Note: Descriptions are shown in the official language in which they were submitted.
CA 02281947 1999-08-17
WO 98/37066 PCT/US98/01006
IMPROVED POLYAMIDES FOR BINDING IN THE MINOR
GROOVE OF DOUBLE STRANDED DNA
The U.S. Government has certain rights in this invention pursuant to Grant
Nos. GM
26453, 27681 and 47530 awarded by the National Institute of Health.
CROSS REFERENCE TO RELATED APPLICATIONS
1o This application is a continuation-in-pan of PCT/LJS97/03332 filed February
20, 1997,
Serial No. 08/853,522 filed May 8, 1997 and PCT/LJS 97/12722 filed July 21,
1997 which are
continuation-in-part applications of Serial No. 08/837,524, filed April 21,
1997, Serial No.
08/607,078, filed February 26, 1996, provisional application Serial No.
60/042,022, filed April
16, 1997 and provisional application Serial No. 60/043,444, filed April 8,
1997.
BACKGROUND OF THE INVENTION
Field of the Invention
2o This invention relates to polyamides which bind to predetermined sequences
in the
minor groove of double stranded DNA.
Description of the Related Art
The design of synthetic ligands that read the information stored in the DNA
double helix
has been a long standing goal of chemistry. Cell-permeable small molecules
which target
predetermined DNA sequences are useful for the regulation of gene-expression.
Oligodeoxynucleotides that recognize the major groove of double-helical DNA
via triple-helix
formation bind to a broad range of sequences with high affinity and
specificity. Although
oligonucleotides and their analogs have been shown to interfere with gene
expression, the triple
helix approach is limited to purine tracks and suffers from poor cellular
uptake. The
development of pairing rules for minor groove binding polyamides derived from
N-
methylpyrrole (Py) and N-methylimidazole (Im) amino acids provides another
code to control
sequence specificity. An Im/Py pair distinguishes G~C from C~G and both of
these from A~T
or T~A base pairs. Wade, W.S., Mrksich, M. & Dervan, P.B. describes the design
of peptides
that bind in the minor groove of DNA at S'-(A,T)G(A,T)C(A,T)-3' sequences by a
dimeric
side-by-side motif. J. Am. Chem. Soc. 114, 8783-8794 (1992); Mrksich, M. et
al. describes
antiparallel side-by-side motif for sequence specific-recognition in the minor
groove of DNA by
the designed peptide 1-methylimidazole-2-carboxamidenetropsin. Proc. Natl.
Acad. Sci. USA
89, 7586-7590 (1992); Trauger, J.W., Baird, E. E. Dervan, P.B. describes the
recognition of
DNA by designed ligands at subnanomolar concentrations. Nature 382, 559-561
(1996). A
CA 02281947 1999-08-17
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Py/Py pair specifies A~T from G~C but does not distinguish A~T from T~A.
Pelton, J.G. &
Wemmer, D.E. describes the structural characterization of a 2-1 distamycin A-
d(CGCAAATTTGGC) complex by two-dimensional NMR. Proc. Natl. Acad. Sci. USA 8b,
5723-5727 (1989); White, S., Baird, E. E. & Dervan, P.B. Describes the effects
of the A~T/T~A
degeneracy of pyrrole-imidazole polyamide recognition in the minor groove of
DNA.
Biochemistry 35, 6147-6152 (1996); White, S., Baird, E. E. & Dervan, P. B.
describes the
pairing rules for recognition in the minor groove of DNA by pyrrole-imidazoie
polyamides.
Chem. & Biol. 4, 569-578 ( 1997); White, S., Baird, E. E. & Dervan, P.B.
describes the 5'-3' N-
C orientation preference for polyamide binding in the minor groove. In order
to break this
1o degeneracy, a new aromatic amino acid, 3-hydroxy-N-methylpyrrole (Hp)
incorporated into a
polyamide and paired opposite Py, has been found to discriminate A~T from T~A.
The
replacement of a single hydrogen atom on the pyrrole with a hydroxy group in a
Hp/Py pair
regulates affinity and specificity of a polyamide by an order of magnitude.
Utilizing Hp
together with Py and Im in polyamides to form four aromatic amino acid pairs
(Im/Py, Py/Im,
Hp/Py, and Py/Hp) provides a code to distinguish all four Watson-Crick base
pairs in the minor
groove of DNA.
SUMMARY OF THE INVENT10N
The invention encompasses improved polyamides for binding to the minor groove
of
double stranded ("duplex") DNA. The polyamides are in the form of a hairpin
comprising two
groups of at least three consecutive carboxamide residues, the two groups
covalently linked by
an aliphatic amino acid residue, preferably y-aminobutyric acid or 2,4
diaminobutyric acid, the
consecutive carboxamide residues of the first group pairing in an antiparallel
manner with the
consecutive carboxamide residues of the second group in the minor groove of
double stranded
DNA. The improvement relates to the inclusion of a binding pair of Hp/Py
carboxamides in the
polyamide to bind to a T~A base pair in the minor groove of double stranded
DNA or Py/Hp
carboxamide binding pair in the polyamide to bind to an A~T base pair in the
minor groove of
double stranded DNA. The improved polyamides have at least three consecutive
carboxamide
3o pairs for binding to at least three DNA base pairs in the minor groove of a
duplex DNA
sequence that has at least one A~T or T~A DNA base pair, the improvement
comprising
selecting a Hp/Py carboxamide pair to correspond to a T~A base pair in the
minor groove or a
Py/Hp carboxamide pair to bind to an A~T DNA base pair in the minor groove.
Preferably the
binding of the carboxamide pairs to the DNA base pairs modulates the
expression of a gene.
In one preferred embodiment, the polyamide includes at least four consecutive
carboxamide pairs for binding to at least four base pairs in a duplex DNA
sequence. In another
preferred embodiment, the polyamide includes at least five consecutive
carboxamide pairs for
binding to at least five base pairs in a duplex DNA sequence. In yet another
preferred
2
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WO 98/37066 PCT/US98/01006
embodiment, the polyamide includes at least six consecutive carboxamide pairs
for binding to at
least six base pairs in a duplex DNA sequence. In one preferred embodiment,
the improved
polyamides have four carboxamide binding pairs that will distinguish A~T, T~A,
C~G and G~C
base pairs in the minor groove of a duplex DNA sequence. The duplex DNA
sequence can be a
regulatory sequence, such as a promoter sequence or an enhancer sequence, or a
gene sequence,
such as a coding sequence or a non-coding sequence. Preferably, the duplex DNA
sequence is a
promoter sequence.
The preparation and the use of polyamides for binding in the minor groove of
double
to stranded DNA are extensively described in the art. This invention is an
improvement of the
existing technology that uses 3-hydroxy-N-methylpyrrole to provide carboxamide
binding pairs
for DNA binding polyamides.
The invention encompasses poiyamides having y-aminobutyric acid or a
substituted y-
aminobutvric acid to form a hairpin with a member of each carboxamide pairing
on each side of
it. Preferably the substituted y-aminobutyric acid is a chiral substituted y-
aminobutvric acid
such as (R}-2,4-diaminobutyric acid. In addition, the polyamides may contain
an aliphatic
amino acid residue, preferably a ~i-alanine residue, in place of a non-Hp
carboxamide. The ~i-
alanine residue is represented in fotzrtulas as Vii. The (3-alanine residue
becomes a member of a
2o carboxamide binding pair. The invention further includes the substitution
as a ~3~~ binding pair
for non-Hp containing binding pair. Thus, binding pairs in addition to the
Hp/Py and Py/Hp are
Im/(3, ~3/Im, PY/~i, ~i/PY, and ~i/~i.
