Combinatorial Chemistry and Drug Discovery Lab Jasmine ... - haspi

Combinatorial
Chemistry and Drug
Discovery Lab
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

Background
This lab protocol was
adapted from the original
work of Scott Wolkenberg
and Andrew Su of The
Scripps Research Institute
in La Jolla, California. The
experiment was originally
published in the June 2001
issue of the Journal of
Chemical Education and
implemented in the San
Diego area from May 2002
thru 2005.
CITATION:
Wolkenberg, Scott E.; Su, Andrew I. J Chem.Educ.
2001 78 784

Overview
• In this lab students will identify a drug that
kills bacteria by producing libraries of
compounds based on the A-B model.
• They will test the mixtures for antibiotic
activity and then isolate the individual
compound(s) which possess antibiotic
properties.
• Students t will screen the mixtures by utilizing
i
techniques used to conduct Kirby-Bauer and
Ouchterlony tests.
t

California
Science Content
Standards
Subject Area
Content Standard
Chemistry
Biology
Genetics (Molecular Biology)
Genetics (Biotechnology)
Evolution
Physiology
2.a. Chemical Bonds – formation of ionic and covalent (peptide) bonds
3.g. Stoichiometry – redox reactions, dehydration synthesis (condensation)
10.b. Organic Chemistry – bonding characteristics of carbon
10.e. Functional Groups – formation of a hydrazone from an aldehyde d and a
hydrazine, identification and analysis of amine groups
1.a. Membrane Regulation – membrane structure and function
1.c. Prokaryotic and Eukaryotic Cells – structure and function
1.h. Macromolecules – structure and function
4.c. Mutations – antibiotic expression
4.e. Proteins – structure
4.f. Proteins – function/chemical properties
5.c. Biotechnology – production of novel biomedical and agricultural
products
7.a. Natural Selection – phenotype vs. genotype
7.d. Genetic Variation – influence of environmental factors on the natural
selection of adaptive traits
8.a. Natural Selection – selective fitness; differential survival of groups
10.b. Immune Response – antibody/antigen response
10.d. Bacterial Infections – use of antibiotics in treating bacterial infections;
use of antibacterial agents to control the growth of bacteria

National
Science Education
Standards
Category
Content Standard
Unifying Concepts and Processes
Science as Inquiry
Life Science
Science and Technology
Science in Personal and Social
Perspectives
History and Nature of Science
Change, constancy and measurement – process of generating chemical
libraries and screening for effective compounds, combinatorial data
Evidence, models, and explanation – Kirby-Bauer & Ouchterlony Tests
Form and dfunction –cells, macromolecules, l prokaryotes (E.coli )
Abilities necessary to do scientific inquiry – production of mixtures
(libraries) of compounds using the A-B model; deconvolution (separation)
of the mixtures to identify the compound(s) with antibiotic properties
The Cell
– types of cells, cell membrane structure and function
Biological evolution – development of antibiotic resistance
Behavior of organisms – growing bacterial cultures (lag vs. log phase),
interpretation of bacterial plates (deconvolution)
Understandings about science and technology – process and design in
combinatorial chemistry; bringing a new pharmaceutical to market
Personal and community health – drug discovery, pharmacology,
diagnosis of human disease and course of treatment
Natural and human-induced hazards – aseptic (sterile) technique,
safety protocols including Material Safety Data Sheets (MSDS)
Science as a human endeavor – biomedical research, clinical trials,
bioethics of the biotechnology industry

Chemicals

Chemicals
CHEMICAL VENDOR CATALOG # PRICE*
A1: 2-nitrobenzaldehyde Sigma-Aldrich 772780-50g $116.50
A2: 5-nitro-2-furaldehyde Sigma-Aldrich 170968-25g $102.50
A3: 3-nitrobenzaldehyde Sigma-Aldrich
B1: 4-bromophenylhydrazine
hydrochloride
Sigma-Aldrich 143219-10g $101.50
B2: 4-cyanophenylhydrazine
hydrochloride
Sigma-Aldrich 453471-5g $42.20
B3: aminoguanidine bicarbonate Sigma-Aldrich
N10845-
100g $17.80
109266-
100g
$15.00
Total: $395.50
50

Suggested Materials

Presenter Notes
Preparing Overnight Culture of E. coli
1. Transfer 10ml of sterile LB broth in a culture tube.
2. To obtain a sample of E.coli, take the inoculating loop and dip into the frozen E. coli
stock. Be certain that some of the stock has adhered to the loop.
3. Dip the inoculating loop into the culture tube containing the 10ml of LB broth. Stir the
broth using the loop to ensure that the E. coli is thoroughly mixed in the broth.
4. Cap the tube and incubate the E. coli culture overnight at 37°C with agitation.

