FORM 2
THE PATENTS ACT, 1970
(39 of 1970)
AND
THE PATENTS RULES, 2003
COMPLETE SPECIFICATION
(See section 10; rule 13)
TITLE OF THE INVENTION
"A method for analytical detection and quantification of polycyclic aromatic hydrocarbons and a process for synthesizing
1,3-Di-naphthalimide conjugate of calix [4] arene"
APPLICANT
Indian Institute of Technology, Bombay,
Department of Chemistry,
Powai, Mumbai-400 076
Maharashtra.India;
The following specification particularly describes the invention and the manner in which it is to be performed

FIELD OF INVENTION
This invention relates to a method for analytical detection and quantification of polycyclic aromatic hydrocarbons and a process for synthesizing 1,3-Di-naphthalimide conjugate of calix [4] arene.
BACKGROUND AND TECHNICAL FIELD OF INVENTION
Polycyclic aromatic hydrocarbons (PAHs) are known to be hazardous for human kind because of their carcinogenic and mutagenic properties. PAHs possess condensed benzene structures and are widespread organic environmental pollutants which are formed during the incomplete combustion of carbon containing fuels apart from their occurrence in natural crude oil, fossil fuels and coal deposits. Owing to their lipophilic nature, their solubility in water is very poor and hence persist in the environment for a longer period. However, these are degraded by microbial in a manner of converting these to some of their oxidative products and hence becomes soluble. So it is important to find effective receptors to detect these even at very low concentrations. Detection of PAHs is also relevant in oil spills. In the literature, there are limited reports which employ Raman spectroscopy and chromatography for the detection of PAHs. However, there are no reports on the selective recognition of PAHs by calixarene derivative in solution using absorption and emission spectroscopy techniques. It is now found that 1,3-di-naphthalimide derivative of calix[4]arene (L) is a potential molecule for the analytical detection and quantification of environmental pollutants of anthracenyl and higher aromatics.
This invention also includes a novel process for synthesizing 1,3-Di-naphthalimide conjugate of calix [4] arene in good yield and purity.

BRIEF DESCRIPTION OF INVENTION
This invention relates to a method for analytical detection and quantification of polycyclic aromatic hydrocarbon and their derivatives in samples which comprises the steps of fluorescence titration of 1.3-Di-naphthalimide conjugate of calix[4]arene with said sample and assessing the emission band thereof for fluorescence quenching, intensity of fluorescence and absorption spectra decreasing with the increase in concentration of polycyclic aromatic hydrocarbons and their derivatives in the samples.
This invention also includes a novel process for synthesizing 1,3-Di-naphthalimide conjugate of Calix[4}arene which comprises the steps of converting tert-butylcalix-butylcahx [4]arene (L1) to corresponding dinitrile (L2) and thereafter to diamine (L3); reacting said diamine with naphthalic anhydride as shown in the following reaction scheme.

DETAILED DESCRIPTION OF THE INVENTION
Fluorescence titration: 1.3-Di-naphthalimide conjugate of calix[4]arene has been titrated with different aromatic derivatives (viz., guest molecules) shown in Chart 1 to explore its selectivity. All the titrations were carried out in acetonitrile by

adding incremental concentrations of guest molecules to L (10μM). The emission band observed at 378nm for 1,3-Di-naphthalimide conjugate of calix[4]arene gradually decreases as the concentration of the added guest species increases, among all the guest molecules, pyrenaldehyde showed maximum quenching of 378 nm band of L.
I.J KX^ I.XXJ
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CHQ CMO C^O
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OHO CHO
O C^ XX
-,J* *,b II II
.rk^Myk ir-k ^
4n 4b I -tc
NO,
OH ^H ^.CH
5r0M OCrOM CCO
?r* £1> ?c
fYVTYVl
ob 6c
Chort I. Aromaric hydrocarbons and their derivatives ued in the fluorescence and absorptionsrudies. 5o-1- (D-Glucopyrcmosyl-2-deoxy-2-iminomethy-hydioxybenzene (Giu-2-SI) 5b -l-(D-Gkiropvranosyl-2-deoxy-2-iminomethy-2-hydioxynaptJialene (Glu-2-NI). 5c -l-(D-glucopyanosy1-2-deoxy2-iminoinethyl)-a3itliiacene (Glu-2-AI). 6b -N-{2-hydroxynapthylid-2-ine)-burylamine (BNI). 6c -N-(2-hydroxyaathracenylid-2-ine)-burylamine (BAI).
As the emission has some contribution from the added guest, like that happens in case of anthracenyl moiety (the emission intensity is indeed low when the guest

