Chemical Papers 65 (6) 909–912 (2011)DOI: 10.2478/s11696-011-0082-6

SHORT COMMUNICATION

2-Alkylsulphanyl-4-pyridinecarbothioamides – inhibitorsof oxygen evolution in freshwater alga ��������� ������

aKatarína Kráľová*, aFrantišek Šeršeň, bVěra Klimešová, bKarel Waisser

aInstitute of Chemistry, Faculty of Natural Sciences, Comenius University, SK-842 15 Bratislava, Slovakia

bDepartment of Inorganic and Organic Chemistry, Faculty of Pharmacy, Charles University,

CZ-501 65 Hradec Králové, Czech Republic

Received 7 June 2011; Revised 19 July 2011; Accepted 20 July 2011

The inhibition of the oxygen evolution rate (OER) in Chlorella vulgaris by 2-alkylsulphanyl-4-pyridinecarbothioamides (APCTs; alkyl = methyl up to hexadecyl) was studied. APCTs werefound to inhibit photosynthetic electron transport (PET) which resulted in the inhibition of OERin algae. The inhibitory activity of APCTs was highly dependent on the alkyl chain length of the2-alkylsulphanyl substituent and the corresponding dependence showed a bilinear course with thedecyl derivative as being the most active inhibitor. Using EPR spectroscopy, the site of APCT actionin the algal photosynthetic apparatus was determined. It was confirmed that APCT interactedmainly with the D. intermediate, i.e. with tyrosine radical (TyrD) occurring at the 161st positionin D2 protein which is situated on the donor side of photosystem 2.c© 2011 Institute of Chemistry, Slovak Academy of Sciences

Keywords: alkyl length, Chlorella vulgaris, EPR spectroscopy, photosynthetic electron transport,inhibition, oxygen electrode

Many substituted heterocyclic compounds, includ-ing pyridine derivatives, exhibit a wide spectrumof biological effects, e.g. antibacterial (Okazaki etal., 1997), herbicidal (Ren et al., 2010), neurotropic(Krauze et al., 1999), or protein kinase inhibition(Chioua et al., 2009). Some alkyl- and alkylsul-phanyl derivatives of 4-pyridinecarboxylic acid werefound to be biologically active compounds show-ing antifungal (Klimešová, 1996a; Waisser et al.,1996a), antimycobacterial (Waisser et al., 1996a), andphotosynthesis-inhibiting activities (Kráľová et al.,1997). The dependence of the antimycobacterial ac-tivity of 2-alkylsulphanyl-4-pyridinecarbothioamides(APCTs) against some mycobacterial strains on thelipophilicity of the alkyl substituent showed twomaxima of inhibitory activity, indicating the inter-action of both pharmacophore groups (the alkyl-sulphanyl and thioamide groups) with the biolog-ical subject (Waisser et al., 1996b). The antimy-cobacterial and antifungal activities of alkylsulphanyl

derivatives of pyridinecarbothioamides were foundto be associated with the ortho and para posi-tions of the substituent on the pyridine moiety,whereby APCTs showed a higher inhibitory effi-ciency than the corresponding 4-alkylsulphanyl-2-pyridinecarbothioamide isomers (Klimešová et al.,1996b; Waisser et al., 1996a, 1996b).Using electron paramagnetic resonance (EPR)

spectroscopy for observation of the inhibition ofphotosynthetic electron transport (PET) in spinachchloroplasts, it was found that APCT interacted pre-dominantly with the intermediate D. (TyrD) (Kráľováet al., 1997), whereas anilides of 2-alkyl-substituted 4-pyridinecarboxylic acid interacted with both Z. andD. intermediates which are situated in D1 and D2proteins on the donor-side of photosystem (PS) 2(Kráľová et al., 1998a).The present paper is aimed at investigating the in-

hibitory effect of APCTs on photosynthetic electrontransport in Chlorella vulgaris and at determining the

*Corresponding author, e-mail: kralova@fns.uniba.sk

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site of APCT action in the algal photosynthetic appa-ratus using EPR spectroscopy.The homologous series of the APCTs (alkyl =