Presenter Notes
Growing E. Coli into Log Phase
1. Transfer 25ml of sterile LB broth in a 50ml Erlenmeyer flask.
2. Take 1ml of the prepared overnight E. coli culture and transfer the
sample into the Erlenmeyer flask containing 25ml of LB broth.
3. Cap the Erlenmeyer flask and incubate at 37°C with agitation.
ti

Presenter Notes
Spectrophotometer
1. Turn on the spectrophotometer and set to O.D. 550.
2. Take a 3ml sample of the E. coli culture and transfer into a test tube. Wipe and handle the
test tube with Kimwipes and place into the spectrophotometer.
3. Note the reading. The culture will reach log phase once the O.D. 550 reaches 0.3-0.4.
4. If the reading has not reached log phase, repeat steps 2 and 3 using a clean test tube
and waiting 30 minutes between each new reading.
5. Once the culture has reached log phase, note the total volume of culture that remains in
the flask. According to the volume, add glycerol in the amount of 15% of the total volume
to the flask and immediately transfer 1 ml aliquots of the log phase culture into cryogenic
tubes and freeze at -80ºC.

Instructor Notes
Prepare stock solutions
You have been provided 6 uniquely labeled conical tubes, each
containing i a specific chemical. Add 12 mL of deionized i d water to
each tube and shake vigorously for approximately ten seconds.

Instructor Notes
If mixtures are not completely dissolving
using a hot water bath may be effective.

Combinatorial
Chemistry and Drug
Discovery Lab
Student Protocol

Tips for handling E.coli:
Lab Safety
1. Wipe down the lab bench or station with a 10% bleach solution or 70%
isopropanol solution at the beginning and end of each laboratory session.
2. When creating mixtures and transferring chemical solutions or liquid bacterial
cultures, keep nose and mouth away from the opening of the tube to avoid
inhaling any aerosols that may be created.
3. All spills should be reported to your instructor and cleaned up immediately
according to the Material Safety Data Sheets (MSDS) for each chemical used in
the experiment. Be sure to wear proper footwear (closed toe) to prevent injury.
4. Dispose of any materials that have come in contact with bacterial cultures (i.e.
tubes, pipettes) in special waste containers as provided d by your instructor.
t
5. Wash hands with soap and water before leaving the lab.

Overview
• This lab protocol was adapted
from the original work of Scott
Wolkenberg and Andrew Su of
The Scripps Research Institute
in La Jolla, CA. The experiment
was originally published in the
June 2001 issue of the Journal of
Chemical Education.
• 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.
CITATION:
Wolkenberg, Scott E.; Su, Andrew I. J Chem. Educ. 2001 78 784

During the
EXPONENTIAL or LOG
PHASE bacteria are
growing and dividing
at the maximal rate
given their genetic
potential, nature of the
medium and conditions
under which they are
growing.
In the STATIONARY
PHASE, the total number
of viable bacteria
a
remains constant. This
may result from a
balance between cell
division and cell death or
cells may cease to divide
while remaining
metabolically active.
When microorganisms are
introduced into fresh culture
medium usually no immediate
increase in cell number occurs
therefore this is referred to as
the LAG PHASE.
Detrimental environmental
conditions such as lack of
nutrients and waste buildup
lead to the decline, usually
logarithmic, in the number of
viable cells. This is characteristic
of the DEATH PHASE.
What limiting factors would cause a microbial
population to enter the stationary phase?

•Disk-diffusion method used
for routine testing in a clinical
laboratory in which an isolated
microbe is tested t for
susceptibility to numerous
antibiotics.
Kirby-Bauer
Test
•The isolated organism is
uniformly placed on an agar
plate with paper disk of fixed
concentrations of antibiotics.
•Growth of the organism and
diffusion of the antibiotic
occur simultaneously resulting
in a circular zone of inhibition
if the antibiotic has
antibacterial properties.
Measuring Antibiotic
Resistance

Ouchterlony Test
•A double diffusion technique developed by Organ
Ouchterlony more than 40 years ago.
•AA technique in which reaction partners, antigen and
antibody, are allowed to diffuse to each other in an agar
gel in a precipitation reaction.
•Classical procedure used to detect the presence of
antibodies and determine their specificity by
visualization of "lines of identity" or precipitin lines.

