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Cyanide scavengers: kinetics of the reactions of cyanide with a water soluble cobalt(III) porphyrin
Cyanide Scavengers: Kinetics of the Reactions of Cyanide with a Water Soluble Cobalt(II1) Porphyrin Peter Hambright and Robert Langley PH. Department of Chemistry, Howard University, Washington, D.C.-RL.
Department
of Chemistry, Lincoln University, Lincoln, PA
ABSTRACT Theequilibrium and kinetic aspects of the interaction of cyanide with a model anticyanide drug cobalt(lII)tetrakis(4-sulfonatophenyl)porphyrin [Co-P] were studied at 25°C I = 0.1 (NaNOs). At the physiologic pH of 7.4, 1.9 f 0.1 mol of cyanide were rapidly bound per molecule of Co-P. The dissociation constant of cyanide from Co-P(Hr0) (CN) was < 10-t2, and the formation constant of Co-P(CN)r from CoP(H20) (CN) and CN- was 3.5 x 106. From pH 4 to 10.5, the kinetics of mono-cyano Co-P formation were first order in cyanide and porphyrin, with the following specific rate constants (units M-t s-r): CoP(HIO)z/CN-, 3.1 x 102; Co-P(H*O) (OH)/CN-, 2.4 x 103; Co-P(OH)JCN-, 5.1 x 10’ and CoP(H*O)/HCN, 3.1 x 10e3. At pH 7.4, a second cyanide molecule adds more rapidly than the first: CoP(H*O) (CN)/CN- , 3 x lo4 M- I s- I. It is concluded that low molecular weight water soluble cobalt(m) porphyrins might be used as effectively and at lower dose levels than hydroxocobalamin (Bm,), a known in vivo anticyanide agent.
INTRODUCTION Several procedures are currently used, and a number of others have been suggested [l] to combat the rapid acting poison, cyanide, which complexes (among other places [2]) to iron and copper sites in cytochrome oxidase and leads to the inhibition of cellular respiration. In the United States, amyl nitrite is inhaled and sodium nitrite is given i.v. to oxidize about 30% of the iron hemoglobin into the high cyanide affinity iron@) methemoglobin form [3]. This is followed by an injection of sodium thiosulfate, which serves as a massive sulfane sulfur source for the enzyme rhodanese [4], which converts CN- into the more benign SCN-. In Europe and elsewhere, dicobaltAddress reprint requests to Peter Hambright, D.C. 20059.
Department of Chemistry,
Howard University,
Journal of Inorganic Biochemistry 32, 197-205 (1988) 0 1988 Elsevier Science Publishing Co., Inc., 52 Vanderbilt Ave., New York, NY 10017
Washington,
197 01624134/88/$3.50
198 P. Hambright and Robert Langley
ethylenediaminetetraaceticacid ([Co1*(H20)&01’(EDTA)] *2 HZ0 in the solid state [5]) is the method of choice [6], where cobalt(I1) scavenges cyanide. Both procedures may have dangerous consequences. The rapid production of methemoglobin makes the patient even more hypoxic [7], and cobalt(I1) ions are cardiotoxic. In fact, the toxicity of dicobalt-EDTA is reciprocally neutralized by cyanide [8]. Potential cyanohydrin formers such as pyruvic [9] or alpha-ketoglutaric acid [lo] or stroma-free methemoglobin itself [ 1 l] have been used in animal studies of cyanide intoxication. Since nitrite oxidations are relatively slow, the compound 4-dimethylaminophenol has applications as a more rapid methemoglobin former [ 121. The vitamin Bi2 derivative, hydroxocobalamin (Bm,), is a cobalt(II1) complex having the corrin ring structure. The drug is considered nontoxic, and animal work indicates that it has action against cyanide [13-151. Unfortunately, rather large quantities of the agent need to be administered. Around 1,350 mg of hydroxocobalamin react in vivo with 26 mg of CN- ,and lower molecular weight compounds acting in the same manner are desired. The cobalt(II1) oxidation state can be stabilized by water soluble porphyrins, which in principle can be designed to have lower formula weights than found in Bm,. To this end, we report the kinetic and equilibrium parameters of the interactions of cyanide with a model metalloporphyrin, cobalt(II1) tetrakis(4-sulfonatophenyl)porphyrin (Co-TPPS). The results are compared to Bm,, and related anation reactions of trivalent metalloporphyrins. EXPERIMENTAL The Co(III)-TPPS was made following the heterogeneous metal-insertion procedure of Herrmann and coworkers [ 161. In air, 1 .O g of H2 TPPS. 12 Hz0 [ 171 and 5 g of Co0 (Strem Chemicals) were refluxed overnight in 500 ml of water. The oxide was filtered off, the solution evaporated to 50 ml, and then refiltered through a Millipore Metricel GA-6 0.45 pm filter. The solution was then frozen and lyophilized. For CoC,_,N4H2&S40itNas* 12 HzO; Calcd; Co, 4.63; C, 41.52; N, 4.40; H, 3.80, S, 10.08. Found: Co, 4.49, C, 41.68, N, 4.40, H, 3.29, S, 10.37. The same degree of hydration was noted before by Krishnamurthy and coworkers [18], who prepared this complex by a mercury(B) displacement reaction. Neta [ 191 demonstrated that heating certain cobalt(II1) porphyrins at 120°C causes partial reduction to the cobalt(B) state, and this might explain the production of Co(II)-TPPS using this same metal oxide procedure, where the waters of hydration were removed at 250°C before analysis
U61. The kinetics were followed at 25°C on either a Durrum-Gibson stopped-flow apparatus, or with a Beckman Acta III recording spectrophotometer, and pHs were monitored with a Radiometer PHM 64 Research pH meter. The buffers Tris, Hepes, Pipes, Mes, HOAc/NaOAc, and chloroacetic acid were at lo-mM levels, and the ionic strength was maintained at 0.1 with NaN03, in both the kinetic and equilibrium sections. A Radiometer Ion 83 ion /meter with a cyanide selective electrode was employed for the cyanide uptake work. Cyanide (KCN) was analyzed with silver nitrate by the Liebig-Deniges method [20]. RESULTS
Cyanide Binding The number of cyanide ligands bound by Co(III)-TPPS was studied at pH 7.4 (I = 0.2; 0.1 in phosphate buffer and 0.1 M NaN03), using an ion-selective cyanide
CYANIDE
1
2
3
4
lo4 [ Co-TPPS],M
SCAVENGERS
199
FIGURE 1. Plot of the decrease in total cyanide concentration with added Co(III)-TPPS at pH 7.4, I = 0.2, 25°C.
