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The action of diazomethane on haemin chlorides
150
BIOCHIMICAET BIOPHYSICAAC'IA BBA 3918
T H E ACTION OF D I A Z O M E T H A N E ON HAEM1N C H L O R I D E S P. s. CLEZY ANt) D. B. MORELL School of Chemistry, University of New South Wales and ]nstitui~ of Medical Research, Royal North Shore Hospital, Sydney (,4 ustralia)
(Received July 25th, I962)
SUMMARY Haemin chlorides have been shown to react with diazomethane in ethereal solution to give a spectroscopically identifiable species with an ab,:-o.-~tion maximum near 6oo ml~. This compound is decomposed by acid to give the porphy~-n corresponding to the original haemin chloride. Alkaline hacmatins or porphyrin-copper complexes are not affected by diazomethane.
INTRODUCTION The most common and convenient method of preparing the methyl esters of porphyrins involves the use of diazomethane in ethereal solution. No side reactions have been reported during such methylation procedures which take place practically instantaneously, although it is known that long contact of diazomethane with formyl porphyrins in methanol produces a compound whose properties are consistent with those of a substituted ethylene oxide 1. Attempts to employ diazomethane for the methylation of carboxylic acid groups in haemin chlorides has surprisingly led to major spectral alterations. Fortunately, however, haemins can be methylated by the more time-consuming method using anhydrous methanol containing hydrogen chloride or sulphuric acid. Ethereal solution of haemins, when allowed to come into contact with diazomethane, immediately lose their characteristic red-brown colour and become greenishyellow. This colour change is brought about by the disappearance of haemin absorption bands and their replacement by an absorption maximum in the vicinity of 600 m/,. This paper discusses, in some detail, the interaction of diazomethane with haemin chlorides and in particular describes the characterisation and properties of the product formed from the action of diazomethane on deuterohaemin chloride. METHODS AND bIATERIALS S[.ectroscopy: Visible spectroscopic measurements were made with a Perkin-Elmer
"35 °" Spectrophotometer, the wavelength scale being standardized against reduced cytochrome c (55° m~). Infrared spectra were recorded as Nujol mulls using a PerkinElmer "Infracord" Spectrophotometer. Biochim: Biophys. Aaa, 7t (I963) x5o--x56
ACTION CF DIAZOMETHANE ON HAEMINS
I5I
Melting points : Melting points are corrected and were determdned using a Griffin a n d Tatlock hot-stage microscope. Paper chromatograpt~v. The method of CHu, GREEN AND CHU°- with slight modifications was used. Kerosene - chloroform (4.0 : 2.6) and kerosene - propanol (6 : I) were used as solvent systems. Solvents: All solvents were distilled prior to use with the exception of ether which did not contain a reducing agent and was used as supplied. Hydrochloric acid: All percent concentrations refer to w/v. Ethereal diazomethane: ]'his was prepared b y the gradual addition of 2o g ol N-nitrosomethylurea 3 to 2oo ml of 2o % aqueous potassium hydroxide 1,~yered under 5oo ml of ether. When all the urea had reacted with the alkali the ethereal solution was separated from the aqueous phase and used as such. 29reparation of porphyrins and haemins : Deuterohaemin, protohaemin and monoformylhaemin chloride were prepared as their dimethyl esters by methods previou.~ly described ~. Deuteroporphyrin dimethyl ester was obtained by the diazomethane metbylation of the porphyrin free acid prev:~red by the removal of iron from deuterohaemin using the method of MORELL AND STEWART~. EXPERIMENTAL AND RESULTS
Action of diazomethane on haemin chlorides A b o u t 0.3-0.4 mg of haemin chloride methyl ester was dissolved in 2 drops of chloroform and the solution diluted to 3 ml with ether. This was treated in a cuvette with I ml of ethereal diazomethane (see METHODS AND MATERIALS) and the reaction followed spectrophotometricaily taking readings against a blank prepared b y the addition of I ml of ethereol diazomethane to 3 ml of ether. R e p e a t e d scanning of the visible spectrum showed t h a t the reaction was substantially complete after 15 rain. T h e / t a e m i n absorption m a x i m a had disappeared and a new absorption m a x i m u m was a p p a r e n t in the vicinity of 600 m/~, the actual position being dependent on the n a t a r e of electrophilic groups in conjugation with the tetrapyrrolic ring system. Monoformyl-, proto- and deuterohaemins after reaction with diazomethane developed m a x i m a at 612 m/,, 595 mt z and 588 m/~ respectively. The use of ether, previously washed with ferrous sulphate to remove peroxides, or the careful drying of the ethereal diazomethane solution over potassium hydroxide pellets did not alter these results. The h y d r o x y haematins (alkaline haematins) and the copper complexes o_f porphyriils were unaffected b y ethereal diazomethane for periods up to I h. As deuterohaemin appeared less likely to undergo side reactions v,dth diazomethane a n d was available in large quantities, this haemin was selected for the further examination of this reaction.
