Investigation of The Three-Dimensional Solution Structure of Melanostatin Using One - and Two-Dimensional NMR Spectroscopy and Molecular Dynamics Calculations

Investigation of the three-dimensional solution
structure of melanostatin using one- and

two-dimensional NMR spectroscopy and molecular
dynamics calculations
Ryan McQuillen
Department of Physics, Lafayette College, Hugel Science Center, Easton, PA 18042
Advisor: Professor Bradley C. Antanaitis
May 9, 2012
Investigation of the three-dimensional solution structure of
melanostatin using one- and two-dimensional NMR spectroscopy
and molecular dynamics calculations
Ryan J. McQuillen
Department of Physics, Lafayette College, Hugel Science Center, Easton, PA 18042
Advisor: Professor Bradley C. Antanaitis
May 9, 2012
Abstract
Two cyclic nonapeptides derived from alpha-fetoprotein (AFP) have been synthe-
sized by researchers at Albany Medical College and shown to inhibit the growth of
estrogen-receptor (ER) positive breast cancer while exhibiting minimal toxicity. How-
ever, the three-dimensional structures of these nonapetides in solution are unknown
and their biological receptors remain to be identied. A method has been developed
to elucidate their solution structures using a combination of NMR spectroscopy and
molecular dynamics. Because of the complexity of the nonapeptides NMR spectra
and the large number of conformations presumably available to them, it was deemed
prudent to rst test the methodology on simpler compounds whose structures are rea-
sonably well characterized in the literature. For this purpose melanostatin (Pro-Leu-
Gly), a tripeptide having two amino acid residues in common with the nonapeptides
was chosen. Using a custom suite of sophisticated one- and two-dimensional NMR
spectroscopic protocols all resonance peaks in melanostatin and its three constituent
amino acids were identied. These assignments combined with calculations made by
HyperChem, a molecular dynamics program, allowed the prediction of the dominant
solution conformations of melanostatin. In principle, similar techniques applied to the
nonapeptides should give complete and unambiguous resonance assignments of their
complex NMR spectra and identify their dominant solution structures. In turn, that
information will serve as input for molecular docking studies designed to identify the
biological receptors of these promising anti-breast cancer compounds and thus shed
light on their mode of action.
Keywords: melanostatin, hydrogen bond, solution structure
1
Contents
1 Introduction 9
1.1 Motivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.2 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.3 General Protein Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
1.4 Melanostatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
2 Materials and Methods 13
2.1 Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
2.2 Nuclear Magnetic Resonance (NMR) Spectroscopy . . . . . . . . . . . . . . . 13
2.2.1 NMR Suite for Structural Determination of Polypeptides . . . . . . . 14
2.2.2 Solvent Solutions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
2.3 Molecular Dynamics Calculations . . . . . . . . . . . . . . . . . . . . . . . . 16
2.3.1 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
2.3.2 Understanding the AMBER Force Field . . . . . . . . . . . . . . . . 18
2.3.3 Simulated Annealing and Temperature Mimicking Scheme . . . . . . 23
2.4 Running a Simulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23
3 Results 24
3.1
1
H and
13
C NMR Spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
3.1.1 1D Carbon-13 NMR . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
3.1.2 1D and 2D Proton NMR . . . . . . . . . . . . . . . . . . . . . . . . . 35
3.2 Solution Conformations Predicted by Molecular Dynamics . . . . . . . . . . 60
4 Conclusion & Discussion 70
4.1 Future Work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
A Nuclear Magnetic Resonance (NMR) Spectroscopy 74
A.1 Physical Basis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
A.2 Magnetic Resonance Condition . . . . . . . . . . . . . . . . . . . . . . . . . 79
A.3 Saturation and T
1
Relaxation Processes . . . . . . . . . . . . . . . . . . . . 79
A.4 The Bloch Equations: A Classical Description . . . . . . . . . . . . . . . . . 81
A.4.1 The Non-Rotating Reference Frame . . . . . . . . . . . . . . . . . . . 81
A.4.2 The Rotating Reference Frame . . . . . . . . . . . . . . . . . . . . . 85
A.5 Introduction of the Transverse Magnetic Field . . . . . . . . . . . . . . . . . 88
A.5.1 Nutation of M by B
1
. . . . . . . . . . . . . . . . . . . . . . . . . . . 91
A.5.2 Pulse Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
A.6 Recording a Signal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
A.7 The Chemical Shift . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96
A.7.1 Contributions to Nuclear Shielding . . . . . . . . . . . . . . . . . . . 98
A.7.2 The Chemical Shift Scale . . . . . . . . . . . . . . . . . . . . . . . . . 101
A.7.3 The Reference Compound . . . . . . . . . . . . . . . . . . . . . . . . 101
A.8 First-Order (Weak) Spin-Spin Coupling . . . . . . . . . . . . . . . . . . . . . 103
A.8.1 Singly Coupled (AX) Nuclei . . . . . . . . . . . . . . . . . . . . . . . 104
2
A.8.2 Coupling to Two Inequivalent Nuclei (AMX) . . . . . . . . . . . . . . 105
A.8.3 Coupling to Two Equivalent Nuclei (AX
2
) . . . . . . . . . . . . . . . 105
A.8.4 Coupling to n Equivalent Nuclei (AX
n
) . . . . . . . . . . . . . . . . . 107
A.9 Pulse Sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
A.9.1 The
1
H and
13
C Free-Induction-Decay (FID) Pulse Sequence . . . . . 108
A.9.2 The
13
C Single Pulse Broadband Decoupling Pulse Sequence . . . . . 109
A.9.3 The
1
H Single Pulse Watergate Suppression Pulse Sequence . . . . . 110
A.9.4 The
13
C Attached Proton Test (APT) Pulse Sequence . . . . . . . . . 110
A.9.5 The
13
C Distortionless Enhancement by Polarization Transfer (DEPT)
Pulse Sequence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
A.9.6 The Correlation Spectroscopy (COSY) Pulse Sequence . . . . . . . . 112
B The Berendsen Thermostat 113
3
List of Figures
1.1 Illustrations of the cyclic AFP-derived nonapeptide. . . . . . . . . . . . . . . 10
1.2 The three amino acid constituents of melanostatin and their representative
structures.[12] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
1.3 A ribbon model representing the secondary structure of a -bended molecule.[13] 13
2.1 Experimental process for a given system or model.[27] . . . . . . . . . . . . . 16
2.2 Mass-spring model of two atoms (i and j) separated by a single (M
1
) bond. . 19
2.3 The angle between two atoms (i and j) separated by two (M
2
) bonds. . . . . 19
2.4 Torsion angle of the bond (j,k) between two bonded pairs (M
3
) of atoms (i,j
and k,l). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
2.5 Plot of the LJ Potential between two atoms (i and j) separated by three or
more bonds (M
4
) in a ctitious molecule. . . . . . . . . . . . . . . . . . . . . 21
2.6 Plot of the Hydrogen Bond Potential between two atoms (i and j) separated
by three or more bonds (M
4
) in a ctitious molecule. . . . . . . . . . . . . . 22
2.7 Plot of the electrostatic interaction potential as a function of atomic separation
for two atoms (i and j) separated by three or more bonds (M
4
) in a ctitious
molecule. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
2.8 The free energy surface of Beta3s (a mini-protein) and assigned conformational
states.[34] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24
3.1 Glycine 100mM 90% D
2
O/10% H
2
O
13
C with Broadband Decoupling spectra. 26
3.2 L-4-Hydroxyproline 100mM
13
C with Broadband Decoupling spectra. . . . . 28
3.3 Leucine 100mM 90% H
2
O/10% D
2
O
13
C with Broadband Decoupling Spectra. 30
3.4 Melanostatin 3mM 90%H
2
O/10% D
2
O
13
C with Broadband Decoupling spectra. 32
3.5 Hypothetical 1D proton spectrum of melanostatin resulting from superpo-
sition of L-4-hydroxyproline, leucine, and glycine assuming no inter-residue
interactions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
3.6
1
H Single Pulse with Water Suppression Spectra of Glycine 100mM in both
solvent mixtures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
3.7 L-4-Hydroxyproline 100mM 90% H
2
O/10% D
2
O
1
H Single Pulse with Water
Suppression Spectrum. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
3.8 Enlarged detailed regions of the L-4-Hydroxyproline 100mM 90% H
2
O/10%
D
2
O
1
H Single Pulse with Water Suppression Spectra. . . . . . . . . . . . . . 40
3.9 L-4-Hydroxyproline 100mM 90% D
2
O/10% H
2
O
1
H Single Pulse with Water
Suppression Spectrum. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
3.10 Enlarged detailed regions of the L-4-Hydroxyproline 100mM 90% D
2
O/10%
H
2
O
1
H Single Pulse with Water Suppression Spectra. . . . . . . . . . . . . . 42
3.11 Leucine 100mM 90% H
2
O/10% D
2
O
1
H Single Pulse with Water Suppression
Spectrum. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
3.12 Enlarged detailed regions of the Leucine 100mM 90% H
2
O/10% D
2
O
1
H Single
Pulse with Water Suppression Spectra. . . . . . . . . . . . . . . . . . . . . . 45
3.13 Leucine 100mM 90% D
2
O/10% H
2
O
1
Spectrum. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
3.14 Leucine 100mM 90% D
2
O/10% H
2
O
1
Spectrum with detailed regions enlarged. . . . . . . . . . . . . . . . . . . . . 47
4
3.15 Melanostatin 3mM 90% H
2
O/10% D
2
O
1
H Single Pulse with Water Suppres-
sion Spectrum. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
2
O/10% D
2
O
1
sion Spectrum with detailed regions enlarged. . . . . . . . . . . . . . . . . . 50
2
O/10% D
2
O
1
sion Spectrum with detailed regions enlarged (continued). . . . . . . . . . . . 51
3.18 Melanostatin 3mM 90% D
2
O/10% H
2
O
1
sion Spectrum. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
2
O/10% H
2
O
1
sion Spectrum with detailed regions enlarged. . . . . . . . . . . . . . . . . . 53
2
O/10% H
2
O
1
sion Spectrum with detailed regions enlarged (continued). . . . . . . . . . . . 54
3.21 Glycine Correlated Spectroscopy (COSY) Spectra . . . . . . . . . . . . . . . 56
3.22 L-4-Hydroxyproline Correlated Spectroscopy (COSY) Spectra . . . . . . . . 57
3.23 Leucine Correlated Spectroscopy (COSY) Spectra . . . . . . . . . . . . . . . 58
3.24 Melanostatin Correlated Spectroscopy (COSY) Spectra . . . . . . . . . . . . 59
3.25 Two dierent conformations of melanostatin generated using HyperChem. . . 60
3.26 Diagrammatic representation of the melanostatin and its dihedral angles.[14] 61
3.27 Karplus plot for a dihedral angle using the empirical constants A = 7.13,
B = 1.31, and C = 1.56. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
4.1 Peptide backbone of a protein with the expected correlations from COSY and
NOESY experiments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
4.2 Two advanced features of HyperChem to utilize in future studies. . . . . . . 71
A.1 Splitting of spin states for a spin-
1
2
nuclide. . . . . . . . . . . . . . . . . . . . 76
A.2 The motion of a spinning top (left) with the force of gravity acting on it. The
motion of a nucleus (right) with the force of an external magnetic eld acting
on it. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77
A.3 A system of homogeneous nuclei (left) whose average s add up to M
0
(right). 77
A.4 The bulk magnetic moment vector of a system of I =
1
2
nuclides.[21] . . . . . 78
A.5 (a) Graphical depiction of the T
1
relaxation process. (b) Exaggerated pictorial
view of each step. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
A.6 If several spins (ms) precess in the xy plane at slightly dierent rates, the
total spin amplitude decreases due to dephasing.[22] . . . . . . . . . . . . . . 83
A.7 The path traced by the tip of the bulk magnetic moment vector M as it relaxes
according to Equations A.31A.33.[22] . . . . . . . . . . . . . . . . . . . . . 85
A.8 Coordinate system dened by x
, y
, and z rotating about the z axis. . . . . 85

