Pulse radiolysis study of reactions of tetracycline with radiolytically generated reducing species

Radiat. Phys. Chem. Vol. 44, No. 5. pp. 507-511,

1994

Copyright 0 1994 Elsevicr Science Ltd Printed in Great Britain. All rights reserved 0969-806X/94 $7.00 + 0.00

Pergamon

PULSE RADIOLYSIS STUDY OF REACTIONS OF TETRACYCLINE WITH RADIOLYTICALLY GENERATED REDUCING SPECIES S. SABHARWAL’

and K. KISHORE’

‘Isotope Division and *Applied Chemistry Division, Bhabha Atomic Research Centre, Trombay, Bombay 400 085, India (Received 14 January 1993; accepted 28 July 1993) Abstract-The

transients involved in the reaction of tetracycline (TC) with reducing radicals such as etq. (CH,),COH and COi_ have been characterized by the pulse radiolysis technique. The semi-reduced spectes formed (2, = 630 nm, r = 3.4 x IO’ dm’ mol-’ cm-‘) has been found to be a strong reductant with reduction potential lying in the range -0.450 to - 1.40 V vs NHE. TC reacts with e4 at diffusion-controlled rates and the rate constant, depending upon the ionic form of TC existing at a particular pH, varies from 1.2 x 1O’Oto 2.8 x lOtodm3 mot-’ s-‘. Based on these results a plausible site of electron addition has been suggested. Reaction of H atoms with TC gives rise to a transient which exhibits spectral and kinetic features different from that of semi-reduced species.

INTRODUCTION

Radiation chemistry of tetracycline (TC) is of relevance in the context of radiation sterilization of its pharmaceutical preparations. TC in the dry state is stable to radiation but undergoes degradation when irradiated in aqueous solutions (Jacob, 1983). It has been suggested that radiolytic degradation of pharmaceuticals, when irradiated in aqueous solutions, can be minimized or eliminated either by incorporating suitable additives or by irradiating at low temperature (Kishore et al., 1976, Pikaev, 1985). This requires a thorough understanding of the radiation chemistry of the drug, particularly its reactivity towards various primary species of water-radiolysis. The reactivity of TC with OH radicals and e; has been determined by steady-state y radiolysis (Dziegielewski, 1977; Guedes and Vasconcelles, 1986; Dziegielewski and Glowacki, 1982). However, no systematic investigation appears to have been carried out to elucidate the nature of transient species produced during radiolysis, which are involved in the process of degradation. We have carried out the pulse radiolysis study of this compound in aqueous solutions with the objective of understanding the nature and characteristics of such intermediates. In the present paper, reactions of TC with reducing species produced by pulse radiolysis of aqueous solutions are reported. EXPERIMENTAL

TC was obtained from Sigma Chemicals and used as received. All other reagents were AnalaR grade and solutions were prepared with water having a

conductivity of < 0.1 p S cm-‘. The pH values of the solutions were adjusted using H2S04, KH2P04, Na,HP0,2H,O, Na,B,O,IOH,O and NaOH in appropriate concentrations. Stock TC solutions were prepared fresh daily, kept in the dark and refrigerated to avoid photodecomposition and dehydration. A 7-MeV pulsed linear electron accelerator (Ray Technologies, England) was used for pulse radiolysis work. Air saturated decimolar KSCN solution was used as dosimeter (G .c = 2.23 x low4 m* J-’ at 500 nm) (Fielden, 1982). Depending upon the need either 50 or 500 ns single pulses were employed. The absorbed doses were about 20 Gy for 50 ns pulse and 40 Gy for 500 ns pulse. A matrix containing isopropyl alcohol (1 mol dme3) and acetone (0.2 mol dm-‘) was used for generating isopropylketyl radicals while NzO saturated sodium formate solutions were used for generating CO; radicals. In neutral solutions H atoms were generated by reaction of e, with H2PG; (Ye and Schuler, 1986). Details regarding the detection facility, transient recorder and dosimetry are published elsewhere (Guha et al., 1987). Kinetic analysis were carried out with the help of on-line data acquisition and analysis using Iwatsu TS 8 123 storage oscilloscope coupled to an IBM PC.

