Coumarin derivative for detection of cysteine and process for the synthesis thereof

Abstract

The present invention relates to a coumarin derivative of Formula (L) for detection of cysteine and process for preparation thereof. The present invention further relates to a process of detection of cysteine residues present in protein as well as the cysteine released by the enzymatic action of aminoacylase 1 by using coumarin derivative of Formula (L). ##STR00001##

Claims

1. A Coumarin derivative of Formula (L) ##STR00012## Wherein R.sub.1 and R.sub.2 are same or different, straight chain alkyl groups, branched chain alkyl groups.

2. The compound as claimed in claim 1, wherein said compound is selected from 7-(diethylamino)-3-((E)-4-((E)-2-nitrovinyl)styryl)-2H-chromen-2-one and 7-(dimethylamino)-3-((E)-4-((E)-2-nitrovinyl)styryl)-2H-chromen-2-one.

3. A process for the preparation of coumarin derivatives of Formula (L) as in claim 1 comprising the steps of: a) adding N,N-Dicyclohexylcarbodiimide (DCC) to a solution of vinyl acetic acid in dicholoromethane at temperature ranging from 0 C. to 5 C. for the time period ranging from 1-4 hrs followed by addition of substituted salicylaldehyde and 4-Dimethylaminopyridine (DMAP) and stirring the resultant solution at the temperature ranging from 25 C. to 30 C. for the time period ranging from 2-3 hrs; b) adding caesium carbonate (CS.sub.2CO.sub.3) to the filtrate of step (a) followed by stirring the reaction mixture for the time period ranging from 12-14 hrs at the temperature ranging from 25 C. to 30 C. to obtain compound 1; c) adding aldehyde, coupling agents to a solution of compound 1 of step (b) in dimethylformamide followed by heating the reaction mixture for the time period ranging from 12-14 hrs at the temperature ranging from 80 C. to 90 C. under inert atmosphere to obtain compound 2; d) adding ammonium acetate to a solution of compound 2 of step (c) in nitromethane followed by refluxing for the time period ranging from 3-4 hrs at the temperature ranging from 80 C. to 90 C. to obtain heterocyclic derivatives of Formula (L).

4. The process as claimed in claim 3, wherein said substituted salicylaldehyde is selected from 4-(diehtylamino) salicylaldehyde and 4-(dimethylamino) salicylaldehyde.

5. The process as claimed in claim 3, wherein said coupling agents of step (c) are sodium acetate, triphenyl phosphine and Palladium (II) acetate.

6. A process of detection of cysteine comprising treating compound formula (L) as claimed in claim 1 with natural amino acids in HEPES: ACN (9:1) at pH 7 characterized in that the selectivity of compound (L) to cysteine is 100%, wherein said amino acids is selected from tryptophan (Trp), leucine (Leu), isoleucine (Ile), methionine (Met), threonine (Thr), tyrosine (Tyr), valine (Val), alanine (Ala), serine (Ser), glycine (Gly), cysteine (Cys), glutathione (GSH), homocysteine (Hcy), proline (Pro) and arginine.

7. A process of enzymatic estimation of cysteine using compound (L) as claimed in claim 1 comprising the steps of: a) adding N-Acetyl-Cysteine to the probe L in HEPES:CH.sub.3CN (9:1) followed by adding different concentration of Amino acylase-1 solution; b) incubating resulting solution at 37 C. for 45 min.

8. The process as claimed in claim 7, wherein said process is taking place at pH 7.

9. A visual test for detection of cysteine using compound (L) as claimed in claim 1 comprising the steps of: a) preparing thin layer chromatography test strips by coating probe L solution in acetonitrile on silica TLC plates; b) adding Cystein in aq.HEPES buffer (pH7) on it; c) drying for 5-10 mins and observing visual as well as fluorescence colour changes; said test is used in enzymatic reaction on TLC plate by using of NAC and amino acylase-1 enzyme.

10. A process of detection of cysteine in biological fluids and raw milk (whey) using compound (L) as claimed in claim 1 comprising the steps of: a) adding different amount of whey to probe L in HEPES: ACN at pH 7; b) adding different concentrations of N-Acetyl Cysteine to each of the above solution of step (a); and c) incubating at 37 C. for 45 minutes and recording luminescence changes.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: (A) UV-Vis spectra of L (10 M) in the absence and presence of various amino acids (AAs); (B) UV-vis changes upon addition of various concentration of Cys (0-100 equiv.).

(2) FIG. 2: (A) Luminescence response of L (10 M) in the absence and presence of various amino acids (AAs); (B) Luminescence changes upon addition of various concentration of Cys (0-1000 equiv.).