Student/Group Lab Set-up
3 Luria Broth (LB) agar plates
6, 15mL conical tubes
containing stock solutions:
•A1: 2-nitrobenzaldehyde
•A2: 5-nitro-2-furaldehyde
•A3: 3-nitrobenzaldehyde
•B1: 4-bromophenylhydrazine hydrochloride
•B2: 4-cyanophenylhydrazine hydrochloride
•B3: aminoguanidine bicarbonate
1 cryotube (orange cap)
containing 1.0 mL E. coli
1 cell spreader
15 disposable transfer
pipettes or P-1000 micropipette
9 eppendorf tubes
1 plastic straw, wrapped
1 sterile wrapped transfer pipette
Conical i l tube rack
Sharpie Marker

Label transfer pipettes
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 M1, M2, M3, M4, M5, M6, A#B1, A#B2, A#B3. These
will be used to transfer your compounds onto the plates. Each student or
group will be assigned mixtures to test for confirmation of antibiotic
activity (i.e. # = 1, 2, or 3 for A1B1, A1B2, A1B3; A2B2… ).

Label epitubes

Label Petri dishes
M1 M2
M4 M5
A#B1 A#B2
M3
M6
A#B3
Each student or group will be
assigned mixtures to test for
confirmation of antibiotic activity.
(i.e. # = 1, 2, or 3 for A1B1, A1B2,
A1B3; A2B2...).
M1 M2 M4 M5 A#B1 A#B2
M3 M6 A#B3
Create wells in plates

Carefully invert the tube containing i 10
1.0
ml of thawed E. coli several times before
opening. Spread the E. coli culture evenly
on the surface of each agar plate.

Prepare mixtures
(libraries)
1. Make sure that the
proper transfer pipette is
used for each solution to
avoid contamination.
2. Add solutions in the
order indicated in the table.
Record your observations.
Tube # Add 5 drops Then 5 drops
Then 5
drops
Then 15 drops
M1 B1 B2 B3 A1
M2 B1 B2 B3 A2
NOTE:
15 drops = ~ 750µL
5 drops = ~ 250µL
1 drop = .05 mL = 50 µL
M3 B1 B2 B3 A3
M4 A1 A2 A3 B1
M5 A1 A2 A3 B2
M6 A1 A2 A3 B3

Add compounds to wells and
wait approximately 15-20
minutes for liquid to absorb.

Incubate at 37ºC or store at
room temperature overnight.
(48 hours is optimal for room temperature
option)
1. If no incubator is
available: Plates can
be stored overnight at
room temp. with the
agar side down.
2. If incubator is available:
Allow compounds to
absorb into agar then
incubate agar side up.

Chemical Structures
A carbonyl group
at the end of the
carbon skeleton
indicates that the
compound is an
ALDEHYDE
The amino group
(-NH 2 ) consists of
a nitrogen atom
bonded dto two
hydrogen atoms;
the compound is
a HYDRAZINE
Wolkenberg, Scott E.; Su, Andrew I. J Chem. Educ. 2001 78 784

Chemical Structures of
Synthesized Compounds
A special type of
covalent bond called a
peptide bond forms
between the carbon
and the nitrogen
creating a newly
synthesized
compound called a
HYDRAZONE.
The oxygen from the carbonyl
group of the aldehyde leaves
to bond with the hydrogen
from the amino group of the
hydrazine to form water. This
represents a condensation
reaction or dehydration
synthesis.
A-B Model
Wolkenberg, Scott E.; Su, Andrew I. J Chem. Educ. 2001 78 784

Combinatorial
Chemistry and Drug
Discovery Lab
Data Analysis/
Results

Data Analysis
- + = no inhibition of growth = zone of inhibition of growth
Mixture Contents Result
M1
A1, B1, B2, B3
-
M2
A2, B1, B2, B3
+
-
M3
A3, B1, B2, B3
M4 B1, A1, A2, A3 -
M5
B2, A1, A2, A3
-
+
M6 B3, A1, A2, A3

Data Analysis
Mixtures
(Libraries) M1 M2 M3
M4
A1-B1
A2-B1
A3-B1
M5
A1-B2 A2-B2 A3-B2
M6 A1-B3 A2-B3 A3-B3
Using this table, we can determine which compound in
our active mixtures has antibiotic properties. First,
shade in the column to indicate the mixture that shows
antibiotic activity for compounds M1 thru M3. Next,
shade in the row that corresponds to the mixture M4
thru M6 that shows antibiotic activity. The position in
the table where the shaded column and shaded
row intersect is the active compound.

