NOVEL ECO-FRIENDLY INSECTICIDES BASED ON SILICON

COMPOUNDS AND PROCESS OF PREPARATION THEREOF FIELD OF INVENTION

The present invention relates to a novel biodegradable active insecticides. More particularly, the present invention provides novel insecticidal actives, silicon-based compounds or a salt thereof and process for preparation thereof. The present invention also provides the compositions comprising said novel biodegradable active insecticides and methods for the control and/or prevention of insect/pest using said compositions.

BACKGROUND OF THE INVENTION

There is a whole host of chemicals employed in plant protection practices around the world for pest and disease control. The organochlorine insecticide, DDT [ 1, 1, 1- Trichloro-2,2-bis(4-chlorophenyl)ethane] is globally known for its insecticidal property and to control malaria vector population. The discovery of DDT in 1948, and thereafter particularly after 1962 due to a book published by Rachel Carson, DDT came into limelight for its hazardous nature. Apart from the success of DDT and its application as an insecticide, the demerit of DDT is that it gets accumulated in the body of animal particularly predators, including human being. In addition to this, because of non-biodegradability nature, it gets trasported through the various food chains, and causes breast cancer, pancreatic cancer, reproductive disorder, etc. A variety of synthetic pesticides were discovered, designed, and evaluated for their pesticidal activity. The organochlorines were followed by even more powerful insecticides such as organophosphate, carbamate, and other groups of active agents. Although these insecticides provided excellent control of major pests of crops, they posed considerable environmental problems, such as adverse effects on aquatic life like fish and wildlife, high toxicity to mammals, biomagnification, and persistence in water, soil, and food crops. Hence, a need exists for the development of new eco- friendly insecticide compounds. Responding to these concerns, experts and stakeholders in plant protection and disease vector control programs shifted their research focus to develop and synthesize new environment-friendly compounds, use as an alternative active agents. Thus, a new era for the development and practical uses of safe and bio-rational products dawned.

To overcome the biodegradation problems regarding DDT [l, l, l-Trichloro-2,2- bis(4-chlorophenyl)ethane], the inventors of present invention developed an efficient and novel synthesis of eco-friendly silicon-based isosteres of DDT. The said novel compounds are biodegradable and are effective as an insecticides as well as insect repellents.

The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

OBJECTIVES OF THE INVENTION:

• The primary objective of the present invention aims to provide an eco-friendly insecticides.

• One more objective of the present invention is to provide a novel silicon-based compounds possessing insecticidal and insect repellent properties.

• One more objective of the present invention to provide a biodegradable and safe isostere of l, l, l-Trichloro-2,2-bis(4-chlorophenyl)ethane (DDT).

• Another objective of the present invention is to provide an insecticidal compounds with a significant reduction in ecological and toxicological problems thereby overall making the compounds environment-friendly.

• Another objective of the present invention is to provide a process for the synthesis of novel silicon-based compounds possessing insecticidal and insect repellent properties.

• Yet another objective is to provide a compositions and formulations of said silicon-based compounds for effective pest control and insect repellent. SUMMARY OF THE INVENTION:

The present invention provides a novel silicon-based compounds or salt thereof with the general formula (I);

FORMULA (I)

Where, and Rj" represent halogen, hydrogen, alkyl, aikoxy or aryi group. In. a specific embodiment of the invention, R.i and R2 represent chlorine, fluorine or metltoxy group.

in yet another specific embodiment of the invention, R3 represents chlorine or hydroxy! group.

in yet another specific embodiment, of the invention, .R , R* and Rs represents chlorine or hydrogen a torsi,

One more embodiment, of the present invention relate to processes for Synthesis of a compounds according to formula (I) comprising the following steps;

Step 1) Chloriiiaiion of compound of formula (H) to give mixture of chloromethyl trichlorosilane, dieh!oromethyl trichlorosilane and trichioromethyi triehiorositane compound, as represented by formula III, IV and V, respectively;

Step 2) Separation of chloromethyl trichlorosilane, _. diehloromethyl trichlorosilane and tri chloromethyl trichlorosilane by distillation technique;

Step 3) The reaction of a compound of formula (III or IV or V) obtained in step 2) with a compound of formula (VI) i the presence of an organic solvent at a temperature range from 0 to 30 °G to form a compound of formula (I).

