Modulation of ESPT Dynamics in a Model Lactim-Lactam Tautomeric

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C: Physical Processes in Nanomaterials and Nanostructures

Modulation of ESPT Dynamics in a Model Lactim-Lactam Tautomeric System by Anisotropic Gold Nanoparticles Debarati Ray, Arghyadeep Bhattacharyya, Subhash Chandra Bhattacharya, and Nikhil Guchhait J. Phys. Chem. C, Just Accepted Manuscript • DOI: 10.1021/acs.jpcc.8b05481 • Publication Date (Web): 02 Jul 2018 Downloaded from http://pubs.acs.org on July 3, 2018

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The Journal of Physical Chemistry

Modulation of ESPT Dynamics in a Model Lactim-Lactam Tautomeric System by Anisotropic Gold Nanoparticles Debarati Raya, Arghyadeep Bhattacharyyab, Subhash Chandra Bhattacharyaa*, Nikhil Guchhaitb* a

Department of Chemistry, Jadavpur University,188, Raja S. C. Mallick Road, Kolkata-700032,

India b

Department of Chemistry, University of Calcutta, 92 A. P. C. Road, Kolkata-700009, India

*To

whom

correspondence

should

be

addressed.

E-mail:

[email protected],

[email protected] (S.C.B.) Tel.: +91-33-2350-8386. Fax: +91-33-2351-9755. E-mail: [email protected] (N.G.).

ACS Paragon Plus Environment

The Journal of Physical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Abstract The past few decades have witnessed significant advances in the development of functionalized gold nanoparticles for diverse applications in various fields such as chemistry, biology, pharmacy and physics. But a recent in vivo toxicity study of GNPs in Drosophila melanogaster demonstrated GNPs are capable of inducing mutagenesis. Considering mutagenicity and tautomerism (lactim-lactam and amine-imine) of DNA base pair are correlated to each other, a recent theoretical study reveal the modification of double proton transfer (SDPT) process of guanine-cytosine (GC) DNA base pairs upon interaction with gold surface (Au(111)). But there is dearth of experimental data about the PT process in isolated base pairs. In this particular work we have chosen a model lactim-lactam tautomeric system namely 1-(2-hydroxy-5-chlorophenyl)-3,5-dioxo-1H-imidazo-[3,4-b] isoindole (ADCL) and carried out for the first time a comprehensive study of the modification of excited state proton transfer (ESPT) dynamics in presence of synthesized isotropic (spherical) and anisotropic (trianglular, rod and trigonal bipyramidal) gold nanoparticles (GNPs) by means of steady state and time resolved fluorescence spectroscopy. The PT process operative in ADCL was experimentally found to be accelerated on anisotropic gold nanosurface whereas spherical shaped GNPs showed little impact on the dynamics of the above mentioned photo-physical phenomenon. Therefore our experimental results regarding the modification of lactim-lactam tautomerism of a model compound on gold nanosurface is an indirect evidence of mutation caused by GNPs.


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1. Introduction Over the past few years there have been many studies devoted to the application of functionalized nanomaterials in biochemistry, microbiology, immunology, cytology, plant physiology, morphology, etc.1-8 Among the various nanomaterials, gold nanoparticles (GNPs) have been widely used in modern medical and biological sciences; in particular, it comprises genomics, biosensors, immune analysis, clinical chemistry, detection and photothermolysis of microorganisms and cancer cells; the targeted delivery of drugs, DNA and antigens; optical bioimaging and monitoring of cells and tissues using modern registration systems etc.1-8 The growing interest in colloidal gold nanoparticles in the various fields lends a progressive impetus to the development of their novel preparation methods for different shapes, sizes with surface modification.9-14 However, before exploring the wide potentiality of GNPs it is necessary to assess their effects upon interaction with living systems. Recently, in vivo toxicity study of GNPs in Drosophila melanogaster by Vecchio et al. revealed a phenotype modification in the subsequent generations of Drosophila which lent them to conclude that the GNPs are capable of inducing mutagenesis.15 It is proposed that the tautomeric equilibrium i.e., lactim (enol)-lactam (keto) tautomerism (in thymine and guanine) and amine-imine tautomerism (in adenine and cytosine) occurring in the nitrogen bases of DNA is one of the main reason behind spontaneous mutation.16 Inspired by the aforesaid work by Vecchio et al, Freitas et al. have investigated the impact of gold surface (Au(111)) on the simultaneous double proton transfer (SDPT) process of guanine-cytosine (GC) DNA base pairs by means of first principle computational simulations.17 They observed significant differences in the electronic structure, energetics and kinetics of the tautomeric process of the GC pair during the contact with an inert gold surface which can influence the SDPT mechanism.17 They have also determined a reduction of about 31 % in the