A.9 Graphical depiction of the precession of the nuclear spins and M when both
the static magnetic eld B
0
and the oscillating magnetic eld B
1
are present. 88
A.10 Decomposition of the linear oscillating eld B
1
. . . . . . . . . . . . . . . . . 89
A.11 The path traced by the tip of the magnetic moment vector M as it relaxes
back to M
0
.[22] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
A.12 Illustration of how the linearly oscillating magnetic eld B
1
is created in an
NMR spectrometer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
A.13 Two dierent illustrations of the pulsed magnetic eld B
1
. . . . . . . . . . . 93
5
A.14 Direction of the magnetic moment vector, M, in the rotating frame after a) a
pulse of arbitrary angle , b) after a 90
pulse, and c) after a 180
pulse.[21] 94
A.15 A dipole (m) along the x-axis generates a ux through the shaded region (coil)
in the xy-plane that is equal and opposite to that through the hemispherical
cap.[22] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
A.16 Consequences of electron shielding for a given nuclide in an NMR sample. . . 97
A.17 The circulation of electronic charge brought about by the mixing of electronic
wavefunctions by an external eld B
0
. This paramagnetic current generates
a small local magnetic eld that acts to deshield the nucleus at the center of
the electron density.[19] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99
A.18 Dipolar lines of ux generated by an induced magnetic moment at the center
of a cylindrically symmetric neighboring group (i.e., an aromatic ring) with
two protons A and B nearby. Proton A is more shielded by the paramagnetic
eect while proton B is deshielded. . . . . . . . . . . . . . . . . . . . . . . . 100
A.19 NMR spectra are conventionally plotted with chemical shift increasing from
right to left. Nucleus A, which is more strongly shielded than nucleus B, thus
appears to the right of B, has a lower resonance frequency than B, and is
sometimes referred as being upeld of B.[19] . . . . . . . . . . . . . . . . . 102
A.20 Two common well-shielded NMR standards. . . . . . . . . . . . . . . . . . . 102
A.21 The eect of
1
H
13
C scalar coupling in H
13
CO
2
on the energy levels and
spectrum of the
1
H nucleus. For clarity, the energy-level shifts due to the J-
coupling have been greatly exaggerated. The central pair of energy levels and
the upper spectrum are appropriate in the absence of a J-coupling interaction.
Scalar coupling produces the energy levels on the left and right, and the lower
spectrum. Also, you can see here that J
AX
is the coupling constant measured
in Hz[19] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
A.22 NMR spectrum of nucleus A in an AMX spin system. The four components of
the multiplet, a doublet of doublets, arise from the four combinations of M and
X spins, indicated (m = +
1
2
) and (m =
1
2
). Drawn for J
AM
> J
AX
> 0.[19]106
A.23 NMR spectrum of nucleus A in an AX
2
spin system. The triplet arises from
the four combinations of the two X spins, as indicated. Drawn for J
AX
> 0.[19] 106
A.24 Pascals triangle showing the binomial coecients in the expansion of (1+x)
n
.
The rows give the relative intensities of the (n + 1) lines in the A multiplet
of an AX
n
spin system (n = 06), where X is a spin-
1
2
nucleus. The columns
give the positions of the lines, relative to the chemical shift position, in units
of J
AX
.[19] . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
A.25 Multiplet patterns for the A nucleus in various spin systems. M and X are spin-
1
2
unless otherwise stated. Weak coupling is assumed throughout. Spectra are
drawn for [J
AX
[ > [J
AM
[.[19] . . . . . . . . . . . . . . . . . . . . . . . . . . . 107
A.26 Pulse sequence and signal for a free-induction-decay measurement.[22] . . . . 108
A.27 Pulse sequence and signal for a heteronuclear spin decoupling measurement. 109
A.28 Depiction of the elds and their associated eects on the bulk magnetic mo-
ment vectors of the carbon, M
C
, and the the hydrogen, M
H
. . . . . . . . . . 109
A.29 Pulse sequence and signal for a APT measurement. . . . . . . . . . . . . . . 111
A.30 Pulse sequence and signal for a DEPT measurement. . . . . . . . . . . . . . 111
6
A.31 Pulse sequence and signal for a COSY measurement. . . . . . . . . . . . . . 112
List of Tables
3.1 NMR peak data for Glycine 90% D
2
O/10% H
2
O
13
C Single Pulse Experiment
with Broadband Decoupling . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
2
O/10% H
2
O
13
C Attached Proton Test
(APT) Experiment with Broadband Decoupling . . . . . . . . . . . . . . . . 27
2
O/10% H
2
O
13
C Distortionless Enhance-
ment by Polarization Transfer (DEPT) Experiment with Broadband Decoupling 27
3.4 NMR peak data for L-4-Hydroxyproline 100mM 90% D
2
O/10% H
2
O
13
C Sin-
gle Pulse Experiment with Broadband Decoupling . . . . . . . . . . . . . . . 29
3.5 NMR peak data for L-4-Hydroxyproline 100mM 90% H
2
O/10% D
2
O
13
C At-
tached Proton Test (APT) Experiment with Broadband Decoupling . . . . . 29
3.6 NMR peak data for L-4-Hydroxyproline 100mM 90% H
2
O/10% D
2
O
13
C Dis-
tortionless Enhancement by Polarization Transfer Experiment with Broad-
band Decoupling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
3.7 NMR peak data for Leucine 90% H
2
O/10% D
2
O
13
with Broadband Decoupling. . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
3.8 NMR peak data for Leucine 90% D
2
O/10% H
2
O
13
(APT) Experiment with Broadband Decoupling. . . . . . . . . . . . . . . . . 31
2
O/10% D
2
O
13
C Distortionless Enhance-
ment by Polarization Transfer (DEPT) Experiment with Broadband Decoupling. 31
3.10 NMR peak data for Melanostatin 90% H
2
O/10% D
2
O
13
C Single Pulse Ex-
periment with Broadband Decoupling. . . . . . . . . . . . . . . . . . . . . . 33
3.11 NMR peak data for Melanostatin 90% D
2
O/10% H
2
O
13
C Attached Proton
Test (APT) Experiment with Broadband Decoupling. . . . . . . . . . . . . . 33
3.12 NMR peak data for Melanostain 90% H
2
O/10% D
2
O
13
C Distortionless En-
hancement by Polarization Transfer (DEPT) Experiment with Broadband De-
coupling. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
3.13 NMR peak data for Glycine 90% H
2
O/10% D
2
O
1
H Single Pulse Experiment
with Water Suppression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
2
O/10% H
2
O
1
3.15 NMR peak data for 4-L-OH-Proline 90% H
2
O/10% D
2
O
1
H Single Pulse Ex-
periment with Water Suppression . . . . . . . . . . . . . . . . . . . . . . . . 43
3.16 NMR peak data for 4-L-OH-Proline 90% D
2
O/10% H
2
O
1
H Single Pulse Ex-
periment with Water Suppression . . . . . . . . . . . . . . . . . . . . . . . . 43
2
O/10% D
2
O
1
3.18 NMR peak data for Leucine 90% D
2
O/10% H
2
O
1
3.19 NMR peak data for Melanostatin 90% H
2
O/10% D
2
O
1
H Single Pulse Exper-
iment with Water Suppression . . . . . . . . . . . . . . . . . . . . . . . . . . 55
7
3.20 NMR peak data for Melanostain 90% D
2
O/10% H
2
O
1
iment with Water Suppression . . . . . . . . . . . . . . . . . . . . . . . . . . 55
3.21 Best t dihedral angles and their associated parameters from MD simulations.
All energies are in kJ/mol and all angles are in degrees. . . . . . . . . . . . . 62
3.22 Values for the various energies and dihedral angles obtained from MD simu-
lations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
A.1 Nuclear spin quantum numbers (I) of some commonly occurring nuclides.[19] 74
8
1 Introduction
1.1 Motivation
Of all the human scourges, cancer remains one of the most intractable, recalcitrant, and
deadly. In spite of large sums of money spent in research and treatment, the over-all mortality
rate for the past 40 years has changed little.[1] To this day the best outcomes for all forms
of cancer involve early detection followed by complete surgical removal of all visible tumors.
Unfortunately, by the the time symptoms appear it is often too late because the tumor has
metastasized and become inoperable. One of the few eective treatments of metastasized
cancer involves chemotherapy, the systemic administration of cytotoxins that kill or slow the
growth of rapidly dividing cells. But this also wreaks havoc with healthy tissue, especially
tissue composed of rapidly growing cells, for example, hair, blood cells and mucosa. Even
though chemotherapy may win remission in the short run, it typically fails as the cancer
cells eventually develop a resistance to chemotherapy. For these reasons, researchers are
constantly developing new chemotherapeutic agents, always with an aim of reducing toxicity
and improving long-term ecacy.
1.2 Background
Alpha-fetoprotein (AFP) is an extensively studied protein found in pregnant women, fe-
tuses, and individuals with cancer, the last observation making the protein of great interest
to the medical community. Specically, AFP has been shown to prevent the growth of
estrogen-receptor positive breast cancer cells in vitro and thus shows promise as a poten-
tial chemotherapeutic agent in the ght against breast cancer.[2][3][4] Unfortunately, whole
proteins tend to be unwieldy, dicult to administer, inherently unstable, and expensive to
isolate. These are properties that cause pharmaceutical companies to shy away from them
as front line therapeutic agents. Recently, researchers at Albany Medical College have iden-
tied and synthesized cyclized nonapeptide derivatives, United States patent 7132400, from
9
the active site of AFP that prevent the growth of carcinogen-induced mammary tumors in
rats and inhibit the growth of estrogen-receptor positive human breast cancer xenografts
in mice.[4][5][7] Furthermore, these compounds (see Figure 1.1) retain their oncostatic ef-
cacy when taken orally, show no toxicity at levels administered to date, and evidently
have a mode of action that diers from the currently preferred chemotherapeutic agent,
tamoxifen.[2][5][6][8] Understandably, research on these and related oligopeptides, focusing
on their potential as anticancer drugs, is proceeding apace. Professor Antanaitis of the
Physics Department has recently acquired small amounts of these compounds for structural
determination. The ultimate goal of his research is the elucidation of the three-dimensional
solution structure of these compounds and identication of potential biological receptors.
However, even a cursory examination of the aqueous NMR spectra of these nonapeptides
reveals considerable complexity, making unambiguous assignment of all resonances, the rst
step in solving any structure by NMR spectroscopy, very challenging. That observation
combined with the likelihood that the polypeptides conformational space is large suggest
the structural determination project be broken down into smaller projects, each with its own
specic goals.
(a) 2D Depiction of the nona-
paptides chemical structure.
(b) 1D Depiction of the nonapaptides chemical structure.
Figure 1.1: Illustrations of the cyclic AFP-derived nonapeptide.
The current research project focuses on the development and execution of a research
protocol for structural determination of melanostatin, a tripeptide that has two amino acids
10
in common with the nonapeptides and, like the nonapeptides, is water soluble. In partic-
ular, the solution structure of melanostatin was determined by an approach that combines
information derived from a suite of one- and two-dimensional proton and carbon thirteen
NMR experiments with the predictive and illustrative power of computational molecular
modeling. For the most part, the structure of melanostatin is already well-dened in the
literature, except for a nagging dispute about the existence of an intramolecular hydrogen
bond. Bearing this in mind, this preliminary exercise will provide the means for elucidation
of the structure of the nonapeptides and also will help resolve the hydrogen bond dispute.
Key results of both the NMR and molecular modeling studies along with the techniques
developed are given below.
1.3 General Protein Structure
Proteins are linear polymer-like molecules consisting of a string of individual units called
amino acids. Twenty dierent naturally occurring amino acids have been discovered to
date; nine of which humans derive from food. Figure 1.2 gives the representative structures
of the three amino acids making up melanostatin. The three-dimensional structure and
thus the function of proteins depends on the number and sequence of the amino acids in
the chain. Because structure and function are so closely related, much research has gone
into both identifying and predicting their structures. Experimental methods, such as X-ray
crystallography and nuclear magnetic resonance (NMR) spectroscopy, have been employed
in an attempt to accurately delineate the structure of proteins.[11]
1.4 Melanostatin
To test the accuracy and feasibility of the methodology that will eventually be applied to
the nonapeptides a small, water soluble tripeptide, melanostatin, was chosen. Melanostatin,
which contains the amino acids: proline, leucine, and glycine, was thought to be appropriate
not only because it is small, but also because two of its three amino acid constituents (the
11
Figure 1.2: The three amino acid constituents of melanostatin and their representative
structures.[12]
hydroxylated form of proline and glycine) are contained in the AFP-derived nonapeptides.
Further it has been observed that even though a number of papers have been published
regarding the solution structure of melanostatin, its actual three-dimensional conformation
in solution is still in dispute. Some groups claim that an intramolecular hydrogen bond exists,
and others claim it does not.[16][14] Initial X-ray crystallographic studies of melanostatin
found a -bend structure characterized by a hydrogen bond from one of the terminal amide
hydrogen atoms to the prolyl carbonyl oxygen atom (see Figure 1.3).[14][15] However, X-
ray crystallography requires that the oligopeptide be in a crystalline state; whereas we are
interested in the structure of the protein in solution. Initial molecular dynamics studies
of the tripeptide in solution claimed that the turn structure is one of the most stable
conformations of melanostatin.[16] In contrast, a later study suggested that the -bend
conformation, and thus the hydrogen bond between the N-H of the amide group and the
C=O of proline, is highly unlikely in solution.[14] Making the choice all the more interesting
is the observation that melanostatin is the tripeptide tail of the neurohormone oxytocin, a
12
compound recently discovered to have some anticancer properties of its own.[17][18]
Figure 1.3: A ribbon model representing the secondary structure of a -bended molecule.[13]
2 Materials and Methods
2.1 Materials
Reagent grade L-4-hydroxyproline, L-leucine (pH = 5.19), glycine, and melanostatin (pH
= 8.97) were obtained from the Sigma-Aldrich Chemical Company and were used without
further purication. Two 10mM solutions of the amino acids were prepared, one in 1:9 and
one in 9:1, H
2
O:D
2
O solvent volume ratios. Similarly, 3mM solutions of melanostatin were
prepared in both 1:9 and 9:1 H
2
O:D
2
O solvent volume ratios. Because both L-leucine and
melanostatin have low solubilities, they were gently heated above room temperature using a
hot plate to bring them into solution. Samples were then placed in NMR tubes, evacuated
using a water aspirator, pressurized with ultra-pure argon gas, and stored at 4
C. We chose
4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) as an internal standard because of its low
chemical shift, sharp signal and low reactivity. The DSS standard was added in a 10:1 volume
ratio.
2.2 Nuclear Magnetic Resonance (NMR) Spectroscopy
NMR spectra were recorded with an JEOL Eclipse+ 400MHz spectrometer and analyzed
using both JEOL Delta (v. 4.36 or 5.01) and Mnova (v. 7.1.2) software. All spectra are
13
reported as intensity vs. chemical shift. Chemical shifts are reported in parts per million
(ppm) relative to DSS. The ensuing discussion is intended to briey describe the ideas behind
NMR spectroscopy. For a more detailed explanation of the theory behind NMR spectroscopy,
refer to Appendix A.
2.2.1 NMR Suite for Structural Determination of Polypeptides
A main goal of this study is to devise a method for the unambiguous assignment of all
resonances in the 1D
1
H and
13
C spectra of a given polypeptide. To complete this task,
a number of NMR pulse sequences (experiments) were used. An outline of the approach
follows:
Step 1: Initial
13
C Peak Assignments and Carbon Counting Before looking at the
1D proton spectrum of a given amino acid or polypeptide, the 1D
13
C single pulse Free
Induction Decay (FID) spectrum was analyzed. Peaks were counted to conrm that the
number of resonances corresponded to the number of carbons in the compound. Then with
the aid of amino acid data tables in reference [24], preliminary peak assignments were made
based on the relative shielding of a given carbon atom in the molecule from well-shielded
carbons with low chemical shifts to less shielded atoms with higher chemical shifts.
Step 2: Conrmation of
13
C Peak Assignments Preliminary peak assignments in the
1D
13
C single pulse FID spectrum were conrmed using both Attached Proton Test (APT)
and Distortionless Enhancement by Polarization Transfer (DEPT) spectra which identify
carbon resonances via their phase angle with respect to the chemical shift axis. Information
gleaned from the APT and DEPT spectra allow us to more condently assign resonances to
each of the carbons. Specically, in an APT experiment, C and CH
2
groups are displayed
+90
with respect to the chemical shift axis whereas CH

3
and CH groups are displayed 90
with respect to the chemical shift axis. In a DEPT experiment, CH and CH