electrode. A solution ca. 1 mM in cyanide was titrated with Co-TPPS in the same buffer. The reaction was fast, and the potentials stabilized within one min of mixing. Figure 1 is a plot of the change in the total cyanide concentration with added Co-TPPS. The results are that 1.9 + 0.1 mol of cyanide are bound per mole of Co-TPPS at pH 7.4. Cyanide was not taken up by metal free H*-TPPS. Absorption
Spectra
The spectrum of CO-TPPS(H~O)~ at pH 2 (I = 0.1 NaNOs) is similar to that reported by Ashley and Au-Young [21]; A,,,423 nm (E = 2.2 x lo5 M-’ cm-‘), 537 nm (1.4 x 104) and 565 nm (sh, 5.6 x 103). If a 3 x 10e6 M solution of Co-TPPS is made from 0.1-5 mM in cyanide at pH 9, the dicyano Co-TPPS(CN)2 is fully formed; 442 nm (1.7 x 105), 536 nm (8.3 x 103), and 608 nm (9.0 x 103). When HNOs is added to bring the solution to pH 2, the mono-cyano Co-TPPS(H20)(CN) is produced: 427 nm (1.8 x 105), 542 nm (1.2 x 104), and 580 nm (sh, 4.4 x 103). The absorption maxima of the cyano complexes are similar to those found in the Co-TPPS/SCNstudy [21], with Co-TPPS(SCN)z at 442 nm and Co-TPPS(H20)(SCN) at 428 nm. Equilibria In order to estimate the dissociation constant &, of Co-TPPS(H,O)(CN) (Eq. solution 2 x 10e6 M in Co-TPPS was made 2 x 10e4 A4 is cyanide at pH 8 and dicyano formation, the pH was lowered to 2. The only complex present after 2 equilibration was the mono-cyano derivative; no spectral evidence for the diaquo was found. Thus Ka, < lo-i2. HZ0 + Co-TPPS
(H20) (CN)-
= CO-TPPS(H~O)~ + CN-
K,,,
1), a after days form
(1)
The equilibrium constant Ki2 for the formation of Co-TPPS (CN)2 from CoTPPS(H20) (CN) (Eq. 2) was measured by a spectrophotometric method. Known concentrations of Co-TPPS and cyanide were mixed at pH 9 forming the dicyano adduct. Acid CN- + Co-TPPS (H20) (CN)-
= Co-TPPS (CN)22- + Hz0
K12
(2)
was then added to a given pH, and the spectra was monitored after 5 min when equilibrium had been attained.The dissociation constant of HCN has pK, = 9.14 [22]. It was assumed that the total porphyrin, Pr, was in either the mono- or dicyano forms.
200
P. Hambright and Robert Langley
TABLE
1. Formation Constant of Co-TPPS(CN)z from Co-TPPS(H20) (CN)
PH”
Y = [Co-TPPS(CN)JPTJ
10’ (CN-), Mb
5.24 4.92 4.60 4.35 4.08 3.81 3.58 3.34
0.891 0.810 0.696 0.537 0.389 0.246 0.157 0.0962
25.3 12.1 3.96 3.26 1.75 0.939 0.553 0.318
1O-6 K,2C 3.2 3.5 3.9 3.6 3.6 3.5 3.4 3.4 Average 3.5 + 0.2
0 T = 25”C, I = 0.1 (NaNO,). b [CN-JT = 2.3 x lo-* M. CK,2 = [Y/(1 - Y)](CN-)-I.
From the spectra, the fraction Y = [Co-TPPS(CN)JPT] could be calculated and knowing Y, PT, pK,, and [CN-1, the free unbound cyanide concentration, (CN-), could be determined. The equilibrium constant Kr2 = [Y/(1 - Y)] (CN-)-I. Typical results are shown in Table 1, where a single Klz represented the data from pH 5.3 to 3.3, as the fraction of the dicyano porphyrin varied from 89 % to 9 % . For CoTPPS, Kr2 = (3.5 + 0.2) x 106. Similar studies were done on cobalt(III)-tetrakis(NmethykLpyridyl)porphyrin, [Co-TMPyP, Kr2 = (5.6 k 0.3) x IO’], and cobalt(III)tetrakis(4-N,N,N-trimethylanilinium)porphyrin, [Co-TAP, Kr2 = (2.2 f 0.4) x 1061.
