Combinatorial Chemistry Lab B - Creating Compound Libraries

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Objective

Students will identify a drug that kills bacteria by producing libraries of compounds based on the A-B model.

Big Idea

Techniques used in combinatorial chemistry fulfill key goals of the pharmaceutical industry.

Introduction

10 minutes

Combinatorial Chemistry is a technique used to synthesize a library of compounds and screen for a desired property. Instead of screening one compound at a time, the compounds are screened more efficiently in mixtures which maximize the use of resources while increasing the possibility of identifying an active compound.  This is a key goal of the pharmaceutical industry and thus a common practice in drug discovery.

This lab protocol was adapted from the original work of Scott Wolkenberg and Andrew Su of The Scripps Research Institute (TSRI) in La Jolla, California. The experiment was originally published in the June 2001 issue of the Journal of Chemical Education (Wolkenberg, Scott E.; Su, Andrew I. J Chem.Educ. 2001 78 784) and implemented in the San Diego area from May 2002 thru 2005.  Through collaboration between the Southern California Biotechnology Center at Miramar College, Junipero Serra High School Biotechnology and Dr. Floyd Romesberg, an expert in the area of antibiotic resistance and professor of chemistry at TSRI, the protocol was revised for implementation in a high school setting.

Revision by: Jasmine Erfe Miramar College/Lab Technician, Ericka Senegar-Mitchell Science in the City/Director; Junipero Serra High School/Science Educator, Sandra Slivka Southern CA Biotechnology Center @Miramar College/Director

Objective(s):

  • Students will successfully perform mathematical calculations required to prepare the media, reagents, and compounds needed for the research and development of a novel drug therapy based on techniques used in the field of Combinatorial Chemistry. (Lab A)
  • Students will identify a drug that kills bacteria by producing libraries of compounds based on the A-B model. (Lab B)
  • Students will test the mixtures for antibiotic activity and then isolate the individual compound(s) which possess antibiotic properties. (Lab C)
  • Students will screen the mixtures by utilizing techniques used to conduct Kirby-Bauer and Ouchterlony tests. (Lab C)

 

NOTE: Instructor's are encouraged to complete the pre-lab activities found in the lesson entitled, "Combinatorial Chemistry - Designing Novel Drug Therapies to Combat Antibiotic Resistance" before completing this laboratory investigation series.

Materials

10 minutes

For each Student Lab Group:

 - 3 LB agar plates

 - Six 15mL conical tubes (blue cap), labeled, containing the following chemicals:

 A1: 2-nitrobenzaldehyde (54 mg)

 A2: 5-nitro-2-furaldehyde (51 mg)

 A3: 3-nitrobenzaldehyde (54 mg)

 B1: 4-bromophenylhydrazine hydrochloride (80 mg)

 B2: 4-cyanophenylhydrazine hydrochloride (61 mg)

 B3: aminoguanidine bicarbonate (49 mg)

 - 1 cryotube (orange cap) containing 1.0 mL E. coli

 - 1 cell spreader

 - 15 transfer pipettes (6 will be used to prepare compounds, 9 will be used to transfer compounds onto wells and 1 will be used to transfer the E. coli culture onto the plates)

 - 1 sterile wrapped transfer pipette

 - 9 eppendorf tubes

 - 1 plastic straw, wrapped

 - Conical tube rack

 - Sharpie Marker

Methods

45 minutes

For the Instructor:

Labeling Materials

The correct labeling of the tubes and pipettes used in this investigation is critical to preventing cross contamination of the chemicals. Therefore, I walk each student group through the proper labeling of these lab materials during a whole group pre-lab discussion. Say or write the following instructions: 

1. Label 6 of the transfer pipettes A1, A2, A3, B1, B2, B3. These will be used to prepare your compounds.

2. Label 9 transfer pipettes tubes M1, M2, M3, M4, M5, M6, A#B1, A#B2, A#B3. These will be used to transfer your compounds onto the LB agar plates.

3. Label the 9 eppendorf tubes in the same manner as the transfer pipettes in step 2.

4. Set aside, being mindful to maintain aseptic conditions, until ready to use. 

For each Student Lab Group:

Preparing the Compound Library

1. Using the chart below, begin adding the required amount of each solution to the corresponding eppendorf tube. Make sure that the proper transfer pipette is used for each solution to avoid contamination.

2. Cap each eppendorf tube and shake for approximately 10 seconds.

3. Make sure that the solutions are added in the order indicated in the table and record your observations while adding the solutions and after shaking the tubes. For example, for tube M1, add 5 drops of B1, then 5 drops of B2, then 5 drops of B3, then finally 15 drops of A1.

Tube #

Add 5 drops

Then 5 drops

Then 5 drops

Then 15 drops

M1

B1

B2

B3

A1

M2

B1

B2

B3

A2

M3

B1

B2

B3

A3

M4

A1

A2

A3

B1

M5

A1

A2

A3

B2

M6

A1

A2

A3

B3

Tube #

Add 15 drops

Then 15 drops

 

A#B1

A#

B1

 

A#B2

A#

B2

 

A#B3

A#

B3

 

 

 

 

 

 

 

 

 

 

 

 

 


Results

15 minutes

At the end of this segment of the investigation students should have created nine brightly colored compounds. Please refer to the Combi Chem Lab PowerPoint Presentation SLIDE 17 for an illustration of the prepared compounds. 

Discussion

30 minutes

Instructors are encouraged to allow the chemicals to set overnight before proceeding to the next phase of the investigation. If time permits, students can discuss as a whole group the triumphs and tribulations experienced as they completed Combi Chem Lab B. Each student group can also edit and revise their group lab report during this time. Group Lab Report A and Lab Report B are examples of lab reports completed by students.