Synthesis of Different Penicillin Derivatives Using Acylation and the Effects of Functional
Group on Bioinhibitory Functionality
Karel Kletetschka
Chemistry 431W
Prof. Dr. Katherine Masters
Pennsylvania State University
Department of Chemistry
Department of Microbiology
Chemistry 431W
October 25, 2014
Karel Kletetschka
Abstract:
Understanding the functionality of different derivatives of penicillin can be critical
information considering how ubiquitous antibiotics have become in treating bacterial illnesses.
Using acylation, modification of 6-aminopenicillanic acid (6-APA) can allow for the synthesis of
penicillin derivatives 3 with different functional groups. Penicillin G, penicillin V, and phenyl
penicillin were synthesized and compared in terms of Minimum Inhibitory Concentration (MIC)
against Streptococcus aureus and Escherichia coli. Penicillin G and phenyl penicillin were more
effective against the gram-positive S. aureus with values between 100-400 ng/ml and penicillin
V showed more effectiveness toward the gram-negatice E. coli, suggesting the added stability of
the structure is more effective against gram-negative bacteria.
Introduction:
Antibiotics are biochemical compounds that act to inhibit bacterial processes necessary
for their survival. First identified in 1928, penicillin started a revolution of medicinal research in
curing infections such as staphylococcus aureus and now there are over 3000 known compounds
with antibacterial properties2. The structure-activity relationship (SAR) describes how the
specific chemical structure of a compound has profound effects on biological activity and thus
modifying functional groups or any aspect of the three-dimensional structure of a large molecule
can act to inhibit or promote the functionality of the biochemical's.1
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Penicillin, a β-Lactam antibiotic, is characterized by a covalent bond between cysteine
and valine. This is essential to its functionality as it binds to the enzyme DD-transpeptidase
which serves as a catalyst for the formation of peptidoglycan cross-links in the cell wall of
bacteria. Ultimately, this forms an imbalance since the hydrolyzing enzymes in the
peptidoglycan cross links are still active whereas the enzymes that facilitate formation of the
cross links are not which leads to deterioration of the cell wall and subsequently an osmotic
pressure imbalance that leads to the death of the cell.2 The core skeleton of penicillin, often
referred to as "penam", is the basis of antibiotic activity, however, modifying the functional
group attached to this core skeleton can vitally effect potency based on the SAR principle3.
Using acid halide acylation, the functional group attached to the penam core can be
modified with relative ease. The primary agent used is 6-aminopenicillic acid (2) which can be
reacted with different acid chlorides. This can potentially allow for the synthesis of numerous
derivatives of penicillin, all with potentially varying bioactivities.
Figure 1. Targeted synthetic penicillin derivatives: penicillin G (left), penicillin V (middle), and
phenyl penicillin (right)
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Karel Kletetschka
Penicillin G (3a) is known as one of the most potent derivatives of penicillin, and is particularly
effective against gram-positive organisms when compared to penicillin V (3b). Penicillin V,
however is rather resistant to hydrolysis by acidic secretions in the body and is thus often taken
orally.4 Phenyl penicillin (3c) has been synthesized but is not often used in vivo.
The primary focus was to explore synthetic routes for derivatives of penicillin,
particularly acid halide acylation reactions. Understanding the intricacies of these penicillin
derivatives and their syntheses can shed light on bioactivity which is critical considering the
degree to which these pharmaceuticals are utilized in medicine.
Results and Discussion:
Scheme 1. Acylation Synthetic Route to Penicillin G
The synthesis of penicillin G was performed by acylation of 2 with the phenylacetyl chloride 1a
(Scheme 1). Crystallization occurred immediately after the introduction of the sodium-2ethylhexanoate indicating the formation of the sodium carboxylate salt of penicillin G (0.427g,
48% yield)
Characterization
Proton NMR spectra indicate successful synthesis of 3a, There appears to be some impurity at
3.3-3.6 ppm which may be some remnant starting reagent 1a since that is where hydrogen's
adjacent to chlorine would appear. Carbon NMR values seem to correspond to the literature
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values well. Infrared Spectroscopy (IR) showed the presence of a likely amine functional group
at 3352 cm-1, aromatic and alkyl hydrogen's in the 2900-3100 region and three signals in the
carbonyl (1600-1800 cm-1) region which corresponds to 3a.