(3) FIG. 3: Change in fluorescence response of L (10 M) in 10 mM HEPES:CH.sub.3CN (9:1 v/v) with Cys, Hcy and GSH upon increase in pH values .sub.Ext=445 nm.

(4) FIG. 4: (A) Time dependent fluorescence response of L (10 M) with biothiols (Cys, Hcy and GSH) (B) Micro plate fluorescence reading of L with different amino acids.

(5) FIG. 5: Fluorescence response of L (10 M) with NAC (200 equiv.) in presence of amino acylase-1.

(6) FIG. 6: Fluorescence response of probe (10 M) with Cys and N-acetyl cysteine.

(7) FIG. 7: (A) Visual and (B) Fluorescent color changes of L (5 M) coated on TLC plates upon addition of different analytes.

(8) FIG. 8: Fluorescence intensity at 524 nm upon addition of Cys (0-10 M)

(9) FIG. 9: Fluorescence response of L (10 M) with Whey protein (500 l) after hydrolysis with NAC (0-500 l).

DETAILED DESCRIPTION OF THE INVENTION

(10) The invention will now be described in detail in connection with certain preferred and optional embodiments, so that various aspects thereof may be more fully understood and appreciated.

(11) For the purpose of this invention, the expression probe L and coumarin derivatives of Formula (L) are used interchangeably throughout the specification and both having the same meaning.

(12) The present invention provides a novel coumarin derivative of Formula (L) i.e. 7-(dialkylamino)-3-((E)-4-((E)-2-nitrovinyl)styryl)-2H-chromen-2-one which are useful for the selective detection of cysteine.

(13) ##STR00004##

(14) Wherein R.sub.1 and R.sub.2 are same or different, straight chain alkyl groups, branched chain alkyl groups, preferably selected from methyl and ethyl.

(15) In an embodiment, said coumarine derivatives of Formula (L) are selected from 7-(diethylamino)-3-((E)-4-((E)-2-nitrovinyl)styryl)-2H-chromen-2-one, and 7-(dimethylamino)-3-((E)-4-((E)-2-nitrovinyl)styryl)-2H-chromen-2-one.

(16) ##STR00005##

(17) In another embodiment, said coumarin derivatives of Formula (L) is useful for selective sensing of Cysteine (Cys).

(18) In still another embodiment, said coumarin derivatives of Formula (L) can be used to monitor the drug metabolism in living liver cells.

(19) In yet another embodiment, said coumarin derivatives of Formula (L) can be used for monitoring the enzymatic activity of aminoacylase 1, an important enzyme used exclusively in industries for the synthesis of chiral amino acids.

(20) In still yet another embodiment, said coumarin derivatives of Formula (L) is sensitive enough to detect the Cysteine levels in nanomolar range. So, it can be applied to measure the Cysteine levels in bio fluids like blood plasma. Since, any abnormality in Cysteine level is an indicator for many diseases, so this reagent has significance in clinical diagnosis.

(21) In still yet another embodiment, said coumarin derivatives of Formula (L) can detect Cysteine on simple silica coated plate, this is much cheaper and one doesn't need any instrumental techniques for detection.

(22) In still yet another embodiment, said coumarin derivatives of Formula (L) can give visually observable colour change from reddish brown to green upon reaction with Cysteine. This helps in easy detection.

(23) In still yet another embodiment, said coumarin derivatives of Formula (L) can specifically binds to Cysteine present in proteins, this is more advantageous for studying the structure and conformational changes occur in proteins.

(24) In still yet another embodiment, the present invention provides the comparison study of of said coumarin derivatives of Formula (L) with previous prior arts coumarin derivative.

(25) TABLE-US-00001 Coumarin derivatives of formula (L) L Previous prior art Previous prior art 1. This compound is Detect only micromolar range Not specific to Cys useful for selective sample. Not sensitive. Studies were sensing of Cysteine No colorimetric response. performed (Cys). No enzymatic application. in 50% acetonitrile, 2. It could be used to No protein labelling which is monitor the drug application. not good. metabolism in living Useful only in solution. No enaymatic liver cells. Cannot detect Cys with cheap application. 3. It is useful for test strips. No protein labelling monitoring the Application studies enzymatic activity of No Test strips. aminoacylase 1, an important enzyme used exclusively in industries for the synthesis of chiral amino acids. 4. This reagent is sensitive enough to detect the Cys levels in nanomolar range. So, it could be applied to measure the Cys levels in bio fluids like bllod plasma. Since, any abnormality in Cys level is an indicator for many diseases, so this reagent has significance in clinical diagnosis. 5. This reagent can detect Cys on simple silica coated plate, this is much cheaper and one doesn't need any instrumental techniques for detection. 6. This reagent gives visually observable colour change from reddish brown to green upon reaction with Cys. This helps in easy detection. 7. This reagent specifically binds to Cys present in proteins, this is more advantageous for studying the structure and conformational changes occur in proteins.