Kinetics The kinetics of cyanide addition to Co-TPPS were studied from pH 4 to 10.5. The reactions were followed in the Soret region with porphyrin concentrations ca. 3 x 10m6 A4, and the total cyanide levels were at least 20 times those of the porphyrin. Under such conditions, the reactions were first order in porphyrin over 3 half-lives, with an observed pseudo first order rate constant kobsd, which was independent of whether the disappearance of the aquo/hydroxy or appearance of the dicyano porphyrin bands were monitored. Decreasing the buffer concentration from 10 mM to 1 mM had no significant effects on k.,bsd. The insert in Figure 1 shows that the reaction is also first order in cyanide, from 7.5 x 10d5 M to 7.5 x 10m4 M, at pH 8.1. The reactions were found to be biphasic above pH 11, perhaps partially due to the presence of slowly reacting cobalt porphyrin dimers and polymers in equilibrium with dihydroxy monomers [23]. Figure 1 gives the pH profile of the reaction, as a plot of kobsd/(CN-) versus pH. The following reactions are considered: HCN=H+
+CN-
CO-TPPS(H~O)~ = Co-TPPS(H*O) Co-TPPS(H*O)
(OH)-
K, (OH) - + H +
= CO-TPPS(OH)~~-
CO-TPPS(H~O)~ + CN- -Co-TPPS(H20)
(3)
+ H+
(CN)-
+ Hz0
K,,
(4)
Kti
(5) kcN
(6)
CYANIDE
2
4
SCAVENGERS
201
6
/-
I-
I
I
1
I
4
5
6
7
I
I
I
0
9
10
PH FIGURE 2. pH profile of the kinetics of cyanide addition to Co(BI)-TPPS, I = 0.1 (NaN03), 25°C. The dots are experimental points, and the solid curve was calculated from Eq. 12. The insert shows that the reaction is first order in cyanide.
Co-TPPS(H20)
(OH)-
CO-TPPS(OH)~~-
+ CN- -Co-TPPS(CN)
+ CN- --Co-TPPS(CN)
CO-TPPS(H~O)~ + HCN-rCo-TPPS(H20) Co-TPPS(OH) Co-TPPS(H20)
(CN)2(CN)-
(OH)‘-
+ Hz0
kz
(OH)2- + OH(NCH) + Hz0
+ H+ = Co-TPPS(H20)
(CN)-
+ CN- -CO-TPPS(CN)~~-
(7)
k3
(8)
kncN
(9)
fast fast
(10) (11)
Essentially, CN- reacts with the diaquo (I
202 P. Hambright and Robert Langley
The observed
rate law is thus of the form:
kobsd/(cN-)=(kcN+_4(~+)+B/(~+)+C/(~+)2)/(D) withA
= kucN/Ka, B = k2 Kal, C = k3 K,i Kti, andD
(12) = (1 + K&H+)
+ K,, Kti/
(H+)*). From related work [26] at I = 1.0 (NaC104), pK,i = 7.02, and pKti = 9.76. Each term in Equation 12 is important in a given pH range, and limiting forms of the overall equation were developed and iterations were done to obtain the equilibrium and specific rate constants. We find pK,, = 7.1 + O.l,pKaz = 9.4 f O.l,kcN = (3.1 + 0.4) x 102M-‘s-l, kucN = (3.1 f 0.3) x 10-3M-1s-1, k2 = (2.4 + 0.3) x lo3 M-’ s-’ and k3 = (5.0 f 0.8) x 10’ M-’ s-l. The solid line in Figure 1 was calculated from these parameters using Equation 12, and the overall agreement with the observed data is satisfactory. Brief experiments were done from pH 6.5 to 8.0 with cyanide reacting with preformed Co-TPPS(H20) (CN). The reactions were first order in cyanide from 1.4 x 10m3 Mto 8.7 x 10e5 M, with a specific rate constant of 3 x lo4 M-l s-r, which was independent of pH. Thus, as assumed, addition of the second cyanide occurs about 10 times more rapidly than the first. DISCUSSION Table 2 shows a comparison of the anation rate constants of the diaquo and monohydroxyl forms of Co-TPPS with CN- , SCN- , and I-. The remarkable similarity in the values found indicates that Co-TPPS reacts by a dissociative interchange mechanism, as postulated before for various cobalt(II1) porphyrins [24, 26-291 and BIZa [25]. The effect of the axial ligand in labilizing the opposite coordinated water molecule is in the order CN- > OH- > HzO, as ca 200:20:1. Anion substitution into the diaquo forms of the metalloporphyrins [30, 3 I] appears to be in the order Co(II1) > Rh(II1) > Cr(II1). About 67% of the monohydroxy CoTPPS is present at pH 7.4, and it complexes more rapidly with cyanide than do the
TABLE 2. Rate Constant Comparisons, 25°C Reaction Co(III)-TPPSKNCo(III)-TPPS/SCNm Co(III)-TPPS/ICo(III)-TPPS/HCN Rh(III)-TPPS/CNRh(lII)-TPPS/HCN Cr(III)-TPPWSCN H20-B,Z,/CNm H20-B,2,/HCN NC-B,* (base-on)/CNNC-B,* (base-off)/CNCo(III)-TPPS(H,O)(CN)/CN 0 This work. b Units M-’ s-l.