Bioactivity
The minimum inhibitory concentration (MIC) values indicate the minimum concentration
necessary to visibly inhibit a microorganism after overnight incubation5 and the penicillin G
samples as a whole that were synthesized by various members of the team showed more of an
inhibitory potential on the streptococcus aureus (S. aureus) strain than the escherichia coli
bacterium with a 4.2 fold difference on average in bioactivity for the first section trial and a 1.2
fold average difference for the second sections trials. This is expected as literature indicates that
penicillin G is especially active against gram-positive bacteria; S. aureus being gram-positive
and E. coli being gram-negative.
Scheme 2. Acylation Synthetic Route to Penicillin V
Penicillin V was also synthesized using the acylation method, with phenoxyacetyl chloride 1b
directing the synthesis towards 3b. Crystallization for this reaction also occurred spontaneously,
yielding the sodium carboxylate salt of 3b with a yield of 0.499 g, 57%.
Characterization
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The proton NMR values are very similar to the literature penicillin V values. There is a rather
significant impurity peak at 3.5 ppm with an integral value of 71 with respect to the other peaks
which suggests that the sample was dilute. IR spectra indicates that the correct functional groups
are present.
Bioactivity
The MIC values for the penicillin V samples showed the opposite trend when compared to the
penicillin G with the penicillin V being 3 times more active against E. coli in the first section
trial and ~9 times more active in the second section trials on average. Both penicillin G and V
are known to be active against gram-negative bacteria such as E. coli, however perhaps the
added stability of penicillin V due to the phenyl ether caused the added potency in inhibitory
function.
Scheme 3. Acylation Synthetic Route to Phenyl Penicillin
The phenyl penicillin 3c was also synthesized using acylation and proved the most difficult to
properly synthesize. Crystallization of the phenyl penicillin sodium salt occurred a few minutes
after the introduction of the sodium-2-ethylhexanoate however it was difficult to dry the crystals
and a somewhat gelatinous substance was obtained until dry clumps were finally obtained. The
final product showed a 0.325 g, 38% yield.
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Characterization
The phenyl penicillin derivative NMR spectra matched what was expected however some
impurities were evident. The peaks at 2.9, 2.1, and 1.2-1.4 indicate contamination with nbutanol, evidently form the final addition of the procedure. The carbon NMR also shows peaks
that could be associated with n-butanol in the 20-35 ppm region and otherwise indicates the
general penicillin pattern seen before. The IR spectra frequency at the 3300 cm-1 range is rather
broader than for the other two penicillin derivatives which is likely from the alcohol group on the
proposed n-butanol contaminant. Otherwise, all of the proper functional groups are present on
the IR spectrum.
Bioactivity
The MIC values of the phenyl penicillin derivative suggest that it is a very potent inhibitor of S.
aureus and was 4.2 times more potent on average towards S. aureus than E. coli for the first
section trials and 5 times more potent for the second section trial. This suggests that the lack of
the carbon separating the phenyl and the carbonyl is significant.
Both penicillin G and the phenyl penicillin derivatives were more effective against the
gram positive S. aureus strain of bacteria. This suggests that the ether group on the penicillin V
may be significant in determining potency towards certain bacteria, more specifically gram
negative bacteria. This may be due to the added stability that the ether group provides the
molecule. Some factors that may have obscured the data are impurities, as well as time between
synthesis and bioassay analysis. There were also differences in temperature for each synthesis.
Future endeavors could involve exploring additional functional groups such as phenyl esthers or
longer carbon chains or distance between the phenyl group and the amine group since that may
have very well had an impact on the bioactivity and stability of the different derivatives.