The polyamides of the invention can have additional moieties attached
covalently to the
polyamide. Preferably the additional moieties are attached as substituents at
the amino terminus
of the polyamide, the carboxy terminus of the polyamide, or at a chiral (R)-
2,4-diaminobutyric
acid residue. Suitable additional moieties include a detectable labeling group
such as a dye,
biotin or a hapten. Other suitable additional moieties are DNA reactive
moieties that provide
for sequence specific cleavage of the duplex DNA.
Brief Description of the Drawiag_s
Figure I illustrates the structure of polyamide 1 2 and 3_.
Figure 2 illustrates the pairing of polyamides to DNA base pairs.
3s Figure 3 illustrates the DNase footprint titration of compounds 2 and 3.
Figure 4 illustrates a list of the structures of representative Hp containing
polyamides.
Figure 5 illustrates the synthesis of a protected Hp monomer for solid phase
synthesis.
Figure 6 illustrates the solid phase synthesis of polyamide 2.
Figure 7 illustrates the 1H-NMR characterization of polyamide 2.
3
CA 02281947 1999-08-17
W0 98/37066 PCT/US98/01006
Figure 8 illustrates the Mass spectral characterization of polyamide 2.
Figure.9 illustrates 1H-NMR characterization of synthesis purity.
Figure 10 illustrates DNaseI footprint titration experiment.
Figure 11 illustrates the synthesis of bifunctional conjugate of polyamide 2.
Figure 12 illustrates affinity cleaving evidence for oriented hairpin
formation.
Figure 13 illustrates increased sequence specificity of Hp/Py containing
polyamides.
Figure 14 illustrates 8-ring hairpin polyamides which target 5'-WGTNNW-3'
sites.
Figure 15 illustrates 8-ring hairpin polyamides which target 5'-WGANNW-3'
sites.
Figure 16 illustrates 8-ring hairpin polyamides which target 5'-WGGNNW-3'
sites.
to Figure 17 illustrates 8-ring hairpin polyamides which target 5'-WGCNNW-3'
sites.
DETAILED DESCRIPT10N OF THE INVENT10N
Within this application, unless otherwise stated, definitions of the terms and
illustration
1 ~ of the techniques of this application may be found in any of several well-
known references such
as: Sambrook, J., et al., Molecular Cloning: ;f Laboraton~ Manual, Cold Spring
Harbor
Laboratory Press ( 1989); Goeddel, D., ed., Gene Expression Technologn,
Methods in
Enzvmologv, 185, Academic Press, San Diego, CA ( 1991 ); "Guide to Protein
Purification" in
Deutshcer, M.P., ed., Methods in Enzymoloy, Academic Press, San Diego, CA
(1989); Innis, et
2o al., PCR Protocols: A Guide to Methods and Applications, Academic Press,
San Diego, CA
( 1990); Freshney, R.L, Culture of Animal Cells: A Manual of Basic Technique,
Z"° Ed., Alan
Liss, Inc. New York, NY (1987); Murray, E.J., ed., Gene Transfer and
Expression Protocols,
pp. 109-128, The Humana Press Inc., Clifton, NJ and Lewin, B., Genes vl,
Oxford University
Press, New York ( I 997).
For the purposes of this application, a promoter is a regulatory sequence of
DNA that is
involved in the binding of RNA polymerase to initiate transcription of a gene.
A gene is a
segment of DNA involved in producing a peptide, polypeptide or protein,
including the coding
region, non-coding regions preceding ("leader") and following ("trailer") the
coding region, as
3o well as intervening non-coding sequences ("introns") between individual
coding segments
("exons"). Coding refers to the representation of amino acids, start and stop
signals in a three
base "triplet" code. Promoters are often upstream (" 'S to") the transcription
initiation site of
the corresponding gene. Other regulatory sequences of DNA in addition to
promoters are
known, including sequences involved with the binding of transcription factors,
including
response elements that are the DNA sequences bound by inducible factors.
Enhancers comprise
4
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WO 98/37066 PCT/US98/01006
yet another group of regulatory sequences of DNA that can increase the
utilization of
promoters, and can function in either orientation (5'-3' or 3'-5') and in any
location (upstream
or downstream) relative to the promoter. Preferably, the regulatory sequence
has a positive
activity, i.e., binding of an endogeneous ligand (e.g. a transcription factor)
to the regulatory
sequence increases transcription, thereby resulting in increased expression of
the corresponding
target gene. In such a case, interference with transcription by binding a
polyamide to a
regulatory sequence would reduce or abolish expression of a gene.
The promoter may also include or be adjacent to a regulatory sequence known in
the art
l0 as a silencer. A silencer sequence generally has a negative regulatory
effect on expression of
the gene. In such a case, expression of a gene may be increased directly by
using a polyamide
to prevent binding of a factor to a silencer regulatory sequence or
indirectly, by using a
polyamide to block transcription of a factor to a silencer regulatory
sequence.
t5 It is to be understood that the polyamides of this invention bind to double
stranded DNA
in a sequence specific manner. The function of a segment of DNA of a given
sequence, such as
5'-TATAAA-3', depends on its position relative to other functional regions in
the DNA
sequence. In this case, if the sequence S'-TATAAA-3' on the coding strand of
DNA is
positioned about 30 base pairs upstream of the transcription start site, the
sequence forms part
20 of the promoter region (Lewin, Genes Vl, pp. 831-835). On the other hand,
if the sequence S'-
TATAA.A-3' is downstream of the transcription start site in a coding region
and in proper
register with the reading frame, the sequence encodes the tyrosyl and lysyl
amino acid residues
(Lewin, Genes Vl, pp. 213-215).
While not being held to one hypothesis, it is believed that the binding of the
polyamides
of this invention modulate gene expression by altering the binding of DNA
binding proteins,
such as RNA polymerase, transcription factors, TBF, TFIIIB and other proteins.
The effect on
gene expression of polyamide binding to a segment of double stranded DNA is
believed to be
3o related to the function, e.g., promoter, of that segment of DNA.
It is to be understood by one skilled in the art that the improved polyamides
of the
present invention may bind to any of the above-described DNA sequences or any
other
sequence having a desired effect upon expression of a gene. In addition, U.S.
Patent No.
5
CA 02281947 1999-08-17
WO 98/37066 PCT/US98/01006
5,578,444 describes numerous promoter targeting sequences from which base pair
sequences
for targeting an improved polyamide of the present invention may be
identified.
It is generally understood by those skilled in the art that the basic
structure of DNA in a
living cell includes both major and a minor groove. For the purposes of
describing the present
invention, the minor groove is the narrow groove of DNA as illustrated in
common molecular
biology references such as Lewin, B., Genes VI, Oxford University Press, New
York (1997).
To affect gene expression in a cell, which may include causing an increase or
a decrease
to in gene expression, a effective quantity of one or more polyamide is
contacted with the cell and
internalized by the cell. The cell may be contacted in vivo or in vitro.
Effective extracellular
concentrations of polyamides that can modulate gene expression range from
about 10
nanomolar to about 1 micromolar. Gottesfeld, J.M., et al., Nature 387 202-205
(1997). To
determine effective amounts and concentrations of polyamides in vitro, a
suitable number of
cells is plated on tissue culture plates and various quantities of one or more
polyamide are
added to separate wells. Gene expression following exposure to a polyamide can
be monitored
in the cells or medium by detecting the amount of the protein gene product
present as
determined by various techniques utilizing specific antibodies, including
ELISA and western
blot. Alternatively, gene expression following exposure to a polyamide can be
monitored by
2o detecting the amount of messenger RNA present as determined by various
techniques, including
northern blot and RT-PCR.
