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Synthesis and interaction of some dipeptide amides with deoxyribonucleic acid

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Title:
Synthesis and interaction of some dipeptide amides with deoxyribonucleic acid
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Sheardy, Richard Dean
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English
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xi, 116 leaves : ill. ; 28 cm.

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Subjects / Keywords:
Amides ( jstor )
Amino acids ( jstor )
DNA ( jstor )
Esters ( jstor )
Hydrochlorides ( jstor )
Hydrogen ( jstor )
Molecules ( jstor )
Protons ( jstor )
Signals ( jstor )
Viscosity ( jstor )
Amides ( lcsh )
Chemistry thesis Ph. D
DNA ( lcsh )
Dissertations, Academic -- Chemistry -- UF
City of Gainesville ( local )
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bibliography ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis--University of Florida.
Bibliography:
Bibliography: leaves 109-115.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Richard Dean Sheardy.

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University of Florida
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Full Text


SYNTHESIS AND INTERACTION OF
SOME DIPEPTIDE AMIDES
WITH DEOXYRIBONUCLEIC ACID
BY
RICHARD DEAN SHEARDY
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1979


TO MOM AND DAD.
AND ARTIE.


ACKNOWLEDGEMENTS
The author extends his appreciation to Professor
Edmond J. "Eddie" Gabbay for his guidance, continuous
support and friendship during the past five years, until
the time of his death. He was a good friend and excellent
scientist -- and he will be missed very much.
Thanks are also extended to all those who have
helped in the author's graduate studies, especially
Mr. M. Adkins, Mr. B. Arvan, Mr. T. Baugh, Mr. C. Cromwell,
Mr. B. Griggs, Mr. D. King, Dr. R. King, Dr. D. McRitchie,
Dr. S. Pearce and Mr. T. Rigl. Special thanks also go to
Dr. M. Battiste, whose friendship and support during our
time of sadness this past summer is greatly appreciated.
Thanks also go to Dr. W. D. Wilson for being an outside
reader. Very special thanks to Ms. Joanne IJpham, whose
kindness, patience, and love are immeasurable, for her
excellent typing.
The completion of this course of study would not have
been possible without the support and encouragement of the
author's family.
i i i


For Eddie
There was a man-
I knew him well.
He lived his life
With dreams to sell.
I know that some
Can't understand.
But dreams can oft
Turn into sand.
And now my eyes
Fill with sorrow,
For he will see
No tomorrow.
He was a friend-
I know that well;
A helpful hand-
A joke to tell.
He had to go-
As all things must;
To fly away
And turn to dust.
And though my tears
May dry so fast,
My mind will not
Forget the past.
What is a life-
A game to play
Until it's time
To fade away?
Or is it more-
A heart to give
To those we love
That they may live?
And though there's fear
Throughout the night,
The morning sun
Is shining bright.
Richard Dean Sheardy
October 17,1979
IV


TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS Ui
LIST OF TABLES vi
LIST OF FIGURES vii
ABSTRACT x
CHAPTER I INTRODUCTION 1
Structure of DNA 2
Physical Properties of DNA 9
Evidence for Hydrogen Bonding 13
Stacking Forces 17
Electrostatic Effects 20
Electronic Effects 20
Dynamic Structure of Nucleic Acids 23
Chromatin 24
Small Molecule-Nucleic Acid Interactions 25
Statement of Problem 30
CHAPTER II RESULTS AND DISCUSSION 31
1H NMR Studies 31
Viscosity Studies 40
Ultraviolet Absorption Studies 46
Binding Studies 46
Circular Dichroism Studies 52
Equilibrium Dialysis 52
Melting Temperature Studies 56
Discussion 58
CHAPTER III EXPERIMENTAL 69
Synthesis 70
Analytical Methods 101
REFERENCES 109
BIOGRAPHICAL SKETCH 116
v


LIST OF TABLES
Table
Page
1.
The Effect of Increasing Length of the
Polynucleotide (Ap) A on the UV Absorp
tion Spectrum. n
20
2.
Absorption and Hypochromicity Data on
the Dipeptide Amides.
47
3.
Typical Output for McGhee-von Hippel
Binding Isotherms for Various Values
of n.
49
4.
Binding Isotherms for DNP and Nitrotolyl
Containing Dipeptides.
51
5.
Apparent Binding Affinity (Ka) of the
Dipeptides to Salmon Sperm DNA.
55
6.
The Effect of the Dipeptides on the ATm
on the Helix-Coil Transition of Salmon
Sperm DNA, Poly I Poly C, and Poly
d(A-T).
57
vi


LIST OF FIGURES
Figure Page
1. Schematic Representation of the Watson-
Crick Double Helix of DNA. 3
2. Watson-Crick Base Pairs. 4
3. Keto-Enol Tautomers for the Bases Guanine
and Cytosine. 4
4. Structure of a Section of a DNA Chain. 6
5. Absorption-Temperature Profile for DNA. 10
6. Intrinsic Viscosity-pH Profile for DNA. 10
7. Acid-Base Titration Curve for DNA. 11
8. a) Hoogsteen Adenine-Thymine Base Pair.
b) Anit-Hoogsteen Adenine-Thymine Base Pair. 14
9. Shielding of the Aromatic Protons Caused
by Vertical Stacking. 18
10. Geometry of Stacked Nucleosides. 19
11. Exciton Splitting of Energy Levels. 21
12. Intensity Interchange Between Two Inter
acting Transition Moments. 22
13. Reporter Molecule I. 28
14. Schematic illustration of a segment of DNA
double helix which can either partially
intercalate a molecule or fully intercalate
a molecule. 28
15. Schematic illustration of a DNA segment
showing a possible mechanism whereby the c
and a amino groups of the N-terminal L-lysyl
residue are stereospecifically anchored and
thus dictating the positioning of the aro
matic ring of the diastereomeric dipeptides
in the DNA-peptide complex. 29
Vll


Page
Figure
16. The Dipeptide Amides Synthesized and
Studied.
17. The Partial NMR Spectra of DNA at
Various Temperatures.
18. NMR Signal of the Aromatic Protons of
Dipeptides _1 and 2_ in the Presence and
Absence of DNA at Various Temperatures.
19. NMR Signal of the Aromatic Protons of
Dipeptides _3 and _4 in the Presence and
Absence of DNA at Various Temperatures.
20. NMR Signal of the Aromatic Protons of
Dipeptides .5 and 6_ in the Presence and
Absence of DNA at Various Temperatures.
21. NMR Signal of the Aromatic Protons of
Dipeptides 7_ and 8^ in the Presence and
Absence of DNA at Various Temperatures.
22. NMR Signal of the Aromatic Protons of
Dipeptides 9^ and 10. in the Presence and
Absence of DNA at Various Temperatures.
23. The Effect of Dipeptides 1_ and 2_ on the
Relative Specific Viscosity of Salmon
Sperm DNA.
24. The Effect of Dipeptides 3., 4., 7. and 8^
on the Relative Specific Viscosity of
Salmon Sperm DNA.
25. The Effect of Dipeptides 5. and 6 on the
Relative Specific Viscosity of Salmon
Sperm DNA.
26. The Effect of Dipeptides 9^ and _10_ on the
Relative Specific Viscosity of Salmon
Sperm DNA.
27. A Plot of v/L Versus v From Data in
Table 3.
28. The CD Spectra of Dipeptides _5, 6^ 9^
and ^0 in the Absence and Presence of DNA.
The CD Spectra of Dipeptides 3^ and 4- in
the Absence and Presence of DNA.
viii
32
33
34
35
36
37
38
42
43
44
45
50
53
29.
54


Figure
Page
30 .
Liquid HF Apparatus.
74
31.
Diagram of the Viscometer with the Photo
electric Device in Place.
103
IX


Abstract of Dissertation Presented to the
Graduate Council of the University of Florida
in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy
SYNTHESIS AND INTERACTION OF
SOME DIPEPTIDE AMIDES
WITH DEOXYRIBONUCLEIC ACID
By
Richard Dean Sheardy
December 1979
Chairman: Edmond J. Gabbay (deceased), Merle A. Battiste
Major Department: Chemistry
This thesis discusses the synthesis and interaction
specificities of ten dipeptide amides with salmon sperm
DNA. For each dipeptide, the N-terminal amino acid was
an L-lysyl residue, while the C-terminal amino acid had
an a carbon of defined chirality (either L or D) and an
aromatic moiety a certain distance from the a carbon. The
rationale for this series was to determine if intercalation
or partial intercalation is a function of both the chirality
of the C-terminal amino acid a carbon as well as the dis
tance of the aromatic moiety from the a carbon. The ex
tent of intercalation was determined by a variety of tech
niques: 1) ^H NMR studies; 2) viscometric titration; and
3) UV absorption and CD studies. It was found that partial
x


intercalation occurs with the peptide containing a C-ter-
minal L-pNO^-phenylalanine residue with no intercalation
for its diastereomeric dipeptide amide, indicating a
dependence on the chirality of the C-terminal amino acid.
Six of the remaining dipeptides fully intercalated, sug
gesting the necessity to evaluate electronic and hydro-
phobic effects on intercalation. The two diastereomeric
dipeptide amides containing an S-p-nitrobenzylcysteinyl
moiety gave conflicting results. Possible explanations
for the above phenomenon are provided.
xi


CHAPTER I
INTRODUCTION
Undoubtedly, one of the greatest advances of biological
science in this century was the determination of deoxyribo-
1 2
nucleic acid fiber structure by Watson and Crick in 1953.
Since that time, volumes of work have been published at the
expense of many man hours to either confirm or disprove
certain theoretical aspects of the Watson-Crick structure.
Certainly, knowledge of DNA structure may lead to the
treatment of inherited diseases, or those diseases caused
by viruses and bacteria, and ultimately to find cures for
cancer. This knowledge is not easy to obtain due to the
immense complexity of the genetic code.
It has been suggested that a single chromosome may be
just one DNA molecule having a molecular weight of up to
9
4 x 10 daltons and an overall length of over 2 centi
meters.^ Unfortunately, it is difficult to isolate a com
plete DNA molecule since its structure is: 1) sensitive to
changes in temperature^^ and pH;^ and 2) subject to breakage
7
under minimal shearing forces.
Commercially available DNA is usually derived from calf
thymus or salmon testes. Also, numerous synthetic DNA's are
available with definite sequence and molecular weight. The
concentration of a nucleic acid solution is determined from
1


2
the absorption of the bases at 260 nm using, for example, a
molar extinction coefficient of e = 6,500 for salmon sperm
P
O
DNA, and has the dimensions of moles of phosphate per liter.
Structure of DNA
Watson and Crick proposed a right-handed double helix
(Figure 1), comprised of two anti-parallel sugar-phosphate
backbones held together by specific hydrogen bonds between
the complementary bases and by hydrophobic forces that
1 2
favor stacking of the bases. Their model was based on
X-ray diffraction data on DNA fibers as well as the chemical
g
evidence of Chargaff and Lipschitz. Due to the low resolu
tion of X-ray analysis of fibers, much of the evidence came
from the chemical approach.
Chargaff and Lipschitz used a variety of naturally
occurring DNA to determine the relative number of purines
9
and pyrimidines. They found that: 1) the amount of
guanine (G) is equal to the amount of cytosine (C); 2) the
amount of adenine (A) is equal to the amount of thymine (T);
and 3) the A-T/G-C ratio is constant for a particular species.
The base pairing scheme of Watson and Crick, seen in Figure
2, accounts for these observations.
The free bases can exist in one of two tautomeric
forms as shown in Figure 3 for guanine and cytosine.
However, in the Watson-Crick scheme, the bases are in their
keto forms which allow for two hydrogen bonds between the
A-T base pair and three hydrogen bonds between the G-C base


3
ma j or
groove
minor
groove
Figure 1. Schematic Representation of the Watson-Crick
Double Helix of DNA. The outer helical strands
represent the sugar-phosphate backbone, while
the horizontal lines represent the base pairs
and the vertical line is the helical axis.


4
major groove
major groove
minor groove
A-T
Figure 2. Watson-Crick Base Pairs.
Figure 3. Keto-Enol Tautomers for the Bases Guanine and
Cytosine.


5
pair. Watson and Crick used molecular models to fit the
base pairing scheme to the dimensions of the molecule as
obtained from X-ray analysis. They found that an A-G base
pair was too large to fit these dimensions, whereas a T-C
pairing was too small. In their scheme (i.e., A-T and G-C
base pairing], it was found that the distance between the
sugar phosphate backbone for each set of base pairs was
identical, lending further support to their base pairing
scheme.
The individual strands of DNA are enzymatically joined
monomer nucleoside phosphates resulting in an alternating
sugar-phosphate-sugar backbone in which the bases are
stacked on top of one another (Figure 4). The D-deoxyribose
sugar, in the furanoside form, is numbered as shown in
Figure 4 and has two hydroxyl groups at the 3' and 5' posi
tions, respectively, and the base at the 1' position. The
two complementary strands are placed anti-parallei in order
to attain maximum symmetry. In other words, one strand has
its sugar-phosphate backbone directed 3' -> 5 while the
other backbone is directed 5' - 3' This aspect of the
double helix was proven correct by Josse et al. in 1961 using
. ,, 10
a nearest neighbor analysis.
The linkage between two successive sugars is formally
a phosphate diester. At neutral pH, the phosphate is mono
anionic and thus, the oxygens are directed away from the
helix into solution. Futhermore, the two oxygens are not
equivalent since one lies parallel to the helical axis


6
Figure 4. Structure of a Section of a DNA Chain.


(axial) whereas the other oxygen lies perpendicular to the
helical axis (equatorial).
7
Langridge and coworkers used X-ray diffraction data to
determine that the double helix makes one complete turn
11
every 34 A; this is known as the pitch. With ten base
O
pairs per pitch, there is a translation of 3.4 A between
successive base pairs. Since each turn of the double helix
requires 360 of rotation, the angle between successive
base pairs must be 36.
Inspection of Figures 1 and 2 reveals that two grooves,
one large (major) and one small (minor), are formed as a
consequence of the twist of the double helix. As a result
of the N-9 purine glycosidic bond and the N-l pyrimidine
glycosidic bond, the sugar phosphate backbones on each base
lie on the same side of each base pair, thus giving rise to
two distances between the backbone. As seen in Figure 2,
the third hydrogen bond of the G-C base pairs is located in
the minor groove, while the methyl group of thymine is lo
cated in the major groove. These two structural features
affect the binding of small molecules to DNA.
X-ray work with DNA requires the use of DNA in its
fiber state, which does not allow enough definable data
points to give a definite arrangement of the atoms. Usually,
molecular models are built and fitted to the data, allowing
for elimination of those models which are inconsistent with
the data or stereochemically unfeasible. This process even
tually leads to a model that is consistent with the data.


8
Conclusions from this type of analysis must be taken
with caution. For example, X-ray analysis of DNA fibers
drawn from different media (i.e., Li+, K+, Na+ or Mg+ salts)
have yielded slightly different structures for the same DNA
molecule.^ Also, at relative humidity above 801, X-ray
data suggest that the DNA is in the B form, which has a
O
pitch of 34 A with 10 residues per turn with the base pairs
perpendicular to the helical axis. However, the X-ray
studies of this fiber cannot distinguish between a left- or
12
right-handed helix. Below 80% relative humidity, the DNA
structure changes to the A form, in which the base pairs are
tilted 15-20 from the perpendicular and the helical pitch
O
is lowered to 28 A with 11 bases per turn. X-ray analysis
of the A form shows it to be a right-handed helix, and from
this, it can be assumed that the B form is also right-
handed. It has been suggested that there are at least eight
different structural forms of natural DNA (i.e., A, B, C,
13
P, P^, J, J? and S), all varying in the angle of the tilt
of the base pairs from the perpendicular and the pitch of
the helix. Furthermore, it has been shown that the A-T/G-C
ratio will cause a change in DNA structure, suggesting that
the secondary structure of DNA is a function of the primary
. 13
structure.
It is apparent that the structure of DNA in the fiber
state is sensitive to many variables and thus the conclu
sions of X-ray analysis can be questioned. In fact,
Donahue^^ ^ and Arnott^ have suggested that X-ray


9
techniques not be used. Furthermore, extrapolation from
the fiber structure to that of solution can only be done if
one assumes that there is no structural change of the DNA
molecule upon solvation. In fact, Bram used the low angle
X-ray scattering technique on DNA in solution to support
the contention that the fiber and solution structures of
DNA are different.^^
Physical Properties of DNA
It was mentioned earlier that DNA is a delicate mole
cule whose structure is subject to breakage due to shearing
forces and is sensitive to changes in temperature and pH.
For example, if the absorption at 260 nm is monitored as the
temperature of a DNA solution is gradually increased, a
sigmoidal curve, as shown in Figure 5, will be observed.^^
Concomitantly, the molar ellipticity and viscosity
versus temperature profile of the DNA solution also shows
a dramatic change. These observations can be explained in
terms of a molecular transformation from a double helix to
a random coil. This helix-coil transition is known as de-
naturation (and/or "melting out") and is usually irrever
sible, indicating that permanent alteration of the native
DNA structure has occurred upon melting.
The structure of DNA is affected by hydrogen-ion concen
tration as depicted in the viscosity-pH profile of a DNA
solution in Figure 6.^ In the pH range of 4.5 to 11.5, the
viscosity of the solution remains relatively constant,


10
Figure 5. Absorption-Temperature Profile for DNA.
Figure 6.
Intrinsic Viscosity-pH Profile for DNA.


indicating no major structural changes. However, at pH
below 4.5 or above 11.5, the viscosity decreases rapidly,
indicative of a "melting out" of the DNA structure. There
fore, DNA solutions are always prepared in a
pH lies between 6 and 8.
A titration curve for DNA is shown in F
Curve A represents the titration starting at
titrating with either acid or alkali. Curve
titration starting from either pH 2.5 or 12.
It is apparent that: 1) DNA has both acidic
tratable groups; and 2) the titration curves
sible. The back titrations indicate that we
and basic groups not available in the first
available in the second (denatured DNA).
buffer whose
- 19
igure 7.
pH 6.9 and
B is the back
5 to neutral,
and basic ti-
are not rever
akly acidic
titration are
PH
Figure 7. Acid-Base Titration Curve for DNA.


12
The DNA structure (i.e., native or denatured) is also
20
dependent upon salt concentration. Thomson et al. have
shown that DNA in solution is irreversibly denatured when
_ 3
the sodium chloride concentration falls below 10 M or when
the magnesium chloride concentration falls below 10 ^ M.
Due to the irreversibility of the denaturation, care must
be taken in preparing DNA solutions to keep the ionic
strength at a minimum of 1 mM.
The hydrogen bonding capacity and the dielectric con
stant of the solution medium are also important for main
taining the DNA structure. Lower alcohols (i.e., methanol
and ethanol) cause reversible denaturation of DNA, while
organic solvents such as formamide, DMF or DMSO cause
21
irreversible denaturation. Since DNA is not soluble in
the above solvents, these experiments are performed by
adding the organic solvent to an aqueous DNA solution.
It is obvious from the above studies that the stability
of the DNA double helix is subject to many types of forces.
An understanding of how these forces influence the structure
of DNA in solution is prerequisite to gain insight into the
mechanisms by which other molecules interact with DNA.
Therefore, analysis of these systems must be at the molecular
level. Since DNA is a very large molecule, classical chem
ical approaches, which are successful for small molecules,
are not always feasible when studying macromolecules. The
usual practice is to construct model systems using monomer
or oligomer units and to extrapolate the data to the larger


13
molecules. Some of the more revealing studies in this re
gard are now examined with the intention of specifying the
forces that control the structure of nucleic acid in solu
tion .
Evidence for Hydrogen Bonding
Watson and Crick proposed that the hydrogen bonding
scheme depicted in Figure 2 is mandatory to account for
the base pairing interactions, as well as to help maintain
1 2
the double helical structure. It has been demonstrated
that, based on the geometrical restraints of colinear
hydrogen bonds (i.e., A-H B) and H-bonding distances
O
of 2.80 3.00 A, 29 different base pairs connected by two
or three hydrogen bonds could be formed between the four
2 2 2 3
nucleosides found in nucleic acids. However, only
three distinct schemes have been found in the crystalline
state. Calculations based on dipole dipole interactions by
Nash and Bradley have shown that only the Watson-Crick scheme
is favored for guanine-cytosine base pairing, but adenosine-
uracil pairs can exist in three forms with two of these forms
of approximately equal energy.^ In fact, for adenine and
thymine derivatives, two different hydrogen bonding schemes
have been found, neither of which is the Watson-Crick type.
Hoogsteen postulated the hydrogen bonding scheme in Figure 8a
2 5 26
for cocrystals of 9-methyladenine and 1-methylthymine.^
Since the sugar-phosphate backbones would be closer together
in a Hoogsteen type base pairing than in the Watson-Crick


14
Figure 8. a) Hoogsteen Adenine-Thymine Base Pair.
b) Anti-Hoogsteen Adenine-Thymine Base Pair.
type, the authors suggested that the Watson-Crick scheme
is favored due to less electronic repulsions between the
strands. This type of hydrogen bonding has also been ob-
2 7
served for 9-ethyladenine-1-methylthymine cocrystals .
Moreover, cocrystals of adenosine and 5-bromouridine have
been found to assume a different hydrogen bonding scheme:
2 Q 7 Q
the anti-Hoogsteen type (Figure 8b). This type of
hydrogen bonding scheme has also been observed for other
30 31
systems. On the other hand, X-ray analysis of single
crystals of derivatives of guanine and cytosine has shown
that only the expected Watson-Crick base pairing is oper-
32-34
ative in these base pairs. This is presumably due to


15
the fact that the pairing scheme for guanine-cytosine has
three hydrogen bonds, whereas the others (Hoogsteen and anti-
Hoogsteen) have only two. Thus, in the crystalline state,
only three out of a possible twenty-nine base pairing com
binations have been observed.
The question naturally arises as to what happens in
solution. Tuppy and Kuebler covalently attached a specific
nucleoside to an Amberlite support and used this modified
35
support in a column. It was found that when a solution
of the other three nucleosides was passed through this
column, only the complement to the covalently bound nucleo
side was retarded. Furthermore, this association was
abolished when the column was eluted with an aqueous-urea
solution, strongly suggesting that the retention of the
complementary nucleoside was due to hydrogen bonding for
mation.
Infrared spectroscopy has been used to determine
hydrogen bonding between derivatized purines and pyrimidines
by monitoring the appearance of new absorption bands be
tween 3500 and 3000 cm ^ due to N-H stretching frequency
3 6
changes when this group is involved in hydrogen bonding.
When solutions of 9-ethyladenine and 1-cyclohexyl-uracil
were mixed, two new bands at 3490 and 3330 cm ^ appeared
and were maximized when the components were mixed at a
ratio of 1:1. Similar results have been obtained with de-
37-39
rivativesof guanine and cytosine.


16
The 4H NMR has also been used to investigate hydrogen
bonding between bases in nonaqueous solution by monitoring
the downfield chemical shift of the hydrogen bonded N-H pro
ton due to a decrease in the electron density about the pro
ton nucleus upon hydrogen bonding. Katz and Penman found
that: 1) base pairing decreases as the hydrogen bonding
ability of the solvent increases; and 2) in nonhydroxylic
solvents, the base pairing is quite specific.For
example, the downfield chemical shift of the N-l proton of
guanosine was -134.7 Hz in the presence of the complemen
tary cytosine, whereas, there was relatively little or no
downfield shift in the presence of the other nucleosides.
A similar observation was seen for the N-3 proton of uridine
in the presence of adenine.4'* Shoup et al. have also em
ployed an NMR study of the four purine and pyrimidine com-
42
ponents of DNA and have obtained similar results.
The above studies reveal that hydrogen bonding between
base pairs is quite specific in nonhydroxylic solvents.
However, in aqueous media, competition for the hydrogen
bonding sites on the bases by water would minimize the im
portance of these forces. Thus, one might conclude that
hydrogen bonding forces are not significant in terms of
stabilizing the double helical structure. This dilemma is
resolved by the realization that the interior of the double
helix, in which the base pairs are located, is very non
polar. The exclusion of water from this region due to the
hydrophobic nature of the bases provides an excellent


17
environment for the formation of hydrogen bonds between the
bases.
Stacking Forces
It was suggested as early as 1958 that hydrogen
bonding between bases was probably not the sole source of
4 3 44
DNA stability. This was based on the observation that
at a pH low enough to break hydrogen bonds, the DNA helix
could be kept intact, providing a sufficiently low temper
ature was maintained. Further work has proposed that a
major stabilizing factor for DNA is a hydrophobic inter
action between the bases. Since the bases are essentially
nonpolar, they tend to stack in aqueous media. Since the
stacked bases are less hydrated than the individual bases,
45
the phenomenon of stacking is thought to be entropy driven.
Ts'O has determined the osmotic coefficients for
several purine and pyrimidine systems and calculated
activity coefficients by the Gibb-Duhem relationship. The
data clearly indicated that the properties of the bases and
nucleosides in solution were far from ideal, indicating
extensive interaction of the nucleosides in aqueous solu
tion. ^ Hydrogen bonding was ruled out for two reasons:
1) the nucleosides associate much more extensively than
urea, which is known to be one of the best hydrogen bonding
agents in water; and 2) methylation of hydrogen bond donor
sites on the nucleosides enhances association.
A more direct method to study the association of bases
and nucleosides in solution is through NMR. Chan et al.


18
found that as the concentration of purine increased, the
resonances of the three protons of purine became in-
46 1
creasingly shifted to higher fields. From the H NMR
theory on ring current anisotropy (Figure 9), it is
possible to predict that the association results from
47
vertical stacking of the purine bases.
Figure 9. Shielding of the Aromatic Protons Caused by
Vertical Stacking.
From the magnitude of the Hi NMR chemical shifts for
the various protons of adenosine, it was found that the
five-membered ring experiences less shielding than the six-
membered ring. Broom et al. proposed two models (Figure 10)
for the preferred average orientation of the stacked
4 8
nucleoside bases to account for these observations.
It has been found that substitution with an amino
group on the 6 position of purine to give adenosine en
hances association. Furthermore, 5-bromo uridine asso
ciates to a greater extent than thymidine. These obser
vations lead to the conclusion that a hydrophobic force


19
R
Figure 10. Geometry of Stacked Nucleosides.
is not solely responsible for the stacking interaction.^^
It has been suggested that there is a correlation between
the stacking and the polarizability of the bases. The con
clusion by Hanlon that London dispersion forces are respon
sible for the stability of the DNA helix is in line with
49
this concept.
Further insight into stacking interactions has been
obtained from studies of dinucleoside phosphates. Using
NMR, Chan and Nelson showed that ApA exists in a 3'-
anti-5'-anti right-handed stack.^ It should be noted
that the Watson-Crick model requires that the bases be in
the anti conformation. It has also been observed that
different dinucleoside phosphates differ in their stacking
ability.^ For example, ApA's structure persisted upon
elevation of temperature, whereas UpU lost its ordered
structure. This has been attributed to different stacking
interactions for the two dinucleoside phosphates.


20
Electrostatic Effects
At neutral pH, the backbone phosphate groups of the
DNA double helix are negatively charged, creating a poly
anion. Therefore, at low ionic strength, there is a large
interstrand Coulombic repulsion between the "naked" anionic
phosphates. There are also intrastrand phosphate inter
actions. Thus, at low ionic strength, there is an increased
propensity for the DNA to denature. Gabbay has determined
that the distance between adjacent phosphate groups along
the same chain of the Watson-Crick double helix is approxi-
0 52
mately 7 A. As the ionic strength of a DNA solution in
creases, the intrinsic viscosity decreases (the intrinsic
viscosity is related to the length of rod-like DNA). It
has been suggested that the decrease in viscosity is re
lated to a decrease in the length of rod-like DNA due to
less inter- and intrastrand repulsions of the phosphates
5 3
as they are neutralized by added electrolyte.
Electronic Effects
The fact that the bases interact electronically is
well known. Examination of Table 1 reveals that as the
Table 1. The Effect of Increasing Length of the Polynucleo
tide (Ap)nA on the UV Absorption Spectrum.
n
X
E /monome
max
max
0
260
15,000
1
257
13,600
2
257
12,600
3
257
11,300
4
257
10,800
300 (poly A)
256
9,000


21
length of the molecule increases, there is a concomitant
decrease in the extinction coefficient of the adenine bases
as well as a blue shift in the wave length maximum.^^^^
A theoretical treatment, employing the exciton theory,
was used to explain the results.^ ^ The theory states
that for a molecular crystal, the absorption of light is
not limited to a single molecule, but rather, it is dis
tributed over many. Since a polynucleotide can be con
sidered a one dimensional crystal, the exciton theory
can be applied to nucleic acids. According to the theory,
the excited states of the bases would be split into, say,
two new levels (Figure 11). The higher energy level has
the electronic vectors of the bases parallel, whereas the
lower energy level has the vectors anti-parallel. Quantum
mechanical selection rules forbid transitions to the lower
<
<
>
Figure 11. Exciton Splitting of Energy Levels.


22
excited state. Therefore, the allowed transition to the
higher energy level is one of energy higher than the origi
nal uncoupled transition and results in a blue shift for
the absorption maximum.
The observed hypochromism is a result of an intensity
interchange of coupled transition moments as illustrated in
Figure 12. Since the transition moments are in a card
stacked orientation in a DNA molecule, the higher energy
transition is hyperchromic, while the lower energy transi
tion is hypochromic.
(a)Card Stack Arrangement
(b)Head-to-Tail Arrangement
A is hypochromic
B is hyperchromic
(c)Herringbone Arrangement
A is hyperchromic
B is hypochromic
A
No intensity interchange
Figure 12. Intensity Interchange Between Two Interacting
Transition Moments.


23
Studies utilizing circular dichroism have also shown
59
the theory to be correct. The selection rules for cir
cular dichroism allow both transitions to occur, resulting
in both a red shifted band and a blue shifted band relative
to the absorption maximum of the monomer. However, without
more complicated quantum mechanical considerations, the
signs of the CD bands cannot be predicted.^ it should be
noted, though, that the two bands are of opposite signs,
resulting in a distinctive double cotton effect for the
polynucleotide.
Dynamic Structure of Nucleic Acids
All of the above factors (i.e., hydrogen bonding,
stacking forces, electronic and electrostatic forces) con
tribute to various extents to the overall stability of the
double helical structure. However, the double helical
structure is not a static one, but rather a dynamic one.
For example, von Hippel and coworkers incubated nucleic
acids in tritiated water, and after separation of the poly
nucleotide from radioactive solvent via gel filtration,
found that tritium was incorporated into the nucleic acids
with kinetics of exchange much faster than with protein
61 ~ 6 3
systems. It was also shown that the observable ex
change took place with those protons involved in the base-
base hydrogen bonding of the double helix. This led
von Hippel and McConnel to propose three models to account
for the phonemenon: 1) unstacking without hydrogen bond


24
breakage; 2) hydrogen bond breakage without unstacking; and
3) hydrogen bond breakage with partial or complete unstacking
62 63
and strand separation. Based on tritium exchange and
pH studies, it was concluded that the "breathing model" (3)
is correct. Kinetic studies of intercalation by Muller and
Crothers^ and Gabbay et al.^ agree with this hypothesis.
Chromatin
As stated before, one DNA molecule may be up to 2 cm
3
long if fully extended. However, a typical animal cell
3 66
nucleus has a diameter of ^5 pm and a volume of ^65 ynr .
The question obviously arises as to how such a long mole
cule fits into such a small package. The answer lies in
the substituents of the nuclear material, chromatin.
Chromatin contains 151 DNA, 10% RNA and 75% protein. Some
of this protein contains enzymes or repressors, but a por
tion is what is known as histones. Histones are rich in
basic amino acid and are tightly associated with DNA pre
sumably by neutralization of the negatively charged phos
phates. There are 5 classes of histones with molecular
weights in the range of ^11,000 21,500.^ They are:
1) HI (lysine rich); 2) H2a and H2b (moderately lysine
rich); and 3) H5 and H4 (arginine rich).
Electron micrographs of chromatin reveal a regular
repeating structure resembling a string of beads. Each
bead is 7-10 nm in diameter and there is a spacer section
of DNA 2-14 nm between each bead.^^;^^^
Each bead


25
contains approximately 200 base pairs of DNA folded around
a histone octamer containing H2a, H2b, fL and H^. This
would effectively condense a 68 nm DNA chain into a 10 nm
bead. It has thus been concluded that histones act to fold
74
DNA so that it will fit into the nucleus.
Of course, histones are not the only proteins which
interact with DNA. Repressor proteins bind to specific
sections of DNA to prevent transcription of a particular
gene. It is still not clear how these interactions take
place.
Small Molecule-Nucleic Acid Interactions
The recognition process between protein systems and
nucleic acids has generated much research in the past ten
years. However, it is not feasible to study the interac
tion specificity between two macromolecules due to the
immense complexity of these interactions. A more sim
plified approach involves the use of model systems whereby
the interaction specificity of small molecules with DNA
is considered. With the results of these studies, one can
extrapolate to more complex systems.
There are several forces operative when a small mole
cule binds to DNA. These interactions involve electrostatic
interaction between positively charged groups on the mole
cule and the anionic backbone phosphates, hydrogen bonding
between available hydrogen bonding sites on the molecule and
the functional groups of the purine and pyrimidine bases,


26
and hydrophobic forces. Three types of hydrophobic-type
interactions have been noted: 1) intercalation between
75-79
base pairs of DNA by aromatic cations; 2) "partial"
insertion between base pairs by sterically restricted com-
pounds containing an aromatic moiety; and 3) ex
ternal hydrophobic-type binding as exemplified by steroidal
amine-nucleic acid complexes.^^ It has been proposed
that the nucleic acids may use all or any combination of
8 7
the above forces to specifically bind polypeptide chains.
Intercalation of aromatic amino acids with double and
single stranded nucleic acids was observed with L-phenyl-
alanine, L-tryptophan, and L-histidine using poly A. The
NMR studies showed that the H-2 and H-8 protons of adenine
were downfield shifted due to the destacking of the adenine
bases as a result of intercalation of the aromatic ring of
8 8
the amino acids. Poly A is also destacked and inter
calated by derivatives of tyramide, tyrosine and tryptamine
since it was found that the aromatic protons of the latter
were upfield shifted in the poly A complex due to ring
89-91
current anisotropy.
Brown observed a very large stabilizing effect on the
Tm of DNA in the presence of arginyltryptophan methyl ester
when compared to other similarly charged dipeptide deriva-
92
tives. He therefore suggested that the aromatic rings of
amino acids behave as "bookmarks" which anchor the proteins
to specific sequences of nucleic acids via an intercalation
mode of binding.