Step 4) Purification to obtain pure of -final product.

Other embodiments of the present invention relate to processes for preparing a compound according to formula (I) as a free base, acid, or salts thereof. -Further, the embodiments relate to any synthetic intermediates, which are useful in the synthesis of a compound of formula (I) as a free base, acid, or salts thereof.

BRIEF DESCRIPTION OF DRAWINGS:

For a more complete understanding of the features and advantages of the present invention, reference is no made to the detailed description of the invention along with the accompanying figures and in which:

Figure 1: Illustrate Ί ΐ-NMR spectra of Chlorobis(4-chlorophenyl) (trichloromethyl)silane synthesized in Example 1 (compound 1).

Figure 2: Illustrate ¹³ C-NMR spectra of Chlorobis(4-chlorophenyl) (trichloromefhyl)silane synthesized in Example 1 (compound 1).

Figure 3: Illustrate IR spectra of Chlorobis(4-chlorophenyl) (trichloromethyl)silane synthesized in Example 1 (compound 1).

Figure 4: Illustrate MS spectra of C h 1 orob i s ( 4- eh 1 orophen y 1 ) (trichloromethyl)silane synthesized in Example 1 (compound 1).

Figure 5: Illustrate a bioefficacy of insecticide Chlorobis(4-chlorophcnyl) (trichloromethyl)silane (compound 1).

Figure 6: Illustrate a residual efficacy of insecticide Chlorobis(4-chlorophenyl) (trichloromethyl )silane (compound 1).

Figure 7: Illustrate a bioefficacy of Chloro(ehloromethyl)bis(4- ch 1 or opheny 1 )s i lan e (compound 3).

Figure 8: Illustrate a biodegradation studies for Chlorobis (4- chlor opheny 1) (trichloromethyl)silane (compound 1).

Figure 9: Illustrate a biodegradation studies for l, l, l-Trichloro-2,2-bis(4- chIorophenyl)ethane, [commercial available DDT] .

Figure 10: Illustrate general structure of Formula (I)

DETAILED DESCRIPTION OF THE INVENTION:

Whils the making and using of various embodiments of the present invention are discussed in detail below, it should be appreciated thai the present invention provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts. The specific embodiments discussed herein are merely illustrative of. specific ways t© make and use the invention and do not delimit the scope of the invention.

To facilitate the understanding of this invention, a number of ^' terms are defined below. Terms defined herein have meanings, as commonly understood by a person of ordinary skill in the areas relevant to the present invention. Terms such as "a", "an" and "the" are not, intended to refer to only a singular entity but include- the- general class of which a specific example may be used for illustration. The terminology herein, is used to describe specific embodiments of the -invention, but their usage does -not limit the invention, except as outlined in the claims.

The term 'salt thereof is intended to mean salts formed by the addition. of an acids, such as organic or inorganic acids, any suitable bases or hydrates and alcoholates. The organic acid may be, but is not lim ited to, acetic, formic, propanoic, methane sulfonic, benzene sulfonic, lactic, malic, citric, tartaric, succinic or maleic acid. The inorganic acid may be, but is not limited to, hydrochloric, hydrobromic, sulfuric, nitric acid or phosphoric acid. The base may be but is not. limited to, ammonia and hydroxides of alkali, or alkaline earth metals.

As _. used herein, 'halogen' refers to fluorine, chlorine, bromine, and iodine. The present invention provides a novel silicon>-based compounds or salt thereof with the general formula (Ϊ ):

FORMULA (I)

Where, "Ri and R2" independently represent the halogen, hydrogen, alkyl, aikoxy or aryl group.