(26) In another embodiment, the present invention provides process for the preparation of novel coumarin derivatives of Formula (L) comprising the steps of: a) adding N,N-Dicyclohexylcarbodiimide (DCC) to a solution of acid in solvent at temperature ranging from 0 C. to 5 C. for the time period ranging from 1-2 hrs followed by addition of substituted salicylaldehyde and 4-Dimethylaminopyridine (DMAP) and stirring the resultant solution at the temperature ranging from 25 C. to 30 C. for the time period ranging from 2-3 hrs; b) adding cesium carbonate (CS.sub.2CO.sub.3) to the filtrate of step (a) followed by stirring the reaction mixture for the time period ranging from 12-14 hrs at the temperature ranging from 25 C. to 30 C. to obtain compound 1; c) adding aldehyde, to a solution of compound 1 of step (b) in solvent followed by heating the reaction mixture for the time period of ranging from 12-14 hrs at the temperature ranging from 80 C. to 90 C. under inert atmosphere to obtain compound 2. d) adding base to a solution of compound 2 of step (c) in nitroalkane followed by refluxing for the time period ranging from 3-4 hrs at the temperature ranging from 80 C. to 90 C. to obtain heterocyclic derivatives of Formula (L).

(27) In a preferred embodiment, said substituted salicylaldehyde is selected from 4-(diehtylamino) salicylaldehyde and 4-(dimethylamino) salicylaldehyde,

(28) In another preferred embodiment, said acid is selected from vinyl acetic acid.

(29) In still another preferred embodiment, said solvent of step (a) is dichloromethane.

(30) In yet another preferred embodiment, said coupling agents of step (c) are sodium acetate, triphenyl phosphine and Palladium (II) acetate.

(31) In still yet another preferred embodiment, said solvent of step (c) is dimethylformamide.

(32) In still yet another preferred embodiment, said base of step (d) is ammonium acetate.

(33) In still yet another preferred embodiment, said nitroalkane is selected from nitromethane.

(34) The process for the synthesis for novel coumarin derivatives of Formula (L) is as depicted in Scheme (I);

(35) ##STR00009##

(36) The process for the synthesis for 7-(diethylamino)-3-((E)-4-((E)-2-nitrovinyl)styryl)-2H-chromen-2-one is as depicted in Scheme (II);

(37) ##STR00010##

(38) The process comprises DCC coupling of vinyl acetic acid with 4-(diethylamino) salicylaldehyde followed by cyclization in presence of cesium carbonate to result in the formation of vinyl coumarin. Extension of conjugation achieved by Heck coupling of vinyl coumarin with bromo benzaldehyde, which resulted in an extended aldehyde (compound 2). Incorporation of nitro olefin unit to the parent chromophore was achieved by condensation of aldehyde with nitro methane to get probe L or compound of Formula (L).

(39) In another embodiment, the present invention provides a process of detection of cysteine comprises treating said coumarin derivative of Formula (L) with natural amino acids in HEPES:CH.sub.3CN (9:1) at pH 7 characterized in that the selectivity of compound to cysteine is 100%.

(40) In a preferred embodiment, said amino acids are selected from tryptophan (Trp), leucine (Leu), isoleucine (Ile), methionine (Met), threonine (Thr), tyrosine (Tyr), valine (Val), alanine (Ala), serine (Ser), glycine (Gly), cysteine (Cys), glutathione (GSH), homocysteine (Hcy), proline (Pro) and arginine.

(41) Derivatives of coumarin have different substituent at the Nitrogen center (like dimethyl, diethyl etc.). Since the molecule is built for selective sensing purpose, any major change in the heterocyclic coumarin core drastically affects the selectivity and sensitivity.

(42) All the figures provided are showing the sensing behaviour of 7-(diethylamino)-3-((E)-4-((E)-2-nitrovinyl)styryl)-2H-chromen-2-one.

(43) The probe L (10 M) is treated with natural amino acids (100 equiv. each) in HEPES: ACN (9:1) at pH 7, Only Cys gives an observable change in absorption spectra whereas the other amino acids does not give any significant changes in absorption (FIG. 1A). With the addition of Cys (0-100 equiv.), absorption band at 468 nm gradually decreases and a new band appeared at 438 nm with a visible colour change from red to green (FIG. 1B). A hypsochromic shift (30 nm) with three well defined isosbestic points are observed at 445 nm, 372 nm and 325 nm. This Cys-induced hypsochromic shift indicates the suppression of ICT process from donor diethylamino unit to acceptor nitroalkene unit.

