Characterization of penicillin.pptx

Penicillin, one of the first and still one of the most widely used antibiotic agents, is 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.
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.

Instrumental methods of characterization:
FTIR
MASS
C13-NMR
1H-NMR
FTIR: -
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.
Collision-Induced Dissociation (CID) technique
MASS:-
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. 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 observed at m/z 334.
C13-NMR: -
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-). The chemic shift for C-5 is 67.2 ppm. The chemic shift for C-6 is 58.4 ppm.
The lactam group shows its chemical shift at 175.0 ppm
Amino group

1. Characterization of Natural Compounds: Penicillin 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. 3

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 C 13 -NMR 1 H-NMR 7

8. Characterization of Penicillin by FT-IR: All solutions are prepared at 0.1 M concentration in D 2 O and DMSO. The samples for linear and non-linear experiments are prepared by squeezing a small amount of solution (40 μ L) between 2 mm thick CaF 2 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 D 2 O DMSO

10. FT-IR Spectra analysis: Penicillin G molecule and its IR spectra in D 2 O and in DMSO. Spectra are characterized by the presence of three intense bands. β - lactam CO stretching observe at 1761 cm -1 in D 2 O 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: C ollision-Induced D issociation (CID) technique A high-resolution, hybrid tandem mass spectrometer was used to obtain CID spectra. The CID spectra were acquired by: M ass selecting the precursor ions using the first mass spectrometer . I njecting the ions into the first quadrupole (collision cell ) where they undergo CID. M ass-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 13 C-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 13 C spectra. 13 C-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 D 2 0. Carbon chemical shifts were measured relative to internal 1,4-dioxane and adjusted to the Me 4 Si scale by the relation. 14

15. 13 C-NMR Spectra: 175.0 δ (lactam) 174.5 δ (COOH) 173.9 δ (amido ) C2 = 64.9 δ C3 = 73.6 δ C5 = 67.2 δ C6 = 58.4 δ 1 2 3 4 5 6 7 2 α -. 2 β -Me 27.0 δ 31.4 δ C7 42.5 δ Benzene ring C1’ = 134.8 δ C2’ = 129.8 δ C3’ = 129.3 δ C4’ = 127.7 δ C5’ = 129.3 δ C6’ = 129.8 δ 15 (C 16 H 18 N 2 O 4 S)

16. 13 C-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 1 H-NMR spectroscopy: 17 1 H-NMR spectra of solutions of methyl esters of penicillin's in CDCI 2 at 60 MHz a) benzylpenicillin b) Phenoxymethylpenicillin c) Methicillin d) cloxacillin methyl ester derivative penicillin

18. Characterization of penicillin by 1 H-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, New York, 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. 22

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