A orion of diazomethane on deuterohaemin chloride dimethyl ester Small scale reaction: IOO mg of deuterohaemin chloride dimethyl ester was dissolved in 25 ml chloroform a n d trea ted with an eqaal volume of ethereal diazomethane. An immediate change in the absorption spectrum of the solution was noted. The spectrum of tb, i~ dia:~odeuterohaematin, which was formed b y a!!o,.~.'ng deuterohaemin
Biockim. Bicphys. Acta, 7t (x963) 1.5o-I5o
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P.S. CLEZY, D.B. MORELL
to remain in contact with diazomethane for I h is givetp in Fig. I. q his compound appears quitc stable in ethereal solution and has been kept for several weeks at room temperature withoat a p p a r e n t change. It gives a pyridine haemochrorne in 20 % aqueous pyridine with sodium dithionite as reducing agent, and this haemochrome is spectroscopically identical with pyridine deuterohacmochiome. The diazodeuterohaematin is readily soluble in ~5 % HCI whereupon the two-banded diazohaenmtin spectrum is converted into a three-banded spectrum (maxima at 595 m/z, 566 mt~ and 55o m/~, Fig. I). W i t h time it was observed that the 566-m/, maximum.
6~o
66o'
~o
'
~6o
575 525 Wavelength (rn,u)
'
475
Fig. I. Spectra : ], diazodeuterohaematin in ether ; 2, diazodeuterohaematin shortly after extraction into z5 °o HCI showing the development of acid porphyrin bands; 3, the acid porp_hyrin bands produced after 3 h in dilute HCI. The pigment concentrations are not equivalent. originally the most intense band, slowly faded while the m a x i m a at 595 m/~ a n d 550 m/~ became stronger. This change was greatly accelerated upon dilution of the acidic solution. A d j u s t m e n t of the p H of the solution with sodium acetate enabled a porphyrin to be e x t r a c t e d into ether. Spectroscopically and paper chromatographically this porphyrin was identical with deuteroporphyrin. The aqueous phase left after extlaction of the porphyrin contained yellow by-products devoid of s h a r p spectral bands. This fraction was not investigated. La~,e scale reaction : 1.3 g of deuterohaernin chloride dimethyl ester was dissolved in I I of chloroform which was then diluted b y the addition of 3 1 of ether. To this mixture was added 500 ml of ethereal diazomethane and the whole allowed to s t a n d for x5 min, when the h a n d spectroscope showed at this stage only one major b a n d (appio~. 585 m/z). The ethereal solution was then extracted repeatedly with ¢5 % H(?I until the extracts were no longer coloured. The ethereal phase which contained unaltered haemin was washed once with water, e v a p o r a t e d to half its volume a n d again t r e a t e d with 500 ml of ethereal diazomethane as above. Recovered haemin from this t r e a t m e n t , after washing and reduction of solution volume, was again subjected to
Biochim. Biophys..4 eta, 71 (1963) t 5o-156
ACTION OF I)IAZO.METHANE ON HAEMINS
I53
fresh ethere'd diazomethane as above. No significant amounts of haemin were recovered after this treatment. All 25 % HCI extracts, which were dark violet in colour and showed the characteristic three-banded spectrum referred to above, were combined and diluted with 2 volumes of water. After 3 h tile 5(75 m/, m a x i m u m had disappeared leaving the 595 mr* and 55 ° mt~ max;ma which are typical of an acid porphyrin spectrum. After careful p H adjustment, first with sodium hydroxide and finally with sodium acetate, an ethereal solution of a porphyrin was obtained. This ethereal solution was washed repeatedly with water to free it of acetic acid. The porphyrin was then purified by acid extraction (~ - 2 %; ". 5 % H~!) which left a smal! -'_tmoont of haemin in the ether. "l'his was rejected. The porphyrin was returned to ether with aqueous sodium acetate and the ethereal solution again freed af acetic acid b y repeated water washings. After a brief t r e a t m e n t with ethereal diazomethane to ensure that the porphyrin was fully esterified the ethereal porphyrin solution was washed with aqueous sodium carbonate and water and dried (Na2SO4). Removal of the solvent in vaclto gave a residue which was obtained crystalline from chloroform-methanol. Repeated recrystallization from the same solvent mixture gave o.5 g of fine needles, m.p. 220-222 ° undeplessed by admixture with deuteroporphyrin dimethyl ester, m.p. 22a-224 °. Tke visible and infrared spectra of this porphyrin were identical with those of deuteroporphyrin dimethvl ester and furthermore the two porphyrins had the same RF vames in two solvent systems (see METHOI)SAND ?dATERIALS). (Found : C, 7o.7 ; tt, 6.2; N, lO.45; O, II.80o. Ca.,Ha4()4N ~ requires C, 71.35; H, 6.4; N, lO.4; O, I I . 9 %. )