(44) The fluorescence changes of L (10 M) upon treatment with various amino acids (200 equiv.) in HEPES:CH.sub.3CN (9:1) at pH 7 were displayed in FIG. 2A). Only Cys gives an enhancement in the fluorescence compare to all other amino acids. In the absence of Cys, probe L is almost non fluorescent (.sub.f=0.06), upon addition of Cys (0-1000 equiv.), fluorescence intensity is increased remarkably up to 7 fold with an emission band centered at 524 nm (.sub.Ext=445 nm) (FIG. 2B). In addition to this, the addition of Cys to solution of L resulted in an observable colour change from non-emissive red to highly emissive green when excited with a hand held 365 nm UV lamp (FIG. 2B, inset). The quantum yield of probe after the addition of Cys increases (.sub.f=0.32) with respect to fluorescein (.sub.f=0.92). The fluorescence turn on response of the probe upon addition of Cys is due to the formation of 1,4 conjugate addition product, which blocks the PET quenching by nitro olefin and also ICT process from diethylamino to nitro olefin unit. The results were in well agreement with our proposed hypothesis. Under identical conditions, other biological thiols such as Hcy and GSH does not give any significant changes in fluorescence (FIG. 2A), which indicates the selectivity of probe towards Cys. The fluorescence intensities of the probe at 524 nm show a good linear relationship with the concentration of Cys between 0 to 1000 M. The detection limit of Cys is determined as 23.65 nM (23.6510.sup.9) based on S/N=3. (FIG. 8)

(45) Since the pK.sub.a values of Cys, Hcy and GSH are different (8.3, 10.0 and 9.5 respectively), pH dependent study carried out in order to investigate the effect of pH. And the pH studies clearly indicates the selectivity of probe to Cys at pH 7 and the interference from GSH starts with increase in the pH values (FIG. 3). Cys being a low pK.sub.a protein thiol, at neutral pH, the thiolate/thiol ratio is higher for Cys than for Hcy and GSH, which results in greater reactivity of Cys with the probe. This could be the reason for getting selectivity to Cys over Hcy and GSH at pH 7. Also pH 7, being neutral, much suitable for biological studies. Hence, all the studies were carried out at pH 7.

(46) Time dependent fluorescence response of probe L or compound of Formula (L) is carried out with 200 equiv of Cys. The kinetic study showed that the reaction is completed in 25 minutes. The kinetic study further extended to check the potential interference of Hcy and GSH but no significant changes in the fluorescence observed, which indicates the selectivity of probe towards Cys over Hcy and GSH and it is supported additionally by microplate reading experiment where different amino acid solutions with 10 M probe in HEPES:ACN (9:1) were taken in a 46 well microplate and the data indicates that only cysteine is giving significant emission changes with respect to other amino acids. (FIG. 4).

(47) In still another embodiment, the present invention provides a kit for the selective detection of cysteine characterized in that the selectivity of novel coumarin derivatives of Formula (L) to cysteine is 100% and a process for detection using the kit.

(48) In preferred embodiment, the TLC test strips are prepared and with the addition of Cys, the colour of probe coated TLC plates changes from red to yellowish green and becomes highly fluorescent when observed under 365 nm UV lamp (FIG. 7). But Hcy and GSH does not give any observable visible as well as fluorescence changes. The same strategy is further extended to detect Cys in blood plasma and the Cys released in an enzymatic reaction as well. The probe quoted test strips showed visible as well as fluorescent colour changes to Cys as well as Cys present in blood plasma. The same methodology is further extended to detect the enzymatically generated Cys from NAC.

(49) In yet another embodiment, the present invention provides a process for the detection of cysteine present in natural food sources.

(50) In preferred embodiment, the present invention provides a process of detection of cysteine in boilogical fluids and raw milk wherein said process comprising novel coumarin derivatives of Formula (L), characterized in that the selectivity of compound to cysteine is 100%.

(51) In still yet another embodiment, the present invention provides a visual test for detection of cysteine using novel coumarin derivatives of Formula (L).

(52) In preferred embodiment, the present invention provides the nitro olefin based colorimetric as well as fluorescent probe for selective discrimination of Cys over Hcy and GSH. The probe L exhibits high selectivity to Cys, over other amino acids through Michael addition reaction of Cys with probe. The detection limit is found to be 23.65 nM. The probe L which is successfully utilized for the detection of enzymatically generated Cys.

(53) In still yet another embodiment, the present invention provides a method of the detection of cysteine generated in an enzymatic reaction.