activation energy barrier of the proton transfer (PT) process in GC/Au (111) system which points towards the faster PT process on the gold surface. However, there is dearth of experimental data about the PT process in isolated base pairs till date. In this particular work we have strategically chosen an imidazole based hetero cyclic molecule namely,1-(2-hydroxy-5-chloro-phenyl)-3,5-dioxo-1H-imidazo-[3,4-b]isoindole(ADCL) where lactim-lactam tautomerism happens

18

and carried out for the first time a comprehensive

study of the modification of excited state proton transfer (ESIPT) dynamics in presence of synthesized isotropic (spherical) and anisotropic (trianglular, rod and trigonal bipyramidal) gold nanoparticles (GNP) by means of steady state and time resolved fluorescence spectroscopy. The very molecule ADCL contains a four member intramolecular hydrogen bonding network which closely resembles the basic building block of DNA base, thymine and guanine. From the previous photophysical study of ADCL in various solvents reveal that the molecule remains both in lactim (enol) and lactam (keto) form in the ground state and upon photo excitation of the lactim form it exhibits dual emissions attributed to the excited normal (or lactim emission, N*) and a tautomer (or, lactam emission, T*) form that originates from an ESIPT (N*→T*, Scheme 1) reaction. 18 The ESIPT rate of this molecule is quite slower in organic solvents in comparison to the other PT system due to the high lactim*→lactam* energy barrier through the four member intramolecular hydrogen bonding unit.18 Therefore any modification in the tautomeric energy barrier in presence of the GNPs must influence the dynamics of the PT process. Recently, Kyrychenko et al. have studied the interaction of 3-hydroxychromone (an ESIPT probe) with dodecanethiol protected gold nanoparticle (spherical shaped) in order to understand the extent of hydrophobicity of the surface of the nanoparticle but they concluded nothing about the modification of PT dynamics of the probe.19 Nag et al. have also investigated the effect of


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nanopores in porous gold nanoparticles (Au NPs) on excited-state double proton transfer (DPT) in [2,2′-bipyridyl]-3,3′-diol (BP(OH)2) in an aqueous environment but their main focus is the understanding of the mechanism of the DPT process.

20

Till date there is no such experimental

evidence present regarding the influence of GNPs on the kinetics of the PT process of any molecule. On the other hand the title compound has biological interest also because this kind of molecule is widely used as drug template in medicinal chemistry. 21,22 As GNPs are widely used as drug carrier, the interaction of ADCL with various kind of gold nanoparticle is an essential task for the future application in medicinal or biological chemistry. 2. Experimental Section 2.1. Materials The title compound 1-(2-hydroxy-5-chloro-phenyl)-3,5-dioxo-1H-imidazo-[3,4-b] isoindole (ADCL) was synthesized according to the reported literature procedure.

22

The structure of

ADCL is presented in Scheme 1. Tetrachloro aurate hydrate(HAuCl4.3H2O), Cetyltrimethyl ammonium bromide (CTAB) were purchased from Sigma-Aldrich, and the other chemicals like Trisodium citrate, Sodium borohydride dehydrate (NaBH4), ascorbic acid, concentrated hydrochloric acid (HCl), silver nitrate (AgNO3) were purchased from Emarck, Germany, and were used as received. The water used was treated in a Milli-Q water purification system. The spectroscopic grade solvents acetonitrile (ACN) and methanol (MeOH) were purchased from Spectrochem (India) and were used after proper distillation whenever required. 2.2. Synthesis of different sized and shaped gold nanoparticles. (a) Spherical Shaped Gold nanoparticles:



