Fluorescence quenching of

Fluorescence quenching of ?uoroquinolone antibiotics by 4-hydroxy-TEMPO in aqueous solution

q

Krzysztof _Zamojc

′?,Wies?aw Wiczk,Bart?omiej Zaborowski,Mariusz Makowski,Joanna Pranczk,Dagmara Jacewicz,Lech Chmurzyn

′ski University of Gdansk,Faculty of Chemistry,Wita Stwosza 63,80-308Gdansk,Poland

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

●

a r t i c l e i n f o Article history:

Received 29November 2013

Received in revised form 11June 2014Accepted 21June 2014

Available online 28June 2014Keywords:

Fluoroquinolone antibiotics 4-Hydroxy-TEMPO

Fluorescence quenching Dynamic quenching

a b s t r a c t

The ?uorescence quenching of nor?oxacin,dano?oxacin,enro?oxacin and levo?oxacin,belonging to a group of ?uoroquinolone antibiotics,by 4-hydroxy-TEMPO was studied in aqueous solutions with the use of steady-state,time-resolved ?uorescence spectroscopy as well as UV–VIS absorption spectroscopy methods.In order to understand the mechanism of quenching the absorption and ?uorescence emission spectra of all ?uoroquinolone antibiotics studied as well as decreases of their ?uorescence were regis-tered as a function of the 4-hydroxy-TEMPO concentration.No deviations from a linearity in the Stern–Volmer plots (determined from both,steady-state and time-resolved measurements)were observed.The ?uorescence quenching mechanism was proved to be totally dynamic,what was addition-ally con?rmed by the registration of Stern–Volmer plots at 5temperatures ranging from 15to 55°C.On the basis of theoretical calculations of ?uoroquinolones’molecular radii and ionization potentials the mechanism of electron transfer was rejected.It seems that the ?uorescence quenching is diffusion-lim-ited and is caused by the increase of nonradiative processes,such as internal conversion or intersystem crossing.The Stern–Volmer quenching constants and bimolecular quenching constants were determined at the room temperature for all ?uoroquinolone antibiotics studied.

ó2014Elsevier B.V.All rights reserved.

Introduction

The ?uoroquinolones are antibiotics that play an important role in the antimicrobial therapy.Although the mechanism of action of all ?uoroquinolones is very similar,there are many

signi?cant dif-ferences in their antimicrobial spectrum of activity,pharmacoki-

https://www.360docs.net/doc/944996637.html,/10.1016/j.saa.2014.06.127

1386-1425/ó2014Elsevier B.V.All rights reserved.

q

Selected paper presented at XIIth International Conference on Molecular spectroscopy,Kraków –Bialka Tatrzanska,Poland,September 8–12,2013.?

Corresponding author.Tel.:+48(58)5235057;fax:+48(58)5235012.

E-mail address:krzysztof.zamojc@https://www.360docs.net/doc/944996637.html,.pl (K._Zamojc ′).

netic characteristics and safety pro?les.These compounds are gen-erally highly effective against aerobic Gram-negative and many Gram-positive bacteria.The?uoroquinolones are very helpful in the treatment of a wide range of infections,including uncompli-cated or complicated urinary tract infections,respiratory tract infections,bacterial gastroenteritis and soft tissue infections due to Gram-negative organisms.Many of the?uoroquinolone antibi-otics can be administered orally.As all of the?uoroquinolones have long half-lives they may be administered once or twice daily. They are widely distributed in the body,attaining therapeutic con-centrations in many tissues.On the other hand,these antibiotics have been proved to damage juvenile weight-bearing joints in animals.Thus?uoroquinolones should be used with caution, especially in children[1–4].

Fluorescence quenching attributes to any process that decreases the?uorescence intensity,lifetime or quantum yield of luminescent species as a result of the interactions with other chemical compounds.Although the studies on the?uorescence quenching(performed with steady state and transient methods) provide much information about?uorescent dyes and their quenchers[5–13],such a technique has not been widely studied for?uoroquinolones yet[14–16].

4-hydroxy-TEMPO(4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl)is a stable membrane permeable radical scavenger which effectively protects cells and tissues from damages caused by oxi-dative and nitrosative stress conditions[17].It has been proven that4-hydroxy-TEMPO acts also as a superoxide dismutase mimic [18].

In the present study we have used a UV–VIS absorption method along with steady-state and time-resolved?uorescence measure-ments to investigate the?uorescence quenching of four?uoro-quinolone antibiotics by4-hydroxy-TEMPO in aqueous solutions with a view to understand the mechanism of quenching involved in that system.