(54) In another preferred embodiment, the detection of Cys generated in an enzymatic reaction is studied. N-acetyl-cysteine (NAC) is employed as substrate and amino acylase-1 as an enzyme. N-acetyl-cysteine, an acetylated form of Cys, a well known pro-drug used as a cysteine supplement in treatment of glutathione replenishment, acetaminophen over dose, HIV patients etc. NAC supplement releases the Cys inside the cells. Since both Cys as well as NAC has free sulfhydril groups, ideally both should bind with the probe but the higher pK.sub.a value of NAC (pK.sub.a=9.5) hinders its reactivity towards the probe at pH 7. Hence, the interference from NAC could be avoided at pH 7 (FIG. 6). Amino acylases are a class of enzymes which specifically hydrolyses the N-acetylated amino acids to release the free amino acids. There are many amino acylases known for de-acetylation of acetylated amino acids, among them Amino acylase-1 is well known for its ability to hydrolyse acetylated cysteine. In the current study, it can be utilized the luminescence ON response for L-Cys formation due to a reaction between L and Cys, released by enzymatic action of aminoacylase-1 on NAC (FIG. 5) In absence of any aminoacylase-1, no change in emission intensity was observed at 520 nm (FIG. 5). After the addition of aminoacylase-1, fluorescent intensity gradually increases, which indicates the efficient hydrolysis of NAC by aminoacylase-1.

(55) Suitability of this reagent for specific recognition and estimation of Cys-residues present in natural milk proteins was also explored. For this study whey protein was isolated from milk. Initially some fluorescence was observed, which is due to the presence of free Cys. Then NAC tablets are used to reduce the disulfides present in whey. Upon addition of different concentrations of whey and NAC to L, the fluorescence intensity of L gradually increases, indicating the reduction of disulfides by NAC to release Cysteine (FIG. 9). So, this reagent could be used as protein labeling agent to specifically label the Cys present in proteins.

(56) ##STR00011##

(57) FIG. 1 depicts (A) UV-Vis spectra of L (10 M) in the absence and presence of various amino acids (AAs); e.g. Tryptophan (Trp), Leucine (Leu), Isoleucine (Ile), Methionine (Met), Threonine (Thr), Tyrosine (Tyr), Valine (Val), Alanine (Ala), Serine (Ser), Glycine (Gly), Cysteine (Cys), Glutathione (GSH), Homocysteine (Hcy), Proline (Pro) and Arginine (Arg); (B) UV-vis changes upon addition of various concentration of Cys (0-100 equiv.). Inset-Visible colour change observed upon addition of Cys. All studies were performed in 10 mM HEPES:CH.sub.3CN (9:1, v/v) at pH 7 at 298 K.

(58) FIG. 2 depicts (A) Luminescence response of L (10 M) in the absence and presence of various amino acids (AAs); (B) Luminescence changes upon addition of various concentration of Cys (0-1000 equiv.). Inset: fluorescence colour change observed under hand held 365 nm UV lamp. All studies were performed in 10 mM HEPES: ACN (9:1) v/v at pH 7 at 298K

(59) FIG. 3 depicts change in fluorescence response of L (10 M) in 10 mM HEPES:CH.sub.3CN (9:1, v/v) with Cys, Hcy and GSH upon increase in pH values .sub.Ext=445 nm.

(60) FIG. 4 depicts (A) Time dependent fluorescence response of L (10 M) with biothiols (Cys, Hcy and GSH) 200 equiv. each in 10 mM HEPES:CH.sub.3CN (9:1, v/v) at pH7, .sub.Ext=445 nm; (B) Micro plate fluorescence reading of L with different amino acids. Inset-fluorescent color change observed under UV lamp. (From 1-12. L only, cys, Hcy, GSH, Met, His, Tryp, Ph-al, Gly, Val, Tyr, Ser)

(61) FIG. 5 depicts the mechanism of hydrolysis of NAC by aminoacylase-1 and the corresponding fluorescence response of L (10 M) with NAC (200 equiv.) in presence of amino acylase-1.

(62) FIG. 6 depicts fluorescence response of probe (10 M) with Cys and N-acetyl cysteine.

(63) FIG. 7 depicts (A) Visual and (B) Fluorescent color changes of L (5 M) coated on TLC plates upon addition of different analytes. Fluorescent color changes were observed under hand held 365 nm UV lamp.

(64) FIG. 8 depicts fluorescence intensity at 524 nm upon addition of Cys (0-10 M).

(65) FIG. 9 depicts the mode of reaction of L with whey protein and fluorescence response of L (10 M) with Whey protein (500 l) after hydrolysis with NAC (0-500 l).

(66) The following examples, which include preferred embodiments, will serve to illustrate the practice of this invention, it being understood that the particulars shown are by way of example and for purpose of illustrative discussion of preferred embodiments of the invention.

