Product yields and mechanism of penicillamine radiolysis at p H 5

Product yields and mechanism of penicillamine radiolysis at p H 5

GREESH CHAND G O Y A L ~ AND DAVID ANTHONY ARMSTRONG Deporttnet~t of Clietnisrr)~, Utiirersily of Calgary, Colgrrry, Alto., Crrt~rrdo T2N IN4

Received November 14, 1975

GREESEI CHAND GOYAL and DAVID ANTHONY ARMSTRONG. Can. J. Chem. 54, 1938 (1976). The major products from the radiolysis of penicillamine in N20-saturated solutions a t

p H 5 are penicillamine disulfide, penicillamine trisulfide, and valine with yields of 2.0, 1.2, and 1.5 molecules per 100 eV in lo-' M solutions and 1.8, 0.94, and 0.60 molecules per 100 eV in 10-3 M solutions respectively. These yields remain essentially the same when the major attacking radical is changed from .OH to .CH20H, .Br2- or .(CNS)?-, and it is concluded that all four species produce predominantly pens. radicals on attacking penicillamine. The formation of trisulfide and an equivalent amount of valine is attributed to secondary reactions of pens. radicals and discussed in the light of recent pulse radiolysis experiments and other work.

In deaerated solutions hydrogen and H2S were observed in significant yields and the valine yield increased. These observations can be accounted for quantitatively by the reactions:

[To] . H + penSH + pens. + H z

171 . H + penSH + pen. + H2S

[I31 e,,- + penSH + pen. + SH-

[251 pen. + penSH+penH + pens. The yields of minor (G < 0.5 molecules per 100 eV) products like NH3 and CH2=C(CH3)-

CH(NH,+)COO- indicated that fragmentation of p e n and other secondary radicals from penSH was more important than for cys. and other radicals from cysSH.

GREESH CHAND GOYAL et DAVID ANTHONY ARMSTRONG. Can. J. Chem. 54, 1938 (1976) Les produits majeurs de la radiolyse de la pinicillamine dans des solutions saturees de NzO 2

un p H de 5 sont le disulfure de ~Cnicillamine, le trisulfure de pCnicillamine et la valine avec des rendements respectifs de 2.0, 1.2 et 1.5 molCcules par 100 eV pour des solutions B 10-2 M et de 1.8, 0.94 et 0.60 molicules par 100eV pour des solutions B 10-3 M. Ces rendements ne changent pratiquement pas quand le radical provoquant l'attaque majeure change de .OH B .CHIOH, .Br2- ou .(CNS)2-et on en conclut que les quatre espkces prod~~isent principalement des radicaux pens. par attaque sur la pCnicillamine. On attribue la formation du trisulfure et une quantiti equivalent de valine B des reactions secondaires des radicaux pens. et on discute de cette formation en fonction dlexpCriences ricentes de radiolyse pulsie et d'autres travaux.

Dans des solutions ne contenant pas d'air, on a observC la formation d'hydrogkne et de H2S avec des rendements importants et le rendement de valine augmente. On peut expliquer ces observations par les reactions:

[701 . H + penSH + pens. + H2

[71 . H + penSH + pen. + H,S el,,- + penSH + pen. + SH- pen. + penSH + penH + pens

Les rendements des produits forrnis en quantitis mineures, (G < 0.5 molecules par 100 eV) comme NH3 et CH2=C(CHl)CH(NHl+)C00-, indiquent que la fragmentation du pen. et des autres radicaux secondaires B partir du penSH est plus importante que pour cys. et d'autres radicaux provenant de cysSH.

[Traduit par le journal]

Introduction efficient itz uivo radiation protectors (1). However ~h~ sulfhydryl amino acid cysteine (HS- penicillamine, which differs from cysteine in

CH2CH(NH3+)Co2-) and the 1,2-amino thiols possessing two methyl groups adjacent to the related to it are generally among the most sulfhydryl (viz. HS-C(CH~)~CH(NH~-I-)CO~-),

is an exception to this rule and in some situations

I Present address : Biophysics Laboratory, Department may evenLact as a radiation sensitizer (2, 3). The of Physics. Illinois Institute of Technology, Chicago, ex~lanatioll for the lack protective ability in Illinois. penicillamine is clearly of fundamental impor-

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GOYAL AND ARMSTRONG 1939

tance to an understanding of the mechanism of sulfhydryl protection. For this reason and also because of the significant role played by molecules related to penicillamine in medicinal chemistry, the present investigation of the products of degradation of penicillamine by free radicals was undertaken. For convenience the formula of the molecule is henceforth abbre- viated to penSH.