Preparation of copper complex 5o mg of porphytin dissolved in 5o nil of acetic acid was heated on a steam bath for IO rain in the presence of excess cupric acetate. After cooling this solution, the copper complex was transferred to ether and the ethereal solution washed with water and 5 % HCI to remove acetic acid and p , r p h y r i n respectively. The bright red copper complex was purified by recrvstallisation from c h l o r o f o r m - m e t h a n o l , m.p. 229-232 undepressed upon a d m i x t u r e with the copper complex of deuteroporphyrin dimethvl ester, m.p. 223-235 °. The visible and infrared spectra of these two complexes were identical. (Found: C, 03.8; H, 5.25; N, q.3°,g. C3.,H3~O~N4Cu requires C, 64.o; H, 5.4; N, 9-3 %.) DISCUSSION Haemin chlorides when subjected to diazomethane in ethereal solution are almost instantaneously transformed into a ~nocio~ which can be identified spectroscopically b y an absorption m a x i m u m near 6oo nl~. Since porphyrins or their copper complexes are not affected b y this reagent it can be assumed that the diazomethane attacks the iron of the haemin chloride. Furthermore since a pyridine haemochrome is produced from diazohaematins bv the action of aqueous pyridine in the presence of dithionite it would seem that the iron is still present in such a compound. This leads to the conclusion t h a t diazomethane displaces the chloride from the iron. The spectroscopic properties of the diazohaematin are also in accord with this conclusion. On this basis then it is not surprising that copper complexes and alkaline haematins are not affected b y diazomethane. Copper. co-ordinated with tetrapyrrolic
Biochim. Biophys. Acta, 71 (I963) I5o-i56
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P.S. CLEZY, D.B. MORELL
molecules does n o t react w i t h ot~,er ligands, while u n d e r tile n e u t r a l conditions o f this reaction it is to be e x p e c t e d t h a t t h e chloride of a h a e m i n chloride w o u l d be displaced m u c h m o r e r e a d i l y t h a n t h e h y d r o x y l g r o u p of an alkaline h a e m a t i n . H o w e v e r , t h e d i a z o h a e m a t i n is r e m a r k a b l e in t h a t a q u e o u s acid r e m o v e s iron f r o m t h e molecule to yield t h e p o r p h y r i n c o r r e s p o n d i n g to t h e original h a e m a t i n . T h e overall reaction then, brings a b o u t t h e r e m o v a l of iron from t h e h a e m i n chloride. N o r m a l l y t h e r e m o v a l of iron f r o m a F e 3 ~- p o r p h y r i n c o m p l e x is e x t r e m e l y difficult a n d can o n l y be a c h i e v e d in rather special cases a n d w h e n v e r y s t r o n g acids are used, e g. m o n o f o r m y l d e u t e r o h a e m i n is c o n v e r t e d to its p o r p h y r i n b y 36 N sulphuric acid s. T h e m o r e usual p r o c e d u r e in such a t r a n s f o r m a t i o n is to reduce t h e iron to t h e ferrous s t a t e w h i c h r e a d i l y gives up its m e t a l u n d e r acid conditions. F e r r o u s a c e t a t e in acetic acid a t 80 ° (see ref. 7) or t h e m o r e c o n v e n i e n t a n d m i l d e r ferrous s u l p h a t e p r o c e d u r e of MORELL AND STEWART 5 are t h e r e d u c i n g a g e n t s g e n e r a l l y employed. In t h e p r e s e n t case. since t h e iron a t o m can be r e m o v e d from t h e d i a z o h a e m a t i n b y a q u e o u s acid it a p p e a r s t h a t at s o m e stage of this r e a c t i o n t h e o x i d a t i o n s t a t e of t h e iron is c h a n g e d f r o m F e 3+ to F e 2+. Most of t h e findings p r e s e n t e d here can be rationalised in t e r m s of t h e following mechanism. T h e s t r u c t u r e of d i a z o m e t h a n e is u s u a l l y r e p r e s e n t e d as a h y b r i d of t h e f o r m s : --
--N
<----> C H 2
= ~" = "~
a n d the d i a z o h a e m a t i n m i g h t be formed in t h e following w a y : "\,,3+, / -, "".,3+/ ~ ~-, Fe - CI . . . . . . --* Fe - N = N = CH 2
/
',,,,, ;, •~
........,
"',.3~/" -----> Fc - N = N - CH 2 CI
,,
+ = N = CH a
/
",,
C1
This species w o u l d t h e n be responsible for t h e a b s o r p t i o n m a x i m u m near 600 m l , characteristic of d i a z o h a e m a t i n s . In a q u e o u s acids t h e d i a z o h a e m a t i n m i g h t be i m a g i n e a to d e c o m p o s e b y t h e follm~dng s c h e m e : " ,, 3+/"
v,.