In a specific embodiment of the invention, Ri and ¾> represent chlorine, fluorine or meihoxy group. In yet another specific embodiment of the invention, R3 -represents chlorine or hydroxyl group.

In yet another specific embodiment of the invention, R4, Rs and « represents chlorine or hydrogen atom.

Further, the invention most specifically relates, hut it is not limited to the following compounds of formula (I), refer .Figure .10;

Compound 1 : ^■ Chiorobis(4-chiorophenyl)(triehlorGmeti yl)silane;

Compound 2: Chiorobis(4-ehlorophenyl)(dichloromethyl)silane;

Compound 3 ; Chiorobis(4-chIorophenyi)(chloroHf! ethyl)silane;

Compound 4: Ghiorobis(4 luorophenyl)(trichlQramethyl)siIane;

Compound 5: Chlorobis(4-ftuorophcnyl)(dichloiOin ethyl) sihme;

Compound 6: Chlorobis(4-fiuorophenyj.)(chlororaethyl)silatie;

Compound 7: Chlorobis(4-m.e ioxyphenyi)(triehloro5nethyl)silane;

Compound 8: -Chiorobis(4-methoxyphenylXdichlorometJiyl)siiaii-e;

Compound 9: CJ iorobis(4-niiethosyphenyl)(chloromethyl)silaRe;

Compoun d 0: Chi rob is(4- chl orophenyl )( .meth 1) sil ane;

Compound 11 : Chloi bis(4-il' orophenyl)(methyl)silane;

Compound 12: Clilorobis{4-metl ?xyphefiyi)(metliyl)silafte;

Compound 13 : ChiorodiphenyiftricMoromethyljsilans;

Compound 14: Chloro(dichloromelhyl)di-p-tolylsilane.

Other embodiments .of the present -invention.. relate to processes tor synthesis, of a compounds having general formula (I) as a tree base, acid, or salts thereof. Further, additionally, embodiments relate to synthetic intermediates, which are useful in the synthesis of a compounds of formula (I) as a free base, acid, or salts thereof.

FORMULA (!) Wherein,

⁴ Rr and 'Ra eac independently represents any of chlorine,- fluorine arid methoxy group;

¾ ' represents any of chlorine and hydroxy! group;

'R4*, 'Rs' and each independently represents chlorine.

The acceptable salts of the compounds of the present invention include, for example, salts with hydrochloric acid, sulfuric acid, phosphoric acid, tartaric acid, methanesulfonic acid and the like are included. These salts can be formed by the usual known methods. The compounds of the present inventio or its acceptable salts may form solvates (e.g., hydrates or the like) and/or crystal polymorphs. The present invention, encompasses those various solvates and crystal polymorphs. "Solvates" may be those wherein any numbers of solvent molecules (e.g., water molecules or the like) are coordinated with the compounds of the present invention.

Process for synthesis of compounds of the present invention:

A generalize process steps for synthesis ^' of novel silicon-based compounds having insectieidal properties as disclosed in present invention is exemplified below. Further, the technique of extraction, purification and/or crystallisation, which is performed in a normal experiment of organic chemistry may be conducted.

The method process for synthesis of novel silicon-based compounds or salt thereof having general formula (I);

FORMULA (!) Wherein,

'Ri' arid 'R 2 each independently represents any of chlorine, fluorine and methoxy group;

¾' represents any of chlorine and hydroxy! group;

¾5 ^: and 'Re' each independently represents chlorine; comprising the following steps;

Step 1) Chlormation of compound of formula (II) using of chlorine gas in the presence of UV light to give -mixture of ehloromethyl trichiorosilane, dichioromeihyl irichlorosilane. and Iriehloromethyl irichlorosiiane (Compounds of formula 111, T.V and V respectively).