Spherical shaped GNPs were synthesized through normal citrate reduction method. 19 mL of aqueous 0.526 mM HAuCl4.3H2O( Hydrogen tetrachloroaurate (III) trihydrate) was brought to boil at ~ 65-700C while stirring, and then 1.0 mL of 30 mM aqueous tri-sodium citrate (TSC) was added. The boiling and stirring was continued for 25 min. (b) Synthesis procedure of Gold nanorods: i) Preparation of Seed solution: The seed solution was prepared according to the literature method9: 5 ml aqueous solution of 0.5 mM HAuCl4 was mixed with 5 mL of 200 mM CTAB solution and the mixture was stirred for 2 mins to obtain an amber coloured solution. Then, 600 µL ice cold aqueous solution of NaBH4 (10 mM) was added and the mixture was stirred vigorously and was allowed to stand for few minutes and then used for the subsequent synthesis of gold nanorods. ii) Preparation of Gold nanorods For the preparation of the growth solution 5 ml 1 mM HAuCl4, and 5 ml 200 mM CTAB were mixed with proper stirring. To the mixture 350 µL AgNO3 and 90 µL ascorbic acid were added to obtain a colourless solution. Then 18 µL of seed solution was injected to the growth solution and left for 24 hrs for gold nano rod at room temperature. (c) Synthesis of Gold Nanotriangles 7.5 ml 1.7 mM trisodium citrate was refluxed at 1000C with stirring. 5 ml aqueous solution containing 1.25 mM HAuCl4 and 7.5 mM CTAB were heated to 50 0C and then injected to the hot trisodium citrate. After 5 mins the flask was separated from heating mantle and cooled to room temperature11. (d) Synthesis of Gold trigonalbipyramids


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The trigonal bipyramids were grown in a solution containing 10 ml 0.1 M CTAB, 0.5 ml 0.01 M HAuCl4, 0.1 ml of 0.01 M AgNO3, 0.2 ml 1 N HCl, 0.08 ml of 0.1 M ascorbic acid and 120 µL seed solution. The solution was gently stirred at 400 rpm, 30 0C for 2 hrs10.

Lactim form Cl

Cl

H

H

O

O

N N

N N

O

H O

H

O

O

Open form

Closed form

Lactam form Cl

H O H N N

O

O

Scheme 1. Schematic Presentation of ADCL Molecule (Both Lactim and Lactam Form). 2.2. Preparation of nanoparticle solutions Four different synthesized nanoparticles (spherical, trianglular, rod and TBP shaped) reaction mixture were centrifuged at 7000 rpm for 20 min and washed with millipore water and the washing process were performed thrice. Then the resulted solids (nanoparticles) were repeatedly washed with MeOH solvent twice to remove water. After that MeOH solvent was added in the dried nanoparticle and redispersed in methanolic medium by ultrasound sonification. The same



procedure was followed in case of nanoparticle solution in ACN medium. But it is found that the triangular shaped nanoparticles were extremely unstable in ACN medium as it cannot be redispersed in this medium. Therefore in our present study we have used four different nanoparticle solutions (spherical, triangular, rod and TBP shaped) in MeOH medium and three different nanoparticle solutions (spherical, rod and TBP shaped) in ACN medium. The concentration of gold nanorod and spherical shaped nanoparticles are calculated by considering the available molar extinction coefficient value of the corresponding wavelength 23,24 whereas the concentration of GNP of triangle and TBP shaped in a typical solution were estimated by presuming that all of the HAuCl4 used in the preparation were converted to nanoparticle of exactly the same size. 2.3. Instrumentation and procedure Steady-State Spectral Measurements.

The steady state UV-vis absorption spectra and

fluorescence emission spectra were recorded in a Hitachi UV-Vis U-3501 spectrophotometer. and Perkin-Elmer LS-55 fluorimeter, respectively. For spectral measurement only freshly prepared solutions are used and every time proper background corrections have been done. All experiments were carried out at room temperature (303 K). Time-Resolved Fluorescence Decay. Fluorescence lifetimes of all solutions were carried out with a Horiba Jobin Yvon Fluorocube-01-NL time correlated single photon counting (TCSPC) setup. A nano LED operating at λex = 340 nm with a repetition rate of 1 MHz is used as the excitation source. The width of the instrument function, which was limited by the fwhm of the excitation source, was 1 ns. The lamp profile was recorded by placing a scatterer in place of the sample. The signals were collected at the magic angle of 54.7ο. The decays were analyzed using