-X=X-CH.,CI+-H
.........
"\ 3*/"
'F~"
-X=ktiCCHoCl }1
,., 2 + / "\., 3 + / ! Fe + N z -e It" 4 - - - - - - Fe -- N = NH +-. . . . . . . . ' - . . . . . . . -+ CH~.CI / / .,. /. . ---.
i
+OH 2
HCHO HCI H+
a n d t h e ferrous c o m p l e x so o b t a i n e d c o n v e r t e d u n d e r t h e acid c o n d i t i o n s to t h e free porphyfin.
\a+/ T h e free radical d e c o m p o s i t i o n of
/
Fe
\
-- -~" = .'~H is analogous to t h e p r o p o s e d Biochim. Biophys. Acla, 71 (1963) 15o--x5~J
ACTION OF I)IAZOMETHANE ON HAEMINS
I55
mechanism of phenylation of aromatic hydrocarbons bv phenylhydrazine in the presence of silver oxide s. t'h -- NH -- Nil,, :}_g2([_~ "£1'h - N = N}t . . . . . .
l'h" + N._,~- H"
When diazodeuterohaematin is dissolved in 25 °o hydrochloric acid a threebanded spectrum is obtained. Two of these maxima are almost certainly due t(~ small amounts of acid porphyrin. It is not possible to say with certainty whether the third maximum, at 566 m~ is due to diazodeuterobaematin ltseli (the change from 588 mtL being a t t r i b u t e d to solvent difference) or whether it is due to another intermediate along the .decomposition pathway. A consideration of general intelest is that in this reaction a haematin is converted to a porphyrin in an aqueous medium. MORELL AND STEWART~ have reported earlier that removal of iron from baematins in acetic acid is seriously retarded as the water concentration rises. If the mechanism proposed here for the decomposition of diazohacmatin is essentially correct it might be supposed that the presence of water affects not the actual removal of iron but its reduction by ferrous sulphate.
F \ 650
600
550 500 575 525 WQvelength(m/u)
4-)5
Fig. 2. "File spectrum of dcuteroporphyrin dimethyl ester in ether demonstrating band !X" as a doublet. One last point deserves comment. The spectrum of deuteroporph3Tin presented in this paper (Fig. 2) shows very clearly that b a n d IV is a doublet. This does not appear to have been reported before. Haematoporpbyrin and mesoporphyrin also show this spectroscopic feature but protoporphyrin, monoformyldeuteroporphyrin and diacetyldeuteroporphyrin, all of which are electrophilically substituted, exhibit only a single band. ACKNOWLEDGEMENTS One of US (D.B.M.) w~shes to acknowledge personal support from the National Health a n d Medical Research Council of Australia. These spectrophotometri:c studies were Biochim. Biophys...Iota, 71 [t963) x5o--I5~*
156
P . s . CLEZY, D. B. MORELL
g r e a t l y f a c i l i t a t e d b y t h e u s e of a P e r k i n - E l m e r " 3 5 o ' ' s p e c t r o p h o t o m e t e r p u r c h a s e d from funds s u p p l i e d b y the a b o v e Council a n d the P o s t - G r a d u a t e Medical R e s e a r c h F o u n d a t i o n in S y d n e y . REFERENCES z 1'. S. ('LEZV unpublished observation. T. C. Cm', A. G, GREEN AND E. J, CHU, J, Biol. Chem., 19o (t951i (>433 l:..\RNDT, Organic Syntheses, Collective Vol. 2 (1943) 40I. a I>. S. CLEZ'; AND D. B. MORELL, Biochim. Biophys. Acta, 71 (t963) x65. :' l). I{..~,IORELL AND M. *t~TEIA,ARI..~*~.,i, t d l a n ,]. ExplI. I3iol..'lled. Sci.. 34 (";5 ¢') ,~t '; H. I:ISCHER ANt) G. ~VEcKF.R, Z. Physiol. Chem., 272 (I~t42) I. 7 (). \VARBURG AND E. NEGELEIN, I3iochem. Z., 244 (193-) t~. " l/. 1,. t-IARDIE AND li. 1t. THO.~tSON, ]. Chem. Soc., (I957) 2512.
