The document provides a comprehensive overview of penicillin, detailing its discovery, classification, chemistry, structural characteristics, and instrumental methods for characterization including FT-IR, mass spectrometry, and NMR spectroscopy. It highlights the historical context of penicillin's isolation and purification, as well as specific analytical results related to its molecular structure. The document concludes with references for further reading on the topic.

1. Presented by -
Pranav Kumar Ambast
M. Pharm(Pharmaceutical Chemistry)
SPER-JAMIA HAMDARD
1

2. Contents
Topics Page no.
Penicillin 3.
Classification of penicillin 4.
Chemistry of penicillin 5.
Structures of penicillin's 6.
Characterization of penicillin's 7.
Characterization of penicillin's by FT-IR 8.
Characterization of penicillin's by mass 11.
Characterization of penicillin's by carbon 13 NMR 14.
Characterization of penicillin's by H-1 NMR 17.
Reference 21.
2

3. Penicillin:
Penicillin, one of the first and still one of the most widely
used antibiotic agents, derived from the penicillium mold. In 1928 scottish
bacteriologist alexander fleming in a contaminated green mold penicillium
notatum. He isolated the mold, grew it in a fluid medium, and found that it
produced a substance capable of killing many of the common bacteria that infect
humans. Australian pathologist howard florey and British biochemist ernst boris
chain isolated and purified penicillin in the late 1930s, and by 1941 an injectable
form of the drug was available for therapeutic use.
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4. 4

5. Chemistry of penicillin:
Penicillin's are beta lactam antibiotics and characterized by three fundamental
structural requirements
• The fused beta-lactam and thiazolidine ring structure.
• free carboxylic acid group.
• And one or more substituted acylamino side chain.
• Penam nucleus: 7-oxo-l-thia-4-azabicyclo [3.2.0] heptane
• Absolute configuration: 3-S, 5-R, 6-R.
5

6. Structures:
6

7. Characterization of penicillin:
 Instrumental methods of characterization:
• FTIR
• MASS
• C13-NMR
• 1H-NMR
7

8. Characterization of Penicillin by FT-IR:
 All solutions are prepared at 0.1 M concentration in D2O and DMSO. The
samples for linear and non-linear experiments are prepared by squeezing a small
amount of solution (40 μL) between 2 mm thick CaF2 windows separated by a
50 micron Teflon spacer. The average absorbance at the peak is 0.5.
experiments are run at laboratory temperature (22 C).
 Stationary IR spectra are recorded on the same sample cell in a Bruker Alpha
FT-IR spectrometer, with a resolution of 2 cm-1.
8
Amide
β – lactam
carbonyl group
Carboxylic acid

9. FT-IR Spectra:
Experimental and calculated FT-IR frequencies (cm-1) of the three investigated vibrational
modes and their assignment
9
D2O DMSO

10. FT-IR Spectra analysis:
 Penicillin G molecule and its IR spectra in D2 O and in DMSO. Spectra are
characterized by the presence of three intense bands.
 β- lactam CO stretching observe at 1761 cm-1 in D2O and 1762 cm-1 in DMSO
solution.
 Amide group is observe at 1640 cm-1 in D2O and 1674 cm-1 in DMSO solution.
 Asymmetric stretching of carboxylate group is observe at 1601 cm-1 in D20 and
1615 cm-1 in DMSO solution.
 A large red shift of amide , out of the frequency window, is observed upon proton
exchange in DMSO.
10

11. Characterization of penicillin by MS/MS
Spectrometer:
 Collision-Induced Dissociation (CID) technique
 A high-resolution, hybrid tandem mass spectrometer was used to obtain CID
spectra. The CID spectra were acquired by:
 Mass selecting the precursor ions using the first mass spectrometer.
 Injecting the ions into the first quadrupole (collision cell) where they undergo
CID.
 Mass-analyzing the fragment ions produced using the second quadrupole.
 Argon was used as the collision gas, and the pressure in the collision cell was
adjusted to attenuate the precursor ion intensity to 20-50% of the original
intensity. The collision energy of the ions ranged from 160 to 180 eV.
11

12. CID MS/MS Spectra:
collision-induced dissociation (CID) spectrum of protonated benzylpenicillin (m/z
335). The inset shows a postulated structure of this ion and the cleavage to form
the major fragment ion
217-
289
128
12

13. CID MS/MS Spectral Analysis:
 The mass spectra shown abundant fragmentations at m/z 160 and m/z 176
that were reported to arise from cleavage of the β-lactam ring.
 protonated benzyl penicillin exhibits abundant fragment ions at m/z 160,
m/z 176, m/z 217, m/z 128 and m/z 289. The most abundant CID fragment
at m/z 160 and the molecular ion peak was observe at m/z 334.
13

14. Characterization of penicillin by 13C-NMR:
 Spectra were measured at natural abundance on JEOL PFT-100
multinuclear spectrometers, using the SD-HC heteronuclear decoupler.
Data were (collected into the JEOL EC-100 computer. Operating
frequencies were 25.03 MHz for 13C spectra.
 13C-NMR resonance assignments are based on chemical shifts, Off-
resonance and single-frequency decoupling experiments, relative peak
height, and partial exchange experiments.
 Solvent use for the characterization were D20.
 Carbon chemical shifts were measured relative to internal 1,4-dioxane and
adjusted to the Me4Si scale by the relation.
14