Similarly, to determine effective amounts and concentrations of polyamides for
in vivo
administration, a sample of body tissue or fluid, such as plasma, blood,
urine, cerebrospinal
fluid, saliva, or biopsy of skin, muscle, liver, brain or other appropriate
tissue source is
analyzed. Gene expression following exposure to a polyamide can be monitored
by detecting
the amount of the protein gene product present as determined by various
techniques utilizing
specific antibodies, including ELISA and western blot. Alternatively, gene
expression
following exposure to a polyamide can be monitored by the detecting the amount
of messenger
3o RNA present as determined by various techniques, including northern blot
and RT-PCR.
The polyamides of this invention may be formulated into diagnostic and
therapeutic
compositions for in vivo or in vitro use. Representative methods of
formulation may be found
6
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in Remington: The Science and Practice of Pharmacy, 19th ed., Mack Publishing
Co., Easton,
PA {1995).
For in vivo use, the poiyamides may be incorporated into a physiologically
acceptable
pharmaceutical composition that is administered to a patient in need of
treatment or an animal
for medical or research purposes. The polyamide composition comprises
pharmaceutically
acceptable carriers, excipients, adjuvants, stabilizers, and vehicles. The
composition may be in
solid, liquid, gel, or aerosol form. The polyamide composition of the present
invention may be
administered in various dosage forms orally, parentally, by inhalation spray,
rectally, or
to topically. The term parenteral as used herein includes, subcutaneous,
intravenous,
intramuscular, intrastemal, infusion techniques or intraperitoneally.
The selection of the precise concentration, composition, and delivery regimen
is
influenced by, inter alia, the specific pharmacological properties of the
particular selected
t5 compound, the intended use, the nature and severity of the condition being
treated or diagnosed,
the age, weight, gender, physical condition and mental acuity of the intended
recipient as well
as the route of administration. Such considerations are within the purview of
the skilled artisan.
Thus, the dosage regimen may vary widely, but can be determined routinely
using standard
methods.
Polyamides of the present invention are also useful for detecting the presence
of double
stranded DNA of a specific sequence for diagnostic or preparative purposes.
The sample
containing the double stranded DNA can be contacted by polyamide linked to a
solid substrate,
thereby isolating DNA comprising a desired sequence. Alternatively, polyamides
linked to a
suitable detectable marker, such as biotin, a hapten, a radioisotope or a dye
molecule, can be
contacted by a sample containing double stranded DNA.
The design of bifunctional sequence specific DNA binding molecules requires
the
integration of two separate entities: recognition and functional activity.
Polyamides that
3o specifically bind with subnanomolar affinity to the minor groove of a
predetermined sequence
of double stranded DNA are linked to a functional molecule, providing the
corresponding
bifunctional conjugates useful in molecular biology, genornic sequencing, and
human medicine.
Polyamides of this invention can be conjugated to a variety of functional
molecules, which can
be independently chosen from but is not limited to arylboronic acids, biotins,
polyhistidines
7
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WO 98/37066 PCT/US98/01006
comprised from about 2 to 8 amino acids, haptens to which an antibody binds,
solid phase
supports, oligodeoxynucleotides, N-ethylnitrosourea, fluorescein,
bromoacetamide,
iodoacetamide, DL-a-lipoic acid, acridine, captothesin, pyrene, mitomycin,
texas red,
anthracene, anthrinilic acid, avidin, DAPI, isosulfan blue, malachite green,
psoralen, ethyl red,
4-(psoraen-8-yloxy)-butyrate, tartaric acid, (+)-a-tocopheral, psoralen, EDTA,
methidium,
acridine, Ni(II)~Gly-Gly-His, TO, Dansyl, pyrene, N-bromoacetamide, and gold
particles. Such
bifunctional polyamides are useful for DNA affinity capture, covalent DNA
modification,
oxidative DNA cleavage, DNA photocleavage. Such bifunctional polyamides are
useful for
DNA detection by providing a polyamide linked to a detectable label. Detailed
instructions for
1o synthesis of such bifunctional polyamides can be found in copending U.S.
provisional
application 60/043,444, the teachings of which are incorporated by reference.
DNA complexed to a labeled polyamide can then be determined using the
appropriate
detection system as is well known to one skilled in the art. For example, DNA
associated with
~ 5 a polyamide linked to biotin can be detected by a streptavidin / alkaline
phosphatase system.
The present invention also describes a diagnostic system, preferably in kit
form, for
assaying for the presence of the double stranded DNA sequence bound by the
polyamide of this
invention in a body sample, such brain tissue, cell suspensions or tissue
sections, or body fluid
2o samples such as CSF, blood, plasma or serum, where it is desirable to
detect the presence, and
preferably the amount, of the double stranded DNA sequence bound by the
polyamide in the
sample according to the diagnostic methods described herein.
The diagnostic system includes, in an amount sufficient to perform at least
one
25 assay, a specific polyamide as a separately packaged reagent. Instructions
for use of the
packaged reagents) are also typically included. As used herein, the term
"package" refers
to a solid matrix or material such as glass, plastic (e.g., polyethylene,
polypropylene or
polycarbonate), paper, foil and the like capable of holding within fixed
limits a polyamide of
the present invention. Thus, for example, a package can be a glass vial used
to contain
3o milligram quantities of a contemplated polyamide or it can be a microliter
plate well to which
microgram quantities of a contemplated polypamide have been operatively
affixed, i.e., linked
so as to be capable of being bound by the target DNA sequence. "Instructions
for use" typically
include a tangible expression describing the reagent concentration or at least
one assay method
parameter such as the relative amounts of reagent and sample to be admixed,
maintenance time
g
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periods for reagent or sample admixtures, temperature, buffer conditions and
the like. A
diagnostic system of the present invention preferably also includes a
detectable label and a
detecting or indicating means capable of signaling the binding of the
contemplated polyamide
of the present invention to the target DNA sequence. As noted above, numerous
detectable
labels, such as biotin,- and detecting or indicating means, such as enzyme-
linked (direct or
indirect) streptavidin, are well known in the art.
Figure 1 shows representative structures of polyamides. ImImPyPy-y-ImPyPyPy-~i-
Dp
(1), ImImPyPy-y-ImHpPyPy-~3-Dp (2), and ImImHpPy-y-ImPyPyPy-(3-Dp (3). (Hp = 3-
1o hydroxy-N-methylpyrrole, Im = N-methylimidazole, Py = N-methylpyrrole, (3 =
~3-alanine, y =
y-aminobutyric acid, Dp = Dimethylaminopropylamide). Polyamides were
synthesized by solid
phase methods using Boc-protected 3-methoxypyrrole. imidazole, and pyrrole
aromatic amino
acids, cleaved from the support by aminoiysis, deprotected with sodium
thiophenoxidc, and
purified by reversed phase HPLC. Baird, E. E. & Dervan, P. B. describes the
solid phase
t5 synthesis of polyamides containing imidazole and pyrrole amino acids. J.
Am. Chem. Soc. 118,
6141-6146 ( 1996); also see PCT US 97/003332. The identity and purity of the
polyamides
were verified by 'H NMR, analytical HPLC, and matrix-assisted laser-desorption
ionization
time-of flight mass spectrometry (MALDI-TOF MS-monoisotopic): 1 1223.6 (I223.6
calculated), 2 1239.6 (1239.6 calculated); 3 1239.6 (1239.6 calculated).