Earlier esr studies (4) in flow systems have led to the conclusion that the radicals formed by OH attack are predominantly thiyl radicals:

More recent research on the pulse radiolysis of nitrous oxide-saturated solutions is in agreement with this (5-8) and has shown (6) that . CH20H radicals also produce pens. radicals:

At pH's where the pens- concentration is significant ( p H 2 7 ; PK,,,,~, = 7.9 (6)) pens. radicals form penSS-pen radical anions, which possess a strong optical absorption with A,,, at 450 nm (5-8). The absorption attributed to

pens. radicals is much weaker and has a maxi- mum at 330 nm (5-8). The kinetics of the decays of the optical absorptions were consistent with the following radical-radical reactions:

and

[5] pens. + pens>-pen + pens- + penSSpen

However, there was also evidence for a first order decay of penSS-pen at low doses per pulse (6), which was tentatively attributed to reaction 6.

[6] penSS-pen (+ HzO) + penSS. + penH + OH-

Purdie, Gillis, and Klassen (6) have reported yields of penSSpen, penSSSpen, penH, and H2S from the pulse and O0Co 7 radiolysis of nitrous oxide-saturated solutions at p H 5 and 8. The hydrogen sulfide (G(H2S) = 0.5 to 0.7) was attributed primarily to reaction 7

which had previously been observed at p H = 1 by Peterson (9) and investigated by competition kinetics by Tung and Kuntz (10). The formation

of trisulfide was attributed to reactions 6, 8, and 9:

with the tetrasulfide reacting with penSH prob- ably in reaction 10:

The rate of this reaction was such that any tetrasulfide produced would have disappeared in the time which elapsed between radiolysis and analysis (approximately 1 h) in the experiments of ref. 6. The penSSH from reaction 10 may reasonably be expected to form further trisulfide in reaction 11 :

Purdie et al. (6) found that the yield of trisulfide increased with increasing pH, and this supports the involvement of penS.S-pen ions iil the formation of trisulfide. The yield also increased at lower dose rates which is consistent with the longer time available for reaction 6. However, the increase was not as great as expected for quantitative agreement with the rates of reactions 3 to 6 at p H 8.

Apart from the above studies and the hydrogen yield determinations of Tung and Kuntz (10) no systematic investigations of product yields from penicillamine radiolysis have been reported. The present study was undertaken to obtain a more complete picture of the products resulting from the reactions of e,,-, . H, and . OH at p H 5 where the pens- concentration is small. Experiments were also performed with CHzOH, . (Br)2-, and .(CNS)2-, since it seemed important to determine whether the production of trisulfide occurred only with - H and . OH.

Experimental Reager~ts

D-Penicillamine and L-norleucine from the Sigma Chemical Co., D-valine from Mann Research Labora- tories, grade 'A' p-hydroxyvaline from Calbiochem, and D-penicillamine disulfide and 5,5'-dithiobis-(2-nitrobenzoic acid) from the Aldrich Chemical Co. were used without further purification. The penicillamine trisulfide was kindly supplied by J. W. Purdie of the Defence Research Board in Ottawa. Only the free base forms of amino acids were used in this work to avoid possible formation of .Clz- from the hydrochloride salts.

The sources of potassium bromide, sodium thiocyanate,

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1940 CAN. J. CHEM.

perchloric acid, and methanol were as given in ref. 11, which also states the source and method of purification ofthe nitrous oxide. All other chemicals were of analar or reagent grade.

Procedures arrd Ar~ai)*ses Penicillamine solutions were prepared in fresh triply

distilled nitrogen-purged water which contained the solutes needed for the production of the radicals being investigated. The use of buffers was avoided, the solutions being allowed to assume their natural pH's which lay in the range 4.8 to 5.4. Aliquots of 5 ml were transferred to the carefully cleaned Pyrex cells and attached to the vacuum line. After thorough degassing (12) the samples were equilibrated with N,O a t 760 torr and sealed. One served as an unirradiated standard and was injected immediately into the Technicon amino acid analyser. The remaining samples were stored in a refrigerator prior to irradiation. Immediately after the irradiation a small accurately measured volume (usually 1 ml) was taken and the analysis for amino-containing products was performed on the amino acid analyser. Norleucine was used as an internal standard and elution buffers of p H 3.18,3.80, and 5.00 were employed in succession for periods of 130, 90, and 80 min respectively. These were first passed through a Hi-Rez Type DC-3 resin (Pierce Chemical Co.) to remove ammonia impurities. Standardisation procedures and other details of the amino acid analyses may be found in refs. 12 and 13. As the vinyl compound (CH2= CH(CH3)CH(NH3+)COy) has been carefully identified as a product of the radiolysis of penSSpen (14) and since no standard sample of it was available, it was prepared by irradiating a solution of the disulfide. This solution was injected into the analyser and used to determine the elution time for the vinyl compound, which is also produced from penSH but in a relatively small yield.

