Synthesis of Penicillin Derivatives
Adapted from R. D. Whitaker, et al., Journal of Chemical Education, 87, 6, 634-636, 2010
Penicillins are a class of antibiotics that are used to treat some Gram-negative and Gram-positive bacteria. The
interaction between penicillium glaucum and E.coli was first published in a dissertation by French physician,
Ernest Duchesne, in 1897, but was largely overlooked by the scientific community. Sir Alexander Fleming is
credited with the discovery of penicillin in 1928 and shared a Nobel Prize with Howard Florey and Ernst Boris
Chain in 1945 for their research on penicillin. Penicillins were first manufactured on a large-scale during WWII
by fermentation, but after the structure was identified, it was realized that different laboratories were producing
compounds with a common core (+) 6-aminopenicillanic acid (6-APA), but different amide side chains (Figures 1
and 2). In 1957, Dr. John C. Sheehan first synthesized Penicillin V which has a phenoxymethyl side chain on the
core backbone which contains a -lactam (a cyclic amide). Since that time, a large class of -lactam antibiotics
has been synthesized.
-Lactam antibiotics are known to prevent the formation of peptidoglycan crosslinks in the bacterial cell wall, but
some bacteria produce an enzyme called -lactamase that hydrolyzes the lactam amide functional group rendering
it inactive as an antibiotic. The core backbone of penicillin (6-APA) contains multiple functional groups that can
be chemically modified in hopes of making more effective antibiotics. Most commonly, acylation of the amine
functional group has occurred (Figure 2), but esterification of the carboxylic acid has also been studied.
In this experiment, you will synthesize a penicillin compound as a part of a parallel combinatorial synthesis
scheme by performing an acylation reaction on the amine functional group of 6-APA using an acyl chloride and
then characterizing its biological efficacy as an antibiotic. The amine can act as a nucleophile attacking the
carbonyl carbon of the R–COCl group, eliminating HCl to form the new N-C amide bond (Figure 2). Amines are
also bases and if protonation of the amine occurs as HCl is produced, the reaction will cease. To prevent this
from occurring, a non-nucleophilic base (NaHCO3) will be added to react with the HCl formed.
Combinatorial chemistry is often used in drug discovery research since it allows for the simultaneous synthesis of
many potentially active compounds. The products are then screened for biological activity. In this experiment
students working in pairs will choose an acyl chloride (see list below) to react with 6-APA forming a penicillin
compound whose biological efficacy as an antibiotic can be tested in subsequent laboratory periods. To
emphasize the interdisciplinarity of science research, students from the organic chemistry laboratories will team
up with students from the microbiology laboratories to test the new penicillin compounds and the results from the
entire class will be compiled for analysis. Each new compound will be classified according to efficacy (high,
medium, and low/none), and structure-function relationships based on its effectiveness at inhibiting bacterial
growth. There is not a predetermined outcome of the experiment. By choosing to include penicillins that are not
commercially available, this experiment is meant to resemble a drug discovery process with the possibility of
creating and identifying new types of penicillin.
Experimental
You will participate in planning your experiment by choosing an acyl chloride (Table 1, below) and calculating
the amount that you need to react with the 6-APA. You will need to sign-up for which acyl chloride you will be
using. Most acyl chlorides are foul-smelling, insoluble in water and somewhat expensive, so be sure to work in
your hoods and understand the synthetic procedure prior to starting.
(Caution: Acyl chlorides are lachrymators. Keep them away from eyes, nose and mouth).
Synthesis: Week 1
Dissolve 1.08 g (0.005 mol) of (+) 6-aminopenicillanic acid (6-APA) in 3 mL of acetone in a 50 ml Erlenmeyer
flask with a stir bar. While stirring, add 20 mL of 1.07 M NaHCO3 and continue stirring for 10 minutes until
everything is dissolved. The sodium bicarbonate will deprotonate the carboxylic acid of the 6-APA making it
water soluble, and it will also neutralize the HCl formed as the reaction occurs.
Separately, place 0.01 mol of the selected acyl chloride in a medium test tube and add 1 ml of acetone. Mix the
solution vigorously until dissolved by flicking the tube (add more acetone, if needed). Slowly add the acyl
chloride solution drop-wise to the reaction flask containing the 6-APA over a 5 minute period with stirring using a
Pasteur pipet, and then stir the reaction vigorously for 40 minutes.
While the reaction is stirring, place 10 mL of DI water, 2 mL of 5 M sulfuric acid and 6 mL of n-butyl acetate in 3
separately labeled test tubes and cool them in an ice bath (to be used below). Obtain another 18 mL of n-butyl
acetate in a labeled test tube that will remain at room temperature.
When the reaction is over, pour the reaction mixture into a separatory funnel and extract three times
with 6 mL portions of the room temperature n-butyl acetate to remove any unreacted acyl chloride.
