Green Chemiluminescence of Highly Fluorescent Symmetrical Azo-based Luminol Derivative

A symmetrical azo-based Luminol derivative 2 was synthesized using catalytic dehydrogenative coupling reaction procedure. This symmetrical molecule shows blue fluorescence in polar aprotic solvents (λmax = 450 nm). With an increase in aqueous content 2 was exhibiting aggregation caused fluorescence quenching (ACQ) with a red shift of 30 nm (λmax = 480 nm). Distinct emission features were observed for this molecule with a variation in the pH range. In basic medium, deprotonated form of 2 shows fluorescence quenching followed by enhancement of green chemiluminescence (λmax = 490 nm) with H2O2/Fe 3+catalytic system. The fluorescence and chemiluminescent (CL) properties of 2 were compared with its parent luminol 1, The CL emission intensity of 2 was found to be about 16 times higher than that of 1. In addition, HOMO-LUMO energy gap for 2 was also calculated using density functional theory (DFT) studies.


INTRODUCTION
Luminol 1 is a classic chemiluminescent (CL) reagent 1 first reported by Albrecht in 1928 2 , extensively used for different analytical applications 3,4 The basic solution of Luminol exhibits a striking blue glow when mixed with an oxidizing agent and an Iron catalyst. 1][11][12][13][14] and White et al., have synthesized alkyl derivatives of luminol to maximize the CL (λmax = 410 nm) quantum yield. 120][11][12][13][14] Zaitsu et al., have synthesized N-(4-substituted benzyl) isoluminol with substituents like, 4-bromo,4-methoxy and 4-nitro groups.The chemiluminescent intensities of these compounds were 0.03-4.7 times that of luminol. 12Naphthalene analogues of luminol have an extended aromaticity which leads to green chemiluminescence (λmax = 490 to 550nm). 15t is noteworthy that green chemiluminescence in longer wavelength region is of great interest for biological applications. 16The increase of aromaticity leads to a redshift with a green CL.Balaban et al., have synthesized 5-(azo-para-phenylene-N-aza-15-crown-5)-phthalhydrazide, an azo derivative of Luminol 17 which shows a weak fluorescence but a significant chemiluminescence at 425nm.But the chemiluminescent emission was found around 425nm similar only to its parent luminol.Another azo derivative of luminol, phthalhydrazide-5-azo-2-naphthol, synthesized by coupling diazotized 5aminophthalhydrazide with 2-naphthol which shows fluorescence at 350nm. 18This derivative was used to complex with metal ions, but chemiluminescent studies were not reported.] The catalytic system employed was Cu(I)Br along with DMAP (Dimethylamino pyridine) and AIBN (Azobisisobutyronitrile).Luminol 1 is also basically an aromatic amine with a phthalhydrazide group.Dehydrogenative homo coupling of luminol would also result in a symmetrical azo product of luminol, which is actually a dimer with an extended conjugation between two luminol units through an azo link 2. To the best of our knowledge, this kind of homocoupling of luminol was not known.So we have decided to synthesize a symmetrical luminol derivative with an objective to increase the CL intensity and a significant red shift.In the present work, we have performed the homo coupling of 1 and optimized the conditions for a maximum yield.Azobenzene and its derivatives are commonly used as dyes and fluorescent materials.Linking chemiluminescent luminol units through an azo group would also result in interesting fluorescent properties through the short and compact π-conjugated structure.The presence of two phthalhydrazide groups guarantee its chemiluminescent nature, which can be taken advantage for applications like biosensing.

Synthesis of Compound 2
Compound 2 was synthesized according to the procedure earlier reported by Reddy et al., [19][20] Accurately weighed Luminol (250 mg, 1.0 mmol) was taken in a dry 100 ml two necked round bottom flask followed by the addition of 4-dimethyl aminopyridine (DMAP) (150 mg, 1.2 mmol), cuprous bromide (14.3 mg, 0.10 mmol) and Azobisisobutyronitrile (AIBN) (50 mg, 0.3 mmol) under an inert atmosphere condition.To these contents, 3 ml of DMF was added.The reaction mixture was allowed to stir under inert conditions for a period of 7 h at 50 °C, after which the reaction mass, was filtered.The filtrate obtained was concentrated under high vacuum and the resultant sticky crude product was purified using column chromatography (silica gel, hexane/ethyl acetate, 85/15 as an eluent).The product was obtained in a pure form as a yellow colour solid in 92% of isolated yield and its structure was established using various analytical techniques. 1 H NMR (400 MHz, DMSO-d6, TMS) 6.90 (1H, d); 6.95 (1H, d); 7.42 (1H, t) 13   (1-9μM) was added to this solution.Solutions of same concentrations were used for all absorbance, fluorescence and CL spectroscopic studies unless otherwise specified.

General procedure for calculating Density functional theory studies:
Density functional theory calculations were carried out with B3LYP level using Gaussian 09 program.The dihedral angle (Φ) between the plane of the optimized energy structure is found to be 50° The Frontier molecular orbitals of compound 2 obtained from DFT calculations.