FORMULA (II)

FORMULA (III) FORMULA (IV) FORMULA (V) Step 2) Separation of chlorinated compounds by distillation under nitrogen purging: The separation of ehloiOfflethyl trichiorosiiane and dichioromethyl irichlorosilane was done at temperature range between 100-120 °C and 140- 155 °C respectively. The compound, trichloromethyl Irichlorosilane was left as a whits waxy solid in reaction, vessel. Step 3) The reaction of specific chlorinated compound obtained in step 2, (compound of formula III, formula ^' IV or form la V) with a Grignard reagent having general formula (VI) in the presence of an -organic solvent at a temperature range from 0 to 30 °C to obtain a compound of formula (I).

FORMULA (VI)

Wherein

~R ^' represents halogen, hydrogen, alky!. aikoxy or aryl group;

And 'X' represents any halogen.

Farther, the Grignard reagent optionally he synthesized .in-sita lor this step, magnesium turning was taken in glass reactor and dry THF was added in it. To this refl xing suspension l-brotno-4-cWorobenzene was added with crystal of. -iodine. The reaction was stirred for 3 h.

Step 4) Purification by any suitable method.

In an embodimentof the process disclosed in the present invention, the chlorinatio in step 1) is selected from any conventionally available method such as ehlorittaiion of methyl trichlorosilane using chlorine gas in the presence of UV light/visible light or addition of suitable chlorinating agents.

In an embodiment of the process of the present invention, the reaction of step 1) is carried out for 0.5- 15 h between temperature range from 50 to 200 "C. i an. embodiment, of the process of the present invention, -the reaction of step 3} is carried out for 0.5- 15 h. A general method of the present invention i represented by the following representative scheme:

Step 1: Chlorination

Step 2: Separation

Step 3 : Reaction with Grigna ^' reagent

Step 4: Purification.

Alternatively, in accordance to one more embodiment, the present invention provides a compounds of general fommla (V) and. rocess of preparation thereof;

FORMULA (V)

Wherein, and ' j' independently represent the halogen, hydrogen, aJkyl, alkoxy or aryl group;

¾' represents chlorine or hydroxyl group. The process for synthesis of compounds of general formula (V) comprising the following steps; Reaction of methyl trichlorosilane having formula (II) with Grignard reagent of formula (VI) in the presence of an organic solvent at a temperature range from 0 to 30 °C to form a compound of formula (V). The general reaction is shown as follows.

In an embodiment of the process of the present invention, the organic solvent is selected from any conventionally used organic solvent such as but not limiting to tetrahydrofuran, diethyl ether, dimethyl ether, isopropyl ether, toluene, and xylene.

In a preferred embodiment, the process for the preparation of the novel silicon- based compounds of formula (I) is carried out in a nitrogen or argon atmosphere.

The compounds as disclosed in the present invention is having all of the following excellent characteristics :

i) Insecticidal activity

ii) Pesticidal activity

iii) Pest repellent activity

For the purpose of treating the plants, insects and pests, the compounds of the present invention may be formulated as a powder, a granule, tablets, capsules, pills, a liquid and any suitable formulation thereof. The effective doses of the present compounds may be mixed with excipients suitable for the dosage form, such as fillers, binders, disintegrators, and surfactants, as appropriate, to form stable preparations. In accordance to another embodiment of the invention also involves the application of the novel silicon-based compounds of formula (I) and its used in a method to treat or prevent pest or insect. In another specific embodiment, the formulation comprising any selected compound having general formula (I) and additives selected from any of the conventionally used additives, but not limiting to fillers, binders, disintegrators, and surfactants, pH modifying agents, thickening agents, viscosity modifying agents, surfactants and mixture thereof.

The compounds of the present invention can be used in combination with other agents/active agents or the like (hereinafter referred to as combination formulation) to increase the activity of the compounds, reduce the dose of the compounds, or the like.

The present invention is further described with the help of the following examples, which are given by way of illustration.