2o
Figure 2 illustrates binding models for polyamides 1-3 in complex with 5'-
TGGTCA-3'
and 5'-TGGACA-3' (A~T and T~A in fourth position highlighted). Filled and
unfilled circles
represent imidazole and pyrrole rings respectively; circles cornaining an H
represent 3-
hydroxypyrrole, the curved line connecting the polyamide subunits represents y-
aminobutyric
25 acid, the diamond represents (3-alanine, and the + represents the
positively charged
dimethylaminopropylamide tail group.
Figure 3 shows quantitative DNase I footprint titration experiments with
polyamides 2
and 3 on the 3' 3zP labeled 250-by pJK6 EcoRIlPvuII restriction fragment. Lane
l, intact DNA;
30 lanes 2-11 DNase I digestion products in the presence of 100, 50, 20, 10,
5, 2, 1, 0.5, 0.2, 0.1
nM polyamide, respectively; lane 12, DNase I digestion products in the absence
of polyamide;
lane 13, adenine-specific chemical sequencing. Iverson, B. L. & Dervan, P. B.
describes an
adenine-specific DNA chemical sequencing reaction. Methods Enrymol. 15, 7823-
7830 ( 1987).
9
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WO 98/37066 PCT/US98/01006
All reactions were done in a total volume of 400 ~L. A polyamide stock
solution or H20 was
added to an assay buffer containing radiolabeied restriction fragment, with
the fnal solution
conditions of 10 mM Tris-HC1, 10 mM KC1, 10 mM MgClz, 5 mM CaCIZ, pH 7Ø
Solutions
were allowed to equilibrate for 4-12 h at 22 °C before initiation of
footprinting reactions.
Footprinting reactions, separation of cleavage products, and data analysis
were carried out as
described. White, S., Baird, E. E. & Dervan, P. B. Effects of the A~T/T~A
degeneracy of
pyrrole-imidazole polyamide recognition in the minor groove of DNA.
Biochemistry 35, 6147-
6152 (1996).
Figure 4 shows the structure and equilibrium dissociation constant for
numerous
compounds of the present invention. Polyamides are shown in complex with their
respective
match site. Filled and unfilled circles represent imidazole (Im) and pyrrole
(Py) rings,
respectively; circles containing an H represent 3-hydroxypyrrole (Hp), the
curved line
connecting the polyamide subunits represents y-aminobutyric acid (y), the
diamond represents
(3-alanine (~3), and the + represents the positively charged
dimethylaminopropylamide tail group
(Dp). The equilibrium dissociation constants are the average values obtained
from three DNase
I footprint titration experiments. The standard deviation for each set is less
than 15% of the
reported number. Assays were carried out in the presence of 10 mM Tris~HC1, 10
mM KCI, 10
mM MgCl2, and S mM CaCI, at pH 7.0 and 22°C.
Figure 5 shows the synthetic scheme for 3-O-methyl-N-Boc protected pyrrole-2-
carboxylate. The hydroxypytrole monoester can be prepared in 0.5 kg quantity
using published
procedures on enlarged scale.
Figure 6 shows the solid phase synthetic scheme for ImImPyPy-y-ImHpPyPy-~3-Dp
starting from commercially available Boc-~i-Pam-Resin: (i) 80% TFA/DCM, 0.4 M
PhSH; (ii)
Boc-Py-OBt, DIEA, DMF; (iii) 80% TFA/DCM, 0.4 M PhSH; (iv) Boc-Py-OBt, DIEA,
DMF;
(v) 80% TFA/DCM, 0.4 M PhSH; (vi) Boc-3-OMe-Py-OH, HBTU, DMF, DIEA; (vii) 80%
TFA/DCM, 0.4 M PhSH; {viii) Boc-Im-OH, DCC, HOBt; (ix) 80% TFA/DCM, 0.4 M
PhSH;
(x) Boc-y-aminobutyric acid, DIEA, DMF; (xi) 80% TFA/DCM, 0.4 M PhSH; (xii)
Boc-Py-
OBt, DIEA, DMF; (xiii) 80% TFA/DCM, 0.4 M PhSH; (xiv) Boc-Py-OBt, DMF, DIEA;
(xv)
80% TFA/DCM, 0.4 M PhSH; (vxi) Boc-Im-OH, DCC, HOBt (xvii) 80% TFA/DCM, 0.4 M
PhSH; (xviii) imidazole-2-carboxylic acid, HBTU, DIEA; (xviv)
dimethylaminopropylamine,
55 °C, 18h. Purification by reversed phase HPLC provides ImImPyPy-y-
ImOpPyPy-(3-Dp. (Op
= 3-methoxypyrrole). Treatment of the 3-methyoxypyrrole polyamide with
thiophenol, NaH,
DMF, at 100 °C for 120 min provides polyamide 2 after reverse phase
HPLC purification.
CA 02281947 1999-08-17
WO 98/37066 PCT/US98/01006
Figure 7 shows the aromatic region from 7-11 ppm for the 1H-NMR spectrum
determined at 300 MHz for ImImPyPy-y-ImOpPyPy-~3-Dp and ImImPyPy-y-ImHpPyPy-~i-
Dp.
This region of the spectrum may be used to determine compound identity and
purity.
Figure 8 shows_the MALDI-TOF mass spectrum determined in positive ion mode
with a
monoisotopic detector for the polyamides for ImImPyPy-y-ImOpPyPy-~i-Dp and
ImImPyPy-y-
ImHpPyPy-~i-Dp. This spectrum may be used to determine compound identity and
purity.
Figure 9 shows the methyl group region from 3.5-4.0 ppm for the 1 H-NMR
spectrum
to determined at 300 MHz for ImPyPy-y-OpPyPy-~3-Dp and ImPyPy-y-HpPyPy-(3-Dp.
This region
of the spectrum may be used to directly follow the progress for conversion of
3-methoxypyrrole
to 3-hydroxypyrrole.
Fig. 10 shows quantitative DNase I footprint titration experiments with the
polyamides
~ 5 ImPyPy-y-PyHpPy-(3-Dp and ImHpPy-y-PyPyPy-~i-Dp on the 3'-3zP labeled 370-
by pDEH 1
EcoRI/Pw~II restriction fragment. Intact lane, labeled restriction fragment no
polyamide or
DNase I added; lanes 1-10, DNase I digestion products in the presence of 10
pM, 5 pM, 2 pM,
1 pM, 500 nM, 200 nM, 100 nM, 50 nM, 20 nM, 10 nM ImPyPy-y-PyPyPy-~i-Dp,
respectively
or 1 pM, 500 nM, 200 nM, 100 nM, 50 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM ImHpPy-
y-
2o PyPyPy-~3-Dp, respectively; DNase I lane, DNase I digestion products in the
absence of
polyamide; A lane, adenine-specific chemical sequencing. Iverson, B. L. &
Dervan, P. B.
describes an adenine-specific DNA chemical sequencing reaction. Methods
Enrymol. 15, 7823-
7830 ( 1987). All reactions were done in a total volume of 40 uL. A polyamide
stock solution
or Hz0 was added to an assay buffer containing radiolabeled restriction
fragment, with the final
25 solution conditions of 10 mM Tris-HC1. 10 mM KC1, 10 mM MgClz, 5 mM CaCl2,
pH 7Ø
Solutions were allowed to equilibrate for 4-12 h at 22 °C before
initiation of footprinting
reactions. Footprinting reactions, separation of cleavage products, and data
analysis were
carried out as described. White, S., Baird, E. E. & Dervan, P. describe the
pairing rules for
recognition in the minor groove of DNA by pyrrole-imidazole polyamides.
