Molecular Modelling in the Undergraduate Chemistry Curriculum: The Use of p-lactams as a Case Study Neil S Flingan' and Lisa Grayson Molecular and Life Sciences Department, University of Abertay Dundee, Bell Street, Dundee, DDl lHG,United Kingdom
As the concepts of stereochemistry and molecular structure become more important to the understanding of modern chemistry, traditional methods for teaching stereochemistry in the undergraduate curriculum are being reconsidered. Skeletal models (e.g., Dreiding models) are useful for providing a basic background to the three-dimensional structure of molecules and for introducing the concept of chirality. The major difficulty of these models is, however, the problem of illustrating these concepts in the lecture and tutorial setting. I n the past 2-3 years, rapid advances in the price to performance ratio of desktop computers has made molecular graphics and molecular modelling available on personal computers, such a s the Apple Macintosh I1 family and IBM-PC compatibles ( I , 2). Consequently, molecular graphics and modelling are no longer iust research tools with costly hardware an: software.'l"nky are now teaching aids aGailable to virtually all educational establishments. As the search for novel biologically active agents continues apace, corn~uter-uidsdmolecular design (CAMDI is becoming increasingly important in the drug-design strategies of pharmaceutical companies. Many graduates working in the pharmaceutical industry will encounter CAMD, either first hand or by reading the scientific literature. I n order to ensure that graduates are exposed to the concepts and terminology of this discipline, a discussion of the applications of CAMD is a useful addition to our finalyear BSc. honours courses in chemistry and biochemistry. The overall objective of this development was to give students a n opportunity for hands-on use of a molecular modelling package to examine a specific problem from a n appropriate area of chemistry and biochemistry. This would allow them to investigate concepts, such a s chirality and conformational analysis, which were previously covered from a more theoretical approach. We wanted to exemplify the complete spectrum of activities normally associated with solving a real problem using molecular modelling. We 'Author to whom correspondence should be addressed.
856
Journal of Chemical Education
have developed this case study for undergraduate chemistry and biochemistry students to provide a n overview of the process. Development of the Case Study The principal features we wished to exemplify were
.
use of a structural database to retrieve X-ray coordinates
energy minimization using molecular mechanics conformational analysis of molecules fitting two molecules together to examine similarities
Figure 1. Repeating unit of peptidoglycan
The examnle chosen as the basis for this case studv was the biological mbde of action of penicillins. We have conkderable expertise (3)within this department in studying the chemistry of beta-lactams. The crystal structures of a number of oenicillins and c e ~ h a l o s ~ o r i nhave s been solved. Consequently, the steredchemi& of the beta-lactam nucleus is well-documented ( 4 , 5 ) . The Biological Mode of Action of Penicillins
The molecular basis of the biolo&cal mode of action of penicillins is known to be via inhihtion of bacterial cellwall biosynthesis (6).The bacterial cell wall is composed of a peptidoglycan matrix. The glycan portion comprises N-acetvlelucosamine and N-acetvlmuramate units, connected through p1,4 linkages. A ientapeptide (LALA-D-GLU-LLYS-D-ALA-D-ALA)is attached to the glycan, and the cell wall is cross-linked by a transpeptidase &&me joining these ~ e.o t i d cchains to form two-dimensional &hects.The repeat. ing unit of peptidoglycan is shown in Figure 1. Penicillins are believed (7) to mimic the terminal D-ALAD-ALAresidues of the oenta~eotidein the erowine - o.e.~ t i doglycan cell wall. ~ o n s e ~ u e n t & the , transpeptidase forms a n acvlenzvme intermediate with the oenicillin, not with the p&ultimate D-ALA residue in the peptidoglycan. This gives rise to a covalently bonded antihiotic-enzyme complex, which cannot undefgo further reaction. The cell wall remains incomplete, and the bacterium is no longer viable. The Student Exercise
The overall aim of the exercise was to construct a representative model of the D-ALA-D-ALAportion of the pentapeptide of the peptidoglycan and to compare this model to the crystal structure of a biologically active penicillin. The case study to be explored by the students was arranged into the following sections. Modelling the System
The initial stages of the project were split into three subsections for the students to carry out.
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construction and enerw minimization of the peptidoglycan prptide system retrieval of a biologically active penicillin structure From a crystallog~phicdataha8~ comparison of the structures of the dipeptide and the penicillin
Analysis of Results
After the oeotide svstem had been modelled and compared to the penicillin, the overall aim was to consider whether the atoms of the peptide system and the penicillin would lie in a similar three-dimensional arrangement. If so then it is likely that the penicillin does act a s a mimic of the naturally occumng peptide. Without the three-dimensional crystal structure of the full peptidoglycan system, however. it would not he nossible to state cateeoricallv that the penikllin does mimi; the natural substrGe. The Case Study Modefling the System
The molecular-modelling software used during the development of this case study was the Nemesis package from Oxford Molecular (I, 8 ) running on a n Apple Macintosh IIci. Students used the same package on a n 80486-PC running under Windows. This case study could be implemented on any of the Macintosh or PC-based molecularmodelling packages currently available (1).
Construction and Energy Minimization of the Peptide System Initially we proposed to model the complete pentapeptide portion of peptidoglycan in order to ensure that the model system would have the same flexibility and conformational constraints a s the real svstem. Nemesis includes a fragment library containing ali amino acid residues a s the L-enantiomers. I n order to construct the pentapeptide, the chirality of a n alanine and a glutamic acid residue were inverted (using a function in the software). Then the peptide was constructed using these and the library amino acids. An N-acyl group was added to the initial L-alanine residue in order to mimic the bond between the peptide sidechain and the carbohydrate backbone of the peptidoglycan. After construction of the peptide, the COSMIC (9) molecular-mechanics energy minimizer supplied with Nemesis was used to oroduce a low-enerw conformation of the molecule. ,41thoigh molecular mech&cs is a quick method for obtaining low-energy structures, and the COSMIC force field is applicable to a diversity of systems, this technique does suffer from a number of drawbacks. The molecule is treated a s a n isolated entity in vacua, rather than in the aqueous environment found in vivo. This limitation was accepted because the alternative techniques available to produce a low-energy structure in aqueous conditions (e.g., molecular dynamics in a solvated periodic box) would be too time-consuming with ~e computing resources available. Also, this was not considered to be a n essential part of this study. Attempts to minimize the initial pentapeptide structure were unsuccessful. One of the parameters in a molecularmechanics energy minimization relates to Van der Waals interactions between atoms in the structure. I n Nemesis a Van der Waals Morse potential algorithm is used (10). If the number of interactions between atoms is too great for the program to handle, then the molecular-mechanics algorithm decreases the distance a t which Van der Waals interactions are deemed to be valid. I n Nemesis the default interaction distance is 15 A, which is decreased in 2-A steps until the number of interactions can be processed. For the pentapeptide under studv. . . the interaction distance was decreased to such an extent that many posd)lu interactions ~ w u l dnot he considered. Thus, no reliabihtv could be olaced on the results. A similar situation existed for the ckresponding ~ - a c ~ l tetrapeptide (i.e., not considering the initial L-alanineresidue). However, no significant decrease in the Van der Waals interaction distance was observed with the N-acylL-LYS-D-ALA-D-ALAtripeptide. I t is believed (7)that the beta-lactam mimics the terminal D-ALA-D-ALAfragment of the pentapeptide, so energy minimization of N-acyl-DALA-D-ALAwas carried out. vieldina a structure with a virtually identical conformati& to &at of the minimized trioeotide around the alanine residues. In order to render the screen display a s legible a s possible for student use and because the two conformations were so. similar, the N-acyl-D-ALA-D-ALAdipeptide was selected for further use by the students. Retrieval of a Beta-Lactam Crystal Structure Crystal structures were obtained from the Cambridge Structural Database (CSD), which was accessed on the Science and Engineering Research Council (SERC) VAX a t Daresbury Laboratories, England. This database has an associated search and retrieval system called Crystal Structure Search and Retrieval (CSSR), which was used by the students. This query language allows the user to scan the database using either keywords or a query structure. In order to identify the range of penicillins for which crysVolume 71
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rms deviation between the two molecules being fitted. The initial rms difference between the molecules was 0.201. Because the peptide system contains a certain degree of conformational flexibility, the final stage of the modelling process was designed to include a conformational analysis in order to study the conformational space that the peptide could encompass. The four torsion angles indicated i n F i g u r e 3 were defined within Nemesis and a comprehensive conformational analysis was undertaken, involving
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rotation of single bonds simultaneous rotation of pairs of bonds simultaneous rotation of all four bands
Penicillin G
N-Acetyl D-ALA-D-ALA Hydrogens are omitted for clarity Figure 2. Atoms used to fit Nacetyl-D-ALA-0-ALAandpenicillin G.