27
In order to investigate the extent of insertion of an
aromatic ring between base pairs of DNA, Gabbay and Kapicak
examined the interaction specificity of reporter molecule I
7 8
(Figure 13) with DNA. They found that when n = 1, there
is a decrease in viscosity of the DNA-reporter molecule (due
to shortening of the DNA helix), whereas there was an in
creased viscosity of the DNA-reporter complex for n = 2, 3
and 4 (due to an increase in the effective length of the
DNA helix). Furthermore, NMR studies reveal that the
aromatic protons H. and HD (in DNA-1 complex when n = 1)
experience upfield chemical shifts of 10 and 6 Hz, respec
tively, but that the aromatic protons of the DNA-1 complex
when n = 2, 3 and 4 are completely broadened and indis
tinguishable from baseline noise.
It was therefore proposed that the p-nitropheny1 ring
of I (when n = 1) is partially inserted between base pairs
7 8
of DNA (Figure 14). This model accounts for: 1) the
decrease in the effective length of DNA-I caused by bending
at the point of insertion; and 2) the different upfield
chemical shifts of the aromatic protons and Hg (i.e.,
experiences a larger ring current anisotropy than Hg as a
result of "partial" insertion. Presumably, the single
methylene between the aromatic moiety and the quaternary
ammonium group on the side chain is not sufficient to allow
for "full" insertion and lengthening of the helix.
Further evidence for "partial" intercalation came from
experiments using the diastereomeric dipeptides


(CH2)nN(CH3)2(CH2)3NCCH3)3 2Br
Figure 14. Schematic illustration of a segment of DNA double helix (a) which
can either partially intercalate a molecule (b) or fully intercalate
a molecule (c) .
K)
CO


29
H
H
NH--C-CONH-C-CONH
(^2)4 ch2
+nh7
L-lys-L-pheA
CH,
NFU -C-CONH-C-CONH9
(ch9)
2d
+NH,
.t)
L-lys-D-pheA
Figure 15. Schematic illustration of a DNA segment showing
a possible mechanism whereby the e and a amino
groups of the N-terminal L-lysyl residue are
stereospecifically anchored and thus dictating
the positioning of the aromatic ring of the
diastereomeric dipeptides in the DNA-peptide
complex.


30
L-lysyl-L-phenylalanine amide (L-lys-L-pheA) and L-lysyl-
8 2-84
D-phenylalanine amide (L-lys-D-pheA). In particular,
the NMR, viscometric, and flow dichroism data suggest that
the aromatic ring of L-lys-L-pheA is partially inserted
between the base pairs of DNA, whereas the aromatic ring
of L-lys-D-pheA points out toward the solvent (Figure 15).
It was concluded that this specificity arises from a
stereospecific interaction of the e- and a-amino groups of
the N-terminal L-lysyl residue which dictates the posi
tioning of the aromatic ring of the C-terminal phenyl
alanine residue of the L-lys-L-pheA and L-lys-D-pheA. The
specificity observed with L-lys-L-pheA and L-lys-D-pheA
cannot be attributed to the chirality of the phenylalanine
moiety itself since NMR studies reveal no significant
differences in the binding of L- and D-phenylalanine amides
to DNA.
Statement of Problem
In this dissertation, the synthesis of several dipep
tide amides was undertaken to determine the effect of the
chirality of the C-terminal amino acid a carbon and the
length of the "arm" between the a carbon and the aromatic
ring on the extent of intercalation of the aromatic ring
(used in this case as a probe) between the base pairs of
DNA.


CHAPTER II
RESULTS AND DISCUSSION
The interactions of the dipeptide amides (Figure 16)
with DNA has been examined by pulsed Fourier transform
NMR, viscosity, UV absorption and binding studies, circular
dichroism, equilibrium dialysis, and melting temperature
(Tm of helix-coil transition) studies. The results are pre
sented below.
NMR Studies
The NMR studies conducted in this work were used
to determine the effect of binding of the peptide to DNA
on: 1) the line broadening of the aromatic proton NMR
signals of the peptide; and 2) the upfield chemical shift
of the aromatic protons of the peptide. The change in
chemical shifts is related to the proximity of the aromatic
protons to the ring current of the DNA base pairs. The
NMR line broadening can be explained by several mechanisms:
1) slow rate of exchange between various DNA binding sites;
2) restricted tumbling of the aromatic ring; 3) larger dif
ferences in the chemical shifts experienced by the ortho,
para, and meta protons; or 4) a combination of all three
mechanisms. In these experiments, the peptides are fully
bound as determined by equilibrium dialysis. The results
31


1 L-lys-L-pN02-pheA
H H
+ I I
NH,-C-CONll-C-CONH,
CH2-Q>'N2
Nil,
6 L lys -1), L-DNP-DABA
NII,-C-CONH-CH-CONH,
3 | | 2
(cn2)4 cn2cn2-Nil
Nil,
-NO,
NO,
2 L-lys-D-pN02pheAa
H CH,-<( )) NO
NH,-C-CONH-C-CONH,
3 | ,2
(CH2). II
I
Nil,
7 L-lys-L-S-pN02bz-cysAc
H II
NII,-C-CONH-C-CONH-
3 | | 2
(CII214 Cll2 S-CII2
Nil,
NO,
3 L-lys-L-S-NT-cysAa
4 L-lys-D-S-NT-cysA
NO,
NO,
NH, C CONII C CONII,
3 | ,2
(Cll2)4 II
NH,
8 L-lys-D-S-pNOjbz-cysAc
9 L-lys-L-DNP-ornA
H CH, S- Cll,
+ I I 2
NH, C- CONII C CONII,
3 i
Abb
NO,
(Cll2)4 H
NH,
H II
* I I
Nil, C CONII C CONII,
3 | | 2
(CH2)4 CH2CI12CH2NH-
Nil, NO,
-NO,
5 L-lys-L-DNP-DABA
H II
NII,-C-CONH-C-CONII,
3 t i 2
CCH2)4 CH2CH2-NH-rt)VN02
NH,
NO,
10 L-lys-D-DNP-ornA
Figure 16. The Dipeptide Amides Synthesized and Studied.
a) dihydrobromide salt; b) dihydroacetate salt.
H cii,ch2cii2nh-(( j)-no
NHj-C-CONH-C-CONII,
(Cll2)4 11
Nil j
O'!
hJ


33
of these studies are shown in Figures 17 22, and lead to
the following observations.
First, the extent of NMR signal line broadening of
the aromatic protons depends in general on the chirality of
the C-terminal amino acid a carbon and the length of the
"arm" between the aromatic ring and the a carbon. For
example, the signal of the aromatic protons of L-lys-
L-pNO^-pheA (_1) are broadened more than those of the
c
1
__J L I I
2.5 1.5 ppm
The Partial NMR Spectra of DNA at Various
Temperatures. The temperatures are: a) 37C;
b) 70C; and c) 90C.
Figure 17.


34
8.0 7.0 ppm 8.0 7.0 ppm
L- lys-L-pNO^-pheA (1) L- lys -D-pN09-pheA (2)
Figure 18. NMR Signal o£ the Aromatic Protons of Dipep
tides _1 and 2_ in the Presence and Absence of
DNA at Various Temperatures. The DNA base pair
to dipeptide ratio is: a) 0 (37C) ; b) 10 (37C) ;
c) 7 (37C) ; d 5.5 (37C); e) 5.5 (70C); and
f) 5.5 (90C).


35
Figure 19. H NMR Signal of the Aromatic Protons of Dipep
tides _3 and £ in the Presence and Absence of
DNA at Various Temperatures. The DNA base pair
to dipeptide ratio is: a) 0 (37C) ; b) 15 (37C);
c) 10 (37C) ; d) 7 (37C) ; e) 7 (70C); and
f) 7 (90C).


36
H NMR Signal of the Aromatic Protons of Dipep
tides 5^ and 6^ in the Presence and Absence of
DNA at Various Temperatures. The DNA base pai
to dipeptide ratio is: a) 0 (37C); b) 15 (37
c) 10 (37C); d) 7 (37C); e) 7 (70C) ; and
f) 7 (90C) .
Figure 20.
O t-j


37
Figure 21. H NMR Signal of the Aromatic Protons of Dipep
tides 1_ and 8^ in the Presence and Absence of
DNA at Various Temperatures. The DNA base pair
to dipeptide ratio is: a) 0 (37C); b) 15 (37C) ;
c) 10 (37C) ; d) 7 (37C) ; e) 7 (70C) ; and
f) 7 (90C).


38
£
8.5 7.5 ppm
L-lys-D-DNP-ornA (10)
H NMR Signal o£ the Aromatic Protons of Dipep
tides 9^ and 10_ in the Presence and Absence of
DNA at Various Temperatures. The DNA base pai
to dipeptide ratio is: a) 0 (37C); b) 15 (37
c) 10 (37C) ; d) 7 (37C) ; e) 7 (70C) ; and
£) 7 (90C).
Figure 22.
o p-j


39
diastereomeric L-lys-D-pNC^-pheA (2^. The NMR signal for
the aromatic protons of L-lys-L-S-NT-cysA (3), L-lys-D-S-NT-
cysA (4), L-lys-L-DNP-DABA (5), L-lys-D,L-DNP-DABA (6),
L-lys-L-DNP-ornA and L-lys-D-DNP-ornA (10_) are com
pletely broadened into the base line and are indistinguish
able from base line noise at all PNA base pair to peptide
ratios. For compounds L-lys-L-S-pNC^bz-cysA {!) and L-lys-
D-S-pNO^bz-cysA (8^, there is considerable line broadening,
with perhaps slightly more broadening for peptide 7_.
It should be noted that the line broadening decreases
at higher temperatures as the DNA begins to "melt-out,"
freeing bound peptide. Furthermore, the effect of the vis
cosity of the DNA solution on the line broadening is found
to be small [e.g., the signal line width of internal stan
dard 2,2,3,5-d^-3-trimethylsilylproprionate (TSP) is found
to be 1.2 +_ 0.2 and 2.1 + 0.3 Hz in the absence and presence
of DNA, respectively, at 37C]. In addition, examination
of Figure 17 reveals that some of the structure of the NMR
spectra in the region of 6 7.0 8.5 ppm is due to DNA base
protons.
Secondly, the extent of the upfield chemical shift of
the NMR signal of the aromatic protons also depends on
the chirality of the C-terminal amino acid a carbon and the
distance from it to the aromatic ring. The spectra for
L-lys-L-pNC^-pheA (IT) is considerably more complicated in
the DNA complex as opposed to free peptide. Examination of
Figure 18 shows two peaks at approximately 45 Hz upfield


40
from the original peaks. Interpretation of this spectra is
difficult due to the complexity of the splitting. However,
for L-lys-D-pNO^-pheA (2_), there is little or no upfield
shifting of the aromatic proton signals. The NMR spectra
of L-lys-L-S-pNO?bz-cysA (7) and L-lys-D-S-pNO?bz-cysA (8)
also become more complex in the presence of DNA (Figure 21).
There is considerable upfield shifting of the aromatic sig
nals for both compounds, with more shifting for compound 7_
than _8. In fact, both spectra resemble that of L-lys-L-
pNC^-pheA (_1) .
Since the NMR signals for the aromatic protons
of compounds L-lys-L-S-NT-cysA (3), L-lys-D-S-NT-cysA (£) ,
L-lys-L-DNP-DABA (5), L-lys-D,L-DNP-DABA (6), L-lys-L-DNP-
ornA (9_) and L-lys-D-DNP-ornA (10) are completely broadened
into the baseline, nothing can be concluded about upfield
shifting of these protons in the DNA complex. However,
in all cases, the resolution of the signals increases as
the temperature increases, indicative of "release" of the
dipeptide from the DNA complex upon denaturation of the
DNA.
Viscosity Studies
It has been shown by a number of investigators that
planar aromatic compounds (e.g., ethidium bromide, dauno-
rubicin, and the acridine dyes), intercalate between the
base pairs of DNA. The DNA complex increases in viscosity
relative to free DNA due to an increase in the effective


41
length of the DNA molecule. To prove that the DNA length
increases, the determination of the intrinsic viscosity,
(p), of the solution is necessary. Intrinsic viscosity
is defined by
r ^ Limit ^sp
(,1) = c 0 c
where p is the specific viscosity of a DNA solution and
sp r
c is the concentration of DNA. Unfortunately, these experi
ments are uninformative since the peptide dissociates from
the DNA complex at high dilution (probably due to the low
binding constant of peptide to DNA) and thus, the intrinsic
viscosity of the complex approaches that of free DNA.
Therefore, the effect of increasing the concentration
of the dipeptide amides on the relative specific viscosity,
p /p (where p and p are the specific viscosities
sp spo v sp spo ^
of the DNA solution in the presence and absence of dipeptide
amide, respectively) at 3.0 x 10 ^ DNA phosphate/liter (in
10 mM MES, 5 mM Na+, pH 6.2) was studied at 37C using the
low shear Zimm viscometer. Since the study was carried out
at low DNA concentration, the relative values of p /p
sp spo
are close approximations of the relative values of the
intrinsic viscosity of a DNA-dipeptide complex to free DNA
([n]/[p] ). The results are shown in Figures 23 through 26,
and indicate that peptides ]^, 2_, 7_ and 8^ decrease the spe
cific viscosity of a DNA solution, with peptide (L decreasing
the viscosity to a much greater extent than 2, 7_ and _8.
Furthermore, peptides 3, 4, 5, 6, 9 and 10 increase the


n

1E
spo
Figure 23. The Effect of Dipeptides 1^ and _2 on the Relative Specific
Viscosity of Salmon Sperm DNA.


Figure 24. The Effect of Dipeptides 3, 4_, 7_ and 8^ on the Relative Specific
Viscosity of Salmon Sperm DNA.


Figure 25. The Effect of Dipeptides 5^ and 6^ on the Relative Specific
Viscosity of Salmon Sperm DNA.
A


Figure 26. The Effect of Dipeptides 9^ and 10_ on the Relative Specific
Viscosity of Salmon Sperm DNA.
Ul


46
specific viscosity of a DNA solution, the effect being
greatest for _5, 9_ and _10 with only a slight increase for
peptides £ and 4_.
Ultraviolet Absorption Studies
Absorption data for the dipeptides are shown in Table
2. All peptides have a well defined X in the range of
260 400 nm with extinction coefficients on the order of
8.5 x 10^ for compounds 1, 2, 3, 4, 7 and 8 and 15.5 x 10
for compounds _5, 6_, 9_ and 1C£ For those compounds with
X greater than 300 nm, two effects can be seen on the
absorption spectra upon binding to DNA. First, large
hypochromic effects are seen for all DNP derivatives
(namely £, £, 9_ and 10) with a considerable hypochromic
effect for one of the nitrotolyl derivatives, peptide 3.
Furthermore, there is a large bathochromic shift for X
in £ and 4_ in the DNA complex (i.e., from 340 nm to 355 nm)
and a small bathochromic shift for the DNP derivatives £,
£, £ and £0 (i.e., from 360 nm to 365 nm).
Binding Studies
The binding of those peptides with X greater than
300 nm to salmon sperm DNA was determined by spectral
titration. The effect of increasing concentration of DNA
on the absorption of peptides 3, 4, 5, 6, 9 and 10 at 340
nm for the nitrotolyl containing peptides or at 360 nm for
the DNP containing peptides was studied in 6.6 mM phosphate
buffer (pH 7.2) at three ionic strengths (5 mM, 10 mM and


Table 2. Absorption and Hypochromicity Data on the Dipeptide Amides.
Free
DNA complex
Compound3
A
max
e
max
A
max
e
max
U\h
L-lys-L-pNC^-pheA (1)
275
8.75
x 103
ND
ND
ND
L-lys-D-pNO.,-pheA (2)
275
8.75
x 103
ND
ND
ND
L-lys-L-S-NT-cysA (3)
340
7. 88
x 103
355
6.3 x 10
25
L-lys-D-S-NT-cysA (4)
340
7.88
x 103
355
7.5 x 103
5.1
L-lys-L-DNP-DABA (5)
360
15.5
x 103
365
1.0 x 104
55
L-lys-D,L-DNP-DABA (6)
360
15.5
x 103
365
1.0 x 104
55
L-lys-L-S-pNO^bz-cysA (7)
275
8.50
x 103
ND
ND
ND
L-lys-D-S-pNO^bz-cysA (8)
275
8.50
x 103
ND
ND
ND
L-lys-L-DNP-ornA (9)
360
15.5
x 103
365
1.0 x 104
55
L-lys-D-DNP-ornA (10)
360
15.5
x 103
365
1.0 x 104
55
Measurements were carried
Cary-17D spectrophotometer
out in 10 mM
at 25C using
MES buffer (5
salmon sperm
mM Na+, pH
DMA. ND =
6.2) with the
not determined.
= percent hypochromicity.


48
and 50 mM Na+) in a 1 or 5 cm cell which was thermostated
at 210 C .
The spectral data were analyzed by the McGhee-von Hippel
9 3
technique according to the following equation
n-1
where v is the number of moles of peptide bound per DNA base
pair, L is the concentration of free peptide, K is the
apparent binding constant, and n is the number of base pairs
per binding site. To calculate the above parameters, the
raw absorption data are fed to a Pet 2001 microcomputer
which then calculates v and v/L (see experimental chapter).
For each pair of v and v/L, a value of K is calculated as
a function of N. The programmer inputs various values of n
until the smallest deviation in the set of K's is obtained.
The average value of the K's is then taken as (e.g., see
Table 3) .
A plot of v/L versus v is shown in Figure 27. It can
be seen that only a small range of v's was obtained from
the spectral titrations. Furthermore, most of the points
are on the straight part of the graph, with only a few
points on the curved part. Therefore, the values of K and
n should be taken with caution. More accurate values of
K and n could only be obtained with a broader range of
3
v's. However, the values of K
a
tive binding strengths.
in Table 4 do reflect rela-


49
Table 3. Typical Output for McGhee-von Hippel Binding Iso
therms for Various Values of na.
K
a
V
v/L
n= 3
n=4
n= 5
. 405
.052
7379
9894
11347
13203
.421
. 051
7384
9814
11201
12958
.445
.050
7620
10096
11505
13283
. 465
. 049
7782
10261
11662
13423
.488
.049
8077
10628
12065
13868
. 508
. 048
8298
10875
12321
14127
. 524
.048
8435
10997
12424
14198
. 541
. 047
8599
11157
12573
14325
. 561
. 046
8908
11527
12973
14757
.581
. 046
9254
11948
13430
15254
. 601
.046
9643
12423
13948
15823
.617
. 045
9939
12760
14302
16190
.631
. 044
10126
12942
14471
16337
.657
. 043
10585
13420
14944
16790
.681
. 042
11001
13835
15343
17155
aPerformed
by a Commodore Pet
2001 microcomputer fo
r L-lys-
L-DNP-ornA
(9) (10
mM
Na ) where a = ratio
of bound
peptide
v = moles
of bound
peptide/mole of DNA base pairs,
L =
concentration of free
peptide,
n = number
of base p
airs per
binding site, and
K =
apparent
binding constant for
that se
of v and v/L.


50
The values of K and n for the dipeptides 3, 4, 5, 6,
9^ and 10_ are found in Table 4. A number of observations
can be made.
1) In general, K and n decrease with increasing
ionic strength.
2) Peptides _5, 9^ and _10_ bind very strongly to DNA,
while compounds 4_ and 6_ exhibit fairly weak binding.
3) Peptide 3^ binds stronger than its diastereomer,
peptide 4_, and peptide 5_ binds stronger than the diastereo-
meric mixture 6.


Table 4. Binding Isotherms for DNP and Nitrotolyl Containing
Dipeptides.
5 mM
Na +
10 mM Na+
5 0 mM
Na+
Compound3
K x 10'
5
n
K x 10~5
n
K x 10'4
n
3.
8.
L-lys-L-S-NT-cysA (3)
1.3
4.0
0.32
3.3
L-lys-D-S-NT-cysA (4)
0.53
3.2
0.024
2.7
L-lys-L-DNP-DABA (5)
2.5
4.6
2.4
4.0
1. 8
2 .1
L-lys-D,L-DNP-DABA (6)
0.81
2.6
1. 2
2.6
0.18
2.1
L-lys-L-DNP-ornA (9)
1.9
4.5
1.1
3.9
1.1
2.5
L-lys-D-DNP-ornA (10)
2.3
4.5
2.2
4.0
1.1
2 5
Measurements were carried out on a Cary-17D
or 340 nm (nitrotolyl derivatives) in 6.6 mM
spectrophotometer
phosphate buffer
at 360 nm
(pH 7.2) in
(DNP derivatives)
a 1 or 5 cm
cell at 21C.


52
4) All binding constants are higher than those ob
tained from equilibrium dialysis, but these constants were
calculated at different peptide and DNA concentrations.
Circular Dichroism Studies
The effect of increasing concentration of salmon
sperm DNA on the circular dichroism spectra of compounds
3, 4, _5, (3, 9^ and 10. in the range of 300 400 nm was
studied. The results, shown in Figures 28 and 29, show
that there is an induced CD spectra for all compounds in
the range of 300 400 nm. The extent of the induced CD
in the presence of excess DNA (i.e., at a DNA base pair to
peptide ratio of 12) follows the following order:
1) L-lys-L-DNP-ornA (9) L-lys-D-DNP-ornA (10) = L-lys-L-
DNP-DABA (5) > L-lys-D,L-DNP-DABA (6) > L-lys-L-S-NT-cysA (3)
> L-lys-D-S-NT-cysA (4.) .
Equilibrium Dialysis
Equilibrium dialysis was utilized to perform direct
binding studies of the dipeptide amides with salmon sperm
DNA. The quantitative analysis of the dipeptide amide was
accomplished by UV absorption data at the X of the
various dipeptides. These studies were carried out in
duplicate at a single peptide and DNA concentration (i.e.,
at 3.0 x 10 ^ M DNA phosphate/liter and either 2.5 x 10 ^ M
or 5.0 x 10 ^ M peptide) using a 10 mM MES buffer (5 mM Na+,
pH 6.2). The apparent binding constant, K ,
3.
from
was evaluated


Figure 28. The CD Spectra of Dipeptides _5, 6, 9 and 10_ in the Absence and
Presence of DNA. The DNA base pair to peptide ratio is: a) 0;
b) 1.5'; and c) 12.


54
Figure 29. The CD Spectra of Dipeptides 3 and £ in the
Absence and Presence of DNA. The DNA base
pair to dipeptide ratio is: a) 0; b) 1.5;
and c) 12.
R,
K =
a (Pt Rb)Rf
where R^ and R^ is the concentration of free and bound
dipeptide, respectively, and P is the total DNA phosphate
concentration. This equation can be used assuming that
each DNA phosphate binds independently to a peptide mole
cule and that the maximum number of binding sites per DNA
phosphate is one. Even though this may not be true, the
binding data from the interactions of peptides to nucleic
acids are evaluated in this way and thus the Ka values in


5 5
Table 5. Apparent Binding Affinity (K ) of the Dipeptides
to Salmon Sperm DNAa. a
Peptide System
L-lys-L-pNC>2-pheA (1_)
L-lys-D-pNC^-pheA (2^
L-lys-L-S-NT-cysA (3)
L-lys-D-S-NT-cysA (40
L- lys-L-DNP-DABA (_5)
L-lys-D,L-DNP- DABA (6)
L-lys-L-S-pNO^bz-cysA (2)
L-lys-D-S-pNC^bz-cysA (80
L-lys-L-DNP-ornA (9)
L-lys-D-DNP-ornA (10)
Ki
K2
5.3
X
102
5.4
X
102
6.8
X
102
4.9
X
102
3.6
X
103
1.3
X
103
2.7
X
3
io-3
8.7
X
103
3.0
X
103
1.3
X
10
1.3
X
103
1.3
X
103
CO
X
2
10*
3.2
X
102
2.2
X
2
10*
6.6
X
101
2.7
X
103
7.9
X
2
10*
3.1
X
103
1.6
X
IQ0
3. A
Equilibrium dialysis was carried out with 3.0 x 10 M
salmon sperm DMA in 10 mM MES buffer (5 mM Na+ pH 6.2)
with peptide concentration of either 2.5 x 10 "4 M (K,)
or 5.0 x IQ'4 M (K2).


56
Table 5 do reflect differences in binding strength.
Examination of Table 3 reveals that, in general, dipeptides
containing a nitrotolyl moiety (i.e., 3^ and 4) or a DNP
moiety (i.e., _5, 6^, 9_ and 10) bind more strongly than
those compounds containing a p-nitrophenyl moiety (i.e., 1_,
2_, 1_ and 8^) .
Melting Temperature Studies
The effect of increasing concentration of peptide on
the Tm (melting temperature) of the helix-coil transition
for three different nucleic acids was determined. Several
observations can be made from examination of the Tm data
in Table 4.
1) All peptides stabilize the helix relative to the
random coil and the transition exhibits a monophasic
melting behavior. Furthermore, the DNP derivatives _5, 6,
9^ and 10_ and the nitrotolyl containing peptide stabilize
the helix to a greater extent than the other peptides.
2) All peptides, in general, stabilize poly d(A-T)
to a greater extent than poly I poly C, indicative of
preferential binding to A-T sites.
3) For a given set of diastereomeric dipeptides,
the compound having the L configuration for the C-terminal
amino acid a carbon stabilizes the helix to a greater ex
tent than the peptide with the D configuration at the C-
terminal amino acid a carbon (e.g., 3 > 4, 9 > 10, etc.).


Table 6. The Effect of the Dipeptides on the AT of the Helix-Coil Transition
of Salmon Sperm DNA, Poly I Poly C, and Poly d(A-T) (ATm = Tjn T ,
where Tm and T are the melting temperatures of the nucleic acids mo
in the presence and absence of dipeptide).
Peptide System3 ATm
Salmon Sperm DNA
Poly I -
Poly C
Poly
d(A-T)
250 yM
500 yM
250 yM
5 00 yM
250 yM
500 yM
L-lys-L-pNO?-pheA (1)
1.8
2.3
3.8
5.7
4.0
6.8
L-lys-D-pNC^-pheA (2)
0.9
3.5
0.4
6.8
1. 2
2.4
L-lys-L-S-NT-cysA (3)
5.1
11.0
2.6
5.3
14.0
17.1
L-lys-D-S-NT-cysA (4)
2.4
3.3
1.9
4.4
6.2
12.7
L-lys-L-DNP-DABA (5)
7.5
15.6
7.0
11.8
10.6
22.9
L-lys-D,L-DNP-DABA (6)
4.3
8.4
4.3
7.3
7.7
18.3
L-lys-L-S-pNC^bz-cysA (7)
0.2
0 3
1.7
3.5
3.8
6.1
L-lys-D-S-pNO?bz-cysA (8)
0.3
1.2
2.6
2.9
2.3
3.7
L-lys-L-DNP-ornA (9)
>30.0
>30.0
6.7
11. 5
14.3
23.3
L-lys-D-DNP-ornA (10)
14.9
>30.0
1.1
5.7
7.0
19.3
Tm studies were carried out in 0
and peptide concentrations of 250
.01 M MES
and 500
buffer,
yM.
pH 6.2, using
126 yM P/L
of DNA


5 8
The interpretation of these data is complicated by
the fact that the helix-coil transition involves the inter
action of the dipeptide (P), not only with the helix (H),
but also with the coil (C) as shown in the following
equation:
KH KC
H + P + H- P + C- P + C + P.
It can be assumed that K is greater than K since T is
increased in the presence of the dipeptide system. How
ever, Gabbay and Kleinman have pointed out that this con
clusion is valid only when and are determined
9 4
directly.
Discussion
Much research has been devoted to the study of the
recognition specificity of proteins for nucleic acids.
Due to the several types of forces operating at several
sites along the nucleic acid and protein, this type of
study is of immense complexity. Furthermore, recent
studies on chromatin reveal that protein-DNA binding
specificity is not only a dynamic process, continuously
changing during the cell cycle, but may also involve spe
cific protein aggregates-DNA recognition.^
The problem can be simplified by studying the inter
action specificities of small dipeptides with DNA as under
taken in this work. As stated before, small molecules may
bind to DNA by electrostatic, hydrogen bonding, hydrophobic


59
8 7
forces, or any combination of these. Electrostatic
forces are not expected to lead to specific recognition
between a small molecule and DNA, since this type of
interaction may occur at each of the DNA phosphate groups.
Furthermore, hydrogen bonding may occur at any base pair;
so, it can be assumed that hydrogen bonding forces would
not lead to specific recognition.
It has therefore been suggested that hydrophobic-type
interaction (which has been noted in the binding of aro
matic and hydrophobic amino acids to DNA preferentially at
A-T sites) is primarily responsible for the recognition
8 7
process. Gabbay has shown that the "partial" intercala
tion of the aromatic ring of the side chain of phenylalanine
with native DNA (which has been shown to possess A-T
clusters'*^ ^^) is consistent with this interpretation.
From the work with reporter molecule I and the dia-
stereomeric dipeptides L-lys-L-pheA and L-lys-D-pheA, two
criteria were established to dictate whether full, partial
78 82 84
or no intercalation will operate. From the re
porter molecule work, it was shown that there is a depend
ence of length between the aromatic ring and the quaternary
ammonium group, i.e., if the length was long enough, full
intercalation occurs; if the length was short enough, only
partial intercalation occurs. The dependence on the
chirality of the C-terminal amino acid a carbon for partial
intercalation was shown by the work with L-lys-L-pheA and
L-lys-D-pheA. The results suggest that the dipeptide is


60
stereospecifically anchored to the DNA by an electrostatic
interaction between the N-terminal lysyl a and e amino
groups and that as a result of this binding, the orienta
tion of the aromatic ring of the C-terminal amino acid is
established. When the C-terminal amino acid is of the L
configuration, the aromatic ring is pointed toward the in
terior of the helix; whereas, in the case of the D config
uration, the aromatic ring is pointed out toward the sol
vent (Figure 15).
In an attempt to provide additional experimental
evidence for (or against) these two criteria, the synthesis
and study of the interaction specificities of a series of
dipeptide amides were undertaken. The series consisted of
pairs of diastereomeric dipeptides (differing in chirality
of the C-terminal amino acid a carbon) with an aromatic
ring of various distances from the C-terminal amino acid
a carbon.
From the above considerations, it was assumed that
the aromatic ring of L-lys-L-pNC^-pheA (_1) would partially
intercalate while that of L-lys-L-S-NT-cysA (3) may par
tially or fully intercalate and that there would be full
intercalation for L-lys-L-DNP-DABA (5), L-lys-L-S-pNO^bz-
cysA (7_) and L-lys-L-DNP-ornA (£) On the other hand, no
intercalation of the aromatic ring was expected for those
peptides whose C-terminal amino acid a carbon was of the
D configuration [i.e., L-lys-D-pN0o-pheA (2), L-lys-D-S-NT-
cysA (4-), L-lys-D-S-pNO^bz cysA (8^) and L lys D-DNP ornA (10) ] .


61
A "mixed" behavior was expected for the diastereomeric mix
ture L-lys- D, L -DNP-DABA (6).
Several types of experimental data can be obtained to
determine the extent of intercalation. From NMR studies,
the extent of line broadening and upfield shifting of the
aromatic proton signals is indicative of the extent of in
tercalation. Full intercalation results in total line
broadening, while partial intercalation results in broad
ening and upfield shifting of the aromatic peaks. No up
field shifting is observed in the absence of intercalation.
Inspection of the NMR spectra of the peptides in the
presence of DNA (Figures 18 22) reveals some interesting
points. First, the NMR signals of aromatic protons of com
pounds 3^, 4_, _5, 6_, 9^ and _10 are completely broadened,
indicative of full intercalation. The aromatic signals for
peptides 1, 1_ and 8^ are upfield shifted and broadened,
which would suggest partial intercalation. On the other
hand, the NMR signals for the aromatic protons on compound
2 are only slightly shifted and broadened. This can be in
terpreted as the absence of intercalation. The greater
extent of line broadening for 1^ as compared to 2_ can be
explained by several mechanisms: 1) slower tumbling rates
of the aromatic ring of _1 in the DNA complex as compared
to 2_; 2) slower exchange between the various DNA binding
sites for DNA-^L as compared to the DNA-2^ complex; 3) larger
differences in the ortho and meta protons of the aromatic
ring of 1 in the DNA complex as compared to 2_\ or 4) a


62
combination of all three mechanisms. Since the value of
the spin lattice relaxation time (T^) is dependent (among
other things) on the correlation time (Tc) and mean resi
dence time (t ), determination of T. for 1 and 2 in the
presence and absence of DNA could help discriminate be
tween the three mechanisms. Unfortunately, due to the
complexity of the spectra and inability to accurately meas
ure peak intensities in the spectra, values could
not be obtained for the DNA-1^ complex. However, previous
work by Gabbay^'7 on L-lys-L-pheA-DNA and L-lys-D-pheA-DNA
complex revealed that the of the aromatic protons of
both peptides in the DNA complex were nearly identical
(T^ 0.65 sec.), suggesting that the tumbling rate (1/t )
and the chemical exchange rate (1/t^) of the aromatic pro
tons of the peptides in the DNA complex were very similar
in magnitude. ^ It was concluded that the greater
signal line broadening observed for the aromatic protons
of the L-lys-L-pheA-DNA complex as compared to the L-lys-
D-pheA-DNA complex could only be due to large differences
in the chemical shifts experienced by the ortho, meta, and
para protons. It can therefore be assumed that the greater
line signal broadening of the aromatic protons of L-lys-L-
pNO^-pheA in the DNA complex as compared to that of the
L-lys-D-pNO^-pheA-DNA complex can only be due to differences
in the chemical shifts experienced by the ortho and meta
protons.