Chemistry ~
3o Biology 4, 569-578 (1997).
Figure 11 shows the synthesis of a bifunctional polyamide which incorporates
the Hp/Py
pair. Treatment of a sample of ImImPyPy-y-ImHpPyPy-~i-Pam-resin (see Figure 6)
with 3,3'-
diamino-N methyldipropylamine, 55°C, 18 h followed by reverse phase
HPLC purification
35 provides the Op polyamide with a free primary amine group which can be
coupled to an
activated carboxylic acid derivative. Treatment with (i) EDTA-dianhydride,
DMSO/NMP,
DIEA, SS °C; (ii) O.1M NaOH, followed by reverse phase HPLC
purification provides the Op-
Py-Im-polyamide-EDTA conjugate. Treatment of the 3-methyoxypyrrole polyamide
with
CA 02281947 1999-08-17
WO 98/37066 PCT/LTS98/01006
thiophenol, NaH, DMF, at 100 °C for 120 min provides polyamide 2 after
reverse phase HPLC
purification.
Figure 12 shows the determination of the binding orientation of hairpin
polyamides
ImImPyPy-y-ImHpPypy-~3-Dp-EDTA~Fe(II) 2-E~Fe(II) and ImImHpPy-y-ImPyPypy-(3-Dp-
EDTA~Fe(II) 3-E~Fe(II) by affinity cleaving footprint titration. Top and
bottom left: Affinity
cleavage experiments on a 3' 3zP labeled 250-by pJK6 EcoRIl Pvu II restriction
fragment. The
5'-TGGACA-3' and 5'-TGGTCA-3' sites are shown on the right side of the
autoradiogram.
Top left: lane l, adenine-specific chemical sequencing reaction; lanes 2-6,
6.5 ItM, 1.0 pM,
to 100 nM, 10 nM, 1 nM polyamide 2-E~Fe(II); lane 7, intact restriction
fragment, no polyamide
added. Bottom left: lane 1, A reaction; lanes 2-6, 8.5 pM, 1.0 pM, 100 nM, 10
nM, 1 nM
poIyamide 3-E~Fe(II); lane 7, intact DNA. All reactions were carried out in a
total volume of
40 pL. A stock solution of polyamide or H,0 was added to a solution containing
20 kcpm
labeled restriction fragment, affording final solution conditions of 25 mM
Tris-Acetate, 20 mM
NaCI, 100 pM/ by calf thymus DNA, at pH 7Ø Solutions were allowed to
equilibrate for a
minimum of 4 h at 22°K before initiation of reactions. Affinity
cleavage reactions were carried
out as described White, S., Baird, E.E. & Dervan, P.B. Effects of the A~TfT~A
degeneracy of
pyrrole-imidazole polyamide recognition in the minor groove of DNA.
Biochemisrn~ 35, 6147
6152 ( 1996). Top and bottom right: Affinity cleavage patterns of 2-E~Fe(II)
and 3-E~Fe(II) ai
100 nM bound to 5'-TGGACA-3' and 5'-TGGTCA-3'. Bar heights are proportional to
the
relative cleavage intensities at each base pair. Shaded and nonshaded circles
denote imidazole
and pyrrole carboxamides, respectively. Nonshaded diamonds represent the (3-
alanine moiety.
A curved line represents the y-aminobutyric acid, and the + represents the
positively charged
dimethylaminopropylamide tail group. The boxed Fe denotes the EDTA~Fe(II)
cleavage
moiety.
Figure 13 shows quantitative DNase I footprint titration experiments with the
polyamides ImPyPyPyPy-y-ImPyPypyPy-~3-Dp and ImHpPyPyPy-y-ImHpPyPyPy-(3-Dp on
the
3' 3zP labeled 252-by pJK7 EcoRIl Pvu II restriction fragment. For ImPyPyPyPy-
Y-
3o ImPyPyPyPy-~3-Dp gel (left): lane 1, DNase I digestion products in the
absence of polyamide;
lanes 2-18, DNase I digestion products in the presence of 1.0 ~tM, 500 nM,
200, 100, 65, 40, 25,
15, 10, 6.5, 4.0, 2.5, 1.5, 1.0, 0.5, 0.2, 0.1 nM polyamide; lane 19, DNase I
digestion products in
the absence of polyamide; lane 20, intact restriction fragment; lane 21,
guanine-specific
chemical sequencing reaction; lane 22, adenine-specific chemical sequencing
reaction. For
ImHpPyPyPy-y-ImHpPypyPy-~i_Dp gel (right): lane 1, intact DNA; lane 2, DNase I
digestion
products in the absence of poiyamide; lanes 3-19, 1.0 pM, 500 nM, 200, 100,
S0, 20, 10, 5, 2, 1,
0.5, 0.2. 0.1, 0.05, 0.01, 0.005, O.OOI nM polyamide; lane 20, DNase I
digestion products in the
absence of polyamide; lane 21, A reaction. All reactions were done in a total
volume of 400
t2
CA 02281947 1999-08-17
WO 98/37066 PCTNS98/01006
wL. A polyamide stock solution or H,0 was added to an assay buffer containing-
radiolabeled
restriction fragment, with the final solution conditions of 10 mM Tris-HCI, 10
mM KCI, 10 mM
MgCl2, 5 mM CaCl2, pH 7Ø Solutions were allowed to equilibrate for 4-12 h at
22°C before
initiation of footprinting reactions. Footprinting reactions, separation of
cleavage products, and
data analysis were carried as described. White, S., Baird, E.E. & Dervan, P.B.
Effects of the
A~T/T~A degeneracy of pyrrole-imidazole polyamide recognition in the minor
groove of DNA.
Biochemistry 35, 6147-6152 (1996).
Fig. 14 shows the 8-ring Hp-Py-Im-polyamide hairpins described by the pairing
code of
to the present invention. The eight ring hairpin template is shown at the top.
A polyamide having
the formula X, XzX3X4-Y-XSX6X~X8 wherein y is the -NH-CHZ-CHz-CHZ-CONH-
hairpin
linkage derived from y-aminobutyric acid or a chiral hairpin linkage derived
from R-2,4-
diaminobutyric acid; X4/X5, X3/X6, Xz/X~, and X~/Xe represent carboxamide
binding pairs
which bind the DNA base pairs. The minor groove sequence to be bound is
represented as 5'-
WGTNNW-3', where the 5'-GTNN-3' core sequence is defined as position a, b, c,
and d (W =
A or T, N = A, G, C, or T). A linear sequence of aromatic amino acids fills
the hairpin template
in order to satisfy the ring pairing requirements to correspond to the DNA
base pairs in the
minor groove to be bound. The ring pairing code as applied is listed in Table
2. The 16 unique
hairpin polyamides which target 16 5'-WGTNNW-3' sequences are drawn as binding
models
where filled and unfilled circles represent imidazole and pvrrole rings
respectively; circles
containing an H represent 3-hydroxypyrrole, and the curved line connecting the
polyamide
subunits represents y-aminobutyric acid.
