Synthesis of 2,4-Dnp: Nucleophilic Aromatic Substitution

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While in the case of 4-chloroanisole, since it has methoxy substituent which is an electron donating group, it will destabilize the intermediate. The electronegative oxygen will activate the aromatic ring by increasing the electron density on the ring through a resonance donating effect (Carey, 2000). Since the ring is partially negative in charge, it will repel the nucleophile, thus an increase in the energy will be needed for the reaction to proceed.

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B. Characterization of the product

- Melting point determination

Melting point of the product was determined to know the index of purity of the synthesis product. Oil-bath method was used. The red-orange product was placed inside a capillary tube, then the melting point was observed. The experimental value was 184-188 deg. It was a few points lower than the theoretical melting point of 2,4-DNP, which is around 198-200 deg. This temperature difference might be caused by presence of 1-chloro-2,4-dinitrobenzene, which has a theoretical melting point of 53 deg.

- Reaction with carbonyl compounds

A solution of 2,4-dinitrophenylhydrazine in a mixture of methanol and sulfuric acid is known as Brady's reagent. This is used to qualitatively detect the carbonyl functionality of a ketone or aldehyde functional group.

In the experiment, small amount of the synthesized 2,4-DNP was dissolved in 1.5 mL concentrated H2SO4, added to a solution of 2mL water and 7mL 95% ethanol. The mixture was mixed thoroughly and filtered. The prepared reagent was used to test the its reaction with acetone and benzaldehyde. About 1 mL of the prepared reagent was added to a solution of 1 drop of acetone or benzaldehyde in 2 mL of 95% ethanol. The mixture was then mixed vigorously by shaking. The initial appearance of both acetone and benzaldehyde mixtures was still yellow in color. So the two test tubes were stood for a while at room temperature. After, there are precipitates observed from the two test tubes. The test tube with the benzaldehyde had orange precipitates while in the test tube with acetone, yellow precipitates were observed.

This difference in the color intensity of the 2,4-dinitrophenylhydrazones formed was due to the number of double bond conjugation of the molecule (Reusch, 2003). Since the benzaldehyde-2,4-dinitrophenylhydrazone derivative has more double bond conjugation than the 2,4-dinitrophenylhydrazone derivative of the acetone, as seen in the reactions below, the color of the benzaldehyde is deeper than that of the acetone.

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***revise to benzaldehyde

Aldehydes and ketones react with ammonia derivatives to give analogous adduct. The products of the reaction of acetaphenone with; ammonia, hydroxylamine, hydrazine and phenylhydrazine are shown below.

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REFERENCES:

Carey, FA. 2000. Organic Chemistry.4th ed. McGraw-Hill College, Boston, MA

Carey FA, RJ Sundberg. 2007. Advanced Organic Chemistry, Part A- Structure _ and Mechanisms. 5th ed. Springer US.

Gallardo I, Guirado G, Marquet J. 2002. Journal of Organic Chemistry. vol. 67. p.2548 - 2555

Hill G, Holman J. 2000. Chemistry in Context. 4th ed. Nelson Thornes.

King, C.J. 1980. Separation Processes. 2nd ed. McGraw Hill.

Reusch, W. 2003 “Visible and Ultraviolet Spectroscopy”. Retrieved January 29, 2017 from https://www2.chemistry.msu.edu/faculty/reusch/virttxtjml/spectrpy/

uv-vis/spectrum.htm

Vollhardt KP, NE Schore. 2011. Organic Chemistry. 6th ed. W. H. Freeman.

Wells FB, CFH Allen. 1935. "2,4-DINITROANILINE". Organic Syntheses, Inc.Retrieved 13October2013from http://orgsyn.org/demo.aspx?prep=CV2P0221.

APPLICATION

The research aims to develop a quick method for the preliminarily screening of mutant strains that can accumulate 9α-hydroxyandrost-4-ene-3,17-dione (9α-OH-AD), a high-throughput screening method was presented by applying the principle that 2,4-dinitrophenylhydrazine (DNPH) can react with ketones to produce precipitation. The optimal color assay conditions were the substrate androst-4-ene-3,17-dione (AD) concentration at 2.0 g/L, the ratio of AD to DNPH solution at 1:4, and the sulfuric acid and ethanol solution percentages in DNPH solution at 2% and 35%, respectively. This method was used to preliminarily screen the mutants of Rhodococcus rhodochrous DSM43269, from which the three ones obtained could produce more 9α-OH-AD. This DNPH color assay method not only broadens screening methods and increases screening efficiency in microbial mutation breeding but also establishes a good foundation for obtaining strains for industrial application. The different concentrations of DNPH with the substrate AD reacting have different results. The red precipitation became increasingly evident as the AD to DNPH solution volume ratio decreased. According to the exp, the wild type R. rhodochrous DSM43269 can completely decompose 2.0 g/L of AD. Moreover, 2.0 g/L of AD can produce distinct red precipitation with DNPH. Thus, the optimal substrate concentration to screen mutants in a short time was set as 2.0 g/L.

Method:

The color assay with DNPH was carried out as follows: R. rhodochrous DSM43269 was initially incubated in a 250 mL shake flask with 20 mL of the seed medium at 200 rpm and 30°C for about 20 h (OD600 2.0±0.1). Afterward, 1 mL of culture was transferred into a new 250 mL shake flask with 20 mL of the seed medium to continue growth for approximately 20 h under the same condition. The substrate AD dissolved in methanol was added into the culture at the final concentration of 2.0 g/L. The transformation was monitored by thin layer chromatography (TLC), and 300 μL of the culture was extracted and fully mixed with 300 μL of ethyl acetate to obtain the organic solutions. Afterward, 0.4 g DNPH was dissolved in 3.0 mL of concentrated sulfuric acid, slowly poured into 30 mL of ethanol solution (95% ethanol), and diluted with deionized