Biochim. Biophys. Acla, 71 (t963) I5O-156
BIOCHIMICAET BIOPHYSICAAC'IA BBA 3918
T H E ACTION OF D I A Z O M E T H A N E ON HAEM1N C H L O R I D E S P. s. CLEZY ANt) D. B. MORELL School of Chemistry, University of New South Wales and ]nstitui~ of Medical Research, Royal North Shore Hospital, Sydney (,4 ustralia)
(Received July 25th, I962)
SUMMARY Haemin chlorides have been shown to react with diazomethane in ethereal solution to give a spectroscopically identifiable species with an ab,:-o.-~tion maximum near 6oo ml~. This compound is decomposed by acid to give the porphy~-n corresponding to the original haemin chloride. Alkaline hacmatins or porphyrin-copper complexes are not affected by diazomethane.
INTRODUCTION The most common and convenient method of preparing the methyl esters of porphyrins involves the use of diazomethane in ethereal solution. No side reactions have been reported during such methylation procedures which take place practically instantaneously, although it is known that long contact of diazomethane with formyl porphyrins in methanol produces a compound whose properties are consistent with those of a substituted ethylene oxide 1. Attempts to employ diazomethane for the methylation of carboxylic acid groups in haemin chlorides has surprisingly led to major spectral alterations. Fortunately, however, haemins can be methylated by the more time-consuming method using anhydrous methanol containing hydrogen chloride or sulphuric acid. Ethereal solution of haemins, when allowed to come into contact with diazomethane, immediately lose their characteristic red-brown colour and become greenishyellow. This colour change is brought about by the disappearance of haemin absorption bands and their replacement by an absorption maximum in the vicinity of 600 m/,. This paper discusses, in some detail, the interaction of diazomethane with haemin chlorides and in particular describes the characterisation and properties of the product formed from the action of diazomethane on deuterohaemin chloride. METHODS AND bIATERIALS S[.ectroscopy: Visible spectroscopic measurements were made with a Perkin-Elmer
"35 °" Spectrophotometer, the wavelength scale being standardized against reduced cytochrome c (55° m~). Infrared spectra were recorded as Nujol mulls using a PerkinElmer "Infracord" Spectrophotometer. Biochim: Biophys. Aaa, 7t (I963) x5o--x56
ACTION CF DIAZOMETHANE ON HAEMINS
I5I
Melting points : Melting points are corrected and were determdned using a Griffin a n d Tatlock hot-stage microscope. Paper chromatograpt~v. The method of CHu, GREEN AND CHU°- with slight modifications was used. Kerosene - chloroform (4.0 : 2.6) and kerosene - propanol (6 : I) were used as solvent systems. Solvents: All solvents were distilled prior to use with the exception of ether which did not contain a reducing agent and was used as supplied. Hydrochloric acid: All percent concentrations refer to w/v. Ethereal diazomethane: ]'his was prepared b y the gradual addition of 2o g ol N-nitrosomethylurea 3 to 2oo ml of 2o % aqueous potassium hydroxide 1,~yered under 5oo ml of ether. When all the urea had reacted with the alkali the ethereal solution was separated from the aqueous phase and used as such. 29reparation of porphyrins and haemins : Deuterohaemin, protohaemin and monoformylhaemin chloride were prepared as their dimethyl esters by methods previou.~ly described ~. Deuteroporphyrin dimethyl ester was obtained by the diazomethane metbylation of the porphyrin free acid prev:~red by the removal of iron from deuterohaemin using the method of MORELL AND STEWART~. EXPERIMENTAL AND RESULTS
Action of diazomethane on haemin chlorides A b o u t 0.3-0.4 mg of haemin chloride methyl ester was dissolved in 2 drops of chloroform and the solution diluted to 3 ml with ether. This was treated in a cuvette with I ml of ethereal diazomethane (see METHODS AND MATERIALS) and the reaction followed spectrophotometricaily taking readings against a blank prepared b y the addition of I ml of ethereol diazomethane to 3 ml of ether. R e p e a t e d scanning of the visible spectrum showed t h a t the reaction was substantially complete after 15 rain. T h e / t a e m i n absorption m a x i m a had disappeared and a new absorption m a x i m u m was a p p a r e n t in the vicinity of 600 m/~, the actual position being dependent on the n a t a r e of electrophilic groups in conjugation with the tetrapyrrolic ring system. Monoformyl-, proto- and deuterohaemins after reaction with diazomethane developed m a x i m a at 612 m/,, 595 mt z and 588 m/~ respectively. The use of ether, previously washed with ferrous sulphate to remove peroxides, or the careful drying of the ethereal diazomethane solution over potassium hydroxide pellets did not alter these results. The h y d r o x y haematins (alkaline haematins) and the copper complexes o_f porphyriils were unaffected b y ethereal diazomethane for periods up to I h. As deuterohaemin appeared less likely to undergo side reactions v,dth diazomethane a n d was available in large quantities, this haemin was selected for the further examination of this reaction.
