Comparison between the Molecular and Crystal Structures of a Benzylpenicillin Ester and its Corresponding Sulfoxide with Drastically Reduced Biological Activity Harald Labischinski*, Dieter Naumann Robert-Koch-Institut des Bundesgesundheitsamtes, Nordufer 20, D-1000 Berlin (West) 65 Gerhard Barnickel3, Wolfgang Dreißig, Wojciech Gruszecki, Andreas Hofer, and Hans Bradaczek Institut für Kristallographie, Freie Universität Berlin, Takustraße 6, D-1000 Berlin (West) 33 Z. Naturforsch. 42b, 367-375 (1987); received June 30/0ctober 14, 1986 X-Ray, Penicillin G, Penicillin G Sulfoxide, Thiazolidine Ring Conformation, Structure Activity Relationships A comparative X-ray structure determination was performed to elucidate possible conforma- tional differences between penicillins and penicillin sulfoxides. Penicillin-G-acetoxy-methylester and its Iß-oxide were used as model substances, because the only chemical difference between both compounds resides in the thiazolidine ring sulfur oxidation. On the basis of the X-ray data as well as of infrared measurements it is discussed that the drastically reduced biological activity of the penicillin-G-sulfoxide might be related to conformational differences in thiazolidine ring puckering or, even more simply, to the geometric position of the sulfoxide oxygen atom, both of which may hamper the proper reaction of the sulfoxide with its target enzyme(s). Introduction transformation, i.e. no penetration problems nor /3-Lactam antibiotics, although being in therapeut- any destruction by ß-lactamases should occur): First, ical use for more than 40 years, still provide the most the chemical reactivity leading to the acylation of the widely used antibacterial drugs. An enormous target enzyme might be changed or, secondly the amount of chemical modifications of the originally ability for proper interaction with the protein might discovered penicillin G structure has been syn- be affected by conformational factors. In order to thesized and tested, and several empirical rules were distinguish between these possibilities, the investiga- elucidated about which modifications possible could tion of the three-dimensional structures and chemical lead to more active or, on the other hand, inactive reactivities of those drugs involving minimal chemi- compounds (for review see [1]). One of these rules cal deviations but large variation in biological activity states that oxydation of the penam moiety at position would be highly desirable. 1 to its corresponding sulfoxide normally leads to a We report here on a comparison of the crystal and drastically reduced activity of the drug [1]. Since it is molecular structures of two penicillin G derivatives now well known, that an important prerequisite for differing only in sulfur oxidation, the results of which the bacterial activity of a /3-lactam antibiotic is its compare well with those obtained using similar inves- binding to and subsequent acylation of those raem- tigations on two other ß-lactam sulfoxides, namely brane-associated bacterial proteins involved in the on penicillin-V-sulfoxide [6] and on cloxacillin sulf- final steps of bacterial cell wall assembly (the so oxide [13]. For both latter compounds a thiazolidine called penicillin binding proteins, PBP's, for review ring conformation (3-a-COOH-equatorial) opposed see [2, 3]), there seem to exist two most obvious to that of their parent compounds (3-a-COOH-axial) possibilities of explaining a reduction in drug activity was observed, exactly as it will be reported here for induced by chemical modifications (provided that the the case of penicillin G and its sulfoxide. drug actually may reach the target without chemical Experimental a Present address: E. Merck, D-6100 Darmstadt, Germany. * Reprint requests to Dr. Harald Labischinski. X-ray investigations Verlag der Zeitschrift für Naturforschung, D-7400 Tübingen Penamecillin (penicillin-G-acetoxymethylester 0340 - 5087/87/0300 - 391/$ 01.00/0 (1)) and its lß-oxide (2), were prepared following Dieses Werk wurde im Jahr 2013 vom Verlag Zeitschrift für Naturforschung This work has been digitalized and published in 2013 by Verlag Zeitschrift in Zusammenarbeit mit der Max-Planck-Gesellschaft zur Förderung der für Naturforschung in cooperation with the Max Planck Society for the Wissenschaften e.V. digitalisiert und unter folgender Lizenz veröffentlicht: Advancement of Science under a Creative Commons Attribution Creative Commons Namensnennung 4.0 Lizenz. 4.0 International License. 368 H. Labischinski et al. • X-Ray Study on Benzylpenicillin and its Sulfoxide the route described in [4]. Crystals suitable for X-ray H-atoms were included in the refinements with diffraction were obtained from isopropanole/acetone isotropic temperature factors. 