Combine the organic fractions in a labeled beaker and set them aside in your hood. Return the aqueous
solution to the separatory funnel and add 6 mL of cold n-butyl acetate. Slowly add 2 mL of the cold 5 M
sulfuric acid using a Pasteur pipet, shake and check the pH of the lower aqueous layer by taking a
small amount of the aqueous liquid out using a Pasteur pipet. Add more sulfuric acid if a pH of 2
is not achieved, and retest the pH of the lower aqueous layer. The addition of acid will reprotonate the carboxylic acid functional group on the product creating a neutral molecule that
favors the organic layer over the aqueous layer. Remove the aqueous layer and wash the
organic layer twice with 5 mL of cold DI water. Remove the aqueous layer and place the
organic layer into an Erlenmeyer flask. Inspect for water before drying over sufficient
anhydrous sodium sulfate for 15 minutes.
Using a Pasteur pipet, filter the dried solution through a slug of glass wool loosely packed
in a second Pasteur pipet (Figure 3) to remove the drying agent.
into
Add 2 mL of potassium 2-ethylhexanoate solution (50% w/v in 1-butanol) to induce
crystallization forming the potassium salt of the penicillin compound. Place the beaker in
an ice bath after crystallization occurs, before vacuum filtering over a Hirsch funnel. Rinse
the crystals with 1 mL of dry acetone. (If you do not get crystals, see the instructor). Label using the
acyl chloride #, date and your initials and store your crystals until next week.
Characterize the product by taking a melting point range and an IR spectrum. If you have less than 0.0200 g of
dried product, please let us know!
TABLE 1: ACYL CHLORIDES
#
Name
Structure
#
Name
1
benzoyl chloride
7
Diphenylacetyl
chloride
2
p-nitro benzoyl
chloride
8
phenylacetyl
chloride
3
3,5-dinitro benzoyl
chloride
9
hydrocinnamoyl
chloride
4
p-anisoyl chloride
10 cinnamoyl chloride
5
4-(phenolazo)
benzoyl chloride
11 4-heptyloxy
6
Structure
benzoyl chloride
o-acetylsalicyloyl
chloride
Penicillin Synthesis Data Sheet
Please complete the following and hand it in on a separate piece of paper during the next laboratory period. This
will be used to assess and improve the various aspects of this laboratory experiment. This sheet is separate from
your laboratory report.
1.
2.
3.
4.
5.
6.
Your name (first and last):
Your lab partner(s) name(s) (first and last):
Your lab section (day and time):
Your acyl chloride number and name:
Grams of dried product; Percent yield; Melting Point Range; IR Spectrum:
Discussion: Please sign your name at bottom if you are willing to have your % yield and comments
published anonymously for research purposes.
a. Sources of error: If anything went wrong, what went wrong,
b. Any changes in the procedure
c. Comments on the lab (i.e. likes and dislikes about this experiment):
Antimicrobial Susceptibility Testing
Adapted from Microbiology Lab Manual, BIOL 275, Introduction to Microbiology, Anne Kruchten, Ph.D., Fall 2014,
Linfield College, Department of Biology, pp. 72-75
A disc diffusion method of assaying antimicrobial susceptibility will be used to test the different penicillin
derivatives synthesized in the organic chemistry laboratory. The results will be analyzed and compiled to
hypothesize structure-function relationships based on the effectiveness at inhibiting bacterial growth for Gramnegative and Gram-positive bacteria.
Discs containing known amounts of antibiotics are placed on agar plates coated with Gram-negative and Grampositive bacteria allowing the antibiotic to diffuse out of the disc. The zone of inhibition around the disc is
measured after 24 h and compared with known antibiotics using the same method (see published values below) to
determine if the size of the zone corresponds to resistance, intermediate susceptibility, moderate susceptibility or
susceptibility of the microorganism to the antibiotic being assayed.
A unit of measure that is commonly used for drugs is the International Unit (IU). This unit depends on both the
concentration and the potency of the substance, thus, the exact measurement of one IU of a substance is
established by an international agreement for each substance organized by the WHO Expert Committee on
Biological Standardization. The conversion factor for penicillin G potassium salt is 15980 IU for a 10 mg/ml
solution (0.0100 g in 1 ml). We will use this conversion factor for our penicillin derivatives.
Preparation of Antibiotic Solutions by Serial Dilution: Week 2, Part A.
Obtain a micro-centrifuge tube rack, 8 micro-centrifuge tubes, a test tube of Millipore DI water, 1000 l
micropipette, a 100 l micropipette and a permanent marker.
Wipe down your workbench with ethanol and a paper towel, and then wash hands with warm soapy water.
Weigh 0.0200 g of the antibiotic into a micro-centrifuge tube and add 1000 l of Millipore DI water (18M) to
prepare a 20.0 mg/ml High concentration stock solution (see instructor for demonstration on using micropipettes).
Cap the micro-centrifuge tube and vortex to ensure the solid has completely dissolved (if not, see instructor).