M(III)-TPPS(H20)2 b 3.1 3.2 1.2 3.1 2.3 4.3 2.5 8.0 8.4 3
x 102 x lo* x 10’ x 10-3 5.0 x 10-j x 10-3 x 102 x IO’ 2.0 x lo4 x 104
M(III)-TPPS(H,O) (OH) b 2.4 x IO3 1.4 x 10’
1om2 2.1 x 10’ -
-
Ref. d 26 21 a 30 30 31 25 2.5 25 25 B
CYANIDE
SCAVENGERS
203
other hydrated species. Interestingly, Rh-TPPS(H*O) (OH) shows minimal reactivity towards cyanide [30]. Thus, at pH 7.4 the specific rate constant for cyanide uptake is 1.4 M-r s-i for Rh(III)-TPPS and 1.6 x lo3 M-l s-i for Co(III)-TPPS, virtually eliminating the rhodium derivative as a potential anticyanide drug. The specific rate constants (in units of M- ’ s- ‘) for the first cyanide addition at pH 7.4 are similar for the various cobalt(II1) derivatives: 1.1 x lo3 for Co-TMPyP, 1.6 x lo3 for Co-TPPS, 2.2 x lo3 for Co-TAP and 2.2 x lo3 for H20-B12 (B&. The HO-Bi2(Bm,) does not react with cyanide (the pK, for the Biza/Bizb reaction is 8.1), and at pH 7.4,90% of cyanide uptake is via the undissociated HCN/Bi2, pathway [25]. In contrast, Co-TPPS reacts predominantly with the CN- anion mainly through the diaquo and mono-hydroxy species at this pH. The cyanide in cyanocobalamin is considered irreversibly bound [32] to the cobalt 7 x lo- ls) [33], and many oral Vitamin B12 supplements are given in this wol form. The cyanide is also tightly bonded in Co-TPPS(H20) (CN), with an estimated dissociation constant of less than lo- ‘*. The addition of a second cyanide to CoTPPS(H*O) (CN) at pH 7.4 is rapid, and the formation constant for this process is 3.5 x 106. In contrast, K for cyanide addition to base-on cyanocobalamin is 5 X lo3 M- ’ (pH 11.7), with a relatively slow formation rate constant of 2.0 M- I s- ’ at pH 9 [25]. Unlike Co-TPPS, B12a reacts more rapidly with the first cyanide than the second. All of the in vitro work presented here indicates that Co-TPPS at the physiological pH of 7.4 is as efficient a cyanide scavenging agent as hydroxocobalamin. Most cases of cyanide intoxication arise from accidental poisoning or suicide, and as noted earlier, the NaN02/Na2S203 or dicobalt-EDTA regimes are relatively dangerous. The use of hydroxocobalamin as an anticyanide drug in animals has been known for over 35 years [14]. In conjunction with dicobalt-EDTA [34], 5-g doses of B12bhave been administered to three adults having cyanide poisoning, with successful results. Six guinea pigs each receiving a lethal i.v. dose (4 mg/kg NaCN) of cyanide immediately followed by twice the molar concentration of hydroxocobalamin were healthy for at least 36 h, while the control group receiving saline all died within 30 min [ 131. In vivo studies found cyanocobalamin to be useless as an anticyanide agent, and only B1zb had antidotal activity [14]. Although no toxic effects were noted [13] in animals receiving 1 g/kg of B12b, little work has been done on the effects of large doses in humans [8]. Hydroxocobalamin has a rather high molecular weight (1,346 g), and related cobalt(II1) porphyrins having formula weights around 600 g should lower the effective drug dose by a factor of four, where two ligands are bound. Relatively little is known about the toxicity of water soluble metalloporphyrins. Mn(III)-TPPS, used for magnetic resonance tumor imaging work [35], is less toxic than Mn(III)-TMPyP or Mn(III)-TAP. We have shown before that a variety of cobalt(II1) porphyrins have a high affinity for cyanide at pH 7.5 [36]. In addition to TPPS, TAP and TMPyP (and the 3 and 2-N methylated forms), the list includes proto, hemato, copro and uroporphyrins, the tetrakis(4-(and3)-carboxyphenyl)porphyrins, 2,4-disulfonated deuteroporphyrin, as well as the tetrakis(N-methyl_4(and3)quinolyl)-porphyrins. Both the cobalt(II1) and cobalt(I1) tetrasulfonated phthalocyanines also bind cyanide under these conditions. Certain of these porphyrins will no doubt have less harmful in vivo effects than others, and some might approach the lack of toxicity so far noted for B12b. However, it has been well established [37] that high concentrations of natural porphyrins cause central nervous system damage, and this could limit the usefulness of these proposed compounds. While none of these agents can be taken orally to provide
204
P. Hambright and Robert Langley
long-term porphylactic anticyanide protection, probably be administered intramuscularly.
the less toxic
derivatives
could
This work was supported by the U.S. Army Medical Research Acquisition Activity, Contract DAMDl7-85-C-5086, and is Contribution Number 1827 to the U.S. Army Drug Development Program.
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17, 1383 (1978). 6. J. Nagler, R. A. Provoust, and G. Parizel, J. Occup. Med. 20, 414 (1978). 7. C. M. Berlin, Pediatrics, 46, 99 (1970). 8. B. H. Rumack, in Proc. Symp. on Respiratory Care of Chemical Casualties, U.S. Army Medical Research and Development Command, Report No. USAMRICD-P-2784, 1983 pp. 183-196. 9. C. Schwartz, R. L. Morgan, L. M. Way, and J. L. Way, Toxicol. Appl. Pharmacol.
57, 437 (1979). 10. S. J. Moore, J. C. Norris, I. K. Ho, and A. S. Hume, Toxicol. Appl. Pharmacol.
82,
40 (1986). and W. E. Ottinger, Am. J. Emerg. Med. 3, 5 19 (1985). 12. N. P. Weger, Fundam. App. Toxicol. 3, 387 (1983). 13. M. A. Posner, R. E. Tobey, and H. McElroy, Anesthesiology, 44, 157 (1976). 14. C. Mushett, K. Kelley, and C. Boxer, Proc. Sot. Exp. Biol. Med. 81, 234 (1952). 15. R. H. Drew, Vet. Hum. Toxicol. 25, 342 (1983). 16. 0. Herrmann, S. Mehdi, and A. Corsini, Can. J. Chem. 56, 1084 (1978). 17. M. Krishnamurthy, Znorg. Chim. Acta. 25, 205 (1977). 18. J. Abwao-Konya, A. Cappelli, L. Jacobs, M. Krishnamurthy, and M. Smith, Transition Met. Chem. 9, 270 (1981). 19. P. Neta, J. Phys. Chem. 85, 3678 (1981). 20. J. Bassett, R. C. Denney, G. H. Jeffery, and J. Mendham, Eds, Vogel’s Textbook of Quantitative Inorganic AnaIysis, 4th ed., Longman Scientific & Technical, Essex, 11. R. P. Ten Eyck, A. D. Schaerdel,
Harlow, England, 1978. 21. K. R. Ashley and S. Au-Young, J. Znorg. Chem. 15, 1937 (1976). 22. R. M. Smith and A. E. Martell, Critical Stability Constants, VoIume 4: Inorganic Complexes, Plenum Press, New York, 1976. 23. R. F. Pasternack and G. R. Parr, J. Znorg. Chem. 15, 3087 (1976). 24. R. F. Pasternack and M. A. Cobb, J. Znorg. Nucl. Chem. 35, 4327 (1973); Biochem. Biophys. Res. Comm. 51, 507 (1973). 25. W. W. Reenstra and W. P. Jencks, J. Amer. Chem. Sot. 101, 5780 (1979). 26. K. R. Ashley and J. G. Leipoldt, J. Znorg. Chem. 20, 2326 (1981). 27. G. J. Lamprecht, J. G. Leipoldt, and T. W. Swaddle, Znorg. Chim. Acta. 129, 21
(1987).