tal structures had been deposited in the database, query structure 1was created by the students during the search session and used to scan the database for matches.
m
The step size by which torsions were rotated depended on the total number of bonds being rotated and varied from 5' for single-bond rotations to 40' for all four bonds rotating together. Graphs of torsion angle against energy were obtained for rotation of individual bonds, and energy contour maps for bond pair rotations were printed for subsequent analysis. A best-fit peptide was modelled by rotation of individual torsion angles so that atoms in the dipeptide and corre-
0
1
From a total of 35 hits in the database, penicillin G (21, one of the simplest beta-ladams in clinical usage, was selected for examination. The crystal structure was elucidated in 1949 hy Crowfoot and coworkers (11) and was most recently refined in 1978 (12). Penicillin G is still in clinical use to treat infections of the respiratory and genitourinary tracts, though i t has limited usefulness against strains of bacteria that are resistant to beta-lactams.
p h c H 2 c 0 N ~ & :
0
CO~H
0
Figure 3. Bonds rotated during conformational analysis. Hydrogens are omitted for clarity
2
The three-dimensional coordinates of the structure were transferred from the SERC VAX onto students' own workstations over the Joint Academic Network (JANET). Comparison of Structures I n order to gain a n approximate view of how the minimized dipeptide and the crystal structure of the penicillin are related in terms of conformation, both molecules were read into the Nemesis workspace and fitted (treating both as rigid bodies) using the atoms indicated in Figure 2. As a measure of "goodness of fit", Nemesis calculates (13) an 858
Journal of Chemical Education
List of Torsion Angles in Best-Fit and MinimumEnergy Conformations of NAcetyl-D-ALA-D-ALA
Bond
Best Fit
Minimum Energy
1
(C-N-GC)
141.29
46.89
2
(N-C-GN)
150.68
178.41
3
(C-GN-C)
148.99
165.92
4
(C-N-GC)
-150.48
19.56
Penicillin G
/"
N-Acetyl D-ALA-D-ALA
Hydrogens are omitted for clarity Figure 4. Superimposition of penicillin G and best-fit conformationof Nacetvl-o-ALA-o-ALA. sponding atoms of the penicillin were superimposed. The fitting rms fell *om 0.201 to 0.016, indicating a significant improvement in fit. Because the best-fit was highly subjective and was determined by individual students' perceptions of how well the two structures were superimposed, variations on the rms fitting deviation of 0.016 were observed. The penicillin crystal structure and the best-fit conformation of the peptide are shown in Figure 4.
This case study has proved to be a valuable tool inintroducing students to the concepts of molecular modelling and computational chemistry. This exercise was not designed to be a rigorous investigation into the mode of action of beta-lactam drugs, so the results are unlikely to be of any real value to such understandmg. Beta-lactams are highly strained molecules that require significantlymore intensive computational techniques to obtain minimum-energy structures than those used in a study of this type (14). The use of a molecular-mechanics minimized structure for the dipeptide system was accepted as necessary due to t h e time constraints and computer power available. Clearly, these structures do not reflect the aqueous environment of the natural system. Also, no adjustment was made to account for the packing forces evident in the crystal structure of the penicillin, though clearly these forces are absent in the natural environment. The case study was undertaken by students over a period of five weeks with 4 h of class time per week. This time was initially used to introduce the computer package being used and to present the theory behind the problem being modelled. Most students spent time in addition to scheduled time because unrestricted computer access was available. Conclusion This exercise proved popular with the students and allowed them to develop a working knowledge of some of the important aspects of computer-aided molecular design. As a teaching aid, the exercise proved useful in illustrating the three-dimensional differences between pairs of enantiomers, exemplifying the concepts of torsion angle and conformational analysis. Students also had considerable opportunity for experimentation with the system to explore their own ideas.