3
groups are
displayed +90
with respect to the chemical shift axis whereas CH

2
groups are displayed
14
90
with respect to the chemical shift axis. In DEPT carbon spectra, carbons with no
hydrogen coordination are not present.
Step 3: Initial
1
H Peak Assignments After assigning carbon resonance peaks, the 1D
1
H Single Pulse FID spectrum of the amino acid or polypeptide is analyzed. Preliminary
resonance assignments are made based on chemical shifts, multiplet structure, relative peak
intensities, and when appropriate spectral linewidths.
Step 4: Conrmation of
1
H Resonances To conrm the initial peak assignments made
on the 1D proton spectrum, a combination of 2D experiments were carried out, specically,
COSY (Correlated Spectroscopy), NOESY (Nuclear Overhauser Spectroscopy), and TOCSY
(Total Correlated Spectroscopy). Specically, proton-proton COSY spectra were used to
identify sets of protons coupled via two (geminal coupling) or three (vicinal coupling) covalent
bonds. TOCSY is similar to COSY but identies not only those sets of protons coupled
through two or three covalent bonds but those connected by coupling relays. Thus, TOCSY
allows one to map out the entire spin tree of a given amino acid residue.
Step 5: HETCOR (Heteronuclear Spin Coupling) HETCOR establishes links be-
tween specic carbon resonances in the
13
C spectrum and proton multiplets in the 1D
1
H
spectrum.
Step 6: 1D Proton Spectrum Simulation and Peak Deconvolution HyperChem
v. 8.1 has two very useful modules for interpreting proton NMR spectra: a proton spectral
simulation program called NMR and a deconvolution program for resolving overlapping
peaks. The programs can be used to clear up any remaining uncertainties in a spectral
assignment. An example of each is shown in Figures 4.2a and 4.2b respectively.
15
2.2.2 Solvent Solutions
As mentioned in Section 2.1, two dierent solvent solutions (90% H
2
O/10% D
2
O and 90%D
2
O/10%
H
2
O) were used in the NMR experiments. These solvents allow one to readily identify labile
protons, i.e., those that are exchanged with the solvent. In 90:10 H
2
O/D
2
O such resonances
are usually present, but disappear in 10:90 H
2
O/D
2
O.
2.3 Molecular Dynamics Calculations
All simulations were carried out using HyperChem (v. 8.1.). Custom Excel macros were
written to run repeated simulations and record data.
2.3.1 Background
Molecular Dynamics (MD) simulations attempt to mimic the behavior of a real system by
manipulating observables and making calculations based on a simplied model of the system
(see Figure 2.1).[27] All MD simulations involve making approximations of physical theories
with the goal of producing the most accurate results within a reasonable computational time
frame. There are many dierent forms of molecular dynamics, and choosing the form that
is best suited for a given study depends on a number of factors including: type of system,
degrees of freedom, number of particles, how they interact, phenomena being observed, and
computing power available. Basically, the available methods fall into two broad categories:
quantum mechanical (QM) simulations and classical simulations.
Manipulate & control Measure or compute
certain observables SYSTEM other observables
(inputs) (outputs)
Figure 2.1: Experimental process for a given system or model.[27]
Two of the most common QM methods are ab-initio and hybrid Classical/QM (semi-
classical) simulations. Both are very useful and the decision to use one method or the
16
other often comes down to the complexity of the system being studied and the available
computing power. Given the complexity of melanostatin and the limitations of computers
readily available, it is not practical to use QM-based calculations.[28]
The simulations used in the present study fall into the fully classical mechanical domain.
Classical simulations are well suited for modeling the behavior of large biomolecules as they
can eciently handle a large number of particles because their computations scale as O(N
2
)
compared to the scaling of O(N
7
) for QM methods. The behavior of melanostatin, consisting
of 43 atoms, can easily be simulated using an empirical force eld. The semi-empirical
molecular simulations used involve a two-part model: one for intramolecular interactions
and one for system-environment interactions.[27] In these simulations the molecule is treated
classically and is coupled to an external bath to maintain a constant temperature. This
treatment causes the system to reside in the Canonical regime with each atom obeying
Newtons laws. As is customary in such treatments, the system is assumed to obey the
ergodic hypothesis which states that the time averages for quantities of interest are, to a good
approximation, equal to their associated ensemble averages. Although the present treatment
of the tripeptide does not exactly meet the qualications of the Canonical ensemble, one may
treat it as such because no analytical partition function is calculated.
To describe the interactions between various atoms in melanostatin an empirical force
eld consisting of a sum of various potential energies is used. A standard (and complete)
potential function may be written as follows:
V (r) = V
stretch
+ V
bend
+ V
oop
+ V
tors
+ V
cross
+ V
vdW
+ V
e
(2.1)
with V
stretch
describing individual bond lengths, V
bend
describing angles between two consec-
utive bonds, V
oop
describing the out of plane bending of rings, V
tors
describing the various
torsional angles within the molecule, V
cross
describing the cross terms that come from the
potentials previously mentioned, V
vdW
describing the long-range Van der Waals interactions
17
of non-bonded atoms, and V
e
which describes the long-range electrostatic interactions of
non-bonded atoms.[25]
The Assisted Model Building and Energy Renement (AMBER) force eld package in
the latest version of HyperChem (v8.1) was chosen for this study because it was designed
specically for use with biomolecules. The AMBER potential function is similar to the one
described by Equation 2.1 except it neglects the V
oop
and V
cross
potential function components
as previous studies suggest that these interactions are unimportant for most amino acid
residues.[29]
2.3.2 Understanding the AMBER Force Field
The AMBER potential consists of ve terms, each describing a dierent interaction within
the system. For a more detailed discussion on the evaluation schemes of these potentials,
see references [30] and[31]. Following is an abridged version of Equation 2.1 containing only
those potential components used in the AMBER force eld.
V (r) =
(i,j)M
1
f
r
+
(i,j)M
2
f
(i,j)M
3
f
(i,j)M
4
f
HB
+
(i,j)M
4
f
vdW
+
(i,j)M
4
f
e
(2.2)
It is important to note that in Equation 2.2, M
1
, M
2
, M
3
, and M
4
in the indices of the
summations above refer to atoms separated by one, two, three, or more than three cova-
lent bonds respectively and the fs denote dierent components of the complete potential
function.
Bond Length Potential Atoms separated by one bond can be visualized as connected
by a spring with an associated spring constant K
r
ij
specic to the bond type (Figure 2.2
below). The bond length potential can then be written as a linear approximation in the
form of Hookes law:
f
r
=
(i,j)M
1
K
r
ij
(r
ij
r
0
ij
)
2
, (2.3)
18
where r
ij
= [r
j
r
i
[ is the bond length of interest, r
0
ij
is the preferred bond length as if the
diatomic system were alone in vacuo, and K
ij
is the associated spring constant associated
with each bond. The eective spring constants for each bond type have been predetermined
by the creators of AMBER and are based on a large body of experimental data.[30] Though
not exact, a linear approximation is sucient for present purposes since one is predicting
near-equilibrium states of the tripeptide.
Figure 2.2: Mass-spring model of two atoms (i and j) separated by a single (M
1
) bond.
Bond Angle Potential The bond angle portion of the potential function is the angular
analog of the bond length potential as shown below:
f
(i,k)M
2
K
ik
(
ik
0
ik
)
2
(2.4)
Note the denition of the indices here:
ik
refers to two atoms (i and k) bound to a third
atom (j) as indicated in Figure 2.3. In this potential,
0
ik
is the equilibrium bond angle and
K
ik
is the angular spring constant associated with the bonded pairs.
Figure 2.3: The angle between two atoms (i and j) separated by two (M
2
) bonds.
19
Torsion Angle Potential The torsion potential (sometimes referred to as the dihedral
angle potential) as depicted in Figure 2.4 is modeled by the periodic potential function:
f
(i,l)M
3
1
2
V
n
[1 + cos(n
il
il
il
)] (2.5)
where V
n
is the equilibrium potential of the three-bond component,
il
is the phase angle
between bonds ij and kl (see Figure 2.4), n
il
is the number of bonds separating atoms i
and l, and
il
is the dihedral angle optimized on the simplest possible molecule.[30][31] This
potential is usually applicable only for molecules with C symmetry; however, with a simple
phase shift resulting in an alteration to V
n
, the potential can be transformed to work in
asymmetric molecules such as the tripeptide being examined as displayed in Equation 2.4
above.
Figure 2.4: Torsion angle of the bond (j,k) between two bonded pairs (M
3
) of atoms (i,j
and k,l).
Lennard-Jones (LJ) Potential Atoms more than three bonds away are considered to
interact through long-range interactions, and can be described by the Lennard-Jones (LJ)
potential shown in Figure 2.5:
f
LJ
=
(i,j)M
4
_
A
ij
R
12
ij
B
ij
R
6
ij
_
(2.6)
where R
ij
is the distance between atoms i and j, and A
ij
and B
ij
are interaction terms for
these two atoms derived from tting to experimental data.[30] The LJ potential accurately
models legitimate physical interactions and provides for ease of calculation. In particular,
20
the 1/R
12
term accounts approximately for the Pauli repulsion forces resulting from over-
lapping electron orbitals and the 1/R
6
term describes Van der Waals attraction (i.e., dipole
uctuations).
Figure 2.5: Plot of the LJ Potential between two atoms (i and j) separated by three or more
bonds (M
4
) in a ctitious molecule.
Hydrogen Bond Potential In addition to the Lennard-Jones potential, AMBER adds
a second long-range interaction term (Equation 2.7). This is a 1012 potential that applies
only to pairs of atoms that can participate in hydrogen bonding.
f
HB
=
(i,j)M
4
D
ij
_
5
_
0
ij
ij
_
12
6
_
0
ij
ij
_
10
_
(2.7)
In this potential (shown in Figure 2.6),
ij
is the distance between the hydrogen atom
and the atom in questions,
0
ij
is the equilibrium hydrogen bond distance, and D
ij
is the
hydrogen bond energy for the given atom pair specied by the creators of AMBER. Despite
its name, the creators of AMBER state that the hydrogen bond potential does not con-
tribute signicantly to the hydrogen bonding interactions between two atoms; rather, it is
implemented to ne-tune the distances between these atoms.[32]
21
Figure 2.6: Plot of the Hydrogen Bond Potential between two atoms (i and j) separated by
three or more bonds (M
4
Electrostatic Potential The electrostatic portion the potential function, shown in Figure
2.7, describes the long-range charge-charge interactions of non-bonded atoms and is modeled
by
f
e
=
(i,j)M
4
q
i
q
j
r
ij
, (2.8)
which is simply Coulombs Law for point charges. Here, is the dielectric constant of the
baths medium dened by the user.
Figure 2.7: Plot of the electrostatic interaction potential as a function of atomic separation
for two atoms (i and j) separated by three or more bonds (M
4
22
2.3.3 Simulated Annealing and Temperature Mimicking Scheme
The simulated annealing process involves steadily raising the temperature of the system well
beyond the desired simulation temperature after each simulation iteration before allowing
the tripeptide to equilibrate at the desired simulation temperature.
During the entire simulation, the temperature of the system was simulated by scaling
the velocities of each of the atoms at each time step by a factor of . This scaling factor is
dened as
=
_
1 +
t
_
T
0
T
1
__1
2
(2.9)
where T
0
is the temperature of the bath, T is the instantaneous temperature of each indi-
vidual atom related to its kinetic energy, t is the length of each time step dened by the
experimenter, and is the temperature relaxation time of the bath.
The origin of this scaling term has its roots in the Langevin equation of motion using
a Gaussian stochastic term to model the random forces on the atoms of the system due to
uctuations of the solution. This process of scaling the velocities of the atoms is known as
coupling the system to an external bath. A detailed discussion of this treatment is presented
by Berendsen et al. in Reference [33]. A more extensive explanation of the derivation
described in this paper can be found in Appendix B.
2.4 Running a Simulation
Multiple iterations of MD simulations of the tripeptide were run using simulated annealing
to prevent the tripeptide from becoming trapped in a local minimum conformations of the
energy landscape (see Figure 2.8). A dielectric constant of = 4 was used to simulate the
chemical environment in solution. The system was rst raised from 298 K to 600 K over a
period 50 ps, then allowed to equilibrate at 600 K for 100 ps before being slowly cooled back
to 298 K over a period of 200 ps. Tight coupling to the bath at a value of = 0.1 ps was
employed as we were concerned only with equilibrium conformations and not the dynamics
23
of the tripeptide during the simulation period.[35] Values for various energies and torsion
angles were collected after each simulation.
Figure 2.8: The free energy surface of Beta3s (a mini-protein) and assigned conformational
states.[34]
3 Results
This section contains 1D and 2D proton and carbon NMR spectra with key information sum-
marized in tables. There is also a subsection devoted to predictions made by MD calculations
and another joining the two.
3.1
1
H and
13
C NMR Spectra
3.1.1 1D Carbon-13 NMR
The
13
C-NMR spectra yielded no surprises with the number and type of carbons in each
amino acid and the tripeptide easily identiable. Specically, glycine depicted in Figure 3.1a
showed two sharp, well-separated peaks at 172.58 ppm and 41.74 ppm with the lower one
representing the methylene alpha carbon and the upper one the carbon of an ionized carboxyl
24
group. For this simple amino acid containing only one type of protonated carbon, APT
and DEPT experiments, shown in Figure 3.1b and 3.1c, respectively, provide no additional
information (both spectra are consistent with this result).
L-4-hydroxyproline yields to a similar analysis with the
13
C spectrum, Figure 3.2a, show-
ing ve sharp peaks easily identied on the basis of their relative chemical shift. Both APT
in Figure 3.2b and the DEPT spectra in Figure 3.2c conrm these assignments, with the
metheylene carbons having positive phase and the methines having opposite phase. As usual
the carboxyl carbon has the same phase as the methylene carbons and appears far upeld.
These results are summarized in Tables 3.43.6.
The leucine spectrum in Figure 3.3a, though more complicated, still yields to this type
of analysis, with six sharp peaks each corresponding to a specic carbon environment. Here
APT and DEPT spectra in Figures 3.3b and 3.3c respectively substantiate the tentative
assignments based on relative chemical shift. In particular the APT spectrum has four
sharp peaks below the chemical shift axis and one sharp peak above, with the CH
2
carbon
having a positive phase and the methyl and methine carbons having a negative phase. The
DEPT spectrum in Figure 3.18c conrms these assignments. These results are summarized
in Tables 3.73.9.
Even the crowded
13
C spectrum for melanostatin in Figure 3.4a allows tentative assign-
ments of all resonances aided by the fact that it is a virtual superposition of peaks found in
the spectra of its three constituent amino acids. Any ambiguity about resonance assignments
is claried by both the APT spectrum in Figure 3.4b and the DEPT spectrum 3.4c. Again,
these results are summarized in Tables 3.103.12.
25
-
2
0
-
1
0
0
1
0
2
0
3
0
4
0
5
0
6
0
7
0
8
0
9
0
1
0
0
1
1
0
1
2
0
1
3
0
1
4
0
1
5
0
1
6
0
1
7
0
1
8
0
1
9
0
2
0
0
2
1
0
2
2
0
C
h
e
m
ic
a
l
S
h
if
t

(
p
p
m
)
-
2
0
0
0
0
0
-
1
0
0
0
0
0
0 1
0
0
0
0
0
2
0
0
0
0
0
3
0
0
0
0
0
4
0
0
0
0
0
5
0
0
0
0
0
6
0
0
0
0
0
7
0
0
0
0
0
8
0
0
0
0
0
9
0
0
0
0
0
I n t e n s i t y
4 1 . 7 4 [ 4 ]
1 7 2 . 5 8 [ 1 ]
4
1
C
1
O
2
N
H
2
3
C
H
2
4
O
H
5
G
ly
c
i
n
e

1
0
0
m
M

9
0
%

D
2
O
/
1
0
%

H
2
O

1
3
C

S
i
n
g
le

P
u
ls
e

E
x
p
e
r
i
m
e
n
t

w
i
t
h

B
r
o
a
d
b
a
n
d

D
e
c
o
u
p
li
n
g
(
a
)
S
i
n
g
l
e
P
u
l
s
e
S
p
e
c
t
r
u
m
.
-
2
0
-
1
0
0
1
0
2
0
3
0
4
0
5
0
6
0
7
0
8
0
9
0
1
0
0
1
1
0
1
2
0
1
3
0
1
4
0
1
5
0
1
6
0
1
7
0
1
8
0
1
9
0
2
0
0
2
1
0
2
2
0
C
h
e
m
ic
a
l
S
h
if
t

(
p
p
m
)
-
1
5
0
0
0
0
-
1
0
0
0
0
0
-
5
0
0
0
0
0 5
0
0
0
0
1
0
0
0
0
0
1
5
0
0
0
0
2
0
0
0
0
0
2
5
0
0
0
0
3
0
0
0
0
0
3
5
0
0
0
0
4
0
0
0
0
0
4
5
0
0
0
0
5
0
0
0
0
0
5
5
0
0
0
0
6
0
0
0
0
0
6
5
0
0
0
0
7
0
0
0
0
0
I n t e n s i t y
4 1 . 7 5
4
C
1
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H
2
3
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2
4
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5
G
ly
c
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7
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8
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4
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I n t e n s i t y
4 1 . 7 5
4
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p
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.
26
Carbon Group Number
center
C COOH 1 172.52
C
CH
2
4 41.73
Table 3.1: NMR peak data for Glycine 90% D
2
O/10% H
2
O
13
with Broadband Decoupling
Carbon Group Number
center
C
CH
2
4 41.75
2
O/10% H
2
O
13
C Attached Proton Test (APT)
Experiment with Broadband Decoupling
Carbon Group Number
center
C
CH
2
4 41.75
2
O/10% H
2
O
13
C Distortionless Enhancement
by Polarization Transfer (DEPT) Experiment with Broadband Decoupling
27
-
2
0
-
1
0
0
1
0
2
0
3
0
4
0
5
0
6
0
7
0
8
0
9
0
1
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0
1
1
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1
2
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1
3
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1
4
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5
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9
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0
I n t e n s i t y
3 7 . 5 3
5 3 . 0 5
5 9 . 9 3
7 0 . 1 7
1 7 4 . 3 8
7
6
3
8
9
O
1
O
H
2
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9
0
1
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1
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1
2
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3
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4
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0
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0
I n t e n s i t y
3 7 . 6 0
5 3 . 2 4
6 0 . 0 6
7 0 . 2 9
1 7 4 . 4 4
3
7
9
6
8
O
1
O
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4
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1
1
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2
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4
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5
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0
5
0
0
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0
0
0
I n t e n s i t y
3 7 . 5 9 [ 7 ]
5 3 . 2 3 [ 3 ]
6 0 . 0 4 [ 8 ]
7 0 . 2 8 [ 6 ]
6
8
3
7
L
-
4
-
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(
D
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P
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)
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1
O
H
2
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H
2
3
N
H
4
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H
5
C
H
6
C
H
2
7
C
H
8
C
9
(
c
)
9
0
%
H
2
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/
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s
t
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a
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a
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3
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2
:
L
-
4
-
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y
d
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p
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l
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n
e
1
0
0
m
M
1
3
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w
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t
h
B
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a
d
b
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d
D
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c
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t
r
a
.
28
Carbon Group Number
center
C COOH 9 174.38
C
CH 8 59.93
C
CH
2
7 37.53
C
CH 6 70.17
C
CH
2
3 53.05
Table 3.4: NMR peak data for L-4-Hydroxyproline 100mM 90% D
2
O/10% H
2
O
13
C Single
Pulse Experiment with Broadband Decoupling
Carbon Group Number
center
C COOH 9 174.44
C
CH 8 60.06
C
CH
2
7 37.60
C
CH 6 70.29
C
CH
2
3 53.24
Table 3.5: NMR peak data for L-4-Hydroxyproline 100mM 90% H
2
O/10% D
2
O
13
C Attached
Proton Test (APT) Experiment with Broadband Decoupling
Carbon Group Number
center
C
CH 8 60.04
C
CH
2
7 37.59
C
CH 6 70.28
C
CH
2
3 53.23
Table 3.6: NMR peak data for L-4-Hydroxyproline 100mM 90% H
2
O/10% D
2
O
13
C Distor-
tionless Enhancement by Polarization Transfer Experiment with Broadband Decoupling
29
2
0
3
0
4
0
5
0
6
0
7
0
8
0
9
0
1
0
0
1
1
0
1
2
0
1
3
0
1
4
0
1
5
0
1
6
0
1
7
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1
8
0
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h
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if
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(
p
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)
-
1
0
0
0
0
0
0 1
0
0
0
0
0
2
0
0
0
0
0
3
0
0
0
0
0
4
0
0
0
0
0
5
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6
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7
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0
0
0
0
1
0
0
0
0
0
0
1
1
0
0
0
0
0
I n t e n s i t y
2 1 . 0 3
2 1 . 9 6
2 4 . 0 2
4 0 . 0 8
5 1 . 9 9 [ 2 ]
1 7 3 . 2 5
8
6
7
4
2
1
C
1
C
H
2
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3
C
H
2
4
N
H
2
5
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H
6
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H
3
7
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H
3
8
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9
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1
0
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m
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0
%

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2
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/
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%

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1
3
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w
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4
0
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4
5
0
0
0
0
5
0
0
0
0
0
I n t e n s i t y
2 1 . 0 4 [ 7 ]
2 1 . 9 7 [ 8 ]
2 4 . 0 3 [ 6 ]
4 0 . 0 9 [ 4 ]
5 2 . 0 0 [ 2 ]
1 7 3 . 2 5 [ 1 ]
4
1
2
7 8
6
C
1
C
H
2
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3
C
H
2
4
N
H
2
5
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6
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7
0
8
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1
1
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2
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3
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4
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5
0
0
0
0
0
I n t e n s i t y
2 1 . 0 1 [ 7 , 8 ]
2 4 . 0 2 [ 6 ]
4 0 . 0 9 [ 4 ]
5 1 . 9 7 [ 2 ]
6
2
7
,
8
4
C
1
C
H
2
O
3
C
H
2
4
N
H
2
5
C
H
6
C
H
3
7
C
H
3
8
O
H
9
L
e
u
c
i
n
e

1
0
0
m
M

9
0
%

H
2
O
/
1
0
%

D
2
O

1
3
C

D
i
s
t
o
r
t
i
o
n
le
s
s

E
n
h
a
n
c
e
m
e
n
t

b
y

P
o
la
r
i
z
a
t
i
o
n

T
r
a
n
s
f
e
r

(
D
E
P
T
)
(
c
)
D
i
s
t
o
r
t
i
o
n
l
e
s
s
E
n
h
a
n
c
e
m
e
n
t
b
y
P
o
l
a
r
i
z
a
t
i
o
n
T
r
a
n
s
f
e
r
(
D
E
P
T
)
S
p
e
c
t
r
u
m
F
i
g
u
r
e
3
.
3
:
L
e
u
c
i
n
e
1
0
0
m
M
9
0
%
H
2
O
/
1
0
%
D
2
O
1
3
C
w
i
t
h
B
r
o
a
d
b
a
n
d
D
e
c
o
u
p
l
i
n
g
S
p
e
c
t
r
a
.
30
Carbon Group Number
center
C COOH 1 173.25
C
CH 2 51.99
C
CH
2
4 40.08
C
CH 6 24.02
C
1
CH
3
7 21.03
C
2
CH
3
8 21.96
Table 3.7: NMR peak data for Leucine 90% H
2
O/10% D
2
O
13
with Broadband Decoupling.
Carbon Group Number
center
C
CH 2 52.00
C
CH
2
4 40.09
C
CH 6 24.03
C
1
CH
3
7 21.04
C
2
CH
3
8 21.97
Table 3.8: NMR peak data for Leucine 90% D
2
O/10% H
2
O
13
C Attached Proton Test (APT)
Experiment with Broadband Decoupling.
Carbon Group Number
center
C
CH 2 51.97
C
CH
2
4 40.09
C
CH 6 24.02
C
1
CH
3
7 21.01
C
2
CH
3
8 21.01
2
O/10% D
2
O
13
C Distortionless Enhancement
by Polarization Transfer (DEPT) Experiment with Broadband Decoupling.
31
-
2
0
-
1
0
0
1
0
2
0
3
0
4
0
5
0
6
0
7
0
8
0
9
0
1
0
0
1
1
0
1
2
0
1
3
0
1
4
0
1
5
0
1
6
0
1
7
0
1
8
0
1
9
0
2
0
0
2
1
0
2
2
0
C
h
e
m
ic
a
l
S
h
if
t