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DAS-6 decay analysis software and the acceptability of the fits was judged by χ2 criteria. The time-resolved fluorescence decay (I(t)) is described by the following expression 25:

I (t ) = ∑α i exp(−t / τ i )

(1a)

i

Where τi is the characteristic decay time constant of the ith component with relative amplitude of αi. For the time-resolved anisotropy measurement, the fluorescence decay curves were recorded at vertical and horizontal positions of the polarizer and analyzed by the following equations 25:

I II (t ) = I (t )

[1 + 2r (t )] 3

(2a)

I ⊥ (t ) = I (t )

[1 − r (t )] 3

(2b)

Where, III and I┴ are the intensities collected at emission polarizations parallel and perpendicular, respectively, to the polarization axis of the excitation beam. I(t) and r(t) are the time-dependent intensity (at the magic angle) and anisotropy decay, respectively. Thus, the anisotropy decay function r(t) has been constructed from these I||(t) and I⊥(t) decays 25:

r (t ) =

I II (t ) − G ⋅ I ⊥ (t ) I II (t ) + 2 ⋅ G ⋅ I ⊥ (t )

(3)

G is the correction factor for the detector sensitivity to the polarization detection of the emission and it is defined by



G=

I ⊥ (t ) I II (t )

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(3a)

Transmission electron microscopy The transmission electron microscopy (TEM) images were obtained using a JEOL-TEM-2010 transmission electron microscope with an operating voltage of 200 kV. Samples for microscopic investigations were prepared by evaporation of MeOH of its dilute nanoparticle solution on the surface of carbon films.

3. Results and Discussions 3.1. Characterizations of synthesized GNPs 3.1.1. UV-Vis study The gold nanoparticles of different shapes (spherical, triangular, rod and trigonalbipyramid) were synthesized according to the available literature procedures

9-12

and the formations of isotropic

and anisotropic GNPs were confirmed from UV-visible spectroscopic analysis (Figure 1). The plasmon band of spherical shaped GNPs is centered at ~ 510 nm and triangular nanoplates show a strong surface plasmon resonance band at ~ 565 nm and a broad but weak band in the nearinfrared (near-IR) region. In case of rod shaped gold nanoparticle characteristics transverse and longitudinal plasmon peaks appear at ~ 600 nm and ~715 nm, respectively. The extinction spectrum of TBP shaped GNPs shows absorption maxima at ~ 550 nm (transverse peak) and ~ 827nm (longitudinal peak). These results are in good agreement with the available literature reports.9-12


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Norm. Absorbance (O.D.)

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1.2

Rod Spherical TBP Triangular

0.8 0.4 0.0

600 800 1000 Wavelength (nm)

Figure 1: Absorption spectra of spherical, rod, triangular and TBP shaped gold nanoparticles in MeOH medium

3.1.2. Transmission electron microscopy (TEM) study We have also performed transmission electron microscopy (TEM) for further confirmation regarding the shape and the size of the synthesized nanoparticles. Figure 2 represents the TEM images of synthesized spherical gold nanoparticles, nanorods, nano triangular, and nanotrigonal bipyramids. Figure 2 (a) and (b) represent spherical and triangular shaped gold nanoparticles of size range 2–4 nm and 60-65 nm, respectively. Gold nanorods of size (longitudinal length) in the range of 20-22 nm are given in Figure 2(c). Figure 2(d) represents trigonal bipyramidal shaped GNPs.



Figure 2: TEM images of (a) spherical (b) triangular, (c) rod and (d) TBP shaped gold nanoparticles.

3.2. Interaction of ADCL with synthesized GNPs 3.2.1. Steady State Absorption and Emission Spectroscopic Studies The absorption profile of ADCL in MeOH and ACN shows two bands centered at λabs∼ 320 nm and ∼400 nm, characteristic absorption maxima of the lactim and lactam form of ADCL, respectively.

18

In presence of gold nanoparticles (both isotropic and anisotropic) in methanolic

and acetonitrilic media no change in the absorption profile of ADCL is observed (figure not given).