The analysis obtained in each reference example was carried out on following instruments:

NMR: analysis were recorded on an Agilent 500 MHz nuclear magnetic resonance Spectrometer.

HR-LCMS: Column zorbax SB c l8, (2.1 mm x 150 mm 1.8 μιη). Agilent Technologies, USA: 1290 Infinity UHPLC System, 1260 infinity Nano HPLC with Chip cube, 6550 iFunnel Q-TOFs.

FTIR:

Fourier transfer infrared characterization was recorded on Shimadzu 8400s spectrometer. All the spectra were measured at a 4 cm ^"1 resolution and scanned from 4000 to 400 cm ^"1 EXAMPLES:

Example 1: Synthesis of Compounds from 1 to 9

Step 1: Chlorination: Chlorination reaction assembly was completely dried and flushed using nitrogen gas. 25 g of methyl trichlorosilane (formula II) was taken in a glass reactor. Chlorination of methyl trichlorosilane was conducted at temperature between 70 °C to 155 °C in the presence of ultraviolet/visible light by passing chlorine gas (40 ml min) which was dried by passing through sulphuric acid. The unreacted chlorine gas and generated hydrochloric acid gas were passed into empty scrubber and further neutralized in sodium hydroxide solution and then in water. The reaction was conducted for 3 to 15 h under reflux condition. After completiong of reaction, the reaction mass consist of a mixture of chloromethyl trichlorosilane, dichloromethyl trichlorosilane and trichloromethyl trichlorosilane. The effect of reaction temperature on the selectivity for chlorinated compounds shown in table 1. The selectivity of the products were studies using HPLC analysis.

Table 1. Temperature optimization for chlorination for selectivity towards trichloromethyl trichlorosilane:

Distillation % Selectivity

Temperature chloromethyl dichloromethyl trichloromethyl

(°C) trichlorosilane trichlorosilane trichlorosilane

70-80 80 20 -

110- 120 - 25 75

135- 155 - 7 93

Step 2. Separation of chlorinated compounds: Separation of chloromethyl trichlorosilane, dichloromethyl trichlorosilane and trichloromethyl trichlorosilane from reaction mass was done by distillation at different temperatures. The fractions of distillate were collected in three moisture-free conical flasks in series which were cooled in an ice bath. First collection was at 60 to 80 °C of unreacted methyl trichlorosilane, second collection was at 100-120 °C of chloromethyl trichlorosilane, third collection was at 140-155 °C of dichloromethyl trichlorosilane and remaining in the round bottom flask was white waxy solid of trichloromethyl trichlorosilane.

Step 3: Reaction assembly was completely dried and flushed using nitrogen to remove moisture. Schlenk technique was used for the moisture free reaction. Freshly dried tetrahydrofuran was placed in glass reactor and added chlorinated product (chloromethyl trichlorosilane, dichloromethyl trichlorosilane or trichloromethyl trichlorosilane). Grignard reagent was added drop wise via cannula through the addition funnel at temperature 0 °C under constant stirring. The molar ratio of chlorinated compound to Grignard reagent was 1 :2. After complete addition of Grignard reagent, the temperature of reaction mass was slowly increased up to 30 °C and kept constant for 0.5-15 h.

Step 4: Purification of the product: Ethyl acetate was added to the reaction mixture. White precipitate of magnesium salt was formed and separated by centrifuge. Ethyl acetate and THF was evaporated on a rotary evaporator. Yield and purity of different final products are given in table 2. The characterization data of final products is given in table 3.

Table 2. Yield and purity of synthesized compounds

Table 3. Characterization of compounds:

Example 2: Synthesis of Compound 10 (Chlorobis(4-chlorophenyl)(methyl) silane)

Step 1: Reaction assembly was completely dried and flushed using nitrogen to remove moisture. Schlenk technique was used for the moisture free reaction. Freshly dried tetrahydrofuran was placed in glass reactor and added Methyltrichlorosilane. To this, 4-chlorophenyl magnesium bromide was added drop wise via cannula through the addition funnel at temperature 0 °C under constant stirring. The molar ratio of Methyltrichlorosilane to 4-chlorophenyl magnesium bromide was 1:2. After complete addition of 4-chlorophenyl magnesium bromide, the temperature of reaction mass was slowly increased up to 30 °C and kept constant for 0.5-15 h.