Label the tube with your acyl chloride number, the letter “H” and your initials for High concentration stock
solution (20.0 mg/ml).
Pipet 500 l of the 20.0 mg/ml High concentration stock solution into a micro-centrifuge tube and add 500 l of
Millipore DI water (18M) to prepare a 10.0 mg/ml Medium concentration stock solution. Cap the microcentrifuge tube and vortex to ensure mixing. Label the tube with your acyl chloride number, the letter “M” and
your initials for Medium concentration solution (10.0 mg/ml).
Pipet 100 l of the 1.0 mg/ml Medium concentration solution into a micro-centrifuge tube and add 900 l of
Millipore DI water (18M) to prepare a 1 mg/ml Low concentration solution. Cap the micro-centrifuge tube and
vortex to ensure mixing. Label the tube with your acyl chloride number, the letter “L” and your initials for the
Low concentration solution (1.0 mg/ml).
**If you do not have 0.0200 g of product see instructor.
TABLE 2: Concentration of Test Solutions Prepared and Amount to Load onto Discs
SOLUTIONS
High conc. test soln
Medium conc. test soln
Low conc. test soln
LABEL Conc. (mg/ml)
#H Int.
#M Int.
#L Int.
20.0
10.0
1.0
Conc. (IU)
31960
15980
1598
Conc. (IU/l) (IU) in 25l
31.96
15.98
1.598
799
399.5
39.95
Loading of Discs with Test Solutions: Week 2, Part B.
Wipe your workbench down again with ethanol and a paper towel, and then wash hands with warm soapy water.
Place a paper towel on your workbench. Using a soft flame, sterilize some forceps and let them cool.
Obtain 32 paper discs using sterile forceps. Arrange them in 4 rows of 8 on a paper towel and label each row
Low, Medium, High, and Control.
Using a micropipette with a clean tip, slowly drizzle 25 l of your Low concentration test solution on each of the
discs in the row labeled Low.
Using a micropipette with a new tip, slowly drizzle 25 l of your Medium concentration test solution on each of
the discs in the row labeled Medium.
Using a micropipette with a new tip, slowly drizzle 25 l of your High concentration test solution on each of the
discs in the row labeled High.
Using a micropipette with a new tip, slowly drizzle 25 l of Millipore DI water on each of the discs in the row
labeled Control.
Allow the discs to air dry, and then place all 8 of the Low concentration discs into a micro-centrifuge tube labeled
with the #L Int., all the Medium concentration discs into a micro-centrifuge tube labeled with the #M Int., all the
High concentration discs into a micro-centrifuge tube labeled with the #H Int. and all the control discs into a
micro-centrifuge tube labeled #C Int.
Place all of the solutions and the discs you prepared in the labeled micro-centrifuge tube racks on the back counter
until next week and begin the Identification of a Single Unknown Experiment.
Antimicrobial Susceptibility Testing: Week 3
Students in the organic chemistry laboratories will team-up with students in the microbiology laboratories to test
the antimicrobial susceptibility of the penicillin derivatives that have been synthesized. Before lab, prepare a brief
handout to describe the structure of penicillin, the acylation reaction performed, and the penicillin derivative that
was synthesized. At the beginning of the laboratory period, spend 5 minutes briefly explaining the chemistry and
structure of the penicillin derivative that you synthesized to the microbiology students that you will be working
with for this experiment. Determine a time that you will be able to come in the following day with these students
to analyze and interpret your results from this assay.
The microbiology students will instruct you how to do the following in a sterile environment:
1. Make a bacterial solution of the correct concentration.
2. Prepare a lawn of Gram-positive and Gram-negative bacteria on an Agar plate.
3. Apply the antibiotics using the disc diffusion method.
4. Incubate and grow bacteria overnight.
5. Analyze the results by measuring and interpreting inhibition zones on the Agar plates and comparing
them to known values the following day.
(Be sure to take good notes and observations).
Penicillin Biological Assay Data Sheet
Please complete the following and hand it in on a separate piece of paper during the next laboratory period. This
will be used to assess and improve the various aspects of this laboratory experiment. This sheet is separate from
your laboratory report.
1)
2)
3)
4)
5)
6)
7)
Your name (first and last):
Your lab partner(s) name(s) (first and last) including microbiology students:
Your lab section (day and time):
Your acyl chloride number and name:
A table of your exact high, medium and low concentrations (mg/ml) and your data measurements:
Photos or drawings of your lab results with data included:
Discussion: Please sign your name at bottom if you are willing to have your data and comments published
anonymously for research purposes.
a. Sources of error: If anything went wrong, what went wrong,
b. Suggestions for any changes to be made in the procedure
c. Comments on the experiment (i.e. likes and dislikes about it):
We will compile all of the biological assay results and send them out to the entire class before your final lab
report is due, so you can comment on each new compound according to efficacy (high, medium, and low/none),
and structure-function relationships based on its effectiveness at inhibiting bacterial growth.
Sample Results to measure should be included: