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).
Bacteria are small prokaryotic organisms that vary widely in shape and may be seen as individual cells or as a group of cells.2
Escherichia 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 (their outer membrane protects them from the 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.
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 NNH2.5 The functional nitrogen atom in C=N has a lone pair of electrons. The nitrogen atoms 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 Experiment 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 cup. Traditional 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 (Fig.2), 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 (Fig.1). It is advised to perform the experiment in a fume cabinet to avoid the inhalation of 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:
The combinatorial method 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 tests are carried out on mixtures of compounds instead of on individual compounds.
Combinatorial synthesis could make both the identification and the lead optimization process faster and more efficient.4 (Cabri, W. 2000)
However, once it has been determined that a mixture has the desired property, the problem changes to identifying which of the compounds in the mixture is the active one in the process. This process is called Deconvolution.
This method includes the preparation of the compound library by combinatorial means. Table 1 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 sessionIf 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. Table 2 shows that mixtures M2, M4 and M6 have diameter if the inhibited are larger or equal to 1.4cm, which means that one of those mixtures contains an active drug.Table 3 shows that 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. M4 and M6 mixtures 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 in Table 4 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. The drugs that are active belong to a family of antibiotics called nitrofurans. Both A2-B1 and A2-B3 molecules show antibiotic activity and inhibit the growth of the bacteria. A2 is a 5-nitro-2-furaldehyde that has a similar molecular structure to Nitrofuran – both molecules consist of a five-membered ring with four carbon and one oxygen atoms and a NO2 as a side chain. However, unlike A2 react, the furan is an aromatic ring with many resonance structures which makes it more stable.