RESULTS AND DISCUSSION
In the earlier work by Reddy et al., homocoupling of simple and substituted aromatic amines at ambient temperature under mild conditions was reported [19][20] .Luminol is a basic aromatic amine with a phthalhydrazide group.Following the mentioned reported procedure dehydrogenative homocoupling of luminol 1 was done using the catalytic system.The catalytic system employed was 10 mol% CuBr, 0.3 mmol of AIBN and 1.2 mmol of DMAP at room temperature as well as 50° C which resulted in 2, a symmetric molecule 2 (Table 1) has two phthalhydrazide groups which can produce chemiluminescence as like the parent luminol molecule.The yield of coupling product was less when reactions were performed at room temperature.(Entries 1, 4, 6 ).To increase the yield slightly higher temperature a 50 °C was used with DMF(Entries 2).The yield of the desired coupling product 2 was 92%.At higher temperature of 80 °C (Entry 3) the yield was not found to be increasing (72%).At this stage it was almost confirming the better activity of the catalytic system only at 50 °C.The reaction was also performed in DMSO at ambient temperature (Entry 4) as well as (Entry 5) 50 °C.The yield was higher at 50 °C (83%) compared to room temperature (27%) but still lower than that in DMF (Entry 2).The reaction was also performed in acetonitrile at ambient temperature (Entry 6) and 50 °C (Entry 7) as well as DCM at 50 °C (Entry 8).Only with the DMF solvent, the highest conversion was obtained.General procedure for the absorbance, Fluorescence and Chemiluminescence (CL) Studies 1 (1.8×10 -5 M) was dissolved in DMF whereas 2 (2.5×10 -5 M) was dissolved in various solvents like DMF, DMSO, ethyl acetate and THF.For CL studies, both 2 (2.5×10 -5 M) and 1 (1.8×10 -5 M) were dissolved in DMF. 2 mL of these solutions were taken in a quartz cuvette, followed by addition of 100μL of potassium ferricyanide (1.8 ×10 -5 M) in 9μM sodium hydroxide solution.To initiate CL, H 2 O 2 The product 2 is symmetrical and shows fluorescence because of the extended conjugation as two phthalhydrazide groups are also present, it is obvious to expect chemiluminescence also. 2 was found to be highly soluble in polar solvents like DMF, DMSO, Ethyl acetate and THF.These solutions were fluorescent with varying intensities.Compared with 2, the parent luminol molecule was found to be non-fluorescent when dissolved in DMF. (Fig. 1. black background under UV lamp).
Solutions of 2 with various solvents were prepared and the absorption and fluorescence spectra were recorded using these solutions.The absorption spectra of 2 in different solvents are given in (Fig. 2) Compared with parent luminol (DMF), the absorbance maxima of 2 shows red shifted due to the increase in conjugation. 1 shows two absorption bands at 300nm and 350nm due to π−π* transitions.2 shows two absorption bands one in the wavelength range of 250-300 nm due to π−π* transitions of aromatic group and a shoulder in the range of 300-390 nm which is due to π−π* transitions of −N=Ngroup with all solvents.The intensity of absorption spectra was found to be varying in accordance with the dielectic constant of the solvents employed. 21High dielectic constant, solvents like DMSO (ε =46.7) and DMF, (ε = 36.7),2] Insolvents with low dielectric constant like THF (ε 7.52) and Ethyl acetate (ε = 6.02), 2 show two absorption band with a λ max at 253 and a shoulder at 315, 253 and 375nm respectively which is evident for negative solvatochromism.This type of observation may be due to more polarisation of 2 in polar solvents with a high dielectric constant.The compound 2, is a symmetrical homocoupling product of Luminol and was found to be highly fluorescent in polar solvents.Compared with parent luminol, 2 shows blue fluorescence with a λ max at 458 nm in DMF (Fig. 3).In other polar solvents like DMSO, Ethyl acetate, and THF, maximum fluorescence was found at 467, 445 and 449 nm respectively.(Fig. 4) A clear redshift was noticed in DMSO.This may be due to the intermolecular charge transfer taking place between the solvents and compound 2 and also the compound 2 is more polar in the excited state 21 .In Ethyl acetate a blue fluorescence with a λ max at 445 nm.(Fig. 4).which is due the negative solvatochromism appeared in compound 2. In less dielectic constant solvent like Ethyl acetate (ε=6.02) and THF (ε=7.02) Self-aggregation is a well known phenomenon among the azo dyes caused due to hydrophobic and/or vanderwaals forces, which results in fluorescence quenching, prevalently called as aggregation caused quenching (ACQ) 23 which affect its development as a solid-state fluorescent materials.The symmetric luminol azo derivative 2 was also expected to show ACQ.Compound 2 in their respective solvents were prepared.By varying the proportion of water 10-90% v/v using these solutions the Fluorescence spectra were recorded.(Fig. 4) With an increase in water content the fluorescence bands got in different solvents gets quenched and also get red shifted to 480 nm.In aqueous media the intramolecular rotations are limited due to the formation of aggregates (ACQ) which block the radiative excitations getting the molecule quenched. 24In the case of 2a/water, 2b/water mixtures fluorescence intensity of 2 gradually decreases with an increase in water upto 10-40% v/v.Further increase in the percentage of water to 50%v/v, ACQ breaks quenched intensity of the fluorescence band gets improved.(Fig. 5 & 6 ).But furthermore addition of water from 60% -90% v/v results in a gradual quenching of fluorescence.So for all other studies like effect of pH and CL emission 50% v/v DMF-water mixture of 2 was used.