63
The magnitude and the sign of the change in the vis
cosity of a DNA-peptide complex can also be informative as
to the extent of intercalation. An increase in the vis
cosity of a DNA-small molecule solution is noted for full
intercalation of the aromatic ring of the small molecule
between the base pairs of DNA, while a decrease in viscos
ity is observed for "partial" intercalation. It should be
noted that electrostatic binding (without intercalation)
of cationic molecules to DNA also decreases the viscosity
of a DNA-small molecule solution due to neutralization of
the anionic phosphates and subsequent shortening of the
DNA chain. However, the degree of decrease in the viscos
ity of the DNA solution is not as great as when intercala
tion also takes place.
Consistent with the NMR data, peptides _3, £, 5^, (3, 9.
and 1_0 increase the viscosity of the DNA-peptide solution
(i.e., full intercalation), while peptides 1, 2_, 7_ and 8^
decrease the viscosity of the DNA-peptide solution, the de
crease greatest with L-lys-L-pNO^-pheA (1) .
Further evidence for full intercalation of the aromatic
rings of compounds 3^, 4_, 5_, (3, 9_ and 1^0 comes from the UV
and CD studies. From the UV studies, a red shift in A
max
and hypochromism is observed for these peptides in the
presence of DNA. The red shift arises from an electronic
interaction between the aromatic ring and the purine and
pyrimidine base resulting in a splitting of the excited
state energy levels of the peptide aromatic ring producing


64
a lower excited state energy level and the hypochromism
arises from an intensity exchange between the coupled
transition moments of the aromatic ring and the purine
and pyrimidine bases. There is also an induced CD in the
region of X of the peptides in the DNA complex. This
can be explained by intercalation of the aromatic ring
into the interior of the DNA double helix, which is
assymetric (thus, an induced CD).
When considering the binding of these peptides to DNA,
it must be kept in mind that two types of interaction are
operative, namely electrostatic binding of the N-terminal
amino acid lysyl residue via the a and e amino groups and
intercalation of the aromatic ring of the C-terminal amino
acid. Furthermore, one type of binding may predominate
depending on the relative strengths of the two possible
interactions. In general, electrostatic binding is favored
at low ionic strength, while intercalation is favored at
higher ionic strength. The apparent binding constant, K ,
is reflective of this phenomenon.
Those compounds which have been shown to intercalate
from NMR and viscosity studies also have a higher K than
those compounds which only partially intercalate or do not
intercalate at all (Table 5). This is also borne out from
the Tm studies. The compounds that fully intercalate
stabilize the DNA double helix to a greater extent than
those that only partially intercalate. The Tm data also
show that poly d(A-T) is stabilized more than poly I -
poly C, indicating the peptides show a preference for A-T sites.


65
The results from these studies were pretty much in
line with what was predicted. However, a few unexpected
results were obtained.
1) The aromatic ring of L-lys-D-S-NT-cysA (£) inter
calates. It was predicted that this would not be the case,
since the aromatic ring of _4 should point out toward the
solvent. However, if a strong electronic interaction
exists between the aromatic ring of £ and the base pairs
of DNA, intercalation would be favored even at low salt.
Hydrophobic-type interactions would also favor intercala
tion. One or both of these factors is presumably opera
tive for this molecule.
2) The aromatic ring of L-lys-D-DNP-ornA (10) also
fully intercalates. This result can be explained by two
mechanisms: 1) intercalation is favored for the same
reasons as noted above (i.e., electronic or hydrophobic);
or 2) electrostatic binding and intercalation as a result
of the flexibility of the "arm" between the a carbon and
aromatic moiety. In other words, the arm is long enough,
after electrostatic binding, to allow the aromatic ring,
which is seeking a nonpolar environment, to "swing" around
and intercalate. This is not unreasonable in light of the
results from L-lys-D,L-DNP-DABA (£) The extent of in
duced CD and increase in viscosity for this peptide is not
as great as for L-lys-L-DNP-DABA (£) However, L-lys-D-
DNP-ornA (10) behaves exactly like L-lys-L-DNP-ornA (9).
With regard to mechanism 2, it would be tempting to assume,


66
in the case of the L-lys-D,L-DNP-DABA (6) peptide, that
electrostatic binding is taking place, and the aromatic
ring of the L-lys-L-DNP-DABA diastereomer is fully inter
calating (as in peptide _5) while that of the L-lys-D-
DNP-DABA is only partially intercalating since the arm
is not long enough (as opposed to the case of L-lys-D-
DNP-ornA, 10_) to allow full intercalation; thus, the
"mixed" results.
Model building studies can be used to support
mechanism 2. For example, for reporter molecule I with
n = 1, the distance between the quaternary ammonium
nitrogen and the center of the aromatic ring is on the
O O
order of 4 A, while this distance is on the order of 5 A
for reporter molecule I with n = 2. Furthermore, the
distance between the center of the aromatic ring and the
assymetric carbon of the phenylalanyl residue of L-lys-L-
O
pheA is also approximately 4 A. Therefore, the critical
distance between the aromatic ring and the a carbon to
O
which it is attached must be between 4 and 5 A to allow
for full intercalation. For L-lys-L-DNP-DABA (5) and
L-lys-L-DNP-ornA (9), this distance is approximately 6
O
and 7.5 A, respectively, which is certainly long enough
to allow full intercalation of the aromatic ring for
each compound. For L-lys-D-DNP-ornA (1_0) the distance
between the aromatic ring and the a carbon to which it is
O
attached is about 4.5 A after swinging the aromatic ring
around and pointing it toward the interior of the helix.


67
This distance is apparently long enough to allow for full
intercalation. However, for L-lys-D-DNP-DABA, this dis-
O
tance is only on the order of 3 A, which is insufficient
for full intercalation. It should be noted that the models
were built assuming a certain rigidity in the peptides
after binding. More experimental evidence is needed to
support this mechanism, however.
3) Finally, the results from the studies with L-lys-
L-S-pNO^bz-cysA (7) and L-lys-D-S-pNO^bz-cysA (8^ are
very difficult to interpret. It was hoped that studies
with these two compounds would resolve any anomolies
created by L-lys-D,L-DNP-DABA (60 Instead, the entire
situation becomes more complicated. It is evident from
these studies that full intercalation is not taking place
even for L-lys-L-S-pNC^bz-cysA (7). The NMR suggests that
there is at least partial intercalation for both 1_ and 8/,
yet neither of these peptides significantly decreases the
viscosity of a DNA-peptide solution [relative to L-lys-
L-pNO^-pheA (1) ] The fact that there is no full intercala
tion for L-lys-L-S-pNO^bz-cysA (7) can be explained by two
possible effects: 1) the sulfur in the middle of the side
chain is creating a steric effect by positioning the aro
matic ring away from the interior of the helix; or 2) there
is insufficient electronic interaction between the aro
matic ring and the base pairs of DNA. This last explana
tion is unlikely in light of the fact that the nitrophenyl
ring of reporter molecule I (where n = 2 4) fully


68
intercalates. In order to resolve this dilemma, a more
in-depth study of these compounds and derivatives is re
quired. Furthermore, with regard to the first explana
tion (i.e., steric effects), model building studies were
uninformative.
In conclusion, this work has shown the partial depend
ence of chirality of the N-terminal amino acid a carbon and
the distance between the aromatic ring and this center of
chirality for intercalation. It has also been suggested,
though, that electronic and hydrophobic effects can also
predominate in terms of intercalation, and must be con
sidered in any study of the interaction of small molecules
with DNA.


CHAPTER III
EXPERIMENTAL
All starting amino acids were purchased from either
Sigma Chemical Co. or Vega Biochemicals, and were used with
out further purification. Salmon sperm DNA was purchased
from Worthington Biochemical Corp. Elemental analysis of
all products was performed by Atlantic Microlab, Inc.,
Atlanta, Georgia. TLC plates with fluorescent indicator
were purchased from either Kodak Chemical Co. or Scientific
Products, Inc. Melting points were taken on a Mel-Temp appa
ratus and the measurements are uncorrected. All solutions
containing peptides or DNA were prepared in buffers made
with deionized water.
The NMR spectra were recorded with either a Varian
A-60A or a Jeol-JNM-FX100 Fourier Transform NMR spectro
meter. Chemical shifts were determined relative to the
internal standard trimethylsilane (TMS) for spectra taken
with organic solvents or 2,2,3,3-d^-3-trimethylsilylpro-
prionate (TSP) for those determined in deuterium oxide.
Ultraviolet and visible absorption spectra were recorded
with a Cary-17D UV/Vis spectrophotometer. A Jasco J-20
spectropolarimeter was used to record circular dichroism
measurements. Viscosity measurements were performed with
a low shear Zimm viscometer (Beckman Instrument Co.).
69


70
Synthesis
All of the prepared dipeptides, and the required inter
mediates leading up to the final products, were analyzed for
purity by two or more of the following: TLC, NMR or ele
mental analysis. All reactions are known to proceed without
racemization. The various abbreviations used in this sec
tion are: 1) THF (tetrahydrofuran); 2) CBZ (carbobenzoxy);
3) t-Boc (t-butyloxycarbonyl); 4) NT (nitrotolyl); 5) DNP
(dinitropheny1); and 6) TFA (triflouroacetic acid). In some
cases, a molecule or two of water of hydration has to be in
cluded for the elemental analysis. This is reasonable since
the final compounds, and some of the precursors, are quite
hygroscopic.
Preparation of L-lysyl-L-pNO-,-phenylalanine amide dihydro
bromide, 1.
L-pNC^-phenylalanine ethyl ester hydrochloride was syn
thesized by the Curtius and Goebel esterification proce
dure: L-pNO^-phenylalanine (1.0 g, 4.7 mmol) was placed
in a 3 neck flask equipped with a magnetic stirrer, a drying
tube and a gas dispersing tube. Then, %100 mL of ethanol
was added and a stream of dry hydrogen chloride was passed
through the solution at room temperature until all the solid
dissolved. Upon dissolution, the flask was placed in an ice/
acetone bath and the bubbling continued to saturation. The
tube was removed and the stoppered flask was allowed to stand
at room temperature for 4 hours. The solution was then evap
orated by a stream of dry nitrogen. The solid obtained was


71
recrystallized from ethanol/ethyl ether to yield 1.1 g (4.0
mmol, 851 yield) of L-pN07-phenylalanine ethyl ester hydro
chloride. The NMR in D2O showed a 3H methyl triplet at
6 1.3 ppm, a 2H methylene doublet at 6 3.5 ppm, a 3H multi-
plet at 6 4.4 ppm and two 2H doublets at 6 7.6 ppm and 6.
8.3 ppm, respectively, for the aromatic protons.
Anal. calculated for C-j^H-^^O^Cl; C, 48.00 ; H, 5.46.
Found: C, 48.06; H, 5.51.
The ester was then coupled to DiCBZ-L-lysine by the
112
general method of Anderson et al. DiCBZ-L-lysine
(0.60 g, 1.4 mmol) in 20 mL freshly distilled THF was placed
in a 3 neck flask equipped with a magnetic stirrer and a
calcium sulfate drying tube. A stream of dry nitrogen was
passed over the solution to keep the atmosphere free of
moisture. The flask was then placed in an ice/acetone bath
and 1.5 mmol of triethylamine was added to the solution,
followed by 1.5 mmol of isobutylchloroformate. The solution
was allowed to stir for 15 minutes, after which time a pre
cooled solution of L-pNC^-phenylalanine ethyl ester hydro
chloride (0.40 g, 1.4 mmol) in 10 mL of 1:1 THF/DMF was
added. An additional 1.5 mmol of triethylamine was added
and the reaction was allowed to stir at -10C for 2 hours.
Upon completion of the reaction, the ice bath was removed
and the solvent evaporated with a stream of dry nitrogen.
The resulting solid was dissolved in ethyl acetate/saturated
NaCl solution, and the organic phase was washed 3 times with
1 M HC1, 3 times with saturated sodium bicarbonate, and 2


72
times with saturated sodium chloride. The organic phase
was dried over sodium sulfate, filtered and evaporated.
The resultant solid was recrystallized from ethyl acetate/
hexane to yield 0.74 g (1.2 mmol, 86% yield) of DiCBZ-L-
lysyl-L-pNC^-phenylalanine ethyl ester. The NMR in
CDCl^ showed a broad 9H multiplet at 6 1.6 ppm, a broad 4H
multiplet at 6 3.2 ppm, a 2H methylene quartet at 5 4.2 ppm,
a broad 6H multiplet at 6 5.0 ppm, a broad 1H (NH) doublet
at 6 5.5 ppm, a broad 1H (NH) doublet at 6 6.8 ppm, a 13H
multiplet at 6 7.3 ppm, and a 2H doublet at 6 8.1 ppm.
Anal. calculated for gN^OgH?0; C, 60.71; H, 6.18.
Found: C, 60.80; H, 6.19.
The diprotected dipeptide ester (0.69 g, 1.1 mmol) was
dissolved in 70 mL methanol and saturated with ammonia gas
at 0C in a high pressure bottle. The bottle was securely
corked and allowed to stand at room temperature for 48 hours.
The cork was removed and the solvent evaporated by a stream
of dry nitrogen. The solid obtained was recrystallized from
ethyl acetate/hexane to yield 0.53 g (0.89 mmol, 81% yield)
of DiCBZ-L-lysyl-L-pNC^-phenylalanine amide. The NMR in
D^-DMSO showed a broad 6H multiplet at 6 1.4 ppm, a broad 2H
multiplet at 6 3.0 ppm, a very broad 2H multiplet centered
at 6 4.4 ppm, a 4H singlet at 6 5.1 ppm, a broad 17H multi
plet at 6 7.4 ppm, and a 2H doublet at 6 8.2 ppm.
Anal. calculated for C3iN58H33 *1C, 59.97; H, 5.71.
Found: C, 59.97; H, 5.71.


73
To cleave the CBZ group, 0.60 g (1.02 mmol) of the di-
protected dipeptide amide was placed in a reaction reservoir
of the HF generator (Figure 30) with a small stir bar and
0.5 mL of anisle. Liquid HF was placed into the main reser
voir and distilled, using a water jacket at 90C, into the
reaction reservoir which had been placed in a dewar of dry
ice/acetone. After 20 mL of HF had been distilled over, the
reaction vessel was allowed to warm to 0C and stirred in an
ice water bath for an additional 1 hour. The HF was evapo
rated and the residue dissolved in 0.1 M HCl/ethyl ether.
The organic layer was discarded and the aqueous layer washed
2 more times with ethyl ether. The aqueous layer was
lypholyzed.
The residue was then dissolved in 2 mL of 0.1 M NH^OAc
and placed on a CM-Sepharose (CL-6B) cation exchange column.
The column was eluted with 0.1 M NH^OAc and fractions col
lected. Those fractions absorbing at 295 nm were pooled and
lypholyzed. The residue was placed in a drying oven (40C)
for 12 hours, lypholyzed, and dried in an oven for an addi
tional 12 hours.
The dipeptide amide dihydroacetate was dissolved in
2 mL H?0 and placed on an Amberlite CG-400 anion exchange
column (bromide form) and eluted with H?0. Fractions
absorbing at 275 nm were pooled and lypholyzed.
The residue was dissolved in MeOH, transferred to a
tared, labeled vial, evaporated and placed in a P,,0j. dessi-
cator for 48 hours. The solid was broken into a fine powder


74
Figure 30. Liquid HF Apparatus. With valve Di open and all
others closed, liquid HF is drained from the HF
tank (A) into the distillation reservoir (B).
Valve D-^ is closed and valves I>2 are opened,
and liquid HF is distilled over into reaction
reservoirs (C). The reaction is stirred by mag
netic stirrers (E) until completion. Upon com
pletion, valves I>2, D3, and D4 are opened and
the HF is evaporated.


and the vial placed in a drying pistol for 12 hours to
yield 133 mg (0.27 mmol, 26% yield) of L-lysyl-L-pNO^-
phenylalanine amide dihydrobromide. The NMR in D2O
showed a 6H multiplet at 6 1.6 ppm, a 2H methylene triplet
at 6 3.0 ppm, a 2H methylene doublet at 6 3.3 ppm, a 1H
methine triplet at 6 3.9 ppm, a 2H doublet at 6 7.5 ppm and
6 8.2 ppm, respectively, for the aromatic protons.
Anal. calculated for ^N^O^Br1.51^0; C, 34.22 ; H, 5.32.
Found: C, 34.26; H, 5.34. M.P. 189 192C.
Preparation of L-lysyl-D-pNO^-phenylalanine amide dihydro
bromide 2.
The dipeptide amide was prepared in the same manner as
compound 1, but starting with D-pNC^-phenylalanine. D-pNC^-
phenylalanine (1.0 g, 4.7 mmol) was converted to 1.1 g of
D-pNO.,-phenylalanine ethyl ester hydrochloride (4.0 mmol,
851 yield) by the action of absolute ethanol and dry hydrogen
chloride. The dipeptide ester, DiCBZ-L-lysyl-D-pNO?-phenyl-
alanine ethyl ester was prepared by reacting DiCBZ-L-lysine
(0.60 g, 1.4 mmol) with 1.5 mmol triethylamine and 1.5 mmol
of isobutylchloroformate followed by addition of D-pNO,,-
phenylalanine ethyl ester hydrochloride (0.40 g, 1.4 mmol)
and 1.5 mmol of triethylamine. Workup and recrystallization
yielded 0.70 g (1.1 mmol, 791 yield) of DiCBZ-L-lysy1-D-pN0?-
phenylalanine ethyl ester. The NMR in CDC1, showed a
broad 9H multiplet at 6 1.6 ppm, a broad 4H multiplet at 6
3.2 ppm, a 2H quartet at 6 4.2 ppm, a broad 6H multiplet at
6 5.0 ppm, a broad 1H doublet at 6 5.5 ppm, a broad 1H doublet


76
at 5 6.8 ppm, a 13H multiplet at 5 7.3 ppm, and a 2H doublet
at 5 8.1 ppm.
Anal. calculated for 5H?0; C, 61.58; H, 6.11.
Found: C, 61.56; H, 6.11.
The diprotected dipeptide ester (0.59 g, 0.93 mmol) was
converted to 0.40 g (.67 mmol, 12% yield) of DiCBZ-L-lysyl-
D-pNC^-phenylalanine amide by the action of MeOH saturated
with NHj and subsequent workup and recrystallization. The
NMR in Dg-DMSO showed a broad 6H multiplet at 6 1.4 ppm,
a broad 2H multiplet at 6 3.0 ppm, a very broad 2H multiplet
centered at 6 4.4 ppm, a 4H singlet at 6 5.1 ppm, a broad 17H
multiplet at 6 7.4 ppm, and a 2H doublet at 6 8.2 ppm.
Anal. calculated for Cg^NgOgH-^; C, 60.88; H, 5.63.
Found: C, 60.67; H, 5.68.
The diprotected dipeptide amide (0.60 g, 1.02 mmol) was
cleaved with liquid HF (20 mL) as previously described. The
lypholyzed residue obtained was passed through a CM-Sepharose
(CL-6B) column, eluting with 0.1 M NH^OAc and collecting
fractions absorbing at 275 nm as before. After lypholyza-
tion of the appropriate fractions and removal of all residual
NH^OAc, the solid was dissolved in 2 mL F^O and passed through
an Amberlite CG-400 anion exchange column (bromide form).
The fraction absorbing at 275 nm was then lypholyzed. The
resultant residue was dissolved in MeOH, transferred to a
tared vial, evaporated and placed in a P90g dessicator and
drying pistol for 48 and 24 hours, respectively. The resul
tant powder was 375 mg of L-lysyl D-pNO.,-phenylalanine amide


77
dihydrobromide (0.75 mmol, 73.5%). The NMR in D^O
showed a 6H multiplet at 6 1.6 ppm, a 4H multiplet at 6 3.2
ppm, a 1H methine triplet at 3.9 ppm, and a 2H doublet at
7.5 ppm and 6 8.2 ppm, respectively, for the aromatic pro
tons .
Anal. calculated for C-^l^ 4Ho0; C, 31.52; H, 5.78.
Found: C, 31.50; H, 5.83. M.P. 194 197C.
Preparation of L-lysyl-L-S-nitrotolyl-cysteine amide dihydro-
bromide, 5.
L-S-nitrotolyl-cysteine hydrochloride was prepared by
dissolving 4.0 g (2.55 mmol) of L-cysteine hydrochloride
in 60 mL of 1 M NaOH. The solution was cooled to 0C and
a solution of 2.0 g (12.7 mmol) of 2-flouro-5-nitrotoluene
in 40 mL of dioxane was added over a period of 2 hours.
Stirring at 0C was continued for 2 more hours after which
time the reaction was stirred at room temperature for 3-4
hours. The reaction mixture was washed 3 times with ethyl
ether, acidified with concentrated HC1 and evaporated. The
resultant solid was recrystallized from water to yield 2.84
g (9.7 mmol, 76.4% yield) of L-S-nitrotolyl-cysteine hydro
chloride. The *'H NMR in D^-DMSO showed a sharp 3H methyl
singlet at 6 2.4 ppm, a broad 3H multiplet at 6 3.8 ppm, a
1H doublet at 7.6 ppm, and a 2H multiplet at 6 8.1 ppm.
Anal, calculated for C1()H13N204SC1 -F^O; C, 38.64; H, 4.83.
Found: C, 38.70; H, 4.89.
L-S-NT-cysteine hydrochloride (1.5 g, 5.1 mmol) was
esterified with ethanol saturated with dry HC1. Subsequent


78
workup and recrystallization from ethanol/ethyl ether yielded
1.3 g (4.1 mmol, 80.41 yield) of L-S-NT-cysteine ethyl ester
hydrochloride.
The NMR in D?0 showed a 3H methyl triplet at 6 1.3
ppm, a sharp 3H methyl singlet at 6 2.5 ppm, a 2H methylene
doublet at 6 3.9 ppm, a 3H multiplet at 6 4.4 ppm, a 1H
doublet at 6 7.6 ppm, and a 2H multiplet at 6 8.1 ppm.
Anal. calculated for C-^ y^O^SCl H?0; C, 42.60; H, 5.62.
Found: C, 42.71; H, 5.70.
Di-t-Boc-L-lysine (1.3 g, 3.7 mmol) was dissolved in
dry THF and treated with 4.0 mmol of Et^N and isobutyl-
chloroformate, respectively, followed by 1.2 g of L-S-NT-
cysteine ethyl ester (3.7 mmol) and an additional 4.0 mmol
of Et^N. Subsequent workup and recrystallization from THF/
hexane yielded 1.2 g (2.0 mmol, 54.1% yield) of Di-t-Boc-
L-lysyl-L-S-NT-cysteine ethyl ester. The NMR in CDCl^
showed a broad 27H multiplet at 6 1.4 ppm, a 3H methyl
singlet at 6 2.4 ppm, a broad 2H multiplet at 6 3.1 ppm,
a broad 2H multiplet at 6 3.5 ppm, a 3H multiplet at 6 4.2
ppm, a broad 3H multiplet at 6 5.0 ppm, a 1H (NH) doublet
at 6 7.1 ppm, a 1H doublet at 6 7.4 ppm, and a 2H multiplet
at 6 8.0 ppm.
Anal. calculated for C? gH^N^OgS; C, 54.90 ; H, 7.19.
Found: C, 54.79; H, 7.27.
The diprotected dipeptide ethyl ester was converted
to the amide by ammoniolysis of 1.0 g (1.6 mmol) of Di-t-
Boc-lysyl-L-S-NT-cysteine ethyl ester in methanol.


79
Subsequent workup and recrystallization from ethyl acetate/
hexane yielded 0.37 g (0.63 mmol, 39.4% yield) of Di-t-Boc-
L-lysyl-L-S-NT-cysteine amide. The NMR in CDCl, showed
a 24H multiplet at 1.4 ppm, a 3H methyl singlet at 2.4
ppm, a 2H multiplet at 3.1 ppm, a 2H multiplet at 6 3.5
ppm, a 1H methine multiplet at 6 4.1 ppm, a 1H methine multi
plet at 6 4.8 ppm, a 2H multiplet at 6 5.8 ppm, a 4H multi
plet at 6 7.3 ppm, and a 2H multiplet at 6 8.0 ppm.
Anal. calculated for C^H^N^OgS; C, 53.52; H, 7.03.
Found: C, 52.19; H, 6.96.
In the deprotection reaction, 0.23 g (0.39 mmol) of
Di-t-Boc-L-lysyl-L-S-NT-cysteine amide was treated with 30
mL of a solution of 25% TFA in CH2C1?. The reaction was
allowed to proceed for 45 minutes at room temperature, and
then evaporated. The resultant residue was dissolved in
0.1 M HC1 and washed 3 times with ethyl ether. The aqueous
layer was lypholyzed to yield a solid which was subsequently
dissolved in 2 mL L^O and passed through an Amberlite CG-400
anion exchange column (bromide form). The fraction absorbing
at 275 nm was lypholyzed to yield a solid that was pure by
TLC. The solid was dissolved in methanol, and the methanol
solution was transferred to a tared vial and evaporated.
The vial was placed in a P20,- dessicator for 48 hours, and
the resultant solid broken into a fine powder. The solid
was identified by NMR and elemental analysis as 0.17 g (0.31
mmol, 79.5% yield) of L-lysyl-L-S-NT-cysteine amide dihydro
bromide. The NMR in D20 showed a 6H multiplet at 6 1.5


80
ppm, a 3H methyl singlet at 6 2.4 ppm, a 2H multiplet at 6
2.9 ppm, a 2H multiplet at 6 3.6 ppm, a 1H methine triplet
at 6 4.1 ppm, a 1H doublet at 7.5 ppm, and a 2H multiplet
at 6 8.1 ppm.
Anal. calculated for Ci6^2 7^5<",4^r7l"^2<1 C, 32.43 ; H, 5.07.
Found: C, 32.63; H, 5.48. M.P. 209 211C.
Preparation of L-lvsyl-D-S-nitrotolyl-cysteine amide dihydro
bromide 4.
D-S-nitrotolyl-cysteine hydrochloride was prepared
from 4.0 g (25.5 mmol) of D-cysteine hydrochloride and 2.0
g (12.7 mmol) of 2-flouro-5-nitrotoluene as previously des
cribed. The resultant solid was recrystallized from water
to yield 1.9 g (6.5 mmol, 51.21 yield) of D-S-NT-cysteine
hydrochloride. The NMR in D^-DMSO showed a sharp 3H
methyl singlet at 6 2.4 ppm, a broad 3H multiplet at 3.8
ppm, a 1H doublet at 6 7.6 ppm, and a 2H multiplet at 6 8.1
ppm.
Anal, calculated for C^qH-^^O^SCI*; C, 38.64; H, 4.83.
Found: C, 38.66; H, 4.89.
D-S-NT-cysteine hydrochloride (1.7 g, 5.8 mmol) was
esterified with ethanol/HCl to yield, upon recrystallization
from ethanol/ethyl ether, 1.4 g (4.4 mmol, 75.9% yield) of
D-S-NT-cysteine ethyl ester hydrochloride. The NMR in
D2O showed a 3H methyl triplet at 6 1.3 ppm, a 3H methyl
singlet at 6 2.5 ppm, a 2H methylene doublet at 6 3.9 ppm,
a 3H multiplet at 6 4.4 ppm, a 1H doublet at 6 7.6 ppm, and
a 2H multiplet at 6 8.1 ppm.


81
Anal. calculated for C^?H^ ^^O^SCl H^O; C, 42.60; H, 5.62.
Found: C, 42.53; H, 5.68.
D-S-NT-cysteine ethyl ester hydrochloride (1.2 g, 3.7
mmol) was coupled to Di-t-Boc-L-lysine (1.3 g, 3.8 mmol)
using 3.9 mmol of Et,N and isobutylchloroformate, respec
tively, in the mixed anhydride reaction. Subsequent workup
and recrystallization from ethyl acetate/hexane yielded 1.5
g (2.5 mmol, 67.61 yield) of Di-t-Boc-L-lysy1-D-S-NT-
cysteine ethyl ester. The NMR in CDCl^ showed a broad
27H multiplet at 6 1.4 ppm, a 3H methyl singlet at 6 2.4
ppm, a broad 2H multiplet at 6 3.1 ppm, a broad 2H multiplet
at 6 3.5 ppm, a 3H multiplet at 6 4.2 ppm, a broad 3H
multiplet at 6 5.0 ppm, a 1H (NH) doublet at 6 7.1 ppm, a
1H doublet at 6 7.6 ppm, and a 2H multiplet at 5 8.0
ppm.
Anal. calculated for C^gH^^N^OgS; C, 54.90; H, 7.19.
Found: C, 54.65; H, 7.27.
Ammoniolysis of 1.3 g (2.1 mmol) of Di-t-Boc-L-lysyl-
D-S-NT-cysteine ethyl ester, and subsequent recrystalliza
tion, yielded 300 mg (0.51 mmol, 24.3% yield) of Di-t-Boc-
L-lysyl-D-S-NT-cysteine amide. The NMR in CDCl^ showed
a 24H multiplet at 6 1.4 ppm, a 3H methyl singlet at 6 2.4
ppm, a 2H multiplet at 6 3.1 ppm, a 2H multiplet at 6 3.5
ppm, a 1H methine multiplet at 6 4.1 ppm, a 1H methine
multiplet at 4.8 ppm, a 2H multiplet at 6 5.8 ppm, a 4H
multiplet at 6 7.3 ppm, and a 2H multiplet at 6 8.0 ppm.
The TLC on this compound indicated that it was only on the


82
order of 95% pure; therefore, no elemental analysis was
obtained, and the compound was cleaved without further
purification.
For the cleavage reaction, 200 mg (0.34 mmol) of Di-
t-Boc-L-lysyl-D-S-NT-cysteine amide was treated with TFA/
CF^C^ as before. After the reaction was completed, the
solvent was evaporated and the resultant residue was dis
solved in 0.1 M HC1, washed 3 times with ethyl ether, and
the aqueous layer lypholyzed. The solid obtained was dis
solved in 1 mL of H-,0 and passed through an Amberlite
CG-400 anion exchange column (bromide form). The fraction
absorbing at 275 nm was lypholyzed to yield a solid that
was pure by TLC, and was identified by NMR as 75 mg (0.13
mmol, 38.2% yield) of L-lysyl-D-S-NT-cysteine amide di
hydrobromide. The NMR in D2O showed a 6H multiplet at
6 1.6 ppm, a sharp 3H methyl singlet at 6 2.4 ppm, a 2H
multiplet at 6 2.9 ppm, a 2H multiplet at 6 3.6 ppm, a 1H
methine triplet at 6 4.1 ppm, a 1H doublet at 6 7.5 ppm,
and a 2H multiplet at 6 8.1 ppm.
Anal, calculated for C-^F^yNj-O^SB^ 5H?0; C, 33.45 ; H, 4.88.
Found: C, 33.46; H, 5.36. M.P. 214 217C.
Preparation of L-lysyl-L-DNP-diaminobutyric acid amide
dihydrobromide, TT
L-N,y-DNP-diaminobutyric acid was synthesized according
113
to the general method of Sanger. L-diaminobutyric acid
dihydrochloride (2.0 g, 10.5 mmol) was dissolved in 75 mL
hot water and excess CuC0~ (5.0 g, 40.7 mmol) was added.


83
The solution was filtered and the volume reduced to 30 mL;
NaHCO- (5.7 g, 68.7 mmol) was added followed by dinitro-
flourobenzene (6.0 g, 32.2 mmol) in 20 mL ethanol. The
solution was stirred for 2 hours at room temperature, during
which time a green precipitate formed, which was filtered
and dried. The solid was subsequently dissolved in 100 mL
of 1 M HC1, followed by addition of thioacetamide (7.8 g,
10.4 mmol) dissolved in water. The solution was heated
for 1/2 hour on a steam bath to precipitate CuS. Activated
charcoal was added and the solution filtered. The filtrate
was evaporated and the solid obtained recrystallized from
1 M HC1 to yield 1.4 g (4.4 mmol, 421 yield) of L-N,y-DNP-
diaminobutyric acid hydrochloride. The NMR in D^-DMSO
showed a broad 2H multiplet at 6 2.3 ppm, a broad 3H multi-
plet at 6 3.9 ppm, a 1H doublet at 6 7.5 ppm, and a broad 4H
multiplet at 6 9.0 ppm.
Anal. calculated for C-^gH-^N^OgCl; C, 37.44 ; H, 4.08;
N, 17.47. Found: C, 37.40; H, 4.12; N, 17.45.
L-N,y-DNP-diaminobutyric acid (1.0 g, 3.1 mmol) was
converted to the ester via the normal procedure utilizing
absolute ethanol saturated with dry hydrogen chloride.
After evaporating the solvent, the resultant solid was re
crystallized from ethanol/ethyl ether to yield 0.82 g
(2.4 mmol, 77.41 yield) of L-N,y-DNP-diaminobutyric acid
ethyl ester hydrochloride. The NMR in D2O showed a
3H methyl triplet at 6 1.3 ppm, a 2H methylene quartet at
6 2.4 ppm, a 2H methylene triplet at 6 3.8 ppm, a 3H


84
multiplet at 6 4.4 ppm, a 1H doublet at 6 7.3 ppm, a 1H
doublet of doublets at 6 8.4 ppm, and a broad 1H doublet
at 6 9.0 ppm.
Since the addition of the DNP group could go to either
the a amine nitrogen or the y amine nitrogen, it was neces
sary to prove that only the y amine group reacted. The
ester hydrochloride was treated with aqueous NaHCCU and
extracted into CDC1-. The NMR revealed a 3H methyl
triplet at 6 1.3 ppm, a 4H multiplet at 6 2.1 ppm, a 3H
multiplet at 6 3.7 ppm, a 2H methylene quartet at 6 4.2
ppm, a 1H doublet at 6 7.0 ppm, a 1H doublet of doublets
at 6 8.3 ppm, and a broad 2H doublet at 5 9.0 ppm.
In the protonated ester, the a proton was buried under
the 3H multiplet at 6 4.4 ppm, with the other two protons
coming from the ester ethyl methylene. The y methylene was
a 2H triplet at 6 3.8 ppm in the protonated ester. Upon
neutralization of the positive charge, only one proton,
namely the a proton, shifted (from 4.4 ppm to 3.7 ppm),
thus indicating that the addition of the DNP group did not
occur at the a amine group. Furthermore, the y methylene
did not shift significantly, indicating that addition did
occur on the y amine group.
Anal. calculated for C-pH-^N^OgCl; C, 41.32; H, 4.88.
Found: C, 39.46; H, 4.83.
The TLC and elemental analysis indicated that this
compound was only on the order of 95% pure; however, it was
used without further purification. L-N,y-DNP-diaminobutyric


85
acid ethyl ester (0.82 g, 2.4 mmol) was converted to the
amide through the use of methanol saturated with ammonia.
Workup and recrystallization gave 0.52 g (1.6 mmol, 67%
yield) of L-N,y-DNP-diaminobutyric acid amide hydrochloride.
The NMR in D^-DMSO/D^O showed a broad 2H multiplet at 6
2.1 ppm, a broad 3H multiplet at 6 3.8 ppm, a broad 1H
doublet at 6 7.3 ppm, a 1H doublet of doublets at 6 8.3
ppm, and a 1H doublet at 6 9.1 ppm.
Anal. calculated for C^qH-^^N^O^CI E^O; C, 35.55; H, 4.74.
Found: C, 35.47; H, 4.63.
The L-N,y-DNP-diaminobutyric acid amide hydrochloride
was then coupled to DiCBZ-L-lysine by the normal procedure.
DiCBZ-L-lysine (0.32 g, 0.77 mmol) was dissolved in THF
and 0.78 mmol of Et~N and 0.78 mmol of isobutylchloroformate
added. A precooled solution of L-N,y-DNP-diaminobutyric
acid amide hydrochloride (0.25 g, 0.77 mmol) in DMF/THF was
added followed by an additional 0.78 mmol of Et^N. Subse
quent workup and recrystallization afforded 0.25 g (0.37
mmol, 48% yield) of DiCBZ-L-lysyl-L-DNP-diaminobutyric acid
amide. The NMR in D^-DMSO showed a broad 8H multiplet
at 6 1.4 ppm, a broad 4H multiplet at 6 3.0 ppm, a very
broad 2H multiplet at 6 4.1 ppm, a 4H singlet at 6 5.1 ppm,
a broad 15H multiplet at 7.4 ppm, a very broad 2H multi
plet at 6 8.2 ppm, and a broad 2H doublet at 9.1 ppm.
Anal, calculated for C,9H,yNyO^Q; C > 56.55; H, 5.49.
Found: C, 56.43; H, 5.53.


86
The diprotected dipeptide amide (200 mg, 29 mmol) was
stirred in 50 mL acetic acid that had been saturated with
dry hydrogen bromide (351 HBr by weight) for 2 hours. The
solution was poured into 150 mL of dry ethyl ether and
placed in the refrigerator overnight. The precipitate was
filtered and recrystallized from MeOH/ethyl ether to yield
100 mg (0.17 mmol, 58.91 yield) of L-lysyl-L-DNP-diamino-
butyric acid amide dihydrobromide. The NMR in D2O
showed a broad 8H multiplet at 6 1.6 ppm, a 2H methylene
triplet at 6 3.0 ppm, a 2H methylene triplet at 3.6 ppm,
a 1H methine triplet at 4.0 ppm, a 1H doublet at 6 7.1
ppm, a 1H doublet of doublets at 6 8.3 ppm, and a 1H doublet
at 6 9.1 ppm.
Anal. calculated for ^NyO^B^3H90; C, 30.62; H, 5.31.
Found: C, 30.62; H, 5.33. M.P. 170 173C.
Preparation of L-lysyl-D,L-DNP-diaminobutyric acid amide
dihydroacetate, 61.
The DNP derivative of D,L-diaminobutyric acid was pre
pared as described previously. D,L-diaminobutyric acid di
hydrochloride (5.0 g, 26.2 mmol) was dissolved in hot water
and treated with excess CuCO^ and NaHCO^- Then, 2,4-dinitro-
flourobenzene (15.0 g, 80.6 mmol) in ethanol was added.
The greenish solid obtained was dissolved in HC1 and thio-
acetamide (2.0 g, 26.6 mmol) was added. After filtering
and evaporation, the yellow solid obtained was recrystal
lized from 1 M HC1 to yield 5.32 g (16.6 mmol, 63.4% yield)
of D,L-N,y-DNP-diaminobutyric acid. The NMR in D^-DMSO


87
showed a broad 2H multiplet at 6 2.3 ppm, a broad 3H multi-
plet at 6 3.9 ppm, a 1H doublet at 5 7.5 ppm, and a broad
4H multiplet at 6 9.0 ppm.
Anal. calculated for C-^qH^ ^N^O^Cl; C, 37.44; H, 4.08.
Found: C, 37.40; H, 4.12.
The D,L-N,y-DNP-diaminobutyric acid hydrochloride
(4.5 g, 14.0 mmol) was treated with absolute ethanol satu
rated with dry hydrogen chloride. Workup and recrystalliza
tion from ethanol/ethyl ether yielded 4.8 g (13.2 mmol,
94% yield) of D,L-N,y-DNP-diaminobutyric acid ethyl ester
hydrochloride. The NMR in D?0 showed a 3H triplet at
6 1.3 ppm, a 2H quartet at 6 2.4 ppm, a 2H triplet at 6
3.8 ppm, a 3H multiplet at 6 4.4 ppm, a 1H doublet at 6
7.3 ppm, a 1H doublet of doublets at 6 8.4 ppm, and a broad
1H doublet at 6 9.0 ppm.
Anal. calculated for Cig^yN^O^Cl; 41.38; H, 4.92.
Found: C, 41.39; H, 4.94.
The D,L-N,y-DNP-diaminobutyric acid ethyl ester hydro
chloride (0.30 g, 0.86 mmol) was converted to 0.21 g (0.66
mmol, 771 yield) of D,L-N,y-DNP-diaminobutyric acid amide
hydrochloride by the action of methanol saturated with
ammonia and subsequent workup and recrystallization from
methanol/ethyl ether. The "'H NMR in Dg-DMS0/D?0 showed a
broad 2H multiplet at 6 2.1 ppm, a broad 3H multiplet at 6
3.8 ppm, a broad 1H doublet at 6 7.3 ppm, a 1H doublet of
doublets at 6 8.3 ppm, and a 1H doublet at 6 9.1 ppm.


Anal. calculated for C-^qH^^N^O^CI 2H?0; C, 33.73; H, 5.10.
Found: C, 33.55; H, 4.70.
For the coupling reaction, DiCBZ-L-lysine (0.39 g,
0.94 mmol) was dissolved in THF and 1.0 mmol of Et^N and
1.0 mmol of isobutylchloroformate added. D,L-N,y-DNP-
diaminobutyric acid amide hydrochloride (0.30 g, 0.94
mmol) in DMF/THF was added followed by an additional 1.0
mmol of Et^N. Subsequent workup and recrystallization
yielded 0.36 g (0.53 mmol, 561 yield) of DiCBZ-D,L-DNP-
diaminobutyric acid amide. The NMR in D^-DMSO showed
a broad 8H multiplet at 6 1.4 ppm, a broad 4H multiplet at
6 3.0 ppm, a very broad 2H multiplet at 6 4.1 ppm, a 4H
singlet at 6 5.1 ppm, a broad 15H multiplet at 6 7.4 ppm,
a very broad 2H multiplet at 6 8.2 ppm, and a broad 2H
doublet at 6 9.1 ppm.
Anal. calculated for ; C, 56.55; H, 5.45.
Found: C, 57.29; H, 5.74.
The diprotected dipeptide amide was deprotected by
the action of 20 mL of liquid HF on DiCBZ-L-lysyl-D,L-DNP-
diaminobutyric acid amide (0.90 g, 1.3 mmol). After
lypholyzation, the residue was dissolved in 2 mL of 0.2 M
NH^OAc and placed on a CM-Sepharose (CL-6B) cation exchang
column and eluted with 0.2 M NH^OAc. The fractions ab
sorbing at 480 nm were pooled and lypholyzed. After re
peated drying and lypholyzation, 150 mg of L-lysyl-D,L-DNP
diaminobutyric acid amide dihydroacetate (0.28 mmol, 21.51
yield) was obtained. The NMR in Do0 showed a broad 8H


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V\QWKH6M6MQ HUDF22VKHD



SYNTHESIS AND INTERACTION OF
SOME DIPEPTIDE AMIDES
WITH DEOXYRIBONUCLEIC ACID
BY
RICHARD DEAN SHEARDY
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1979

TO MOM AND DAD.
AND ARTIE.

ACKNOWLEDGEMENTS
The author extends his appreciation to Professor
Edmond J. "Eddie" Gabbay for his guidance, continuous
support and friendship during the past five years, until
the time of his death. He was a good friend and excellent
scientist -- and he will be missed very much.
Thanks are also extended to all those who have
helped in the author's graduate studies, especially
Mr. M. Adkins, Mr. B. Arvan, Mr. T. Baugh, Mr. C. Cromwell,
Mr. B. Griggs, Mr. D. King, Dr. R. King, Dr. D. McRitchie,
Dr. S. Pearce and Mr. T. Rigl. Special thanks also go to
Dr. M. Battiste, whose friendship and support during our
time of sadness this past summer is greatly appreciated.
Thanks also go to Dr. W. D. Wilson for being an outside
reader. Very special thanks to Ms. Joanne IJpham, whose
kindness, patience, and love are immeasurable, for her
excellent typing.
The completion of this course of study would not have
been possible without the support and encouragement of the
author's family.
i i i

For Eddie
There was a man-
I knew him well.
He lived his life
With dreams to sell.
I know that some
Can't understand.
But dreams can oft
Turn into sand.
And now my eyes
Fill with sorrow,
For he will see
No tomorrow.
He was a friend-
I know that well;
A helpful hand-
A joke to tell.
He had to go-
As all things must;
To fly away
And turn to dust.
And though my tears
May dry so fast,
My mind will not
Forget the past.
What is a life-
A game to play
Until it's time
To fade away?
Or is it more-
A heart to give
To those we love
That they may live?
And though there's fear
Throughout the night,
The morning sun
Is shining bright.
Richard Dean Sheardy
October 17,1979
IV

TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS iii
LIST OF TABLES vi
LIST OF FIGURES vii
ABSTRACT x
CHAPTER I - INTRODUCTION 1
Structure of DNA 2
Physical Properties of DNA 9
Evidence for Hydrogen Bonding 13
Stacking Forces 17
Electrostatic Effects 20
Electronic Effects 20
Dynamic Structure of Nucleic Acids 23
Chromatin 24
Small Molecule-Nucleic Acid Interactions 25
Statement of Problem 30
CHAPTER II - RESULTS AND DISCUSSION 31
1H NMR Studies 31
Viscosity Studies 40
Ultraviolet Absorption Studies 46
Binding Studies 46
Circular Dichroism Studies 52
Equilibrium Dialysis 52
Melting Temperature Studies 56
Discussion 58
CHAPTER III - EXPERIMENTAL 69
Synthesis 70
Analytical Methods 101
REFERENCES 109
BIOGRAPHICAL SKETCH 116
v

LIST OF TABLES
Table
Page
1.
The Effect of Increasing Length of the
Polynucleotide (Ap) A on the UV Absorp¬
tion Spectrum. n
20
2.
Absorption and Hypochromicity Data on
the Dipeptide Amides.
47
3.
Typical Output for McGhee-von Hippel
Binding Isotherms for Various Values
of n.
49
4.
Binding Isotherms for DNP and Nitrotolyl
Containing Dipeptides.
51
5.
Apparent Binding Affinity (Ka) of the
Dipeptides to Salmon Sperm DNA.
55
6.
The Effect of the Dipeptides on the ATm
on the Helix-Coil Transition of Salmon
Sperm DNA, Poly I - Poly C, and Poly
d(A-T).
57
vi

LIST OF FIGURES
Figure Page
1. Schematic Representation of the Watson-
Crick Double Helix of DNA. 3
2. Watson-Crick Base Pairs. 4
3. Keto-Enol Tautomers for the Bases Guanine
and Cytosine. 4
4. Structure of a Section of a DNA Chain. 6
5. Absorption-Temperature Profile for DNA. 10
6. Intrinsic Viscosity-pH Profile for DNA. 10
7. Acid-Base Titration Curve for DNA. 11
8. a) Hoogsteen Adenine-Thymine Base Pair.
b) Anit-Hoogsteen Adenine-Thymine Base Pair. 14
9. Shielding of the Aromatic Protons Caused
by Vertical Stacking. 18
10. Geometry of Stacked Nucleosides. 19
11. Exciton Splitting of Energy Levels. 21
12. Intensity Interchange Between Two Inter¬
acting Transition Moments. 22
13. Reporter Molecule I. 28
14. Schematic illustration of a segment of DNA
double helix which can either partially
intercalate a molecule or fully intercalate
a molecule. 28
15. Schematic illustration of a DNA segment
showing a possible mechanism whereby the c
and a amino groups of the N-terminal L-lysyl
residue are stereospecifically anchored and
thus dictating the positioning of the aro¬
matic ring of the diastereomeric dipeptides
in the DNA-peptide complex. 29
Vll

Page
Figure
16. The Dipeptide Amides Synthesized and
Studied.
17. The Partial NMR Spectra of DNA at
Various Temperatures.
18. NMR Signal of the Aromatic Protons of
Dipeptides _1 and 2_ in the Presence and
Absence of DNA at Various Temperatures.
19. NMR Signal of the Aromatic Protons of
Dipeptides _3 and _4 in the Presence and
Absence of DNA at Various Temperatures.
20. NMR Signal of the Aromatic Protons of
Dipeptides .5 and 6_ in the Presence and
Absence of DNA at Various Temperatures.
21. NMR Signal of the Aromatic Protons of
Dipeptides 7_ and 8^ in the Presence and
Absence of DNA at Various Temperatures.
22. NMR Signal of the Aromatic Protons of
Dipeptides 9^ and 10. in the Presence and
Absence of DNA at Various Temperatures.
23. The Effect of Dipeptides 1_ and 2_ on the
Relative Specific Viscosity of Salmon
Sperm DNA.
24. The Effect of Dipeptides 3., 4., 7. and 8^
on the Relative Specific Viscosity of
Salmon Sperm DNA.
25. The Effect of Dipeptides 5. and 6 on the
Relative Specific Viscosity of Salmon
Sperm DNA.
26. The Effect of Dipeptides 9^ and _10_ on the
Relative Specific Viscosity of Salmon
Sperm DNA.
27. A Plot of v/L Versus v From Data in
Table 3.
28. The CD Spectra of Dipeptides _5, 6., 9^
and ^0 in the Absence and Presence of DNA.
The CD Spectra of Dipeptides 3^ and 4- in
the Absence and Presence of DNA.
viii
32
33
34
35
36
37
38
42
43
44
45
50
53
29.
54

Figure
Page
30 .
Liquid HF Apparatus.
74
31.
Diagram of the Viscometer with the Photo¬
electric Device in Place.
103
IX

Abstract of Dissertation Presented to the
Graduate Council of the University of Florida
in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy
SYNTHESIS AND INTERACTION OF
SOME DIPEPTIDE AMIDES
WITH DEOXYRIBONUCLEIC ACID
By
Richard Dean Sheardy
December 1979
Chairman: Edmond J. Gabbay (deceased), Merle A. Battiste
Major Department: Chemistry
This thesis discusses the synthesis and interaction
specificities of ten dipeptide amides with salmon sperm
DNA. For each dipeptide, the N-terminal amino acid was
an L-lysyl residue, while the C-terminal amino acid had
an a carbon of defined chirality (either L or D) and an
aromatic moiety a certain distance from the a carbon. The
rationale for this series was to determine if intercalation
or partial intercalation is a function of both the chirality
of the C-terminal amino acid a carbon as well as the dis¬
tance of the aromatic moiety from the a carbon. The ex¬
tent of intercalation was determined by a variety of tech¬
niques: 1) ^H NMR studies; 2) viscometric titration; and
3) UV absorption and CD studies. It was found that partial
x

intercalation occurs with the peptide containing a C-ter-
minal L-pNO^-phenylalanine residue with no intercalation
for its diastereomeric dipeptide amide, indicating a
dependence on the chirality of the C-terminal amino acid.
Six of the remaining dipeptides fully intercalated, sug¬
gesting the necessity to evaluate electronic and hydro-
phobic effects on intercalation. The two diastereomeric
dipeptide amides containing an S-p-nitrobenzylcysteinyl
moiety gave conflicting results. Possible explanations
for the above phenomenon are provided.
xi

CHAPTER I
INTRODUCTION
Undoubtedly, one of the greatest advances of biological
science in this century was the determination of deoxyribo-
1 2
nucleic acid fiber structure by Watson and Crick in 1953. ’
Since that time, volumes of work have been published at the
expense of many man hours to either confirm or disprove
certain theoretical aspects of the Watson-Crick structure.
Certainly, knowledge of DNA structure may lead to the
treatment of inherited diseases, or those diseases caused
by viruses and bacteria, and ultimately to find cures for
cancer. This knowledge is not easy to obtain due to the
immense complexity of the genetic code.
It has been suggested that a single chromosome may be
just one DNA molecule having a molecular weight of up to
9
4 x 10 daltons and an overall length of over 2 centi¬
meters.^ Unfortunately, it is difficult to isolate a com¬
plete DNA molecule since its structure is: 1) sensitive to
changes in temperature^’^ and pH;^ and 2) subject to breakage
7
under minimal shearing forces.
Commercially available DNA is usually derived from calf
thymus or salmon testes. Also, numerous synthetic DNA's are
available with definite sequence and molecular weight. The
concentration of a nucleic acid solution is determined from
1

2
the absorption of the bases at 260 nm using, for example, a
molar extinction coefficient of e = 6,500 for salmon sperm
P
O
DNA, and has the dimensions of moles of phosphate per liter.
Structure of DNA
Watson and Crick proposed a right-handed double helix
(Figure 1), comprised of two anti-parallel sugar-phosphate
backbones held together by specific hydrogen bonds between
the complementary bases and by hydrophobic forces that
1 2
favor stacking of the bases. ’ Their model was based on
X-ray diffraction data on DNA fibers as well as the chemical
g
evidence of Chargaff and Lipschitz. Due to the low resolu¬
tion of X-ray analysis of fibers, much of the evidence came
from the chemical approach.
Chargaff and Lipschitz used a variety of naturally
occurring DNA to determine the relative number of purines
9
and pyrimidines. They found that: 1) the amount of
guanine (G) is equal to the amount of cytosine (C); 2) the
amount of adenine (A) is equal to the amount of thymine (T);
and 3) the A-T/G-C ratio is constant for a particular species.
The base pairing scheme of Watson and Crick, seen in Figure
2, accounts for these observations.
The free bases can exist in one of two tautomeric
forms as shown in Figure 3 for guanine and cytosine.
However, in the Watson-Crick scheme, the bases are in their
keto forms which allow for two hydrogen bonds between the
A-T base pair and three hydrogen bonds between the G-C base

3
ma j or
groove
minor
groove
Figure 1. Schematic Representation of the Watson-Crick
Double Helix of DNA. The outer helical strands
represent the sugar-phosphate backbone, while
the horizontal lines represent the base pairs
and the vertical line is the helical axis.

4
major groove
major groove
minor groove
A-T
Figure 2. Watson-Crick Base Pairs.
Figure 3. Keto-Enol Tautomers for the Bases Guanine and
Cytosine.

5
pair. Watson and Crick used molecular models to fit the
base pairing scheme to the dimensions of the molecule as
obtained from X-ray analysis. They found that an A-G base
pair was too large to fit these dimensions, whereas a T-C
pairing was too small. In their scheme (i.e., A-T and G-C
base pairing], it was found that the distance between the
sugar phosphate backbone for each set of base pairs was
identical, lending further support to their base pairing
scheme.
The individual strands of DNA are enzymatically joined
monomer nucleoside phosphates resulting in an alternating
sugar-phosphate-sugar backbone in which the bases are
stacked on top of one another (Figure 4). The D-deoxyribose
sugar, in the furanoside form, is numbered as shown in
Figure 4 and has two hydroxyl groups at the 3' and 5' posi¬
tions, respectively, and the base at the 1' position. The
two complementary strands are placed anti-parallei in order
to attain maximum symmetry. In other words, one strand has
its sugar-phosphate backbone directed 3' -> 5 * , while the
other backbone is directed 5' -*•3'. This aspect of the
double helix was proven correct by Josse et al. in 1961 using
. . ,, , . 10
a nearest neighbor analysis.
The linkage between two successive sugars is formally
a phosphate diester. At neutral pH, the phosphate is mono¬
anionic and thus, the oxygens are directed away from the
helix into solution. Futhermore, the two oxygens are not
equivalent since one lies parallel to the helical axis

6
Figure 4. Structure of a Section of a DNA Chain.

(axial) whereas the other oxygen lies perpendicular to the
helical axis (equatorial).
7
Langridge and coworkers used X-ray diffraction data to
determine that the double helix makes one complete turn
° 11
every 34 A; this is known as the pitch. With ten base
O
pairs per pitch, there is a translation of 3.4 A between
successive base pairs. Since each turn of the double helix
requires 360° of rotation, the angle between successive
base pairs must be 36°.
Inspection of Figures 1 and 2 reveals that two grooves,
one large (major) and one small (minor), are formed as a
consequence of the twist of the double helix. As a result
of the N-9 purine glycosidic bond and the N-l pyrimidine
glycosidic bond, the sugar phosphate backbones on each base
lie on the same side of each base pair, thus giving rise to
two distances between the backbone. As seen in Figure 2,
the third hydrogen bond of the G-C base pairs is located in
the minor groove, while the methyl group of thymine is lo¬
cated in the major groove. These two structural features
affect the binding of small molecules to DNA.
X-ray work with DNA requires the use of DNA in its
fiber state, which does not allow enough definable data
points to give a definite arrangement of the atoms. Usually,
molecular models are built and fitted to the data, allowing
for elimination of those models which are inconsistent with
the data or stereochemically unfeasible. This process even¬
tually leads to a model that is consistent with the data.

8
Conclusions from this type of analysis must be taken
with caution. For example, X-ray analysis of DNA fibers
drawn from different media (i.e., Li , K , Na or Mg salts)
have yielded slightly different structures for the same DNA
molecule.^ Also, at relative humidity above 801, X-ray
data suggest that the DNA is in the B form, which has a
O
pitch of 34 A with 10 residues per turn with the base pairs
perpendicular to the helical axis. However, the X-ray
studies of this fiber cannot distinguish between a left- or
12
right-handed helix. Below 80% relative humidity, the DNA
structure changes to the A form, in which the base pairs are
tilted 15-20° from the perpendicular and the helical pitch
O
is lowered to 28 A with 11 bases per turn. X-ray analysis
of the A form shows it to be a right-handed helix, and from
this, it can be assumed that the B form is also right-
handed. It has been suggested that there are at least eight
different structural forms of natural DNA (i.e., A, B, C,
13
P, P^, J, J? and S), all varying in the angle of the tilt
of the base pairs from the perpendicular and the pitch of
the helix. Furthermore, it has been shown that the A-T/G-C
ratio will cause a change in DNA structure, suggesting that
the secondary structure of DNA is a function of the primary
. . 13
structure.
It is apparent that the structure of DNA in the fiber
state is sensitive to many variables and thus the conclu¬
sions of X-ray analysis can be questioned. In fact,
Donahue^^^ and Arnott^ have suggested that X-ray

9
techniques not be used. Furthermore, extrapolation from
the fiber structure to that of solution can only be done if
one assumes that there is no structural change of the DNA
molecule upon solvation. In fact, Bram used the low angle
X-ray scattering technique on DNA in solution to support
the contention that the fiber and solution structures of
DNA are different.^^^
Physical Properties of DNA
It was mentioned earlier that DNA is a delicate mole¬
cule whose structure is subject to breakage due to shearing
forces and is sensitive to changes in temperature and pH.
For example, if the absorption at 260 nm is monitored as the
temperature of a DNA solution is gradually increased, a
sigmoidal curve, as shown in Figure 5, will be observed.^’^
Concomitantly, the molar ellipticity and viscosity
versus temperature profile of the DNA solution also shows
a dramatic change. These observations can be explained in
terms of a molecular transformation from a double helix to
a random coil. This helix-coil transition is known as de-
naturation (and/or "melting out") and is usually irrever¬
sible, indicating that permanent alteration of the native
DNA structure has occurred upon melting.
The structure of DNA is affected by hydrogen-ion concen¬
tration as depicted in the viscosity-pH profile of a DNA
solution in Figure 6.^ In the pH range of 4.5 to 11.5, the
viscosity of the solution remains relatively constant,

10
Figure 5. Absorption-Temperature Profile for DNA.
Figure 6.
Intrinsic Viscosity-pH Profile for DNA.

indicating no major structural changes. However, at pH
below 4.5 or above 11.5, the viscosity decreases rapidly,
indicative of a "melting out" of the DNA structure. There
fore, DNA solutions are always prepared in a buffer whose
pH lies between 6 and 8.
19
A titration curve for DNA is shown in Figure 7.
Curve A represents the titration starting at pH 6.9 and
titrating with either acid or alkali. Curve B is the back
titration starting from either pH 2.5 or 12.5 to neutral.
It is apparent that: 1) DNA has both acidic and basic ti-
tratable groups; and 2) the titration curves are not rever
sible. The back titrations indicate that weakly acidic
and basic groups not available in the first titration are
available in the second (denatured DNA).
PH
Figure 7. Acid-Base Titration Curve for DNA.

12
The DNA structure (i.e., native or denatured) is also
20
dependent upon salt concentration. Thomson et al. have
shown that DNA in solution is irreversibly denatured when
_ 3
the sodium chloride concentration falls below 10 M or when
the magnesium chloride concentration falls below 10 ^ M.
Due to the irreversibility of the denaturation, care must
be taken in preparing DNA solutions to keep the ionic
strength at a minimum of 1 mM.
The hydrogen bonding capacity and the dielectric con¬
stant of the solution medium are also important for main¬
taining the DNA structure. Lower alcohols (i.e., methanol
and ethanol) cause reversible denaturation of DNA, while
organic solvents such as formamide, DMF or DMSO cause
21
irreversible denaturation. Since DNA is not soluble in
the above solvents, these experiments are performed by
adding the organic solvent to an aqueous DNA solution.
It is obvious from the above studies that the stability
of the DNA double helix is subject to many types of forces.
An understanding of how these forces influence the structure
of DNA in solution is prerequisite to gain insight into the
mechanisms by which other molecules interact with DNA.
Therefore, analysis of these systems must be at the molecular
level. Since DNA is a very large molecule, classical chem¬
ical approaches, which are successful for small molecules,
are not always feasible when studying macromolecules. The
usual practice is to construct model systems using monomer
or oligomer units and to extrapolate the data to the larger

13
molecules. Some of the more revealing studies in this re¬
gard are now examined with the intention of specifying the
forces that control the structure of nucleic acid in solu¬
tion.
Evidence for Hydrogen Bonding
Watson and Crick proposed that the hydrogen bonding
scheme depicted in Figure 2 is mandatory to account for
the base pairing interactions, as well as to help maintain
1 2
the double helical structure. ’ It has been demonstrated
that, based on the geometrical restraints of colinear
hydrogen bonds (i.e., A-H • • • B) and H-bonding distances
O
of 2.80 - 3.00 A, 29 different base pairs connected by two
or three hydrogen bonds could be formed between the four
2 2 2 3
nucleosides found in nucleic acids. ’ However, only
three distinct schemes have been found in the crystalline
state. Calculations based on dipole - dipole interactions by
Nash and Bradley have shown that only the Watson-Crick scheme
is favored for guanine-cytosine base pairing, but adenosine-
uracil pairs can exist in three forms with two of these forms
of approximately equal energy.^ In fact, for adenine and
thymine derivatives, two different hydrogen bonding schemes
have been found, neither of which is the Watson-Crick type.
Hoogsteen postulated the hydrogen bonding scheme in Figure 8a
2 5 26
for cocrystals of 9-methyladenine and 1-methylthymine.^ ’
Since the sugar-phosphate backbones would be closer together
in a Hoogsteen type base pairing than in the Watson-Crick

14
Figure 8. a) Hoogsteen Adenine-Thymine Base Pair.
b) Anti-Hoogsteen Adenine-Thymine Base Pair.
type, the authors suggested that the Watson-Crick scheme
is favored due to less electronic repulsions between the
strands. This type of hydrogen bonding has also been ob-
2 7
served for 9-ethyladenine-1-methylthymine cocrystals .
Moreover, cocrystals of adenosine and 5-bromouridine have
been found to assume a different hydrogen bonding scheme:
9 O 9 Q
the anti-Hoogsteen type (Figure 8b). ’ This type of
hydrogen bonding scheme has also been observed for other
30 31
systems. ’ On the other hand, X-ray analysis of single
crystals of derivatives of guanine and cytosine has shown
that only the expected Watson-Crick base pairing is oper-
32-34
ative in these base pairs. This is presumably due to

15
the fact that the pairing scheme for guanine-cytosine has
three hydrogen bonds, whereas the others (Hoogsteen and anti-
Hoogsteen) have only two. Thus, in the crystalline state,
only three out of a possible twenty-nine base pairing com¬
binations have been observed.
The question naturally arises as to what happens in
solution. Tuppy and Kuebler covalently attached a specific
nucleoside to an Amberlite support and used this modified
35
support in a column. It was found that when a solution
of the other three nucleosides was passed through this
column, only the complement to the covalently bound nucleo¬
side was retarded. Furthermore, this association was
abolished when the column was eluted with an aqueous-urea
solution, strongly suggesting that the retention of the
complementary nucleoside was due to hydrogen bonding for¬
mation.
Infrared spectroscopy has been used to determine
hydrogen bonding between derivatized purines and pyrimidines
by monitoring the appearance of new absorption bands be¬
tween 3500 and 3000 cm ^ due to N-H stretching frequency
3 6
changes when this group is involved in hydrogen bonding.‘
When solutions of 9-ethyladenine and 1-cyclohexyl-uracil
were mixed, two new bands at 3490 and 3330 cm ^ appeared
and were maximized when the components were mixed at a
ratio of 1:1. Similar results have been obtained with de-
37-39
rivativesof guanine and cytosine.

16
The NMR has also been used to investigate hydrogen
bonding between bases in nonaqueous solution by monitoring
the downfield chemical shift of the hydrogen bonded N-H pro¬
ton due to a decrease in the electron density about the pro¬
ton nucleus upon hydrogen bonding. Katz and Penman found
that: 1) base pairing decreases as the hydrogen bonding
ability of the solvent increases; and 2) in nonhydroxylic
solvents, the base pairing is quite specific.^ For
example, the downfield chemical shift of the N-l proton of
guanosine was -134.7 Hz in the presence of the complemen¬
tary cytosine, whereas, there was relatively little or no
downfield shift in the presence of the other nucleosides.
A similar observation was seen for the N-3 proton of uridine
in the presence of adenine.4^ Shoup et al. have also em¬
ployed an NMR study of the four purine and pyrimidine com-
42
ponents of DNA and have obtained similar results.
The above studies reveal that hydrogen bonding between
base pairs is quite specific in nonhydroxylic solvents.
However, in aqueous media, competition for the hydrogen
bonding sites on the bases by water would minimize the im¬
portance of these forces. Thus, one might conclude that
hydrogen bonding forces are not significant in terms of
stabilizing the double helical structure. This dilemma is
resolved by the realization that the interior of the double
helix, in which the base pairs are located, is very non¬
polar. The exclusion of water from this region due to the
hydrophobic nature of the bases provides an excellent

17
environment for the formation of hydrogen bonds between the
bases.
Stacking Forces
It was suggested as early as 1958 that hydrogen
bonding between bases was probably not the sole source of
4 3 44
DNA stability. ’ This was based on the observation that
at a pH low enough to break hydrogen bonds, the DNA helix
could be kept intact, providing a sufficiently low temper¬
ature was maintained. Further work has proposed that a
major stabilizing factor for DNA is a hydrophobic inter¬
action between the bases. Since the bases are essentially
nonpolar, they tend to stack in aqueous media. Since the
stacked bases are less hydrated than the individual bases,
45
the phenomenon of stacking is thought to be entropy driven.
Ts'O has determined the osmotic coefficients for
several purine and pyrimidine systems and calculated
activity coefficients by the Gibb-Duhem relationship. The
data clearly indicated that the properties of the bases and
nucleosides in solution were far from ideal, indicating
extensive interaction of the nucleosides in aqueous solu¬
tion. ^ Hydrogen bonding was ruled out for two reasons:
1) the nucleosides associate much more extensively than
urea, which is known to be one of the best hydrogen bonding
agents in water; and 2) methylation of hydrogen bond donor
sites on the nucleosides enhances association.
A more direct method to study the association of bases
and nucleosides in solution is through NMR. Chan et al.