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The emission profile of ADCL in MeOH and ACN is characterized by a large Stokes’ shifted, broad band at λem∼ 490 nm upon excitation at λex∼ 320nm, which is attributed to the

Figure 3: Steady-state fluorescence (λex= 320 nm) quenching profiles of ADCL (6 µM) in presence of (a) spherical where (i)→(xii) [GNP] = 0, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20, 22.5, 25 and 30 µM, (b) triangular where (i)→(xi) [GNP] = 0, 3, 5, 8, 11, 13, 15, 18, 21, 24 and 27 µM (c) rod where (i)→(xi) [GNP] = 0, 2, 4, 6, 8,10, 12, 15, 17, 20, 22 and 25 µM and (d) TBP where (i)→(xi) [GNP] = 0, 2, 4, 6, 8,10, 12, 15, 17, 20, 22 and 25 µM shaped GNPs in MeOH medium. phototautomer (lactam form) of ADCL due to the operation of ESIPT phenomenon (lactim*→lactam*).18 In presence of various shape of gold nanoparticles (spherical, triangle , rod and TBP) in MeOH medium the fluorescence intensity of ADCL is found to be quenched without any shifting of emission maxima (Figure 3). Similar results were also obtained in ACN



medium (figure not given). Generally quenching of emission intensity of any probe upon interaction with the metal nanoparticles is a result of nanometal surface energy transfer (NSET) mechanism from molecular donor to metal nanomaterial (acceptor).26-31 Basically NSET is a fluorescence resonance energy transfer (FRET) type process which originates from the interaction of the electromagnetic field of the donor dipole with the conduction band's delocalized electrons of the acceptor.26-31 The fundamental difference is that, unlike FRET, NSET does not require resonant electronic transitions.26-31 FRET is used as a powerful optical tool for the determination of the distances between donor and acceptor, upto only 80 Å, mostly when they are covalently labeled at the two specific sites of a rigid biological macromolecule whereas NSET study allows determining the distance nearly twice than that obtained from FRET mechanism.26-31 The necessary condition for the operation of this long range energy transfer process is that there should be an considerable amount of overlapping between the absorption profile of the acceptor (here, gold nanoparticle) and the emission profile of the donor (here, ADCL).26-31 It is quite evident from the emission profile of ADCL that apart from the spherical shaped gold nanoparticles other shaped nanoparticles would not have considerable amount of overlapping, which was very helpful to understand long range energy transfer. Therefore, with this analysis except spherical GNPs we can negate the possibility of operation of NSET mechanism between anisotropic gold nanoparticles and ADCL molecule for the quenching of the


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Figure 4: Stern-Volmer plots for the fluorescence quenching of ADCL in presence GNPs of varying shape. emission of the molecule. From the fluorescence intensity data of ADCL in presence of varying concentration of GNPs of different shapes we have determined the Stern−Volmer quenching constant (KSV) using the following Stern−Volmer relationship25 between the fluorescence intensity and concentration of metal nanoparticles as quencher:

(4)

1

where, F0 and F are the steady state fluorescence intensity in the absence and presence of quencher, respectively, and [Q] is the concentration of quencher i.e., GNPs. The calculated Stern-Volmer quenching constants from the Figure 4 in presence of spherical, triangular, rod and TBP shaped GNPs are 22.95 µM-1, 14.62 µM-1 , 89.27 µM-1 and 51.56 µM-1 , respectively at 303 K. The nature of quenching mechanism i.e., static or dynamic of ADCL with anisotropic GNPs can be confirmly obtained from the time resolved fluorescence study 25 that will be discussed in the following section. One question may arise that whether the modification of the fluorescence intensity of ADCL in presence of GNP is due to the GNP surface stabilizing agent, CTAB (for anisotropic) and citrate