Hydrogen sulfide yields were determined with the molybdate reagent (15) using previously described cali- bration procedures (12), and hydrogen yields by the technique outlined in ref. 12.

The loss of penSH was normally calculated from the reduction in the height of the penSH peak on the amino acid chromatogram for the irradiated solution. However, in some cases the loss of penSH was confirmed with Ellman's reagent (5,s'-dithiobis-(2-nitrobenzoic acid)) after purging the irradiated solution with purified nitrogen to remove H2S. The procedure has already been described (16, 17).

In preliminary experiments the number of amino- and sulfur-containing products was established by high voltage ionophoresis. Small aliquots of the irradiated solutions were applied to Whatmann 3 M M paper as a band and crystal violet dye marker was applied as a spot. Amino acid markers ( R and T) were applied as 5/11 aliquots to a 1 cm band. After vertical ionophoresis at p H 2 and 4 kV for 30 min the dye had migrated 18 cm and the paper was dried and cut into strips. Amino acids were detected by treating one of the penSH strips with colli- dine-ninhydrin reagent (18). The presence of sulfur- containing products was established by treating a second strip with freshly prepared platinic iodide reagent (19).

Irradiatiorrs All irradiations were performed a t 23 "C in a 'Gamma

VOL. 54, 1976

Cell 220' purchased from Atomic Energy of Canada. The dose rate was determined several times during the course of the study using ferrous sulfate dosimetry and assuming G(Fel+) = 15.6 (p. 217 of ref. 20). The mean value over the duration of the investigation was 5 X 1016 eV ml-1 min-1.

Results

Iclent$catiot2 of Atnitlo Proh~c i s In addition to the peaks for residual penicil-

lamine and the internal standard norleucine, the amino acid chromatograms of irradiated solutions exhibited six other peaks. Comparison of their elution times with those of standards led to the following identifications, which are given in the order of appearance: P-hydroxyvaline (a very small peak), vinyl compound (CHFCH(CH3)- CH(NH3+)C02-, a small peak), penSH, penH, penSSpen, norleucine, penSSSpen, and ammonia (a small peak). There are therefore two major amino products containing sulfur (pen- SSpen and penSSSpen) and one without it (penH). The formation of the two sulfur-containing amino products agreed with the results of the ionophoresis experiments. These also gave evidence for the vinyl compound and penH, but did not detect the very small yield of 8-hydroxyvaline or the ammonia.

Particular care was taken to substantiate the identification of the trisulfide peak since it was not expected as a product in the radiolysis of a monosulfhydryl. In one experiment an aliquot of an irradiated penicillamine solution was analysed to obtain the area of the peak assigned to penSSSpen. A second aliquot was then mixed with a standard solution of penSSSpen and also chromatographed. Only one uniform peak attributable to penSSSpen was observed and its area had increased to that expected for the combined penSSSpen concentrations. In a second experiment which was kindly performed for us by P. J. Krueger and G. Lee the Raman spectrum of an irradiated solution was taken on a Cary model 82 laser Raman spectrometer and analysed. This solution exhibited a band at 510 cm-l, which has been assigned to trisulfide vibrations (21). Other bands at 590 and 560 cm-I due to the C-S and S-S stretching modes were also observed. These occur in both di- and trisulfides.

Proclz~ct Yields For every system the number of radicals of

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T A B L ~ 1. G values for radicals reactlng with penicillamine and radical balances for deaerated and N 2 0 saturated 10-I M penicillam~ne solutions at p H 4.8 to 5 4

G(Rad1ca1s attacking penSH)

Pr~mary H1O radical species

Solute Total of Z[G(RSSR)+G(RSSSR)] Solute system radicals go,, gcnq- glr all radicals for M penSH

Deaerated solutions lo-' M penSH only - 2 . 8 p 2 .7p O.Goh 6.2 6 .0(977h)L

N 2 0 saturated solutions (i) lo-' M penSH only - 5 .4p 0.5f 0.60b 6 .6 6.5(98 C/; )

(6.0)" ( ~ i ) 10-2 M penSH with

1 M CH?OH gr1,,o,,=5.91C 0.&' 0.52C 0.3?' 7 .O 6.3(90C/,)

(iii) 10-' M penSH with 0.15 11.1 KBr ~ ( ~ ~ , ~ - = 4 . 4 , / 1 .09/ 0.SLC 0.50n 6.6 6.3(953,)

(iv) lo-' M penSH with 0.10 M NaCNS g(css)2-=5.74'L 0.Z411 0 . 5 ~ ~ 0 . 5 ~ g 7.0 6.5(93 yo)

"From yield-react~v~ty curves (25) using k,.,-+,..sH X (penSH) = 5.1 X lo's-' and ~oH+~. ,su x (penSH) = 4 5 X lo7 s-l. k,.,-+,.,sH = 5.1 X 109 M-I s" was taken from refs. 23 and 24 and ko~~+~..stl = 4.5 X 109 M-I s-I from refs. 4 and 6; average of 4.7 X 109 M-I s'l and 4.3 X 109 M-I s".