18
found that as the concentration of purine increased, the
resonances of the three protons of purine became in-
46 1
creasingly shifted to higher fields. From the H NMR
theory on ring current anisotropy (Figure 9), it is
possible to predict that the association results from
47
vertical stacking of the purine bases.
Figure 9. Shielding of the Aromatic Protons Caused by
Vertical Stacking.
From the magnitude of the H NMR chemical shifts for
the various protons of adenosine, it was found that the
five-membered ring experiences less shielding than the six-
membered ring. Broom et al. proposed two models (Figure 10)
for the preferred average orientation of the stacked
4 8
nucleoside bases to account for these observations.
It has been found that substitution with an amino
group on the 6 position of purine to give adenosine en¬
hances association. Furthermore, 5-bromo uridine asso¬
ciates to a greater extent than thymidine. These obser¬
vations lead to the conclusion that a hydrophobic force

19
R
Figure 10. Geometry of Stacked Nucleosides.
is not solely responsible for the stacking interaction.^^
It has been suggested that there is a correlation between
the stacking and the polarizability of the bases. The con¬
clusion by Hanlon that London dispersion forces are respon¬
sible for the stability of the DNA helix is in line with
49
this concept.
Further insight into stacking interactions has been
obtained from studies of dinucleoside phosphates. Using
NMR, Chan and Nelson showed that ApA exists in a 3'-
anti-5'-anti right-handed stack.^ It should be noted
that the Watson-Crick model requires that the bases be in
the anti conformation. It has also been observed that
different dinucleoside phosphates differ in their stacking
ability.^ For example, ApA's structure persisted upon
elevation of temperature, whereas UpU lost its ordered
structure. This has been attributed to different stacking
interactions for the two dinucleoside phosphates.

20
Electrostatic Effects
At neutral pH, the backbone phosphate groups of the
DNA double helix are negatively charged, creating a poly¬
anion. Therefore, at low ionic strength, there is a large
interstrand Coulombic repulsion between the "naked" anionic
phosphates. There are also intrastrand phosphate inter¬
actions. Thus, at low ionic strength, there is an increased
propensity for the DNA to denature. Gabbay has determined
that the distance between adjacent phosphate groups along
the same chain of the Watson-Crick double helix is approxi-
0 52
mately 7 A. As the ionic strength of a DNA solution in¬
creases, the intrinsic viscosity decreases (the intrinsic
viscosity is related to the length of rod-like DNA). It
has been suggested that the decrease in viscosity is re¬
lated to a decrease in the length of rod-like DNA due to
less inter- and intrastrand repulsions of the phosphates
5 3
as they are neutralized by added electrolyte.
Electronic Effects
The fact that the bases interact electronically is
well known. Examination of Table 1 reveals that as the
Table 1. The Effect of Increasing Length of the Polynucleo¬
tide (Ap)nA on the UV Absorption Spectrum.
n
X
E /monome
max
max
0
260
15,000
1
257
13,600
2
257
12,600
3
257
11,300
4
257
10,800
300 (poly A)
256
9,000

21
length of the molecule increases, there is a concomitant
decrease in the extinction coefficient of the adenine bases
as well as a blue shift in the wave length maximum.^^^^
A theoretical treatment, employing the exciton theory,
was used to explain the results.^ ^ The theory states
that for a molecular crystal, the absorption of light is
not limited to a single molecule, but rather, it is dis¬
tributed over many. Since a polynucleotide can be con¬
sidered a one - dimensional crystal, the exciton theory
can be applied to nucleic acids. According to the theory,
the excited states of the bases would be split into, say,
two new levels (Figure 11). The higher energy level has
the electronic vectors of the bases parallel, whereas the
lower energy level has the vectors anti-parallel. Quantum
mechanical selection rules forbid transitions to the lower
<â– 
<
>
Figure 11. Exciton Splitting of Energy Levels.

22
excited state. Therefore, the allowed transition to the
higher energy level is one of energy higher than the origi¬
nal uncoupled transition and results in a blue shift for
the absorption maximum.
The observed hypochromism is a result of an intensity
interchange of coupled transition moments as illustrated in
Figure 12. Since the transition moments are in a card
stacked orientation in a DNA molecule, the higher energy
transition is hyperchromic, while the lower energy transi¬
tion is hypochromic.
(a)Card Stack Arrangement
(b)Head-to-Tail Arrangement
A is hypochromic
B is hyperchromic
(c)Herringbone Arrangement
A is hyperchromic
B is hypochromic
A
No intensity interchange
Figure 12. Intensity Interchange Between Two Interacting
Transition Moments.

23
Studies utilizing circular dichroism have also shown
59
the theory to be correct. The selection rules for cir¬
cular dichroism allow both transitions to occur, resulting
in both a red shifted band and a blue shifted band relative
to the absorption maximum of the monomer. However, without
more complicated quantum mechanical considerations, the
signs of the CD bands cannot be predicted.^ It should be
noted, though, that the two bands are of opposite signs,
resulting in a distinctive double cotton effect for the
polynucleotide.
Dynamic Structure of Nucleic Acids
All of the above factors (i.e., hydrogen bonding,
stacking forces, electronic and electrostatic forces) con¬
tribute to various extents to the overall stability of the
double helical structure. However, the double helical
structure is not a static one, but rather a dynamic one.
For example, von Hippel and coworkers incubated nucleic
acids in tritiated water, and after separation of the poly¬
nucleotide from radioactive solvent via gel filtration,
found that tritium was incorporated into the nucleic acids
with kinetics of exchange much faster than with protein
61 ~ 6 3
systems. It was also shown that the observable ex¬
change took place with those protons involved in the base-
base hydrogen bonding of the double helix. This led
von Hippel and McConnel to propose three models to account
for the phonemenon: 1) unstacking without hydrogen bond

24
breakage; 2) hydrogen bond breakage without unstacking; and
3) hydrogen bond breakage with partial or complete unstacking
62 63
and strand separation. ’ Based on tritium exchange and
pH studies, it was concluded that the "breathing model" (3)
is correct. Kinetic studies of intercalation by Muller and
Crothers^ and Gabbay et al.^ agree with this hypothesis.
Chromatin
As stated before, one DNA molecule may be up to 2 cm
3
long if fully extended. However, a typical animal cell
3 66
nucleus has a diameter of ^5 ym and a volume of ^65 ynr .
The question obviously arises as to how such a long mole¬
cule fits into such a small package. The answer lies in
the substituents of the nuclear material, chromatin.
Chromatin contains 151 DNA, 10% RNA and 75% protein. Some
of this protein contains enzymes or repressors, but a por¬
tion is what is known as histones. Histones are rich in
basic amino acid and are tightly associated with DNA pre¬
sumably by neutralization of the negatively charged phos¬
phates. There are 5 classes of histones with molecular
weights in the range of ^11,000 - 21,500.^ They are:
1) HI (lysine rich); 2) H2a and H2b (moderately lysine
rich) ; and 3) H5 and H4 (arginine rich).
Electron micrographs of chromatin reveal a regular
repeating structure resembling a string of beads. Each
bead is 7-10 nm in diameter and there is a spacer section
of DNA 2-14 nm between each bead.^^’;^^^
Each bead

25
contains approximately 200 base pairs of DNA folded around
a histone octamer containing H2a, H2b, fL and H^. This
would effectively condense a 68 nm DNA chain into a 10 nm
bead. It has thus been concluded that histones act to fold
74
DNA so that it will fit into the nucleus.
Of course, histones are not the only proteins which
interact with DNA. Repressor proteins bind to specific
sections of DNA to prevent transcription of a particular
gene. It is still not clear how these interactions take
place.
Small Molecule-Nucleic Acid Interactions
The recognition process between protein systems and
nucleic acids has generated much research in the past ten
years. However, it is not feasible to study the interac¬
tion specificity between two macromolecules due to the
immense complexity of these interactions. A more sim¬
plified approach involves the use of model systems whereby
the interaction specificity of small molecules with DNA
is considered. With the results of these studies, one can
extrapolate to more complex systems.
There are several forces operative when a small mole¬
cule binds to DNA. These interactions involve electrostatic
interaction between positively charged groups on the mole¬
cule and the anionic backbone phosphates, hydrogen bonding
between available hydrogen bonding sites on the molecule and
the functional groups of the purine and pyrimidine bases,

26
and hydrophobic forces. Three types of hydrophobic-type
interactions have been noted: 1) intercalation between
75-79
base pairs of DNA by aromatic cations; 2) "partial"
insertion between base pairs by sterically restricted com-
pounds containing an aromatic moiety; ’ and 3) ex¬
ternal hydrophobic-type binding as exemplified by steroidal
amine-nucleic acid complexes.^^ It has been proposed
that the nucleic acids may use all or any combination of
8 7
the above forces to specifically bind polypeptide chains.
Intercalation of aromatic amino acids with double and
single stranded nucleic acids was observed with L-phenyl-
alanine, L-tryptophan, and L-histidine using poly A. The
NMR studies showed that the H-2 and H-8 protons of adenine
were downfield shifted due to the destacking of the adenine
bases as a result of intercalation of the aromatic ring of
8 8
the amino acids. Poly A is also destacked and inter¬
calated by derivatives of tyramide, tyrosine and tryptamine
since it was found that the aromatic protons of the latter
were upfield shifted in the poly A complex due to ring
89-91
current anisotropy.
Brown observed a very large stabilizing effect on the
Tm of DNA in the presence of arginyltryptophan methyl ester
when compared to other similarly charged dipeptide deriva-
92
tives. He therefore suggested that the aromatic rings of
amino acids behave as "bookmarks" which anchor the proteins
to specific sequences of nucleic acids via an intercalation
mode of binding.

27
In order to investigate the extent of insertion of an
aromatic ring between base pairs of DNA, Gabbay and Kapicak
examined the interaction specificity of reporter molecule I
7 8
(Figure 13) with DNA. They found that when n = 1, there
is a decrease in viscosity of the DNA-reporter molecule (due
to shortening of the DNA helix), whereas there was an in¬
creased viscosity of the DNA-reporter complex for n = 2, 3
and 4 (due to an increase in the effective length of the
DNA helix). Furthermore, NMR studies reveal that the
aromatic protons H. and HD (in DNA-1 complex when n = 1)
experience upfield chemical shifts of 10 and 6 Hz, respec¬
tively, but that the aromatic protons of the DNA-1 complex
when n = 2, 3 and 4 are completely broadened and indis¬
tinguishable from baseline noise.
It was therefore proposed that the p-nitropheny1 ring
of I (when n = 1) is partially inserted between base pairs
7 8
of DNA (Figure 14). This model accounts for: 1) the
decrease in the effective length of DNA-I caused by bending
at the point of insertion; and 2) the different upfield
chemical shifts of the aromatic protons and Hg (i.e.,
experiences a larger ring current anisotropy than Hg as a
result of "partial" insertion. Presumably, the single
methylene between the aromatic moiety and the quaternary
ammonium group on the side chain is not sufficient to allow
for "full" insertion and lengthening of the helix.
Further evidence for "partial" intercalation came from
experiments using the diastereomeric dipeptides

(CH2)nN(CH3)2(CH2)3N(CH3)3 2Br
Figure 13. Reporter Molecule I (where n = 1, 2, 3, or 4).
Figure 14. Schematic illustration of a segment of DNA double helix (a) which
can either partially intercalate a molecule (b) or fully intercalate
a molecule (c) .
K)
CO

29
H
H
NH--C-CONH-C-CONH
^H2^4 CH2
+nh7
L-lys-L-pheA
CH,
NFU"C- CONK -C-CONH9
(ch9)
2-4
+NH,
.5
L-lys-D-pheA
Figure 15. Schematic illustration of a DNA segment showing
a possible mechanism whereby the e and a amino
groups of the N-terminal L-lysyl residue are
stereospecifically anchored and thus dictating
the positioning of the aromatic ring of the
diastereomeric dipeptides in the DNA-peptide
complex.

30
L-lysyl-L-phenylalanine amide (L-lys-L-pheA) and L-lysyl-
8 2-84
D-phenylalanine amide (L-lys-D-pheA). In particular,
the NMR, viscometric, and flow dichroism data suggest that
the aromatic ring of L-lys-L-pheA is partially inserted
between the base pairs of DNA, whereas the aromatic ring
of L-lys-D-pheA points out toward the solvent (Figure 15).
It was concluded that this specificity arises from a
stereospecific interaction of the e- and a-amino groups of
the N-terminal L-lysyl residue which dictates the posi¬
tioning of the aromatic ring of the C-terminal phenyl¬
alanine residue of the L-lys-L-pheA and L-lys-D-pheA. The
specificity observed with L-lys-L-pheA and L-lys-D-pheA
cannot be attributed to the chirality of the phenylalanine
moiety itself since NMR studies reveal no significant
differences in the binding of L- and D-phenylalanine amides
to DNA.
Statement of Problem
In this dissertation, the synthesis of several dipep¬
tide amides was undertaken to determine the effect of the
chirality of the C-terminal amino acid a carbon and the
length of the "arm" between the a carbon and the aromatic
ring on the extent of intercalation of the aromatic ring
(used in this case as a probe) between the base pairs of
DNA.

CHAPTER II
RESULTS AND DISCUSSION
The interactions of the dipeptide amides (Figure 16)
with DNA has been examined by pulsed Fourier transform
NMR, viscosity, UV absorption and binding studies, circular
dichroism, equilibrium dialysis, and melting temperature
(Tm of helix-coil transition) studies. The results are pre¬
sented below.
NMR Studies
The NMR studies conducted in this work were used
to determine the effect of binding of the peptide to DNA
on: 1) the line broadening of the aromatic proton NMR
signals of the peptide; and 2) the upfield chemical shift
of the aromatic protons of the peptide. The change in
chemical shifts is related to the proximity of the aromatic
protons to the ring current of the DNA base pairs. The
NMR line broadening can be explained by several mechanisms:
1) slow rate of exchange between various DNA binding sites;
2) restricted tumbling of the aromatic ring; 3) larger dif¬
ferences in the chemical shifts experienced by the ortho,
para, and meta protons; or 4) a combination of all three
mechanisms. In these experiments, the peptides are fully
bound as determined by equilibrium dialysis. The results
31

1 L-lys-L-pN02-pheA
H H
+ I I
NH,-C-CONH-C-CONH,
4 CH2-Q>'N02
Nil,
6 L - lys -1), L-DNP-DABA
NII,-C-CONH-CH-CONH-
3 | | 2
(cn2)4 ch2cii2-nM'
Nil,
-NO,
NO,
2 L-lys-D-pN02pheAa
H CH,-<( )) - NO
NH,-C-CONH-C-CONH,
3 | ,2
(CH2). H
I
NH,
7 L-lys-L-S-pN02bz-cysAc
H II
nii,-c-conh-c-conh,
3 | | 2
(CII2)4 CII2-S-CII2
Nil,
NO,
3 L-lys-L-S-NT-cysAa
4 L-lys-D-S-NT-cysA
•NO,
•NO,
NH, - C - CONII - C - CONII,
3 | ,2
(Cll2)4 H
NH,
8 L-lys-D-S-pNOjbz-cysAc
9 L-lys-L-DNP-ornA
H CH,-S-CM,
+ I I 2
NH. - C- CONII - C - CONII,
3 , i ¿
Abb
â– NO,
(C.l2)4 H
NH,
H II
* I I
NH, - C - CONII - C - CONII,
3 | | 2
(CH2)4 CII2CH2CH2NH-
Nil, NO,
-NO,
5 L-lys-L-DNP-DABA
H II
Nil,-C-CONH-C-CONII,
3 i i 2
(CH2)4 ch2ch2-nh-C()Vno2
NH,
NO,
10 L-lys-D-DNP-ornA
Figure 16. The Dipeptide Amides Synthesized and Studied.
a) dihydrobromide salt; b) dihydroacetate salt.
i! ch2ch2ch2nh-(( j)-no
NH j-C-CONH-C-CONIIj
(CH2)4 11
Nllj
CaJ
K)

33
of these studies are shown in Figures 17 - 22, and lead to
the following observations.
First, the extent of NMR signal line broadening of
the aromatic protons depends in general on the chirality of
the C-terminal amino acid a carbon and the length of the
"arm" between the aromatic ring and the a carbon. For
example, the signal of the aromatic protons of L-lys-
L-pNC^-pheA (_1) are broadened more than those of the
c
1_ I
2.5
1 I I
1.5 ppm
The Partial NMR Spectra of DNA at Various
Temperatures. The temperatures are: a) 37°C;
b) 70°C; and c) 90°C.
Figure 17.

8.0
L-lys-L-pNO^-pheA (1)
7.0 ppm
8.0
L-lys-D-pNC^-pheA (2^
Figure 18. H NMR Signal of the Aromatic Protons of Dipep¬
tides 1 and 2_ in the Presence and Absence of
DNA at Various Temperatures. The DNA base pair
to dipeptide ratio is: a) 0 (37°C) ; b) 10 (37°C)
c) 7 (37°C) ; d 5.5 (37°C) ; e) 5.5 (70°C); and
f) 5.5 (90°C).

35
Figure 19. H NMR Signal of the Aromatic Protons of Dipep¬
tides _3 and £ in the Presence and Absence of
DNA at Various Temperatures. The DNA base pair
to dipeptide ratio is: a) 0 (37°C) ; b) 15 (37°C);
c) 10 (37°C) ; d) 7 (37°C); e) 7 (70°C); and
f) 7 (90°C).

36
H NMR Signal of the Aromatic Protons of Dipep¬
tides 5^ and 6^ in the Presence and Absence of
DNA at Various Temperatures. The DNA base pai
to dipeptide ratio is: a) 0 (37°C); b) 15 (37
c) 10 (37°C); d) 7 (37°C); e) 7 (70°C); and
f) 7 (90°C) .
Figure 20.
o t-j

37
Figure 21. H NMR Signal of the Aromatic Protons of Dipep¬
tides 1_ and 8^ in the Presence and Absence of
DNA at Various Temperatures. The DNA base pair
to dipeptide ratio is: a) 0 (37°C); b) 15 (37°C) ;
c) 10 (37°C) ; d) 7 (37°C) ; e) 7 (70°C) ; and
f) 7 (90°C).

38
£
8.5 7.5 ppm
L-lys-D-DNP-ornA (10)
Figure 22. H NMR Signal of the Aromatic Protons of Dipep¬
tides 9^ and 10_ in the Presence and Absence of
DNA at Various Temperatures. The DNA base pai
to dipeptide ratio is: a) 0 (37°C); b) 15 (37
c) 10 (37°C) ; d) 7 (37°C) ; e) 7 (70°C) ; and
f) 7 (90°C).
O

39
diastereomeric L-lys-D-pNC^-pheA (2^. The NMR signal for
the aromatic protons of L-lys-L-S-NT-cysA (3), L-lys-D-S-NT-
cysA (4), L-lys-L-DNP-DABA (5), L-lys-D,L-DNP-DABA (6),
L-lys-L-DNP-ornA and L-lys-D-DNP-ornA (10_) are com¬
pletely broadened into the base line and are indistinguish¬
able from base line noise at all PNA base pair to peptide
ratios. For compounds L-lys-L-S-pNC^bz-cysA {!) and L-lys-
D-S-pNO^bz-cysA (8), there is considerable line broadening,
with perhaps slightly more broadening for peptide 7_.
It should be noted that the line broadening decreases
at higher temperatures as the DNA begins to "melt-out,"
freeing bound peptide. Furthermore, the effect of the vis¬
cosity of the DNA solution on the line broadening is found
to be small [e.g., the signal line width of internal stan¬
dard 2,2,3,5-d^-3-trimethylsilylproprionate (TSP) is found
to be 1.2 +_ 0.2 and 2.1 + 0.3 Hz in the absence and presence
of DNA, respectively, at 37°C]. In addition, examination
of Figure 17 reveals that some of the structure of the NMR
spectra in the region of 6 7.0 - 8.5 ppm is due to DNA base
protons.
Secondly, the extent of the upfield chemical shift of
the NMR signal of the aromatic protons also depends on
the chirality of the C-terminal amino acid a carbon and the
distance from it to the aromatic ring. The spectra for
L-lys-L-pNC^-pheA (IT) is considerably more complicated in
the DNA complex as opposed to free peptide. Examination of
Figure 18 shows two peaks at approximately 45 Hz upfield

40
from the original peaks. Interpretation of this spectra is
difficult due to the complexity of the splitting. However,
for L-lys-D-pNO^-pheA (2_), there is little or no upfield
shifting of the aromatic proton signals. The NMR spectra
of L-lys-L-S-pNO?bz-cysA (7) and L-lys-D-S-pNO?bz-cysA (8^
also become more complex in the presence of DNA (Figure 21).
There is considerable upfield shifting of the aromatic sig¬
nals for both compounds, with more shifting for compound 7_
than _8. In fact, both spectra resemble that of L-lys-L-
pNC^-pheA (_1) .
Since the NMR signals for the aromatic protons
of compounds L-lys-L-S-NT-cysA (3), L-lys-D-S-NT-cysA (4_) ,
L-lys-L-DNP-DABA (5), L-lys-D,L-DNP-DABA (6), L-lys-L-DNP-
ornA (9_) , and L-lys-D-DNP-ornA (10) are completely broadened
into the baseline, nothing can be concluded about upfield
shifting of these protons in the DNA complex. However,
in all cases, the resolution of the signals increases as
the temperature increases, indicative of "release" of the
dipeptide from the DNA complex upon denaturation of the
DNA.
Viscosity Studies
It has been shown by a number of investigators that
planar aromatic compounds (e.g., ethidium bromide, dauno-
rubicin, and the acridine dyes), intercalate between the
base pairs of DNA. The DNA complex increases in viscosity
relative to free DNA due to an increase in the effective

41
length of the DNA molecule. To prove that the DNA length
increases, the determination of the intrinsic viscosity,
(p), of the solution is necessary. Intrinsic viscosity
is defined by
r ^ Limit ^sp
(,1) = c * 0 c
where p is the specific viscosity of a DNA solution and
sp r
c is the concentration of DNA. Unfortunately, these experi¬
ments are uninformative since the peptide dissociates from
the DNA complex at high dilution (probably due to the low
binding constant of peptide to DNA) and thus, the intrinsic
viscosity of the complex approaches that of free DNA.
Therefore, the effect of increasing the concentration
of the dipeptide amides on the relative specific viscosity,
p /p (where p and p are the specific viscosities
sp spo v sp spo ^
of the DNA solution in the presence and absence of dipeptide
amide, respectively) at 3.0 x 10 ^ DNA phosphate/liter (in
10 mM MES, 5 mM Na+, pH 6.2) was studied at 37°C using the
low shear Zimm viscometer. Since the study was carried out
at low DNA concentration, the relative values of p /p
’ sp spo
are close approximations of the relative values of the
intrinsic viscosity of a DNA-dipeptide complex to free DNA
C [nl/[p] ). The results are shown in Figures 23 through 26,
and indicate that peptides ]^, 2_, 7_ and 8^ decrease the spe¬
cific viscosity of a DNA solution, with peptide (L decreasing
the viscosity to a much greater extent than 2, 7_ and _8.
Furthermore, peptides 3, 4, 5, 6, 9 and 10 increase the

Figure 23. The Effect of Dipeptides 1^ and 2_ on the Relative Specific
Viscosity of Salmon Sperm DNA.

Figure 24. The Effect of Dipeptides 3, 4_, 1_ and 8^ on the Relative Specific
Viscosity of Salmon Sperm DNA.

Figure 25. The Effect of Dipeptides 5^ and 6^ on the Relative Specific
Viscosity of Salmon Sperm DNA.
A

Figure 26. The Effect of Dipeptides 9^ and 10_ on the Relative Specific
Viscosity of Salmon Sperm DNA.
Ul

46
specific viscosity of a DNA solution, the effect being
greatest for _5, 9_ and _10 with only a slight increase for
peptides £ and 4_.
Ultraviolet Absorption Studies
Absorption data for the dipeptides are shown in Table
2. All peptides have a well defined X in the range of
260 - 400 nm with extinction coefficients on the order of
8.5 x 10^ for compounds 1, 2, 3, 4, 7 and 8 and 15.5 x 10
for compounds _5, 6_, 9_ and ICh For those compounds with
X greater than 300 nm, two effects can be seen on the
absorption spectra upon binding to DNA. First, large
hypochromic effects are seen for all DNP derivatives
(namely _5, £, 9_ and 10) , with a considerable hypochromic
effect for one of the nitrotolyl derivatives, peptide 3.
Furthermore, there is a large bathochromic shift for X
in 3_ and £ in the DNA complex (i.e., from 340 nm to 355 nm)
and a small bathochromic shift for the DNP derivatives £,
£, £ and 10_ (i.e., from 360 nm to 365 nm) .
Binding Studies
The binding of those peptides with X greater than
300 nm to salmon sperm DNA was determined by spectral
titration. The effect of increasing concentration of DNA
on the absorption of peptides 3, 4, 5, 6, 9 and 10 at 340
nm for the nitrotolyl containing peptides or at 360 nm for
the DNP containing peptides was studied in 6.6 mM phosphate
buffer (pH 7.2) at three ionic strengths (5 mM, 10 mM and

Table 2. Absorption and Hypochromicity Data on the Dipeptide Amides.
Free
DNA complex
Compound3
A
max
e
max
A
max
£
max
U\h
L-lys-L-pNC^-pheA (1)
275
8.75
x 103
ND
ND
ND
L-lys-D-pNO^-pheA (2)
275
8.75
x 103
ND
ND
ND
L-lys-L-S-NT-cysA (3)
340
7. 88
x 103
355
6.3 x 10ó
25
L-lys-D-S-NT-cysA (4)
340
7.88
x 103
355
7.5 x 103
5.1
L-lys-L-DNP-DABA (5)
360
15.5
x 103
365
1.0 x 104
55
L-lys-D,L-DNP-DABA (6)
360
15.5
x 103
365
1.0 x 104
55
L-lys-L-S-pNO^bz-cysA (7)
275
8.50
x 103
ND
ND
ND
L-lys-D-S-pNO^bz-cysA (8)
275
8.50
x 103
ND
ND
ND
L-lys-L-DNP-ornA (9)
360
15.5
x 103
365
1.0 x 104
55
L-lys-D-DNP-ornA (10)
360
15.5
x 103
365
1.0 x 104
55
Measurements were carried
Cary-17D spectrophotometer
out in 10 mM
at 25°C using
MES buffer (5
salmon sperm
mM Na+, pH
DMA. ND =
6.2) with the
not determined.
= percent hypochromicity.

48
and 50 mM Na+) in a 1 or 5 cm cell which was thermostated
at 210 C .
The spectral data were analyzed by the McGhee-von Hippel
9 3
technique according to the following equation
n-1
where v is the number of moles of peptide bound per DNA base
pair, L is the concentration of free peptide, K is the
apparent binding constant, and n is the number of base pairs
per binding site. To calculate the above parameters, the
raw absorption data are fed to a Pet 2001 microcomputer
which then calculates v and v/L (see experimental chapter).
For each pair of v and v/L, a value of K is calculated as
a function of N. The programmer inputs various values of n
until the smallest deviation in the set of K's is obtained.
The average value of the K's is then taken as (e.g., see
Table 3) .
A plot of v/L versus v is shown in Figure 27. It can
be seen that only a small range of v's was obtained from
the spectral titrations. Furthermore, most of the points
are on the straight part of the graph, with only a few
points on the curved part. Therefore, the values of K and
n should be taken with caution. More accurate values of
K and n could only be obtained with a broader range of
3
v's. However, the values of K
’ a
tive binding strengths.
in Table 4 do reflect rela-

49
Table 3. Typical Output for McGhee-von Hippel Binding Iso¬
therms for Various Values of na.
K
a
V
v/L
n= 3
n=4
n= 5
. 405
.052
7379
9894
11347
13203
.421
. 051
7384
9814
11201
12958
.445
.050
7620
10096
11505
13283
. 465
. 049
7782
10261
11662
13423
.488
.049
8077
10628
12065
13868
. 508
. 048
8298
10875
12321
14127
. 524
.048
8435
10997
12424
14198
. 541
. 047
8599
11157
12573
14325
. 561
. 046
8908
11527
12973
14757
.581
. 046
9254
11948
13430
15254
. 601
.046
9643
12423
13948
15823
.617
. 045
9939
12760
14302
16190
.631
. 044
10126
12942
14471
16337
.657
. 043
10585
13420
14944
16790
.681
. 042
11001
13835
15343
17155
aPerformed
by a Commodore Pet
2001 microcomputer fo
r L-lys-
L-DNP-ornA
(9) (10
mM
Na ) where a = ratio
of bound
peptide
v = moles
of bound
peptide/mole of DNA base pairs,
L =
concentration of free
peptide,
n = number
of base p
airs per
binding site, and
K =
apparent
binding constant for
that se
of v and v/L.

50
The values of K and n for the dipeptides 3, 4, 5, 6,
9^ and 10_ are found in Table 4. A number of observations
can be made.
1) In general, K and n decrease with increasing
ionic strength.
2) Peptides _5, 9^ and _10_ bind very strongly to DNA,
while compounds 4_ and 6_ exhibit fairly weak binding.
3) Peptide 3^ binds stronger than its diastereomer,
peptide 4_, and peptide .5 binds stronger than the diastereo-
meric mixture 6.

Table 4. Binding Isotherms for DNP and Nitrotolyl Containing
Dipeptides.
5 mM
Na +
10 mM Na+
5 0 mM
Na+
Compound3
K x 10'
5
n
K x 10~5
n
K x 10'4
n
3.
8.
L-lys-L-S-NT-cysA (3)
1.3
4.0
0.32
3.3
L-lys-D-S-NT-cysA (4)
0.53
3.2
0.024
2.7
L-lys-L-DNP-DABA (5)
2.5
4.6
2.4
4.0
1. 8
2 .1
L-lys-D,L-DNP-DABA (6)
0.81
2.6
1. 2
2.6
0.18
2.1
L-lys-L-DNP-ornA (9)
1.9
4.5
1.1
3.9
1.1
2.5
L-lys-D-DNP-ornA (10)
2.3
4.5
2.2
4.0
1.1
2 . 5
Measurements were carried out on a Cary-17D
or 340 nm (nitrotolyl derivatives) in 6.6 mM
spectrophotometer
phosphate buffer
at 360 nm
(pH 7.2) in
(DNP derivatives)
a 1 or 5 cm
cell at 21°C.

52
4) All binding constants are higher than those ob¬
tained from equilibrium dialysis, but these constants were
calculated at different peptide and DNA concentrations.
Circular Dichroism Studies
The effect of increasing concentration of salmon
sperm DNA on the circular dichroism spectra of compounds
3, 4, _5, (3, 9^ and 10. in the range of 300 - 400 nm was
studied. The results, shown in Figures 28 and 29, show
that there is an induced CD spectra for all compounds in
the range of 300 - 400 nm. The extent of the induced CD
in the presence of excess DNA (i.e., at a DNA base pair to
peptide ratio of 12) follows the following order:
1) L-lys-L-DNP-ornA (9) - L-lys-D-DNP-ornA (10) = L-lys-L-
DNP-DABA (5) > L-lys-D,L-DNP-DABA (6) > L-lys-L-S-NT-cysA (3)
> L-lys-D-S-NT-cysA (4.) .
Equilibrium Dialysis
Equilibrium dialysis was utilized to perform direct
binding studies of the dipeptide amides with salmon sperm
DNA. The quantitative analysis of the dipeptide amide was
accomplished by UV absorption data at the \ of the
various dipeptides. These studies were carried out in
duplicate at a single peptide and DNA concentration (i.e.,
at 3.0 x 10 ^ M DNA phosphate/liter and either 2.5 x 10 ^ M
or 5.0 x 10 ^ M peptide) using a 10 mM MES buffer (5 mM Na+,
pH 6.2). The apparent binding constant, K ,
3.
from
was evaluated

Figure 28. The CD Spectra of Dipeptides _5, 6, 9 and 10_ in the Absence and
Presence of DNA. The DNA base pair to peptide ratio is: a) 0;
b) 1.5'; and c) 12.

54
Figure 29. The CD Spectra of Dipeptides 3 and £ in the
Absence and Presence of DNA. The DNA base
pair to dipeptide ratio is: a) 0; b) 1.5;
and c) 12.
R,
K =
a - (Pt - Rb)Rf
where R^ and is the concentration of free and bound
dipeptide, respectively, and P is the total DNA phosphate
concentration. This equation can be used assuming that
each DNA phosphate binds independently to a peptide mole¬
cule and that the maximum number of binding sites per DNA
phosphate is one. Even though this may not be true, the
binding data from the interactions of peptides to nucleic
acids are evaluated in this way and thus the Ka values in

5 5
Table 5. Apparent Binding Affinity (K ) of the Dipeptides
to Salmon Sperm DNAa. a
Peptide System
L-lys-L-pNC>2-pheA (1_)
L-lys-D-pNC^-pheA (2^
L-lys-L-S-NT-cysA (3)
L-lys-D-S-NT-cysA (40
L- lys-L-DNP-DABA (_5)
L-lys-D,L-DNP- DABA (6)
L-lys-L-S-pNO^bz-cysA (2)
L-lys-D-S-pNC^bz-cysA (80
L-lys-L-DNP-ornA (9)
L-lys-D-DNP-ornA (10)
Ki
K2
5.3
X
102
5.4
X
102
6.8
X
102
4.9
X
102
3.6
X
103
1.3
X
103
2.7
X
3
10-3
8.7
X
103
3.0
X
103
1.3
X
3
10°
1.3
X
103
1.3
X
103
CO
X
2
10*
3.2
X
102
2.2
X
2
10*
6.6
X
101
2.7
X
103
7.9
X
2
10*
3.1
X
103
1.6
X
3
IQ0
3. _ A
Equilibrium dialysis was carried out with 3.0 x 10 M
salmon sperm DMA in 10 mM MES buffer (5 mM Na + pH 6.2)
with peptide concentration of either 2.5 x 10'4 M (K.)
or 5.0 x 10"4 M (K2).