15. 13C-NMR Spectra:
175.0δ (lactam)
174.5δ (COOH)
173.9δ (amido)
C2 = 64.9δ
C3 = 73.6δ
C5 = 67.2δ
C6 = 58.4δ
1
2
3
4
5
6
7
2α-. 2β-Me
27.0 δ
31.4 δ
C7
42.5δ
Benzene ring
C1’ = 134.8δ
C2’ = 129.8δ
C3’ = 129.3δ
C4’ = 127.7δ
C5’ = 129.3δ
C6’ = 129.8δ
15
(C16H18N2O4S)

16. 13C-NMR Spectral analysis:
 The four sp3 ring carbons give rise to resonances in the decreasing chemical shift
order C-3, C-5, C-2 and C-6.
 Chemical shift for C-2 is 64.9 ppm and the substituents attached with it are α-
methyl 27.0 ppm and β-methyl 31.4 ppm. Chemical shift for C-3 is 73.6 ppm and
174.5 ppm for carboxylate functions (reflecting the smaller de-shielding influence
of COOH over that of COO-). Chemical shift for C-5 is 67.2 ppm. Chemical shift
for C-6 is 58.4 ppm.
 The lactam group shows its chemical shift at 175.0 ppm
 Amino group attached to C6 shows the chemical shift at 173.9 ppm.
 For Benzene ring C1’ (134.8ppm), C2’ (129.8ppm), C3’ (129.3ppm), C4’
(127.7ppm), C5’ (129.3ppm), C6’ (129.8ppm).
 The benzylic carbon C7’ shows the chemical shift at 42.5ppm.
16

17. Characterization of penicillin by 1H-
NMR spectroscopy:
17
1H-NMR spectra of solutions of methyl esters of penicillin's in CDCI2 at 60 MHz
a) benzylpenicillin
b) Phenoxymethylpenicillin
c) Methicillin
d) cloxacillin
methyl ester derivative penicillin

18. Characterization of penicillin by 1H-NMR
spectroscopy:
 The measurements were conducted on the JEOL Co. spectrometers of the JNM-C60
and JNM-4H-100 types at working frequencies of 60 and 100 MHz, respectively.
Tetramethylsilane was used as an internal standard. The concentration of the
solutions comprised 1-2 mole %. This concentration was selected as the optimum
after an investigation of the concentration dependence of the chemical shifts of
penicillin's.
 The value of the coupling constant (J) of the interaction between the β-lactam
protons of benzylpenicillins and of other penicillin's is from 4 to 5 Hz And The
constant of interaction of the protons (N8 )H and (C6)H lies within the range 8-10
Hz.
18

19. 1H-NMR Spectra of penicillin derivatives:
19
intensity
Penicillin g
Penicillin v
Methicillin
cloxacillin

20. 1H-NMR Spectral analysis of penicillin
derivatives:
S.no compound Chemical shift (δ)
C2
[(CH3)2]
OCH3 H(C3) H(C5) H(C6) H(N8)
Substituents
Protons
1. Penicillin g 1.45 1.45 3.76 4.38 5.50 5.65 6.14
CH2 = 3.64
C6H8 = 7.31
2. Penicillin v 1.50 1.61 3.79 4.38 5.59 5.74 7.39
CH2 = 4.57
C6H5 = 6.8—7.3
3. Methicillin 1.52 1.67 3.80 4.47 5.65 6.01 6.65
(OCH3)2 = 3.82
C6H3 m-H 6.62
p-H 7.30
4. Cloxacillin 1.36 1.40 3.73 4.27 5.42 5.74 6.01
CH3= 2.79
C6H4= 7.5
20

21. References:
21
 Barnes, R.B.; Gore, R.C.; Williams, E.F.; Linsley, S.G.; Petersen E.M. Infrared Analysis of
Crystalline Penicillins. Anal. Chem. 1947, 19, 620–627.
 Le Sueur, A.L.; Horness, R.E.; Thielges, M.C. Applications of two-dimensional infrared
spectroscopy. Analyst 2015, 140, 4336–4349.[CrossRef] [PubMed].
 Fritzsch, R.; Hume, S.; Minnes, L.; Baker, M.J.; Burley, G.A. ; Hunt, N.T. Two-dimensional
infrared spectroscopy: An emerging analytical tool? Analyst 2020, 145, 2014–2024. [CrossRef]
[PubMed].
 Petti, M K.; Lomont, J.P.; Maj, M.; Zanni, M.T. Two-Dimensional Spectroscopy Is Being Used to
Address Core Scientific Questions in Biology and Materials Science. J. Phys. Chem. B 2018, 122,
1771–1780. [CrossRef] [PubMed].
 W. Richter and K. Biemann, Monatsh. Chem., 95,766 (1964).
 (2) V. Bochkarev, N. Ovchinnikova, N. S. Vulfson,E. M. Kleiner, and A. S. Khokhlov, Dokl. Akad.
Nauk. SSSR, 172,1079 (1967).
 (8) M. Ohashi, S. Yamada, H. Kudo, and N. Nakayama, Biomed. Mass Spectrom., 5,578 (1978).

22. References:
 P. V. Demarco and R. Nagarajan in "Cephalosporins and Penicillins. Chemistry and
Biology", E. H. Flynn, Ed., Academic Press, New York, N.Y., 1972, Chapter 8.
 S. Kukolja, N. D. Jones, M. 0. Chaney, T. K. Elzey. M. R. Gleissner, J. W. Paschal, and D.
E. Dorman. J. Org. Chem., 40, 2388 (1975).
 J. E. Stothers, "Carbon-13 NMR Spectroscopy", Academic Press, NewYork, N.Y., 1972;
(b) E. L. Eliel et al., J. Am Chem. SOC.. 97, 322 (1975).
 J.R. Johnson, R. B. Woodward, and R. Robinson, The Chemistry of Penicillins, Princeton
Univ. Press (1949), p. 440.
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