Step 2: Purification of the product: Ethyl acetate was added to the reaction mixture and solid magnesium salt was separated by centrifuge. Ethyl acetate was evaporated on a rotary evaporator. Yield-84 % and Purity-92 %. IR: 3024.76, 2961.47 and 2904.16 cm ^"1 (Aromatic C-H), 1643.76, 1578.93 and 1484.29 (Si-Ph), 1078.62 and 1015.04 cm ^"1 (Si-CH3), 785.56 cm ^"1 (Aliphatic C-Cl stretching), 651.98 cm ^"1 (Si-Cl).

Example 3: Evaluation of bio-efficacy of synthesized compounds w.r.t. DDT:

WHO cone bioassay method: The test solutions of different concentration were prepared in THF (0.05 % w/w, 0.10 % w/w and 0.20 % w/w) and sprayed on the surface (Cement surface) and air dried. Two mosquitoes species namely A nopheles stephensi and Anopheles Culicifacies, (laboratory bread susceptible, nonblood feed) of aged 2-5 days old were introduced into WHO plastic cone ( 12 cm diameter) placed surface treated with active agent (10 mosquitoes for each bioassay/cone). Mosquitoes were exposed for 30 min on the every treated surfaces and observed knockdown at 5 minutes interval up to 30 minutes. The mosquitoes were transferred to 150 ml plastic cup having 10 % sugar/sucrose solution. The plastic cup was placed in room maintained at standard temperature 27 °C ± 2 °C and relative humidity of 80 % ± 10 % RH. Percent mortality were calculated after 24 h.

For control study, the same procedure followed as mentioned above, wherein the surface was not sprayed with test compound.

I) Bioefficacy of chlorobis(4-chlorophenyl)(trichloromethyl)silane (Refer as ID) synthesized compound 1 w.r.t. DDT (Refer as D):

a) % Mortality: As depicted in Figure 5, the chlorobis(4- chlorophenyl)(trichloromethyl)silane and DDT showed 100 % mortality for both species i.e. A. Stephens! and A. Culicifacies.

Comparing to DDT, mortality of newly synthesized compound 1 as disclosed in present invention, have the same mortality ( 100 %), and this proves that this newly synthesized compound is as effective as DDT. b) Residual Efficacy:

This study was carried out for one week. Results are depicted in Figure 6.

• For Concentration 0.05 % w/w, mortality for chlorobis(4- chlorophenyl)(trichloromethyl)silane was about 70 % for both species; whereas mortality for DDT for A. Stephensi for was 60 % and for A.

Culicifacies 70 %.

• For Concentration 0.10 % w/w, mortality for chlorobis(4- chlorophenyl)(trichloromethyl)silane was about 70 % for A. Stephensi and 60 % for A. Culicifacies; whereas mortality for DDT was 60 % for both species

• For Concentration 0.20 % w/w, mortality for chlorobis(4- chlorophenyl)(trichloromethyl)silane was about 80 % for A. Stephensi and 70 % for A. Culicifacies; whereas mortality for DDT was 70 % for both species Hence, newly synthesized compound (Compound 1) have better mortality as compared to standard DDT. The said compound can be effectivily useful as an insecticide/pesticide for alternative to non- biodegradable, hazardous DDT.

