instance, gives a triplet (—CH 3 split by the adjacent —CH 2 −group into three peaks)
and a quartet (—CH2—split into four peaks by the —CH 3 ). The magnitude of the cou-
pling constant for two protons is also influenced by the dihedral angle of the X—Y
bond in an H—X—Y—H structure, and can be used in conformational analysis.
In peptides, the coupling constants of the —CH— and —NH— protons show a corre-
lation with the dihedral angle. This, however, can be ambiguous, since some coupling
constants can be assigned to four different dihedral angles. Additional structural informa-
tion can be obtained from the coupling constant of the H—^13 C—N—H structure or
H—C—C—^15 N arrangement, giving correlations that do not overlap with the H—X—
Y—H curve.
Much NMR work has been done on the interaction of small molecules with macro-
molecules, which is obviously of great interest in drug–receptor binding studies as well
as in enzymology. In principle, the small-molecule resonances are easy to follow, pro-
vided they are not overlapped by the very complex and broad spectra of the macromol-
ecules in the same solution. This technique was used to gain information on drug
binding to serum albumin, and in some cases the binding moieties of the small mole-
cule could be recognized by increased relaxation rates of some of the protons. It is
much more difficult to obtain data on the dynamics of the binding of a macromolecule,
such as an enzyme.
The advent of high magnetic field spectrometers and two-dimensional spectroscopy
techniques has facilitated the utility of NMR spectroscopy in determining molecular
structure. In a typical experiment, the key to using NMR spectroscopy in determining
molecular structure lies in the information obtained from interactions among protons in
the drug molecule. As part of this process, it is mandatory to assign all of the individ-
ual resonances in the NMR spectrum to specific protons in the drug molecule. The
assignment of resonances is greatly simplified by two-dimensional NMR experiments:
COSY (correlatedspectroscopy), which provides information about through-bond
interactions between protons; and NOESY (nuclearOverhauser enhancementspec-
troscopy), which provides information about through-space interactions. These tech-
niques are permitting NMR spectroscopy to provide valuable structural information
about drug molecules and even about the macromolecular peptidic receptors with which
the drug molecules interact.
1.6.6.3 Comparison of Experimental Techniques
In comparing the use of experimental techniques such as NMR and X-ray crystallogra-
phy against theoretical techniques such as quantum mechanics and molecular mechan-
ics, a number of strengths and weaknesses must be considered. In general, experimental
techniques have the strength of being applied to “real molecules” and being “less
abstract.” X-ray crystallography is the “gold standard” and the “method of choice” for
determining the structure of a drug molecule. However, before X-ray crystallography
can be used, the compound must be synthesized, purified, and crystallized. Also, X-ray
crystallography provides structural information about a solid-state form of the drug
molecule. This solid-state geometry and conformation may bear no resemblance what-
soever to the solution phase structure of the drug. (Determining how a drug interacts
with its receptor by using solid state X-ray data is analogous to determining how geese
60 MEDICINAL CHEMISTRY