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28. S. Funahashi, M. Inamo, K. Ishihara, and M. Tanaka, J. Inorg. Chem. 21, 447 (1982). 29. G. N. Williams and P. Hambright, J. Inorg. Chem. 17, 2687 (1978). 30. P. Hambright and R. Langley, Inorg. Chim. Acfu. 137, 209 (1987). 31. K. R. Ashley, J. G. Leipoldt, and V. K. Joshi, J. Inorg. Chem. 19, 1609 (1980). 32. G. C. Hayward, H. A. 0. Hill, J. M. Pratt, N. J. Vanston, and R. J. P. Williams, J. Chem. Sot. 6485 (1965). 33. H. M. Marques, D. A. Baldwin, and J. M. Pratt, J. Inorg. Biochemistry, 29, 79
(1987). 34. F. Lutier, P. Dusoleil, and J. De Montgros, Arch. Med. Prof. 32, 683 (1972). 35. R. C. Lyon, P. J. Faustino, J. S. Cohen, A. Katz, F. Momex, D. Colcher, C. Baglin, S. H. Koenig, and P. Hambright, Mug. Reson. Med. 4, 24 (1987). 36. H. H. Newball, P. Hambright, S. di Stefano, D. R. Franz, and S. I. Baskin, Proc. Symp. on Respiratory Care of Chemical Casualties, U.S. Army Medical Research and Development Command, Report No. USAMRICD-P-27-84, 1983, pp. 197-212. 37. M. Wintrope, Clinical Hematology, Lee and Febiger, Philadelphia, 1981, 8th ed., chap. 44.
Department
of Chemistry, Lincoln University, Lincoln, PA
ABSTRACT Theequilibrium and kinetic aspects of the interaction of cyanide with a model anticyanide drug cobalt(lII)tetrakis(4-sulfonatophenyl)porphyrin [Co-P] were studied at 25°C I = 0.1 (NaNOs). At the physiologic pH of 7.4, 1.9 f 0.1 mol of cyanide were rapidly bound per molecule of Co-P. The dissociation constant of cyanide from Co-P(Hr0) (CN) was < 10-t2, and the formation constant of Co-P(CN)r from CoP(H20) (CN) and CN- was 3.5 x 106. From pH 4 to 10.5, the kinetics of mono-cyano Co-P formation were first order in cyanide and porphyrin, with the following specific rate constants (units M-t s-r): CoP(HIO)z/CN-, 3.1 x 102; Co-P(H*O) (OH)/CN-, 2.4 x 103; Co-P(OH)JCN-, 5.1 x 10’ and CoP(H*O)/HCN, 3.1 x 10e3. At pH 7.4, a second cyanide molecule adds more rapidly than the first: CoP(H*O) (CN)/CN- , 3 x lo4 M- I s- I. It is concluded that low molecular weight water soluble cobalt(m) porphyrins might be used as effectively and at lower dose levels than hydroxocobalamin (Bm,), a known in vivo anticyanide agent.
INTRODUCTION Several procedures are currently used, and a number of others have been suggested [l] to combat the rapid acting poison, cyanide, which complexes (among other places [2]) to iron and copper sites in cytochrome oxidase and leads to the inhibition of cellular respiration. In the United States, amyl nitrite is inhaled and sodium nitrite is given i.v. to oxidize about 30% of the iron hemoglobin into the high cyanide affinity iron@) methemoglobin form [3]. This is followed by an injection of sodium thiosulfate, which serves as a massive sulfane sulfur source for the enzyme rhodanese [4], which converts CN- into the more benign SCN-. In Europe and elsewhere, dicobaltAddress reprint requests to Peter Hambright, D.C. 20059.