(
p
p
m
)
-
1
5
0
0
0
0
-
1
0
0
0
0
0
-
5
0
0
0
0
0 5
0
0
0
0
1
0
0
0
0
0
1
5
0
0
0
0
2
0
0
0
0
0
2
5
0
0
0
0
3
0
0
0
0
0
3
5
0
0
0
0
4
0
0
0
0
0
4
5
0
0
0
0
5
0
0
0
0
0
5
5
0
0
0
0
6
0
0
0
0
0
6
5
0
0
0
0
7
0
0
0
0
0
7
5
0
0
0
0
I n t e n s i t y
2 0 . 7 9 [ 1 8 ]
2 2 . 2 1 [ 2 0 ]
2 4 . 4 6 [ 1 9 ]
2 5 . 3 0 [ 4 ]
3 0 . 5 4 [ 1 7 ]
3 9 . 7 0 [ 5 ]
4 2 . 2 1 [ 1 3 ]
4 6 . 5 9 [ 2 ]
5 2 . 7 0 [ 9 ]
5 9 . 9 5 [ 3 ]
1 7 4 . 2 2 [ 1 0 ]
1 7 5 . 5 9 [ 6 ]
1 7 7 . 3 0 [ 1 4 ]
1
8
1
9
3
2
0
4
1
7
6
9
2
1
3
5
1
0
1
4
N
H
1
C
H
2
2
C
H
3
C
H
2
4
C
H
2
5
C 6
O 7 N
H
8
C
H
9
C 1
0
O 1
1
N
H
1
2
C
H
2
1
3
C 1
4
O 1
5
N
H
2
1
6
C
H
2
1
7
C
H
3
1
8
C
H
1
9
C
H
3
2
0
M
e
la
n
o
s
t
a
t
i
n

3
m
M

9
0
%

H
2
O
/
1
0
%

D
2
O

1
3
C

S
i
n
g
le

P
u
ls
e

E
x
p
e
r
i
m
e
n
t

w
i
t
h

B
r
o
a
d
b
a
n
d

D
e
c
o
u
p
li
n
g
(
a
)
S
i
n
g
l
e
P
u
l
s
e
S
p
e
c
t
r
u
m
-
2
0
-
1
0
0
1
0
2
0
3
0
4
0
5
0
6
0
7
0
8
0
9
0
1
0
0
1
1
0
1
2
0
1
3
0
1
4
0
1
5
0
1
6
0
1
7
0
1
8
0
1
9
0
2
0
0
2
1
0
2
2
0
C
h
e
m
ic
a
l
S
h
if
t

(
p
p
m
)
-
5
0
0
0
0
0
-
4
5
0
0
0
0
-
4
0
0
0
0
0
-
3
5
0
0
0
0
-
3
0
0
0
0
0
-
2
5
0
0
0
0
-
2
0
0
0
0
0
-
1
5
0
0
0
0
-
1
0
0
0
0
0
-
5
0
0
0
0
0 5
0
0
0
0
1
0
0
0
0
0
1
5
0
0
0
0
2
0
0
0
0
0
2
5
0
0
0
0
3
0
0
0
0
0
3
5
0
0
0
0
4
0
0
0
0
0
4
5
0
0
0
0
5
0
0
0
0
0
I n t e n s i t y
2 0 . 7 9 [ 1 8 , 2 0 ]
2 4 . 4 7 [ 1 9 ]
2 5 . 3 0 [ 4 ]
3 0 . 5 5 [ 1 7 ]
3 9 . 7 1 [ 5 ]
4 2 . 2 1 [ 1 3 ]
4 6 . 5 9 [ 2 ]
5 2 . 7 0 [ 9 ]
5 9 . 9 5 [ 3 ]
1
7
2
1
3
5
4
3
9
1
9
1
8
,
2
0
N
H
1
C
H
2
2
C
H
3
C
H
2
4
C
H
2
5
C 6
O 7 N
H
8
C
H
9
C 1
0
O 1
1
N
H
1
2
C
H
2
1
3
C 1
4
O 1
5
N
H
2
1
6
C
H
2
1
7
C
H
3
1
8
C
H
1
9
C
H
3
2
0
M
e
la
n
o
s
t
a
t
i
n

3
m
M

9
0
%

H
2
O
/
1
0
%

D
2
O

1
3
C
A
t
t
a
c
h
e
d

P
r
o
t
o
n

T
e
s
t

(
A
P
T
)
(
b
)
A
t
t
a
c
h
e
d
P
r
o
t
o
n
T
e
s
t
(
A
P
T
)
S
p
e
c
t
r
u
m
1
5
2
0
2
5
3
0
3
5
4
0
4
5
5
0
5
5
6
0
6
5
7
0
7
5
8
0
8
5
C
h
e
m
ic
a
l
S
h
if
t

(
p
p
m
)
-
1
0
0
0
0
0
0
-
9
0
0
0
0
0
-
8
0
0
0
0
0
-
7
0
0
0
0
0
-
6
0
0
0
0
0
-
5
0
0
0
0
0
-
4
0
0
0
0
0
-
3
0
0
0
0
0
-
2
0
0
0
0
0
-
1
0
0
0
0
0
0 1
0
0
0
0
0
2
0
0
0
0
0
3
0
0
0
0
0
4
0
0
0
0
0
5
0
0
0
0
0
6
0
0
0
0
0
7
0
0
0
0
0
8
0
0
0
0
0
9
0
0
0
0
0
I n t e n s i t y
2 2 . 1 7 [ 1 8 , 2 0 ]
2 4 . 4 5 [ 1 9 ]
2 4 . 9 3 [ 4 ]
3 0 . 3 9 [ 5 ]
3 9 . 6 7 [ 1 7 ]
4 2 . 1 9 [ 1 3 ]
4 6 . 5 9 [ 2 ]
5 2 . 8 1 [ 9 ]
5 9 . 8 6 [ 3 ]
3
9
4 1
9
1
8
,
2
0
1
3
2
1
7
5
M
e
la
n
o
s
t
a
t
i
n

3
m
M

9
0
%

H
2
O
/
1
0
%

D
2
O

1
3
C

D
i
s
t
o
r
t
i
o
n
le
s
s

E
n
c
h
a
n
c
e
m
e
n
t

b
y

P
o
la
r
i
z
a
t
i
o
n

T
r
a
n
s
f
e
r
N
H
1
C
H
2
2
C
H
3
C
H
2
4
C
H
2
5
C 6
O
7 N
H
8
C
H
9
C 1
0
O 1
1
N
H
1
2
C
H
2
1
3
C 1
4
O 1
5
N
H
2
1
6
C
H
2
1
7
C
H
3
1
8
C
H
1
9
C
H
3
2
0
(
c
)
D
i
s
t
o
r
t
i
o
n
l
e
s
s
E
n
h
a
n
c
e
m
e
n
t
b
y
P
o
l
a
r
i
z
a
t
i
o
n
T
r
a
n
s
f
e
r
(
D
E
P
T
)
S
p
e
c
t
r
u
m
F
i
g
u
r
e
3
.
4
:
M
e
l
a
n
o
s
t
a
t
i
n
3
m
M
9
0
%
H
2
O
/
1
0
%
D
2
O
1
3
C
w
i
t
h
B
r
o
a
d
b
a
n
d
D
e
c
o
u
p
l
i
n
g
s
p
e
c
t
r
a
.
32
Carbon Group Number
center
C (G) COOH 14 177.30
C (P) COOH 6 175.59
C (L) COOH 10 174.22
C
(P) CH 3 59.95
C
(L) CH 9 52.70
C
(G) CH
2
13 42.21
C
(P) CH
2
5 39.70
C
(L) CH
2
17 30.54
C
(L) CH 19 24.46
C
(P) CH
2
4 25.30
C
(P) CH
2
2 46.59
C
1
(L) CH
3
20 22.21
C
2
(L) CH
3
18 20.79
Table 3.10: NMR peak data for Melanostatin 90% H
2
O/10% D
2
O
13
C Single Pulse Experi-
ment with Broadband Decoupling.
Carbon Group Number
center
C
(P) CH 3 59.95
C
(L) CH 9 52.70
C
(G) CH
2
13 42.21
C
(P) CH
2
5 39.71
C
(L) CH
2
17 30.55
C
(L) CH 19 24.47
C
(P) CH
2
4 25.30
C
(P) CH
2
2 46.59
C
1
(L) CH
3
20 22.79
C
2
(L) CH
3
18 20.79
Table 3.11: NMR peak data for Melanostatin 90% D
2
O/10% H
2
O
13
(APT) Experiment with Broadband Decoupling.
33
Carbon Group Number
center
C
(P) CH 3 59.95
C
(L) CH 9 52.81
C
(G) CH
2
13 42.19
C
(P) CH
2
5 39.39
C
(L) CH
2
17 36.67
C
(L) CH 19 24.45
C
(P) CH
2
2 46.59
C
1
(L) CH
3
20 22.17
C
2
(L) CH
3
18 22.17
Table 3.12: NMR peak data for Melanostain 90% H
2
O/10% D
2
O
13
C Distortionless En-
hancement by Polarization Transfer (DEPT) Experiment with Broadband Decoupling.
34
3.1.2 1D and 2D Proton NMR
The 1D proton spectrum of melanostatins three constituent amino acids are presented in
the following Figures: glycine (Figures 3.6a and 3.6b), L-4-hydroxyproline (Figures 3.7 and
3.8ad, 3.9, and 3.10ac), and leucine (Figures 3.11, 3.12ad, 3.13, and 3.14ac).
The glycine proton spectrum consists of a single sharp peak at 3.55 ppm with an intensity
corresponding to two protons. Glycines spectral features are independent of solvent. Using
the notation suggested in [24], this is exactly what one would expect for a simple AX spin
system. Because of the inherent simplicity of this spectrum, 2D COSY did not yield any
new information.
A glance at the 1D proton NMR spectrum of L-4-hydroxyproline yields considerable com-
plexity as expected for an A
2
(T
2
)MPX spin system. Relative shielding information allowed
tentative identication of multiplets based on their chemical environments. The combina-
tion of APT and DEPT spectra enabling us to distinguish between methylene protons and
methine protons, as well as the connectivity tree established by COSY allow the assign-
ments appearing in Figure 3.7 and Table 3.15. These assignments were further aided by the
available 1D proton NMR simulation in HyperChem appearing in Figure 4.2a.
Leucines spectrum (an example of an A
3
B
3
MPTX spin system) is every bit as compli-
cated as that of L-4-hydroxyproline, however because of the prominent resonance peaks of
the two groups of methyl protons, its interpretation is somewhat easier. A combination of
relative chemical shifts aided by identication of carbon types by APT and DEPT as well
as COSY connectivities allowed for the assignments that appear in Figures 3.11, 3.12ad,
3.13, 3.14ad and Tables 3.17 and 3.18. Specically, for example, what appears to be a 1:2:1
triplet centered at 0.97 ppm (Figure 3.14a) is actually a superposition of methyl protons
in slightly dierent chemical environments split equally by interaction with a gamma CH
proton. At the other end of the proton spectrum, we see two broad upeld singlets that can
be attributed to protons coupled to nitrogen. Such peaks are broadened by interaction with
the quadrupole moment of nitrogen, with additional broadening of the peak shifted farther
35
upeld attributable to rapid exchange with the aqueous solvent. The identication of these
peaks as labile NH peaks is conrmed by the fact that they do not appear in the 90% D
2
O
spectra. These spectral features will be further investigated in the future by pH dependent
studies. Furthermore, the interesting stereochemistry of leucine suggests that NOESY may
be be used to investigate interactions between methyl protons and NH
2
protons at a later
date.
Figure 3.5: Hypothetical 1D proton spectrum of melanostatin resulting from superposition
of L-4-hydroxyproline, leucine, and glycine assuming no inter-residue interactions.
Even a cursory glance at the 1D proton spectrum of melanostatin reveals its complexity,
but as was noted in the case of its complicated
13
C spectrum, its proton spectrum is, to a rst
approximation, a superposition of the spectra of its three constituent amino acids as shown
in Figure 3.15. Of course a detailed comparison of the hypothetical superposition spectrum
with the actual spectrum of melanostatin reveals a number of dierences highlighted by fea-
36
tures appearing in Figures 3.16ad, 3.17ac, 3.18ac, and 3.19ab. Nowhere is the deviation
more apparent than in the ngerprint region of melanostatin (89 ppm) shown specically
in Figure 3.17c. Here there is a triplet centered at 8.77 ppm resulting from the interaction
of glycines amide proton with its two alpha CH protons. Only glycine could give rise to
this structure as it is the only amino acid present with two alpha carbon protons. It also
appears that there are two unresolved doublets with one centered at 8.8 ppm and the other
at 8.75 ppm. If this hypothesis is valid, that will set an upper limit on the NH alpha carbon
splitting of L-4-hydroxyproline and leucine (these couplings are later correlated with molec-
ular dynamics calculations). Again, the combination of relative chemical shifts, APT and
DEPT carbon dierentiation, as well as COSY connectivities allowed for the assignments
that appear in Figures 3.15 and 3.18 and Tables 3.19 and 3.20. As an example, consider the
four prominent resonances, shown in Figure 3.16a, due to the two methyl groups of leucine.
Tentative resonance assignments have also been made in 1.51.9 ppm region, however, the
complicated overlapping multiplet structure makes these assignments less certain. This is
one region where the deconvolution function of HyperChem (see Figure. 4.2b) would help
sort things out.
Proton Group Number
center
J Intensity
H
CH
2
4 3.55 - 2
Table 3.13: NMR peak data for Glycine 90% H
2
O/10% D
2
O
1
with Water Suppression
Proton Group Number
center
J Intensity
H
CH
2
4 3.55 - 2
2
O/10% H
2
O
1
37
-2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12
Chemical Shift (ppm)
-500000
0
500000
1000000
1500000
2000000
2500000
3000000
3500000
4000000
4500000
5000000
I
n
t
e
n
s
it
y
3
.
5
5
4
DSS
C
1
O
2
N H
2
3
CH
2
4
OH
5
Glycine 100mM 90% H2O/10% D2O
1
(a) Glycine 100mM 90% H
2
O/10% D
2
O
1
Spectrum.
-2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12
-200000
0
200000
400000
600000
800000
1000000
1200000
1400000
1600000
1800000
2000000
2200000
2400000
2600000
2800000
3000000
3200000
3400000
3600000
3800000
4000000
4200000
4400000
I
n
t
e
n
s
it
y
3
.
5
5
4
DSS
C
1
O
2
N H
2
3
CH
2
4
OH
5
(b) Glycine 100mM 90% D
2
O/10% H
2
O
1
H Single Pulse with Water Spectrum.
Figure 3.6:
1
H Single Pulse with Water Suppression Spectra of Glycine 100mM in both
solvent mixtures.
38
-
2
-
1
0
1
2
3
4
5
6
7
8
9
1
0
1
1
1
2
C
h
e
m
i
c
a
l

S
h
i
f
t

(
p
p
m
)
-
1
0
0
0
0
0
0 1
0
0
0
0
0
2
0
0
0
0
0
3
0
0
0
0
0
4
0
0
0
0
0
5
0
0
0
0
0
6
0
0
0
0
0
7
0
0
0
0
0
8
0
0
0
0
0
9
0
0
0
0
0
1
0
0
0
0
0
0
1
1
0
0
0
0
0
1
2
0
0
0
0
0
1
3
0
0
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42
Proton Group Number
center
J Intensity
H
CH 8 4.32, 4.354 7.08, 10.22 -

H
CH
2
7 2.14 4.16, 25.13 -
H
CH 6 2.14 4.09t -
H
CH
2
3 3.47, 3.35 12.53, 12.65 -
Table 3.15: NMR peak data for 4-L-OH-Proline 90% H
2
O/10% D
2
O
1
iment with Water Suppression
Proton Group Number
center
J Intensity
H
CH 8 2.14 4.17 -
H
CH
2
7 2.42, 2.14 14.53, 25.10 -
H
CH 6 4.33 - -
H
CH
2
3 4.34 6.94, 9.98 -
Table 3.16: NMR peak data for 4-L-OH-Proline 90% D
2
O/10% H
2
O
1
iment with Water Suppression
43
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45
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46
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I n t e n s i t y
8

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6

(
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t
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I n t e n s i t y
2