EXAMPLES

(67) General Procedure:

(68) Different substituted salicylaldehyde on DCC coupling with vinyl acetic acid in the presence of catalytic amount of DMAP followed by cyclisation in presence of base gives vinyl coumarin. Heck reaction of vinyl coumarin with halobenzaldehyde gives the intermediate aldehyde, which upon condensation with nitromethane in presence of base gives final product.

(69) Same synthetic methods were followed for the synthesis of other derivatives with different substituted salicylaldehydes.

Example 1: Synthesis of [7-(diethylamino)-3-vinyl-2H-chromen-2-one]

(70) Under N.sub.2 atmosphere, to a solution of vinyl acetic acid (0.28 mL, 3.23 mmol) in dry CH.sub.2Cl.sub.2, DCC (667.45 mg, 3.23 mmol) was added and stirred at 0 C. for 1 hr. To this, 4-(diehtylamino) salicylaldehyde (500 mg, 2.58 mmol) and DMAP (40 mg, 0.32 mmol) were added and it was stirred at room temperature for 3 hrs.

(71) Reaction was monitored by TLC. Once the reaction was completed, the solid was filtered and to the filtrate CS.sub.2CO.sub.3 (843 mg, 2.58 mmol) was added and it was stirred for 12 hrs to complete the reaction. The mixture was washed with H.sub.2O, dried, concentrated under vacuum. The crude product was purified by column chromatography to give vinylcoumarin as greenish yellow solid. Since the compound is labile, it was stored in cold condition. Yield-56%. IR (film) max: 1707 (CO), 1597 (CC), 3017 (CCH) cm.sup.1. .sup.1H NMR (CDCl.sub.3, 400 MHz): (ppm) 1.19 (6H, t, CH.sub.3), 3.38 (4H, q, CH.sub.2), 5.26 (1H, d, J=11.45 Hz, CH), 5.98 (1H, d, J=17.40 Hz), 6.65 (1H, J=17.80 Hz, CH), 7.55 (1H, s), 6.55 (1H, dd, J=8.7 Hz and J=2.75 Hz), 6.45 (1H, d), 7.23 (1H, S). .sup.13C NMR (CDCl.sub.3, 500 MHz): (ppm) 12.48, 44.83, 97.09, 109, 115.75, 117.86, 128.87, 131.16, 135.54, 150.50, 155.80, 161.35. HRMS (ESI): m/z calculated for C.sub.15H.sub.18NO.sub.2 [M+H].sup.+ 244.31 found 244.1331.

Example 2: Synthesis of (E)-4-(2-(7-(diethylamino)-2-oxo-2H-chromen-3-yl)vinyl)benzaldehyde

(72) Vinylcoumarin (75 mg, 0.30 mmol) was taken in dry DMF, to this 4-bromobenzaldehyde (64 mg, 0.36 mMol), sodium acetate (28 mg, 0.33 mmol) and triphenyl phosphine (64.67 mg, 0.24 mmol) was added and it was purged with N.sub.2 and was added Pd(OAc).sub.2 (14 mg, 0.06 mmol). It was heated for 16 hrs at 80 C. under inert atmosphere; the completion of the reaction was monitored by TLC. Reaction mass was washed with H.sub.2O and brine solution, dried, concentrated under vacuum. The crude product was purified by column chromatography to give compound 2 as an orange red solid. Yield-80%. IR (film) max: 1696 (CHO), 1612 (CC), 3021 (CCH) cm.sup.1. .sup.1H NMR (CDCl.sub.3, 400 MHz): (ppm) 1.25 (6H, t, CH.sub.3), 3.46 (4H, q, CH.sub.2), 6.52 (1H, d, J=1.96 Hz), 6.63 (1H, dd, J=8.8 Hz), 7.20 (1H, J=16.14 Hz), 7.28 (1H, t, J=8.80 Hz), 7.55 (1H, d, J=16.38 Hz), 7.65 (2H, d, J=8.07 Hz), 7.73 (1H, s), 7.85 (2H, d, J=8.07 Hz), 9.99 (1H, s, CHO). .sup.13C NMR (CDCl.sub.3, 400 MHz): (ppm) 12.49, 44.93, 97.11, 109.30, 116.83, 126.87, 128.58, 129.15, 130.21, 135.16, 139.77, 143.98, 150.90, 155.89, 161.15, 191.63. HRMS (ESI): m/z calculated for C.sub.22H.sub.21N.sub.2O.sub.3 [M+H].sup.+ 348.42 found 348.1591.