Department of Chemistry,
Howard University,
Journal of Inorganic Biochemistry 32, 197-205 (1988) 0 1988 Elsevier Science Publishing Co., Inc., 52 Vanderbilt Ave., New York, NY 10017
Washington,
197 01624134/88/$3.50
198 P. Hambright and Robert Langley
ethylenediaminetetraaceticacid ([Co1*(H20)&01’(EDTA)] *2 HZ0 in the solid state [5]) is the method of choice [6], where cobalt(I1) scavenges cyanide. Both procedures may have dangerous consequences. The rapid production of methemoglobin makes the patient even more hypoxic [7], and cobalt(I1) ions are cardiotoxic. In fact, the toxicity of dicobalt-EDTA is reciprocally neutralized by cyanide [8]. Potential cyanohydrin formers such as pyruvic [9] or alpha-ketoglutaric acid [lo] or stroma-free methemoglobin itself [ 1 l] have been used in animal studies of cyanide intoxication. Since nitrite oxidations are relatively slow, the compound 4-dimethylaminophenol has applications as a more rapid methemoglobin former [ 121. The vitamin Bi2 derivative, hydroxocobalamin (Bm,), is a cobalt(II1) complex having the corrin ring structure. The drug is considered nontoxic, and animal work indicates that it has action against cyanide [13-151. Unfortunately, rather large quantities of the agent need to be administered. Around 1,350 mg of hydroxocobalamin react in vivo with 26 mg of CN- ,and lower molecular weight compounds acting in the same manner are desired. The cobalt(II1) oxidation state can be stabilized by water soluble porphyrins, which in principle can be designed to have lower formula weights than found in Bm,. To this end, we report the kinetic and equilibrium parameters of the interactions of cyanide with a model metalloporphyrin, cobalt(II1) tetrakis(4-sulfonatophenyl)porphyrin (Co-TPPS). The results are compared to Bm,, and related anation reactions of trivalent metalloporphyrins. EXPERIMENTAL The Co(III)-TPPS was made following the heterogeneous metal-insertion procedure of Herrmann and coworkers [ 161. In air, 1 .O g of H2 TPPS. 12 Hz0 [ 171 and 5 g of Co0 (Strem Chemicals) were refluxed overnight in 500 ml of water. The oxide was filtered off, the solution evaporated to 50 ml, and then refiltered through a Millipore Metricel GA-6 0.45 pm filter. The solution was then frozen and lyophilized. For CoC,_,N4H2&S40itNas* 12 HzO; Calcd; Co, 4.63; C, 41.52; N, 4.40; H, 3.80, S, 10.08. Found: Co, 4.49, C, 41.68, N, 4.40, H, 3.29, S, 10.37. The same degree of hydration was noted before by Krishnamurthy and coworkers [18], who prepared this complex by a mercury(B) displacement reaction. Neta [ 191 demonstrated that heating certain cobalt(II1) porphyrins at 120°C causes partial reduction to the cobalt(B) state, and this might explain the production of Co(II)-TPPS using this same metal oxide procedure, where the waters of hydration were removed at 250°C before analysis
U61. The kinetics were followed at 25°C on either a Durrum-Gibson stopped-flow apparatus, or with a Beckman Acta III recording spectrophotometer, and pHs were monitored with a Radiometer PHM 64 Research pH meter. The buffers Tris, Hepes, Pipes, Mes, HOAc/NaOAc, and chloroacetic acid were at lo-mM levels, and the ionic strength was maintained at 0.1 with NaN03, in both the kinetic and equilibrium sections. A Radiometer Ion 83 ion /meter with a cyanide selective electrode was employed for the cyanide uptake work. Cyanide (KCN) was analyzed with silver nitrate by the Liebig-Deniges method [20]. RESULTS
Cyanide Binding The number of cyanide ligands bound by Co(III)-TPPS was studied at pH 7.4 (I = 0.2; 0.1 in phosphate buffer and 0.1 M NaN03), using an ion-selective cyanide
CYANIDE
1
2
3
4
lo4 [ Co-TPPS],M
SCAVENGERS
199
FIGURE 1. Plot of the decrease in total cyanide concentration with added Co(III)-TPPS at pH 7.4, I = 0.2, 25°C.
electrode. A solution ca. 1 mM in cyanide was titrated with Co-TPPS in the same buffer. The reaction was fast, and the potentials stabilized within one min of mixing. Figure 1 is a plot of the change in the total cyanide concentration with added Co-TPPS. The results are that 1.9 + 0.1 mol of cyanide are bound per mole of Co-TPPS at pH 7.4. Cyanide was not taken up by metal free H*-TPPS. Absorption
Spectra
The spectrum of CO-TPPS(H~O)~ at pH 2 (I = 0.1 NaNOs) is similar to that reported by Ashley and Au-Young [21]; A,,,423 nm (E = 2.2 x lo5 M-’ cm-‘), 537 nm (1.4 x 104) and 565 nm (sh, 5.6 x 103). If a 3 x 10e6 M solution of Co-TPPS is made from 0.1-5 mM in cyanide at pH 9, the dicyano Co-TPPS(CN)2 is fully formed; 442 nm (1.7 x 105), 536 nm (8.3 x 103), and 608 nm (9.0 x 103). When HNOs is added to bring the solution to pH 2, the mono-cyano Co-TPPS(H20)(CN) is produced: 427 nm (1.8 x 105), 542 nm (1.2 x 104), and 580 nm (sh, 4.4 x 103). The absorption maxima of the cyano complexes are similar to those found in the Co-TPPS/SCNstudy [21], with Co-TPPS(SCN)z at 442 nm and Co-TPPS(H20)(SCN) at 428 nm. Equilibria In order to estimate the dissociation constant &, of Co-TPPS(H,O)(CN) (Eq. solution 2 x 10e6 M in Co-TPPS was made 2 x 10e4 A4 is cyanide at pH 8 and dicyano formation, the pH was lowered to 2. The only complex present after 2 equilibration was the mono-cyano derivative; no spectral evidence for the diaquo was found. Thus Ka, < lo-i2. HZ0 + Co-TPPS
(H20) (CN)-
= CO-TPPS(H~O)~ + CN-
K,,,
1), a after days form
(1)
The equilibrium constant Ki2 for the formation of Co-TPPS (CN)2 from CoTPPS(H20) (CN) (Eq. 2) was measured by a spectrophotometric method. Known concentrations of Co-TPPS and cyanide were mixed at pH 9 forming the dicyano adduct. Acid CN- + Co-TPPS (H20) (CN)-
= Co-TPPS (CN)22- + Hz0
K12
(2)
was then added to a given pH, and the spectra was monitored after 5 min when equilibrium had been attained.The dissociation constant of HCN has pK, = 9.14 [22]. It was assumed that the total porphyrin, Pr, was in either the mono- or dicyano forms.