(
d
)
4
.
0
3
J
(
4
.
0
5
) 4
.
0
5
2
C
1
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3
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w
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p
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(
c
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g
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r
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1
4
:
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c
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n
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1
0
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m
M
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0
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2
O
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1
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%
H
2
O
1
H
S
i
n
g
l
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P
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l
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W
a
t
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p
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p
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m
w
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l
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d
.
47
Proton Group Number
center
J Intensity
H
CH 2 4.03 7.74 -
H
CH
2
4 1.74 9.57 -
H
CH 6 1.68 7.83 -
H
1
CH
3
7 0.96 5.36 -
H
2
CH
3
8 0.97 4.34 -
H NH
2
5 7.33 - 0.76
H NH
3
5 7.92 - 1.00
2
O/10% D
2
O
1
Proton Group Number
center
J Intensity
H
CH 2 4.03 4.05 -
H
CH
2
4 1.75 7.19 -
H
CH 2 1.75 7.19 -
H
1
CH
3
7 0.96 4.23 -
H
2
CH
3
8 0.98 4.68 -
Table 3.18: NMR peak data for Leucine 90% D
2
O/10% H
2
O
1
48
-
2
-
1
0
1
2
3
4
5
6
7
8
9
1
0
1
1
1
2
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I n t e n s i t y
8

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d
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7
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(
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8
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d
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(
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1
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7
1
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1
8
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2
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1
7
4
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1
9
2
8
1
2
9
N
H
1
C
H
2
2
C
H
3
C
H
2
4
C
H
2
5
C
6
O
7
N
H
8
C
H
9
C 1
0
O 1
1
N
H
1
2
C
H
2
1
3
C 1
4
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5
N
H
2
1
6
C
H
2
1
7
C
H
3
1
8
C
H
1
9
C
H
3
2
0
D
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3
m
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9
0
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2
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D
2
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3
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1
5
:
M
e
l
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s
t
a
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n
3
m
M
9
0
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H
2
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D
2
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1
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S
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.
49
0
.
8
0
0
.
8
2
0
.
8
4
0
.
8
6
0
.
8
8
0
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9
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9
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0
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9
4
0
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9
6
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9
8
1
.
0
0
1
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0
2
1
.
0
4
1
.
0
6
1
.
0
8
1
.
1
0
1
.
1
2
1
.
1
4
C
h
e
m
ic
a
l
S
h
if
t

(
p
p
m
)
0 5
0
0
0
0
0
1
0
0
0
0
0
0
1
5
0
0
0
0
0
2
0
0
0
0
0
0
2
5
0
0
0
0
0
3
0
0
0
0
0
0
3
5
0
0
0
0
0
4
0
0
0
0
0
0
4
5
0
0
0
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5
0
0
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0
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5
5
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0
0
I n t e n s i t y
2
0

(
d
)
0
.
9
4
J
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6
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1
1
)
1
8

(
d
)
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8
9
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6
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1
2
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6
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1
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6
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1
2
1
8
2
0
N
H
1
C
H
2
2
C
H
3
C
H
2
4
C
H
2
5
C 6
O
7
N
H
8 C
H
9
C 1
0
O 1
1
N
H
1
2
C
H
2
1
3
C 1
4
O 1
5
N
H
2
1
6
C
H
2
1
7
C
H
3
1
8
C
H
1
9
C
H
3
2
0
M
e
la
n
o
s
t
a
t
i
n

3
m
M

9
0
%

D
2
O
/
1
0
%

H
2
O

1
H

S
i
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n
(
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)
1
.
5
7
1
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5
8
1
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5
9
1
.
6
0
1
.
6
1
1
.
6
2
1
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6
3
1
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6
4
1
.
6
5
1
.
6
6
1
.
6
7
1
.
6
8
1
.
6
9
1
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0
1
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1
1
.7
2
1
.7
3
1
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4
1
.7
5
1
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6
1
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7
1
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8
1
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9
1
.
8
0
1
.
8
1
1
.
8
2
1
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8
3
1
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4
1
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8
7
C
h
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if
t

(
p
p
m
)
-
1
0
0
0
0
0
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0
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0
2
0
0
0
0
0
3
0
0
0
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(
c
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t
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)
.
54
Proton Group Number
center
J Intensity
H
(G) CH
2
13 3.90 10.25 -
H
(L) CH 9 4.33 10.71 -

H
(L) CH
2
17 1.76 5.94 -
H
(P) CH
2
5 2.94 6.76 -
H
(L) CH
2
19 2.16 8.37 -
H
(P) CH
2
4 1.65 22.81 -
H
1
(L) CH
3
20 0.94 6.11 -
H
2
(L) CH
3
18 0.89 6.12 -
H
(P) CH
2
2 3.78 3.80, 3.76 -
H (L) NH
2
8 8.77 18.17 -
H (G) NH
2
12 8.77 9.69 -
Table 3.19: NMR peak data for Melanostatin 90% H
2
O/10% D
2
O
1
H Single Pulse Experi-
ment with Water Suppression
Proton Group Number
center
J Intensity
H
(G) CH
2
13 3.90 9.75 -
H
(L) CH 9 4.73 3.63 -

H
(P) CH 3 - - -
H
(L) CH
2
17 1.74 4.65 -
H
(P) CH
2
5 2.92 11.03 -
H
(L) CH
2
19 2.15 12.87 -
H
(P) CH
2
4 1.65 21.34 -
H
1
(L) CH
3
20 0.94 6.11 -
H
2
(L) CH
3
18 0.89 6.12 -
H
(P) CH
2
2 3.77 6.16 -
Table 3.20: NMR peak data for Melanostain 90% D
2
O/10% H
2
O
1
55
-2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12
-2
-1
0
1
2
3
4
5
6
7
8
9
10
11
12
C
h
e
m
ic
a
l
S
h
if
t

(
p
p
m
)
4
4
4
C
1
O
2
N H
2
3
CH
2
4
OH
5
Glycine 100mM 90% H2O/10% D2O
1
H Correlated Spectroscopy (COSY)
DSS
(a) Glycine 100mM 90% H
2
O/10% D
2
O
1
Spectrum.
Glycine 100mM 90% D2O/10% H2O
1
H Correlated Spectroscopy (COSY) with Water Suppression
-2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12
-2
-1
0
1
2
3
4
5
6
7
8
9
10
11
12
C
h
e
m
ic
a
l
S
h
if
t

(
p
p
m
)
4
C
1
O
2
N H
2
3
CH
2
4
OH
5
DSS
Noise
(b) Glycine 100mM 90% D
2
O/10% H
2
O
1
Spectrum.
Figure 3.21: Glycine Correlated Spectroscopy (COSY) Spectra
56
-2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12
-2
-1
0
1
2
3
4
5
6
7
8
9
10
11
12
C
h
e
m
ic
a
l
S
h
if
t

(
p
p
m
)
8 7
3
6
L-4-Hydroxyproline 100mM 90% H2O/10% D2O
1
O
1
OH
2
CH
2
3
NH
4
O H
5
C H
6
CH
2
7
CH
8
C
9
DSS
(a) L-4-Hydroxyproline 100mM 90% H
2
O/10% D
2
O
1
H Correlated Spectroscopy
(COSY) Spectrum.
-2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12
-2
-1
0
1
2
3
4
5
6
7
8
9
10
11
12
C
h
e
m
ic
a
l
S
h
if
t

(
p
p
m
)
8
7
3
6
DSS O
1
OH
2
CH
2
3
NH
4
O H
5
C H
6
CH
2
7
CH
8
C
9
L-4-Hydroxyproline 90% D2O/10% H2O
1
(b) L-4-Hydroxyproline 100mM 90% D
2
O/10% H
2
O
1
H Correlated Spectroscopy
(COSY) Spectrum.
Figure 3.22: L-4-Hydroxyproline Correlated Spectroscopy (COSY) Spectra
57
-2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12
-2
-1
0
1
2
3
4
5
6
7
8
9
10
11
12
C
h
e
m
ic
a
l
S
h
if
t

(
p
p
m
)
5 2 6 8
5
2
6
8
7
8
4
6
2
5
C
1
CH
2
O
3
CH
2
4
NH
2
5
CH
6
C H
3
7
CH
3
8
OH
9
NH3
DSS
Leucine 100mM 90% H2O/10% D2O
1
(a) Leucine 100mM 90% H
2
O/10% D
2
O
1
Spectrum.
-2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12
-2
-1
0
1
2
3
4
5
6
7
8
9
10
11
12
C
h
e
m
ic
a
l
S
h
if
t

(
p
p
m
)
2 4 7
2
4
7
8
7
6
4
2
C
1
CH
2
O
3
CH
2
4
NH
2
5
CH
6
C H
3
7
CH
3
8
OH
9
NH3 and NH2 disappear
DSS
Leucine 100mM 90% D2O/10% H2O
1
(b) Leucine 100mM 90% D
2
O/10% H
2
O
1
Spectrum.
Figure 3.23: Leucine Correlated Spectroscopy (COSY) Spectra
58
-2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12
-2
-1
0
1
2
3
4
5
6
7
8
9
10
11
12
C
h
e
m
ic
a
l
S
h
if
t

(
p
p
m
)
20
18
4
17
19
5
2
13
9
Melanostatin 3mM 90% H2O/10% D2O
1
NH
1
CH
2
2
CH
3
CH
2
4
C H
2
5
C
6
O
7
NH
8 C H
9
C
10
O
11
NH
12
CH
2
13
C
14
O
15
NH
2
16 CH
2
17
C H
3
18
CH
19
CH
3
20
DSS
(a) Melanostatin 3mM 90% H
2
O/10% D
2
O
1
Spectrum.
-2 -1 0 1 2 3 4 5 6 7 8 9 10 11 12
-2
-1
0
1
2
3
4
5
6
7
8
9
10
11
12
C
h
e
m
ic
a
l
S
h
if
t

(
p
p
m
)
20
18
17
4
19
5
13
2
9
Melanostatin 3mM 90% D2O/10% H2O
1
NH
1 C H
2
2
CH
3
C H
2
4 CH
2
5
C
6
O
7
NH
8
C H
9
C
10
O
11
NH
12
CH
2
13
C
14
O
15
NH
2
16
CH
2
17
CH
3
18
C H
19
CH
3
20
DSS
(b) Melanostatin 3mM 90% D
2
O/10% H
2
O
1
Spectrum.
Figure 3.24: Melanostatin Correlated Spectroscopy (COSY) Spectra
59
3.2 Solution Conformations Predicted by Molecular Dynamics
Preliminary analysis of the results of the MD simulations of melanostatin using a constant
dielectric environment suggests one dominant conformation in solution. Figure 3.25a shows
melanostatin in its -bend conguration with a hydrogen bond from the NH of the pro-
line backbone to the COO
end of glycine. Comparing the values for

2
and
3
of this
conformation with those resulting from MD simulations listed in Table 3.22, it can be said
with condence that melanostatin maintains an open structure in solution in accordance
with observations made from NMR data. The average of the absolute values for the simu-
lation dihedral angles are
1
= 155.861
,
2
= 137.951
,
2
= 155.110
, and
3
= 162.686
,
corresponding to an open structure as seen in Figure 3.25b.
(a) Model of melanostatin in -bend conguration.
Here
2
= -75.047
and
3
= 90.723
.
(b) Model of melanostatin created using the
average dihedral angles from MD simula-
tions
Figure 3.25: Two dierent conformations of melanostatin generated using HyperChem.
An elegant way to correlate the experimental NMR data and theoretical information
obtained from MD simulations is to use the Karplus relation (Equation 3.1), a standard
theoretical tool (based on Quantum Mechanics) used by chemists and biochemists alike:
3
J() = Acos
2
+ Bcos + C (3.1)
where = 60
and is a backbone torsion angle.

60
Figure 3.26: Diagrammatic representation of the melanostatin and its dihedral angles.[14]
This equation, derived by Martin Karplus in 1959, uses empirically determined constants
to relate vicinal (
3
J) coupling constants of specic proton groups in protein and peptide
spectra to the dihedral angles of dominant conformations in solution. The vicinal coupling
constants for the amide (NH) peaks listed in Table 3.19 were derived from the proton spec-
trum of melanostatin in 90% H
2
O/10% D
2
as depicted in Figure 3.17c. The values A = 7.134,
B = 1.31, and C = 1.56 were used to determine the values of
2
of leucine and
3
of glycine,
the amide dihedral angles of melanostatins backbone (see Figure 3.20), as per reference [36].
Figure 3.27: Karplus plot for a dihedral angle using the empirical constants A = 7.13,
B = 1.31, and C = 1.56.
The Karplus plot for the prescribed values of A, B, and C is shown in Figure 3.27. Based
on the uncertainty in our spectroscopic measurement, we estimate an error of 1 Hz in our
61
values of
3
J, therefore the values with uncertainties for each of the backbone dihedral angles
are:
2
= (183.462 0.131)
and
3
= (228.494 0.328)
. (3.2)
These values compare favorably with the values of
2
and
3
obtained from the MD
simulations of the tripeptide listed in Table 3.22. As Table 3.21 shows, the values of
2
and
3
that correlate best with those generated by the Karplus equation also correspond to the
lowest energies.
E
total
E
stretch
E
bend
E
tors

1

2

2

3
188.402 24.164 54.793 34.969 146.987 -177.647 153.000 -174.287
189.196 21.850 53.959 33.391 165.811 -175.681 143.205 175.349
190.251 21.074 49.344 38.668 136.362 -117.029 172.007 172.932
191.523 24.911 44.011 40.177 148.980 -143.546 172.797 -166.736
196.539 23.104 44.530 35.466 178.002 -129.377 178.314 -165.029
Table 3.21: Best t dihedral angles and their associated parameters from MD simulations.
All energies are in kJ/mol and all angles are in degrees.
62
T
a
b
l
e
3
.
2
2
:
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.
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q
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a
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2
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3
2
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8
)
.
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63
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4 Conclusion & Discussion
As a result of the current research project a comprehensive NMR suite was developed and ap-
plied to a simple, water soluble tripeptide, melanostatin that shares two amino acid residues
with nonapeptides exhibiting anti-cancer properties and low toxicity. Along with the NMR
studies molecular dynamics calculations were carried out on the same compound revealing
its preference for a solution conformation, that does not even remotely resemble a -turn
structure containing an intramolecular hydrogen bond. Dihedral angle calculations using the
Karplus equation, based on measured backbone vicinal scalar coupling constants in melano-
statin are, to a good approximation, consistent with conclusions drawn from the molecu-
lar dynamics calculations. The absence of additional structure in the ngerprint region of
melanostatin compared with the NMR of its amino acid constituents further substantiates
that conclusion. Preliminary COSY and NOESY measurements made on melanostatin and
its constituent amino acids suggest the feasibility of using these techniques for establishing
connectivities between interacting protons in the cyclic nonpeptides backbone (see Figure
4.1) and further for determining through space proximity of protons attached to residue side
chains.
Figure 4.1: Peptide backbone of a protein with the expected correlations from COSY and
NOESY experiments.
70
4.1 Future Work
The current project is just one part of a much larger one, the aims of which are to deter-
mine the solution structures of the the two cyclicized AFP-derived nonapeptides that show
promise as anti-breast cancer chemotherapeutics, identify their physiological receptors, and
understand their mode of action. Using a relatively simple water soluble tripeptide sharing
two amino acids in common with the nonapeptides, this part of the project sought to develop
and execute both NMR and molecular dynamics protocols that would elucidate the structure
of the simpler compound, melanostatin (a tripeptide). This goal has been largely realized,
however several things remain to be done.
(a) Representative spectra derived from the 1D
1
H NMR spectrum prediction software in Hyper-
Chem.
(b) Complex multiplet structure found in melanos-
tatin. An example where peak deconvolution would
aid in determining the multiplet structure.
Figure 4.2: Two advanced features of HyperChem to utilize in future studies.
Chief among these is perfecting and more fully exploiting some of the 2D NMR techniques
applied to the tripeptide namely, NOESY, ROESY, and HETCOR. Specically, the inter-
residue amide proton-alpha carbon proton NOESY connectivities, complemented by intra-
residue COSY connectivities, will be used to map out the backbone structure of the peptides.
Further, newer 2D-techniques (e.g., HMQC-COSY, and T-ROESY) that can improve the
quality of spectra as well as provide additional information will be explored and integrated
into the nal protocol.[37][38] A second goal of immediate concern is correlating even more
71
information derived from NMR spectra with molecular dynamics simulations while running
such simulations under a wider range of conditions. Lastly, gaining a better understanding
of the deconvolution and NMR simulation capabilities of HyperChem, shown in Figure 4.2,
will aid in the interpretation of complex NMR spectra. Information gleaned from future
work of this type, combined with the methodologies developed in the present project should
yield valuable insights when applied to the structural determination of the nonapeptides and
their interaction with suitable receptors.
72
Acknowledgements
First, I would like to thank my advisor, Professor Brad Antanaitis of the Physics Depart-
ment, for inspiring me to take on this project. All his hard work, eort, and support were
greatly appreciated.
Secondly, I would like to thank Professor Kenneth Haug for his active participation in this
project as my outside advisor from the Department of Chemistry. His helpful insight on
molecular dynamics simulations and overall enthusiasm were essential to the project.
I would also like to thank Professor Andrew Kortyna of the Physics Department for his ac-
tive participation in this project as my departmental reader. His encouragement and support
throughout the process were invaluable to its completion.
Much appreciation also goes to Professor Anthony Novaco of the Physics Department for
many hours spent working out MD theory with me.
Special thanks to Laura Wong Han Chan, class of 2012, for her earlier work in the Biophysics
Research Group, which helped lay the foundation for this project.
I would also like to thank Ashok Krishnaswami, Ph.D. of JEOL Ltd. for providing me with
guidance on running NMR experiments. The insight and pulse sequence les he provided
were an integral part of this project, and his timely help was greatly appreciated.
73
A Nuclear Magnetic Resonance (NMR) Spectroscopy
This appendix draws heavily from the treatments described in references [19], [20] [21], [22],
and [23].
A.1 Physical Basis
NMR spectroscopy is arguably the most important and versatile spectroscopic method used
for structural determination by chemists today. The basis for NMR is the existence of nuclei
that possess magnetic moments proportional to their intrinsic spin angular momentum,