Materials and methods

The?uoroquinolone antibiotics(nor?oxacin,dano?oxacin, enro?oxacin and levo?oxacin)and4-hydroxy-TEMPO were pur-chased from Sigma Aldrich.The molecular structures of4-hydroxy-TEMPO and?uoroquinolones studied are presented in Fig.1.To avoid the self-quenching the solutions of all?uoroquino-lones in deionized water were prepared keeping the concentration constant(1á10à5M).The concentration of the stock solution of4-hydroxy-TEMPO was0.25M.

Absorption spectra were recorded on Perkin Elmer Lambda650 UV–VIS spectrophotometer at the temperature25°C(a slit–2nm).Fluorescence emission spectra were registered with the use of Cary Eclipse Varian spectro?uorimeter at the temperature 25°C(excitation and emission slits–5nm).Both,absorption and emission spectra of all?uoroquinolones(1.9mL,1á10à5M)were recorded in the absence of4-hydroxy-TEMPO and in the presence of its different concentrations in the range of0.0013–0.0125M.In case of emission spectra the solutions of nor?oxacin,dano?oxacin and levo?oxacin were excited at340nm,while the excitation wavelength for the solution of enro?oxacin was set to320nm.

Time-resolved?uorescence measurements were done with the use of Edinburgh CD-900spectro?uorimeter at the room tempera-ture.The?uorescence lifetimes of all?uoroquinolones studied (2.5mL,1á10à5M)were measured using the single photon counting technique in the absence of4-hydroxy-TEMPO and in the presence of its different concentrations in the range of 0.0020–0.0078M.In all the cases the excitation wavelength was set at340nm with the use of Nano Led diode from IBH company. After each addition of4-hydroxy-TEMPO the solution was gently stirred and the?uorescence lifetime measured.The emission wavelength was chosen by monochromator and measured at the wavelength corresponding to the maximum of each?uoroquino-lone emission.

The decreases in?uorescence intensities of all?uoroquinolones after an addition of4-hydroxy-TEMPO were observed at following temperatures:15,25,35,45and55°C.These experiments were performed analogously as the time-resolved?uorescence mea-surements.The?uorescence of each antibiotic solution(2.5mL, 1á10à5M)was measured in the absence of4-hydroxy-TEMPO as well as in the presence of different quencher’s concentrations in the range of0.0020–0.0096M.After each addition of4-hydroxy-TEMPO the solution was gently stirred and the?uorescence inten-sity at the wavelength corresponding to the maximum of each ?uoroquinolone emission was measured.

The semiepirical AM1(Austin Model1)quantum chemistry method[19–21]was applied for the theoretical calculations of the ionization potentials and molecular radii of a series of?uoro-quinolone antibiotics.The AM1quantum-chemical calculations were performed using the MOPAC2012(version12.239L)program package[22,23].Furthermore,the molecular radii were calculated after the preliminary geometry optimization performed by the MMFF94force?eld using Avogadro(version1.1.0)Cross-Platform Computer Program for Building Molecules and Visualizing Struc-ture and Analysis[24].

Results

UV absorption and?uorescence emission spectra of all?uoro-quinolone antibiotics studied were registered in the absence and the presence of the increasing concentrations of4-hydroxy-TEMPO.These spectra are presented in Fig.2on an example of enro?oxacin.

Based on recorded?uorescence spectra the following observa-tions(the same for all?uoroquinolones studied)were made:(a) the higher the concentration of4-hydroxy-TEMPO the lower the ?uorescence intensities;(b)no changes in the shape and the shift of the most long-waved bands in absorption and?uorescence spectra.An increase of absorbance in the range of200–275nm is caused by the addition of4-hydroxy-TEMPO;(c)no changes in the?uorescence intensities in the presence of4-hydroxy-TEMPO were observed.All these?ndings prove that the permanent photo-chemistry has no in?uence on the quenching process and that the type of interactions between?uoroquinolone antibiotics and 4-hydroxy-TEMPO is entirely physical.Furthermore,they indicate that an emissive exciplex is not formed.

In order to understand the nature of quenching of all?uoro-quinolone antibiotics studied,the Stern–Volmer equation was ana-lyzed with the use of a time-resolved?uorescence spectroscopy. The?uorescence lifetimes of each?uorophore were measured in the absence and presence of4-hydroxy-TEMPO at different wave-lengths,corresponding to the maximum of its emission(enro?ox-acin:416nm;dano?oxacin:436nm;levo?oxacin:460nm; nor?oxacin:415nm).The Stern–Volmer plots for the?uorescence quenching of?uoroquinolones studied by4-hydroxy-TEMPO in solutions are presented in Fig.3.

