Phosphate ester derivatives of deuterohemin IX dimethyl ester

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BIOCHEMISTRY

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BIOPHYSICS

163, 398-402 (1972)

COMMUNICATIONS Phosphate

Ester Derivatives

of Deuterohemin

A series of deuterohemin IX dimethyl ester derivatives have been prepared in which dimethylphosphate (DMP), di-(2-ethylhexyl) phosphate (DEHP), and diphenylphosphate (DPP) are the fifth ligands on iron. The assumption that phosphate is bound to iron is supported by method of synthesis, lability of ligand, mass spectra, and the fact that one of these ligands, DEHP, is a reagent for extracting iron (III) from aqueous media into chloroform or hexane. The effects of ligand on absorption in the near infrared and on the chemical shift of the porphyrin methyl groups are presented. The order of increasing iron-phosphate ester interaction appears to be diphenylphosphato < dimethylphosphato < di-(2.ethylhexyl)phosphato. The near infrared evidence indicates that phosphate esters interact more strongly with iron than chloride, bromide, or iodide. On the other hand, nmr evidence yields the order: diphenylphosphato < Br < dimethylphosphato < Cl < di-(2-ethylhexyl)phosphato. Silver phosphate alone does not convert the chlorohemin to phosphatohemin but the addition of 85% phosphoric acid produces a material in solution with a phosphatohemin spectrum. Deuterohemin derivatives with a wide variety of anions in the axial or fifth coordination site have been prepared (1). However, except for our preliminary report (2) no other phosphatohemins have been reported. In order to be able to detect differences in properties, three phosphate esters were chosen which differ widely in size, shape, and unsaturation : dimethylphosphate degree of (DMP), di-(2-ethylhexyl)-phosphate (DEHP), and diphenylphosphate (DPP). Di-(X-ethylhexyl) phosphoric acid is a reagent for extracting iron (III) and other metal ions into nonaaueous media such as chloroform or hexane from aqueous media (3) and it was expected that it would bind readily to iron in hemin in chloroform solution. In addition, this ligand has some resemblance to phos-

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Ester’

pholipid because it has a polar phosphate ester end and long hydrocarbon side-chains. Elemental analyses were performed by Schwarzkopf Analytical Laboratories. Infrared spect,ra were measured in 13-mm KBr disks on the Perkin-Elmer 137 and 621 infrared spectrometers. Electronic spectra were measured on the Cary 14 and Perkin-Elmer 402 and 202 spectrophotometers using a holmium oxide standard. NMR spectra were measured on Bruker 90 MHz and Jeolco 60 MHz spectrometers in deuterochloroform with 1% tetramethylsilane as internal standard. Values for chemical shifts are in parts per million (ppm) from TMS and are the average of three determinations. Maximum deviation from the average was f0.4 ppm. Mass spectra were obtained using a Varian CH-5 spectrometer. Chlorodeuterohemin IX dimethyl ester. Chlorodeuterohemin IX was prepared by the method of Caughey and co-workers (4) from chloroprotohemin IX (bovine, recrystallized, Mann Biochemical Co.). The product was esterified in 5% coned H2S04-95% absolute methanol overnight, worked up with hydrochloric acid by the method of Falk (5), chromatographed on alumina, and crystallized by the method of Caughey and coworkers (S), mp 245-247. The electronic spectrum is described in Tables I and II and Fig. 1. The highest mass spectral peak observed was m/e 592, (deuterohemin dimethyl ester minus ligand) ; also observed were peaks at m/e 519 (deuterohemin minus and dimethyl ester minus ligand CH,COOCH,), m/e 446 (deuterohemin dimethyl ester minus ligand and minus 2 CHZOOCH,), and m/e 36 (HCl). Dimethylphosphatodeuterohemin IX dimethyl ester. A chloroform solution (50 ml) of 100 mg of chlorodeuterohemin IX dimethyl ester was stirred with 2 g of solid sodium dimethylphosphate [prepared from trimethylphosphate (Aldrich) by the method of Bunton (6)] until there was no further change in spectrum in the range of 350-700 nm. The suspension was centrifuged and the supernatant 5uid filtered through Pyrex wool and concentrated. The first addition of petroleum ether (30-60°C) precipitated an oil which later crystallized on refrigeration. The supernatant fraction

1 This investigation was supported by Biomedical Science Support Grant FR-PHS 1505RR07131-01 from the General Research Support Branch, Division of Research Resources, Bureau of Health Professions Education and Manpower Training, Nat,ional Institutes of Health.