%om ref. 20. p. 140. 'Calculated from the yields g, = 3.05 and goa = 2 90 based on the data of ref 26, uslng kc.,-+,..a~ from footnote a and k..,-+N,O = 9.0 + 0 3 X 109

M-I s-I from ref. 22. I I dFrom data of ref. 6

'gou was increased from 2.90(see footnote c) to 3.40 based on y~eld react~v~ty curves (29 , to take account of the Increased reactlvlty lo OH due to kou+c~ ,on X (CHIOH) = 8.4 X IPS-'. We took k o ~ + c a ou = 8.4 X 1 0 M-' s-I In neutral solutions from ref. 27. The proportion of OH reacting with CHIOH to form .CHzOH was calculated using this rate consdnt and that in ". ~ H + C H , O H was taken as 1.6 x l W M-I s-I (28) and k ~ + , . , a ~ as 1.9 X 109 M-I s-I (10).

'goa was increased from 2.90 to 3.00 to take account of reactivity (25) due to 0.15 MBr-. ~ O H + B ~ - X (BI-) = 1 8 X I@ S-I based on ~ O H + B ~ - = I 2 X 109 M-I 5-1 (27).

'ga in Br- and CNS- solutions was reduced by 0.1 to allow for uravenglng of H (29). 'gon was increased by 0.5 to take account of reactivity (25) due to 0.1 M CNS-. k o ~ + c ~ s - x (CNS-) = 1.10 X 109 s-I based on k o ~ + c ~ s - = 1.1 X lot0

M-1 s-1 (27). 'Numbers in parentheses in rlght hand column are percentage radial balances equal to. 100 x 2[G(RSSR) f G(RSSSR)J/G(Total of all radicals).

each type expected to react with penicillamine was calculated from the known rate constants for the competing reactions. For the nitrous oxide- saturated solutions reaction 12 competes with reaction 13,

the rate of which has been measured (8, 22, 23). Also in the presence of added methanol (see Table 1) reactions 14 and 15 are in competition with [l] and [7] plus [7a] respectively,

[~ ( J I .H + penSH -) H2 + pens.

while reactions 16 to 19

1161 .OH + Br- --, OH- + .Br

[I71 .Br + Br- -) .Br2-

[I81 .OH + CNS---, OH- + CNS.

[I91 CNS. + CNS- -) .(cNs),-

occur when KBr and NaCNS are present along with NzO. The numbers of radicals reacting with penSH in each solute system have been calculated assuming straightforward competitions between the solutes and penSH. They are listed in Table 1 along with the rate constants employed in the calculations. Initial experiments showed that at p H - 5 reaction with hydrogel1 peroxide occurred as was observed for cysteine.

However, its rate was slow enough (cf: ref. 13) that any contribution from it could be neglected since the samples were injected into the analyser directly after radiolysis.

G values were calculated from the slopes of product concentration - dose plots. Exa~nples of such plots are shown in Fig. l a for Hz and H2S from M deaerated solutions, and in Fig. 16 for penSSpen and penSSSpen from and M solutions and NH3, penH, and vinyl

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VOL. 54. 1976 1942 CAN. J. CHEM.

I I I 1

2 4 6

Dose (eV ml-' ) x lo-'' FIG. 1. Plots of product concentration cs. dose: (a)

deaerated solutions; 10-3 M penSH: V H2S, V H2; ( 6 ) N 2 0 saturated solutions; Ad penSH: A penSS- pen, 0 penSSSpen; lo-? IM penSH : A penSSpen, C penH, . penSSSpen, u NH,, CH2=C(CH3)CH- (NH3+)C02-.

compound from M solutions, all of which were N20-saturated. As illustrated in Fig. lb, for lo-' M solutions these plots were linear up to at least 6 X 1018 eV ml-I and the G values calculated from them are expected to be within experimental error equal to the initial G values for this concentration. This is borne out by arguments presented in the Discussion.

Detection of most amino products was not feasible below 0.4 X 10Is eV ml-I and for the

M penSH solutions G values were derived from the slopes in the 0 to 2 X 1018 eV ml-I range. Since 1018 eV ml-I corresponds to 10 molyo consumption of the penSH,"hese G values will be lower than initial G values and must be interpreted with caution. Thus our conclusions regarding the mechanism are based primarily on the yields for M solutions. However, the results for M solutions are useful for purposes of comparison for certain products and a few yields for this concentration are therefore reported also.

I t is worth noting that the plots for the penSSSpen concentrations d o not curve upward with dose in either the 1 0 - h r M penSH

'In lo-' M penSH. For lo-' M penSH the m o l x consumption at this dose is <2.