A orion of diazomethane on deuterohaemin chloride dimethyl ester Small scale reaction: IOO mg of deuterohaemin chloride dimethyl ester was dissolved in 25 ml chloroform a n d trea ted with an eqaal volume of ethereal diazomethane. An immediate change in the absorption spectrum of the solution was noted. The spectrum of tb, i~ dia:~odeuterohaematin, which was formed b y a!!o,.~.'ng deuterohaemin
Biockim. Bicphys. Acta, 7t (x963) 1.5o-I5o
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P.S. CLEZY, D.B. MORELL
to remain in contact with diazomethane for I h is givetp in Fig. I. q his compound appears quitc stable in ethereal solution and has been kept for several weeks at room temperature withoat a p p a r e n t change. It gives a pyridine haemochrorne in 20 % aqueous pyridine with sodium dithionite as reducing agent, and this haemochrome is spectroscopically identical with pyridine deuterohacmochiome. The diazodeuterohaematin is readily soluble in ~5 % HCI whereupon the two-banded diazohaenmtin spectrum is converted into a three-banded spectrum (maxima at 595 m/z, 566 mt~ and 55o m/~, Fig. I). W i t h time it was observed that the 566-m/, maximum.
6~o
66o'
~o
'
~6o
575 525 Wavelength (rn,u)
'
475
Fig. I. Spectra : ], diazodeuterohaematin in ether ; 2, diazodeuterohaematin shortly after extraction into z5 °o HCI showing the development of acid porphyrin bands; 3, the acid porp_hyrin bands produced after 3 h in dilute HCI. The pigment concentrations are not equivalent. originally the most intense band, slowly faded while the m a x i m a at 595 m/~ a n d 550 m/~ became stronger. This change was greatly accelerated upon dilution of the acidic solution. A d j u s t m e n t of the p H of the solution with sodium acetate enabled a porphyrin to be e x t r a c t e d into ether. Spectroscopically and paper chromatographically this porphyrin was identical with deuteroporphyrin. The aqueous phase left after extlaction of the porphyrin contained yellow by-products devoid of s h a r p spectral bands. This fraction was not investigated. La~,e scale reaction : 1.3 g of deuterohaernin chloride dimethyl ester was dissolved in I I of chloroform which was then diluted b y the addition of 3 1 of ether. To this mixture was added 500 ml of ethereal diazomethane and the whole allowed to s t a n d for x5 min, when the h a n d spectroscope showed at this stage only one major b a n d (appio~. 585 m/z). The ethereal solution was then extracted repeatedly with ¢5 % H(?I until the extracts were no longer coloured. The ethereal phase which contained unaltered haemin was washed once with water, e v a p o r a t e d to half its volume a n d again t r e a t e d with 500 ml of ethereal diazomethane as above. Recovered haemin from this t r e a t m e n t , after washing and reduction of solution volume, was again subjected to
Biochim. Biophys..4 eta, 71 (1963) t 5o-156
ACTION OF I)IAZO.METHANE ON HAEMINS
I53
fresh ethere'd diazomethane as above. No significant amounts of haemin were recovered after this treatment. All 25 % HCI extracts, which were dark violet in colour and showed the characteristic three-banded spectrum referred to above, were combined and diluted with 2 volumes of water. After 3 h tile 5(75 m/, m a x i m u m had disappeared leaving the 595 mr* and 55 ° mt~ max;ma which are typical of an acid porphyrin spectrum. After careful p H adjustment, first with sodium hydroxide and finally with sodium acetate, an ethereal solution of a porphyrin was obtained. This ethereal solution was washed repeatedly with water to free it of acetic acid. The porphyrin was then purified by acid extraction (~ - 2 %; ". 5 % H~!) which left a smal! -'_tmoont of haemin in the ether. "l'his was rejected. The porphyrin was returned to ether with aqueous sodium acetate and the ethereal solution again freed af acetic acid b y repeated water washings. After a brief t r e a t m e n t with ethereal diazomethane to ensure that the porphyrin was fully esterified the ethereal porphyrin solution was washed with aqueous sodium carbonate and water and dried (Na2SO4). Removal of the solvent in vaclto gave a residue which was obtained crystalline from chloroform-methanol. Repeated recrystallization from the same solvent mixture gave o.5 g of fine needles, m.p. 220-222 ° undeplessed by admixture with deuteroporphyrin dimethyl ester, m.p. 22a-224 °. Tke visible and infrared spectra of this porphyrin were identical with those of deuteroporphyrin dimethvl ester and furthermore the two porphyrins had the same RF vames in two solvent systems (see METHOI)SAND ?dATERIALS). (Found : C, 7o.7 ; tt, 6.2; N, lO.45; O, II.80o. Ca.,Ha4()4N ~ requires C, 71.35; H, 6.4; N, lO.4; O, I I . 9 %. )
