The their outer membrane protects them from many antibiotics.)

The aim of this practical is to synthesise a small library of compounds by both traditional and a combinatorial method and to further evaluate them by testing their antibacterial activity against microorganism (E.coli). By doing this we will gain experience in drug development….Bacteria are small prokaryotic organisms that vary widely in shape and may be seen as individual cells or as a group of cells.2Escherichia coli (E. coli) was first discovered in 1885 by Dr Theodor Escherich, and have been widely used in microbial genetics for cloning the genetic material. It is gram-negative (a group of bacteria that do not retain the crystal violet stain used in the gram-staining method of bacterial differentiation; their outer membrane protects them from many antibiotics.) non-sporing rod, reduce nitrates to nitrites and ferment glucose with the formation of acid and gas.The cell growth rate increases as the temperature rises; normal temperature range for E. coli is between 20°C and 37°C. Survival at a high/low pH mostly depends on the environment in which the E. coli is in.  In the digestive tract, it can pass through stomach acid (pH=2) and reach all the way to the end of the digestive tract at the neutral pH, where the presence of salt kills the organism and not the change in pH. Why hydrazones?In this experiment, we produced a library of hydrazones which are known for having diverse medicinal and pharmacological antimicrobial, anti-inflammatory, analgesic etc properties.Hydrazones are organic compounds which are related to ketones and aldehydes by the replacement of the oxygen with the NNH.These compounds contain C = N bond, which is conjugated with a lone pair of electrons of the functional nitrogen atom.The nitrogen atoms of the hydrazones are nucleophilic and the carbon atom has both electrophilic and nucleophilic nature. The ?-hydrogen of hydrazones is more potent than that of acidic ketones.5 The combination of hydrazones with other functional group leads to compounds with unique physical and chemical character.We used both traditional and combinatorial methods to make the hydrazones.The cup agar diffusion method is used to observe the mixtures for antibiotic activity.  If the solutions contain a compound with antibiotic activity, there will be no growth of the microorganism in a circle surrounding the cupTraditional method:Traditional synthesis usually distinguished by the use of several synthetic steps in the preparation of one compound.3 Usually, it is one compound at a time, in one reaction at a time.In this experiment, three students are chosen to mix each A1, A2 and A3 with all three hydrazines, one at the time e.g. A1/B1, A1/B2, A1/B3 etc. in the condensation reaction to produce 9 hydrazones and water as the excess product. The basic formula for this reaction is A1+B2 = A1-B2 etc. It is advised to perform the experiment in a fume cabinet to avoid the inhalationof harmful fumes that may cause respiratory irritations and breathing difficulties.To prepare each hydrazone, take 1mL of the A solution and add to 1 mL of the B solution in a test tube. Swirl for 10-20 sec.Combinatorial method:Combinatorial is based on efficient, parallel synthesis, in that many more chemical compounds can be generated in a library than the number of steps used in the synthesis.3 Simply saying combinatorial synthesis is the ability to generate large numbers of chemical compounds very quickly.·         The number of tests required for identifying the desired property of the drug is decreased if those assays are carried out on mixtures of compounds instead of on individual compounds.·         Once it has been determined that a mixture has the desired property, the problem changes to identifying which of the many compounds in the mixture is the active one in the process that is called Deconvolution·         Combinatorial synthesis could make both the identification and the lead optimization process faster and more efficient·         Exploratory chem. Section, addressing specifically the early phases of the drug discovery process, has been created. It’s an effective tool to cut the average time needed to bring new medicines to market.4·         Has increased number of hits identified This method includes the preparation of the compound library by combinatorial means.Table 2 illustrates how the reagents are mixed together in a six different test tubes in a specific order and ratio. Using a different pipette for each solution, the appropriate reagents have been added to each of the six test tubes. For this method, it is crucial to be very accurate and precise because volumes of reagents that are required for each test tube are given in drops, and not mL or cm3. The experiment was conducted/ carried out per the script provided in the laboratory session.If the antibiotic was present in the solution, the diameter would be larger than 14 mm. In the mixtures that show inhibition of growth, antibiotic activity, there are three compounds which could be active.E.coli is highly susceptible to A2-B1 A2-B2 and A2-B3 hydrazones since they had large areas of inhibition. The largest A2-B3 having the diameter of 3.8cm.The M2 proximity of growth to antibiotic indicates it may have an antibiotic gene.M2 antibiotic could be bacteriostatic, meaning their growth is inhibited, shown by the dots in the area of inhibition.To analyse the data gathered by the combinatorial method we need to highlight the data that indicates the active compound i.e. the bacterial sample has a large area of inhibition. Highlight both Columns and rows. The position in the table where the highlighted row and highlighted column intersect is the active compound. Both hydrazones A2-B1 and A2-B3 show antibiotic activity i.e. have the biggest inhibition growth.

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