TABLE 2. I 'rod~~ct yields from reactions of OH, en,-, and H with penicillamine in deaerated solutions

Value

p H = j . O i 0 . 1 p H = 5 . 4 i 0 . 1 Parameter [penSH] = 10-I 11.I [penSH] = 10-I M

G(penSSpen) G(penSSSpen) G(penH) G(viny1) G(NH3) G(H2) G(HIS) Total G(-N)" Total G(-S)" G(-penSH)

"G(-N) = Sum of (G value) X (nurnbcr of N ntoms per molecule) for all N-containing products.

*G(-S) = Sun1 of (G \*;~luc) X (number of S ntoms per molcculc) for all S-containing products.

solutions. Such curvature would be expected if disulfide were an intermediate in the formation of penSSSpen. Further evidence against this possibility was derived from experiments in which 5-10 mol% of penSSpen was added to

M penSH solutions before irradiation. While this tended to depress product yields it caused no marked rise in G(penSSSpen).

G values for the products obtained in deaerated and nitrous oxide solutions are list.ed in Tables 2 and 3 respectively. Stoichiometry requires that the number of molecules of peilSH destroyed per 100 eV, G(-penSH), be equal to the total yield of nitrogen, G(-N), appearing in products and also to the total yield of sulfur, G(-S), appearing in products. The degree of agreement between these three quantities provides a test of the completeness of product identification. As seen from Table 2, for the deaerated solutions G(-N), G(-S), and G(-penSH) agreed within 6%. For the nitrous oxide-saturated solutions (see Table 3) G(-N) agreed with G(-penSH) to within 5% except for the S ( B ~ ) ~ - solutions. Here the difference was 10% but it is still within the combined standard deviations. Hydrogen sulfide was not determined by us in nitrous oxide- saturated solutions. However, if one uses G(H2S) = 0.7 per 100 eV as reported for 1 0 - M solutions in ref. 6 G(-S) is 8.4, in an excellent agreement with G(-N) and G(-penSH) in column two of Table 3. In view of the highly satisfactory agreement between G(-penSH), G(-N), and G(-S) for our conditions the con-

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TABLE 3. Product yields from reactions of oxidising radicals with penicillamine in N 2 0 saturated solutions

Value

Parameter (0) (b) (c) (4 (el (f)

G(penSSpen1 2.O4+0.l6 2 .005 0.20 1.9,fo.17 2 . 0 ~ 5 0 . 1 ~ 1.a1+_o.i5 2 . 0 ~ k 0 . 1 , G(penSSSpen) 1.l950.O4 1 . l6 f0 .O4 1 . 1 ~ k o . 0 , l . l , 5 0 . 0 4 O . ~ ~ + O . O ~ 0 . 9 0 + 0 . 0 ~ G(penH) 1.5050.10 1 . 5 ~ 5 0.10 1.4550.10 1.33f 0.05 0.60+_0.04 0.6850.04 G(viny1) 0.155 0.02 0.63+0.07 0 .08f0 .04 0 . 0 5 ~ 0 . 0 3 0.125 0.05 0.'13k0.04 G(NH3) 0.31k 0.03 0.14f 0.03 0.4,+0.05 0.16f 0.05 0.38+0.05 - Total G(-N) 8 .450 .6 8 .7k0 .7 8 .350 .6 8 .1 5 0 . 7 6 . 6 5 0 . 5 7 .0k0 .5 G(-penSH) 8 .850 .8 9 . 0 k 0 . 8 9 . 4 5 1 . 0 8 . 5 5 1 . 0 6 .750 .5 7.1 k 0 . 5

aNo added solute; pH = 5.0 f 0.1; major attacking radical = .OH; [penSH] = 1 0 - 9 f . bAdded solute = I M CHJOH; pH = 5.0 f 0.1; major attacking radical = . CH2OH; [penSH] = lo-' M. CAdded solute = 0.15 M KBr; pH = 4.8 f 0.1; major attacking radical = .(BIZ)-; [penSH] = 10-1 M. dAdded solute = 0.1 M NaCNS; pH = 5.2 f 0.1; major attacking radical = .(CNS)?-; [penSH] = 10-W. eNo added solute; pH = 5.4 + 0.1; major attacking radical = .OH; [penSH] = lo-' M. /Added solute = I M CHIOH; pH = 5.4 f 0.1; major attacking radical = .CH?OH; [penSH] = 10-1 M.

clusion that the total yield of as yet unidentified AT,,KAT ,,,, .CH HH;

products is less than 1 per 100 eV appears to be I ) I [Zl] . X 0enSH ----r XH * HS - C - C - CO;

reasonably well justified.

