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    Combinatorial Chemistry Sample Solution

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    Lab 7: Combinatorial Chemistry

    Ojective:
    - Combinatorial Chemistry will be used to rapidly produce a library of potential drugs and explore their potential as antibiotics.

    This lab is adapted from "Combinatorial Chemistry: Antibiotic Drug Discovery" published in Doxsee and Hutchinson, Green Organic Chemistry: Strategies, Tools and Laboratory Experiments (2004).

    http://greenchem.uoregon.edu/PDFs/GEMsID73.pdf

    Laboratory strains of E. coli are relatively non-toxic, but it is still advisable to be careful. Wear gloves and goggle at all times. Avoid touching your face or any scrapes or cuts with your gloves or instruments. When finished, wipe down the bench with disinfectant and thoroughly wash your hands and arms. Dispose of all waste in the designated container. Some chemicals used in this lab are considered toxic at high concentration. We will be using them at very low concentration, but show them respect.

    The photographs illustrating the techniques were taken by Phillip Lee and Trevor Kilgannon, two Bothell High School students who helped develop this lab using materials and equipment at North.

    Pre-Lab Questions: (To be submitted via ANGEL)
    1. Summary of the procedure in your words.
    2. What is combinatorial chemistry?
    3. What is green chemistry?

    Setup and Preparation:

    1. Obtain two TSA (tryptic soy agar) plates per group. Keep these plates covered whenever possible. The more time they spend uncovered, the greater the chance of contamination.

    2. Draw four divisions on the bottom of two of your plates. [see Figure 2] Label these sectors as M1-M8

    3. Obtain a sterile glass pipette and sterilize an inoculating loop. [see Figure 1] Using a sterile glass pipette and the sterilized inoculating loop, create one hole in each sector on the two marked agar plates for a total of eight holes by pushing the large end of the pipette straight down into the agar and then pulling it straight up. Carefully remove the resulting cylinder of agar with the small end of the pipette or the inoculating loop. [see Figure 2] Sterilize the inoculating loop and dispose of the pipette in the biohazardous waste container when finished.

    Figure 1: Sterilizing the inoculation loop.

    Figure 2: Removing the cylinder of agar with an inoculation loop.

    4. Turn the two plates back over so that the tops are up. Remove the lids. Obtain a sterile cotton swab, and dip the cotton tip into the prepared test tube of E. coli culture. Using a gentle rubbing motion, swab the top of the agar with the cotton tip, trying to distribute the bacteria as evenly as possible. Turn the plate 90 degrees and re-swab, so that these paths cross the original paths perpendicularly. Dispose of the cotton swab in the biohazardous waste container! [see Figure 3]

    Figure 3: Draw divisions and swab plate with E. coli.

    Reaction:

    Solution Concentration Identity
    A1 54mg/12mL 2-nitrobenzaldehyde
    A2 51mg/12mL 5-nitro-2-furaldehyde
    A3 54mg/12mL 3-nitrobenzaldehyde
    A4 54mg/12mL 4-nitrobenzaldehyde
    B1 80mg/12mL 4-bromophenylhydrazine hydrochloride
    B2 61mg/12mL 4-cyanophenylhydrazine hydrochloride
    B3 49mg/12mL aminoguanidine bicarbonate
    B4 64mg/12mL 4-chlorophenylhydrazine hydrochloride

    5. Obtain test tubes and label them M1-M8.

    6. For test tubes M1-M4, place 5 drops of B1 in each tube. Using a new pipette (never use the same pipette for different reagents or else contamination may result) add 5 drops of B2, B3 and B4. Now add 20 drops of A1 to tube M1, 20 drops of A2 to tube M2, 20 drops of A3 to tube M3 and finally 20 drops of A4 to tube M4.

    For test tube M5-M8, place 5 drop of A1, A2, A3 and A4 into each tube. Now add 20 drops of B1 to tube M5, 20 drops of B2 to tube M6, 20 drops of B3 to tube M7 and finally 20 drops of B4 to tube M8

    Gently shake each tube for 10 seconds and record observations regarding any changes that occur.