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IX Dimethyl

399

COMMUNICATIONS TABLE MAXIMA

IN

THY

ELECTRONIC

SPECTRA

I

OF I)BUTEHOHEMIN

IX

DIMETHYL

ESTERS

IN

CHLOROFORM -____

Axial ligand

Absorption

maxima (nm) (extinction

coefficient)

-._____ Cl-

377 (8.59 370 (4.69 367 (4.58 1370 ~(4.15

1)EHP-6 (CH,O)*P&(CsHsO),P02-

x 104) sh x 104) sh X 10’) sh X 104)

.-.__

401 (6.76 395 (7.38 394 (6.14 395 (5.07

Sk 507 X 104) (6.52 498 X 104) (6.09 503 X 104) (5.84 502 X 104) (4.94

533 578 sh X 103) (7.10 X 103) (2.5G X 103) 526 571 sh X 10”) (6.36 X 103) ,(3.01 X 103) ,530 ,576 sh X lo?) ‘jz;l8 X 10”) (2.57 X 103) 567 sh x 10s) i(5.28 X 10”) (2.07 X 10”) -.._________

633 (4.01 X 103) 620 (4.01 X 103) 62G (3.5i X 10”) 627 (3.03 X 103)

ash = shoulder. b Di-(2.et,hylhexyl)phosphate. TABLE

II

NIXK

INFR.WELI THANSITIONS FOR DICUTEROHICMIN IX DIMIYI-HYL ESTISHS IN CHLOROFOIW

Axial ligand

CH30 C&O F CH,COO N3 Cl Br I I)i-(2-ethylhexyl)phosphato

(CH,OM’Oz (CJLOM’O~ (Llteference

Absorption maximum (nm) 7GlG 769O 8008 836= 868G 911 934a 95% 868 880 890

12.

was treated wit,h more petroleum et,her and refrigerated. Blue-black crystals were obtained in quantitative yield, mp 111-112. Anal. Calcd for C32H32N404Fe(CaH604P).HzO: C, 55.4; H, 5.4; Fe, 7.16; P, 4.2. Calcd for C32H32N404Fe (C,H,OIP): C, 56.9; H, 5.3; Fe, 7.8; P, 4.3. Found: C, 55.6; H, 5.3; Fe, 7.9; P, 3.8. Most abundant mass spectral peak: m/e 592 (deuterohemin dimethyl ester minus ligand), also m/e 519 (deuterohemin dimet,hyl ester minus ligand minus CH&OOCHs), m/e 446 (deuterohemin dimethyl ester minus ligand and minus 2 CH,COOCH,), m/e 126 [(CHaO),POOH], m/e 111 [(CH,O)zPOOH minus CH,], m/e [(CH,0)2 POOH minus 2CHaJ. The electronic spectrum is described in Tables I and II. In the infrared spectrum, in addition to all the peaks observed for

X (nm) FIG. 1. Electronic absorption spectrum for chlorodeuterohemin IX dimethyl ester in cholorform. 1.02 X 1W5 molar in Soret region; 1.37 X 1OWmolar in long wavelength region. (--------) Electronic absorption spectrum for di-(2-ethylhexyl) phosphatodeuterohemin dimethyl ester in chloroform. 1.49 X W5 molar in Soret region: 1.49 X lo-’ molar in long wavelength region. (----) deuterohemin dimethyl ester, the follonring peaks charact,eristic of the dimethylphosphate ligand were observed (7) : 1240 cm-l (P=O), 1045 cm-1 (P-O-C asymm), 820 cm-l (P-O-C symm). Diphenylphosphatodeuterohemin IX dimethyl ester. A chloroform solution (50 ml) of 100 mg of chlorodeuterhemin IX dimethyl ester was shaken with 1 g of silver diphenylphosphate (Aldrich) until there was no further change in spectrum. The reaction mixture was worked up in the same manner as for the dimethylphosphate derivative. Blue-black crystals in quantitative yield were obtained from chloroform-petroleum ether (3060”(Z), mp lA7-170. And. Calcd for C,,TI,,O,N,Fe(CI,,H,oO,P) : C, 62.6; H, 5.0; Fe 6.6; P, 3.7. Found: C, 63.2; H, 5.5; Fe, 6.6; P, 3.7.