Preparation of copper complex 5o mg of porphytin dissolved in 5o nil of acetic acid was heated on a steam bath for IO rain in the presence of excess cupric acetate. After cooling this solution, the copper complex was transferred to ether and the ethereal solution washed with water and 5 % HCI to remove acetic acid and p , r p h y r i n respectively. The bright red copper complex was purified by recrvstallisation from c h l o r o f o r m - m e t h a n o l , m.p. 229-232 undepressed upon a d m i x t u r e with the copper complex of deuteroporphyrin dimethvl ester, m.p. 223-235 °. The visible and infrared spectra of these two complexes were identical. (Found: C, 03.8; H, 5.25; N, q.3°,g. C3.,H3~O~N4Cu requires C, 64.o; H, 5.4; N, 9-3 %.) DISCUSSION Haemin chlorides when subjected to diazomethane in ethereal solution are almost instantaneously transformed into a ~nocio~ which can be identified spectroscopically b y an absorption m a x i m u m near 6oo nl~. Since porphyrins or their copper complexes are not affected b y this reagent it can be assumed that the diazomethane attacks the iron of the haemin chloride. Furthermore since a pyridine haemochrome is produced from diazohaematins bv the action of aqueous pyridine in the presence of dithionite it would seem that the iron is still present in such a compound. This leads to the conclusion t h a t diazomethane displaces the chloride from the iron. The spectroscopic properties of the diazohaematin are also in accord with this conclusion. On this basis then it is not surprising that copper complexes and alkaline haematins are not affected b y diazomethane. Copper. co-ordinated with tetrapyrrolic
Biochim. Biophys. Acta, 71 (I963) I5o-i56
I54
P.S. CLEZY, D.B. MORELL
molecules does n o t react w i t h ot~,er ligands, while u n d e r tile n e u t r a l conditions o f this reaction it is to be e x p e c t e d t h a t t h e chloride of a h a e m i n chloride w o u l d be displaced m u c h m o r e r e a d i l y t h a n t h e h y d r o x y l g r o u p of an alkaline h a e m a t i n . H o w e v e r , t h e d i a z o h a e m a t i n is r e m a r k a b l e in t h a t a q u e o u s acid r e m o v e s iron f r o m t h e molecule to yield t h e p o r p h y r i n c o r r e s p o n d i n g to t h e original h a e m a t i n . T h e overall reaction then, brings a b o u t t h e r e m o v a l of iron from t h e h a e m i n chloride. N o r m a l l y t h e r e m o v a l of iron f r o m a F e 3 ~- p o r p h y r i n c o m p l e x is e x t r e m e l y difficult a n d can o n l y be a c h i e v e d in rather special cases a n d w h e n v e r y s t r o n g acids are used, e g. m o n o f o r m y l d e u t e r o h a e m i n is c o n v e r t e d to its p o r p h y r i n b y 36 N sulphuric acid s. T h e m o r e usual p r o c e d u r e in such a t r a n s f o r m a t i o n is to reduce t h e iron to t h e ferrous s t a t e w h i c h r e a d i l y gives up its m e t a l u n d e r acid conditions. F e r r o u s a c e t a t e in acetic acid a t 80 ° (see ref. 7) or t h e m o r e c o n v e n i e n t a n d m i l d e r ferrous s u l p h a t e p r o c e d u r e of MORELL AND STEWART 5 are t h e r e d u c i n g a g e n t s g e n e r a l l y employed. In t h e p r e s e n t case. since t h e iron a t o m can be r e m o v e d from t h e d i a z o h a e m a t i n b y a q u e o u s acid it a p p e a r s t h a t at s o m e stage of this r e a c t i o n t h e o x i d a t i o n s t a t e of t h e iron is c h a n g e d f r o m F e 3+ to F e 2+. Most of t h e findings p r e s e n t e d here can be rationalised in t e r m s of t h e following mechanism. T h e s t r u c t u r e of d i a z o m e t h a n e is u s u a l l y r e p r e s e n t e d as a h y b r i d of t h e f o r m s : --
--N
<----> C H 2
= ~" = "~
a n d the d i a z o h a e m a t i n m i g h t be formed in t h e following w a y : "\,,3+, / -, "".,3+/ ~ ~-, Fe - CI . . . . . . --* Fe - N = N = CH 2
/
',,,,, ;, •~
........,
"',.3~/" -----> Fc - N = N - CH 2 CI
,,
+ = N = CH a
/
",,
C1
This species w o u l d t h e n be responsible for t h e a b s o r p t i o n m a x i m u m near 600 m l , characteristic of d i a z o h a e m a t i n s . In a q u e o u s acids t h e d i a z o h a e m a t i n m i g h t be i m a g i n e a to d e c o m p o s e b y t h e follm~dng s c h e m e : " ,, 3+/"
v,.