As it can be observed from Fig.3the?uorescence quenching shows almost perfectly a linear dependence in the investigated range of4-hydroxy-TEMPO concentrations.It proves that there is no simultaneously present dynamic and static quenching.Further-more,in case of all?uoroquinolones studied,an addition of 4-hydroxy-TEMPO decreases their?uorescence lifetime,what indi-cates that the collisional quenching occurs.Additionally,to con?rm the dynamic mechanism of?uorescence quenching,the in?uence of temperature on Stern–Volmer constants determined on the

888K._Zamojc′et al./Spectrochimica Acta Part A:Molecular and Biomolecular Spectroscopy133(2014)887–891

basis of steady-state measurements was established.The Stern–Volmer plots for the steady-state?uorescence quenching

?uoroquinolone antibiotics by4-hydroxy-TEMPO in aqueous solutions at?ve temperatures(15,25,35,45and55°C)are sented in Fig.4on an example of enro?oxacin.As it can observed from Fig.4,the higher the temperature,the higher slope of these plots,what is characteristic for the dynamic quench-For all?uoroquinolone antibiotics studied the value of obtained from steady-state measurements is higher(in case enro?oxacin approximately two times)than the value of obtained from time-resolved studies.After the correction of rescence intensities for inner?lter effects the F0/F values decreased signi?cantly and are equal to s0/s values in the range of experi-mental error(data not shown).It proves that the absorption some light by the quencher is the main reason of such observation.

In Table1there are gathered the results of time-resolved?uo-rescence quenching studies.Fluorescence lifetimes measured for

Fig.2.Absorption(A)and?uorescence emission(B)spectra of enro?oxacin in presence of increasing concentrations of4-hydroxy-TEMPO(0–0.0125M).

3.Stern–Volmer plots for the?uorescence quenching of?uoroquinolone antibiotics studied by4-hydroxy-TEMPO in aqueous solutions at the temperature.

K._Zamojc′et al./Spectrochimica Acta Part A:Molecular and Biomolecular Spectroscopy133(2014)887–891889

levo?oxacin is in a reasonable agreement with literature data[25]. For other?uoroquinolones no data about lifetimes were found.In order to better understand the mechanism of quenching ionization potentials of all?uoroquinolones studied as well as their molecular radii were determined with the use of theoretical calculations.The results of these studies are collected in Table2.

As it can be observed from Tables1and2the highest bimolec-ular quenching constant was obtained for enro?oxacin which has the highest molecular radius as well as relatively high ionization potential from all antibiotics studies.It proves that the mechanism of electron transfer may be rejected.Furthermore,obtained bimo-lecular quenching constants are of the same order of magnitude as diffusion-controlled value[26](data not shown).It is highly prob-able that the?uorescence quenching process is diffusion-limited caused by the increase of rate constants of nonradiative transitions, such as internal conversion or intersystem crossing.

Conclusions

In this paper the?uorescence quenching of nor?oxacin,dano-?oxacin,enro?oxacin and levo?oxacin by4-hydroxy-TEMPO was investigated.The results show that the?uorescence of all?uoro-quinolones studied is sensitive to the presence of4-hydroxy-TEMPO and these compounds are quenched very effectively.There are no positive deviations in Stern–Volmer plots at high quencher concentrations,indicating the in?uence of only one(collisional) quenching mechanism.Studies performed with the use of steady-state and time-resolved?uorescence measurements along with theoretical calculations let to come to a conclusion that the ?uorescence quenching is diffusion-limited caused by the increase of nonradiative processes,such as internal conversion or intersys-tem crossing.The biological signi?cance of this work is proven by the fact that4-hydroxy-TEMPO is used as the different radicals’scavenger.Its detection under physiological conditions might help to understand the mechanism of an oxidative stress.As there are clearly noticeable differences in the?uorescence quenching of?u-oroquinolones studied,4-hydroxy-TEMPO may be applied for the distinction of these antibiotics.

Acknowledgements

This work was supported by the Polish National Science Center (NCN)under the Grant No.2012/07/B/ST5/00753and by grant for Young Scientists2014from University of Gdansk(538-8232-B342-14).

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Nor?oxacin 5.810.9603 1.29 4.50á109

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