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) .4653(3) Cl .5786(1) -1.5400(4) .9577(3) 08 .4173(2) .1526(4) .5541(3) 08 .5239(1) -1.5028(4) .8974(2) CIO .1759(4) .1487(6) .2741(6) 09 .6564(1) -1.3536(4) 1.2166(2) Cll .0862(2) .4190(8) .1869(4) C10 .6582(3) -1.6436(7) 1.3831(4) C12 .2223(2) .5916(4) .4118(2) Cll .7127(2) -1.8431(5) 1.3119(3) O 13 .2676(2) .7025(3) .3744(2) C12 .5816(1) -1.9087(4) 1.1330(2) O 14 .1373(1) .6087(3) .4653(2) O 13 .5821(1) -2.0121(3) 1.0622(2) C15 .1075(2) .7721(5) .4879(3) O 14 .5625(1) -1.9444(3) 1.2104(2) O 16 .0362(1) .8371(3) .3679(2) C15 .5493(2) -2.1251(4) 1.2192(3) C17 -.0672(2) .7904(4) .3436(3) O 16 .5459(1) -2.1366(3) 1.3262(2) O 18 -.0980(2) .6953(5) .4137(3) C17 .6014(2) -2.1448(5) 1.4396(3) C19 -.1348(3) .8745(9) .2205(5) O 18 .6506(1) -2.1354(6) 1.4474(3) N20 .4825(2) .1299(3) .2689(3) C19 .5927(4) -2.160(1) 1.5468(5) C21 .5706(2) .0533(4) .2512(3) N20 .6472(1) -1.2856(3) .9830(2) 022 .6608(1) .0915(3) .3142(2) C21 .6716(1) -1.1954(4) .9273(2) C23 .5506(3) -.0858(5) .1484(4) 022 .6874(1) -1.2637(3) .8613(2) C24 .4403(2) -.0973(3) .0510(3) C23 .6807(1) -1.0040(4) .9591(3) C25 .4204(3) -.0258(4) -.0770(3) C24 .6485(1) -.8882(4) .8436(2) C26 .3203(3) -.0277(6) -.1661(3) C25 .6813(2) -.8045(5) .8011(3) C27 .2376(3) -.1005(6) -.1298(4) C26 .6507(2) -.6925(6) .6980(4) C28 .2547(3) -.1736(6) -.0029(5) C27 .5865(2) -.6669(6) .6378(4) C29 .3563(3) -.1715(4) .0879(4) C28 .5541(2) -.7518(7) .6784(4) H31 .228(3) .352(4) .476(3) C29 .5842(1) -.8611(5) .7809(3) H51 .435(2) .512(4) .291(3) H31 .573(1) -1.665(5) 1.142(3) H61 .554(3) .302(4) .396(3) H51 .701(1) -1.670(5) 1.087(3) H 101 .164(4) .097(8) .179(6) H61 .644(1) -1.506(5) .905(3) H 102 .115(4) .141(7) .318(5) H 101 .626(2) -1.541(8) 1.356(4) H103 .239(6) .10(1) .332(7) H 102 .691(2) -1.618(9) 1.451(5) Hill .037(3) .402(5) .244(4) H 103 .634(3) -1.741(9) 1.394(5) H 112 .058(4) .345(8) .096(5) Hill .751(2) -1.807(7) 1.397(4) HI 13 .102(3) .539(6) .169(4) H 112 .725(2) -1.868(9) 1.229(5) H 151 .166(3) .836(5) .498(3) H 113 .699(2) -1.941(6) 1.333(4) H152 .070(3) .765(4) .559(3) H 151 .578(1) -2.177(5) 1.223(3) H191 -.102(4) .855(8) .160(5) H 152 .508(2) -2.150(6) 1.144(3) HI 92 -.182(5) .95(1) .247(7) H 191 .571(4) -2.08(1) 1.554(9) H 193 -.189(6) .797(9) .170(7) H192 .632(3) -2.19(1) 1.623(7) H201 .418(3) .112(5) .213(4) H 193 .558(4) -2.24(1) 1.519(7) H231 .603(3) -.085(5) .099(4) H201 .640(1) -1.233(4) 1.035(3) H232 .567(4) -.185(7) .198(5) H231 .669(2) -.983(5) 1.019(3) H251 .476(4) .012(6) -.102(4) H232 .729(2) -.985(7) 1.007(4) H261 .308(3) .016(6) -.256(5) H251 .728(2) -.818(7) .848(4) H271 .155(4) -.098(8) -.201(5) H261 .664(2) -.624(9) .658(5) H281 .208(6) -.213(9) .024(7) H271 .561(2) -.576(8) .564(5) H291 .371(4) -.210(6) .188(5) H281 .503(2) -.750(8) .641(4) H291 .563(2) -.922(7) .816(4)
/^-lactam peptide group deviates from the usual trans- sulfoxide, respectively. The pyramidal nature of ni- planar geometry of unstrained peptides as indicated trogen atom 4 and the amide bond length between by their a>rtorsional angles (for definition cf. atoms 4 and 7 have often been used for correlations Table IV) of 137 and 138° for penamecillin and the to the chemical activity of /Mactam compounds [7]. 370 H. Labischinski et al. • X-Ray Study on Benzylpenicillin and its Sulfoxide
Fig. 1. Stereoscopic view of the molecular conformations of a) penamecillin (1) and b) penamecillin-l-/3-oxide (2) as observed in the crystalline state.
© = s 371 H. Labischinski et al. • X-Ray Study on Benzylpenicillin and its Sulfoxide
( ; = N