    Screening:

    7. Using a clean pipette for each mixture, carefully add one or two drops of each mixture to the appropriately labeled holes (M1-M8) on the agar plates. [see Figure 4] If you spill any reagent on top of the agar or put mixture in the wrong hole, be sure to make note. These incidents could affect your ability to correctly interpret your results.

    Figure 4: Adding the mixture to the agar hole.

    8. Cover and stack your plates, placing them in the designated location. Be sure not to spill any liquid out of the holes while carrying your plates. The plates will incubate overnight at 37 deg C.

    9. Return to the lab and check your results. Sketch or photograph the plates and recording observations. For each division on the plate, decide whether or not there was any growth of the bacteria. Make the answer a simple yes or no. You may have some mixtures with low antibiotic activity, but we are looking for maximum antibiotic activity.

    Results and Post-Lab Questions (20 pts)
    - Sketch of photographs of plates
    - Clear and legible observations about the appearance of the mixtures and the plates.
    - Typed answers to the Post Lab questions. Note that single sentence answers will not suffice. State the answer to the question followed by a brief description of the evidence supporting that answer.

    1. Which of the mixtures M1-M8 appeared to significantly inhibit growth of E. Coli? What was the visual indication of inhibition of growth?

    2. Complete the following table illustrating the composition of each of the mixture M1-M8. Some entries have been made to illustrate how to do this.

    Mixture M1 M2 M3 M4
    Components A1 A2 A3 A4
    M5 B1 A1-B1
    M6 B2
    M7 B3 A3-B3
    M8 B4

    3. Shade the column corresponding to the mixture M1-M4 that displayed antibacterial activity. Shade the row corresponding to the mixture M5-M8 that displayed antibacterial activity. The intersection point gives the identity of the active compound. Draw the structure of this compound.

    4. How many reactions and antibiotic screenings would have been required to carry out this analysis by non-combinatorial means? If we were interested in examining 50 different aldehydes and 50 different hydrazines, how many reactions and screenings would be required to carry out this analysis by non-combinatorial means? How many using combinatorial techniques? Hint: consider the grid that would be created by having 50 A compounds and 50 B compounds.

    5. What is "green" about this experiment or combinatorial chemistry in general?

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    Lab 7: Combinatorial Chemistry

    Ojective:
    - Combinatorial Chemistry will be used to rapidly produce a library of potential drugs and explore their potential as antibiotics.

    This lab is adapted from "Combinatorial Chemistry: Antibiotic Drug Discovery" published in Doxsee and Hutchinson, Green Organic Chemistry: Strategies, Tools and Laboratory Experiments (2004).

    http://greenchem.uoregon.edu/PDFs/GEMsID73.pdf

    Laboratory strains of E. coli are relatively non-toxic, but it is still advisable to be careful. Wear gloves and goggle at all times. Avoid touching your face or any scrapes or cuts with your gloves or instruments. When finished, wipe down the bench with disinfectant and thoroughly wash your hands and arms. Dispose of all waste in the designated container. Some chemicals used in this lab are considered toxic at high concentration. We will be using them at very low concentration, but show them respect.

    The photographs illustrating the techniques were taken by Phillip Lee and Trevor Kilgannon, two Bothell High School students who helped develop this lab using materials and equipment at North.

    Pre-Lab Questions: (To be submitted via ANGEL)
    1. Summary of the procedure in your words.
    2. What is combinatorial chemistry?
    3. What is green chemistry?

    Setup and Preparation:

    1. Obtain two TSA (tryptic soy agar) plates per group. Keep these plates covered whenever possible. The more time they spend uncovered, the greater the chance of contamination.

    2. Draw four divisions on the bottom of two of your plates. [see Figure 2] Label these sectors as M1-M8

    3. Obtain a sterile glass pipette and sterilize an inoculating loop. [see Figure 1] Using a sterile glass pipette and the sterilized inoculating loop, create one hole in each sector on the two marked agar plates for a ...

    Solution Summary

    Combinatorial chemistry sample solution are examined.

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