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Most abundant mass spectral peak: m/e 592 (deuterohemin dimethyl ester minus ligand), also m/e 519 (deuterohemin dimethyl ester minus ligand and minus CH&OOCH,) and m/e 250 [(CsH50)2POOH]. The electronic spectrum is described in Tables I and II. In the infrared spectrum, in addition to all the peaks observed for deuterohemin dimethyl ester, the following peaks characteristic of the axial ligand were observed: 1260 cm-‘, 1200 cm-l, 1170 cm-‘, 1070 cm-l, 925 cm-‘. Silver diphenylphosphate has the following peaks in this region: 1250 cm-‘, 1210 cm-‘, 1060 cm-l, 920 cm-‘, 910 cm+, 900 cm-‘. Di-@ethylhexyl)phosphatodeuterohemin IX dimethyl ester. A chloroform solution (50 ml) of 100 mg chlorodeuterohemin IX dimethyl ester was shaken with 3 g sodium di-(2-ethylhexyl)-phosphate. [The salt had been prepared by neutralizing 32 g (0.1 mole) di-(2-ethylhexyl) phosphoric acid (Union Carbide) with 100 ml of 1 N NaOH and adding excess ether and water until two layers separat,ed. The ether solution was taken to dryness on a rotary evaporator at room temperature, benzene was added repeatedly and taken to dryness, then chloroform and petroleum ether (30-6OO”C) were added and taken to dryness]. The reaction mixture was worked up in the same manner as for the dimethylphosphate derivative. Fine blue-black needles were obtained in quantitative yield, mp 105-106. Anal. Calcd for C32H32N404Fe (C16Hs404P) : C, 63.1, H, 7.2; Fe, 6.1; P, 3.4. Found: C, 62.9; H, 7.4; Fe, 5.9; P, 3.1. The electronic spectrum is described in Tables I and II and Fig. 1. In the infrared spectrum, in addition to all the peaks observed for deuterohemin dimethyl ester, the following peaks characteristic of the axial ligand were observed: 1250 cm-l, 1040 cm-‘. The mass spectrum showed the following peaks : m/e 913 (molecular ion), m/e 592 (deuterohemin dimethyl ester minus ligand), m/e 519 (deuterohemin dimethyl ester minus ligand minus CH,COOCH~), m/e 446 (deuterohemin dimethyl ester minus ligand minus 2CHz COOCH,), m/e 322 (di-(2-etjhylhexyl)phosphoric acid), m/e 113 (CaHn+), m/e 99 (GHl;+), m/e 57 (CaHg+), m/e 44 (C~H.I+), rzo m/e 85 (branched st,ructure precludes Cg fragment). Reaction of chloroprotohemin IX dimethyl ester and chlorodeuterohemin IX dimethyl ester with silver phosphate. Deep-yellow silver phosphate was prepared from silver nitrate and sodium dihydrogen phosphate and washed repeatedly first with water, then acetone, and then air-dried. When 1 g of silver phosphate was suspended in 50 ml of a chloroform solution containing 100 mg chloroprotohemin IX dimethyl ester [prepared from chlorohemin, Mann, and esterified according