1:1 (v/v). Crystal densities were measured by the flo- Two very strong reflections (400 and 001) for the tation method using water/KJ solution. The space sulfoxide presumed to be affected by extinction or groups were determined from oscillation and Wris- measurement error were eliminated from the refine- senberg photographs. Accurate cell parameters were ment at this stage. The final R values for all observed obtained from least-squares refinements of 2 0-values and measured reflections were 0.028 for penamecil- for high-order (>100°) reflections measured on a lin and 0.030 for the sulfoxide. Unit weights were STOE automatic four-circle diffractometer using Ni- used for all reflections. At the end of the refinements filtered CuKn radiation. The intensities of 1851 inde- (A/o) were <0.09 for the non-hydrogen atoms in pendent reflections for penamecillin and 1850 reflec- both structures. tions for its corresponding sulfoxide were collected up to 0 = 65°. Of these. 41 reflections and 17 reflec- Infrared spectroscopy tions, respectively were considered as unobserved All spectra have been recorded on a FT-IR spec- (I<2CT(I)). Crystal and intensity-measurement data trophotometer 10-MXE, Nicolet Instruments, Madi- are shown in Table I. son, USA, applying the KBR-disc as well as the Absorption was ignored. The intensities were cor- -1 nujol-mull technique; resolution was set to 2 cm , rected for Lorentz-polarization effects and anoma- -1 wavenumber accuracy was better than 0.01 cm . lous dispersion corrections were used for S. O and C. The structures were solved by application of the X-ray program system [5]. The E-maps calculated Comparison between the Structures of Penamecillin from the set of phases with the highest combined and its Sulfoxide Derivative in the Crystalline State figures of merit showed all the 28 and 29 non-hydro- gen atoms of the molecules. Several cycles of full- Atomic positional parameters of penamecillin (1) matrix least-squares refinements with CRYLSQ led and its corresponding 1/3-oxide (2) are shown in to conventional R values of 0.069 and 0.063, respec- Tables II and III respectively. A comparative view of tively. A difference Fourier synthesis at this stage the conformations of both molecules is depicted in clearly indicated all hydrogen atom positions. The Fig. 1. The numbering of the atoms (shown in Fig. 2 for both molecules together with the respective val- ues of bond lengths and bond angles) starts on the ring sulphur atom, as usual for /Mactam compounds. Table I. Crystal and intensity-measurement data. Atomic number 9 was reserved for the oxygen con- Penamecillin (1) Penamecillin nected to the sulphur atom, otherwise the numbering l-/3-sulfoxide (2) scheme is identically for both substances. Relevant torsional angles are given in Table IV. Formula C19H22N2O6S C19H22N2O7S Molecular weight 406.322 422.322 In penamecillin. like in all other penicillin G-de- Crystal system monoclinic monoclinic rivatives studied in the crystalline state, no inter- Space group P2, C2 molecular hydrogen bond is formed. Interestingly, Unit cell parameters: there was also no intermolecular hydrogen bonding a 12.912(4) Ä 25.418(6) Ä b 8.087(3) Ä 7.704(3) Ä in both crystal structures. However, in case of the c 10.137(3) Ä 12.543(3) Ä sulfoxide an intramolecular interaction occurs be- ß 105.38(3)° 123.00(3)° tween the NH proton of the 6-aminoacyl side group Unit cell volume 1020.6 A3 2060 A3 Number of mol- and the oxygen 9 at the sulphur atom of the ecules/unit cell 2 4 thiazolidine ring (distances: N20-H201: 0.87 Ä; Wavelength (CuK„) 1.54179 Ä 1.54179 Ä N20---09: 2.86 Ä; 09--H201: 2.28 Ä; angle Absorption N20-H201-09 = 124°) quite similar to what has coefficient 1.70 mm"1 1.74 mm"1 Scattering angle been observed in the crystal structure of penicillin-V- 26 maximum 130° 130° lß-oxide [6]. Scan mode 6-26 6-26 Number of inde- pendent reflections 1851 1850 Geometry of the ß-lactam ring Number of observed Comparing the geometry of the /3-lactam ring both reflections 1792 1833 compounds. 1 and 2, revealed similar features. The 369 H. Labischinski et al. • X-Ray Study on Benzylpenicillin and its Sulfoxide Table II. Fractional atomic coordinates of penamecillin (1). Table III. Fractional atomic coordinates of penamecillin Standard deviations are given in parentheses. l-/3-sulfoxide (2). Presentation analogue to Table II. Atom X y z Atom X y z SI .28847(4) .36650 .15451(5) SI .70019(3) 1.50000 1.24361(6) C2 .1917(2) .3336(4) .2587(3) C2 .6665(1) -1.6917(4) 1.2766(3) C3 .2473(2) .4102(4) .4020(3) C3 .6024(1) -1.7205(4) 1.1473(2) N4 .3618(1) .3895(3) .4207(2) N4 .6120(1) -1.6691(3) 1.0480(2) C5 .4021(2) .4076(4) .3001(2) C5 .6730(1) -1.5929(4) 1.0876(3) C6 .4838(2) .2678(4) .3560(3) C6 .6380(1) -1.4694(4) .9697(3) Cl .4212(2) .2490(4)