Fig. 2. Numbering scheme used for penamecillin 1 (a, c) and its corresponding l-/3-oxide 2 (b, d). In the upper part of the figure (a, b) the bond lengths, in the lower part (c, d) the bond angles as observed in the crystal structures of 1 and 2 are given (standard deviations in brackets). 372 H. Labischinski et al. • X-Ray Study on Benzylpenicillin and its Sulfoxide
Table IV. Relevant torsional an- Penamecillin 1 l-/?-Sulfoxide 2 Atom 1 Atom 2 Atom 3 Atom 4 gles as observed in the crystal angle (deg.) angle (deg.) structures of 1 and 2.
0(9) S(l) C(2) C(3) - 63.6(3)
Definition of torsional angles o)0 = C(23)-C(21)-N(20)-C(6) This view, however, has to be taken with care ac- mined. In both ester group conformations the rota- cording to several recent investigations (for review tional states around the bonds 014—C15 and see [8]). The first parameter is usually described C15 —016 deviated from each other significantly, either by the deviation of nitrogen 4 from the plane too (Is 96.5, 79.9°, 2: 165.5, -80°). defined by carbon atoms 3, 5 and 7 or via the sum The conformation of the thiazolidine ring of both over bond angles at nitrogen 4. The values observed penicillins can be compared using the following were 0.39 and 0.405 Ä, or 337.6 and 335.9 degrees criteria: In case of penamecillin the thiazolidine ring for the penamecillin molecule 1 and its sulfoxide 2, was found in the 3-a-COOH-axial conformation respectively. For the amide bond lengths corre- [9, 13], also characterized by having carbon atom C3 sponding values of 1.379 and 1.395 Ä were found. 0.47 Ä out of the plane defined by atoms 1, 2, 4 and 5, which are nearly coplanar (mean deviation 0.01 Ä). Conformational differences between both compounds In contrast, in case of penamecillin sulfoxide the Comparing those torsional angles common to all thiazolidine ring occupied the 3-a-COOH-equatorial penicillin derivatives, namely co0, cp\, cp2, cou q>2, xp2 conformation having sulfur atom 1 0.89 Ä out of the (for definition see Table IV), there were no signifi- plane formed by atoms 2, 3, 4 and 5 (mean deviation cant deviations between both compounds investi- 0.02 Ä). The angle formed between the thiazolidine gated. The molecular conformation of the sulfoxide 2 ring plane and that of the /^-lactam ring is 63° for differs, however, considerably from its parent com- penamecillin and 53° for its sulfoxide. pound 1 in respect to the orientation of the N-acyl side group as well as the ester group and in respect to the puckering of the thazolidine ring. For the orien- Search for Molecular Parameters Responsible tation of the acyl-group. defined by the torsional for the Different Antibiotic Activities of 1 and 2 angles Gx and d2 (cf. Table IV) in case of compound 2 To elucidate molecular parameters accounting for the values 9] = -103.7°, d2 = -124.6° were found, the distinct antibiotic activity of penicillins and peni- whereas for 10! = 94.8° and 02 = 15.4° were deter- cillin sulfoxides the comparison of penamecillin H. Labischinski et al. • X-Ray Study on Benzylpenicillin and its Sulfoxide 373 with its corresponding Iß sulfoxide seems to be an some conformational flexibility of the /Mactam com- appropriate approach, because the chemical struc- pounds, such that the conformation in the crystalline tures of both compounds are, besides the oxygen state must not correspond to that one present in solu- atom at S 1, identical; nevertheless the conversion of tion. For example, even in the crystalline state penicil- a penicillin to its sulfoxide strongly reduces its anti- lin G can adopt different molecular conformations bacterial activity [2, 10]. The strained /Mactam ring [14]. Furthermore, the analysis of serveral other ß- is usually considered to be a prerequisite for the lactam crystal structures solved before [9, 13, 15] re- biological activity of /Mactam antibiotics. In the veals a certain correlation between the N-acetyl side course of several X-ray studies of /Mactams, geomet- chain conformation and the thiazolidine ring pucker- rical parameters like the bond-length of amide bond ing. We, therefore, consider the differences ob- C7—N4 and the pyrimidality of the nitrogen N4 served between the crystal structures of 1 and 2 in were used for a very rough estimation of the possible respect to side chain conformation to be of minor biological activity [2, 9—12, see, however also 8]. importance. This interpretation is supported by the Comparing these parameters obtained from the crys- well known fact that the side chain of penicillin G tal structures of penamecillin 1 and its corresponding possesses a high conformational flexibility (cf. [16]) sulfoxide 2, no drastic differences between both com- and, as proposed by theoretical calculations [17], ac- pounds were detected. The sulfoxide revealed a tive /Mactam antibiotics should be able to assume a somewhat stronger pyramidal character of its nitro- conformation in solution with a -torsional angle of gen 4 and a slightly enhanced amide bond length. 160—180°, which, according to model calculations This could also be deduced from our infrared meas- would be possible in both compounds, penamecillin urements (Table V). The > C=0 absorption band of as well as its sulfoxide (unpublished results). the /Mactam carbonyl group, which in all /Mactam In respect to the thiazolidine ring puckering, it is compounds is usually observed around 1780 wave- important to notice that penicillin G can adopt both numbers due to the /Mactam ring strain, was found packing types, the 3-a-COOH-axial type (as ob- -1 to be shifted 10 cm to higher values in the spectrum served in our crystal structure of compound 1) as well of the sulfoxide as compared to its parent compound. as the 3-a-COOH-equatorial type (found in the crys- Very similar observations had been made in a com- tal structure of 2) (cf. [13]). parative infrared study of the /Mactams Cloxacillin On the other hand, for all penicillin-sulfoxide com- and Cloxacillin-l-/3-oxide [13]. pounds studied in crystalline state, namely penicillin- The overall molecular structures of both com- V-1-ß-oxide [7], Oxacillin-l-/?-oxide [13] and Cloxa- pounds, 1 and 2, were quite distinct in respect to the cillin-1-ß-oxide [13], the 3-a-COOH-equatorial con- following three aspects: i) conformation of the N- formation was observed, while the corresponding acyl side chain, ii) conformation of the ester group, parent compounds all occupied the 3-a-COOH-axial and iii) puckering of the thiazolidine ring. The dis- thiazolidine ring conformation. cussion of possible correlations of any of these con- Thus, one might consider that active non-sulfoxide formational parameters with the biological activity is penicillins can, within sterically allowed limits, adopt somewhat hampered by the fact, that there exists both conformations, 3-a-COOH-equatorial and -axial, while sulfoxides are forced to the 3-a-COOH- equatorial-type conformation. To check this view, Table V. IR-absorptions bands of amide >C=0 stretching. the following simple model calculation was per- formed similar as described in [8]: we "fixed" the /3-Lactam Exocyclic oxygen 9 atom to the sulfur atom of penamecillin 1 >c=o >c=o using the geometry found in the crystal structure of Penamecillin3 1783 1689 penamecillin and bond lengths as well as bond angles Penamecillin-l-/3-oxidea 1793 1683 as determined from the sulfoxide 2 crystal structure. ab Cloxacillin 1762 1659 In this simulated "3-a-COOH-axial sulfoxide" con- Cloxacillinb