56
Table 5 do reflect differences in binding strength.
Examination of Table 3 reveals that, in general, dipeptides
containing a nitrotolyl moiety (i.e., _3 and 4) or a DNP
moiety (i.e., _5, 6^, 9_ and _10) bind more strongly than
those compounds containing a p-nitrophenyl moiety (i.e., 1_,
2_, 1_ and 8^) .
Melting Temperature Studies
The effect of increasing concentration of peptide on
the Tm (melting temperature) of the helix-coil transition
for three different nucleic acids was determined. Several
observations can be made from examination of the Tm data
in Table 4.
1) All peptides stabilize the helix relative to the
random coil and the transition exhibits a monophasic
melting behavior. Furthermore, the DNP derivatives _5, 6,
9_ and 10_ and the nitrotolyl containing peptide stabilize
the helix to a greater extent than the other peptides.
2) All peptides, in general, stabilize poly d(A-T)
to a greater extent than poly I - poly C, indicative of
preferential binding to A-T sites.
3) For a given set of diastereomeric dipeptides,
the compound having the L configuration for the C-terminal
amino acid a carbon stabilizes the helix to a greater ex¬
tent than the peptide with the D configuration at the C-
terminal amino acid a carbon (e.g., 3 > 4, 9 > 10, etc.).

Table 6. The Effect of the Dipeptides on the AT of the Helix-Coil Transition
of Salmon Sperm DNA, Poly I - Poly C, and Poly d(A-T) (ATm = Tjn - T ,
where Tm and T are the melting temperatures of the nucleic acids mo
in the presence and absence of dipeptide).
Peptide System3 ATm
Salmon Sperm DNA
Poly I -
Poly C
Poly
d(A-T)
250 yM
500 yM
250 yM
5 00 yM
250 yM
500 yM
L-lys-L-pNO?-pheA (1)
1.8
2.3
3.8
5.7
4.0
6.8
L-lys-D-pNC^-pheA (2)
0.9
3.5
0.4
6.8
1.2
2.4
L-lys-L-S-NT-cysA (3)
5.1
11.0
2.6
5.3
14.0
17.1
L-lys-D-S-NT-cysA (4)
2.4
3.3
1.9
4.4
6.2
12.7
L-lys-L-DNP-DABA (5)
7.5
15.6
7.0
11. 8
10.6
22.9
L-lys-D,L-DNP-DABA (6)
4.3
8.4
4.3
7.3
7.7
18.3
L-lys-L-S-pNC^bz-cysA (7)
0.2
0 . 3
1.7
3.5
3.8
6.1
L-lys-D-S-pNO?bz-cysA (8)
0.3
1.2
2.6
2.9
2.3
3.7
L-lys-L-DNP-ornA (9)
>30.0
>30.0
6.7
11. 5
14.3
23.3
L-lys-D-DNP-ornA (10)
14.9
>30.0
1.1
5.7
7.0
19.3
Tm studies were carried out in 0
and peptide concentrations of 250
.01 M MES
and 500
buffer,
yM.
pH 6.2, using
126 yM P/L
of DNA

5 8
The interpretation of these data is complicated by
the fact that the helix-coil transition involves the inter¬
action of the dipeptide (P), not only with the helix (H),
but also with the coil (C) as shown in the following
equation:
KH KC
H + P + H- P + C- P + C + P.
It can be assumed that K„ is greater than K„ since T is
increased in the presence of the dipeptide system. How¬
ever, Gabbay and Kleinman have pointed out that this con¬
clusion is valid only when and are determined
9 4
directly.
Discussion
Much research has been devoted to the study of the
recognition specificity of proteins for nucleic acids.
Due to the several types of forces operating at several
sites along the nucleic acid and protein, this type of
study is of immense complexity. Furthermore, recent
studies on chromatin reveal that protein-DNA binding
specificity is not only a dynamic process, continuously
changing during the cell cycle, but may also involve spe¬
cific protein aggregates-DNA recognition.^
The problem can be simplified by studying the inter¬
action specificities of small dipeptides with DNA as under¬
taken in this work. As stated before, small molecules may
bind to DNA by electrostatic, hydrogen bonding, hydrophobic

59
8 7
forces, or any combination of these. Electrostatic
forces are not expected to lead to specific recognition
between a small molecule and DNA, since this type of
interaction may occur at each of the DNA phosphate groups.
Furthermore, hydrogen bonding may occur at any base pair;
so, it can be assumed that hydrogen bonding forces would
not lead to specific recognition.
It has therefore been suggested that hydrophobic-type
interaction (which has been noted in the binding of aro¬
matic and hydrophobic amino acids to DNA preferentially at
A-T sites) is primarily responsible for the recognition
8 7
process. Gabbay has shown that the "partial" intercala¬
tion of the aromatic ring of the side chain of phenylalanine
with native DNA (which has been shown to possess A-T
clusters^"’ ^^) is consistent with this interpretation.
From the work with reporter molecule I and the dia-
stereomeric dipeptides L-lys-L-pheA and L-lys-D-pheA, two
criteria were established to dictate whether full, partial
78 82 84
or no intercalation will operate. ’ ’ From the re¬
porter molecule work, it was shown that there is a depend¬
ence of length between the aromatic ring and the quaternary
ammonium group, i.e., if the length was long enough, full
intercalation occurs; if the length was short enough, only
partial intercalation occurs. The dependence on the
chirality of the C-terminal amino acid a carbon for partial
intercalation was shown by the work with L-lys-L-pheA and
L-lys-D-pheA. The results suggest that the dipeptide is

60
stereospecifically anchored to the DNA by an electrostatic
interaction between the N-terminal lysyl a and e amino
groups and that as a result of this binding, the orienta¬
tion of the aromatic ring of the C-terminal amino acid is
established. When the C-terminal amino acid is of the L
configuration, the aromatic ring is pointed toward the in¬
terior of the helix; whereas, in the case of the D config¬
uration, the aromatic ring is pointed out toward the sol¬
vent (Figure 15).
In an attempt to provide additional experimental
evidence for (or against) these two criteria, the synthesis
and study of the interaction specificities of a series of
dipeptide amides were undertaken. The series consisted of
pairs of diastereomeric dipeptides (differing in chirality
of the C-terminal amino acid a carbon) with an aromatic
ring of various distances from the C-terminal amino acid
a carbon.
From the above considerations, it was assumed that
the aromatic ring of L-lys-L-pNC^-pheA (_1) would partially
intercalate while that of L-lys-L-S-NT-cysA (3) may par¬
tially or fully intercalate and that there would be full
intercalation for L-lys-L-DNP-DABA (5), L-lys-L-S-pNO^bz-
cysA (7_) and L-lys - L - DNP - ornA (£) . On the other hand, no
intercalation of the aromatic ring was expected for those
peptides whose C-terminal amino acid a carbon was of the
D configuration [i.e., L-lys-D-pN0o-pheA (2), L-lys-D-S-NT-
cysA (4-), L-lys-D-S-pNO^bz - cysA (8^) and L - lys - D-DNP - ornA (10) ] .

61
A "mixed" behavior was expected for the diastereomeric mix¬
ture L-lys- D, L -DNP-DABA (6).
Several types of experimental data can be obtained to
determine the extent of intercalation. From NMR studies,
the extent of line broadening and upfield shifting of the
aromatic proton signals is indicative of the extent of in¬
tercalation. Full intercalation results in total line
broadening, while partial intercalation results in broad¬
ening and upfield shifting of the aromatic peaks. No up¬
field shifting is observed in the absence of intercalation.
Inspection of the NMR spectra of the peptides in the
presence of DNA (Figures 18 - 22) reveals some interesting
points. First, the NMR signals of aromatic protons of com¬
pounds 3^, 4_, _5, 6_, 9^ and _10 are completely broadened,
indicative of full intercalation. The aromatic signals for
peptides 1, 1_ and 8^ are upfield shifted and broadened,
which would suggest partial intercalation. On the other
hand, the NMR signals for the aromatic protons on compound
2 are only slightly shifted and broadened. This can be in¬
terpreted as the absence of intercalation. The greater
extent of line broadening for 1^ as compared to 2_ can be
explained by several mechanisms: 1) slower tumbling rates
of the aromatic ring of 1_ in the DNA complex as compared
to 2_; 2) slower exchange between the various DNA binding
sites for DNA-1^ as compared to the DNA-2^ complex; 3) larger
differences in the ortho and meta protons of the aromatic
ring of 1 in the DNA complex as compared to 2_\ or 4) a

62
combination of all three mechanisms. Since the value of
the spin lattice relaxation time (T^) is dependent (among
other things) on the correlation time (Tc) and mean resi¬
dence time (t ), determination of T. for 1 and 2 in the
presence and absence of DNA could help discriminate be¬
tween the three mechanisms. Unfortunately, due to the
complexity of the spectra and inability to accurately meas¬
ure peak intensities in the spectra, values could
not be obtained for the DNA-1^ complex. However, previous
work by Gabbay^'7 on L-lys-L-pheA-DNA and L-lys-D-pheA-DNA
complex revealed that the of the aromatic protons of
both peptides in the DNA complex were nearly identical
(T^ - 0.65 sec.), suggesting that the tumbling rate (1/t )
and the chemical exchange rate (1/x^) of the aromatic pro¬
tons of the peptides in the DNA complex were very similar
in magnitude. ^ It was concluded that the greater
signal line broadening observed for the aromatic protons
of the L-lys-L-pheA-DNA complex as compared to the L-lys-
D-pheA-DNA complex could only be due to large differences
in the chemical shifts experienced by the ortho, meta, and
para protons. It can therefore be assumed that the greater
line signal broadening of the aromatic protons of L-lys-L-
pNO^-pheA in the DNA complex as compared to that of the
L-lys-D-pNO^-pheA-DNA complex can only be due to differences
in the chemical shifts experienced by the ortho and meta
protons.

63
The magnitude and the sign of the change in the vis¬
cosity of a DNA-peptide complex can also be informative as
to the extent of intercalation. An increase in the vis¬
cosity of a DNA-small molecule solution is noted for full
intercalation of the aromatic ring of the small molecule
between the base pairs of DNA, while a decrease in viscos¬
ity is observed for "partial" intercalation. It should be
noted that electrostatic binding (without intercalation)
of cationic molecules to DNA also decreases the viscosity
of a DNA-small molecule solution due to neutralization of
the anionic phosphates and subsequent shortening of the
DNA chain. However, the degree of decrease in the viscos¬
ity of the DNA solution is not as great as when intercala¬
tion also takes place.
Consistent with the NMR data, peptides _3, £, 5^, (3, 9.
and 1_0 increase the viscosity of the DNA-peptide solution
(i.e., full intercalation), while peptides _1, 2_, 7_ and 8^
decrease the viscosity of the DNA-peptide solution, the de¬
crease greatest with L-lys-L-pNO^-pheA (1) .
Further evidence for full intercalation of the aromatic
rings of compounds 3^, 4_, .5, (3, 9_ and 1^0 comes from the UV
and CD studies. From the UV studies, a red shift in A
’ max
and hypochromism is observed for these peptides in the
presence of DNA. The red shift arises from an electronic
interaction between the aromatic ring and the purine and
pyrimidine base resulting in a splitting of the excited
state energy levels of the peptide aromatic ring producing

64
a lower excited state energy level and the hypochromism
arises from an intensity exchange between the coupled
transition moments of the aromatic ring and the purine
and pyrimidine bases. There is also an induced CD in the
region of X of the peptides in the DNA complex. This
can be explained by intercalation of the aromatic ring
into the interior of the DNA double helix, which is
assymetric (thus, an induced CD).
When considering the binding of these peptides to DNA,
it must be kept in mind that two types of interaction are
operative, namely electrostatic binding of the N-terminal
amino acid lysyl residue via the a and e amino groups and
intercalation of the aromatic ring of the C-terminal amino
acid. Furthermore, one type of binding may predominate
depending on the relative strengths of the two possible
interactions. In general, electrostatic binding is favored
at low ionic strength, while intercalation is favored at
higher ionic strength. The apparent binding constant, K ,
is reflective of this phenomenon.
Those compounds which have been shown to intercalate
from NMR and viscosity studies also have a higher K than
cL
those compounds which only partially intercalate or do not
intercalate at all (Table 5). This is also borne out from
the Tm studies. The compounds that fully intercalate
stabilize the DNA double helix to a greater extent than
those that only partially intercalate. The Tm data also
show that poly d(A-T) is stabilized more than poly I -
poly C, indicating the peptides show a preference for A-T sites.

65
The results from these studies were pretty much in
line with what was predicted. However, a few unexpected
results were obtained.
1) The aromatic ring of L-lys-D-S-NT-cysA (£) inter¬
calates. It was predicted that this would not be the case,
since the aromatic ring of _4 should point out toward the
solvent. However, if a strong electronic interaction
exists between the aromatic ring of £ and the base pairs
of DNA, intercalation would be favored even at low salt.
Hydrophobic-type interactions would also favor intercala¬
tion. One or both of these factors is presumably opera¬
tive for this molecule.
2) The aromatic ring of L-lys-D-DNP-ornA (10) also
fully intercalates. This result can be explained by two
mechanisms: 1) intercalation is favored for the same
reasons as noted above (i.e., electronic or hydrophobic);
or 2) electrostatic binding and intercalation as a result
of the flexibility of the "arm" between the a carbon and
aromatic moiety. In other words, the arm is long enough,
after electrostatic binding, to allow the aromatic ring,
which is seeking a nonpolar environment, to "swing" around
and intercalate. This is not unreasonable in light of the
results from L-lys-D,L-DNP-DABA (£) . The extent of in¬
duced CD and increase in viscosity for this peptide is not
as great as for L-lys-L-DNP-DABA (£) . However, L-lys-D-
DNP-ornA (10) behaves exactly like L-lys-L-DNP-ornA (£) .
With regard to mechanism 2, it would be tempting to assume,

66
in the case of the L-lys-D,L-DNP-DABA (6) peptide, that
electrostatic binding is taking place, and the aromatic
ring of the L-lys-L-DNP-DABA diastereomer is fully inter¬
calating (as in peptide _5) , while that of the L-lys-D-
DNP-DABA is only partially intercalating since the arm
is not long enough (as opposed to the case of L-lys-D-
DNP-ornA, 10_) to allow full intercalation; thus, the
"mixed" results.
Model building studies can be used to support
mechanism 2. For example, for reporter molecule I with
n = 1, the distance between the quaternary ammonium
nitrogen and the center of the aromatic ring is on the
O O
order of 4 A, while this distance is on the order of 5 A
for reporter molecule I with n = 2. Furthermore, the
distance between the center of the aromatic ring and the
assymetric carbon of the phenylalanyl residue of L-lys-L-
O
pheA is also approximately 4 A. Therefore, the critical
distance between the aromatic ring and the a carbon to
O
which it is attached must be between 4 and 5 A to allow
for full intercalation. For L-lys-L-DNP-DABA (5) and
L-lys-L-DNP-ornA (9), this distance is approximately 6
O
and 7.5 A, respectively, which is certainly long enough
to allow full intercalation of the aromatic ring for
each compound. For L-lys-D-DNP-ornA (1_0) , the distance
between the aromatic ring and the a carbon to which it is
O
attached is about 4.5 A after swinging the aromatic ring
around and pointing it toward the interior of the helix.

67
This distance is apparently long enough to allow for full
intercalation. However, for L-lys-D-DNP-DABA, this dis-
O
tance is only on the order of 3 A, which is insufficient
for full intercalation. It should be noted that the models
were built assuming a certain rigidity in the peptides
after binding. More experimental evidence is needed to
support this mechanism, however.
3) Finally, the results from the studies with L-lys-
L-S-pNO^bz-cysA (7) and L-lys-D-S-pNO^bz-cysA (8^ are
very difficult to interpret. It was hoped that studies
with these two compounds would resolve any anomolies
created by L-lys - D , L - DNP-DABA (6). Instead, the entire
situation becomes more complicated. It is evident from
these studies that full intercalation is not taking place
even for L-lys-L-S-pNC^bz-cysA (7). The NMR suggests that
there is at least partial intercalation for both 1_ and 8/,
yet neither of these peptides significantly decreases the
viscosity of a DNA-peptide solution [relative to L-lys-
L-pNO^-pheA (1) ] . The fact that there is no full intercala¬
tion for L-lys-L-S-pNO^bz-cysA (7) can be explained by two
possible effects: 1) the sulfur in the middle of the side
chain is creating a steric effect by positioning the aro¬
matic ring away from the interior of the helix; or 2) there
is insufficient electronic interaction between the aro¬
matic ring and the base pairs of DNA. This last explana¬
tion is unlikely in light of the fact that the nitrophenyl
ring of reporter molecule I (where n = 2 - 4) fully

68
intercalates. In order to resolve this dilemma, a more
in-depth study of these compounds and derivatives is re¬
quired. Furthermore, with regard to the first explana¬
tion (i.e., steric effects), model building studies were
uninformative.
In conclusion, this work has shown the partial depend¬
ence of chirality of the N-terminal amino acid a carbon and
the distance between the aromatic ring and this center of
chirality for intercalation. It has also been suggested,
though, that electronic and hydrophobic effects can also
predominate in terms of intercalation, and must be con¬
sidered in any study of the interaction of small molecules
with DNA.

CHAPTER III
EXPERIMENTAL
All starting amino acids were purchased from either
Sigma Chemical Co. or Vega Biochemicals, and were used with¬
out further purification. Salmon sperm DNA was purchased
from Worthington Biochemical Corp. Elemental analysis of
all products was performed by Atlantic Microlab, Inc.,
Atlanta, Georgia. TLC plates with fluorescent indicator
were purchased from either Kodak Chemical Co. or Scientific
Products, Inc. Melting points were taken on a Mel-Temp appa¬
ratus and the measurements are uncorrected. All solutions
containing peptides or DNA were prepared in buffers made
with deionized water.
The NMR spectra were recorded with either a Varian
A-60A or a Jeol-JNM-FX100 Fourier Transform NMR spectro¬
meter. Chemical shifts were determined relative to the
internal standard trimethylsilane (TMS) for spectra taken
with organic solvents or 2,2,3,3-d^-3-trimethylsilylpro-
prionate (TSP) for those determined in deuterium oxide.
Ultraviolet and visible absorption spectra were recorded
with a Cary-17D UV/Vis spectrophotometer. A Jasco J-20
spectropolarimeter was used to record circular dichroism
measurements. Viscosity measurements were performed with
a low shear Zimm viscometer (Beckman Instrument Co.).
69

70
Synthesis
All of the prepared dipeptides, and the required inter¬
mediates leading up to the final products, were analyzed for
purity by two or more of the following: TLC, NMR or ele¬
mental analysis. All reactions are known to proceed without
racemization. The various abbreviations used in this sec¬
tion are: 1) THF (tetrahydrofuran); 2) CBZ (carbobenzoxy);
3) t-Boc (t-butyloxycarbonyl); 4) NT (nitrotolyl); 5) DNP
(dinitropheny1); and 6) TFA (triflouroacetic acid). In some
cases, a molecule or two of water of hydration has to be in¬
cluded for the elemental analysis. This is reasonable since
the final compounds, and some of the precursors, are quite
hygroscopic.
Preparation of L-lysyl-L-pNO-,-phenylalanine amide dihydro¬
bromide, 1. “
L-pNC^-phenylalanine ethyl ester hydrochloride was syn¬
thesized by the Curtius and Goebel esterification proce¬
dure: 11 L-pNC^-phenylalanine (1.0 g, 4.7 mmol) was placed
in a 3 neck flask equipped with a magnetic stirrer, a drying
tube and a gas dispersing tube. Then, %100 mL of ethanol
was added and a stream of dry hydrogen chloride was passed
through the solution at room temperature until all the solid
dissolved. Upon dissolution, the flask was placed in an ice/
acetone bath and the bubbling continued to saturation. The
tube was removed and the stoppered flask was allowed to stand
at room temperature for 4 hours. The solution was then evap¬
orated by a stream of dry nitrogen. The solid obtained was

71
recrystallized from ethanol/ethyl ether to yield 1.1 g (4.0
mmol, 851 yield) of L-pN07-phenylalanine ethyl ester hydro¬
chloride. The NMR in D2O showed a 3H methyl triplet at
6 1.3 ppm, a 2H methylene doublet at 6 3.5 ppm, a 3H multi-
plet at 6 4.4 ppm and two 2H doublets at 6 7.6 ppm and 6.
8.3 ppm, respectively, for the aromatic protons.
Anal. calculated for C-j^H-^^O^Cl; C, 48.00 ; H, 5.46.
Found: C, 48.06; H, 5.51.
The ester was then coupled to DiCBZ-L-lysine by the
112
general method of Anderson et al. DiCBZ-L-lysine
(0.60 g, 1.4 mmol) in 20 mL freshly distilled THF was placed
in a 3 neck flask equipped with a magnetic stirrer and a
calcium sulfate drying tube. A stream of dry nitrogen was
passed over the solution to keep the atmosphere free of
moisture. The flask was then placed in an ice/acetone bath
and 1.5 mmol of triethylamine was added to the solution,
followed by 1.5 mmol of isobutylchloroformate. The solution
was allowed to stir for 15 minutes, after which time a pre¬
cooled solution of L-pNC^-phenylalanine ethyl ester hydro¬
chloride (0.40 g, 1.4 mmol) in 10 mL of 1:1 THF/DMF was
added. An additional 1.5 mmol of triethylamine was added
and the reaction was allowed to stir at -10°C for 2 hours.
Upon completion of the reaction, the ice bath was removed
and the solvent evaporated with a stream of dry nitrogen.
The resulting solid was dissolved in ethyl acetate/saturated
NaCl solution, and the organic phase was washed 3 times with
1 M HC1, 3 times with saturated sodium bicarbonate, and 2

72
times with saturated sodium chloride. The organic phase
was dried over sodium sulfate, filtered and evaporated.
The resultant solid was recrystallized from ethyl acetate/
hexane to yield 0.74 g (1.2 mmol, 86% yield) of DiCBZ-L-
lysyl-L-pNC^-phenylalanine ethyl ester. The NMR in
CDCl^ showed a broad 9H multiplet at 6 1.6 ppm, a broad 4H
multiplet at 6 3.2 ppm, a 2H methylene quartet at 5 4.2 ppm,
a broad 6H multiplet at 6 5.0 ppm, a broad 1H (NH) doublet
at 6 5.5 ppm, a broad 1H (NH) doublet at 6 6.8 ppm, a 13H
multiplet at 6 7.3 ppm, and a 2H doublet at 6 8.1 ppm.
Anal. calculated for C-^FLgN^Og•H?0; C, 60.71; H, 6.18.
Found: C, 60.80; H, 6.19.
The diprotected dipeptide ester (0.69 g, 1.1 mmol) was
dissolved in 70 mL methanol and saturated with ammonia gas
at 0°C in a high pressure bottle. The bottle was securely
corked and allowed to stand at room temperature for 48 hours.
The cork was removed and the solvent evaporated by a stream
of dry nitrogen. The solid obtained was recrystallized from
ethyl acetate/hexane to yield 0.53 g (0.89 mmol, 81% yield)
of DiCBZ-L-lysyl-L-pNC^-phenylalanine amide. The NMR in
D^-DMSO showed a broad 6H multiplet at 6 1.4 ppm, a broad 2H
multiplet at 6 3.0 ppm, a very broad 2H multiplet centered
at 6 4.4 ppm, a 4H singlet at 6 5.1 ppm, a broad 17H multi¬
plet at 6 7.4 ppm, and a 2H doublet at 6 8.2 ppm.
Anal. calculated for C3iN5°8H33 *1C, 59.97; H, 5.71.
Found: C, 59.97; H, 5.71.

73
To cleave the CBZ group, 0.60 g (1.02 mmol) of the di-
protected dipeptide amide was placed in a reaction reservoir
of the HF generator (Figure 30) with a small stir bar and
0.5 mL of anisóle. Liquid HF was placed into the main reser¬
voir and distilled, using a water jacket at 90°C, into the
reaction reservoir which had been placed in a dewar of dry
ice/acetone. After 20 mL of HF had been distilled over, the
reaction vessel was allowed to warm to 0°C and stirred in an
ice water bath for an additional 1 hour. The HF was evapo¬
rated and the residue dissolved in 0.1 M HCl/ethyl ether.
The organic layer was discarded and the aqueous layer washed
2 more times with ethyl ether. The aqueous layer was
lypholyzed.
The residue was then dissolved in 2 mL of 0.1 M NH^OAc
and placed on a CM-Sepharose (CL-6B) cation exchange column.
The column was eluted with 0.1 M NH^OAc and fractions col¬
lected. Those fractions absorbing at 295 nm were pooled and
lypholyzed. The residue was placed in a drying oven (40°C)
for 12 hours, lypholyzed, and dried in an oven for an addi¬
tional 12 hours.
The dipeptide amide dihydroacetate was dissolved in
2 mL H?0 and placed on an Amberlite CG-400 anion exchange
column (bromide form) and eluted with H?0. Fractions
absorbing at 275 nm were pooled and lypholyzed.
The residue was dissolved in MeOH, transferred to a
tared, labeled vial, evaporated and placed in a P,,0j. dessi-
cator for 48 hours. The solid was broken into a fine powder

74
Figure 30. Liquid HF Apparatus. With valve Di open and all
others closed, liquid HF is drained from the HF
tank (A) into the distillation reservoir (B).
Valve D-l is closed and valves D2 are opened,
and liquid HF is distilled over into reaction
reservoirs (C). The reaction is stirred by mag¬
netic stirrers (E) until completion. Upon com¬
pletion, valves T>2, D3, and D4 are opened and
the HF is evaporated.

and the vial placed in a drying pistol for 12 hours to
yield 133 mg (0.27 mmol, 26% yield) of L-lysyl - L-pNO,-, -
phenylalanine amide dihydrobromide. The NMR in D2O
showed a 6H multiplet at 6 1.6 ppm, a 2H methylene triplet
at 6 3.0 ppm, a 2H methylene doublet at 6 3.3 ppm, a 1H
methine triplet at 6 3.9 ppm, a 2H doublet at 6 7.5 ppm and
6 8.2 ppm, respectively, for the aromatic protons.
Anal. calculated for ^N^O^Br•1.51^0; C, 34.22 ; H, 5.32.
Found: C, 34.26; H, 5.34. M.P. 189 - 192°C.
Preparation of L-lysyl-D-pNO^-phenylalanine amide dihydro¬
bromide , 2.
The dipeptide amide was prepared in the same manner as
compound 1, but starting with D-pNC^-phenylalanine. D-pNC^-
phenylalanine (1.0 g, 4.7 mmol) was converted to 1.1 g of
D-pNO.,-phenylalanine ethyl ester hydrochloride (4.0 mmol,
851 yield) by the action of absolute ethanol and dry hydrogen
chloride. The dipeptide ester, DiCBZ-L-lysyl-D-pNO?-phenyl-
alanine ethyl ester was prepared by reacting DiCBZ-L-lysine
(0.60 g, 1.4 mmol) with 1.5 mmol triethylamine and 1.5 mmol
of isobutylchloroformate followed by addition of D-pN09-
phenylalanine ethyl ester hydrochloride (0.40 g, 1.4 mmol)
and 1.5 mmol of triethylamine. Workup and recrystallization
yielded 0.70 g (1.1 mmol, 791 yield) of DiCBZ-L-lysy1-D-pN0?-
phenylalanine ethyl ester. The NMR in CDC1, showed a
broad 9H multiplet at 6 1.6 ppm, a broad 4H multiplet at 6
3.2 ppm, a 2H quartet at 6 4.2 ppm, a broad 6H multiplet at
6 5.0 ppm, a broad 1H doublet at ó 5.5 ppm, a broad 1H doublet

76
at 5 6.8 ppm, a 13H multiplet at 6 7.3 ppm, and a 2H doublet
at 5 8.1 ppm.
Anal. calculated for C^jH^gN^Og•.5H?0; C, 61.58; H, 6.11.
Found: C, 61.56; H, 6.11.
The diprotected dipeptide ester (0.59 g, 0.93 mmol) was
converted to 0.40 g (.67 mmol, 12% yield) of DiCBZ-L-lysyl-
D-pNC^-phenylalanine amide by the action of MeOH saturated
with NHj and subsequent workup and recrystallization. The
NMR in Dg-DMSO showed a broad 6H multiplet at 6 1.4 ppm,
a broad 2H multiplet at 6 3.0 ppm, a very broad 2H multiplet
centered at 6 4.4 ppm, a 4H singlet at 6 5.1 ppm, a broad 17H
multiplet at 6 7.4 ppm, and a 2H doublet at 6 8.2 ppm.
Anal. calculated for C, 60.88; H, 5.63.
Found: C, 60.67; H, 5.68.
The diprotected dipeptide amide (0.60 g, 1.02 mmol) was
cleaved with liquid HF (20 mL) as previously described. The
lypholyzed residue obtained was passed through a CM-Sepharose
(CL-6B) column, eluting with 0.1 M NH^OAc and collecting
fractions absorbing at 275 nm as before. After lypholyza-
tion of the appropriate fractions and removal of all residual
NH^OAc, the solid was dissolved in 2 mL F^O and passed through
an Amberlite CG-400 anion exchange column (bromide form).
The fraction absorbing at 275 nm was then lypholyzed. The
resultant residue was dissolved in MeOH, transferred to a
tared vial, evaporated and placed in a P-,0,- dessicator and
drying pistol for 48 and 24 hours, respectively. The resul¬
tant powder was 375 mg of L-lysyl-D-pNO-,-phenylalanine amide

77
dihydrobromide (0.75 mmol, 73.5%). The NMR in D^O
showed a 6H multiplet at 6 1.6 ppm, a 4H multiplet at 6 3.2
ppm, a 1H methine triplet at ó 3.9 ppm, and a 2H doublet at
6 7.5 ppm and 6 8.2 ppm, respectively, for the aromatic pro¬
tons .
Anal. calculated for C-^l^ • 4Ho0; C, 31.52; H, 5.78.
Found: C, 31.50; H, 5.83. M.P. 194 - 197°C.
Preparation of L-lysyl-L-S-nitrotolyl-cysteine amide dihydro-
bromide, 3.
L-S-nitrotolyl-cysteine hydrochloride was prepared by
dissolving 4.0 g (2.55 mmol) of L-cysteine hydrochloride
in 60 mL of 1 M NaOH. The solution was cooled to 0°C and
a solution of 2.0 g (12.7 mmol) of 2-flouro-5-nitrotoluene
in 40 mL of dioxane was added over a period of 2 hours.
Stirring at 0°C was continued for 2 more hours after which
time the reaction was stirred at room temperature for 3-4
hours. The reaction mixture was washed 3 times with ethyl
ether, acidified with concentrated HC1 and evaporated. The
resultant solid was recrystallized from water to yield 2.84
g (9.7 mmol, 76.41 yield) of L-S-nitrotolyl-cysteine hydro¬
chloride. The NMR in D^-DMSO showed a sharp 3H methyl
singlet at 6 2.4 ppm, a broad 3H multiplet at 6 3.8 ppm, a
1H doublet at ó 7.6 ppm, and a 2H multiplet at 6 8.1 ppm.
Anal, calculated for C1()H13N204SC1 -F^O; C, 38.64; H, 4.83.
Found: C, 38.70; H, 4.89.
L-S-NT-cysteine hydrochloride (1.5 g, 5.1 mmol) was
esterified with ethanol saturated with dry HC1. Subsequent

78
workup and recrystallization from ethanol/ethyl ether yielded
1.3 g (4.1 mmol, 80.41 yield) of L-S-NT-cysteine ethyl ester
hydrochloride.
The NMR in D?0 showed a 3H methyl triplet at 6 1.3
ppm, a sharp 3H methyl singlet at 6 2.5 ppm, a 2H methylene
doublet at 6 3.9 ppm, a 3H multiplet at 6 4.4 ppm, a 1H
doublet at 6 7.6 ppm, and a 2H multiplet at 6 8.1 ppm.
Anal. calculated for C-^ ?H^ y^O^SCl • H?0; C, 42.60; H, 5.62.
Found: C, 42.71; H, 5.70.
Di-t-Boc-L-lysine (1.3 g, 3.7 mmol) was dissolved in
dry THF and treated with 4.0 mmol of Et^N and isobutyl-
chloroformate, respectively, followed by 1.2 g of L-S-NT-
cysteine ethyl ester (3.7 mmol) and an additional 4.0 mmol
of Et^N. Subsequent workup and recrystallization from THF/
hexane yielded 1.2 g (2.0 mmol, 54.1% yield) of Di-t-Boc-
L-lysyl-L-S-NT-cysteine ethyl ester. The NMR in CDCl^
showed a broad 27H multiplet at 6 1.4 ppm, a 3H methyl
singlet at 6 2.4 ppm, a broad 2H multiplet at ó 3.1 ppm,
a broad 2H multiplet at 6 3.5 ppm, a 3H multiplet at 6 4.2
ppm, a broad 3H multiplet at 6 5.0 ppm, a 1H (NH) doublet
at 6 7.1 ppm, a 1H doublet at 6 7.4 ppm, and a 2H multiplet
at 6 8.0 ppm.
Anal. calculated for C? gH^N^OgS; C, 54.90 ; H, 7.19.
Found: C, 54.79; H, 7.27.
The diprotected dipeptide ethyl ester was converted
to the amide by ammoniolysis of 1.0 g (1.6 mmol) of Di-t-
Boc-lysyl-L-S-NT-cysteine ethyl ester in methanol.