RESULTS AND Reactivity

DISCUSSION

with e&

TC exists in various conjugate solution (Scheme 1) and has pK, 9.69 and 10.7 (Mitscher, 1978). The reactivity of e; with the acid-base forms of TC existing at 507

acid-base forms in values at 3.3, 7.68, different conjugate pH 4.0,6.8,9.0 and

S. SABHARWAL

508

OH

0

OH

and K. KISHORE

OH 3.3 G====

0-

CONHz

CONH2

7.68 e

O’H

0

ii

O’H

TCH+

CONH2

ii TC-

TC

CONHZ

CONH* OH

0

0-

0

TC-’

TC-3

Scheme I

13.0 was determined by monitoring e; absorption at 720 nm. The concentration of TC was varied from 5 x lo-’ to 1 x 10-4mol dm--’ and 0.1 moldmm3 t-butanol was used for scavenging OH radicals. The rate constants for e; reaction with TC at different pH values are presented in Table 1. Expectedly, the rate constant for the reaction of TC with e.& depends upon the state of protonation of TC molecule existing in solution. At pH 6.8, TC exists in the neutral form (TC) and has a high reactivity toward e.q (k = 1.9 x 10”dm3 moll’s-‘) which further increases on decreasing the pH to 4. This is because TC has a pK, at 3.3 where the fi-tricarbonyl group in the ring A (see scheme 1) gets protonated and the molecule exists in the TC+ form. Since it was not possible to study the reaction at pH < 3.3, because H+ starts competing with TC for e;, the rate constant was determined at pH 4 where TC partly exists as TC+ and therefore has a higher rate constant value of 2.5 x 10”dm3 mol-’ s’. As the pH is raised to 9 and then 13, TC exists in the anionic forms and the electrostatic repulsion between negatively charged TC and ea;

Table

I. Spectral and kinetic parameters of the reaction cycline with various reducing species

Reducing species

pH

caq

4.0 6.8 9.2 13

co; (CH,),C H *Calculated tCalculated

OH

Reaction rate constant (dm’mol-‘s-‘) x x x x

c (dm’mol-‘cm-‘)

10’0 10’0 IO” 10’0

580 620 630 630

2.4 3.4 3.7 3.x 3.4

6.8

1.6 x IO”

630

3.2 x IO’t 3.5 x 103t

6.8

4.7 x 10’

630

1.8 5.8

2.5 x IO9 8.9 x IO8

440 450

after correcting after correcting

2.7 1.9 1.6 1.2

I,,,

of tetra-

x x x x x

10’ 103 103 10’ lo’*

2900 2035

for H atom contribution. for CO; bimokcular decay.

of the rate constant. The rate causes lowering constant at pH 13 (1.2 x 10’0dm3mol-’ ss’), although lower than that at pH 4, is not lowered appreciably. The rate of reaction of TC towards ea; obtained in our work is much higher than the previously reported value of 1.5 x lo9 dm3 mol-’ s-’ measured at room temperature by following the competition kinetics (Dziegielewski, 1977) and by ESR measurement at 77 K (Guedes and Vasconcelles, 1986). The difference in the rate constant at 77 K can be attributed to the large difference in temperature as well as lower mobility of hydrated electrons in the rigid matrix. The lower value reported by competition kinetics may be because of a faulty choice of experimental conditions as the rate constant has been determined at pH 1, where the yield of e; will be negligible due to their reaction with H+ ions. Occurrence of back reactions and scavenging of secondary radicals, inherent in steady state radiolysis, may also lead to lower reactivity. It is well known that hydrated electrons are relatively unreactive towards benzene, amines and saturated cyclic compounds with rate constants in the range 105-107dm3moll’s~’ (Buxton et al., 1988; Spinks and Woods, 1990). The rate constants of hydrated electrons are generally high with molecules containing carbonyl group (> lo9 dm3 mol-’ s-l) especially if there is a double bond present adjacent to the carbonyl group. This is attributed to the lowering of the electron density of the double bond because of the n-electron shift (Pikaev, 1971). Therefore, the high reactivity observed in the case of tetracycline may be attributed to the reaction of e.q with the @-tricarbonyl group in the TC molecule, over which the attached electron may get easily delocalized. This also explains the fact that increasing pH from 4 to 13 reduces the reaction rate by a factor of 2 only, as the P-tricarbonyl system has a pK, of 3.3

Pulse radiolysis study of TC

509

0.032

0.028

0.024

d cj d

0.016

x

\\

0.008

\

0. 'a

o atpH4 0.004

x

0

340

I 380

I 420

I 460

I 500

at

1 540

pH 6.8

I 580

Wavelength

x

-0

-0. I 620

I 660

I 700

Y, I 740

I 780

(nM)

Fig. 1. Absorption spectra of the transient formed by reaction of ea; with N, saturated lo-‘mol dmm3 TC solution containing 0.2moldm3 t-butanol at different pH, 4~s after the pulse. Inset: Change of absorbance at 630nm as a function of pH.

and hence electron density around this group essentially remains the same beyond pH 4. Spectral characteristics Figure spectrum