200
P. Hambright and Robert Langley
TABLE
1. Formation Constant of Co-TPPS(CN)z from Co-TPPS(H20) (CN)
PH”
Y = [Co-TPPS(CN)JPTJ
10’ (CN-), Mb
5.24 4.92 4.60 4.35 4.08 3.81 3.58 3.34
0.891 0.810 0.696 0.537 0.389 0.246 0.157 0.0962
25.3 12.1 3.96 3.26 1.75 0.939 0.553 0.318
1O-6 K,2C 3.2 3.5 3.9 3.6 3.6 3.5 3.4 3.4 Average 3.5 + 0.2
0 T = 25”C, I = 0.1 (NaNO,). b [CN-JT = 2.3 x lo-* M. CK,2 = [Y/(1 - Y)](CN-)-I.
From the spectra, the fraction Y = [Co-TPPS(CN)JPT] could be calculated and knowing Y, PT, pK,, and [CN-1, the free unbound cyanide concentration, (CN-), could be determined. The equilibrium constant Kr2 = [Y/(1 - Y)] (CN-)-I. Typical results are shown in Table 1, where a single Klz represented the data from pH 5.3 to 3.3, as the fraction of the dicyano porphyrin varied from 89 % to 9 % . For CoTPPS, Kr2 = (3.5 + 0.2) x 106. Similar studies were done on cobalt(III)-tetrakis(NmethykLpyridyl)porphyrin, [Co-TMPyP, Kr2 = (5.6 k 0.3) x IO’], and cobalt(III)tetrakis(4-N,N,N-trimethylanilinium)porphyrin, [Co-TAP, Kr2 = (2.2 f 0.4) x 1061.
Kinetics The kinetics of cyanide addition to Co-TPPS were studied from pH 4 to 10.5. The reactions were followed in the Soret region with porphyrin concentrations ca. 3 x 10m6 A4, and the total cyanide levels were at least 20 times those of the porphyrin. Under such conditions, the reactions were first order in porphyrin over 3 half-lives, with an observed pseudo first order rate constant kobsd, which was independent of whether the disappearance of the aquo/hydroxy or appearance of the dicyano porphyrin bands were monitored. Decreasing the buffer concentration from 10 mM to 1 mM had no significant effects on k.,bsd. The insert in Figure 1 shows that the reaction is also first order in cyanide, from 7.5 x 10d5 M to 7.5 x 10m4 M, at pH 8.1. The reactions were found to be biphasic above pH 11, perhaps partially due to the presence of slowly reacting cobalt porphyrin dimers and polymers in equilibrium with dihydroxy monomers [23]. Figure 1 gives the pH profile of the reaction, as a plot of kobsd/(CN-) versus pH. The following reactions are considered: HCN=H+
+CN-
CO-TPPS(H~O)~ = Co-TPPS(H*O) Co-TPPS(H*O)
(OH)-
K, (OH) - + H +
= CO-TPPS(OH)~~-
CO-TPPS(H~O)~ + CN- -Co-TPPS(H20)
(3)
+ H+
(CN)-
+ Hz0
K,,
(4)
Kti
(5) kcN
(6)
CYANIDE
2
4
SCAVENGERS
201
6
/-
I-
I
I
1
I
4
5
6
7
I
I
I
0
9
10
PH FIGURE 2. pH profile of the kinetics of cyanide addition to Co(BI)-TPPS, I = 0.1 (NaN03), 25°C. The dots are experimental points, and the solid curve was calculated from Eq. 12. The insert shows that the reaction is first order in cyanide.
Co-TPPS(H20)
(OH)-
CO-TPPS(OH)~~-
+ CN- -Co-TPPS(CN)
+ CN- --Co-TPPS(CN)
CO-TPPS(H~O)~ + HCN-rCo-TPPS(H20) Co-TPPS(OH) Co-TPPS(H20)
(CN)2(CN)-
(OH)‘-
+ Hz0
kz
(OH)2- + OH(NCH) + Hz0
+ H+ = Co-TPPS(H20)
(CN)-
+ CN- -CO-TPPS(CN)~~-
(7)
k3
(8)
kncN
(9)
fast fast
(10) (11)
Essentially, CN- reacts with the diaquo (I
202 P. Hambright and Robert Langley
The observed
rate law is thus of the form:
kobsd/(cN-)=(kcN+_4(~+)+B/(~+)+C/(~+)2)/(D) withA
= kucN/Ka, B = k2 Kal, C = k3 K,i Kti, andD
(12) = (1 + K&H+)
+ K,, Kti/
(H+)*). From related work [26] at I = 1.0 (NaC104), pK,i = 7.02, and pKti = 9.76. Each term in Equation 12 is important in a given pH range, and limiting forms of the overall equation were developed and iterations were done to obtain the equilibrium and specific rate constants. We find pK,, = 7.1 + O.l,pKaz = 9.4 f O.l,kcN = (3.1 + 0.4) x 102M-‘s-l, kucN = (3.1 f 0.3) x 10-3M-1s-1, k2 = (2.4 + 0.3) x lo3 M-’ s-’ and k3 = (5.0 f 0.8) x 10’ M-’ s-l. The solid line in Figure 1 was calculated from these parameters using Equation 12, and the overall agreement with the observed data is satisfactory. Brief experiments were done from pH 6.5 to 8.0 with cyanide reacting with preformed Co-TPPS(H20) (CN). The reactions were first order in cyanide from 1.4 x 10m3 Mto 8.7 x 10e5 M, with a specific rate constant of 3 x lo4 M-l s-r, which was independent of pH. Thus, as assumed, addition of the second cyanide occurs about 10 times more rapidly than the first. DISCUSSION Table 2 shows a comparison of the anation rate constants of the diaquo and monohydroxyl forms of Co-TPPS with CN- , SCN- , and I-. The remarkable similarity in the values found indicates that Co-TPPS reacts by a dissociative interchange mechanism, as postulated before for various cobalt(II1) porphyrins [24, 26-291 and BIZa [25]. The effect of the axial ligand in labilizing the opposite coordinated water molecule is in the order CN- > OH- > HzO, as ca 200:20:1. Anion substitution into the diaquo forms of the metalloporphyrins [30, 3 I] appears to be in the order Co(II1) > Rh(II1) > Cr(II1). About 67% of the monohydroxy CoTPPS is present at pH 7.4, and it complexes more rapidly with cyanide than do the
TABLE 2. Rate Constant Comparisons, 25°C Reaction Co(III)-TPPSKNCo(III)-TPPS/SCNm Co(III)-TPPS/ICo(III)-TPPS/HCN Rh(III)-TPPS/CNRh(lII)-TPPS/HCN Cr(III)-TPPWSCN H20-B,Z,/CNm H20-B,2,/HCN NC-B,* (base-on)/CNNC-B,* (base-off)/CNCo(III)-TPPS(H,O)(CN)/CN 0 This work. b Units M-’ s-l.