II) Bioefficacy of chloro(chloromethyl)bis(4-chlorophenyl)silane synthesized compound 3 :

a) % Mortality: The results are depicted in figure 7. It shows that 20 % mortality was found for 0.05 % w/w of the test compound after 24 h. When 0.10 % w/w compound was sprayed, then up to 30 min mortality was 20 % and up to 24 h mortality was 50 %. For 0.20 % w/w of the test compound, mortality at 20 min, 25 min, 30 min and 24 h was 10 %, 30 %, 60 % and

100 % respectively. Table 4. BioefOcaey of novel compounds synthesized as per present invention for Concentration 0.20 % /w:

No. of mortality

No. of dead mortality

s seeis (One week)

DDT( reference) Example 4: Evaluation and comparison for decomposition rate of synthesized compound chlorobis(4-chlorophenyl)(trichloromethyl)silane (silicon isosteres of DDT) as per present invention and l,l,l-Trichloro-2,2-bis(4- chlorophenyl)ethane (DDT).

Procedure: Degradation of compounds were studied using enzymatic soil-based method.

A. Soil based study

i) Soil treatment: The red lateritic soil was taken from Institute of Chemical Technology, Mumbai, India, garden and dried it in the oven at 100 °C. Then dry soil was sieved through the mesh having size 710 nm for soil uniformity. The organic impurities (if any) were removed by washing 500 g soil with 700 ml acetone (first wash), again washing the soil with 150 ml acetone (second wash) and dried in an oven at 55 °C. Then this soil was washed with 1 L distilled water, dried it and finally washed with a 0. 1 M acetate buffer solution of pH 4.6 and dried at 55 °C and measuring the pH 7.8 which was earlier 8.5. Sieve the soil through 710 nm sieve. This soil was used for degradation study of DDT and silicon isostere of DDT. ii) Preparation of contaminated soil and enzyme loading: Initially, the soil was autoclaved for 1 h in an autoclave and then used for the study. In the volumetric flask, the test compound (0.5 g) was dissolved in 100 ml acetone. For each study, 10 ml test compound solution was used from the volumetric flask. 100 g of soil was taken in the conical flask from prepared autoclaved soil and contaminated it with 10 ml test compound solution, mixed it properly and air dried 3h to evaporate acetone from the soil. Then, 3 ml crude laccase enzyme was added and mixed it properly, 10 ml buffer solution of pH 4.5 was added externally to maintain the acidity of the soil. The prepared soil was added in conical flask and packed by cotton. The flasks were kept at room temperature (30 °C) in a shaker at 160 rpm for 60 days. A control study was also done without enzyme for the confirmation and role of enzyme in the decomposition study. Sampling was done after every 5 days till 60 days.

Sample name: i) Control isostere biodegradation (CIB), ii) Enzymatic isostere biodegradation (EID), iii) Control DDT biodegradation (CDB) and iv) Enzymatic DDT biodegradation (EDB).

Table 5. Soil Treatment

Soil based study

DDT Isostere

With enzyme Control With enzyme Control

Buffer pH 4.6 4.6 4.6 4.6

Enzyme (ml) 20 - 20 -

Buffer (ml) 20 40 20 40

Pesticide Amount (g) 0.05 0.05 0.05 0.05

B. Sam pling procedure for analysis

5 g of soil was taken from each experimental setup separately and washed with 10 ml acetone (4 times). The acetone filtrate was collected and soil discarded. The acetone filtrate was evaporated and the leftover dissolved contents in 1ml ethyl acetate and analyzed in GC and HPLC (For DDT: Gas chromatography and for chlorobis(4-chlorophenyl)(trichloromethyl) silane HPLC) E) Result

The Chromatographic peak was recorded for all samples. The gradual disappearance of isostere peak compared with control study indicate that the isostere (compound syntheized as per present invention) was degraded with time in presence of laccase enzyme shown in figure 8. While in figure 9, the percent peak area of DDT in both case, control and in presence of the enzyme, shows almost same, indicate that isostere is bio-degraded than DDT.

The disappearance of silicon isostere of DDT was observed after 50 days, while DDT was detected even after 60 days. This suggests that silicon isostere is degraded earlier than DDT by laccase enzyme under the same set of conditions.