79
Subsequent workup and recrystallization from ethyl acetate/
hexane yielded 0.37 g (0.63 mmol, 39.4% yield) of Di-t-Boc-
L-lysyl-L-S-NT-cysteine amide. The NMR in CDC1- showed
a 24H multiplet at ó 1.4 ppm, a 3H methyl singlet at ó 2.4
ppm, a 2H multiplet at ó 3.1 ppm, a 2H multiplet at 6 3.5
ppm, a 1H methine multiplet at 6 4.1 ppm, a 1H methine multi¬
plet at 6 4.8 ppm, a 2H multiplet at 6 5.8 ppm, a 4H multi¬
plet at 6 7.3 ppm, and a 2H multiplet at 6 8.0 ppm.
Anal. calculated for C^H^N^OgS; C, 53.52; H, 7.03.
Found: C, 52.19; H, 6.96.
In the deprotection reaction, 0.23 g (0.39 mmol) of
Di-t-Boc-L-lysyl-L-S-NT-cysteine amide was treated with 30
mL of a solution of 25% TFA in CH2C12. The reaction was
allowed to proceed for 45 minutes at room temperature, and
then evaporated. The resultant residue was dissolved in
0.1 M HC1 and washed 3 times with ethyl ether. The aqueous
layer was lypholyzed to yield a solid which was subsequently
dissolved in 2 mL H20 and passed through an Amberlite CG-400
anion exchange column (bromide form). The fraction absorbing
at 275 nm was lypholyzed to yield a solid that was pure by
TLC. The solid was dissolved in methanol, and the methanol
solution was transferred to a tared vial and evaporated.
The vial was placed in a P20,- dessicator for 48 hours, and
the resultant solid broken into a fine powder. The solid
was identified by NMR and elemental analysis as 0.17 g (0.31
mmol, 79.5% yield) of L-lysyl-L-S-NT-cysteine amide dihydro¬
bromide. The NMR in D20 showed a 6H multiplet at 6 1.5

80
ppm, a 3H methyl singlet at 6 2.4 ppm, a 2H multiplet at 6
2.9 ppm, a 2H multiplet at 6 3.6 ppm, a 1H methine triplet
at 6 4.1 ppm, a 1H doublet at ó 7.5 ppm, and a 2H multiplet
at 6 8.1 ppm.
Anal. calculated for C-^gH2yN^O^SBr7*1.5H20; C, 32.43 ; H, 5.07.
Found: C, 32.63; H, 5.48. M.P. 209 - 211°C.
Preparation of L-lvsyl-D-S-nitrotolyl-cysteine amide dihydro¬
bromide , £.
D-S-nitrotolyl-cysteine hydrochloride was prepared
from 4.0 g (25.5 mmol) of D-cysteine hydrochloride and 2.0
g (12.7 mmol) of 2-flouro-5-nitrotoluene as previously des¬
cribed. The resultant solid was recrystallized from water
to yield 1.9 g (6.5 mmol, 51.21 yield) of D-S-NT-cysteine
hydrochloride. The NMR in D^-DMSO showed a sharp 3H
methyl singlet at 6 2.4 ppm, a broad 3H multiplet at ó 3.8
ppm, a 1H doublet at 6 7.6 ppm, and a 2H multiplet at 6 8.1
ppm.
Anal, calculated for C^QH-^NyO^SCl*; C, 38.64; H, 4.83.
Found: C, 38.66; H, 4.89.
D-S-NT-cysteine hydrochloride (1.7 g, 5.8 mmol) was
esterified with ethanol/HCl to yield, upon recrystallization
from ethanol/ethyl ether, 1.4 g (4.4 mmol, 75.9% yield) of
D-S-NT-cysteine ethyl ester hydrochloride. The NMR in
D2O showed a 3H methyl triplet at 6 1.3 ppm, a 3H methyl
singlet at 6 2.5 ppm, a 2H methylene doublet at 6 3.9 ppm,
a 3H multiplet at 6 4.4 ppm, a 1H doublet at 6 7.6 ppm, and
a 2H multiplet at 6 8.1 ppm.

81
Anal. calculated for C^?H^ ^^O^SCl ‘H^O; C, 42.60; H, 5.62.
Found: C, 42.53; H, 5.68.
D-S-NT-cysteine ethyl ester hydrochloride (1.2 g, 3.7
mmol) was coupled to Di-t-Boc-L-lysine (1.3 g, 3.8 mmol)
using 3.9 mmol of Et,N and isobutylchloroformate, respec¬
tively, in the mixed anhydride reaction. Subsequent workup
and recrystallization from ethyl acetate/hexane yielded 1.5
g (2.5 mmol, 67.61 yield) of Di-t-Boc-L-lysy1-D-S-NT-
cysteine ethyl ester. The NMR in CDCl^ showed a broad
27H multiplet at 6 1.4 ppm, a 3H methyl singlet at 6 2.4
ppm, a broad 2H multiplet at 6 3.1 ppm, a broad 2H multiplet
at 6 3.5 ppm, a 3H multiplet at 6 4.2 ppm, a broad 3H
multiplet at 6 5.0 ppm, a 1H (NH) doublet at 6 7.1 ppm, a
1H doublet at 6 7.6 ppm, and a 2H multiplet at 5 8.0
ppm.
Anal. calculated for ’ C, 54.90; H, 7.19.
Found: C, 54.65; H, 7.27.
Ammoniolysis of 1.3 g (2.1 mmol) of Di-t-Boc-L-lysyl-
D-S-NT-cysteine ethyl ester, and subsequent recrystalliza¬
tion, yielded 300 mg (0.51 mmol, 24.3% yield) of Di-t-Boc-
L-lysyl-D-S-NT-cysteine amide. The NMR in CDCl^ showed
a 24H multiplet at 6 1.4 ppm, a 3H methyl singlet at 6 2.4
ppm, a 2H multiplet at 6 3.1 ppm, a 2H multiplet at 6 3.5
ppm, a 1H methine multiplet at 6 4.1 ppm, a 1H methine
multiplet at ó 4.8 ppm, a 2H multiplet at 6 5.8 ppm, a 4H
multiplet at 6 7.3 ppm, and a 2H multiplet at 6 8.0 ppm.
The TLC on this compound indicated that it was only on the

82
order of 95% pure; therefore, no elemental analysis was
obtained, and the compound was cleaved without further
purification.
For the cleavage reaction, 200 mg (0.34 mmol) of Di-
t-Boc-L-lysyl-D-S-NT-cysteine amide was treated with TFA/
CF^C^ as before. After the reaction was completed, the
solvent was evaporated and the resultant residue was dis¬
solved in 0.1 M HC1, washed 3 times with ethyl ether, and
the aqueous layer lypholyzed. The solid obtained was dis¬
solved in 1 mL of Ho0 and passed through an Amberlite
CG-400 anion exchange column (bromide form). The fraction
absorbing at 275 nm was lypholyzed to yield a solid that
was pure by TLC, and was identified by NMR as 75 mg (0.13
mmol, 38.2% yield) of L-lysyl-D-S-NT-cysteine amide di¬
hydrobromide. The NMR in D2O showed a 6H multiplet at
6 1.6 ppm, a sharp 3H methyl singlet at 6 2.4 ppm, a 2H
multiplet at 6 2.9 ppm, a 2H multiplet at 6 3.6 ppm, a 1H
methine triplet at 6 4.1 ppm, a 1H doublet at 6 7.5 ppm,
and a 2H multiplet at 6 8.1 ppm.
Anal, calculated for • . 5H90; C, 33.45 ; H, 4.88.
Found: C, 33.46; H, 5.36. M.P. 214 - 217°C.
Preparation of L-lysyl-L-DNP-diaminobutyric acid amide
dihydrobromide, TT
L-N,y-DNP-diaminobutyric acid was synthesized according
113
to the general method of Sanger. L-diaminobutyric acid
dihydrochloride (2.0 g, 10.5 mmol) was dissolved in 75 mL
hot water and excess CuC0~ (5.0 g, 40.7 mmol) was added.

83
The solution was filtered and the volume reduced to 30 mL;
NaHCO- (5.7 g, 68.7 mmol) was added followed by dinitro-
flourobenzene (6.0 g, 32.2 mmol) in 20 mL ethanol. The
solution was stirred for 2 hours at room temperature, during
which time a green precipitate formed, which was filtered
and dried. The solid was subsequently dissolved in 100 mL
of 1 M HC1, followed by addition of thioacetamide (7.8 g,
10.4 mmol) dissolved in water. The solution was heated
for 1/2 hour on a steam bath to precipitate CuS. Activated
charcoal was added and the solution filtered. The filtrate
was evaporated and the solid obtained recrystallized from
1 M HC1 to yield 1.4 g (4.4 mmol, 421 yield) of L-N,y-DNP-
diaminobutyric acid hydrochloride. The NMR in D^-DMSO
showed a broad 2H multiplet at 6 2.3 ppm, a broad 3H multi-
plet at 6 3.9 ppm, a 1H doublet at 6 7.5 ppm, and a broad 4H
multiplet at 6 9.0 ppm.
Anal. calculated for C^gH^N^OgCl; C, 37.44 ; H, 4.08;
N, 17.47. Found: C, 37.40; H, 4.12; N, 17.45.
L-N,y-DNP-diaminobutyric acid (1.0 g, 3.1 mmol) was
converted to the ester via the normal procedure utilizing
absolute ethanol saturated with dry hydrogen chloride.
After evaporating the solvent, the resultant solid was re¬
crystallized from ethanol/ethyl ether to yield 0.82 g
(2.4 mmol, 77.41 yield) of L-N,y-DNP-diaminobutyric acid
ethyl ester hydrochloride. The NMR in D2O showed a
3H methyl triplet at 6 1.3 ppm, a 2H methylene quartet at
6 2.4 ppm, a 2H methylene triplet at 6 3.8 ppm, a 3H

84
multiplet at 6 4.4 ppm, a 1H doublet at 6 7.3 ppm, a 1H
doublet of doublets at 6 8.4 ppm, and a broad 1H doublet
at 6 9.0 ppm.
Since the addition of the DNP group could go to either
the a amine nitrogen or the y amine nitrogen, it was neces¬
sary to prove that only the y amine group reacted. The
ester hydrochloride was treated with aqueous NaHCCU and
extracted into CDC1-. The NMR revealed a 3H methyl
triplet at 6 1.3 ppm, a 4H multiplet at 6 2.1 ppm, a 3H
multiplet at 6 3.7 ppm, a 2H methylene quartet at 6 4.2
ppm, a 1H doublet at 6 7.0 ppm, a 1H doublet of doublets
at 6 8.3 ppm, and a broad 2H doublet at 5 9.0 ppm.
In the protonated ester, the a proton was buried under
the 3H multiplet at 6 4.4 ppm, with the other two protons
coming from the ester ethyl methylene. The y methylene was
a 2H triplet at 6 3.8 ppm in the protonated ester. Upon
neutralization of the positive charge, only one proton,
namely the a proton, shifted (from 4.4 ppm to 3.7 ppm),
thus indicating that the addition of the DNP group did not
occur at the a amine group. Furthermore, the y methylene
did not shift significantly, indicating that addition did
occur on the y amine group.
Anal. calculated for C-pH^N^O^-Cl; C, 41.32; H, 4.88.
Found: C, 39.46; H, 4.83.
The TLC and elemental analysis indicated that this
compound was only on the order of 95% pure; however, it was
used without further purification. L-N,y-DNP-diaminobutyric

85
acid ethyl ester (0.82 g, 2.4 mmol) was converted to the
amide through the use of methanol saturated with ammonia.
Workup and recrystallization gave 0.52 g (1.6 mmol, 67%
yield) of L-N,y-DNP-diaminobutyric acid amide hydrochloride.
The NMR in D^-DMSO/D^O showed a broad 2H multiplet at 6
2.1 ppm, a broad 3H multiplet at 6 3.8 ppm, a broad 1H
doublet at 6 7.3 ppm, a 1H doublet of doublets at 6 8.3
ppm, and a 1H doublet at 6 9.1 ppm.
Anal. calculated for C^qH-^^N^O^CI *H?0; C, 35.55; H, 4.74.
Found: C, 35.47; H, 4.63.
The L-N,y-DNP-diaminobutyric acid amide hydrochloride
was then coupled to DiCBZ-L-lysine by the normal procedure.
DiCBZ-L-lysine (0.32 g, 0.77 mmol) was dissolved in THF
and 0.78 mmol of Et~N and 0.78 mmol of isobutylchloroformate
added. A precooled solution of L-N,y-DNP-diaminobutyric
acid amide hydrochloride (0.25 g, 0.77 mmol) in DMF/THF was
added followed by an additional 0.78 mmol of Et^N. Subse¬
quent workup and recrystallization afforded 0.25 g (0.37
mmol, 48% yield) of DiCBZ-L-lysyl-L-DNP-diaminobutyric acid
amide. The NMR in D^-DMSO showed a broad 8H multiplet
at 6 1.4 ppm, a broad 4H multiplet at 6 3.0 ppm, a very
broad 2H multiplet at 6 4.1 ppm, a 4H singlet at 6 5.1 ppm,
a broad 15H multiplet at 6 7.4 ppm, a very broad 2H multi¬
plet at 6 8.2 ppm, and a broad 2H doublet at ó 9.1 ppm.
Anal, calculated for C.,9H_yNyO^Q ; C, 56.55; H, 5.49.
Found: C, 56.43; H, 5.53.

86
The diprotected dipeptide amide (200 mg, 29 mmol) was
stirred in 50 mL acetic acid that had been saturated with
dry hydrogen bromide (351 HBr by weight) for 2 hours. The
solution was poured into 150 mL of dry ethyl ether and
placed in the refrigerator overnight. The precipitate was
filtered and recrystallized from MeOH/ethyl ether to yield
100 mg (0.17 mmol, 58.91 yield) of L-lysyl-L-DNP-diamino-
butyric acid amide dihydrobromide. The NMR in D2O
showed a broad 8H multiplet at 6 1.6 ppm, a 2H methylene
triplet at 6 3.0 ppm, a 2H methylene triplet at ó 3.6 ppm,
a 1H methine triplet at ó 4.0 ppm, a 1H doublet at 6 7.1
ppm, a 1H doublet of doublets at 6 8.3 ppm, and a 1H doublet
at 6 9.1 ppm.
Anal. calculated for ^NyO^B^•; C, 30.62; H, 5.31.
Found: C, 30.62; H, 5.33. M.P. 170 - 173°C.
Preparation of L-lysyl-D,L-DNP-diaminobutyric acid amide
dihydroacetate, 61.
The DNP derivative of D,L-diaminobutyric acid was pre¬
pared as described previously. D,L-diaminobutyric acid di¬
hydrochloride (5.0 g, 26.2 mmol) was dissolved in hot water
and treated with excess CuCO^ and NaHCO^- Then, 2,4-dinitro-
flourobenzene (15.0 g, 80.6 mmol) in ethanol was added.
The greenish solid obtained was dissolved in HC1 and thio-
acetamide (2.0 g, 26.6 mmol) was added. After filtering
and evaporation, the yellow solid obtained was recrystal¬
lized from 1 M HC1 to yield 5.32 g (16.6 mmol, 63.41 yield)
of D,L-N,y-DNP-diaminobutyric acid. The NMR in D,-DMSO
6

87
showed a broad 2H multiplet at 6 2.3 ppm, a broad 3H multi-
plet at 6 3.9 ppm, a 1H doublet at 5 7.5 ppm, and a broad
4H multiplet at 6 9.0 ppm.
Anal. calculated for C-^qH^ ^N^O^Cl; C, 37.44; H, 4.08.
Found: C, 37.40; H, 4.12.
The D,L-N,y-DNP-diaminobutyric acid hydrochloride
(4.5 g, 14.0 mmol) was treated with absolute ethanol satu¬
rated with dry hydrogen chloride. Workup and recrystalliza¬
tion from ethanol/ethyl ether yielded 4.8 g (13.2 mmol,
94% yield) of D,L-N,y-DNP-diaminobutyric acid ethyl ester
hydrochloride. The NMR in D?0 showed a 3H triplet at
6 1.3 ppm, a 2H quartet at 6 2.4 ppm, a 2H triplet at 6
3.8 ppm, a 3H multiplet at 6 4.4 ppm, a 1H doublet at 6
7.3 ppm, a 1H doublet of doublets at 6 8.4 ppm, and a broad
1H doublet at 6 9.0 ppm.
Anal. calculated for Cig^yN^O^Cl; 41.38; H, 4.92.
Found: C, 41.39; H, 4.94.
The D,L-N,y-DNP-diaminobutyric acid ethyl ester hydro¬
chloride (0.30 g, 0.86 mmol) was converted to 0.21 g (0.66
mmol, 771 yield) of D,L-N,y-DNP-diaminobutyric acid amide
hydrochloride by the action of methanol saturated with
ammonia and subsequent workup and recrystallization from
methanol/ethyl ether. The '*‘H NMR in Dg-DMS0/D?0 showed a
broad 2H multiplet at 6 2.1 ppm, a broad 3H multiplet at 6
3.8 ppm, a broad 1H doublet at 6 7.3 ppm, a 1H doublet of
doublets at 6 8.3 ppm, and a 1H doublet at 6 9.1 ppm.

Anal. calculated for C-^qH^^N^O^CI • 2H?0; C, 33.73; H, 5.10.
Found: C, 33.55; H, 4.70.
For the coupling reaction, DiCBZ-L-lysine (0.39 g,
0.94 mmol) was dissolved in THF and 1.0 mmol of Et^N and
1.0 mmol of isobutylchloroformate added. D,L-N,y-DNP-
diaminobutyric acid amide hydrochloride (0.30 g, 0.94
mmol) in DMF/THF was added followed by an additional 1.0
mmol of Et^N. Subsequent workup and recrystallization
yielded 0.36 g (0.53 mmol, 561 yield) of DiCBZ-D,L-DNP-
diaminobutyric acid amide. The NMR in D^-DMSO showed
a broad 8H multiplet at 6 1.4 ppm, a broad 4H multiplet at
6 3.0 ppm, a very broad 2H multiplet at 6 4.1 ppm, a 4H
singlet at 6 5.1 ppm, a broad 15H multiplet at 6 7.4 ppm,
a very broad 2H multiplet at 6 8.2 ppm, and a broad 2H
doublet at 6 9.1 ppm.
Anal, calculated for C_?H_; C, 56.55; H, 5.45.
Found: C, 57.29; H, 5.74.
The diprotected dipeptide amide was deprotected by
the action of 20 mL of liquid HF on DiCBZ-L-lysyl-D,L-DNP-
diaminobutyric acid amide (0.90 g, 1.3 mmol). After
lypholyzation, the residue was dissolved in 2 mL of 0.2 M
NH^OAc and placed on a CM-Sepharose (CL-6B) cation exchang
column and eluted with 0.2 M NH^OAc. The fractions ab¬
sorbing at 480 nm were pooled and lypholyzed. After re¬
peated drying and lypholyzation, 150 mg of L-lysyl-D,L-DNP
diaminobutyric acid amide dihydroacetate (0.28 mmol, 21.51
yield) was obtained. The NMR in Do0 showed a broad 8H

89
multiplet at 6 1.6 ppm, a 2H methylene triplet at 6 3.0 ppm,
a 2H methylene triplet at 6 3.6 ppm, a 1H methine triplet at
6 4.0 ppm, a 1H doublet at 6 7.1 ppm, a 1H doublet of doub¬
lets at 6 8.3 ppm, a'nd a 1H doublet at 6 9.1 ppm.
Anal, calculated for .SH^O; C, 44.44; H, 6.30.
Found: C, 44.30; H, 6.38.
Preparation of L-lysyl-L-S-p-nitrobenzylcysteine amide di¬
hydroacetate,
The method developed by Berse et al.^^ was used to
prepare L-S-p-nitrobenzylcysteine. L-cysteine hydrochloride
(3.14 g, 20 mmol) was dissolved in 60 mL of 1 M NaOH at
0°C. Over a period of 30 minutes, a-chloro-p-nitrotoluene
(1.71 g, 10 mmol) in 30 mL dioxane was slowly added. The
reaction was then stirred for an additional 30 minutes at
room temperature. The reaction mixture was then washed 3
times with ethyl ether, acidified with concentrated HC1
and then the solvent evaporated. The resultant solid was
recrystallized from water to yield 2.0 g (8.0 mmol, 80%
yield) of L-S-p-nitrobenzylcysteine. The NMR in D^-DMSO
showed a 2H multiplet at 6 3.0 ppm, a 3H multiplet at 6 4.0
ppm, and a 2H doublet at 6 7.7 ppm and 6 8.2 ppm, respec¬
tively, for the aromatic protons.
Anal. calculated for •! . 51^0; C, 42.40 ; H, 5.30.
Found: C, 42.40; H, 5.35.
L-S-p-nitrobenzylcysteine (2.3 g, 9 mmol) was dissolved
in 10 mL l^O and 19 mmol of Et^N was added. To this solution,
Boc-On^^ (2.1 g, 9 mmol) in 15 mL dioxane was added and the

90
reaction allowed to stir for 3 hours at room temperature.
Excess water was added, and the solution was washed 3 times
with ethyl acetate, acidified with 0.5 M citric acid and
extracted 3 times with ethyl acetate. The organic layer
was dried over Na2SO^, filtered and evaporated to yield
1.4 g (4.0 mmol, 44.41 yield) of an oil identified by
NMR in CDC1- to be N-t-Boc-S-p-nitrobenzylcysteine. The
NMR in CDClj showed a sharp 9H t-Boc singlet at 6 1.5
ppm, a broad 2H methylene doublet at 6 2.9 ppm, a sharp
2H methylene singlet at 5 3.9 ppm, a broad 1H methine
multiplet at 5 4.5 ppm, a broad 1H (NH) multiplet at 6
5.6 ppm, a 2H doublet at 6 7.5 ppm and 6 8.2 ppm, respec¬
tively, and a broad 1H (COOH) singlet at 6 8.7 ppm.
N-t-Boc-S-p-nitrobenzylcysteine (1.7 g, 4.8 mmol) was
dissolved in THF, and 5 mmol of Et_N and isobutylchlorofor-
mate, respectively, were added at -10°C and allowed to stir
for 1 hour. A precooled solution of MeOH saturated with
ammonia (.5 g in 20 mL MeOH) was added and the reaction
mixture was stirred an additional 2 hours. Workup and re¬
crystallization from ethyl acetate/hexane yielded 1.0 g
(2.8 mmol, 58.3% yield) of N-t-Boc-S-p-nitrobenzylcysteine
amide. The NMR in CDCl^ showed a 9H singlet at 6 1.5
ppm, a 2H doublet at 6 2.9 ppm, a 2H singlet at 6 3.9 ppm,
a broad 1H multiplet at 6 4.3 ppm, a broad 1H multiplet at
6 5.4 ppm, a broad 4H multiplet at 6 7.5 ppm, and a 2H
doublet at 6 8.2 ppm.
Anal. calculated for C^^H9-^N„0^S; C, 50.70; H, 5.91.
Found: C, 50.68; H, 5.97.

91
N-t-Boc-S-p-nitrobenzylcysteine amide (0.8 g, 2.2 mmol)
was dissolved in 60 mL of 25% TFA in CH9C1? and stirred for
1 hour at room temperature. The solvent was evaporated;
then, the residue was dissolved in saturated NaHCO^ and ex¬
tracted 2 times with ethyl acetate. The organic layer was
then washed 3 times with saturated NaCl solution, dried over
Na2S0^, filtered and evaporated to yield 0.4 g (1.6 mmol,
72.7% yield) of an oil identified by NMR as L-S-p-nitro-
benzylcysteine amide. The NMR in CDCl^ showed a 2H
amine singlet at 6 1.7 ppm, a 2H methylene doublet at 6
2.9 ppm, a 1H methine multiplet at 6 3.5 ppm, a 2H methylene
singlet at 6 3.9 ppm, a 4H multiplet at 6 7.5 ppm, and a 2H
doublet at 6 8.2 ppm.
L-S-p-nitrobenzylcysteine amide (0.20 g, 0.78 mmol)
was coupled to Di-t-Boc-L-lysine with the aid of 0.79 mmol
of Et^N andisobutylchloroformate by the mixed anhydride
method. After evaporation of the solvent, the residue was
dissolved in ethyl acetate/saturated NaCl solution and the
organic phase was washed 3 times with 0.5 M citric acid,
saturated NaHCO^ and saturated NaCl. The organic layer was
then dried over Na2S0^, filtered and evaporated to give a
solid which was recrystallized from ethyl acetate/hexane to
yield 0.40 g (0.69 mmol, 88.5% yield) of Di-t-Boc-L-lysyl-
L-S-p-nitrobenzylcysteine amide. The NMR in CDC1, showed
a 24H multiplet at 6 3.0 ppm, a 3H multiplet at 6 3.7 ppm,
a 2H multiplet at 6 4.6 ppm, a broad 1H (NH) doublet at 6 5.4
ppm, a broad 1H (NH) doublet at 6 5.6 ppm, a broad 2H (NH2)

92
multiplet at 5 7.0 ppm, and a 2H doublet at 5 7.4 ppm and
6 8.1 ppm, respectively, for the aromatic protons. The
TLC of this compound showed that it was only on the order
of 95% pure; therefore, no elemental analysis was obtained
and it was cleaved without further purification.
For the cleavage reaction, 0.30 g (0.51 mmol) was dis¬
solved in 25% TFA in CH?C1? and stirred for 1 hour at room
temperature. The solvent was evaporated and the residue
dissolved in 0.1 M HC1. The aqueous layer was washed 3 times
with ethyl ether and lypholyzed. The resultant solid was
dissolved in 2 mL 0.1 M NH^OAc and placed on a CM-Sepharose
(CL-6B) cation exchange column. The column was eluted with
a linear gradient from 0.1 M to 0.15 M NH^OAc, followed by
0.15 M NH^OAc. The second fraction absorbing at 275 nm
was lypholyzed, dried in the oven for 48 hours, relypholyzed
and dried again. The resultant residue was dissolved in
methanol and the methanolic solution placed in a preweighed
vial and evaporated. The vial was then placed in a
dessicator for 24 hours and then in a drying pistol for an
additional 24 hours. The resultant solid was 40 mg (8.0 x
- 2
10 mmol, 15.6% yield) of L-lysyl-L-S-p-nitrobenzylcysteine
amide dihydroacetate. The NMR in D^O showed a broad 12H
multiplet at 6 1.7 ppm, a 4H multiplet at 5 3.0 ppm, a 3H
multiplet at 6 3.8 ppm, and a 2H doublet at 6 7.4 ppm and
6 8.2 ppm, respectively.
Anal, calculated for ConH^,Nr0oS•.5Ho0; C, 46.88; H, 6.64.
ZU ¿o bo Z
Found: C, 46.88; H, 7.00.

93
Preparation of L-lysyl-D-S-p-nitrobenzylcysteine amide di¬
hydroacetate, 8.
D-S-p-nitrobenzylcysteine hydrochloride was prepared
from D-cysteine hydrochloride (2.0 g, 12.7 mmol) and
a-chloro-p-nitrotoluene (1.09 g, 6.4 mmol) as before.
Recrystallization from H.,0 yielded 1.4 g (4.8 mmol, 751
yield) of D-S-p-nitrobenzylcysteine hydrochloride. The
NMR in D^-DMSO showed a 2H multiplet at 6 3.0 ppm, a 3H
multiplet at 6 4.0 ppm, a 2H doublet at 6 7.7 ppm, and a
2H doublet at 6 8.2 ppm.
Anal. calculated for C-^qH-^^O^SCI ; C, 41.02; H, 4.44.
Found: C, 41.53; H, 4.58.
D-S-p-nitrobenzylcysteine hydrochloride (3.5 g, 12
mmol) was treated with the Boc-On reagent (2.9 g, 12 mmol)
as previously described. Subsequent workup yielded 1.2 g
(3.4 mmol, 28.3% yield) of an oil identified by NMR as
N-t-Boc-S-p-nitrobenzylcysteine. The ''‘H NMR in CDCl^
showed a sharp 9H singlet at 6 1.5 ppm, a broad 2H doublet
at 6 2.9 ppm, a 2H singlet at 6 3.9 ppm, a broad 1H multi¬
plet at 6 4.5 ppm, a broad 1H multiplet at 6 5.6 ppm, a 2H
doublet at 6 7.5 ppm, a 2H doublet at 6 8.2 ppm, and a
broad 1H singlet at 6 8.9 ppm.
D-N-t-Boc-S-p-nitrobenzylcysteine (1.2 g, 3.4 mmol)
was dissolved in THF and treated with 3.5 mmol of Et^N
and isobutylchloroformate, respectively, at -10°C. A pre¬
cooled solution of methanol saturated with NH^ was added
and the reaction allowed to proceed. Subsequent workup

94
and crystallization yielded 0.61 g (1.7 mmol, 50% yield)
of D-N-t-Boc-S-p-nitrobenzylcysteine amide. The NMR
in CDCl^ showed a sharp 9H singlet at 6 1.5 ppm, a broad
2H doublet at 6 2.9 ppm, a 2H singlet at 6 3.9 ppm, a
broad 1H multiplet at 6 4.3 ppm, a broad 1H multiplet at
6 5.4 ppm, a broad 4H multiplet at 6 7.5 ppm, and a 2H
doublet at 6 8.2 ppm.
Anal. calculated for C, 50.70; H, 5.91.
Found: C, 50.81, H, 5.98.
D-t-Boc-S-p-nitrobenzylcysteine amide (0.50 g, 1.4
mmol) was treated with 25% TFA/CH?C1? for 1 hour, followed
by evaporation of the solvent. The residue was dissolved
in ethyl acetate and the organic layer was washed 3 times
with saturated NaHCCU and saturated NaCl, then dried over
Na2SO^ and evaporated to yield 0.24 g (0.94 mmol, 67.1%
yield) of an oil identified by NMR as D-S-p-nitrobenzyl-
cysteine amide. The NMR in CDCl, showed a 2H singlet
at 6 1.7 ppm, a 2H doublet at 6 2.9 ppm, a 1H multiplet
at 6 3.5 ppm, a 2H singlet at 6 3.9 ppm, a 4H multiplet
at 6 7.5 ppm, and a 2H doublet at 6 8.2 ppm.
For the coupling reaction, Di-t-Boc-L-lysine (0.27 g,
0.78 mmol) was treated with 7.9 mmol of Et^N and isobutyl-
chloroformate, respectively, followed by 0.20 g (0.78 mmol)
of D-S-p-nitrobenzylcysteine amide. Subsequent workup and
recrystallization yielded 0.25 g (0.43 mmol, 55.1% yield)
of Di-t-Boc-L-lysyl-D-S-p-nitrobenzyleysteine amide. The
NMR in CDCl^ showed a 24K multiplet at 6 1.5 ppm, a 4H

95
multiplet at 6 3.0 ppm, a 3H multiplet at 6 3.7 ppm, a 2H
multiplet at 5 4.6 ppm, a broad 1H doublet at 6 5.4 ppm,
a broad 1H doublet at 6 5.6 ppm, a broad 2H multiplet at
6 7.0 ppm, a 2H doublet at 6 7.4 ppm, and a 2H doublet at
S 8.1 ppm.
Anal. calculated for C^^H^^N^OgS; C, 53.52; H, 7.03.
Found: C, 53.65; H, 7.08.
The diprotected dipeptide amide (0.20 g, 0.34 mmol)
was subjected to cleavage by TFA/CHgCl^ for 1 hour. The
solvent was evaporated and the residue dissolved in 0.1
M HC1. The aqueous layer was washed 3 times with ethyl
ether and lypholyzed. The resultant solid was dissolved
in 2 mL of 0.1 M NH^OAc and placed on a CM-Sepharose (CL-6B)
cation exchange column. The column was eluted with a linear
gradient from 0.1 M to 0.15 M NH^OAc, followed by 0.15 M
NH^OAc. The fraction absorbing at 275 nm was lypholyzed.
After complete removal of all excess NH^OAc, the solid was
dissolved in methanol, and the methanol was placed in a
tared vial and evaporated. The vial was placed in a P?0g
dessicator for 48 hours and a drying pistol for 24 hours.
_ 2
The resultant solid was 45 mg (9.0 x 10 mmol, 26.51
yield) of L-lysyl-D-S-p-nitrobenzylcysteine amide dihydro¬
acetate. The NMR in D-,0 showed a broad 12H multiplet
at 6 1.7 ppm, a 4H multiplet at 6 3.0 ppm, a 3H multiplet
at <5 3.8 ppm, a 2H doublet at 6 7.4 ppm, and a 2H doublet
at 5 8.2 ppm.