M(III)-TPPS(H20)2 b 3.1 3.2 1.2 3.1 2.3 4.3 2.5 8.0 8.4 3
x 102 x lo* x 10’ x 10-3 5.0 x 10-j x 10-3 x 102 x IO’ 2.0 x lo4 x 104
M(III)-TPPS(H,O) (OH) b 2.4 x IO3 1.4 x 10’
1om2 2.1 x 10’ -
-
Ref. d 26 21 a 30 30 31 25 2.5 25 25 B
CYANIDE
SCAVENGERS
203
other hydrated species. Interestingly, Rh-TPPS(H*O) (OH) shows minimal reactivity towards cyanide [30]. Thus, at pH 7.4 the specific rate constant for cyanide uptake is 1.4 M-r s-i for Rh(III)-TPPS and 1.6 x lo3 M-l s-i for Co(III)-TPPS, virtually eliminating the rhodium derivative as a potential anticyanide drug. The specific rate constants (in units of M- ’ s- ‘) for the first cyanide addition at pH 7.4 are similar for the various cobalt(II1) derivatives: 1.1 x lo3 for Co-TMPyP, 1.6 x lo3 for Co-TPPS, 2.2 x lo3 for Co-TAP and 2.2 x lo3 for H20-B12 (B&. The HO-Bi2(Bm,) does not react with cyanide (the pK, for the Biza/Bizb reaction is 8.1), and at pH 7.4,90% of cyanide uptake is via the undissociated HCN/Bi2, pathway [25]. In contrast, Co-TPPS reacts predominantly with the CN- anion mainly through the diaquo and mono-hydroxy species at this pH. The cyanide in cyanocobalamin is considered irreversibly bound [32] to the cobalt 7 x lo- ls) [33], and many oral Vitamin B12 supplements are given in this wol form. The cyanide is also tightly bonded in Co-TPPS(H20) (CN), with an estimated dissociation constant of less than lo- ‘*. The addition of a second cyanide to CoTPPS(H*O) (CN) at pH 7.4 is rapid, and the formation constant for this process is 3.5 x 106. In contrast, K for cyanide addition to base-on cyanocobalamin is 5 X lo3 M- ’ (pH 11.7), with a relatively slow formation rate constant of 2.0 M- I s- ’ at pH 9 [25]. Unlike Co-TPPS, B12a reacts more rapidly with the first cyanide than the second. All of the in vitro work presented here indicates that Co-TPPS at the physiological pH of 7.4 is as efficient a cyanide scavenging agent as hydroxocobalamin. Most cases of cyanide intoxication arise from accidental poisoning or suicide, and as noted earlier, the NaN02/Na2S203 or dicobalt-EDTA regimes are relatively dangerous. The use of hydroxocobalamin as an anticyanide drug in animals has been known for over 35 years [14]. In conjunction with dicobalt-EDTA [34], 5-g doses of B12bhave been administered to three adults having cyanide poisoning, with successful results. Six guinea pigs each receiving a lethal i.v. dose (4 mg/kg NaCN) of cyanide immediately followed by twice the molar concentration of hydroxocobalamin were healthy for at least 36 h, while the control group receiving saline all died within 30 min [ 131. In vivo studies found cyanocobalamin to be useless as an anticyanide agent, and only B1zb had antidotal activity [14]. Although no toxic effects were noted [13] in animals receiving 1 g/kg of B12b, little work has been done on the effects of large doses in humans [8]. Hydroxocobalamin has a rather high molecular weight (1,346 g), and related cobalt(II1) porphyrins having formula weights around 600 g should lower the effective drug dose by a factor of four, where two ligands are bound. Relatively little is known about the toxicity of water soluble metalloporphyrins. Mn(III)-TPPS, used for magnetic resonance tumor imaging work [35], is less toxic than Mn(III)-TMPyP or Mn(III)-TAP. We have shown before that a variety of cobalt(II1) porphyrins have a high affinity for cyanide at pH 7.5 [36]. In addition to TPPS, TAP and TMPyP (and the 3 and 2-N methylated forms), the list includes proto, hemato, copro and uroporphyrins, the tetrakis(4-(and3)-carboxyphenyl)porphyrins, 2,4-disulfonated deuteroporphyrin, as well as the tetrakis(N-methyl_4(and3)quinolyl)-porphyrins. Both the cobalt(II1) and cobalt(I1) tetrasulfonated phthalocyanines also bind cyanide under these conditions. Certain of these porphyrins will no doubt have less harmful in vivo effects than others, and some might approach the lack of toxicity so far noted for B12b. However, it has been well established [37] that high concentrations of natural porphyrins cause central nervous system damage, and this could limit the usefulness of these proposed compounds. While none of these agents can be taken orally to provide
204
P. Hambright and Robert Langley
long-term porphylactic anticyanide protection, probably be administered intramuscularly.
the less toxic
derivatives
could
This work was supported by the U.S. Army Medical Research Acquisition Activity, Contract DAMDl7-85-C-5086, and is Contribution Number 1827 to the U.S. Army Drug Development Program.
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