ion (isotropic) or the gold nanosurface itself. In our previous work32 we have studied the modulation of lactim–lactam tautomeric process of an isoindole fused imidazole system namely 1-(2-hydroxy-5-methyl-phenyl)-3,5- dioxo-1H-imidazo-[3,4-b] isoindole (ADII) in three different micellar assemblies, SDS, CTAB and TX 100. We have observed that upon interaction with CTAB in aqueous medium, the lactim emission at ~ 430 nm of ADII dramatically increases whereas the lactam emission quenches up to a certain concentration of CTAB (< 0.2 mM) and then again rises at higher concentration (> 1.0 mM) of CTAB. Basically ADII and ADCL (our present system) are structurally similar with a little dissimilarity in the phenolic unit therefore similar type of results as obtained in case of ADII is also observed in case of ADCL molecule. But in our present work, we found no such increase of lactim emission (vide Figure 3) in presence of anisotropic GNPs. On the other hand, the emission profile of ADCL is practically unchanged in presence of tri sodium citrate (figure not given). Therefore, we can conclude the coated CTAB molecule on the surface of anisotropic GNPs and the citrate anion of isotropic GNPs do not affect the ESPT operation of ADCL.

3.2.2. Time Resolved Fluorescence Study Time resolved fluorescence study is believed to be the most efficient technique to understand various excited state processes.25 When the molecule ADCL in ACN was excited at 340 nm and λem is monitored at 385 nm (lactim emission) the resulting decays are well fitted by a sum of triexponential decay components i.e.; 0.315 ns (τ1, 59 %), 6.53 ns (τ2, 12%) and 1.82 ns (τ3, 29%). On the other hand when lactam emission maxima at 490 nm (T* or lactam emission) have been monitored by 340 nm excitation, the tautomeric emission consists of an initial fast growth features of time constant 0.401 ns (29%) followed by decay component of 6.99 ns (71%) (Table 1) (18). It is found that the fast decay component (τ1) of the normal emission is identical, within


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6000

6000

IRF in MeOH in presence of spherical GNPs

(a)

Counts

Counts

4000

2000

0

6000

0

2

4

6

8

Time (ns)

0

2

4

Time (ns)

6

8

IRF in MeOH in presence of TBP GNPs

(d)

4000

Counts

4000

2000

0

2000

6000

IRF in MeOH in presence of rod GNPs

(c)

IRF in MeOH in presence of triangle GNPs

(b)

4000

0

10

Counts

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


2000

0

2

4

Time (ns)

6

8

0

0

2

4

Time (ns)

6

8

Figure 5: Typical time-resolved fluorescence decay profile of ADCL in presence of different shaped GNPs in MeOH at λex = 336 nm and λmon = 500 nm. experimental error, to the rise time of the tautomer emission, demonstrating that ESIPT dynamics must connect these two species. Therefore the growth component appeared in the lactam emission corresponds to the time constant of the ESIPT (τPT) reaction of ADCL in ACN and we have assigned the long lived components (τ2) is the excited state lifetime of the lactam form and the other two short lived components obtained in the lactim emission, τ1 and τ3, are the excited state lifetime of the closed and the open form of the lactim form, respectively.18 Similar result was also obtained in case ADCL in MeOH but the ESIPT time constant is found to be quite faster (~ 0. 307 ns) than that obtained in ACN (~ 0.401 ns) which is probably due to the involvement of the molecule in hydrogen bonding interaction with the solvent molecules (MeOH).



Figure 5 and Figure 6 present the time resolved decay profile of ADCL (at λex = 340 nm and λem= 500 nm) in presence of gold nanoparticles of different shapes in MeOH and ACN media, respectively and the corresponding data are summarized in (Table 1). The time resolved data

6000

Counts

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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IRF ADCL Rod Spherical TBP

4000

2000

0 0

2

4

6

8

10

Time (ns) Figure 6: Typical time-resolved fluorescence decay profile of ADCL in presence of different shaped GNPs in ACN media at λex = 336 nm and λmon = 500 nm. clearly shows significant modification of ESIPT time constant of ADCL molecule in presence of anisotropic gold nanoparticle (rod, triangular and TBP) in both the organic media. In presence of triangular (only in methanol), rod and TBP shaped gold nanoparticle the ESIPT time constant (τPT) of the molecule decreases but the τPT value is practically unchanged in presence of spherical nanoparticles in both organic media. The decreasing time constant of the ESIPT reaction is a clear indication of the faster proton transfer dynamics of the molecule. In a theoretical study by Freitus et al. suggested that the adsorption of a molecule (here, ADCL) on the surface of any abiogenic minerals (here, gold nanosurface) is governed by the weak van der Waals interaction between the adsorbed molecule and surface atom of the minerals which induces some changes in the electronic structure of the adsorbate.17 Considering the weak van der Waals interaction and SDPT mechanism they obtained a high reduction (of around 31%) in the activation energy barrier of GC→G*C* tautomeric process which actually trigger the occurrence of a spontaneous