Discussion Free radical reactions with the penicillamine

molecule may conveniently be classified in terms of the sites at which chemical damage initially occurs. These are shown in the following diagram,

and Scheme 1 gives sequences of reactions which lead to the formatioil of the vinyl compound, penH, ammonia, and H2S after attack by a radical . X at sites ( I ) , (2), and (4). The rapid elimination o f . SH assumed to occur in reactions 22 and 29 is consistent with the low C-S bond dissociation energy in free radicals (-10 kcal mol-I (30,3 1)). Reactions of oxidising radicals at sites (5 ) and (6) could also lead to . SH elimination and ammonia formation. However, we found less literature evidence for such processes and have not discussed them here. Non-amino products from the fragmentation of the molecule (for example CH(CHs)2COC02- formed in the multistep reaction 27) are not detected by our techniques, but the extent of their formation inferred from the ammonia yields is <0.4 per 100 eV, except for the solutions with (Br)2- as

c - c - ca; 1-1

I I

[24] ck R I.C."S.. .lC I

ATTACK AT SITE 121, CH, HH;

[ZJ] . X - 0lnSH - XSH r

RH

c s , HII;

c - c - ca;

I CHI

I 2 d

A T T K K AT SITE 141 CH, HH;

I I I

major reactant (see Table 3). Abstraction of the H atom at site (3) produces pens. .

The magnitudes of G(penSSpen) and G(pen- SSSpen) for the M solutions in Tables 2 and 3 ark constant and independent of the nature of the attacking radicals. Except for G(penSSpen) in the deaerated solutions this feature also applies to the M solutions. It is best explained if .OH, .CH20H, .(Br)2-, -(CNS)2-,

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1944 CAN. J. CHEM. VOL. 54. 1976

and pen. each produce predominantly pens. radicals, which form the di- and trisulfide in subsequent reactions. Stoichiometry requires that these reactions also contribute to the penH yield and we assume these contributions t o be equal to the mean values of G(penSSSpen) for the and lop3 M solutions, 1.18 and 0.93 per 100 eV respectively. The nature of the secondary reactions of pens. will be discussed later. The reactions of . H and e,,,- and processes producing the vinyl compound and ammonia are considered first.

Reacfions of'. H aizd e,,- Analogy with other low molecular weight

sulfhydryl molecules suggests that initial attack by e,,- will occur at site (2) and by . H at sites (2) or (3). If reactions 7, 7c1, and 13 are the only reactions producing significant yields of hydrogen and hydrogen sulfide then the relation G(H2) + G(H2S) = g,,,,- + grr + GII~" should hold for the deaerated solutions. The results in Table 2 give G(H?) + G(H2S) = 3.6 per 100 eV for the 1 0 - W solutions and 3.0 per lOOeV for the M solutions. These values are very similar to 3.6 and 3.1 per 100 eV for 10-"nd lop3 M cysteine solutions at pH 5.6 (13). The lower magnitude of G(H2) + G(H2S) in the M solutions is attributed to the fact that the radical yields are smaller at the lower concentration and to the greater proportion of primary and secondary radicals reacting with products.

Allowing for the effect of intra-spur scavenging (25, 13) we estimate GI[," = 0.3,~ per 100 eV for the M solutions: Hence, g,,,- + g11 =

G(H2) + G(H2S) - 0.3,~ = 3.3, which is in agreement with 3.35 per 100 for g,,,,- + g,, from Table 1. The observed hydrogen yleld is 0.64 per 100 eV which leaves 0.64 - 0.3,~ = 0.& molecules per 100 eV to be attributed to reaction 7c1. On this basis gIIk7n/(k7a + k7) is 0.34, whence we calculate k7,/k7 = 1.3.

For the deaerated solutions G(H2S) should be equal to [g,,,- + g11k7/(1~7, + k7)I. Using gc,,- = 2.75 and = 0.60 per 100 eV from Table 1, and the above value of I c ~ ~ / ~ ~ one obtains 3.0 per 100 eV, which is again in good agreement with the experimental result in Table 2 for the deaerated 1 0 - W penSH solutions. The significantly smaller value of G(H2S) for the M solutions (2.17) is believed to be caused to a large extent by secondary reactions of this

product with p e n and other radicals, since the H2S concentration - dose plots for these solutions exhibited curvature. For the M nitrous oxide-saturated solutions the number of electrons reacting directly with penSH is 0.52 (see Table 1) and the total H2S yield from [7] and [13] should be 0.78 molecules per 100 eV. This is close to 0.7 molecules per 100 eV reported in ref. 6 for these conditions. Therefore the hydrogen and hydrogen sulfide yields at p H = 5 can be satisfactorily explained by reactions 7a, 7, and 13 with k7a/I~7 = 1.3. This value of k7Jk7 is about three times larger than 0.44 and 0.50 observed at p H = 1 (10) and pH = 0 (32) respectively, suggesting that k7Jk7 is p H de- pendent.