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Figure 2. Bioassay MIC (Average, Range) Data of the Three Penicillin Derivatives
MIC Values (ng/ml)
Penicillin G
Trial 1
S. aureus
243,
17.9-572
E. coli
57.2,
18.3-146
Penicillin V
Phenyl Penicillin
Trial 2
Trial 1
Trial 2
61,
98,
48.4,
357,
429,
35.8-286
2.2-71.5
143-572
286-572
50.3,
293,
439,
85.5,
85.0,
18.3-146.5
2.29-585
146-585
73.2-146
2.29-146
4.5-35.8
Trial 1
Trial 2
Conclusion
All three penicillin derivatives 3a, 3b, and 3c were synthesized according to the melting points,
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H NMR, 13C, and IR spectral data. The bioassay results indicate that the functional groups for
each penicillin derivative may have a significant factor on the degree of inhibitory function each
derivative has for a specific bacteria, as well as the type of bacteria that it is most effective
against. Penicillin G and the phenyl penicillin proved to be more effective against the grampositive S. aureus, whereas the penicillin V derivative was more effective against the gramnegative E. coli, possibly due to the added stability gained by the phenyl ether group. Future
endeavors involve exploring additional functional groups and different distances between the
phenyl group and the amine.
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Experimental:
Synthesis of Penicillin G (3)
To a mixture of acetone:water (12 mL, 1:3) was added sodium bicarbonate (1.05 g, 12.5 mmol)
and the solution was stirred at room temperature until dissolved. 6-aminopenicillanic acid 2
(0.540 g, 2.5 mmol) was added to the mixture. A solution of phenylacetyl chloride 1a (0.387 g,
2.5 mmol) in 1 mL of acetone was added dropwise to the initial mixture over a period of 5
minutes and was then stirred for 40 minutes at room temperature. The reaction solution was
extracted with n-butyl acetate (3 x 6 mL). The aqueous solution was covered with cold n-butyl
acetate and acidified to a pH of 2.0 using cold 5M sulfuric acid. The n-butyl acetate layer was
separated and washed with cold water. The remaining solution was then dried over anhydrous
sodium sulfate (10 min.) and vacuum filtered. To the filtrate was added a 1 mL solution of
sodium 2-ethylhexanoate in butanol (1:1) whereupon crystallization occurred and the crystalline
product 3a was filtered to yield a white powder (0.427g, 48%), . IR (neat) at 3352, 2961, 1773,
1695, 1618 cm-1; 1H NMR (400 MHz, DMSO) δ 8.75 (s, 1H), 7.3 (t, 1H), 7.2 (t, 2H), 7.2 (d, 2H),
5.3 (s, 2H), 3.8 (s, 1H), 3.5 (d, 2H), 3.3 (s, 5H), 2.5 (s, 5H), 1.6 (s, 3H), 1.4 (s, 3H); 13C NMR
(75 MHz, DMSO) δ 173.1, 170.3, 169.2, 136.1, 128.9, 128.2, 126.1, 99.3, 74.09, 66.8, 64.3,
57.5, 39.6, 31.08, 27.3. Mp 223oC (lit. 225oC)
Synthesis of Penicillin V
To a mixture of acetone:water (12 mL, 1:3) was added sodium bicarbonate (1.05 g, 12.5 mmol)
and the solution was stirred at room temperature until dissolved. 6-aminopenicillanic acid 2
(0.540 g, 2.5 mmol) was added to the mixture. A solution of phenoxyacetyl chloride 1b (0.427 g,
2.5 mmol) in 1 mL of acetone was added dropwise to the initial mixture over a period of 5
minutes and was then stirred for 40 minutes at room temperature. The reaction solution was
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extracted with n-butyl acetate (3 x 6 mL). The aqueous solution was covered with cold n-butyl
acetate and acidified to a pH of 2.0 using cold 5M sulfuric acid. The n-butyl acetate layer was