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point mutation during DNA replication.17 Taking into account the above mentioned fact we can conclude that in our case also the high activation energy barrier of the lactim*→lactam* tautomeric process through four member intramolecular hydrogen bonding unit of ADCL also decreases upon adsorption on the anisotropic gold surface by weak van der Waals interaction which results the faster ESIPT dynamics of the probe. On the other hand in presence of spherical shaped gold nanoparticle in both organic media no change in fluorescence decays of ADCL is observed and from the steady state fluorescence study we found a quenching of emission maxima. Similar results were also observed by Kyrychenko et al. during their study of interaction between 3-hydroxychromone (an ESIPT probe) with thiol coated spherical gold nanoparticle.19 As in the earlier section we have mentioned about the possibility of NSET mechanism between ADCL and spherical gold nanoparticle but from the fluorescence decays of ADCL in presence of gold nanoparticle no change in lifetime of the molecule is found out. This results completely negates the possibility of NSET mechanism between these two pair as NSET is an excited state process. Therefore, we can conclude that the interaction between ADCL and spherical gold nanoparticle is of ground state type i.e. static quenching mechanism is operative between them. The nature of quenching mechanism of ADCL fluorescence in presence of different anisotropic gold nanoparticle can also be stated from the data tabulated in (Table 1). As seen in Table 1, the individual time constant of ADCL (specially ESIPT time constant) are found to be modified in presence of triangular, rod and TBP shaped gold nanoparticles suggest the dynamic nature of quenching mechanism among ADCL and other anisotropic GNPs.



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Table 1: Fluorescence Lifetime of ADCL in presence of different shaped GNPs.

ADCL ( ACN)

λem

τ1 (ns)

τ2 (ns)

τ3 (ns)

(nm)

(nm)

(α1)

(α2)

(α3)

340

385

0.315

6.53

1.82

(0.59)

(0.12)

(0.29)

0.303

6.84

1.49

(0.23)

(0.03)

(0.74)

0.401

6.99

-

1.02

(- 0.29)

(0.71)

0.307

6.51

-

1.07

(- 0.25)

(0.75)

0.393

7.01

-

0.98

(- 0.29)

(0.71)

0.316

5.93

-

1.04

(- 0.26)

(0.74)

0.316

7.02

-

0.98

(- 0.23)

(0.77)

0.203

5.67

-

1.2

(- 0.26)

(0.74)

0.251

7.10

-

1.02

(- 0.27)

(0.73)

0.221

6.36

-

1.06

(- 0.20)

(0.80)

0.194

5.30

(- 0.22)

(0.78)

ADCL (MeOH) ADCL (ACN)

340

490

ADCL (MeOH) Spherical (ACN)

340

490

Spherical (MeOH) Rod (ACN)

340

490

Rod (MeOH) TBP (ACN)

340

490

TBP (MeOH) Triangular (MeOH)