Fig. 15 shows the 8-ring Hp-Py-Im-polyamide hairpins described by the pairing
code of
the present invention. The eight ring hairpin template is shown at the top. A
polyamide having
the formula X, X~X3X~-y-XSX~X~Xg wherein y is the -NH-CHz-CHI-CHI-CONH-
hairpin
linkage derived from y-aminobutyric acid or a chiral hairpin linkage derived
from R-2,4-
diaminobutyric acid; X4/X5, X3/X6, XZ/X~, and X~/X$ represent carboxamide
binding pairs
which bind the DNA base pairs. The minor groove sequence to be bound is
represented as S'-
3o WGANNW-3', where the 5'-GANN-3' core sequence is defined as position a, b,
c, and d (W =
A or T, N = A, G, C, or T). A linear sequence of aromatic amino acids fills
the hairpin template
in order to satisfy the ring pairing requirements to correspond to the DNA
base pairs in the
minor groove to be bound. The ring pairing code as applied is listed in Table
2. The 16 unique
hairpin polyamides which target 16 S'-WGA,NN~W-3' sequences are drawn as
binding models
where filled and unfilled circles represent imidazole and pyrrole rings
respectively; circles
containing an H represent 3-hydroxypyrrole, and the curved line connecting the
polyamide
subunits represents y-aminobutyric acid.
13
CA 02281947 1999-08-17
WO 98/37066 PCT/US98/01006
Fig. 16 shows the 8-ring Hp-Py-Im-polyamide hairpins described by the pairing
code of
the present invention. The eight ring hairpin template is shown at the top. A
polyamide having
the formula X, XZX3X4-y-XSX6X~X8 wherein y is the -NH-CHZ-CHz-CHZ-CONH-
hairpin
linkage derived from y-aminobutyric acid or a chiral hairpin linkage derived
from 8-2,4-
diaminobutyric acid; X4/X5, X3/X6, XZ/X~, and X,/Xg represent carboxamide
binding pairs
which bind the DNA base pairs. The minor groove sequence to be bound is
represented as 5'-
WGGNNW-3', where the 5'-GGNN-3' core sequence is defined as position a, b, c,
and d (W =
A or T, N = A, G, C, or T). A linear sequence of aromatic amino acids fills
the hairpin template
in order to satisfy the ring pairing requirements to correspond to the DNA
base pairs in the
minor groove to be bound. The ring pairing code as applied is listed in Table
2. The 16 unique
hairpin polyamides which target 16 5'-WGGNNW-3' sequences are drawn as binding
models
where filled and unfilled circles represent imidazole and pyrroIe rings
respectively; circles
containing an H represent 3-hydroxypyrrole, and the curved line connecting the
polyamide
subunits represents y-aminobutyric acid.
Fig. 17 shows the 8-ring Hp-Py-Im-polyamide hairpins described by the pairing
code of
the present invention. The eight ring hairpin template is shown at the top. A
polyamide having
the formula X,XzX3X.,-y-XSXbX~Xg wherein y is the -NH-CH,-CHz-CH,-CONH-
hairpin
linkage derived from y-aminobutyric acid or a chiral hairpin linkage derived
from 8-2,4-
diaminobutyric acid; X,,/X5, X3/X6, Xz/X7, and X,/X8 represent carboxamide
binding pairs
which bind the DNA base pairs. The minor groove sequence to be bound is
represented as S'-
WGCNNW-3', where the 5'-GCNN-3' core sequence is defined as position a, b, c,
and d (W =
A or T, N = A, G, C, or T). A linear sequence of aromatic amino acids fills
the hairpin template
in order to satisfy the ring pairing requirements to correspond to the DNA
base pairs in the
Z5 minor groove to be bound. The ring pairing code as applied is listed in
Table 2. The 16 unique
hairpin polyamides which target 16 5'-WGCNNW-3' sequences are drawn as binding
models
where filled and unfilled circles represent imidazole and pyrrole rings
respectively; circles
containing an H represent 3-hydroxypyrrole, and the curved line connecting the
polyamide
subunits represents y-aminobutyric acid.
Four-ring polyamide subunits, covalently coupled to form eight-ring hairpin
structures,
bind specifically to 6-by target sequences at subnanomolar concentrations.
Trauger, J.W.,
Baird, E. E. & Dervan, P.B. describe the recognition of DNA by designed
ligands at
subnanomolar concentrations. Nature 382, 559-561 (1996); Swalley, S. E.,
Baird, E. E. &
Dervan, P. B. describe the discrimination of 5'-GGGG-3', 5'-GCGC-3', and 5'-
GGCC'3'
sequences in the minor groove of DNA by eight-ring hairpin polyamides. J. Am.
Chem. Soc.
119, 6953-6961 (1997). The DNA-binding affinities of three eight-ring hairpin
polyamides
shown in Figure 1 as compound 1, 2, and 3 containing pairings of Im/Py, Py/Im
opposite G~C,
14
CA 02281947 1999-08-17
WO 98/37066 PCT/US98/01006
C~G and either Py/Py, Hp/Py, or Py/Hp at a common single point opposite T~A
and A~T has
been determined. Equilibrium dissociation constants (Kd) for IrnInlPyPy-y-
ImPyPyPy-~i-Dp 1,
ImImPyPy-y-ImHpPyPy-~i-Dp 2, ImImHpPy-y-ImPyPyPy-~3-Dp 3 of Figure 1 are shown
in
Table 1. Brenowitz, M., Senear, D. F., Shea, M. A. & Ackers, G. K. describe a
quantitative
DNase footprint titration method for studying protein-DNA interactions.
Methods Enrymol.
130, 132-181 (1986); The ICd values were determined by quantitative DNase I
footprint
titration experiments: on a 3' 32P-labeled 250-by DNA fragment containing the
target sites, 5'-
TGGACA-3' and 5'-TGGTCA-3' which differ by a single A~T base pair in the
fourth position.
The DNase footprint gels are shown in Figure 3.
TABLE
1 Equilibrium
dissociation
constants'
Polyamidet5'-TGGTCA-3' S'-TGGACA-3' Krelx
5'-T T C S'-T G G A
G A-3' C A-3'
G
1 Py/Py + + 2.0
3'-A A G 3'-A C C T
C T-5' G T-5'
C
K~ Kd = 0.15 nM
=
0.077
nM
5'-T T C S'-T G G A
G A-3' C A-3'
G
2 ~~p ~ ' ~
3 A G 3
-A T-5 -A C C T G
C T-5'
C
Kd=ISnh9 Ka=0.83nM
5'-T T C S'-T G G A
G H A-3' C A-3'
G H
3 Hp/Py + + 77
3'-A A G 3'-A C C T
C T-5' G T-5'
C
- Kd Kd =37 nM
=
0.48
nM
'The reported dissociation constants are the average values obtained from
three
DNase 1 footprint titration experiments. The standard deviation for each data
set is
less than 15% of the reported number. Assays were carried out in the presence
of 10
mM Tris~HCI, 10 mM KCI, 10 mM MgCl2, and 5 mM CaCl2 at pH 7.0 and 22
°C.
tRing pairing opposite T~A and A~T in the fourth position.
$Calculated as Kd(5'-TGGACA-3')/Kd(5'-TGGTCA-3').
Based on the pairing rules for polyamide-DNA complexes both of these sequences
are a
match for control polyamide 1 which places a Py/Py pairing opposite
A~T and T~A at both sites. It was determined that in polyamide 1 (Py/Py) binds
to 5'-
TGGTCA-3' and 5'-TGGACA-3' within a factor of 2 (K~ = 0.077 or 0.15 nM
respectively). In
contrast, polyamide 2 (Py/Hp) binds to 5'-TGGT_CA-3' and 5'-TGGACA-3' with
dissociation
constants which differ by a factor of 18 (K,, = 15 nM and 0.83 nM
respectively). By reversing
the pairing in polyamide 3 (Hp/Py) the dissociation constants differ again in
the opposite
direction by a factor of 77 (Ko = 0.48 nM and 37 nNi respectively. Control
experiments
performed on separate DNA fragments; reveal that neither a 5'-TGGC~. CA-3' or
a 5'-TGG~CA-
3' site is bound by polyamide 2 or 3 at concentrations S 100 nM, indicating
that the Hp/Py and
Py/Hp ring pairings do not bind opposite G~C or C~G. The A~T vs. T~A
discrimination is
achieved preferably when the two neighboring base pairs are G~C and C~G (GTC
vs. GAC).