Extension: Kirby–Bauer Test
Determining Class Average Zone Size
1. After 24 hours in the incubator or 48 hours at room temperature check for the
presence of antibiotic activity. This is done by looking for a clear area, called a
zone of inhibition, surrounding a well. Remember to never open the Petri dish
for a better view. Use the agar side to observe and measure any zone of
inhibition.
2. Using a ruler, measure the diameter (in millimeters) of any zone of inhibition and
record your individual group data on the data table provided.
3. Now gather class data to determine the average zones of inhibition, if present,
for each of the different compounds M1 thru M6 and record the averages.
4. After recording the average class data for the diameters of the zones of
inhibition, decide whether your groups sample of E.coli bacteria is susceptible
(sensitive) displaying a CLEAR zone of inhibition, unaffected (resistant)
showing no observable zone of inhibition or intermediate (somewhere in
between) ee for each of the compounds M1 thru M6. Record your conclusion c o in the
final column of the data table.

Extension: Kirby–Bauer Test
Determining Class Average Zone Size
Using a ruler measure the diameter (in millimeters) of any zone of inhibition
Using a ruler, measure the diameter (in millimeters) of any zone of inhibition
and record your individual group data on the data table provided.

Extension: Zone Size Interpretive Chart
ANTIMICROBIAL
AGENT
DISC CODE
R = mm
or less
I = mm
range
MS =
S = mm
or more
amoxicillin (Staph) AMC 19 20
amoxicillin (other
bacteria)
AMC 13 14‐17 18
Concept Review
ampicillin (Staph) AM 28 29
ampicillin (other
AM 11 12‐13 14
Suggest bacteria) a reason why groups may have reported
carbenicillin
different diameters CB for 13 the zones 14‐16 of inhibition 17
(Pseudomonas)
created carbenicillin (other in response to the six compounds tested.
CB 17 18‐22 23
bacteria)
Why do you suppose most antibiotics are allowed a
cefoxatime CTX 14 15‐22 23
cephalothin
range of diameters
CF
for
14their zones
15‐17
of inhibition on
18
chloramphenicol C 12 13‐17 18
the SIR table?
erythromycin E 13 14‐22 23
gentamycin GM 12 13‐14 15
methicillin (used
for Staph only)
M (or DP) 9 10‐1313 14
penicillin P 28 29
streptomycin S 11 12‐14 15
sulfamethoxazole‐
l
trimethoprim
SXT‐TMP 10 11‐15 16
tetracycline TE 14 15‐18 19

Expected Results at 37ºC

Results at Room Temperature

Chemical Structure of the
Active Compound
5-nitro-2-furaldehyde
d
aminoguanidine bicarbonate
One of the resulting compounds created in our library of
mixtures is “discovered” to have antibacterial properties. The
active compound is a known antibiotic called guanofuracin
which was discovered in the 1950’s and developed for both
human and veterinary use.

Combinatorial
Chemistry and Drug
Discovery Lab
Post-lab
Discussion

Autoclaving is the most
effective and most efficient
means of sterilization. All
autoclaves operate on a time/
temperature relationship. The
usual standard temperature/
pressure employed is 121ºC/15
psi for 15 minutes.
Antibacterial
Agents

Three Major Classes of
Antibacterial Agents
Controlling the growth of microorganims usually
involves the use of physical or chemical agents which
either kill or prevent the growth of microorganisms.
1) Bactericidal – agents that kill bacteria are called cidal
agents; also referred to as bactericides.
2) Bacteriostatic – agents which inhibit the growth of cells
(without killing them) are referred to as static agents.
3) Bacteriolytic – agents that have the ability to lyse or
break apart, dissolve, and destroy bacteria by the use of an
enzyme or other agent.
Bacteria have the ability to develop resistance following
repeated or subclinical (insufficient) doses, so more
advanced antibiotics and synthetic antimicrobials are
continually required to overcome them.

Gram – positive
bacteria
Variations in bacterial cell wall structures not only cause
differences in staining i but the anatomy of each cell wall also
leads to differences in the susceptibility of bacteria to antibiotics.
Some antibiotics easily penetrate Gram-positive cell walls while
others are more capable of penetrating Gram-negative cell walls.
Gram – negative
bacteria

Antibacterial Sites of Action

Concept Review
• Analyze the graph by
describing the phase of
growth being represented
by lines A thru E: LAG, LOG,
STATIONARY, or DEATH.
• If the graph shown is the
result of antibiotic resistance
testing, what is the effect of
the antibiotics on the growth
of the bacteria at A thru E:
NORMAL, BACTERIOSTATIC,
RESISTANT or BACTERICIDAL?

Great Job Jasmine Erfe!