χ2

λex

340

490


1.06 1.05

1.15

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3.2.3. Time resolved fluorescence anisotropy study Time-resolved fluorescence anisotropy measurements are widely used to probe the molecular rotational motion that can lead to depolarization of emission following photoselection by polarized excitation.25 This technique is widely applied for the investigation of adsorption of a dye to nanoparticles, polymer matrix, biological macromolecules etc. 33,34 The anisotropy decays of ADCL in MeOH and in the presence of different shaped GNPs are given in Figure 7. Similar kind of results is also obtained in ACN medium (the data not shown here). In pure MeOH medium (absence of GNPs) the fluorescence anisotropy signal of ADCL decays rapidly to zero, as expected for a simple (spherical) emitting rotor in bulk homogenous solution. A single exponential fit of this decay gives a rotational correlation time of ~510 ps which is typical of the fast depolarising rotation of free ADCL in bulk solution. Similar single exponential fit of the time resolved anisotropy decay of ADCL of rotational correlation time ~ 520 ps was obtained in presence of spherical shaped gold nanoparticle, as shown in Figure 7(i). But the situation is quite different in presence of anisotropic gold nanoparticles (triangular, rod and TBP shaped), Figure 7 (ii), (iii) and (iv). In each cases the r(t) profiles attains a minimum thereafter, over a timescale of few nanoseconds, the fluorescence anisotropy again increases with time. This type of complex time resolved anisotropy was previously found in the study of interaction of pyrene with a polymer poly(N-isopropylacrylamide).35 In our case also this complex time resolved fluorescence anisotropy of ADCL in presence of different anisotropic GNPs (triangular, rod and TBP) can only be explained by considering the fact that the probe (ADCL) must experience two distinct environments; one is free in bulk solution and other is adsorbed to the GNPs. When the molecule ADCL is located in the bulk solution rapid rotational mobility happens that corresponds to the fact that r(t) profile attains a minimum. On the other hand when ADCL



(i)

0.4

ADCL in MeOH ADCL + Spherical nanoparticle

0.2

r (t)

r (t)

0.4

-0.2

(ii)

ADCL in MeOH ADCL + Triangle Nanoparticle

0.2

0.0

0.0

0

15

30

45

-0.2

0

10

Time (ns) 0.4

0.4

(iii)

(iv)

ADCL in MeOH ADCL + Rod nanoparticle

r (t)

0.2

30

40

ADCL in MeOH ADCL + TBP Nanoparticle

0.2 0.0

0.0

-0.2

20

Time (ns)

r(t)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 28

0

10

20

30

40

-0.2

0

10

Time (ns)

20

30

Time (ns)

40

Figure 7. Typical time-resolved fluorescence anisotropy decay profile of ADCL in presence of (i) spherical, (ii) Triangular, (iii) rod and (iv) TBP shaped GNPs in MeOH medium at λex = 336 nm and λem = 500 nm. molecules are adsorbed on the GNPs, slower rotational mobility leads to the regaining of the fluorescence anisotropy with time. This result support our previous findings that upon adsorption on the anisotropic GNPs the ESIPT time constant of ADCL molecule becomes faster probably due to the reduction of the high activation energy barrier of Lactim*→Lactam* process. But in presence of spherical shaped gold nanoparticles the interaction of ADCL with GNPs is of ground state type (as found from the time resolved fluorescence decays study), therefore the as obtained single exponential time resolved fluorescence anisotropy decay is due to the free ADCL in bulk ACN medium.


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4. Conclusion The present study deals with the interaction of an biologically important fluorescent molecule ADCL with gold nanoparticles of different shapes (spherical, triangular, rod and TBP) in ACN and MeOH media by means of TEM, steady state absorption and fluorescence (both steady state and time resolved) spectroscopic techniques. From the steady state absorption study of ADCL in presence of different shaped GNPs no such significant results are obtained that can help us to find out the interaction process. In the steady state fluorescence study the large Stokes shifted emission band of ADCL due to the PT process is found to be quenched in presence of GNPs of different shapes. Time resolved fluorescence data reveal the quenching between ADCL with spherical shaped GNPs is of static type while that with other anisotropic GNPs is dynamic in nature. Time resolved fluorescence study reveals the faster PT rate of the molecule in presence of anisotropic gold nanoparticles (triangular, rod and TBP) while PT rate does not alter in presence of spherical GNPs. The adsorption of ADCL on the anisotropic GNPs is evidenced from time resolved fluorescence anisotropic study. Upon adsorption of ADCL on the anisotropic GNPs the PT rate becomes faster, due to probable reduction in high activation energy barrier of the lactim*→lactam* process upon interaction (van der Waals' type) with gold nanosurface. Therefore, in conclusion we can infer that our work is an indirect evidence of the occurrence of a spontaneous mutation by GNPs during DNA replication.

Acknowledgments DR acknowledges University Grant Commission, India for D. S. Kothari Post Doctoral fellowship. AB would like to acknowledge CSIR, India for senior research fellowship. NG would like to acknowledge DST, India (Project No. EMR/2016/004788) for financial support.



Page 24 of 28

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Modulation of ESPT Dynamics in a Model Lactim-Lactam Tautomeric

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