In ethyl acetate and THF also the fluorescence intensity of 2 gradually decreased with the increase in the water fraction (10-90%) and got redshifted to 480nm.(Figure 7 and 8).and a shoulder at 380nm.In neutral pH, the absorption band shows two prominent bands at 280 and 390 nm similar to luminol.In an alkaline pH 9, three bands were observed with the λ max = 280 nm, 340 and 360 nm.The fluorescence spectra of 2 at lower pH 4 gave a single band with λ max = 445nm with a decrease in intensity compared to the band in neutral medium this may be due to the protonation of one nitrogen atom of azo chromophore Scheme 2 (I).There was no change in the structural and electronic environment of solution 2 25 in neutral pH, which was confirmed by a prominent peak at 450 nm Scheme 2(II) In an alkaline pH 9 the fluorescence spectra with a λ max = 477 nm which is due to the anion formed by the deprotonation of the phthalhydrazide groups in 2 Scheme 2 (III).This symmetrical luminol azo derivative 2 exhibits CL with only molecular oxygen and base in aprotic solvents like DMF and DMSO a phenomenon similar to the parent luminol molecule [6][7][8]26 The addition of base in an aprotic solvent to 2 produces a spectral red shift to 477 (Fig. 10) nm and a quenching effect in fluorescence intensity. Whie et al., [6][7][8] have proposed CL studies of Luminol in aprotic solvents like Dimethylsulfoxide (DMSO), Dimethylformamide (DMF) exhibiting the CL with presence of oxygen and based without a need for peroxide/catalyst system.In those solvents, the CL reactions relatively free of complications and amino phthalate ion have been identified as the products of the reaction [6][7][8]26 The fluorescence quenching behavior of compound 2 was studied in DMF and NaOH Solution.To the solution of 2 in DMF taken in a quartz cuvette (1-9μM) NaOH solution was added and the fluorescence spectra were recorded.With the increase in the concentration of NaOH, fluorescence intensity was Fluorescence quenching is quantitatively measured with the Stern-Volmer equation equation.
Where K SV represents Stern-Volmer constants indicating the sensitivity of the quenching effects of compound 2. F 0 and F are the fluorescence intensities before and after the addition of the analytes, and [Q] is the molar concentration of the analyte.Fig. 12 shows the Stern-Volmer plot for quenching effect in OH -ion in compound 2. K SV Value was calculated as 2.8×10 5 M -1 .This suggested that the dynamic nature of quenching process playing a role in higher quencher concentration.The luminescence of Luminol (3-amino-pthalhydrazide) and chemiluminescence during oxidation process have been widely studied.Luminol chemiluminescence has limitations in bioimaging and sensing applications due to its solubility.The fluorescence titration of compound 2 with gradual addition of NaOH quenches the fluorescence intensity progressively at 480 nm.(Fig. 11) due to the increase in the concentration of dianion.But this basic solution can initiate CL reaction, provided the necessary conditions.To initiate the chemiluminescence in 2, (100μL of 1.8 ×10 -5 M) alkaline potassium ferricyanide, followed by varying amounts (1-9μM) of hydrogen peroxide solution were added. 10With an increase in the concentration of peroxide, CL emission intensity was found to be increasing and the lower detection limit (LOD=3σ/s) was found to be 0.76μM.This CL spectra shows a redshift with an emission peak at 490nm and a shoulder from 510nm to 600nm covering the with a green light region (Fig. 13 and 14).An appreciable red shift was shown by 2 (λ max= 490nm) compared to 1 (λ max = 435nm).The CL reaction of 2 produced a fluorescent green colour under UV light (Fig. 16).Compared to the short life time of luminol's blue glow, this fluorescence green colour was long lasting for several hours.The mechanism of CL shown by 2 should be similar to that of parent luminol molecule, 1 (Scheme 3).It should be convincing because of the structural similarity with both these molecules having CL active phthalhydrazide groups.
More structural variations in 2 would further enhancement result in enhancement of CL intensity and longer red shift.With this objective we are now involved in synthesizing the derivatives of 2 with more structural variations and their applications in sensing/imaging.

Density functional theory studies
Density functional theory calculations were carried out with B3LYP level using Gaussian 09 program.The Frontier molecular orbitals of compound 2 obtained from DFT calculations are shown in (Fig. 15) which reveals the energy gap between HOMO and LUMO that reflects the chemical activity of compound 2. HOMO has the ability to donate an electron and LUMO represents an electron acceptor.The calculated energy gap between HOMO and LUMO was 3.502 eV.[30][31] (M+1).