molecule is bound to -CHO or -OH), corresponding emission of the guest species were subtracted before analyzing the data quantitatively.
The number of folds of quenching is observed at saturation for all the cases and the histogram clearly suggests significant quenching in case of the guests possessing aromatic moiety. The quenching is higher when this is anthracenyl or pyrenyl moiety. Thus the extent of fluorescence quenching by different guests follows an order, pyrenyl- > anthracenyl- >>> naphthyl- > phenyl-. The fluorescence quenching is high by ~ 4 - 5 fold on going from phenyl to naphthyl, and -20 fold high on going from naphthyl to anthracenyl, and it is only 2 fold on going from anthracenyl to pyrenyl moiety. All these results suggest that for the recognition, at least a naphthyl or higher size aromatic moiety should be present in the guest species.
It is noted that while phenyl derivatives exhibit minimal quenching, those of the anthracenyl and pyrenyl derivatives exhibit very high quenching, and hence is possible to quantify guests possessing anthracenyl or higher aromatic moiety. Among the aromatic aldehydes, the fluorescene quenching efficiency follows a trend, 2a « 2b < 2c < 2d (Figure 3B) suggesting that as the size of the aromatic ring increases the fluorescence quenching ability also increase. Similar trend was observed in the fluorescence quenching of L, even among the aromatic-glucosyl derivatives (5a « 5b < 5c) (Figure 3C). Based on our study, it may be noted that the increase in quenching is ~5 fold on going from phenyl to naphthyl and about ~3 fold on going from naphthyl to anthracenyl and ~2 fold on going from anthracenyl to pyrenyl. Thus there is about 30-35 fold increase in the quenching of fluorescence intensity on going from phenyl based derivatives to those of the

pyrenyJ based ones. From the fluorescence titration data, Stern-Volmer data has been derived and the same was given in Table 1.
Table 1. Scem-Volmer quenching constant for file titration of L with aromatic guest molecules

Aromatic guest Ksv(M 1) Aromaric guest Kr.-(M"1)
Benzaldehyde (2 a) 773(±S7) 3-Nitroronlene (4b) 922(±96)
Naphthaldehyde (2b) 7S36"(±1S3) 4-Nitrotoulene (4c) 1026(±135>
Antliraldehyde (2c) 37977<±1S65) 2-NitroTonlene (4a) 135o"(±110)
Pyrenaldehyde (2d) 90liS(±4021) Glu-2-Sl(?a) 3595(±170)
SalicyiaWehyae (3a) 6174{±4H) Glu-2-Nl(5V>) 21093(±57>
2-OH uapthaldehyde (3b) 245S6(±PI4) Ghi-2-AI (5c) 36"47S(±1572)
BXI (6 a) 2S552{±322S) BAI (6b) 52000(=164)
Detection limits of naphthyl- and higher PAHs: The detection limits for pyrenaldehyde, 9-anthraldehyde and naphthaldehyde have been determined using fluorescence titration by keeping 1,3-Di-naphthalimide conjugate of calix[4]arene to guest ratio at 1:1 and were found to be, 8.85 ± 0.05 uM, 19.85 ± 0.15 JIM and 33.8 ± 0.4 uM respectively (SI 03). These detection limits are well suited for the detection of PAHs and their derivatives in agricultural soil samples.
Absorption titrations were carried out to support the results obtained from the fluorescence titrations and also to confirm the binding of aromatic guest molecule with 1,3-Di-naphthalimide conjugate of calix[4]arene. Absorbance of 230 nm band decreases upon addition of guest molecule as a characteristic of the K...K

interaction between 1,3-Di-naphthalimide conjugate of calix[4]arene and the guest species. The extent to which the absorbance decreases follows a trend, i.e., pyrenyl > anthracenyl > naphthyl > phenyl and this trend is same as that observed from the fluorescence quenching studies. The presence of incremental levels of π.... π interaction between 1,3-Di-naphthalimide conjugate of calix[4]arene and aldehyde-guests owing to the size of the aromatic portion has been indicated
The following example illustrates the process for synthesizing 1,3-Di-naphthalimide conjugate of calix [4] arene.
(a) Synthesis of the dinitrile
A mixture of p-tert-butylcalix[4]arene, L, (1.0 g, 1.54 mmol), K2C03 (0.85 g, 6.20 mmol), Nal (0.92g, 6.13 mmol), chloroacetonitrile (0.4 mL, 5.33 mmol) was taken in 50 mL dry acetone were refluxed under nitrogen atmosphere for 7h. The reaction mixture was allowed to cool down to room temperature and filtered through celite and washed the celite with dichloromethane to obtain light brown clear solution. This was concentrated to give brown solid, which was recrystallized from chloroform/methanol to give white crystalline solid. Yield (0.87 g) 78 %. 1H NMR: (CDC13, ppm): 0.88, (s, 18H, C(CH3)3), 1.32 (s, 18H, C(CH3)3), 3.45 (d, J = 13.55 Hz, 4H, Ar-CH2-Ar), 4.22 (d, J = 13.55 Hz, 4H, Ar CH2-Ar), 4.81 (s, 4H, OCH2), 6.73 (s, 4H, Ar-H), 7.12 (s, 4H, Ar-H).
(b) Synthesis of diamine:
A solution of the dinitrile of step (a) above (0.75 g, 1.03 mmol) in 30 mL THF, was added to LiAIH4 (0.32 g, 9.12 mmol) at 0° C and the reaction mixture was refluxed for 5 h. After that reaction mixture was allowed to cool down to room temperature, excess LiAlH4 was destroyed by the addition of 20% NaOH solution