methyl up to hexadecyl) studied were synthesisedby using a method described by Klimešová et al.(1996a). Chlorella vulgaris algae used in this studywere cultivated under static conditions (photoperiod16 h light/8 h dark, illumination 12 W m−2 at labo-ratory temperature ((24 ± 1)◦C, pH 7.2) by a methodby Kráľová et al. (1998b). The oxygen evolution rate(OER) in algal suspensions was measured at 24◦C byusing a Clark type electrode (SOPS 31 atp., Chemo-projekt, Prague, Czech Republic) according to Bartošet al. (1975). Illumination was with a 250 W halo-gen lamp (about 100 W m−2). The chlorophyll (Chl)content in the algal suspension was 8–10 mg dm−3.Prior to the OER measurements, the suspensions wereadapted in the dark for 4 h. Because of the low solubil-ity of APCTs in water, the compounds were dissolvedin dimethyl sulphoxide (DMSO). The DMSO concen-tration applied did not exceed 5 vol. % and it hadno effect on the biological activity of the algal chloro-plasts.The EPR spectra were recorded on an ERS 230

apparatus (ZWG AdW, Berlin, Germany) operatingin the X-band at 24◦C. The algal Chl concentrationwas 2.0 g dm−3, microwave power set at 5 mW and themodulation amplitude at 1 mT. The samples were illu-minated directly in the resonator cavity with a 250 Whalogen lamp and they were protected against warm-ing by a water filter (5 cm). The DMSO concentrationapplied did not exceed 10 vol. % and it did not affectthe EPR spectra of the control algal chloroplasts.A decrease in OER in the algal suspension of

Chlorella vulgaris in the presence of APCTs showedthat these compounds inhibited PET. The inhibitoryactivity of APCTs depended strongly on the alkylchain length of the 2-alkylsulphanyl substituent. Lin-ear increase in inhibitory activity with prolongationof the alkyl chain was observed for methyl- up to nor-mal decyl derivatives (IC50 = 1.024 mmol dm−3 formethyl derivative and 34.6 µmol dm−3 for normal de-cyl derivative, respectively). However, with a furtherincrease in the number of carbon atoms in the alkylsubstituent, the OER inhibition by APCTs showeda sharp linear decrease (IC50 = 87.2 µmol dm−3 fornormal dodecyl derivative and 328.8 µmol dm−3 fornormal hexadecyl derivative, respectively) (Fig. 1).It is evident from Fig. 1 that the dependence

of OER-inhibiting activity upon the number of car-bon atoms of the normal alkyl-substituent of APCTsdetermined in algal suspensions exhibits a bilinearcourse. On the other hand, the dependence of PET-inhibiting activity of APCT in spinach chloroplasts onthe lipophilicity of normal alkyl substituent showeda quasi-parabolic course, whereby the most effec-tive inhibitor was the octyl derivative (IC50 = 71.5µmol dm−3) (Kráľová et al., 1997). Such biological

0 4 8 12 16

3.0

3.5

4.0

4.5

log(

1/IC

50/(

mol

dm

–3))

n

Fig. 1. Dependence of IC50 (inhibition of oxygen evolutionrate in Chlorella vulgaris by 2-alkylsulphanyl-4-pyri-dinecarbothioamides) on the number of carbon atomsin the alkyl group.

activity on the alkyl chain length is prevalent for a ho-mologous series of membrane-active compounds and itis known as the “cut-off” effect (Balgavý & Devínsky,1996). The starting enhancement of PET-inhibitingactivity with increasing lipophilicity of APCT can beassociated with its increased concentration in the lipidphase of thylakoid membranes due to increasing thepartition coefficient of APCT between the lipid andaqueous phases. The low concentration of moleculeswith short alkyl-substituent in the lipid phase causesdamage to a relatively small number of photosyntheticproteins, hence the PET inhibition will be low. On theother hand, the molecules with long alkyl-substituent,which are characterised by a high partition coeffi-cient between the lipid and aqueous phases, cannotbe transported in sufficient concentrations through theaqueous regions of the thylakoid membranes and reachthe photosynthetic proteins incorporated in thylakoidmembranes. Consequently, the concentration of suchmolecules in the membrane as well as the resultingdamage to photosynthetic proteins will also be low.Therefore, the highest activity will be exhibited bycompounds with an alkyl chain of medium length al-lowing for a sufficient concentration of the inhibitor inthe thylakoid membranes.Quasi-parabolic or bilinear dependence of OER in-

hibition in Chlorella vulgaris on the total lipophilic-ity of the compound was also observed for anilidesof substituted pyridine-4-carboxylic acid (Kráľová etal., 2001), as well as for chromone-substituted ben-zothiazolium halides with alkyl substituent (Kráľováet al., 1998c); similar dependences were found for theinhibitory activity of alkyl-substituted benzothiazolederivatives related to the reduction of chlorophyll con-tent in freshwater algae Chlorella vulgaris (Kráľová etal., 1992, 1994).It is known that, as is the case with higher plants,

algae also exhibit stable EPR signals in the region of

K. Kráľová et al./Chemical Papers 65 (6) 909–912 (2011) 911

Fig. 2. EPR spectra of untreated algal suspension of Chlorellavulgaris (A) and suspension treated with 0.05 mol dm−3of 2-butylsulphanyl-4-pyridinecarbothioamide (B); thefull lines correspond to chloroplasts kept in the dark,the dashed lines to the illuminated chloroplasts; mod-ulation amplitude: 1.0 mT; Chl content: 2 g dm−3.