If all p e n radicals from reactions 7 and 13 were converted to penH in reaction 25, then for the M deaerated solutions the combined yield from this reaction and the secondary reactions of pens. radicals (1.18 per 100 eV see above) would be 4.18 per 100 eV (= G(H2S) + 1.18). The observed magnitude of G(penH) was 3.5, implying that about 0.7 pen. radicals per 100 eV are lost in reactions other than [25]. Like [13] this is a counterpart of a reaction occurring in the cysteine system, where there is no significant deficit of the corresponding product, CysH (13). The lower bond dissociation energy for the tertiary C-H bond of penH (Dtcrtiary C-II r~ 9 1 kcal mol-I as opposed to DPrimary c-II r~ 95-100 kcal mol-I (33)) formed in the penSH case will decrease the exothermicity of reaction 25. This together with the steric hindrance of the methyl radicals will tend to slow the rate of H atom transfer, permitting a greater proportion of alternative reactions of the pen. radical. Those shown in Scheme 1 ([24] and [26]) yield ammonia and the vinyl compound. Some pen. radicals may combine with pens. to form penspen. However, the absence of significant peaks attributable to penspen indicates that G(penS- pen) < 0.2 per 100 eV.

Recrcfioizs of - OH, . CH?OH, . (Br)2-, clnd . (CNS)2-

Minor Proclrrcts The yield of pen. radicals will be drastically

reduced in nitrous oxide saturated solutions where most ex,- react with N20. Thus the increase in G(NH3) to 0.3 1 in the M nitrous oxide-saturated solutions from 0.19 + 0.03 in

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deaerated solutions of the same concentration means that there are sources of NH3 other than the reactions of pen- radicals. A similar behaviour occurs in the lop3 M solutions (Tables 2 and 3). Reactions 28, 29, and 27 are probable additional sources of ammonia and their analogues in the cysteine system have been pro- posed previously (1 1). Rather interestingly for the lo-' M solutions G(NH3) is greatest for . (Br),, for which the negative charge and strong oxidising power would enhance the rate of reaction at site (4) relative to site (2) or (3). I t is next highest for . O H and comparatively small for . C H 2 0 H and -(CNS)2-. However, for each radical species G(NH3) is larger than was observed with cysteine, and this must be due to steric or inductive effects of the methyl groups, which reduce the likelihood of attack on the SH group of penSH.

From Table 3 vinyl production is most efficient for .CH20H, suggesting an enhanced contribution from reactions 21 and 22 or some other process in this system.

I~erzSSpen arzcl ~xtzSSS~~etz Although the yields of ammonia and vinyl

compound in Table 3 show that radical attacks a t sites other than the sulfhydryl H atom are more frequent with penicillamine than with cysteine (cf: thc smaller ammonia yields in ref. l l ) , they are still small relative to the yields of the major products. This is consistent with the previously mentioned conclusio11 that - O H ,

CH,OH, - (Br)?-, (CNS)?-, and pen- produce predominantly pens. radicals upon reacting with penSH in reactions 1, 2, 31, 32, and 25. In the cysteine system the corresponding radical

[321 .(CNS)Z- + penSH + ZCNS- + H+ + pens

[251 pen. + penSH --, penH + pens.

cysS- disappears primarily by combination to form cysSScys and for any set of conditions the relation 2G(cysSScys) = G(tota1 radicals) holds. If reactions 4-6 and 8-1 I are responsible for the production of penSSpen and penSSSpen in the present system, the corresponding relation would be 2[G(penSSpen) + G(penSSSpen)] = G(tota1 radicals). The values of 2[G(penSSpen) + G- (penSSSpen)] for M solutions in the penul- timate column of Table 1 have been expressed as a percentage of the calculated total radical

yields and are given in the last colunln. These percentages lic in the range 92 to 98%, showing that the relationship holds to a good approximation. Thc small deficiencies in the yields arc expected in view of the probable occurrence of radical disproportion reactions such as [24] and [26] (see Scheme l), which d o not produce penSSpen or penSSSpen.

For thc M nitrous oxide-saturated solutions 2[G(penSSpen) + G(penSSSpen)] = 5.8, with 1 M C H 3 0 H and 5.50 with no solute (i.e. for . O H as the major attacking radical). Thesc quantities are significantly smaller than the estimated total radical yields in Table 1, reflecting the enhanced importance of product-radical reactions a t this concentration. Similarly 2[G- (penSSpen) + (penSSSpen)] = 4.7 for the deaerated lop3 M solutions is much smaller than 6.0 per 100 eV calculated for the total radical yield using g l ~ = 0.6, gp, , - = 2.65 and go11 and 2.70 from yield-reactivity curves in ref. 25. I t is also less than 2G(cysSScys) = 6.0 for lop3 M cysteine, which indicates a higher proportion of radical-product and radical-radical reactions (other than [4] and [5]) in the penSH system.