to Falk (5)] and stirred, the spectrum of the solution changed to: maxima at 401, 466 (sh), 582, and 598 (sh) nm. Evaporation of the solution yielded a solid with strong infrared absorption at 840 cm-’ (Fe-O-Fe) indicating oxodimer structure (1,8). When the dimer was dissolved in chloroform and shaken with coned HCl, the chlorohemin spectrum was obtained and when a chloroform solution of the dimer was shaken with 85% HaPO, , a phosphatohemin spectrum was obtained (2) with maxima at 388 sh, 404, 507, 536, 634 nm. When this solution was filtered through three thicknesses of filter paper, evaporated to dryness, and covered with hexane, a black solid formed which, in addition to the protohemin dimethyl ester infrared lines, also had a strong, broad infrared band centered at 1040 cm-’ (POa). These reactions were also observed when chlorodeuterohemin IX dimethyl ester was used. The corresponding oxo-dimer had the following maxima: 393, 468 fsh) 577 nm. which changed, on shaking with 85oj, H,POa, to the phosphate derivative with maxima at 369 (sh), 391, 498, 524, and 616 nm. Caughey and co-workers (1) effected ligand exchange in hemins by shaking chloroform solutions of acetatodeuterohemin dimethyl ester with nearly saturated aqueous solutions of the sodium salts of the desired ligands. The following anions replaced acetate: Cl-, F-, Br-, I-, N3-, C~HSO-, H,PO,-. When this procedure was applied to sodium dialkylphosphates or sodium phosphat)es, partial reaction, hydrated products, and, in the latter instance, the oxo-bridged dimer, were obtained. However, when the use of water was avoided, and chloroform solutions of chlorodeuterohemin dimethyl ester were equilibrated with salts of the phosphate esters in large excess, until no further changes in visible spectra occurred, quantit,ative yields of the phosphate ester derivatives were obtained. The products were characterized by elemental analyses, infrared spectra, and mass spectra. The infrared spectra showed lines due to deuterohemin dimethyl ester and to the phosphate esters. In the region between 600 and 450 cm-’ where absorption for Fe-O was sought, an unambiguous assignment could not’ be made because of interference by the spectra of the ligands, a difficulty encountered also by Ymythe, Whateley, and Werner (3) in their study of Fe(DEHP)3. However, the change in the antisymmetric PO0 frequency from 1203 cm-’ in di-@ethylhexyl)phosphate ions (9) to 1250 cm-’ in di-(2.ethylhexyl)phosphatodeuterohemin dimethyl ester is analogous to similar shifts observed for hafnium (DEHP)a and interpreted by Peppard and Ferraro as indicative of strong metalLox.ygen binding.

401

COMMUNICATIONS The assumption that the phosphate ester groups are bourld to iron and not to the porphyrin is supported by (a) the ion-exchange method of synthesis; (b) t,he ease of removal of ligand by hydrolysis, and (c) the mass spectra, which showed fragmentation into deuterohemin dimethyl ester and ligands. Except’ for the di-(2-ethylhexyl)phosphat.o compound, which showed a molecular ion at m/e 913, the phosphate ester derivatives gave a major mass spectral peak at m/e 592 (deuterohemin dimethgl ester minus ligand), as well as mass peaks at m/e 519 and 446 corresponding to the fragmentation of the propionic ester groups, respectively. Peaks corresponding to the ligands and to fragmentation of the ligands were observed and described previously. Silver phosphate did not convert chlorohemin to the phosphate. A product with an infrared band at 840 cm-’ (Fe--O--Fe) indicates oxo-dimer formation (1, S). When silver phosphate and phosphoric acid were used in the reaction, the oxobridged dimer was not obtained, but the spectrum of the solution was that of the phosphate derivative (2). Spectral evidence showed that the oxobridged dimer was converted, in chloroform or acetone solution, to the phosphate in the presence of phosphoric acid and to the chlorohemin in the presence of concentrated hydrochloric acid. Phosphate ester derivatives of deuterohemin dimethyl ester hydrolyze slowly in neutral aqueous acetone to yield a solution with a hematin speclrum (1). When the chloride ligand is replaced by phosphate ester the Soret band moves to longer wavelengths and t,he maxima between 450 and 1000 nm are shifted to shorter wavelengt,hs (2). Analytically pure materials were used for spectra and were recoverable unchanged from solution. The maximum near 630 for all of these derivatives, is typical for high-spin iron porphyrins (IO). (Table I and Fig. 1) The near-infrared band has been shown to be sensitive to the nature of the axial ligand (11, 12). Our data and those of Caughey and co-workers (12) are presented in Table II. Caughey and co-workers found a correlation between the maximum in chloroform solution in the near infrared and zero-field splittings (21)) and Mossbauer quadrupole splittings in solids. They concluded that stronger bonding of porphyrin nitrogen to iron and, therefore, weaker iron-axial ligand bonding is reflected in decreased transition energies in the near infrared and increased transition energies in the Soret region (Table II). The order for iron-axial ligand interaction obtained for the ligands studied by Caughey’s group and in this report is (Table II): CH,O > C,H& > F > CH,COO > N3 , di-@ethylhexyl)phosphato > (CHaO)2P02 > (C,H,O)zPO, > Cl > Br > I.