I
( =
I) where

I is a linear vector operator and is the magetogyric ratio. According to
quantum theory nuclear angular momentum behaves, for many purposes, like a vector of
length
_
I(I + 1) where I, the spin quantum number, can assume either integer of half
integer values as shown by the representative nuclei in Table A.1 below. Nuclei with zero
spin angular momentum, and thus no magnetic moment, are NMR silent.
I Nuclide
0
12
C,
16
O
1
2
1
H,
13
C,
15
N,
19
F,
29
Si,
31
P
1
2
H,
14
N
3
2
11
B,
23
Na,
35
Cl,
37
Cl
5
2
17
O,
27
Al
3
10
B
Table A.1: Nuclear spin quantum numbers (I) of some commonly occurring nuclides.[19]
Because

I
2
and

I
z
commute, its possible to construct states that are simultaneously
eigenstates of both

I
2
and

I
z
, for example:

I
2
[Im =
2
I(I + 1) [Im and

I
z
[Im =
74
m
I
[Im where m
I
, the magnetic quantum number, ranges from I to +I in integral
steps (2I + 1 values in all) and [Im are eigenstates of

I
2
and

I
z
with representing the
quantum numbers of other compatible observables required to specify the state of interest.
The application of a strong magnetic eld, B
0
, produces an interaction energy of the
nucleus known as the nuclear Zeeman interaction given by
H = B
0
= B
0
I
z
. (A.1)
The eigenenergies of this Hamiltonian,

H, are multiples of B
0
of the eigenvalues of

I
z
,
namely,
E
m
I
= B
0
m
I
. (A.2)
In the absence of an applied magnetic eld, all 2I +1 magnetic substates are degenerate, but
application of a eld lifts the degeneracy, producing 2I +1 equally spaced states as illustrated
for a spin-1/2 nucleus in Figure A.1. One can induce transitions between quantized energy
levels by applying an oscillating eld of the appropriate frequency perpendicular to the
polarizing eld B
0
. To satisfy conservation of every, the angular frequency of the absorbed
photons must satisfy the following condition
= E = B
0
. (A.3)
Consequently, the allowed transition have angular frequencies given by
= B
0
. (A.4)
Surprisingly, this result is independent of suggesting that a classical picture of the magnetic
resonance phenomenon may be useful.
According to the classical picture, when a nuclear magnetic moment, , is immersed
in a uniform magnetic eld, B
0
, it will precess about the eld at an angle as in Figure
75
Figure A.1: Splitting of spin states for a spin-
1
2
nuclide.
A.2b. This free precession has a mechanical analog; a spinning top immersed in Earths
gravitational eld (see Figure A.2a). In the case of a spinning top, the torque produced by
the earths gravitational eld causes its angular momentum vector, L, to precess similarly.
The underlying reason for this connection is that both systems equations of motion have
similar structure:
top
= r F
gravity

= B
0
, (A.5)
and therefore both systems exhibit similar behavior.
Because the primary focus of this thesis is nuclear magnetic resonance, let us examine
this phenomenon in greater detail. Substituting
for
top
, and m (equal to I for a nucleus
with magnetogyric ratio and spin angular momentum I) for yields
= mB
0
= I B
0
, (A.6)
the equation of motion for a magnetic moment, m, in a uniform magnetic eld B
0
.
The torque exerted on the magnetic moment by B
0
will cause the magnetic moment to
76
Figure A.2: The motion of a spinning top (left) with the force of gravity acting on it. The
motion of a nucleus (right) with the force of an external magnetic eld acting on it.
precess about B
0
with an angular frequency dened by
= B
0
(A.7)
where is called the Larmor frequency of the particular nuclide. Because this observable
is independent of m, all spins of a given nuclide will precess at the same frequency when
immersed in an external magnetic eld.
Figure A.3: A system of homogeneous nuclei (left) whose average s add up to M
0
(right).
Extending this idea to a system consisting of a large number of identical nuclei shows
that the average behavior of the ensemble can be described by a bulk magnetic moment
vector vector, M
0
, oriented in the same direction as the external magnetic eld, B
0
as in
Figure A.3. The magnitude of this vector depends on the relative populations of each of the
77
available spin states for a given nuclear species. Specically, for a I = 1/2 nucleus, there are
two available spin states, m = +1/2 and m = 1/2, giving a Boltzmann population ratio
between the upper (m = 1/2) and lower (m = +1/2) states:
N
upper
N
lower
= e
E
kt
(A.8)
where E = B
0
is the energy gap for a spin-1/2 nucleus, k is Boltazmanns constant, and
T is the absolute temperature of the spin system. At room temperature, E/kT for nuclear
spins will be far less than 1, allowing Equation A.8 to be approximated, to rst order, using
the Taylor series expansion, e
x
1 x such that
N
upper
N
lower
1
E
kT
= 1
B
0
kT
. (A.9)
Because the ratio is so close to 1, it suggests that the sensitivity of any NMR experiment is
enhanced by using larger magnetic elds. As previously mentioned, this population dierence
will directly aect the magnitude of the bulk magnetic moment vector, M
0
(see Figure A.4).
Figure A.4: The bulk magnetic moment vector of a system of I =
1
2
nuclides.[21]
78
A.2 Magnetic Resonance Condition
To detect the presence of quantized energy levels (i.e., those depicted in Figure A.2), requires
the application of a magnetic eld oscillating perpendicular to the static magnetic eld.
Writing the oscillating magnetic eld in terms of an amplitude, B
1
, gives a perturbing term
in the Hamiltonian described in section A.1 of the form
H
pert
= B
1
I
x
cos(t). (A.10)
where the operator

I
x
, has matrix elements between states m and m
, m
I
x
[m, which
vanish unless m
= m1. Consequently, allowed transitions between adjacent energy levels

occur giving: E = = B
0
or simply = B
0
. As shown below, the classical treatment
of the magnetic resonance phenomenon also requires the application of a time-dependent eld
perpendicular to the static magnetic eld B
0
.
A.3 Saturation and T
1
Relaxation Processes
Recall Figure A.3 describing an ensemble of nuclear spins at equilibrium creating the bulk
magnetic moment vector, M
0
. M
0
will be present only if there is a measurable population
dierence between the available spin states (i.e., N
upper
,= N
lower
in our I = 1/2 system).
If in an I = 1/2 system, N
upper
= N
lower
, then M
0
vanishes and the system is said to be
saturated. If the spin system did not interact with its environment, saturation would be an
unavoidable occurrence in any NMR experiment, however this is not the case.
For a system of nuclei in a static magnetic eld, transitions in addition to those induced
by an applied RF eld, B
1
, can occur due to time-dependent interactions of the nuclear
spins with their environment (the lattice) if the spectral density function has components
at the resonance frequency. Radiationless transitions that cause the perturbed system to
return to equilibrium are called longitudinal or spin-lattice relaxation processes and have a
characteristic time constant, T
1
, called the spin-lattice relaxation time. This relaxation time
79
governs the return of the spin system equilibrium according to
N = (N)
eq
_
1 e
t/T
1
_
(A.11)
where (N) is the instantaneous population dierence, (N
eq
) is the equilibrium population
dierence, and T
1
is the longitudinal relaxation time associated with a given system of nuclei.
Figure A.5 gives an exaggerated graphical interpretation of this process.
(a) Here N
2
/N
1
refers to the population of nuclei in the upper
spin state to those in the lower spin state as in Equation A.11.
(b) Step 1: magnet is on at t = 0 nuclei are dispersed
from equilibrium (left), Step: 2 nuclei reach equilibrium
with B
0
o at t = 3T
1
(center), Step 3: B
0
switched on,
nuclei disperse from equilibrium at t = (left)
Figure A.5: (a) Graphical depiction of the T
1
relaxation process. (b) Exaggerated pictorial
view of each step.
80
A.4 The Bloch Equations: A Classical Description
One can gain valuable insight into the magnetic resonance phenomenon by referring to a
set of equations developed by Felix Bloch in 1946. These equations are known as the Bloch
equations and give a classical description of the magnetic resonance phenomenon.
A.4.1 The Non-Rotating Reference Frame
Recall Figure A.2 describing the motion of the magnetic moment vector m immersed in
an external magnetic eld, B
0
. Combining Equations A.1 and A.2, and introducing a new
vector quantity, J = I, describing the total angular momentum of a nucleus, we can write
in accordance with classical electromagnetic theory:
m = J. (A.12)
Furthermore, classical physics tells us that the torque exerted by the magnetic eld on m
equals the time rate of change of the systems angular momentum:
dJ
dt
= mB
0
(A.13)
Multiplying both sides of Equation A.13 by the magnetogyric ratio for the nucleus in question
gives:
dm
dt
= (mB
0
). (A.14)
For a system of identical particles (as in Figure A.3) one can write M =
i
. Applying
this to Equation A.14 above
i
dm
i
dt
=
i
(m
i
B
0
)
81
yeilds
dM
dt
= (MB
0
). (A.15)
Expressing M and B
0
in component form and taking the cross product gives:
MB
0
= (M
y
B
0z
M
z
B
0y
) x + (M
z
B
0x
M
x
B
0z
) y + (M
x
B
0y
M
y
B
0x
) z.
Equating corresponding components on both sides of the equation produces three coupled,
rst-order dierential equations, one for each component of M:
dM
x
dt
= (M
y
B
0z
M
z
B
0y
) (A.16)
dM
y
dt
= (M
z
B
0x
M
x
B
0z
) (A.17)
dM
z
dt
= (M
x
B
0y
M
y
B
0x
). (A.18)
If B
0
is the only eld present and is aligned with the +z axis, then the coupled equations
reduce to the following:
dM
x
dt
= M
y
B
0z
(A.19)
dM
y
dt
= M
x
B
0z
(A.20)
dM
z
dt
= 0 (A.21)
with the possible solution
M
z
= M
(A.22)
M
x
= M
cos(t) (A.23)
M
y
= M
sin(t) (A.24)
where =
L
= B
0
is the angular Larmor frequency, and at t = 0 M
x
= M
, M
y
= 0, and
82
M
z
= M
.
Bloch postulated that due to the exchange of energy with the environment, M
z
would
return to its equilibrium value of M
0
depicted in Figure A.4 at a rate proportional to how
far it is perturbed from equilibrium. The return to equilibrium in the absence of an external
eld can be described by a rst-order rate equation:
dM
z
dt
=
1
T
1
(M
0
M
z
) (A.25)
where T
1
, the longitudinal relaxation time, is also known as the spin-lattice relaxation time.
Figure A.6: If several spins (ms) precess in the xy plane at slightly dierent rates, the total
spin amplitude decreases due to dephasing.[22]
As M
z
returns to its equilibrium value, M
x
and M
y
must also decay to zero as depicted in
Figure A.4. Bloch postulated that processes other than spin-lattice relaxation contribute to
the decay of the in-plane components of M, for example, dephasing of the nuclear magnetic
moments making up M (see Figure A.6). Dephasing can be caused by magnetic interactions
between nearby nuclei or unpaired electrons, or by inhomogeneities in the external eld B
0
.
Whatever the cause, the dephasing eect on M
x
and M
y
in the absence of a static magnetic
83
eld can be written as
dM
x
dt
=
M
x
T
2
(A.26)
dM
y
dt
=
M
y
T
2
(A.27)
where T
2
is called the transverse or spin-spin relaxation time. It should be noted that T
2
T
1
since it is not required that energy be exchanged with the environment for dephasing to occur,
however the magnitude of M
x
and M
y
are indeed coupled to M
z
as Figure A.4 suggests.
The nal step in assembling the Bloch equations is to combine Equation A.15 with the
relaxation Equations A.25-A.27. Doing so yields
dM
z
dt
=
(M
0
M
z
)
T
1
+ (MB
0
)
z
(A.28)
dM
x
dt
=
M
x
T
2
+ (MB
0
)
x
(A.29)
dM
y
dt
=
M
y
T
2
+ (MB
0
)
y
(A.30)
which are formally called the Bloch Equations.
Solving these equations leads to solutions of the form:
M
x
(t) = M
0
e
t/T
2
cos(
L
t) (A.31)
M
y
(t) = M
0
e
t/T
2
sin(
L
t) (A.32)
M
z
(t) = M
0
_
1 e
t/T
1
_
(A.33)
where, again,
L
= B
0
is the angular Larmor frequency. A graphical representation of the
motion of M during the relaxation process is depicted in Figure A.7 below.
84
Figure A.7: The path traced by the tip of the bulk magnetic moment vector M as it relaxes
according to Equations A.31A.33.[22]
A.4.2 The Rotating Reference Frame
In light of the rotational behavior of the bulk magnetic moment vector, M, and the orien-
tation of B
0
along the z-axis, it would seem natural to move into a rotating reference frame
(see Figure A.8 below) in particular one that is rotating at the Larmor frequency of the
nucleus of interest. Doing so leaves only stationary components of M along the x
, y
, and
z
axes.
Figure A.8: Coordinate system dened by x
, y
, and z rotating about the z axis.

Jumping from the rotating frame to the lab frame or vice-versa involves standard trans-
formations (coordinate rotations about the z-z
axes). For the present system, one can simply

85
rewrite the components of M so they reect what is depicted in Figure A.8:
M
x
= M
x
cos M
y
sin (A.34)
M
y
= M
x
sin + M
y
cos (A.35)
M
z
= M
z
. (A.36)
However, noting that the coordinate system in Figure A.8 is rotating about the z-axis
at the Larmor frequency (which is the same frequency at which M is rotating), one can
position M such that it has components on the x
and z
axes only. This operation leaves

no M
y
component of M as shown below:
M
x
= M
x
cos (A.37)
M
y
= M
x
sin (A.38)
M
z
= M
z
(A.39)
or similarly in matrix form
_
_
_
_
_
_
M
x
M
y
M
z
_
_
_
_
_
_
=
_
_
_
_
_
_
cos(
L
t) sin(
L
t) 0
sin(
L
t) cos(
L
t) 0
0 0 1
_
_
_
_
_
_
_
_
_
_
_
_
M
x
0
M
z
_
_
_
_
_
_
(A.40)
where =
L
t = B
0
t and the second matrix is dened as the transformation matrix
(i.e., the matrix that allows transition from the rotating frame back to the inertial frame).
Carrying out the indicated matrix multiplication gives
_
_
_
_
_
_
M
x
(t)
M
y
(t)
M
z
(t)
_
_
_
_
_
_
=
_
_
_
_
_
_
M
cos(
L
t)
M
sin(
0
t)
M
_
_
_
_
_
_
(A.41)
86
namely the same results as Equations A.24A.25. This is just what we would expect as
zeroing the coordinate system at t = 0 is the same as being in a non-rotating frame.
Pure relaxation in the rotating frame has the same form as in the inertial frame (see
Equations A.22-A.24) that is:
dM
x
dt
=
M
x
T
2
(A.42)
dM
y
dt
=
M
y
T
2
(A.43)
dM
z
dt
=
1
T
1
(M
0
M
z
) (A.44)
yielding solutions resembling those in Equations A.31A.33, but without the periodic terms:
M
x
(t) = M
0
e
t/T
2
(A.45)
M
y
(t) = 0 (A.46)
M
z
(t) = M
0
_
1 e
t/T
1
_
. (A.47)
Combining the transformation matrix from Equation A.40 with Equations A.45A.47
provides the solutions to the Bloch Equations found earlier in Equations A.31A.33:
_
_
_
_
_
_
M
x
(t)
M
y
(t)
M
z
(t)
_
_
_
_
_
_
=
_
_
_
_
_
_
cos(
L
t) sin(
L
t) 0
sin(
L
t) cos(
L
t) 0
0 0 1
_
_
_
_
_
_
_
_
_
_
_
_
M
0
e
t/T
2
0
M
0
e
t/T
1
_
_
_
_
_
_
=
_
_
_
_
_
_
M
0
e
t/T
2
cos(
L
t)
M
0
e
t/T
2
sin(
L
t)
M
0
_
1 e
t/T
1
_
_
_
_
_
_
_
. (A.48)
This exercise should now make clear that describing magnetic resonance phenomena in
87
a frame rotating at the Larmor frequency greatly simplies things.
A.5 Introduction of the Transverse Magnetic Field
To produce resonance either classically or quantum mechanically requires application of a
small amplitude magnetic eld, B
1
, oscillating perpendicular to the static eld, B
0
. This
eld will cause all the magnetic moment vectors to bunch together as depicted in Figure A.9.
Notice now that M is at an angle with respect to B
0
.
(a) Precession of nuclides before ex-
posure to B
1
.
(b) Precession of nuclides after expo-
sure to B
1
.
Figure A.9: Graphical depiction of the precession of the nuclear spins and M when both the
static magnetic eld B
0
and the oscillating magnetic eld B
1
are present.
The linear oscillating eld is conveniently viewed as a coherent superposition of two
counterrotating elds (each with half the amplitude of the original) in the xy plane as shown
below:
B
R
=
B
1
2
[cos(t) x + sin(t) y] (A.49)
B
L
=
B
1
2
[cos(t) x sin(t) y]. (A.50)
88
Refer to Figure A.10 below for a graphical depiction of this decomposition.
Figure A.10: Decomposition of the linear oscillating eld B
1
.
It should be clear that only the component of B
1
rotating in the same direction as the
precessing spins can exert a signicant torque on M. With this point in mind, we can focus
on the eects of B
L
and ignore the eects of B
R
; therefore, the magnetic eld components
of interest for magnetic resonance are:
B
x
=
B
1
2
cos t (A.51)
B
y
= 0 (A.52)
B
z
= B
0
. (A.53)
To understand the dynamics of Mdue to both B
0
and B
1
, one must rst rewrite Equation
A.15 to include the eects of the oscillating eld. One can do this simply by recognizing
that B in the non-rotating frame is now the vector sum of B
0
and B
1
. Writing the Bloch
equations with arbitrary B and no relaxation gives
dM
x
dt
= (M
y
B
z
M
z
B
y
) (A.54)
dM
y
dt
= (M
z
B
x
M
x
B
z
) (A.55)
dM
z
dt
= (M
x
B
y
M
y
B
x
). (A.56)
89
Recalling that the rotating frame is rotating at the Larmor frequency, one can transform
these equations into the rotating frame much as was done earlier in Equations A.34A.36.
Using Equation A.34, the equation for the x-component of M can be written as:
dM
x
dt
cos(
L
t)
dM
y
dt
sin(
L
t) +
L
M
x
sin(
L
t)
+
L
M
y
cos(
L
t)
= B
z
[M
x
sin(
L
t) + M
y
cos(
L
t)
The last two terms on the left side of the equation cancel with those on the right, leaving
dM
x
dt
cos(
L
t)
dM
y
dt
sin(
L
t) = 0. (A.57)
A similar approach can be taken for Equations A.55 and A.56: the y component gives
dM
x
dt
sin(
L
t) +
dM
y
dt
cos(
L
t) = B
1
M
z
cos(t) (A.58)
and the z component gives
dM
z
dt
= B
1
M
x
cos(t) sin(
L
t) M
y
cos(t) cos(
L
t). (A.59)
One can eliminate M
x
from Equations A.58 and A.59 by multiplying Equation A.58 by
sin(
L
t), Equation A.59 by cos(
L
t), and then adding, which leaves
dM
y
dt
= B
1
M
z
cos(t) cos(
L
t). (A.60)
In a similar way M
y
can be eliminated from these equations giving
dM
x
dt
= B
1
M
z
cos(t) cos(
L
t). (A.61)
90
A.5.1 Nutation of M by B
1
Equations A.59A.61 provide a basis for understanding how pulsing with B
1
will aect the
motion of the net magnetic moment vector, M, in the rotating frame. Because pulse lengths
are very short compared with typical relaxation times, relaxation eects can safely be ignored
during pulsing. Once the pulse is turned o the solutions of the Bloch Equations for damped
precession pertain.
If B
1
oscillates at the Larmor frequency, Equations A.61A.63 are greatly simplied as
over long periods of time the cos
2
(
L
t) terms average to
1
2
, and the cos(
L
t) sin(
L
t)
terms average to zero leaving only
x
= 0 (A.62)
y
=
B
1
2
M
z
=
1
M
z
(A.63)
z
=
1
M
z
(A.64)
where
1
=
B
1
2
.
Solutions of these equations take the form
M
x
(t) = 0 (A.65)
M
y
(t) = M
0
sin(
1
t) (A.66)
M
z
(t) = M
0
cos(
1
t) (A.67)
which dene nutation of M about the x
axis in the rotating frame.