free radicals (g ∼ 2.0) (Hoff, 1979) which could berecorded by conventional continual wave EPR appa-ratus at laboratory temperature. These signals are as-sociated with photosystems PS 2 and PS 1. Signalsbelonging to PS 2 correspond to tyrosine radicals (de-noted as Z. and D. intermediates) which are situatedin D1 and D2 proteins on the donor side of PS 2. Thenext EPR signal is associated with the cation–radicalof chlorophyll α dimer situated in the core of PS 1.Due to the treatment of plant chloroplasts and algaewith PET inhibitors, the intensity and shape of theEPR signals referred to above could be changed andfrom these changes the site of action of the inhibitorsstudied can be determined. EPR spectroscopy was alsoused for determination of the site of APCT action inthe algal photosynthetic apparatus.Fig. 2A presents signals of the untreated suspen-

sion of Chlorella vulgaris. The signal recorded in thedark (full line) is so-called signal IIslow (g = 2.0046;∆Bpp = 2 mT) belonging to the intermediate D

., i.e.to the radical of tyrosine (TyrD) occurring at the 161stposition in D2 protein on the donor side of PS 2 (De-bus et al., 1988a; Svensson et al., 1991). The EPR sig-nal of the illuminated algal chloroplasts (dashed line)is superimposed from signal IIslow and signal IIvery fast(g = 2.0046; ∆Bpp = 2 mT) and signal I (g = 2.0026;∆Bpp = 0.7 mT). Signal IIvery fast represents approxi-mately the difference between the signals recorded inthe light and in the dark with a small contributionfrom signal I. Signal IIvery fast belongs to the interme-diate Z., i.e. to the radical of tyrosine (TyrZ) occurringat the 161st position in D1 protein on the donor sideof PS 2 (Debus et al., 1988b; Svensson et al., 1991).The intermediate Z. is an electron donor for the coreof PS 2 (P 680). The cation radical of chlorophyll adimer in the core of PS 1 (P 700) is responsible forsignal I (Hoff, 1979). This signal may be observed rela-tively well in the illuminated algal chloroplasts treatedwith APCT (Fig. 2B, dashed line). In the presenceof the normal butyl derivative, the intensity of bothEPR signals II (particularly that of signal IIslow) de-creases (Fig. 2B). Thus, it may be concluded that thecompound studied interacts with the D. intermedi-ate or its close surroundings, in a similar manner tothat found in spinach chloroplasts treated with APCT(Kráľová et al., 1997). Due to this interaction, PET be-tween PS 2 and PS 1 will be limited. Consequently, theintensity of signal I in the illuminated Chlorella vul-garis increases (Fig. 2B, dashed line), reflecting the de-creased photo-reduction of P 700+ (which is oxidisedby PS 1 acceptors in the light) by electrons comingfrom PS 2. It was found that similar effects were alsoexhibited by normal hexyl derivative, but only after24 h treatment. Consequently, it may be assumed thatthe penetration of this inhibitor through the algal thy-lakoid membrane is slower due to its lower solubilityin the aqueous phase.The results may be summarised as follows: APCTs

inhibit the oxygen evolution rate in the freshwateralga Chlorella vulgaris. The site of APCT action issituated on the donor side of PS 2, in the interme-diate D.. Interaction of APCT with the intermediateD. results in the PET interruption. The inhibitory effi-ciency of APCT is highly dependent on the alkyl chainlength of the alkylsulphanyl substituent; the most ef-fective compound is a decyl derivative. The inhibitoryeffect of APCT on PET is associated with their in-teraction with proteins occurring in the lipid regionsof algal photosynthetic membranes. Accordingly, thehighest inhibitory activity is exhibited by compoundshaving the most favourable lipophilicity for enablingsuch partitioning of the compound in the lipid andaqueous regions of biological membranes, which is in-dispensable for interactions with proteins occurring inthe polar and non-polar environments. On the other

912 K. Kráľová et al./Chemical Papers 65 (6) 909–912 (2011)

hand, the potential interaction of the thioamide groupwith residues of amino acids in proteins of the photo-synthetic apparatus should also be taken into consid-eration.

Acknowledgements. This study was supported by the SlovakScientific Grant Agency VEGA, grant No. 1/0612/11.

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