separated and washed with cold water. The remaining solution was then dried over anhydrous
sodium sulfate (10 min.) and vacuum filtered. To the filtrate was added a 1 mL solution of
sodium 2-ethylhexanoate in butanol (1:1) whereupon crystallization occurred and the crystalline
product 3b was filtered to yield a white powder (0.499 g, 57%.). IR (neat) at 3418, 3370, 3352,
2956, 1778, 1698, 1603, 1122 cm-1; 1H NMR (400 MHz, DMSO) δ 8.4 (s, 1H), 7.3 (t, 2H), 7,3
(t, 1H), 7.2 (d, 2H), 5.4 (d, 2H), 4.6 (s, 2H), 3.8 (s, 1H), 3.5 (s, 70H), 2.5 (s, 10 H), 1.5 (s, 3H),
1.4 (s, 3H); 13C NMR (75 MHz, DMSO) δ 173.1, 170.3, 169.2, 159.1, 136.1, 129.3, 121.1, 114.4,
74.0, 66.7, 64.4, 57.1, 39.2, 31.9, 27.2, 17.0. MP 240oC (lit. 242oC)
Synthesis of Phenyl Penicillin
To a mixture of acetone:water (12 mL, 1:3) was added sodium bicarbonate (1.05 g, 12.5 mmol)
and the solution was stirred at room temperature until dissolved. 6-aminopenicillanic acid 2
(0.540 g, 2.5 mmol) was added to the mixture. A solution of benzoyl chloride 1c (0.351 g, 2.5
mmol) in 1 mL of acetone was added dropwise to the initial mixture over a period of 5 minutes
and was then stirred for 40 minutes at room temperature. The reaction solution was extracted
with n-butyl acetate (3 x 6 mL). The aqueous solution was covered with cold n-butyl acetate and
acidified to a pH of 2.0 using cold 5M sulfuric acid. The n-butyl acetate layer was separated and
washed with cold water. The remaining solution was then dried over anhydrous sodium sulfate
(10 min.) and vacuum filtered. To the filtrate was added a 1 mL solution of sodium 2ethylhexanoate in butanol (1:1) whereupon crystallization occurred and the crystalline product 3c
was filtered to yield a white powder (0.325 g, 38% yield). IR (neat) at 3354, 2961, 2931, 2867,
1761, 1670, 1524 cm-1; 1H NMR (400 MHz, DMSO) δ 9.0 (s, 1H), 7.8 (m, 6), 7.5 (t, 1H), 7.47 (t,
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2H), 5.4 (d, 2H), 4.0 (s, 1H), 2.5 (s, 10 H), 2.0 (s, 3H), 1.6 (s, 3H), 1.3 (m, 20H), 1.9 (m, 20H);
C NMR (75 MHz, DMSO) δ 177.7, 169.4, 128.9, 128.7, 127.7, 127.1, 47.5, 31.0, 29.5, 25.2,
13
22.2. Mp 200oC.
Acknowledgments:
I would like to thank Dr. Katherine Masters, and the very helpful TA's for guidance and
supervision throughout this laboratory experiment. I would also like to thank the Pennsylvania
State University, the Department of Chemistry, and the Microbiology faculty that helped
coordinate this interdisciplinary research investigation.
References:
[1] Hansch, C.; Steward, A. R. The use of substituent constants in the analysis of the structureactivity relationship in penicillin derivatives. J. Med. Chem., 1964, 7(6), 691-694.
[2] Park, J. T.; Strominger, J. L. Mode of action of penicillin Biochemical basis for the
mechanism of action of penicillin and for its selective toxicity. Science, 195, 125(3238), 99-101.
[3] Shewale, J. G.; Sivaraman, H. Penicillin acylase: enzyme production and its application in
the manufacture of 6-APA. Process Biochemistry, 1989, 24(4), 146-154.
[4] Cooper, R. D. Structural studies on penicillin derivatives. V. Penicillin sulfoxide-sulfenic
acid equilibrium. J. Am. Chem. Soc., 1970, 92(16), 5010-5011.
[5] Andrews, J. M. Determination of minimum inhibitory concentrations. J. Antimicrobial
Chemotherapy, 2001, 48(suppl 1), 5-16.
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