-X=X-CH.,CI+-H
.........
"\ 3*/"
'F~"
-X=ktiCCHoCl }1
,., 2 + / "\., 3 + / ! Fe + N z -e It" 4 - - - - - - Fe -- N = NH +-. . . . . . . . ' - . . . . . . . -+ CH~.CI / / .,. /. . ---.
i
+OH 2
HCHO HCI H+
a n d t h e ferrous c o m p l e x so o b t a i n e d c o n v e r t e d u n d e r t h e acid c o n d i t i o n s to t h e free porphyfin.
\a+/ T h e free radical d e c o m p o s i t i o n of
/
Fe
\
-- -~" = .'~H is analogous to t h e p r o p o s e d Biochim. Biophys. Acla, 71 (1963) 15o--x5~J
ACTION OF I)IAZOMETHANE ON HAEMINS
I55
mechanism of phenylation of aromatic hydrocarbons bv phenylhydrazine in the presence of silver oxide s. t'h -- NH -- Nil,, :}_g2([_~ "£1'h - N = N}t . . . . . .
l'h" + N._,~- H"
When diazodeuterohaematin is dissolved in 25 °o hydrochloric acid a threebanded spectrum is obtained. Two of these maxima are almost certainly due t(~ small amounts of acid porphyrin. It is not possible to say with certainty whether the third maximum, at 566 m~ is due to diazodeuterobaematin ltseli (the change from 588 mtL being a t t r i b u t e d to solvent difference) or whether it is due to another intermediate along the .decomposition pathway. A consideration of general intelest is that in this reaction a haematin is converted to a porphyrin in an aqueous medium. MORELL AND STEWART~ have reported earlier that removal of iron from baematins in acetic acid is seriously retarded as the water concentration rises. If the mechanism proposed here for the decomposition of diazohacmatin is essentially correct it might be supposed that the presence of water affects not the actual removal of iron but its reduction by ferrous sulphate.
F \ 650
600
550 500 575 525 WQvelength(m/u)
4-)5
Fig. 2. "File spectrum of dcuteroporphyrin dimethyl ester in ether demonstrating band !X" as a doublet. One last point deserves comment. The spectrum of deuteroporph3Tin presented in this paper (Fig. 2) shows very clearly that b a n d IV is a doublet. This does not appear to have been reported before. Haematoporpbyrin and mesoporphyrin also show this spectroscopic feature but protoporphyrin, monoformyldeuteroporphyrin and diacetyldeuteroporphyrin, all of which are electrophilically substituted, exhibit only a single band. ACKNOWLEDGEMENTS One of US (D.B.M.) w~shes to acknowledge personal support from the National Health a n d Medical Research Council of Australia. These spectrophotometri:c studies were Biochim. Biophys...Iota, 71 [t963) x5o--I5~*
156
P . s . CLEZY, D. B. MORELL
g r e a t l y f a c i l i t a t e d b y t h e u s e of a P e r k i n - E l m e r " 3 5 o ' ' s p e c t r o p h o t o m e t e r p u r c h a s e d from funds s u p p l i e d b y the a b o v e Council a n d the P o s t - G r a d u a t e Medical R e s e a r c h F o u n d a t i o n in S y d n e y . REFERENCES z 1'. S. ('LEZV unpublished observation. T. C. Cm', A. G, GREEN AND E. J, CHU, J, Biol. Chem., 19o (t951i (>433 l:..\RNDT, Organic Syntheses, Collective Vol. 2 (1943) 40I. a I>. S. CLEZ'; AND D. B. MORELL, Biochim. Biophys. Acta, 71 (t963) x65. :' l). I{..~,IORELL AND M. *t~TEIA,ARI..~*~.,i, t d l a n ,]. ExplI. I3iol..'lled. Sci.. 34 (";5 ¢') ,~t '; H. I:ISCHER ANt) G. ~VEcKF.R, Z. Physiol. Chem., 272 (I~t42) I. 7 (). \VARBURG AND E. NEGELEIN, I3iochem. Z., 244 (193-) t~. " l/. 1,. t-IARDIE AND li. 1t. THO.~tSON, ]. Chem. Soc., (I957) 2512.
Biochim. Biophys. Acla, 71 (t963) I5O-156