Example 3: Synthesis of 7-(diethylamino)-3-((E)-4-((E)-2-nitrovinyl)styryl)-2H-chromen-2-one (L)

(73) Compound 2 (80 mg, 0.23 mmol) was dissolved in nitro methane (8 mL) and was added ammonium acetate (170 mg, 2.30 mmol). It was refluxed at the 85 C. for 3 hrs and reaction was monitored by TLC. After the completion of reaction, reaction mass was concentrated under vacuum and purified by column chromatography to give compound L as red solid. Yield-49%. IR (film) max: 1701 (CO), 1506 (NO), 1615 (CC), 3024 (CCH) cm.sup.1. .sup.1H NMR (DMSO-d.sub.6, 400 MHz): (ppm) 1.14 (6H, t, CH.sub.3), 3.45 (4H, q, CH.sub.2), 6.57 (1H, s), 6.74 (1H, d, J=8.80 Hz), 7.27 (1H, d, J=16.14 Hz), 7.47 (1H, d, J=8.80 Hz), 7.53 (1H, d, J=16.38 Hz), 7.64 (2H, d, J=8.07 Hz), 7.84 (2H, d, J=8.07 Hz), 8.10 (1H, s), 8.14 (1H, d, J=15.45 Hz), 8.22 (1H, J=13.45 Hz). .sup.13C NMR (DMSO-d.sub.6, 400 MHz): (ppm) 12.83, 44.64, 96.74, 108.85, 109.99, 116.08, 126.78, 127.30, 128.40, 129.66, 130.16, 130.96137.85, 139.43, 141.25, 141.78, 151.21, 155.93, 160.59. HRMS (ESI): m/z calculated for C.sub.23H.sub.22N.sub.2O.sub.4 [M+H].sup.+ 391.45 found 391.1651.

Example 4: Synthesis of 7-(dimethylamino)-3-vinyl-2H-chromen-2-one

(74) 4-(dimethylamino)salicylaldehyde was used as starting compound and same experimental procedure was followed. Yield 53%. .sup.1H NMR (CDCl.sub.3, 400 MHz): (ppm) 3.08 (6H, s, CH.sub.3), 3.38, 7.23 (1H, d, J=11.00 Hz), 6.70 (1H, dd, J=8.70 Hz), 6.61 (1H, J=8.80 Hz), 6.51 (1H, s), 6.07 (1H, d, J=17.61 Hz), 5.32 (1H, d, J=11.25 Hz). .sup.13C NMR (CDCl.sub.3, 100 MHz): (ppm) 40.19, 97.66, 109.31, 116.11, 128.63, 131.10, 138.52, 150.50, 155.80, 161.35.

Example 5: Synthesis of (E)-4-(2-(7-(dimethylamino)-2-oxo-2H-chromen-3-yl)vinyl)benzaldehyde

(75) Vinyl derivative obtained in the above step is coupled with bromobenzaldehyde using the same procedure mentioned previously. Yield 69%. .sup.1H NMR (CDCl.sub.3, 400 MHz): (ppm) 3.06 (6H, s, CH.sub.3), 6.62 (1H, d, J=1.93 Hz), 6.68 (1H, dd, J=8.80 Hz), 7.21 (1H, J=16.16 Hz), 7.29 (1H, t, J=8.78 Hz), 7.53 (1H, d, J=16.33 Hz), 7.68 (2H, d, J=8.17 Hz), 7.70 (1H, s), 7.83 (2H, d, J=8.07 Hz), 9.99 (1H, s, CHO). .sup.13C NMR (CDCl.sub.3, 100 MHz): (ppm) 41.13, 97.16, 109.33, 115.83, 124.87, 127.51, 128.15, 130.11, 134.12, 139.71, 143.93, 150.90, 155.81, 161.16, 191.68.

Example 6: Synthesis of 7-(dimethylamino)-3-((E)-4-((E)-2-nitrovinyl)styryl)-2H-chromen-2-one

(76) Same procedure was followed as mentioned previously. Yield 43%. .sup.1H NMR (DMSO-d.sub.6, 400 MHz): (ppm) 3.05 (6H, s, CH.sub.3), 6.67 (1H, s), 6.80 (1H, d, J=8.80 Hz), 8.22 (1H, d, J=13.69 Hz), 8.13 (2H, d, J=8.80 Hz), 7.85 (2H, d, J=8.80 Hz), 7.66 (2H, d, J=8.07 Hz), 7.54 (2H, d, J=11.25 Hz), 7.49 (1H, d, J=9.05 Hz), 7.28 (1H, d, J=16.38 Hz), 8.22 (1H, J=13.45 Hz). .sup.13C NMR (DMSO-d.sub.6, 400 MHz): (ppm) 41.14, 95.71, 108.80, 108.91, 116.83, 126.70, 127.10, 128.10, 129.16, 130.66, 130.13, 139.43, 141.15, 141.68, 151.12, 155.33, 160.69.