15
CA 02281947 1999-08-17
WO 98/37066 PCT/ITS98/OI006
The specificity of polyamides 2 and 3 for sites which differ by a single
A~T/T~A base
pair results from small chemical changes. Replacing the Py/Py pair in 1 with a
Py/Hp pairing
as in 2, a single substitution of C3-OH for C3-H, destabilizes interaction
with 5'-TGG_TCA-3'
by 191-fold, a free energy difference of 3.1 kcal mol-'. Interaction of 2 with
5'-TGGACA-3' is
destabilized only 6-foW relative to 1, a free energy difference of 1.1 kcal
mol-'. Similarly,
replacing the Py/Py pair in 1 with Hp/Py as in 3 destabilizes interaction with
5'-TGG_ACA-3'
by 252-fold, a free energy difference of 3.2 kcal mol-'. Interaction of 3 with
5'TGG_TCA-3' is
destabilized only 6-fold relative to 1, a free energy difference of 1.0 kcal
mol-'.
to The polyamides of this invention provide for coded targeting of
predetermined DNA
sequences with affinity and specificity comparable to sequence-specific DNA
binding proteins.
Hp, Im, and Py polyamides complete the minor groove recognition code using
three aromatic
amino acids which combine to form four ring pairings (Im/Py, Py/Im, Hp/Py, and
Py/Hp) which
complement the four Watson-Crick base pairs, as shown in TABLE '_'. There are
a possible 240
four base pair sequences which contain at least 1 A~T or T~A base pair and
therefore can
advantageously use an Hp/Py, or Py/Hp carboxamide binding. Polyamides binding
to any of
these sequences can be designed in accordance with the code of TABLE 2.
TABLE 2 Pairing code for minor groove recognition'
Pair G~C C~G T~A A~T
Im/Py + - - -
Py/Im - + - -
Hp/Py - - -i- -
Py/Hp - - - .f-
* favored (+), disfavored (-}
16
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WO 98/37066 PCT/US98/01006
For certain G~C rich sequences the affinity of polyamide~DNA complexes may be
enhanced by substitution of an Im/~i pair for Im/Py at G~C and /3/Im for Py/Im
at C~G. At A~T
and T~A base pairs, either a Py/~i, /3/Py, and ~i/~i may be used. The
alternate aliphatic/aromatic
amino acid pairing code is described in Table 3.
-
TABLE 3 Aliphatic/Aromatic substitution for ring
pairings"
Pair Substitution
Im/Py Imlø
Py/Im ~/Im
Hp/Py PYI~, ~/PY, Hp/~, ~/~
PYMP PY/~, ~~Y. A/HP, 13/13
U. S. Patent 5,578,444 describes numerous promoter region targeting sequences
from
which base pair sequences for targeting a polyamide can be identified.
PCT U.S. 97/00333? describes methods for synthesis of polyamides which are
suitable
for preparing polyamides of this invention. The use of ~i-alanine in place of
a pyrrole amino
acid in the synthetic methods provides aromatic/aliphatic pairing (Im/~3,
~i/Im, Py/~i, and ~3/Py)
and aliphatic/aliphatic pairing ((3/(3} substitution. The use of y-
aminobutyric acid, or a
substituted y-aminobutyric acid such as (R)-2,4 diaminobutyric acid, provides
for preferred
hairpin turns. The following examples illustrate the synthesis of polyamides
of the present
invention.
17
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WO 98/37066 PCT/US98/01006
Example l:
PREPARATION OF A PROTECTED Hp MONOMER FOR SOLID PHASE
SYNTHESIS.
Distamycin and its analogs have previously been considered targets of
traditional
multistep synthetic chemistry. Arcamone, F., Orezzi, P. G., Barbieri, W.,
Nicolella, V. & Penco,
S. describe a solution phase synthesis of distamycin Gazz. Chim. Ital. 1967,
97, 1097. The
repeating amide of distamycin is formed from an aromatic carboxylic acid and
an aromatic
amine. The aromatic acid is often unstable to decarboxyiation, and the
aromatic amines have
been found to be air and light sensitive. Lown, J. W. & Krowicki, K. describe
a solution phase
to synthesis of Distamycin J. Org. Chem. 1985, S0, 3774. The variable coupling
yields, long
reaction times (often >24 h), numerous side products, and reactive
intermediates (acid chlorides
and trichloro ketones) characteristic of the traditional solution phase
coupling reactions make
the synthesis of the aromatic carboxamides problematic. B. Merrifield
describes the solid phase
synthesis of a tetrapeptide J. Am. Chem. Soc. 1963, 85, 2149. In order to
implement an efficient
solid phase methodology for the synthesis of the pyrrole- imidazole
polyamides, the following
components were developed: (1) a synthesis which provides large quantities of
appropriately
protected monomer or dimer building blocks in high purity, (2) optimized
protocols for forming
an amide in high yield from a support-bound aromatic amine and an aromatic
carboxylic acid,
(3) methods for monitoring reactions on the solid support, and (4) a stable
resin linkage agent
2o that can be cleaved in high yield upon completion of the synthesis. Baird,
E. E. & Dervan, P. B.
describes the solid phase synthesis of polyamides containing imidazole and
pyrrole amino
acids. J. Am. Chem. Soc. 118, 6141-6146 (1996); also see PCT US 97/003332. In
order to
prepare polyamides which contain the 3-hydroxypyrrole monomer, a synthesis has
been
developed which allows the appropriately protected Boc-Op acid monomer to be
prepared on 50
g scale. 1H NMR and t3C NMR spectra were recorded on a General Electric-QE 300
NMR
spectrometer in CD30D or DMSO-d6, with chemical shifts reported in parts per
million relative
to residual CHD20D or DMSO-d5, respectively. IR spectra were recorded on a
Perkin-Elmer
FTIR spectrometer. High-resolution mass spectra were recorded using fast atom
bombardment
(FABMS) techniques at the Mass Spectrometry Laboratory at the University of
California,
3o Riverside. Reactions were executed under an inert argon atmosphere. Reagent
grade chemicals
were used as received unless otherwise noted. Still, W. C., Kahn, M. & Mitra,
A. describe flash
column chromatography J. Org. Chem. 1978, 40, 2923-2925. Flash chromatography
was
carried out using EM science Kieselgel 60 (230-400) mesh. Thin-layer
chromatography was
performed on EM Reagents silica gel plates (0.5 mm thickness). All compounds
were
visualized with short-wave ultraviolet light.
18
CA 02281947 1999-08-17
WO '98/37066 PCT/US98/01006
Table 4 :Intermediates for preparation of Boc-protected 3-methoxypyrrole
NAME STRUCTURE
0
OH
Ethyl4-carboxy-3-hydroxy-1- HO /
methylpyrrole-2-carboxylate.
