeiision of thc p x p h y r i i i was washed thoroughly with tlistillctl tvater aii(l iiriini~:liately dried in a vacuum desiccator. The dry celite cake was Ixokeii iiito a fi>icpov-tlrancl packed into a chroinatograph tul)e i!i the i i z u i l iriatiiier. Chloroform was forced throngh thc colunxi u:itler several pountls pressute. I n this way a very coiiceiitratetl solution, free from celite, \vas obtained. T h e filtrate \vas coliccntrated to about 20 nil. and hot inethanol added to i i i cipient precipitation. Oil cooling, the po:-p!iyi-iii separated in fin; glistenitig purple leaves. I t was iilt(~!-et!by suctioii, washed with 20 inl. of 1: 9 chloroforiii-uicttia!iol. aiitl cli-ivgl in a vacuum desiccator; yield 1.7h g. or \ l ~ . t j ' ~ ,i n . ; I . 213.5-2 L A " (cor.). The mother liquors were evaporateti to (1168 ing. of crude b u t erysta!line material. Recrystalliza.tion.-The above 'material w i i dissolved iii 20 ml. of boiling chloroform and 50 ml. of boiling nicrhanol slowly added, care being taken t h a t none of it ran down t h e walls. -1 clear b u t highly supersaturated solution was thus obtained. The glass stopper was immediatrly i l l serted and the flask pliiiiged into a 4-liter bcaker of water hcated to fi:: '. hfter almut a half hour cliister. of sparkling 'gall to appear on the \vdls. Aftci c~),iIiiiq inperature was sIoiv1:~-ticprcsic~l Iiy m l i l i :ially salt. The crystals iverc liltcrctl oiT, washed with 1::) tiiethaiiol-clilorof:)riii aiit1 vacuu.;i clrictl; piclti 1.N g. or 92.2('; ; i n . p . 213.5-3lS.5" ( c o r . ) . Evaporation of the mother liqiiori yielJet1 .42 1115.of crude but crystalline material. Maguifietl 12'5 X , the pure product appeared gated plates shLiwiiigoblique extinction utitlcr c The cryjmls p:)ssess a striki ig parpli