followed by H20(1:3) to the reaction flask and then filtered. The clear filterate was evaporated to dryness to yield diamine, as a paile yellow solid which was recrystallized from chloroform/methanol to give pale yellow crystalline solid. Yield (0.63 mg) 82%. 'H NMR: (CDC13, ppm): 1.10 (s, 18H, C(CH3)3), 1.24 (s, 18H, C(CH3)3), 3.29 (t, J - 4.76, 4.76, 4H, NCH2), 3-37 (d, J = 12.82 Hz, 4H, Ar-CH2- Ar), 4.07 (t, J = 4.76, 4.76 Hz, 4H, OCH2), 4.32 (d, J = 12.82 Hz, 4H, Ar-CH2-Ar), 6.97 (s, 4H, Ar-/H), 7.04 (s, 4H, Ar-H).
Synthesis of 1,3-Di-naphthalimide conjugate of calix[4]arene: To a mixture of the diamine of step (b) (1.5 g, 2.04 mmol) in dry ethanol (150 mL), naphthalic anhydride (1.01 g, 5.09 mmol) was added with stirring and heated at reflux for 12 h under nitrogen atmosphere. The product was. settled upon cooling the reaction mixture and was separated by filtration and was dried under vacuum. The product was purified by chromatographic column filled with silica gel and was eluted with mixture of dichloromethane and methanol (98:2) to give a yield (1.45 g, 65%). 1H NMR (CDC13, 6 ppm): 0.82 (s, 18H, C(CH3)3), 0.88 (s, 18H each, C(CH3)3), 3.28 (d, 4H, Ar-CH2-Ar, J- 13.19 Hz), 4.28 (t, 4H, CH2-CH2,J = 7.2 Hz), 4.34 (d, 4H, Ar-CH2-Ar, J= 13.19 Hz), 4.93 (t, 4H, CH2-CH2-, J= 7.3 Hz), 6.70 (s, 4H, Ar-H), 6.86 (s, 2H, Ar-OH) 6.98 (s, 4H, Ar-H), 7.76 (t, 2H, napthalimide, J = 7.3 Hz), 8.17 (d, 1H, napthalimide, J = 8.06 Hz), 8.68 (d, 1H, napthalimide, J= 7.33 Hz); 13C NMR (CDC13, 100 MHz, 5 ppm)31.1, 31.8, 31.7, 33.9, 34.0, 39.6, 72.1, 122.6, 125.0, 125.6, 127.0, 127.9, 128.3, 131.5, 131.7, 132,4, 134.1, 141.2, 146.8, 150.0, 150.7, 164.2; ESI-MS: m/z = 1118 [M+Na]+, 100%. Anal. Calcd for C74H82N2010 C, 76.66, H, 7.13, N, 2.42. Found: C, 76.24, H, 6.92, N, 2.53.
Obvious alterations and modification known to persons skilled in the art are within the scope and ambit of the appended claims.

WE CLAIM
1) A method for analytical detection and quantification of poly cyclic aromatic hydrocarbon and their derivatives in samples comprising the steps of fluorescence titration of 1,3-Di-naphthalimide conjugate of calix[4]arene with said samples and assessing the emission band thereof for fluorescence quenching; the intensity of fluorescence and absorption spectra decreasing with increase in the concentration of polycyclic aromatic hydrocarbons and their derivatives in the samples.
2) The method as claimed in claim 1, wherein said titration is carried out in a solution of 1,3-Di-naphthalimide conjugate in acetonitrile
3) The method as claimed in claims 1 and 2, wherein anthracenyl and pyrenyl derivatives of polycyclic aromatic hydrocarbon exhibit high rate of fluorescence quenching.
4) A process for synthesizing 1,3-Di-naphthalimide conjugate of calix[4]arene comprising the step of converting tert-buty1 calix [4] arene to corresponding dinitrile and thereafter to diamine, reacting said diamine with naphthalic anhydride as shown in the following reaction scheme.


5) The process as claimed in claim 4, wherein said reaction of diamine. with naphthalic anhydride is carried out in an atmosphere of nitrogen.
6) The process as claimed in claims 4 and 5, wherein the reaction mixture is purified by chromatography and then crystallized.
7) 1,3-Di-naphthalimide conjugate of calix[4]arene whenever synthesized by a process as claimed in claims 4 to 6.
8) A method for analytical detection and quantification of poly cyclic aromatic hydrocarbon substantially as herein described.