Transforming these solutions back into the non-rotating frame one nds that
M
x
(t) = M
0
sin(
1
t) sin(
L
t) (A.68)
M
y
(t) = M
0
sin(
1
t) cos(
L
t) (A.69)
M
z
(t) = M
0
cos(
1
t) (A.70)
91
(a) Relaxation in the rotating
frame.
(b) Relaxation in the non-rotating
frame.
Figure A.11: The path traced by the tip of the magnetic moment vector M as it relaxes
back to M
0
.[22]
where the eects of the static magnetic eld B
0
reside in the terms containing
L
= B
0
and those resulting from B
1
are reected in terms containing
1
=
B
1
2
.
A.5.2 Pulse Types
In the spectrometer, the linearly oscillating magnetic eld B
1
is created by running alter-
nating current through a transmitter coil surrounding the sample as in Figure A.12.
Figure A.12: Illustration of how the linearly oscillating magnetic eld B
1
is created in an
NMR spectrometer.
92
Every nucleus has a unique gyromagnetic ratio, , and therefore a unique resonance
frequency described by
L
=

2
B
0
(A.71)
However this is valid only for an isolated nucleus. Due to variations in the chemical environ-
ment of a nucleus in a sample, the resonance frequency will vary ever so slightly from that
described by Equation A.71. Fortunately, the variation is small and can be eectively dealt
with by using a pulsed-mode method.
(a) Schematic representation of
a pulse. The RF generator is
switched on at t
0
and o at
t
p
.[21]
(b) The monochromatic band of
frequencies centered around
L
as
a result of the RF pulse depicted
to the left.[20]
Figure A.13: Two dierent illustrations of the pulsed magnetic eld B
1
.
In the pulsed-mode NMR experiment, a monochromatic RF pulse at the Larmor fre-
quency is applied to the sample for a brief period of time, t
p
, called the pulse duration
(typically a few s). Fourier analysis asserts that a pulse of short duration contains a range
of frequencies centered around
L
(see Figure A.13). This range of frequencies is wide enough
to induce transitions in nuclei embedded in dierent environments and thus have slightly dif-
ferent resonance frequencies.
In constructing a pulse sequence for most any NMR experiment, two basic pulses are
commonly used: a 90
or /2 pulse, and a 180
or pulse. Their names reect the nutation

or tip angle for M in each case (see Figure A.14).
In Figure A.14, the tip angle corresponds to
1
t in Equations A.66 and A.67.
93
(a) (b) (c)
Figure A.14: Direction of the magnetic moment vector, M, in the rotating frame after a) a
pulse of arbitrary angle , b) after a 90
pulse, and c) after a 180
pulse.[21]
A.6 Recording a Signal
After a 90
pulse has been applied to a molecule immersed in a static magnetic eld B

0
, the
bulk magnetic moment vector of nuclei precesses about the z-axis of the rotating frame as it
relaxes back to equilibrium. It is the weak magnetic eld produced by a precessing M that
is detected by the receiver coil of the spectrometer. Detection of this weak eld occurs by
the same coil of wire that produced the pulsed eld B
1
in Figure A.12. Specically, a time-
varying ux is produced by M as it precesses in the coil and it relaxes in the non-rotating
frame. The time-varying ux produces (in accordance with Faradays Law) an exponentially
damped sine wave called the free induction decay (FID) signal.
To picture this process imagine a single magnetic moment vector, m placed inside a wire
loop Figure A.15. According to classical electrodynamic theory, the the ux through any
closed surface is
_
B dS = 0; however one can calculate the induced voltage, c
ind
, through
the top hemisphere of the loop as
c
ind
=
t
=
t
_ _
B dS
where dS is the innitesimal surface element in spherical coordinates.
94
Figure A.15: A dipole (m) along the x-axis generates a ux through the shaded region (coil)
in the xy-plane that is equal and opposite to that through the hemispherical cap.[22]
First, the ux is calculated as follows:
=
_
2
0
_
/2
0
B
r
r
2
sin dd
=
_
/2
0
B
r
2a
2
sin d
=

o
I
4
_
dl r
r
2
(denition)
=
o
I
4
4
x
a
_
/2
0
cos sin d
=
0
4
2
x
a
=
0
4
2
x
a
e
t/T
2
cos(
L
t)
where
x
is the x-component of m in the xyz-plane.
With m = MV , where the x-component of M is given by Equation A.66 and V is a
nite volume in space, one nds that
c
ind
=
0
4
L
a
2M
0
V e
t/T
2
sin(
L
t).
95
Therefore, the induced voltage, /t is given by
c
ind
=
0
4
0
a
2M
0
V e
t/T
2
_
1
T
2
cos(
L
t) +
L
sin(
L
t)
_
.
Since 1/T
2

L
, this simplies the to:
c
ind
=
0
4
L
a
2M
0
V e
t/T
2
sin(
L
t).
Since the sample usually lls the coil , the signal strength is proportional to a. This
calculation highlights several factors that increase signal strength: 1. sample volume, 2.
sample concentration, and 3. through the Larmor frequency and equilibrium magnetization,
the magnetic moment of the nucleus of interest and the strength of the spectrometer eld.
A.7 The Chemical Shift
In a realistic NMR experiment, the observed nucleus may exist in several dierent chemical
environments. For example in the amino acid glycine (NH
2
CH
2
COOH), protons (hydrogens)
exist in three dierent chemical environments: NH
2
, CH
2
, and OH. Each type of proton
will yield a specic resonance peak in an NMR spectrum reecting the local magnetic eld
experienced. The local eld, B
eff
, can be expressed as
B
eff
= B
0
B
(A.72)
where B
is a eld often produced in opposition to B

0
by the electrons surrounding the
nucleus. This eect is called electron shielding (or electron screening) as shown in Figure
A.15a.
The eld B
eff
produced by the shielding electrons is typically 10
4
10
5
times smaller than
96
(a) The static magnetic eld B
0
causes
electrons in an atom to circulate within
their orbitals. This motion generates
an extra eld B
at the nucleus in the

opposite direction of B
0
.[19]
(b) Alteration of the energy levels of a spin-1/2 nucleus
due to shielding.[19]
Figure A.16: Consequences of electron shielding for a given nuclide in an NMR sample.
the static magnetic eld B
0
. By convention, one usually expresses B
eff
as
B
eff
= B
0
(1 ) (A.73)
where is a proportionality constant called the shielding (or screening) constant. Screening
also leads to a shift in the energy levels between spin states. In particular Equation A.3
becomes
E = B
0
(1 ) (A.74)
as shown in Figure A.16b.
With this modication, the resonance frequency becomes
L
=
B
0
2
(1 ). (A.75)
Thus, the position of resonance peaks in an NMR spectrum depends, among other things,
97
on the extent of local screening.
A.7.1 Contributions to Nuclear Shielding
There are four main sources of nuclear shielding and hence four categorical factors that aect
. Thus, can be written as the sum of four
terms:
= local diamagnetic shielding
+ local paramagnetic shielding
+ eects due to neighboring groups
+ other sources of shielding.
The rst two sources arise from the motion of local electrons (i.e., the electrons surrounding
the nucleus). The third and fourth arise from eects due to electrons orbiting surrounding
nuclei.
Local Diamagnetic Shielding The term diamagnetism refers to the tendency for a ma-
terial to oppose an external magnetic eld. Diamagnetic shielding is a direct consequence of
the motion of electrons within their orbitals as depicted in Figure A.15a and acts to screen a
nucleus from the external magnetic eld, B
0
, by creating an opposing eld, B
d
. The strength
of shielding is proportional to the number of electrons in the ground state of a given nu-
cleus (i.e., the electrons closest to the nucleus). The diamagnetic shielding is fairly easy to
calculate using Lambs formula for individual atoms and Ramseys formula for molecules.[26]
Local Paramagnetic Deshielding Paramagnetism refers to the tendency of a material
to exhibit an induced eld that augments the external eld. This eect is also due to the
motion of electrons in the orbitals surrounding a nucleus; however, due to the mixing of
electron orbitals caused by B
0
, the ground state wavefunction is slightly distorted such that
the motion of the electrons create a eld, B
p
, that adds to the external magnetic eld. For
example, picture a theoretical atom as in Figure A.17 with two electronic states: a ground
98
state p
x
orbital containing two unpaired electrons and an unoccupied excited state p
y
orbital.
In a eld B
0
oriented in the z direction, these two orbitals are slightly admixed such that
their electrons circulate in the xy-plane. This creates a magnetic eld which augments the
external eld and is roughly described by a screening constant having the following functional
dependence:
p