Taking K33 = M (6) and K3 = 300 M-I

[331 penSH = pens- + Ht

(6) one finds [penSS-pen] = 3 X lop3 X [pens.] in lop2 M penSH a t p H = 5, which means that reaction 5 can be neglected. Applying the steady state approximation t o the concentrations of pens- , penSS. and penS,SPpen one then obtains

where p is the total rate of production of pens. radicals. From this relationship it follows that

Also

Assuming reaction 3 maintains an equilibriunl and writing d[penSSSpen]/dt = r , one can derive the expression

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1946 CAN. 1. CHEM. VOL. 54. 1976

FIG. 2. Dependence of normalised trisulfide yields on penicillamine concentration and pH: 0, data of this study at p H 5.4 for 10-3 M penSH and p H 5.0 for 10-2 M penSH and 5 X 1016 eV ml-1 min-1; p, from ref. 6 and

from Purdie2 at pH5.0, varying [penSH] and 4.9 X 1017 eV ml-1 min-1. For definitions of p and p see Discus- sion. The estimated errors in p are larger at 4.9 X 1017 eV ml-1 min-1 because of the much smaller values of G(penSSSpen). Dashed line shows value expected for reaction 6 in 1 0 - U p e n S H at p H 5.

The quantity r./p is equal to G(penSSSpen) divided by G(tota1 pens. radicals formed), which can be assumed to be 6.6 per 100 eV. In Fig. 2 we test expression 34 for p H in the range 5 to 5.4, using the present data and other values of G(penSSSpen) observed a t a tenfold higher dosc rate. One of these was given in ref. 6 and the remainder have been obtained by P ~ r d i e . ~ The left hand side of expression 34 is denoted as PdP. The dashed line passing through the origin shows quantitatively the behaviour expected, assuming the rate constants for reactions 4 and 6 and the equilibrium constant for reaction 3 are p H independent.

The fact that the high and low dose rate results in Fig. 2 are brought into coincidence on multiplying P by dp confirins the dependence of G(penSSSpen) on this parameter and supports the view that a first order reaction of a radical

inadequate to explain the trisulfide formation at p H = 5. Hence it would appear that species other than penSS-pen may be involved, and in this regard the possible intermediacy of the sulfenium radical (penSS(H)pen) cannot be ignored. Although this radical is currently believed to be much less stable than penSS-pen (7, 8) it inay conceivably exist in a high enough concentration a t p H = 5 for reactions 35 to 38

to provide a significant pathway to penSS. formation.

Since the number of penSS radicals is small relative to pens. , reaction 9 was neglected in deriving expression 34. However, the situation is still complicated by the possibility that pen- SSSSpen produced in a significant yield from reaction 9 may react with p e n s radicals.

Reactions of this type have been postulated to occur with sulfanes (34) and methyl polysulfides (35). They will tend to decrease the penSSSpen yield, since they reduce the lifetime of pens. . This could contribute to the fact that for experiments all done a t p H close to 5 (as in Fig. 2) $ is not directly proportional to [penSH]. Likewise it may be a reason why G(penSSSpen) from the y

radiolysis at p H 8 is less than predicted from the rate constants determined from pulse radiolysis at that pH, which indicated that reaction 6 should be the dominant mode of penSS-pen decay (6).

More work on the dependence of G(penSSS- pen) on [Hf] and [penSH] in the p H range 0 to 6 will be required before the mechallisin of trisulfide formation in acid solutions can be settled, and further pulse radiolysis studies of the inter- mediates in the p H = 0 to 6 region would also be of great value. -

intermediate competes with radical combination. "The dependence of P ~ P on [penSH]/[H+] may if

On the other the "lid line F ig does not anything be less than illdicated by tile solid lille in pass through the showillg that a lnech- Fig, 2, since the values of P for low [penSH]/[H+] are anism based only on reactions 3 to 6 and 8 is calculated from G ( ~ e n S S s ~ e n ) for 10-3 M venSH, AS

seen above from a 'compa;ison with radical'yields, the 3We are indebted to Dr. J. W. Purdie for making these product yields in these solutions tend to be smaller than

data available to us. expected.

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GOYAL A N D ARMSTRONG 1917

Acknowledgments

The authors wish to express their thanks to Drs. M. H. Benn, J. W. Purdie, H. Gillis, and N. V. Klassen for helpful discussions, and to Dr. Purdie for other assistance. They are grateful to Drs. P. J. Krueger and G. Lee for the Raman spectral analysis.

Financial support for this work came from Defence Research Board of Canada grant number 957004 and from National Research Council of Canada grant number A 357 1.

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Documents

Product yields and mechanism of penicillamine radiolysis at p H 5