Analysis of Results At the conclusion of the modelling exercise each student had obtained a minimum-energy conformation and a bestfit conformation of the N-~~~-D-ALA-D-ALA dipeptide, along with contour maps and graphs of torsion angle against energy, indicating the conformationalflexibility of the molecule. The final step was to consider whether the best-fit conformation was energetically possible. A single-point COSMIC calculation was carried out to determine the molecular-mechanics energy of the besefit conformation. This resulted in an energy of 25.02 kcal mol-', which compared with an energy of 15.42 kcal mol-' for the minimum-energy conformation calculated by the earlier COSMIC energy-minimization process. An energy difference of this magnitude would be relatively easy to overcome. To ensure that no torsional-energy barriers had to be surmounted in changing between the-two conformations. the enerw contour maos were analvzed. The table lists all four torsion angles studied during the exercise for both best-fit and minimum-energy conformations. In each case there was no barrier to overcome in rotating the bond from the higher-energy to the lower-energy conformation. In theory the dipeptide system could readily adopt a conformation that would place the atoms in similar regions of space to the corresponding atoms of the penicillin, thus making it possible for the penicillin to act as a mimic of the natural pentapeptide system.
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Acknowledgment We would like to thank the European Community for a bursary (to LG) from the European Social Fund; the Science and Engineering Research Council for access to the Cambridge Structural Database; the Research Committee and Industrial Development Fund of University of Abertay Dundee for additional funding, and to David Bremner for advice during the development of this exercise. Literature Cited 1. Dictionary ofckmisfry Softurn 1992;Werr, W ;.Willett, P.;Do-,
G.,Eds.;Cherwell SIientific h b l i s l u n g a n d h r i c a n Chemical Society: (Mord. 1992. 2. Bays, J. P. J C h . Edvf 1992,69,209-215. 3. Bremner,D.H.:Bmm,M.S.;F3rgan,N.S.;lbrranee, J.M.J. Chem. Re$.(SJlsBP. 42M23. Bremner,D. H.; Ringan,N.S.J. Chem. S a , Perkin %inns 11881.1265,9"?
sweet. R M.In Cqklaswrinsand Ponicillirrr: Ckmislry ond Biology; Flw,E. H.,Ed.;Academic: New York, 1973:Chapter 7. 5. Boyd, D. B. In CkmiatryondBiologyafBeto-loefamAntibiofii;Molin. R. B.; G o r man. M., Eds.;Aeademie: New Ymk, 1982:Vol.1,Chapter 5. 6. Frank1in.T J.: Snow.G.ABiockmlslm 4th ed.:. C h a.~ m a n .ofAntimlcmbiolAction. . and nail:&don, i989. 7. Tipper, 0. S.;Stromingsr, J.L.Pmc Natl A d &i. USA 1965.51,113b1141. 8. Oxford Moledar Ltd, Magdalen Centre, W o r d Snence Park, Word, England, 4.
51. lo. Abraham, R J.; Haworth, I S . J CompuL-A&d M d . &sign ISM. 2.125-135. 11. Cmwrmf D.: Bunn,C. w.; Rogera-Law. D. w: TumeFJones, A I" m Ckmistmof Penicillin; Clarke. H. T;Johnson, J. R.; Robinson. R., Eda.;Rineeton University: Princeton. 1949: C'--'-" 12. D e a r , D. D.;van d 13. Maekav. A L.AcIa l
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