1
E
_
1
R
3
_
(A.76)
which is an average over the local electronic distribution where E is the excitation energy
of the electrons, and R is the distance between the nucleus and its surrounding electrons.
Figure A.17: The circulation of electronic charge brought about by the mixing of electronic
wavefunctions by an external eld B
0
. This paramagnetic current generates a small local
magnetic eld that acts to deshield the nucleus at the center of the electron density.[19]
When paramagnetic eects are present, they tend to dominate diamagnetic eects. In
certain cases (i.e., compounds with aromatic rings) induced ring currents can either enhance
or decrease the eect of the applied eld, depending upon whether the aected group is
above, below, or outside the ring. For a case where the associated magnetic eld augments
the external eld, see Figure A.18.
Magnetic Fields of Neighboring Groups Diamagnetic and paramagnetic shielding are
not the only factors that determine the chemical shift of a particular nucleus. Another eect
99
Figure A.18: Dipolar lines of ux generated by an induced magnetic moment at the center of
a cylindrically symmetric neighboring group (i.e., an aromatic ring) with two protons A and
B nearby. Proton A is more shielded by the paramagnetic eect while proton B is deshielded.
to consider would be the motions of electrons around neighboring atoms and atom groups
dependent upon their orientation with respect to a neighboring nucleus. Referring to the
previous example of the benzene ring in Figure A.18 one could imagine that if atom B were
farther away (i.e., part of another group of atoms) the paramagnetic eld lines due to the
ring could be extended and thus still aect atom B. This is an example of how neighboring
groups (or atoms) can interact magnetically with each other. This could be extended to two
atoms very near to each other or two aromatic rings in close quarters.
Other Sources of Chemical Shifts Hydrogen bonding (something that can be prevalent
in medium to large sized molecules) can play a major role in the chemical shift. In eect, it
can be the largest contributor to the chemical shift a given nucleus. For example, the chemical
shift of the hydroxyl proton resonance of ethanol moves about -4 ppm when intermolecular
hydrogen bonding is diluted by a CCl
4
solvent. The reason hydrogen bonding so greatly
aects chemical shifts is still unclear, however it can be invaluable in understanding the
chemical structure of a molecule.
Another source of chemical shifts is local charged or polar groups. These can modify
100
both the paramagnetic and diamagnetic eects on a nucleus by polarizing its electronic
distribution as well as the electronic distributions of nearby atoms or groups. In a similar
way, unpaired electrons can produce strong paramagnetic shifts by creating large dipolar
magnetic elds that either shield or deshield bonded nuclei.
A.7.2 The Chemical Shift Scale
From Equation A.75, one can see that the resonance frequency for a nucleus in a partic-
ular environment is directly proportional to the static magnetic eld, B
0
. Therefore, to
express chemical shifts by the shielding constant would be very inconvenient since the
magnitude of B
0
can vary from spectrometer to spectrometer. To make chemical shift a
universal measurement (independent of spectrometer frequency), one introduces a dimen-
sionless parameter normalized to the spectrometer frequency. Specically, one introduces ,
the standard measure of chemical shift, dened as follows:
=
dierence between a resonance frequency and that of a reference compound
operating frequency of the spectrometer
and mathematically by
= 10
6
(
ref
)
ref
(A.77)
where
ref
is the frequency of a reference compound, is the eld frequency of a resonance
peak in the compound of interest, and 10
6
is a numerical scaling factor used to bring to
a more convenient size. Figure A.19 shows how chemical shifts and other key spectroscopic
factors are related.
A.7.3 The Reference Compound
Reference compounds are generally chosen such that they give a single sharp resonance peak
at a convenient location in the spectrum, often far downeld from most other resonances.
Many reference compounds have been developed to date; two of the most common are
101
Figure A.19: NMR spectra are conventionally plotted with chemical shift increasing from
right to left. Nucleus A, which is more strongly shielded than nucleus B, thus appears to
the right of B, has a lower resonance frequency than B, and is sometimes referred as being
upeld of B.[19]
tetramethylsilane (TMS) and 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS), the latter
especially useful for water solvated compounds. Another desirable property of reference
compounds is their low reactivity towards most compounds under study. A small amount of
the reference compound is added to each sample prior to running an experiment (see Figure
A.20).
(a) TMS (b) DSS
Figure A.20: Two common well-shielded NMR standards.
102
A.8 First-Order (Weak) Spin-Spin Coupling
The fact that identical nuclei in dierent chemical environments produce dierent resonance
peaks is what makes NMR very useful for determining molecular structure. However, these
peaks are not always well dened resonances at a particular chemical shift. Various magnetic
interaction between nuclei produce splittings that encode valuable information about struc-
ture. These interactions are known as spin-spin couplings, J-coupling, or scalar couplings.
These interactions further change the local magnetic eld experienced by a particular nu-
cleus causing it to be either stronger or weaker than it would be in absence of the coupling.
These eects are dependent on both bond distance and bond angle. In an NMR spectrum
J-coupling produces multiplet structure in what would otherwise be a single resonance peak.
In many cases, the peak splitting exhibits an identiable pattern reecting the number and
types of nuclei that are coupled to each other.
The coupling falls into two classes, depending upon whether identical nuclei are inter-
acting with each other or dierent nuclei; these are called homonuclear coupling and het-
eronuclear coupling, respectively. Both heteronuclear and homonuclear coupling act to split
resonance peaks in the same manner.
J-coupling between neighboring nuclei is classied by the number of bonds separating
the two interacting nuclei, with
2
J (geminal coupling) denoting two intervening covalent
bonds, and
3
J (vicinal coupling) denoting three intervening bonds. The splitting in the
NMR spectrum is usually measured in Hertz (Hz). Coupled nuclei are designated by capital
letters and subscripts, for example an AX
n
system indicates that the nucleus of interest, A,
is coupled weakly to n X nuclei. In general, the energy of two weakly coupled nuclei can be
written as
E = hJ
AX
m
A
m
X
(A.78)
where m
A
and m
X
are the magnetic quantum numbers of the two coupled nuclei, and J
AX
is the coupling constant measured in Hz. A J
AX
> 0 implies an anti-parallel (lower energy)
103
conguration whereas J
AX
< 0 implies a parallel (higher energy) conguration of interacting
nuclear spins. Combining this expression for energy with Equation A.76, leads to a modied
resonance condition
A
= B
0
(1
A
)
X=A
J
AX
m
X
(A.79)
where the summation runs over all spins (X) with appreciable coupling to A.
The J-coupling results in multiple splittings of resonance peaks, with each type of cou-
pling producing its own splitting. A convenient example is given in reference [19]: Imagine
a sample containing a number of HCO
2
groups as the subject of a proton NMR experiment.
One might expect to see a single resonance peak representing the single type of hydrogen
atom in the system, but in fact two peaks are observed. The observed doublet results from
the heteronuclear coupling between the H and C atoms in the system. The peaks represent
the transition from spin up to spin down of the hydrogen atom due to its bonded carbon
atom: one peak resulting from HC HC transition and the second resulting from
HC HC transition as depicted in Figure A.21. These two peaks have equal intensity
because the spin-up and spin-down populations of a given nucleus are virtually the same at
room temperature.
It should be noted that in
1
H spectra the eects of weak coupling are very apparent;
however, in
13
C spectra these eects are essentially reduced to zero. This is because the
natural abundance of
13
C is so low compared to
1
H. This greatly simplies
13
C spectra and
as a result makes them signicantly easier to decipher.
A.8.1 Singly Coupled (AX) Nuclei
As previously discussed in the H
13
CO
2
example, the resonance nucleus A coupled to nucleus
X will be split into two peaks of equal intensity separated by a distance J
AX
measured in
Hz. This splitting pattern is appropriately referred to as a doublet. As mentioned previously,
this splitting is symmetrical and would occur in the same way in a spectrum of nucleus X.
104
Figure A.21: The eect of
1
H
13
C scalar coupling in H
13
CO
2
on the energy levels and
spectrum of the
1
H nucleus. For clarity, the energy-level shifts due to the J-coupling have
been greatly exaggerated. The central pair of energy levels and the upper spectrum are
appropriate in the absence of a J-coupling interaction. Scalar coupling produces the energy
levels on the left and right, and the lower spectrum. Also, you can see here that J
AX
is the
coupling constant measured in Hz[19]
A.8.2 Coupling to Two Inequivalent Nuclei (AMX)
In the previous case only coupling between two nuclei in a system was considered. Other
coupling schemes are both possible and commonly observed in NMR. For example, suppose
two dierent nuclei (M and X) are coupled to A; this will produce a characteristic (AMX)
doublet-doublet pattern as depicted in Figure A.22. Here the single resonance is rst split
by coupling with nucleus X forming two peaks of equal intensity separated by a distance
J
AX
. Then these two peaks are each split again by coupling with nucleus M to form two sets
of equal intensity peaks which are separated by a distance of J
AM
.
A.8.3 Coupling to Two Equivalent Nuclei (AX
2
)
In the situation described above, nucleus A was coupled to two dierent nuclei, M and X.
However in certain situations (i.e., the rst H in benzene) nucleus A can be coupled to two
105
Figure A.22: NMR spectrum of nucleus A in an AMX spin system. The four components
of the multiplet, a doublet of doublets, arise from the four combinations of M and X spins,
indicated (m = +
1
2
) and (m =
1
2
). Drawn for J
AM
> J
AX
> 0.[19]
equivalent nuclei such that J
AM
= J
AX
. In this situation, the doublet of doublets forms a
1:2:1 triplet as depicted in Figure A.22. Here the central peaks of the doublet are degenerate
and coincide to form a single peak with an intensity twice as great as the peaks due to the
non-degenerate states.
Figure A.23: NMR spectrum of nucleus A in an AX
2
spin system. The triplet arises from
the four combinations of the two X spins, as indicated. Drawn for J
AX
> 0.[19]
106
A.8.4 Coupling to n Equivalent Nuclei (AX
n
)
It is easy to extend the above treatment to predict the appearance of a spectrum with nucleus
A coupled to n equivalent nuclei. In this situation, the resonance peak of nucleus A will be
split into n+1 equally spaced lines with intensities given by the coecients in the expansion
of (1+x)
n
. This splitting pattern can be visualized using Pascals triangle as in Figure A.24.
Examples of these splitting patterns are given in Figure A.25.
Figure A.24: Pascals triangle showing the binomial coecients in the expansion of (1 +x)
n
.
The rows give the relative intensities of the (n + 1) lines in the A multiplet of an AX
n
spin
system (n = 06), where X is a spin-
1
2
nucleus. The columns give the positions of the lines,
relative to the chemical shift position, in units of J
AX
.[19]
Figure A.25: Multiplet patterns for the A nucleus in various spin systems. M and X are
spin-
1
2
unless otherwise stated. Weak coupling is assumed throughout. Spectra are drawn
for [J
AX
[ > [J
AM
[.[19]
107
A.9 Pulse Sequences
To date hundreds of dierent pulse sequences have been developed, each designed to elicit
dierent kinds of information encoded in an NMR spectrum. Below are detailed the pulse
sequences utilized in the present study. Several factors inuence these choices: (1) the limi-
tations of the local spectrometer, (2) the type of information needed to make unambiguous
resonance assignments in recorded spectra, (3) the limitation of the project itself, and (4)
the knowledge base of the researchers carrying out the experiments.
A.9.1 The
1
H and
13
C Free-Induction-Decay (FID) Pulse Sequence
The FID pulse sequence (Figure A.26) is the most basic one-dimensional NMR experiment
available. Although this exact pulse sequence was not used in the present study, it serves
as a good model for more complex pulse sequences. As described in the preceding sections,
the sample is irradiated by a /2 pulse and the time-dependent behavior of the in-plane
magnetization is recorded by the spectrometer and Fourier transformed. An important
aspect of the FID sequence is that the relaxation delay between pulses is always T
1
so as
to allow the system to relax back to equilibrium; a standard relaxation delay is between 2
and 5 seconds.
Figure A.26: Pulse sequence and signal for a free-induction-decay measurement.[22]
108
A.9.2 The
13
C Single Pulse Broadband Decoupling Pulse Sequence
As described in Section A.8, heteronuclear J-coupling can cause resonance peak splittings
which complicate spectra. One way to simplify a spectrum is to use a technique called
heteronuclear decoupling. This technique eliminates splitting of carbon spectra by coupling
to neighboring protons. The pulse sequence for this experiment is illustrated in Figure A.27.
The net eect of applying a second oscillating eld, B
2
, is to equalize spin-up and spin-down
proton populations such that they do not produce a net eld at the carbon nuclei.
Figure A.27: Pulse sequence and signal for a heteronuclear spin decoupling measurement.
Figure A.28: Depiction of the elds and their associated eects on the bulk magnetic moment
vectors of the carbon, M
C
, and the the hydrogen, M
H
.
109
A.9.3 The
1
H Single Pulse Watergate Suppression Pulse Sequence
Proton NMR experiments run in aqueous media are confounded by an intense peak around
4.79 ppm due to the very high concentration of water protons (110 M) in the sample. This
peak dwarfs all other proton peaks in the spectrum, making them dicult to analyze. For-
tunately this peak can be removed using the same approach outlined in the broadband
decoupling experiment. To remove the water peak, one rst records a standard proton FID
spectrum showing the location (chemical shift) of the water peak. Once the chemical shift of
the water peak is known, one can tailor B
2
to match this frequency and saturate the water
protons thereby eliminating the water peak altogether.
A.9.4 The
13
C Attached Proton Test (APT) Pulse Sequence
To help make peak assignments on a
13
C FID spectrum, a modication of the single pulse
experiment with broadband decoupling called the APT experiment can be employed. The
result of an APT experiment shows, C and CH
2
peaks displayed as +90
with respect to the

chemical shift axis and CH
3
and CH peaks displayed as 90
with respect to the chemical

shift axis.
This experiment takes advantage of the varying dephasing times between these carbon
groups due to their dierent proton couplings. It turns out that their dephasing rates
evolve as a function of their J-coupling constants. This experiment uses a concept called
J-modulated spin echo where the sample is irradiated by a 90
(/2) pulse followed by a

180
() pulse while the decoupling eld, B

2
, is turned o and then allowed to evolve with
the decoupling eld turned back on during detection.
110
Figure A.29: Pulse sequence and signal for a APT measurement.
A.9.5 The
13
C Distortionless Enhancement by Polarization Transfer (DEPT)
Pulse Sequence
Figure A.30: Pulse sequence and signal for a DEPT measurement.
DEPT is comparable to APT in that dierent carbon groups are displayed with dierent
phases on the spectrum. Specically, CH and CH
3
have positive phase relative to the chem-
ical shift axis and CH
2
has negative phase relative to the chemical shift axis. Another key
aspect of a DEPT spectrum is that quaternary carbons do not appear.
111
A.9.6 The Correlation Spectroscopy (COSY) Pulse Sequence
Figure A.31: Pulse sequence and signal for a COSY measurement.
COSY is a basic but very useful type of proton-proton homonuclear 2D spectrum that
shows cross peaks between protons with geminal or vicinal coupling (i.e., separated by two or
three covalent bonds). COSY connectivities play a key role in interpreting any complicated
1
H NMR spectrum.
112
B The Berendsen Thermostat
This appendix outlines the methodology used in the paper titled Molecular dynamics with
coupling to an external bath by Berendsen et al., J. Chem. Phys. 81 (8) to derive , the
thermal coupling constant.
Derivation of this thermal bath constant is based on the Langevin equation of motion
(Equation B.1). The Langevin equation was originally developed to describe the Brownian
motion of molecules in a liquid. Brownian motion refers to the motion of a molecule in
solution due to random collisions with solvent particles. The Langevin equation is
m
i
v
i
= F
i
m
i
i
v
i
+ R(t). (B.1)
Here R
i
is the Gaussian probability term dened by its correlation function
R
i
(t)R
j
(t + ) = 2m
i
i
kT
0
()
ij
(B.2)
where k is the Boltzmann constant, is a damping term related to the viscosity of the
solution, and T
0
is the constant temperature of the thermal bath. This stochastic term
attempts to describe interactions of the molecule with solvent particles. For our purposes,
we will assume
i
= for all atoms.
Before continuing, three relations should be noted: from Equation B.1
v
i
= v
i
(t + t) v
i
(t) (B.3)
=
1
m
i
_
t+t
t
[F
i
(t
) m
i
v
i
(t
) + R
i
(t
)] dt
(B.4)
113
and from Equation B.2
_
t+t
t
dt
_
t+t
t
dt
R
i
(t
)R
i
(t
) = 2m
i
kT
0
t. (B.5)
We will begin by writing the denition for kinetic energy:
dE
k
dt
= lim
t0
_
1
t
_
3N
i=1
1
2
m
i
v
2
i
(t + t)
3N
i=1
1
2
m
i
v
2
i
(t)
__
= lim
t0
_
1
t
_
3N
i=1
1
2
m
i
v
2
i
(t + t) v
2
i
(t)
__
. (B.6)
The key is to stay in second order so, using Equation B.3, we will nd the value for
v
2
(t + t) as follows:
v
2
i
(t + t) = (v
i
(t) + v
i
)
2
= v
2
i
(t) + 2v
i
(t)v
i
+ v
2
i
.
Using this fact we can rewrite Equation B.6 as follows:
dE
k
dt
= lim
t0
_
1
t
_
3N
i=1
1
2
m
i
_
2v
i
(t)v
i
+ v
2
i
__
. (B.7)
114
Now we will use Equations B.4 and B.5 to evaluate this expression:
dE
k
dt
m = lim
t0
_
1
t
_
3N
i=1
1
2
m
i
_
2v
i
(t)
_
1
m
i
__
t+t
t
[F
i
(t
) m
i
v
i
(t
) + R
i
(t
)] dt
+
_
1
m
2
i
__
t+t
t
[F
i
(t
) m
i
v
i
(t
) + R
i
(t
)] dt
_
t+t
t
[F
i
(t
) m
i
v
i
(t
) + R
i
(t
)] dt
___
= lim
t0
_
1
t
_
3N
i=1
1
2
m
i
_
2v
i
(t)
_
1
m
i
_
[F
i
(t) m
i
v
i
(t) + R
i
(t)] t
+
_
1
m
2
i
__
t+t
t
_
t+t
t
_
F
i
(t
)F
i
(t
) m
i
v
i
(t
)F
i
(t
) + R
i
(t
)F
i
(t
) m
2
i
2
i
v
i
(t
)v
i
(t
)
m
i
R
i
(t
) + F
i
(t
)R
i
(t
) m
i
v
i
(t
) + R
i
(t
)R
i
(t
)] dt
dt
]] .
All the uncorrelated
1
terms will go to zero, but we must not forget about Equation B.5.
Noting this you will be left with
dE
k
dt
= lim
t0
_
1
t
_
3N
i=1
v
i
(t) [F
i
(t) m
i
v
i
(t)] t+
3N
i=1
_
1
2m
i
_
__
F
2
i
(t) 2m
i
v
i
(t)F
i
(t) + m
2
i
2
v
2
i
(t)
_
t
2
+ 2m
i
kT
0
t
__
.
All the terms that are second order in t will go to zero when the limit is taken. What
we are left with is a simple sum:
dE
k
dt
=
3N
i=1
_
v
i
(t)F
i
(t) m
i
v
2
i
(t) + kT
0
. (B.8)
1
Two functions are considered to be uncorrelated if their covariance is zero. Covariance is a measure of
how similar or dierent the behavior of two variables is. Similar behavior denotes positive covariance while
opposite behavior denotes negative covariance.
115
Noting that E
k
=
1
2
mv
2
we can simplify further.
=
3N
i=1
[v
i
(t)F
i
(t) 2E
k
+ kT
0
]
=
3N
i=1
v
i
(t)F
i
(t) 2E
k
+ 3NkT
0
=
3N
i=1
v
i
(t)F
i
(t) + 2
_
3N
2
kT
0
E
k
_
. (B.9)
Berendsen paper species the second term in Equation B.9 describes the global coupling
of the system with the bath. This is also an energy term, so knowing that E
k
=
3
2
NkT, you
can write
_
dE
k
dt
_
bath
= 3Nk(T
0
T)
d
dt
_
3N
2
kT
_
bath
= 2
_
3N
2
k(T
0
T)
_
_
dT
dt
_
bath
= 2(T
0
T) (B.10)
which describes the change in temperature of the bath over time. Equation B.10 is know as
the Berendsen Thermostat with the time constant
T
of the coupling equal to (2)
1
.
Therefore Equation B.1 can be rewritten as
m
i
v
i
= F
i
+ m
i
_
T
0
T
1
_
v
i
(B.11)
116
without the stochastic term, where the scaled temperature term is justied by the fact that
dE
k
dt
=
m
i
v
i
v
i
=
3N
i=1
v
i
F
i
m
i
v
2
i
_
T
0
T
1
_
= v
i
F
i
2E
k
_
T
0
T
1
_
= v
i
F
i
2
_
3N
2
kT
__
T
0
T
T
_
= v
i
F
i
3Nk(T
0
T) (B.12)
which is equivalent to Equation B.8.
To derive the associated scaling term, we should rst note that kinetic energy and tem-
perature are related in our system as follows:
E
k
=
1
2
mv
2
=
3
2
NkT
making
T =
mv
2
3Nk
. (B.13)
We can then describe the change in temperature per time step as related to scaling of the
velocity by
T =
m(v)
2
3Nk

mv
2
3Nk
such that
T =
mv
2
3Nk
(
2
1). (B.14)
117
We can now refer back to Equation B.9 which says
_
dT
dt
_
bath
= 2(T
0
T)
where if we modify it to express a change in temperature per time step we get
T =
t
(T
0
T) (B.15)
again where = (2)
1
.
We can now combine Equations B.13 and B.14 and solve for , the velocity scaling factor:
=
_
1 +
t
_
T
0
T
1
__1
2
(B.16)
Q.E.D.
118
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123

Investigation of The Three-Dimensional Solution Structure of Melanostatin Using One - and Two-Dimensional NMR Spectroscopy and Molecular Dynamics Calculations

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Investigation of The Three-Dimensional Solution Structure of Melanostatin Using One - and Two-Dimensional NMR Spectroscopy and Molecular Dynamics Calculations

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Investigation of the three-dimensional solution

structure of melanostatin using one- and

, and z rotating about the z axis. . . . . 85

pulse, and c) after a 180

with respect to the chemical shift axis whereas CH

with respect to the chemical shift axis. In a DEPT experiment, CH and CH

with respect to the chemical shift axis whereas CH

CH 8 4.32, 4.354 7.08, 10.22 -

(L) CH 9 4.33 10.71 -

(L) CH 9 4.73 3.63 -

end of glycine. Comparing the values for

and is a backbone torsion angle.

= m1. Consequently, allowed transitions between adjacent energy levels

, and z rotating about the z axis.

axes). For the present system, one can simply

axes only. This operation leaves

axis in the rotating frame.

or /2 pulse, and a 180

or pulse. Their names reect the nutation

pulse, and c) after a 180

pulse has been applied to a molecule immersed in a static magnetic eld B

is a eld often produced in opposition to B

at the nucleus in the

with respect to the

with respect to the chemical

(/2) pulse followed by a

() pulse while the decoupling eld, B

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