Chemistry 164, Spring, 2007
Molecular Structure and Modeling
Graded Exercise 2
Conformational Analysis of Amphetamine
Most substrates and inhibitors are conformationally flexible and often only a
single conformer or a small range of conformers binds well to the active site of the
enzyme. For energetic and entropic reasons, the distribution of molecules over
conformational states leads to a reduction in its biological activity. Energy is important
when the biologically active conformation is not the global minimum. Entropy is the
decisive factor when the global minimum binds well but there is a large number of nearly
equi-energetic conformers that do not bind well. To solve this problem, many potent
drugs are designed to be conformationally inflexible and adopt a single conformation that
closely matches the biologically active conformation of the native substrate. This issue is
discussed in an essay by M. S. Harrold, “The Influence of Conformational Isomerism on
Drug Action and Design”, Am. J. Pharm. Ed., 60, 192-1997 (1996)
[http://www.ajpe.org/legacy/pdfs/aj6002192.pdf ]. Graded Exercise 2 employs
amphetamine as a case study illustrating the principles discussed in his essay.
The enzyme phenylethanolamine N-methyl transferase (PNMT) [EC 2.1.1.28]
catalyzes the methylation of norephinephrine [CAS 51-42-2] to produce the
neutrotransmitter epinephrine [CAS 51-43-4]. Amphetamine [CAS 300-62-9] is an
inhibitor of PMMT (Ki = 740 μM) but the street drug Euphoria [CAS 29493-77-4] is a
thousand-fold more effective. In a classic paper, Grunewald et al. [G. L. Grunewald, R.
T. Borchardt, M. F. Rafferty, and P. Krass, “Conformational Preferences of
Amphetamine Analogues for Inhibition of Phenylethanolamine N-Methyltransferase”,
Mol. Pharmacol., 20, 377-381 (1981)
[http://molpharm.aspetjournals.org/cgi/reprint/20/2/377] applied the principles of
conformational analysis to the binding of amphetamine and its analogues. His group
addressed the problem via the synthesis and testing of a carefully selected set of
analogues. In this exercise, you will use molecular mechanics to examine amphetamine.
Use the Merck Molecular Force Field (MMFF94) in this exercise. You can complete the
exercise with Spartan but you may find SYBYL to be helpful in some steps.
1) Draw the structure of amphetamine and minimize it via the Equilibrium Geometry
option under Calculations. You will find a local minimum which may or may not be the
global minimum.
2) Next perform a search for conformers using the Conformer Distribution option under
Calculations. Compare the structures and relative energies of these conformers. You
may want to repeat the calculation starting with a different conformation. Search
algorithms sometimes get stuck in a rut and don’t search all of conformational space.
Use the Boltzmann equation to calculate the relative population of each conformer at 298
K and focus your analysis on conformers with a relative abundance of 1% or more.
Compare their structures with those of octopamine [CAS104-14-3] bound to PNMT. The
3D structure of this analogue of norephinephrine has been extracted with the aid of
SYBYL from the crystal structure of the enzyme-substrate complex [pdb file number
2an4] and is available on the course Web page. Compare the various structures and
interpret the results.
3) Discuss whether the proton NMR spectrum of amphetamine would yield unambiguous
information on the presence of any of the conformers.
4) Download the crystal structure of amphetamine from the Cambridge Structural
Database (CSD) and compare it with the structures of the conformers. The CSD freeware
program Mercury [http://www.ccdc.cam.ac.uk/free_services/mercury/ ] allows one to
convert the structure files from a variety of formats, e.g. cif to pdb. This nifty program
also allows one to show how the molecules are packed in the unit cell. Discuss the
similarities and differences.
4) Dr. David A. Gallagher used Fujitsu’s CAChe software to explain the unusual potency
of Euphoria. He concluded the energy is the decisive factor. Euphoria is locked into a
conformation that binds well. The biologically active conformation of amphetamine has
a relatively high energy compared with the global minimum. Do your results support or
refute Gallagher’s conclusions? You may need to perform a conformational analysis of
Euphoria.
5) Obtain an estimate of the energetic barrier(s) between a pair of conformers of
amphetamine. To this end, perform a grid search which is practical since one dihedral
angle defined by the heavy atoms C(1-phenyl)-C-C-N determines most of the its
conformational landscape. A Newman diagram shows that one anti and two gauche
conformers are possible. Choose the gauche/anti pair with the lowest energy. You will
use the Energy Profile option under Calculations. Use a grid spacing , i.e. , of 10.
Observe the orientations of the phenyl ring and the amino group. Are they linked to the
dihedral angle that you varied? [Spartan does not allow one to calculate a twodimensional grid, i.e. a stepwise variation of two dihedral angles. This task can be
performed by SYBYL.]
CH3
H
H
H
NH2
amphetamine
C164_ex2_2007.doc, 24 Jan. 2007, WES