c 1788 1669 formation quite small contact-distances resulted be- bc Cloxacillin-1 -/3-oxide 1804 1675 tween the equatorially oriented oxygen 9 and i) the a Solid state spectra, KBr disks; b taken from Ref. [13]; axial carbon of the ß-methyl group (2.77 Ä) as well c spectra taken from CHC13 solutions. as ii) the nitrogen atom of the acylamido-group 374 H. Labischinski et al. • X-Ray Study on Benzylpenicillin and its Sulfoxide (2.46 Ä), similar to the results of a corresponding COOH-equatorial form, it is, of course, tempting to calculation on cloxacillin sulfoxide [8]. speculate that this result is in favour of the view pro- Although the latter distance might also be taken as posed by Rao's group. an indication for a very strong intramolecular hydro- On the other hand, one has to consider the expla- gen bond, we interpret this very low distance be- nation that already the pure existence of the oxygen tween oxygen 9 and the nitrogen of the acylamido- atom at sulfur atom 1 might be sufficient to induce group as an indication that this conformation is alterations in the interaction between antibiotic and energetically unfavoured. Thus, in an analysis of target enzyme. NH---0 hydrogen bonds using data from reported From studies with model enzymes (for review see crystal structures of amino acids and peptides, a value [3, 9]) it is well known, that the interaction of these of 2.8—2.9 Ä has been found to represent the most enzymes with /3-lactams occurs along the following frequent N---0 distance, while a minimum distance of pathway: 2.6 Ä was observed [21], which still is considerable larger than the 2.46 Ä discussed here. This 2.46 Ä is E + I + P even slightly lower than that obtained in the corre- sponding calculation on cloxacillin sulfoxide [8]. where E = enzyme, I = antibiotic, E—I* = inactive Considering also the fact that in all three crystal complex, P = degradation product, K = dissociation structures of penicillin-sulfoxides now available constant, k3 and k4 = first order rate constants. Thus ([6, 13] this investigation), the 3-a-COOH-equato- the antibiotic first reversibly interacts non-covalently rial thiazolidine ring conformation was observed, it is with the enzyme, then acylates the enzyme irrevers- tempting to speculate that these penicillin sulfoxides ibly. and, eventually, will be degraded by the enzyme may either not be able at all to assume the 3-a- to deliver again active enzyme. Obviously, the anti- COOH-axial conformation or only with considerably biotic capacity of a /3-lactam could be influenced distorted binding geometry, while their biologically either in the initial binding step (the larger the K- more active parent compounds can. It should be value, the less inactive), in acylation velocity (the stressed, however, that the present data do not allow larger k3, the less active) and in capacity to persist in any proof for the existence of the 3-a-COOH- the inactivated complex state (the lower k4, the less equatorial conformation of the sulfoxides also in active). In all these steps, conformational and chemi- aqueous solution. In fact, a relatively weak interac- cal properties of the /Mactam in question could have tion between NH-OS (distance in compound 2 = profound influence. 2.28 Ä) has been taken as an evidence against a 3-a- In this context we are presently undertaking a COOH-equatorial conformation in solution [8]. study on the interaction of both compounds, 1 and 2, Unfortunately, there exist two opposite proposals with the penicillin binding proteins from Staphylo- for the role of thiazolidine ring puckering for the coccus aureus via binding studies. 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