1 shows obtained

of the transient

the transient optical absorption on pulse radiolysis of nitrogen

saturated aqueous solution of TC (1 x lo-’ mol dme3, pH 6.8, containing 0.2m01dm-3 t-butanol) after complete decay of e4 (4~s). Two optical absorption bands with & at 630 and 44Omn were observed. Time resolved spectra showed that whereas absorption at 630 nm decayed with time, the absorption at 440 nm remained more or less the same even after 150 ps indicating the formation of a permanent product at the latter wavelength. Increasing the t-butanol concentration from 0.2 to 2.0 mol drne3 led to a decrease in absorption at 440 nm while the peak at 630 nm remained unaffected. It was, therefore concluded that the weak absorption at 440 nm may be due to the reaction of the small amount of H atoms present in the system and that only one transient species with an absorption maximum at 630 nm with e = 3.4 x 10’ dm3 mol-’ cm-’ is formed by e;lq reaction assuming Ge, value of 3.0. The species decayed by second-order kinetics with 2k = 2.4 x IO9dm3 mol-’ s-i. pK, of the semi-reduced

species

The absorption spectra of the transients obtained at pH 4 and 11 were similar in nature to that at pH 6.8 except that at pH 4 the transient spectrum is blue shifted with maximum at 580 nm (6 = 2.4 x

lo3 dm3 mol-’ cm-‘) whereas at pH 11, the absorption maximum remains unchanged at 630 nm. The increase in absorption at pH > 10 is due to the fact that H atoms react with OH- to yield hydrated electrons (Fig. 1). The dependence of extiction coefficient on pH was made use of to obtain the pK,s of the semi-reduced TC species. By monitoring the changes in normalized absorption at 630 nm as a function of pH, a titration curve (inset in Fig. 1) was obtained, which showed only one inflection point indicating pK,s of the species to be 5.5 in the pH range 4-12. Reaction (CH,),

with

one-electron

reductants

CO;

and

COH

Specific one-electron reducing radicals like CO; and (CH,),COH were able to bring about the reduction of TC. At neutral pH, the transient species formed by both the reductants with TC showed an absorption band at 620-630 nm, confirming that only one transient having absorption in the region 400-800 nm is formed. The rates of reaction of (CH,),COH and CO: with TC were determined by monitoring the formation kinetics of the transient absorption at 630 nm and assuming G,, value of 6.5. The values are presented in Table 1. As expected, the rates of electron-transfer from CO; and (CH,)rCOH radicals to TC are significantly lower as compared to the reaction of es;. The absorption spectrum of the transient obtained with CO; radical is compared with the spectrum obtained by e, reaction with TC (Fig. 2). The two spectra are

510

SSABHARWAL

and K. KISHORE

r

Table 2. Rale constants for the electron transfer reaction from semi-reduced tetracycline to various electron acceptors

0.012

Rate constant Electron acceotor

DH

Thionine

4.0 6.8 4.0 6.8 4.0 6.8

Safranine-T Methyl viologen

380

420

460

500

540

580

Wavelength

620

660

700

spectra of the transients produced by the one-electron reduction of TC (1 x lo-‘mol drn-‘) in aqueous solution by (a) e; (b) COT radicals, 10 ps after the electron pulse, pH 6.8, NzO saturated containing 0.1 mol drn-’ formate.

qualitatively similar except for an additional band at 440 nm in the TC- e, spectrum which has already been attributed to H atoms reaction with TC. However, quantitatively, the absorbance values obtained using CO; as reductant are slightly lower. This is because, as shown in Table I, the rate of COT reaction with TC is two orders of magnitude lower than the ea; reaction with TC, therefore a part of COT radicals may undergo bimolecular decay (2k = 1.5 x lo9 dm3 mol-’ SK) (Simic et al., 1969) resulting in lower absorbance values. In the alkaline pH region, where TC exists in negatively charged form (TC), CO; and (CH,)2COH radicals could not reduce it indicating that the reduction potential of this form is higher than -2.0 V vs NHE, the reduction potential of CO; (Schwarz and Dodson, 1989). of semi-reduced

tetracycline

In order to further probe the nature of the reaction product of e, and TC, its ability to subsequently transfer the electron to various acceptor dyes viz. thionine, safranine-T and methyl viologen was investigated in neutral aqueous solutions TC- +S-+TC+S

2.5 3.2 9.9 1.3 2.1 6.1

x x x x x x

109 IO9 108 109 109 IO’

740

(nm)

Fig. 2. Absorption

Reducing characteristic (TC) species

(dm3mol-‘s~‘)