Example 7: General Experimental Methods for UV-Vis and Fluorescence Studies

(77) 510.sup.3M stock solution of 7-(diethylamino)-3-((E)-4-((E)-2-nitrovinyl)styryl)-2H-chromen-2-one was prepared in CH.sub.3CN and the same solution was used for all the studies after appropriate dilution. Unless and otherwise mentioned, 10 mM and pH 7 solution of aq. HEPES buffer was used for all spectroscopic studies. All amino acid solutions of 1.010.sup.1M were prepared in HEPES buffer (pH 7). For spectroscopic measurements, stock solution of the probe was further diluted by using HEPES:CH.sub.3CN (9:1) mixture and the effective final concentration was made as 10 M. All luminescence measurements were done using .sub.ext=445 nm with an emission slit width of 2 nm. The fluorescence quantum yield was determined according to literature method using fluorescein (in 0.1M NaOH) as reference (.sub.f=0.92).

Example 8: General Procedure for Enzymatic Study

(78) Cipla made effervescent tablets of N-Acetyl-Cysteine were purchased from commercially available sources. Based on the quantity of NAC present in the tablet, 1.010.sup.1M tablet solution was prepared in 10 mM aq.

(79) HEPES buffer solution (pH7). Enzyme solution was prepared according to the requirement by dissolving 1 mg/ml in 10 mM aq.HEPES buffer solution (pH7). A fixed concentration of NAC (200 equiv.) was added to the 10 M probe in HEPES:CH.sub.3CN (9:1, v/v). Since 1 mg of solid enzyme contains 3301 units of protein and 1 unit can hydrolyse 1 M of substrate, accordingly enzyme concentration was varied with respect to the substrate concentration.

(80) 10 M of 7-(diethylamino)-3-((E)-4-((E)-2-nitrovinyl)styryl)-2H-chromen-2-one is treated with 200 equiv. of NAC and different concentration of enzyme (0-2000 units) was added and the resulting solution was incubated at 37 C. for the time period ranging from 45 to 50 mins. Initially there is no fluorescence because NAC cannot bind to probe and fluorescence is turned on gradually with time, which indicates the hydrolysis of NAC by Amino acylase-1 to release of Cys. Fluorescence intensity increases with the increase in concentration of enzyme (0 to 2000 units) as more and more amounts of Cys was released from NAC by the enzymatic activity (FIG. 6). Initially, the same experiment in room temperature but reaction was slow, and took more than 90 min. Incubation at the temperature 37 C. aided in faster reaction, which indicates that enzyme activity was much higher at body temperatures.

Example 9: Preparation of TLC Test Strips

(81) TLC test strips were prepared by coating 5 M of 7-(diethylamino)-3-((E)-4-((E)-2-nitrovinyl)styryl)-2H-chromen-2-one solution in acetonitrile on silica TLC plates. 5 l of Cys (1.010.sup.1M) in 10 mM aq.HEPES buffer (pH7) was added on it, dried and the visual as well as fluorescence colour changes were observed after 5 min. The same was repeated for Hcy and GSH as well. To detect Cys in blood plasma, 20 l of blood plasma was diluted (10 l plasma+10 l buffer) was added on probe quoted TLC plate. Same methodology was repeated for enzymatic reaction on TLC plate by using 10 l of NAC and amino acylase-1 enzyme.

Example 10: General Procedure for Detecting Cysteine from Raw Milk

(82) Raw milk was subjected to fermentation and the liquid whey which was settled above after fermentation process was separated by filtration. A varying amount of whey ranging from 500 l was added to Probe L (7-(diethylamino)-3-((E)-4-((E)-2-nitrovinyl)styryl)-2H-chromen-2-one) (10 M) in HEPES:ACN at pH 7. Varying concentrations of N-Acetyl Cysteine (0-500 l) was added to each of the above solution in order to hydrolyse the bound Cysteine present in whey protein (NAC solution was prepared by dissolving NAC tablets in buffer as mentioned in the enzymatic study). Luminescence changes were recorded after 45-50 minutes of incubation at room temperature. The remaining derivatives of probe L may give similar kind of response.

ADVANTAGES OF INVENTION

(83) a) Simple process of detection b) Selective determination of cysteine c) Economical advantage of material and process d) This method is of much practical significance in real time monitoring as it could be done without the aid of any instruments. e) The present invention provides new pathways for designing the highly selective and sensitive fluorescent probes for Cysteine. f) Reagent could be used for detection of Cysteine present in milk whey. g) Reagent can be used for monitoring enzymatic activity. h) Reagent could be used for labeling the cysteine residues present in proteins, which in turn helps in studying the protein structure and dynamics