Based on this evidence, all of the phosphate esters used as ligands interact more strongly with iron t’han chloride does. The lower transition energies in the Soret region for the phosphate esters compared with chloride also supports this conclusion. Caughey and Johnson (13) have correlated the magnitude of the down-field shift of the peripheral methyl groups on deuterohemin IX dimethyl ester with different axial ligands with t’he order found for zero-field splittings, Mossbauer quadrupole splittings, and near-infrared transitions. The sequence, attributed to the order of increasing iroll--1igand interaction is CFHBO > F > N3 > Cl > Br. The nmr spectra for the deuterohemin dimethyl ester phosphate esters contained the features reported above except, that’ t’he up-field shift, due to meso hydrogens (13) was not observed with our instrument and that the shifts due to porphyrin methyls varied with ligand. The chemical shift,s at maximum peak height for porphyrin methyl groups in phosphate ester derivatives of deuterohemin dimethyl ester are shown in Table III. When these results are combined with the previous work, the order becomes C,HjO > TABLE

III

Axial ligand

Chemical shift”, *

I )Ii:HPc Cl(CH,O),PO,Hr.<” (CsH,O)zPO,-

47.0 -19.4 49.6 51 52.3

u Parts per million downfield from tet,ramethylsilane. b Chemical shift at maximum peak height. c I)i-(2.ethylhexyl)phosphate. fJ Reference 13 at 35°C.

755

535

375

pm

2. Low-field NM11 spectrum phosphatodeuterohemin IS dimethyl FIG.

of diphenylester.

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COMMUNICATIONS

F > di-(2-ethylhexyl)phosphato > Na > Cl > (CHaO)TP02 > Br < (C&HsO)zP02 . This sequence differs from that obtained from near-infrared data where all the phosphate esters appear to interact more strongly with iron than chloride or bromide do. Diphenylphosphate (DPP) produced the largest shift, 52.3 ppm (Fig. 2) and di-(2-ethylhexyl)phosphate (DEHP) had a significantly smaller shift, 47.0 ppm. This would be a consequence of stronger iron-phosphate interaction and weaker iron-porphyrin interaction in the DEHP derivative than in the DPP derivative. In addition, the proximity of the 2-ethylhexyl or phenyl groups to t,he porphyrin methyl groups, could exert some local shielding and deshielding effects, respectively, on them. REFERENCES 1. S~D~VISAN,N.,EBERSPAECHER, H. I., FUCHSMAN, W. I<., AND CAUQHEY, W. S. (1969) Biochemistry 8, 534. 2. RUSSELL, C. S., AND SIEGEL, T. M. (1970) Naturwissenschaften 67, 307. 3. SMYTHE, L. E., WHATELEY, T. L., AND WERNER, 1~. I. (1968) J. Inorg. Nucl. Chem. 30, 1553. 4. CAUGHEY, W. S., ALBEN, J. O., FUJIMOTO,

5. 6. 7. 8.

9. 10. 11. 12.

13.

W. Y., SNU YORK, J. L. (1966) J. Org. Chem. 31, 263. F.ILK, J. E. (1064) Porphyrins and Metalloporphyrins, Elsevier, Amsterdam, Holland. BUNTON, C. A. 0960) J. Ch.em. Sot. 3293. FERRARO, J. R. (1962) Pittsburgh Conf. on Appl. Spectry., paper 162. ALREN, J. O., FUCHSMAN, W.H., BESUDREAU, C. A., AND C~TJGHEY, W. S. (1968) Biochemistry 7, 624. PEPPARD, D. F., AND FERRARO, J. R. (1959) J. Inorg. Nucl. Chem. 10, 275. SMITH, D. W., AND WILLIAMS, It. J. P. (1970) Structure and Bonding 7, 1. DAY, P., SMITH, D. W., AND WILLIAMS,R. J.P. (1967) BiochewLisby 6, 1563. CAUGHEY, W. S., EBERSP~ECHER, H. I., FUCHSMAN, W. H.,McCou, S., AND ALBEN, J. 0. (1969) Ann. N.Y. Acad. Sci. 163, 722. CAUGHEY, W. S., .IND JOHNSON, S. F. (1969) Chem. Commun. 1362.

Department of Chemistry City College of the City University of New York New York, New York 10091 Received May 16, 1972

C. S. IiUSSELL J. LANDIS N. BOCLIN