Table 2, show that the semi-reduced TC species is a stronger reductant at pH 6.8 than at pH 4. These results indicate that the one-electron reduction potential of TC at pH 6.8 is much more negative than -0.450 V vs NHE. Since (CH,)2COH radicals (E A= - 1.40 V vs NHE) bring about reduction of TC, it can therefore be concluded that one-electron reduction potential of TC falls in the range -0.450 to - 1.40 V vs NHE. Reaction

at0m.r

H-atoms were found to react with the protonated form of TC (TCH+, form I) at pH 2, with a rate constant of 2.5 x lo9 dm3 mol-’ s-’ as determined by following the formation of the product transient species at A,,, = 440 nm. OH radicals were scavenged from the system by addition of 0.1 mol dm3 t-butanol. The spectrum of the transient species [Fig. 3(a)] exhibited a major absorption band at 440nm and a very weak band at 630 nm. Time resolved spectra, after 150 p s, showed that 630 nm band decayed completely but the 440 nm band decayed into a permanent product having A,,,,, = 420 nm [Fig. 3(b)]. Taking Gu to be 3.3 and assuming that all the H atoms react to give one transient species, the extinction coefficient of the transient at 440nm was calculated to be 2.9 x 10’ dm3 mol-’ cm-‘. This transient disappeared by a second order reaction with 2k = 3.2 x lO”dm’mol~ s-l. The weak band at 630nm may be attributed to the semi-reduced TC

0.024

(1)

where S = electron acceptor. The one-electron reduction of thionine (TH+, EA = -0.04V vs NHE), safranine-T (SF+, Eh = -0.185 V vs NHE) and methyl viologen (MV+*, E i = - 0.450 V vs NHE) by semi-reduced TC were monitored by following the growth of their respective one-electron reduced species (Swallow, 1982; Guha et al., 1987; Naik and Moorthy, 1990) at 760, 650 and 600nm respectively, both at pH 4 and 6.8. The semi-reduced species formed by e;; reaction with TC was found to transfer electron to these dyes at near diffusion controlled rates. The absorption spectra obtained by this process were similar to those obtained directly by the reaction of the dyes with eaq under identical conditions. These results, presented in

wit/r hydrogen .

r

a

0.020 0.016 d d a

0.012 0.008 0.004

I 380

I 420

I 460

I SO0

I 540

Wavelength

I 580

I 620

I

I

660

700

(nm)

Fig. 3. Absorption spectra of H-atom adduct obtained from N, saturated 1 x 10-j mol dm-’ TC containing 0.1 m t-butanol at pH 2, (a) 4 ps after the pulse, (b) 150 ps after the pulse, (c) 4~s after the pulse at pH 5.8.

511

Pulse radiolysis study of TC with H-atoms reacting partly by electron transfer reaction. Reaction of H-atoms with the neutral form of TC was studied by utilizing the reaction of e; with H,PO; to generate H atoms in the near neutral medium at pH 5.8 (Ye and Schuler, 1986). Under these conditions, the rate constant for the reaction of TC with H atoms was determined to be 8.9 x 105dm3mol-‘s-r. The spectrum of the species at pH 5.8 was found to be similar to that at pH 2 with I,,,,, at 450 and 630 nm [Fig. 3(c)] and t 4M= 2055 dm3 mol-’ s-l. This species also decayed by second-order kinetics. The signal at 630 nm was too weak for any meaningful quantitative analysis. H-atoms can react with organic molecules by different modes viz. addition to the double bond, abstraction of H-atom and one-electron reduction of the molecule. Therefore, the absorption spectrum may be due to more than one transient species. Our results indicate that one-electron reduction is not a major pathway for H-atom reaction with TC. They probably react via addition and/or abstraction reaction. In summary, TC reacts with e; at nearly diffusion controlled rates with the rate constant depending upon the state of protonation of TC. /I-Tricarbonyl group in the ring A appears to be the site of electron addition. The semi-reduced TC is a strong reducing species whose reduction potential lies in the range - 0.450 to - 1.40 V vs NHE. One-electron reduction is not a major pathway for H-atom reactions with TC. REFERENCES

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Spinks J. W. T. and Woods R. J. (1990) An Introduction io Radiation Chemistry, 3rd edn, p. 315. John Wiley, New York. Swallow A. J. (1982) Application of pulse radiolysis to the study of molecules of biological importance. In The Study of Fast Processes and Transienr species by Electron Pulse Rudiolysis (Edited by Baxendale J. H. and Busi F.),

p. 320. D. Reidel, Holland. Ye M. and Schuler R. H. (1986) The reaction of ei with H,PO; as a source of hydrogen atoms for pulse radiolysis studies in neutral solutions. Radiat. Phys. Chem. 28. 223.