96
Anal, calculated for ConH_7Nc0oS; C, 47.71; H, 6.56.
ZU bo o o
Found: C, 47.50; H, 6.64.
Preparation of L-lysyl-L-DNP-ornithine amide dihydrobro-
mide, 9_.
L-N,6-DNP-ornithine hydrochloride was prepared by the
117
general method of Sanger. J L-ornithine hydrochloride
(2.0 g, 11.9 mmol) was dissolved in hot water and treated
with excess CuCO^ (60.0 mmol), and excess NaHCO, (0.10 mol),
followed by an excess of DNFB (36 mmol) in EtOH. The fil¬
tered solid was dissolved in 1 M HC1 and treated with excess
thioacetamide (60 mmol). The Cu9S formed was filtered with
the aid of activated charcoal, and the filtrate was purged
for several hours with N? and then evaporated. The solid
obtained was recrystallized from 1 M HC1 to yield 1.6 g
(4.8 mmol, 40.3% yield) of L-N,6-DNP-ornithine hydrochloride.
The "^H NMR in D^-DMSO showed a broad 4H multiplet at 6 2.0
ppm, a broad 2H methylene multiplet at 6 3.6 ppm, a broad
1H methine multiplet at 6 4.1 ppm, a 1H doublet at ó 7.4
ppm, a 1H doublet of doublets at 6 8.4 ppm, and a 2H doub¬
let at 5 9.1 ppm.
Anal. calculated for C^^H^^N^O^Cl'H?0; C, 37.45; H, 4.82.
Found: C, 37.56; H, 4.90.
The DNP-amino acid was then converted to the ester
by treating L-N,6-DNP-ornithine hydrochloride (1.4 g, 4.2
mmol) with dry ethanol saturated with HC1 gas. Workup and
recrystallization from ethanol/ethyl ether gave 1.3 g (3.6
mmol, 85.7% yield) of L-N,6-DNP-ornithine ethyl ester

97
hydrochloride. The NMR in D?0 showed a 3H methyl triplet
at 6 1.2 ppm, a broad 4H multiplet at 6 1.9 ppm, a broad 2H
methylene multiplet at 6 3.5 ppm, a broad 3H multiplet at
6 4.2 ppm, a 1H doublet at 6 7.4 ppm, a 1H doublet of doub¬
lets at 6 8.5 ppm, and a 1H doublet at 6 9.1 ppm.
Anal. calculated for ,H-^gN^OgCl; C, 43.03; H, 5.24.
Found: C, 42.98; H, 5.30.
L-N,S-DNP-ornithine ethyl ester hydrochloride (1.3 g,
3.6 mmol) was converted to the amide by the action of
ammonia saturated methanol. Workup and recrystallization
from ethanol/ether yielded 0.94 g (2.8 mmol, 77.8% yield)
of L-N , 6 - DNP-ornithine amide hydrochloride. The NMR in
D2O showed a broad 4H multiplet at 6 2.0 ppm, a broad 2H
multiplet at 6 3.6 ppm, a broad 1H methine multiplet at 6
4.2 ppm, a 1H doublet at 6 7.2 ppm, a 1H doublet of doublets
at 6 8.3 ppm, and a 1H doublet at 6 8.9 ppm.
Anal. calculated for C^-^H^^NgOgCl • . 5H90; C, 38.52 ; H, 4.96.
Found: C, 38.66; H, 4.73.
For the coupling reaction, DiCBZ-L-lysine (0.62 g,
1.5 mmol) in THF was treated with 1.65 mmol Et^N and 1.65
mmol of isobutylchloroformate. To this solution was added
L-N,5-DNP-ornithine amide hydrochloride (.5 g, 1.5 mmol) in
DMF/THF, followed by an additional 1.65 mmol of Et^N. Upon
completion of the reaction, the subsequent workup and re¬
crystallization from ethyl acetate/ether yielded 0.70 g
(0.10 mmol, 66.71 yield) of DiCBZ-L-lysyl-L-DNP-ornithine
amide. The NMR in D^-DMSO showed a broad 10H multiplet

98
at 6 1.6 ppm, a broad 2H methylene multiplet at 6 3.0 ppm,
a broad 2H methylene multiplet at 6 3.5 ppm, a very broad
2H multiplet centered at 6 4.2 ppm, a 4H singlet at 6 5.1
ppm, a 15H multiplet at 6 7.4 ppm, a broad 2H multiplet at
ó 8.3 ppm, and a broad 2H multiplet at 6 9.1 ppm.
Anal. calculated for C^NyO-j, qH^q ; C, 56.69; H, 5.77.
Found: C, 56.68; H, 5.72.
DiCBZ-L-lysyl-L-DNP-ornithine amide (500 mg, 0.72 mmol)
was stirred in acetic acid saturated with dry HBr at room
temperature for 1 hour. The solvent was evaporated and
the residue dissolved in water and lypholyzed. The solid
obtained was recrystallized from ethanol/ethyl ether to
yield 200 mg (0.34 mmol, 47.2% yield) of L-lysyl-L-DNP-
ornithine amide dihydrobromide. The NMR in Do0 showed
a broad 10H multiplet at 6 1.6 ppm, a 2H methylene triplet
at 6 3.0 ppm, a 2H methylene triplet at 6 3.6 ppm, a 1H
methine triplet at 6 4.0 ppm, a 1H doublet at 6 7.1 ppm,
a 1H doublet of doublets at 6 8.3 ppm, and a 1H doublet at
6 9.1 ppm.
Anal. calculated for C-j. yF^gNyO^B^ *H?0; C, 33.72; H, 5.12.
Found: C, 33.86; H, 4.88. M.P. 174 - 176°C.
Preparation of L-lysyl-D-DNP-ornithine amide dihydrobro¬
mide , 10 .
D-N,6-DNP-ornithine hydrochloride was prepared as pre¬
viously described from treating D-ornithine hydrochloride
(2.0 g, 12 mmol) with excess CuCO^ (60 mmol) and NaHCO^
(100 mmol) followed by addition of 36 mmol of DNFB.

99
The filtered solid was treated with 60 mmol of thioacetamide.
Subsequent filtration, evaporation, and recrystallization from
1 M HC1 yielded 1.6 g (4.8 mmol, 40.3% yield) of D-N,6-DNP-
ornithine hydrochloride. The NMR in D2O showed a broad
4H multiplet at 6 2.0 ppm, a broad 2H multiplet at 5 3.6
ppm, a broad 1H multiplet at 6 4.1 ppm, a 1H doublet at 6
7.4 ppm, a 1H doublet of doublets at 6 8.4 ppm, and a 1H
doublet at 5 9.1 ppm.
Anal. calculated for C-qH^N^O^CI ‘f^O; C, 37.45 ; H, 4.82.
Found: C, 37.56; H, 4.90.
The D-N,6-DNP-ornithine hydrochloride (1.4 g, 4.2 mmol)
was then treated with ethanol saturated with HC1 gas. Work¬
up and recrystallization yielded 1.3 g (3.6 mmol, 85.7%
yield) of D-N,6-DNP-ornithine ethyl ester hydrochloride.
The '*‘H NMR in D^O showed a 3H triplet at 6 1.2 ppm, a broad
4H multiplet at 6 1.9 ppm, a broad 2H multiplet at 6 3.5
ppm, a broad 3H multiplet at 6 4.2 ppm, a 1H doublet at 6
7.4 ppm, a 1H doublet of doublets at 6 8.5 ppm, and a 1H
doublet at 6 9.1 ppm.
Anal. calculated for C-^ _H^gN^0^Cl; C, 43.03; H, 5.24.
Found: C, 42.96; H, 5.28.
Ammoniolysis of 1.3 g (3.6 mmol) of the above ester
in methanol saturated with ammonia yielded, after workup
and recrystallization, 1.0 g (3.0 mmol, 83.3% yield) of
D-N,6-DNP-ornithine amide hydrochloride.
The "'‘H NMR in D2O showed a 4H multiplet at 6 2.0 ppm,
a broad 2H multiplet at 6 3.6 ppm, a broad 1H multiplet at

100
6 4.2 ppm, a 1H multiplet at 6 7.2 ppm, a 1H doublet of
doublets at 6 8.3 ppm, and a 1H doublet at 6 8.9 ppm.
Anal. calculated for C-^-^H^gN^O^Cl • . SH^O; C, 38.54; H, 4.96.
Found: C, 38.94; H, 4.84.
DiCBZ-L-lysine (0.62 g, 1.5 mmol) in THF was treated
with 1.65 mmol of Et,N and isobutylchloroformate, respec¬
tively, followed by D-N,6-DNP-ornithine amide hydrochloride
(0.50 g, 1.5 mmol) and an additional 1.65 mmol of Et^N.
Workup and recrystallization yielded 0.56 g (0.01 mmol,
541 yield) of DiCBZ-L-lysyl-D-DNP-ornithine amide. The
NMR in D^-DMSO showed a broad 10H multiplet at 6 1.6 ppm,
a broad 2H multiplet at 6 3.0 ppm, a broad 2H multiplet
at 6 3.5 ppm, a very broad 2.H multiplet centered at 6 4.2
ppm, a 4H singlet at 6 5.1 ppm, a 15H multiplet at 6 7.4
ppm, a broad 2H multiplet at 6 8.3 ppm, and a broad 2H multi¬
plet at 6 9.1 ppm.
Anal. calculated for C_*1.51^0; C, 55.00; H, 5.83.
Found: C, 54.97; H, 5.88.
DiCBZ-L-lysyl-D-DNP-ornithine amide (.45 g, .65 mmol)
was stirred in freshly distilled HF at 0°C for 1 hour. After
evaporation of the HF, the residue was dissolved in 0.1 M
HC1, washed with ether, and lypholyzed. The solid obtained
was dissolved in 0.1 M NH^OAc and passed through a CM-
Sepharose (CL-6B) column eluting with 0.1 M NH^OAc. Fractions
absorbing at 450 nm were collected, lypholyzed and dried 2
times. The solid obtained was dissolved in 2 mL H?0 and
passed through an Amberlite CG-400 anion exchange column

101
(bromide form). The fractions absorbing at 450 nm were
pooled and lypholyzed. The resulting solid was dissolved in
methanol, placed in a tared vial and the solvent evaporated.
The vial was then placed in a P2O5 dessicator for 48 hours
and then a drying pistol for 24 hours to yield 190 mg
(0.32 mmol, 49.2% yield) of L-lysyl-D-DNP-ornithine amide
dihydrobromide. The NMR in D^O showed a broad 10H multi-
plet at 5 1.6 ppm, a 2H triplet at 6 3.0 ppm, a 2H triplet
at 6 3.6 ppm, a 1H triplet at 6 4.0 ppm, a 1H doublet at 6
7.1 ppm, and a 1H doublet of doublets at 6 8.3 ppm, and a
1H doublet at 6 9.1 ppm.
Anal. calculated for yH?gN^O^Br^•2.5H90; C, 32.28; H, 5.38.
Found: C, 32.27; H, 5.42. M.P. 180 - 183°C.
Analytical Methods
NMR Experiments
The NMR studies used sonicated salmon sperm DNA
which was prepared and supplied by Dr. P.D. Adawadkar.
A nucleic acid stock solution was prepared by dissolving
90 mg of sonicated DNA in 3 mL of D^O containing 0.2% TSP
(internal standard) to give a solution that was 72 mM
phosphate/liter (3.3 mM Na+, 1 mM phosphate buffer, pD 7.0).
Solutions of each peptide (0.1 M) in D?0 (0.2% TSP) were
prepared and aliquots of the latter were added to 5 mm NMR
tubes containing 0.5 mL of the DNA stock solution. The
concentration of the peptides was varied from 5 mM to 23 mM.
The NMR of the peptides and the DNA-peptide complexes
(at different base pair to peptide ratio) were taken on a

102
Jeol-FXlOO spectrometer at 37°C. At the highest base pair
to peptide ratio, the NMR spectrawere also measured at 70°C
and 90°C. Chemical shifts (in Hz) are reported relative to
the internal standard (TSP) and are reproducible to +_ 0.2 Hz.
Viscosity Apparatus and Measurements
A Beckman rotating cylinder viscometer (Figure 31) was
used employing rotors that were prepared in a manner similar
to that of Zimm and Crothers.A thin strip of styrofoam,
with three sides painted black, was placed in the rotor
during the measurement. As the rotor turned, light from a
nearby light source was reflected from the white side of the
styrofoam strip into a detector as described by Pearce
117
et al. The signal generated was processed and sent to
a recorder.
For the viscosity measurements, the viscometer was
thermostated at 37.5°C with a Haake constant - temperature
- 4
circulator. A stock solution of salmon sperm DNA (3 x 10
P/L) was pipetted into the viscometer. The rotor was then
placed in the viscometer and its height was adjusted rela¬
tive to an etched line on the strator by adding more DNA
solution. The final volume of solution in the viscometer
was 2.5 mL. The viscometer was turned on and 90 rotations
were counted and the length of chart paper measured. This
value was divided by the measured length of 90 rotations for
the buffer solution to obtain the relative viscosity (n -,)
of the DNA solution. The specific viscosity (n ) is then
calculated from n =Prei Aliquots of a 0.01 M peptide

103
Figure 31. Diagram of the Viscometer with the Photoelectric
Device in Place. Components in the Figure are:
A) water jacketed stator; B) stator support;
C) rotor; D) reflector; E) rotating magnet assem¬
bly; F) metal pellet; G) motor; H) photoelectric
device as attached to the stator support.

104
solution were added to the DNA, and after sufficient mixing,
the viscosity of the DNA/peptide solution was determined.
Absorption Studies
Absorption spectra (500 nm - 280 nm) of the DNP pep¬
tides were recorded on a Cary-17D spectrophotometer in a
1 cm cell at room temperature. A 4 x 10 ^ M solution of
the peptide in 10 mM MES buffer was placed in the cell and
the spectra recorded. The spectra of a solution of peptide
with excess DNA, where the concentration of peptide and DNA
were 4 x 10 ^ M and 2.5 x 10 ^ M P/L, respectively, was then
recorded.
Hypochromicity, H, is defined by
H = 100(1 - fb/f£),
where f^ and f£ are the oscillator strengths of DNA bound
and free peptide, respectively. According to quantum mech¬
anical calculations, the oscillator strength, f, is propor¬
tional to the area of the absorption spectra by the following
equation:
f = 4.32 x 10"9/edü
where e is the extinction coefficient of the molecule at
wave number u. The absorption spectra were then re¬
plotted as absorption versus wave number and the area under
the curve determined for free and bound peptide A£ and A^,
respectively. The hypochromicity was then calculated
according to the following:
H = 100(1 - Ab/A£).

105
Binding Studies
The apparent binding constant, Ka, and the maximum
number of base pairs per binding site, n, were determined
by spectral titration on a 5 cm thermostated cell in the
Cary-17D at 21°C. For the DNP derivatives, a 1.20 x 10 ^ M
solution of the dipeptide in 6.6 mM phosphate buffer (pH 7.2,
with 5 mM Na+) was added to the cell and the absorption at
- 3
360 nm determined. Aliquots of a 2.82 x 10 M salmon
sperm DNA P/L and 1.20 x 10 3 M peptide solution were added
and the absorption at 360 nm recorded after each addition
until saturation occurred (i.e., no further change in ab¬
sorption) . The titration was also performed at 10 mM and
50 mM Na+. For the DNP derivatives, peptide concentration
-5 +
was kept at 1.20 x 10 M for both 10 mM and 50 mM Na ,
while the DNA concentrations were 2.82 x 10 3 M (10 mM Na+)
and 5.37 x 10~3 M (50 mM Na+) .
For L-lys-L-S-NT-cysA (3), the peptide concentration
was 2.30 x 10 3 M and the DNA concentration was 6.70 x 10 3 M
at both 5 mM and 10 mM Na+. For L-lys-D-S-NT-cysA (£), the
- 4 +
peptide concentration was 1.14 x 10 M (5 mM Na ) and
- 4 +
1.29 x 10 M (10 mM Na ), while the DNA concentration was
8.4 x 10'3 M (5 mM Na+) and 4.8 x 10'3 M (10 mM Na+). The
titrations for both nitrotolyl derivatives were not performed
at 50 mM Na+ due to weak binding. The absorption at 340 nm
was monitored for these titrations.
The parameters Ka and n were calculated by means of the
93 119
McGhee and von Hippel modification of the Scatchard

106
technique by use of a Commodore Pet 2001 microcomputer with
120
a modified program of D. Wilson. The parameters needed
to make the Scatchard plot u, and u/L are calculated by the
following equations:
D (A - A )
u = 0
(A - A ) B
K o sJ
and
u u
L D - uB
where u is ratio of the concentration of bound peptide to
the concentration of DNA base pairs, L is the concentration
of free peptide, D is the total peptide concentration, Aq,
A , and A are the initial absorption, at saturation, and
for a given aliquot of DNA solution added, respectively,
and B is the concentration of DNA base pairs.
Circular Dichroism Measurements
CD spectra of peptide and peptide/DNA in 10 mM MES
buffer (pH 6.2) solutions were taken in a 5 cm cell with
those peptides whose X was greater than 300 nm. Spectra
v r max B v
were recorded between 500 nm and 300 nm for free peptide
(2.5 x 10 ^ M) and peptide (2.5 x 10 ^ M) in the presence
of a low concentration of DNA (8.4 x 10 ^ M P/L) and a high
concentration of DNA (6 x 10 ^ M P/L) using a Jasco J-20
spectropolarimeter at room temperature.
Equilibrium Dialysis of Dipeptides
The apparent binding affinity of all peptides to DNA
was determined by equilibrium dialysis. The dialysis mem¬
branes were prepared by cutting Visking dialysis tubing

107
(26/100) ft NOJAX castings) into 20 cm strips which were
then cleaned by boiling them in 50% aqueous ethanol con¬
taining 5 x 10'3 M EDTA and 5 x 10'2 M NaHC03 for 1 hour
and repeated 3 times. The membranes were then boiled in
deionized H70 for 5 hours and this was repeated 6 times.
The cleaned membranes were stored in deionized H?0 (2%
CHCl^ as preservative) in the refrigerator.
A pair of Plexiglas blocks with 10 shallow cylindri¬
cal depressions (3 x 0.25 mm) cut into each one were used
in the dialysis experiments. Each block was lightly coated
with silicone grease to prevent leakage, and a clean mem¬
brane was placed between the two blocks, forming two cham¬
bers. The blocks were securely fastened together with 12
screws and 4 C-clamps. To one side of the membrane, 0.2 mL
of 3.0 x 10 ^ M P/L DNA in 10 mM MES buffer (pH 6.2, 5 mM
+ - 4
Na ) was added. Then, 0.2 mL of peptide solution (5 x 10
M or 2.5 x 10 ^ M) in the same buffer was added to the
other side of the membrane. The top of the cells were
sealed with a strip of cellophane tape. The blocks were
placed in a refrigerator (4°C) and equilibrium was allowed
to take place for 48 hours. In one experiment, DNA versus
buffer showed that the absorption at 260 nm was not greater
than 0.005 in the buffer side, indicating no leakage of DNA
across the membrane. Furthermore, in the buffer versus pep¬
tide case, it was found that no binding to membrane had
taken place. The concentration of peptide was determined by
determining the absorption at 360 nm for the DNP containing

IOS
peptide, at 275 nm for the p-nitrophenyl containing peptides,
and at 340 nm for the nitrotolyl containing peptides.
Melting Temperature (T ) Measurements
T measurements were run in 1 mL quantz cuvettes ther-
m
mostated with a Haake constant-temperature circulator
equipped with a constant speed motor to provide a heating
rate of 0.71°C/minute. A Cary-17D equipped with automatic
recording accessories was used and the temperature of the
cell compartment was measured directly with an iron-
constantan thermocouple connected to a chart recorder which
had been standardized at 0°C and 100°C.
The experiment consisted of 2 mL of 1.26 x 10 ^ M
nucleic acid in 10 mM MES buffer (5 mM Na+) and 5, 10, 15
. 3
or 20 ul of a 1 x 10 M peptide solution. The nucleic
acids used were DNA, poly I - poly C, and poly d(A-T).
The solutions were placed in the thermostated compartment
with a buffer blank and allowed to equilibrate for 10
minutes at 45°C (DNA) or 25°C [(poly I - poly C) and (poly
d(A-T)]. Initial absorption was recorded and the temperature
programmer initiated. The temperature and absorption were
recorded continuously throughout the procedure.

REFERENCES
1.
Watson,
(1953) .
J.
D. ,
and
Crick,
F.
H.
C.
Nature, 171, 737
2.
Watson,
(1953) .
J.
D.,
and
Crick,
F.
H.
C.
Nature, 171, 964
3. McBurney, M. W., and Whitmore, G. F. Biochem. Bio-
phys. Res. Comm., 46, 898 (1972).
4. Arnott, J., Fuller, W. , Hodgson, A., and Prutton, I.
Nature, 220, 561 (1968).
5. Doty, P., Boedtker, H., Fresco, J., Haselkorn, R.,
and Litt, M. Proc. Natl. Acad. Sci. U.S., 45, 482
(1959) .
6. Mathieson, A. R., and Matty, S. J. Polymer Sci.,
2_3, 1131 (1957). '
7. Josse, J., and Eigner, J. Ann. Rev. Biochem., 35,
789 (1966).
8. Chargaff, E., Lipschitz, R. , Green, C., and Hodes,
M.E. J. Mol. Biol., 192, 223 (1951).
9.Chargaff, E., and Lipschitz, R. J. Amer. Chem. Soc.,
7_5_, 3658 (1953).
10.Josse, J., Kaiser, A. D., and Kornberg, A. J. Biol.
Chem., 236, 864 (1961).
11.Langridge, R., Wilson, H. R., Hooper, C. W., Wilkins,
M. H. F., and Hamilton, L. P. J. Mol. Biol., 2, 19
(1960) .
12.
Davies, D.
R. Ann. Rev. Biochem
., 36, 321 (1967)
13.
Brams, S.
Biochem. Biophys. Res
. Comm., 48, 1088
(1972).
14.
Donohue, J.
Science, 165, 1091
(1969) .
15.
Donohue, J.
Science, 167, 1700
(1970) .
109

110
16. Arnott, S. J. Mol. Biol. , 59_, 381 (1971).
17. Brams, S. J. Mol. Biol. , 58_, 277 (1971).
18. Brams, S. Nature New Biol., 233, 161 (1971).
19. Gulland, J. M., Jordan, D. 0., and Taylor, H. F. W.
J. Chem. Soc., 1131 (1947).
20. Thomson, J. F., Carttar, M. S., and Tourtellotte,
W. W. Radiation Res., 1, 165 (1954).
21. Helmkamp, G. K., and Ts'O, P. 0. P. J. Amer. Chem.
Soc., 83, 138 (1961).
22. Donohue, J. Proc. Natl. Acad. Sci. U.S., 42, 60
(1956).
23. Donohue, J., and Trueblood, K. N. J. Mol. Biol.,
2, 363 (1960).
24. Nash, H. A., and Bradley, D. F. J. Chem. Phys.,
45, 1380 (1966).
25. Hoogsteen, K. Acta Cryst., 12, 822 (1959).
26. Hoogsteen, K. Acta Cryst., 16, 907 (1963).
27.
Mathews, F. S., and Rich, A. J. Mol.
(1964).
Biol
., 8,
89
28.
Haschemeyer, A. E. V. , and Sobell, H.
Cryst., 18, 525 (1965).
M.
Acta
29.
Haschemeyer, A. E. V. , and Sobell, H.
Acad. Sci. U.S., 50, 782 (1963).
M.
Proc.
Nati.
30.
Sobell, H. M., Tomaita, K., and Rich,
Acad. Sci. U.S., 49, 885 (1963).
A.
Proc.
Nati.
31. Katz, L., Tomaita, K., and Rich, A. J. Mol. Biol.,
13, 340 (1965). â– 
32.
0' Brien,
E.
J.
J. Mol. Biol., 7, 107 (1963).
33.
O'Brien,
E.
J.
J. Mol. Biol., 22, 377 (1966)
34.
O'Brien,
E.
J.
Acta Cryst., 23, 92 (1967).
35. Tuppy, H. T., and Kuebler, E. K. Biophys. Biochem.
Acta, 80_, 669 (1964) .
36. Fritzsche, H. Experientia, 27, 507 (1971).

Ill
37. Pitha, J., Jones, R. N., and Pithova, P. Can. J.
Chem., 44 , 1045 (1966).
38. Küchler, E., and Derkosch, J. Z. Naturforsch, 21b,
209 (1966).
39.
Kyogoku, Y., Lord, R.
518 (1967).
C. ,
and Rich, A.
Science, 154,
40.
Katz, L., and Penman,
(1965) .
S.
J. Mol. Biol.
, 15, 220
41.Hamlin, R. M., Jr., Lord, R. C., and Rich, A.
Science, 148, 1734 (1965).
42. Shoup, R. R., Miles, H. T., and Becker, E. D. Bio-
chem. Biophys. Res. Comm. , 2_3, 194 (1964) .
43. Rice, S. A., Wada, A., and Geiduschek, E. P. Dis-
cussions Faraday Soc., 25, 130 (1958).
44. Sturtevant, J. M., Rice, S. A., and Geiduschek, E, P.
Discussions Faraday Soc. , 2_5_, 138 (1958) .
45. Ts'O, P. 0. P. Molecular Association in Biology,
Pullman, B., Ed., Academic Press, New York, N.Y.
(1968).
46. Chan, S. I., Schweizer, M. P., Ts'O, P. 0. P., and
Helmkamp, G. K. J. Amer. Chem. Soc., 86, 4182 (1964).
47. Pople, J. A., Schneider, W. G., and Bernstein, H. J.
High Resolution Nuclear Magnetic Resonance, McGraw-
Hill Book Co., New York, N.Y. (1959).
48. Broom, A. D., Schweizer, M. P., and Ts'O, P. 0. P.
J. Amer. Chem. Soc. , 89^, 3612 (1967) .
49. Hanlon, S. Biochem. Biophys. Res. Comm., 23, 861
(1966) .
50. Chan, S. I., and Nelson, J. A. J. Amer. Chem. Soc.,
9_1, 168 (1969).
51. Tinoco, I., Jr., Davis, R. C., and Jaskunas, S. R.
Molecular Association in Biology, Pullman, B., Ed.,
Academic Press, New York, N.V. (1968) .
52. Gabbay, E. J. Biochemistry, _5, 3036 (1966).
53. Conway, B. E., and Butler, J. A. V. J. Polymer Sci.,
12, 199 (1954).
54.
Michelson, A. M. Nature, 182 1502 (1958).

112
55. Leng, M., and Felsenfeld, G. J. Mol. Biol., 15, 455
(1964).
56. Tinoco, I., Jr. J. Amer. Chem. Soc., 82, 4785 (1960).
57. Tinoco, I., Jr. J. Chem. Phys., 55, 1552 (1960).
58. DeVoe, H., and Tinoco, I., Jr. J. Mol. Biol., 4,
518 (1962).
59.
Cantor, C. R.,
1059 (1970).
and Warshaw, M. W
Biopolymers, 9,
60.
Tinoco, I., Jr.
J. Amer. Chem.
Soc. ,
86_, 297 (1964)
61.
Printz, M. B. ,
Acad. Sci. U.S.
and von Hippel, P
, 5_5, 565 (1965).
. H.
Proc. Natl.
62.
McConnell, B. ,
5_0_, 297 (1970).
and von Hippel, P
. H.
J. Mol. Biol.,
65.
McConnell, B.,
50_, 517 (1970).
and von Hippel, P
. H.
J. Mol. Biol.,
64.
Muller, W., and
Crothers, D. J.
Mol.
Biol., 55, 251
(1968).
65. Gabbay, E. J., DeStefano, R., and Baxter, C. S.
Biochem. Biophys. Res. Comm., 51, 1085 (1975).
66. Maniloff, J., and Morowitz, H. J. Bacteriol. Rev.,
56, 265 (1972).
67. Elgin, S. C. R., and Weintraub, H. Ann. Rev. Bio-
chem., 44, 725 (1975).
68. Kornberg, R. D., and Thomas, J. 0. Science, 184,
865 (1974).
69. Olson, M. 0. J., Starbuck, W. C., and Busch, H.
The Molecular Biology of Cancer, Busch, H., Ed.,
Academic Press, New York, N.Y. (1974).
70. Olins, D. E., and Olins, A. L. J. Cell. Biol., 55,
715 (1972).
71. Griffith, J. D. Science, 187, 1202 (1975).
72.Langmore, J. P., and Wooley, J. C. Proc. Natl. Acad.
Sci. U.S. , 72_, 2691 (1975).
75. Kolata, G. B. Science, 188, 1092 (1975).

113
74. Metzler, D. E. Biochemistry, Academic Press, New York,
N.Y. (1977).
75. Gabbay, E. J., Glaser, R. , and Gaffney, B. L. Ann.
N.Y. Acad. Sci., 171, 810 (1970).
76. Gabbay, E. J., Scofield, R., and Baxter, C. S.
J. Amer. Chem. Soc., 95, 7850 (1973).
77. Gabbay, E. J., and DePalois, A. J. Amer. Chem. Soc.,
93, 562 (1971).
78. Kapicak, L., and Gabbay, E. J. J. Amer. Chem, Soc.,
97, 403 (1975).
79. Lerman, L. S. J. Mol. Biol. , !5, 18 (1961).
80. Gabbay, E. J., Sanford, K., and Baxter, C. S.
J. Amer. Chem. Soc. , 94-, 2876 (1972) .
81. Gabbay, E. J., and Sanford, K. Bioorg. Chem., 3,
91 (1974).
82. Adawadkar, P. D., Wilson, W. D., Brey, W., and
Gabbay, E. J. J. Amer. Chem. Soc., 97 , 1959 (1975).
83. Gabbay, E. J., Adawadkar, P. D., and Wilson, W. D.
Biochemistry, 15, 146 (1976).
84. Gabbay, E. J., Adawadkar, P. D., Kapicak, L., Pearce,
5., and Wilson, W. D. Biochemistry, 15, 152 (1976).
85. Gabbay, E. J., and Glaser, R. Biochemistry, 10,
1665 (1971).
86. Mahler, H. R., Goutarel, R., Khuong-Huu, G., and
Ho, M. T. Biochemistry, 5_, 1966 (1966).
87. Gabbay, E. J. Bioorganic Chemistry Vol. Ill, van
Tamelen, E. E., Ed., Academic Press, New York (1977).
88. Raska, M., and Mandel, M. Proc. Natl. Acad. Sci. U.S.,
68, 1190 (1971).
89. Helene, C., Montenay-Garestier, T., and Dimicoli, J. L.
Biophys. Biochem. Acta, 254. 349 (1971).
90. Helene, C., Dimicoli, J. L., and Brun, F. Biochemistry,
10., 3802 (1971).
91. Jardetsky, 0., and Jardetsky, C. D. Methods Biochem.
Anal., 9 , 235 (1962) .

114
92.Brown, P. E. Biophys. Biochem. Acta, 213, 282 (1970).
93. McGhee, J. D., and von Hippel, P. H. J. Mol. Biol.,
86, 469 (1974).
94. Gabbay, E. J., and Kleinman, R. Biochem. J., 117,
247 (1970).
95. Gilmour, R. S., and Paul, J. FEBS Lett., 9, 242
(1970).
96. Kleinsmith, L. J., Heidema, J., and Carroll, A.
Nature, 226, 1025 (1970).
97. Spelsberg, T. C., and Hnilica, L. S. Biochem. J.,
120, 435 (1970).
98. Stein, G. S., and Farber, J. Proc. Natl. Acad. Sci.
U.S., 69 , 2918 (1972) .
99. D'Anna, J. A., and Isenberg, I. Biochemistry, 13,
4992 (1974).
100.Kornberg, R. D. Science, 184, 868 (1974).
101. Kornberg, R. D., and Thomas, J. 0. Science, 184,
865 (1974).
102. Olins, A. L., and Olins, D. E. Science, 183, 330
(1974) .
103. Stein, G. S., Spelsberg, T. C., and Kleinsmith, L. J.
Science, 183, 817 (1974).
104. van Holde, K. E., Sahasrabuddhe, C. G., and Shaw, B. R.
Biochem. Biophys. Res. Comm., 70, 1365 (1974).
105. Jones, K. W. Nature, 225, 912 (1970).
106. Yunis, J. J., and Yasmineh, W. G. Science, 168, 263
(1970).
107. Walker, P. M. B. Nature, 219, 228 (1968).
108. Blumenfeld, M., Fox, A. S., and Forest, H. S. Proc.
Natl. Acad. Sci. U.S. , 70, 2772 (1973).
109. Dwek, R. A. Nuclear Magnetic Resonance in Biochemistry,
Clarendon Press, Oxford (1973).
110. Mildvan, A. S., and Cohn, M. Adv. Enzymol. Relat.
Areas Mol, Biol.. 33, 1 (1970).

115
111. Curtius, T., and Goebel, F. J. prakt. Chem., (2)
37150 (1888).
112. Anderson, G. W., Zimmerman, J. E., and Callahan, F. M.
J. Amer. Chem. Soc., 89 , 5012 (1967).
113. Sanger, F. Biochem. J., 39, 507 (1945).
114. Berse, C., Boucher, R., and Piche, L. J. Org. Chem.,
2_2 , 805 (1957).
115. Itoh, M., Hagiwara, D., and Kamiya, T. Tett. Lett.,
49_, 4393 (1975).
116. Zimm, B. H., and Crothers, D. M. Proc. Natl. Acad.
Sci. U.S., 48, 905 (1962).
117. Pearce, S. W., Griggs, B. G., O'Brien, F., and Wilson,
W. D. Chemical Instrumentation, 7_, 33 (1976).
118. Sandorfy, C. Electronic Spectra and Quantum Chem-
istry, Prentice-Hall, Inc., Englewood Cliffs, N.J.
TT964).
119. Scatchard, G. Ann. N.Y. Acad. Sci., 51 , 660 (1949).
120. Wilson, D. W. Personal Communication.

BIOGRAPHICAL SKETCH
Richard Dean Sheardy
1949 Born December 2, in Pontiac, MI.
1968 Graduated from Lake Orion Community High School,
Lake Orion, MI.
1973B.S.T. Degree from Michigan State University in
chemistry education, E. Lansing, MI.
1973 Attended graduate school at University of Florida,
- 74 majored in chemistry, Gainesville, FL.
1974 Taught chemistry and physics at Warren Woods High
- 75 School, Warren Woods, MI.
1975 Attended graduate school at University of Florida,
- 79 majored in chemistry, Gainesville, FL.
1979 Ph.D. Degree, University of Florida, Gainesville, FL.
116

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
" J/¿c c £ O-
Merle A. Battiste, Chairman
Professor of Chemistry
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
X?, ¿.¿¿DtC
George B. Butler
Professor of Chemistry
I certify that
opinion it conforms
presentation and is
I have read this study and that in my
to acceptable standards of scholarly
fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy
William R. Dolbier
Professor of Chemistry

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
Willis B. Person
Professor of Chemistry
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of schoüarly
presentation and is fully adequate, in scope and quality,
as a dissertation for the degree of Doctor of Philosophy.
X>
Philip P<¡>/Sner
Associate Professor of
Physiology
This dissertation was submitted to the Graduate Faculty of
the Department of Chemistry in the College of Liberal Arts
and Sciences and to the Graduate Council, and was accepted
as partial fulfillment of the requirements for the degree
of Doctor of Philosophy.
December 1979
Dean, Graduate School

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