N
O
Ourw OH
Ethy14-[ (Benzyloxycarbonyl )amino)-3-
hydroxy-1-methylpyrrole-2-carboxylate / O
~N
\ ~ ~ O
O N OMe
Ethy14-[(Benzyloxycarbonyi)amino)-3-
methoxy-1-methylpyrrole-2-carboxylate / I O N~O~
\ ~ o
Ethyl4-[(tert-Butyloxvcarbonyl)amino]-3- \/O N OMe
methoxy-I-methylpyrrole-2-carboxylate
N~
O
o N OMe
4-[(tert-Butyloxycarbonvl)amino)-3-methoxy
-1-meth 1 mole-2-carbox lic acid
Y PY Y ~oH
N
O
Ethyl 4-((benzvlo~ycarbonyl)aminoJ-3-hydroxy-I-methylpyrrole-2-carboxylate
Ethyl-4-
carboxy-3-hydroxy-1-methylpyrrole-2-carboxylate (60 g, 281.7 mmol) was
dissolved in 282
mL acetonitrile. TEA (28.53 g, 282 mmol) was added, followed by
diphenylphosphorylazide
{77.61 g, 282 mmol). The mixture was refluxed for 5 hours, followed by
addition of benzyl
alcohol (270 ml) and reflux continued overnight. The solution was cooled and
volitiles
removed in vacuo. The residue was absorbed onto silca and chromatagraphed, 4:1
hexanes
ethyl acetate, to give a white solid (21.58 g, 24%) 1H NMR (DMSO-d6) 8 8.73
(s, 1H), 8.31 (s,
l0 1H), 7.31 (m, 5H), 6.96 (s, 1H), 5.08 (s, 2H), 4.21 (q, 2H, J = 7.1 Hz),
3.66 (s, 3H), 1.25 (t, 3H,
J = 7.1 Hz); MS mle 319.163 (M+H 319.122 calcd. for C16H18~205).
Ethyl 4-~(tert-buto.~ycarbonyl)aminoJ-3-methoxy-I-methylpyrrole-1-
carboxylate. Ethyl
4-[(benzyloxycarbonyl)amino]-3-hydroxy-1-methyipyrrole-2-carboxylate (13.4 g,
42.3 mmol)
was dissolved in 110 mL acetone. Anhydrous K2C03 ( 11.67 g, 84.5 mmoI) was
added,
19
CA 02281947 1999-08-17
WO 98/37066 PCT/US98/01006
followed by methyliodide (5.96 g, 42.3 mmol) and dimethylaminopyridine (0.5 g,
4.23 mmol)
and the mixture stirred overnight. The solid K2C03 was removed by filtration
and 200 ml
water added. Volitiles were removed in vacuo and the solution made acidic with
addition of 1N
H2S04 . The aqueous layer was extracted with diethyl ether. Organic layers
were combined,
washed with 10% H2S04, dried over MgS04, and dried to give a white solid. The
solid was
used without further purification and dissolved in 38 ml DMF. DIEA (11 ml),
Boc anhydride
(9.23 g, 42.3 mmol), and 10 % Pd/C (S00 mg) were added and the solution
stirred under
hydrogen (1 atm) for 2.1 h. The slurry was filtered through celite which was
washed with
methanol. Water 250 ml was added and volitiles removed in vacuo. The aqueous
layer was
extracted with ether. Organic layers were combined, washed with water and
brine, and dried
over MgS04. Solvent was removed in vacuo to give a white solid ( 8.94 g, 71 %)
1 H NMR
{DMSO-d6) 8 8.43 (s, 1H), 7.03 (s, 1H), 4.19 (q, 2H, J = 7.1 Hz), 3.70 (s,
3H), 3.67 (s, 3H),
1.42 (s, 9H), 1.26 (t, 3H, J = 7.1); MS mle 299.161 (M+H 299.153 calcd. for
C14H22N205)~
Ethvl 4-((benzvlox~~carbonyDaminoJ-3-hvdroxv-I-methvlpvrrole-?-carboxtelate
Ethyl 4-
[(tert-butoxycarbonyl)aminoJ-3-methoxy-1-methylpyrrole-2-carboxylate (9.0 g,
30.2 mmol)
was dissolved in 30 mL ethanol. NaOH (30 ml, 1 M, aq) was added and the
solution stirred for
4 days. Water (200 ml) was added and ethanol removed in vacuo. The solution
was extracted
with diethyl ether, aqueous layer acidified to pH = 2-3, and extracted again
with diethyl ether.
2o Organic layers were dried over MgS04, and solvent removed in vacuo to give
a white solid (6.0
g, 20.5 mmol, 87% based on recovered SM) 1 H NMR (DMSO-d6) 8 12.14 (s, 1 H),
8.37 (s,
1H), 6.98 (s, 1H), 3.69 (s, 3H), 3.66 (s, 3H), 1.42 (s, 9H); MS mle 293.112
{M+H 293.104
calcd. forC12H1gN205).
EXAMPLE 2:
SOLID PHASE SYNTHESIS OF 3-HYDROXYPYRROLE POLYAMIDES.
Cycling protocols were optimized to afford, high stepwise coupling yields
(>99%).
Deprotection by aminolysis affords up to 100 mg quantities of polyamide. Solid
phase
3o polyamide synthesis protocols were modified from the in situ neutralization
Boc-chemistry
protocols. Schnolzer, M., Alewood, P., Jones, A., Alewood, D., Kent, S.B.H.
report rapid in situ
neutralization for solid phase peptide synthesis Int. J. Peptide. Protein.
Res. 1992, 40, 180.
Coupling cycles are rapid, 72 min per residue for manual synthesis or 180 min
per residue for
machine-assisted synthesis, and require no special precautions beyond those
used for ordinary
solid phase peptide synthesis. Manual solid phase synthesis of a pyrrole-
imidazole polyamide
consists of a dichloromethane (DCM) wash, removal of the Boc group with
trifluoroacetic acid
(TFA)/DCM/thiophenol (PhSH), a DCM wash, a DMF wash, taking a resin sample for
analysis,
addition of activated monomer, addition of DIEA if necessary, coupling for 45
min, taking a
CA 02281947 1999-08-17
WO 98/37066 PCT/US98/01006
resin sample for analysis, and a final DMF wash (Figure 5, Table I). In
addition, the manual
solid phase protocol for synthesis of pynole-imidazole polyamides has been
adapted for use on
a ABI 430A peptide synthesizer. The aromatic amine of the pyrrole and
imidazole do not react
in the quantitative ninhydrin test. Stepwise cleavage of a sample of resin and
analysis by HPLC
indicates that high stepwise yields (> 99%) are routinely achieved.
Dicyclohexylcarbodiimide (DCC), Hydroxybenzotriazole (HOBt), 2-( 1 H-
Benzotriazole-
1-yl)-1,1,3,3-tetramethyluronium hexa-fluorophosphate (HBTL>) and 0.2
mmol/gram Boc-/3-
alanine-(-4-carboxamidomethyl)-benzyl-ester-copoly(styrene-divinylbenzene)
resin (Boc-~-
1o Pam-Resin) was purchased from Peptides International (0.2 mmol/gram),
NovaBiochem (0.6
mmol/gram), or Peninsula (0.6 mmoUgram). ( (R)-2-Fmoc-4-Boc-diaminobutyric
acid, (S~-2-
Fmoc-4-Boc-diaminobutyric acid, and (R)-2-amino-4-Boc-diaminobutyric acid were
purchased
from Bachem. N,N-diisopropylethylamine (DIEA), N,N-dimethylformamide (DMF), N-
methylpyrrolidone (NMP), DMSO/NMP, Acetic anh dride Ac,
Y ( O), and 0.0002 M potassium
~5 cyanide/pyridine were purchased from Applied Biosystems. Dichloromethane
(DCM) and
triethylamine (TEA) were reagent grade from EM, thiophenol (PhSH),
dimethylaminopropylamine (Dp), Sodium Hydride, (R)-a-methoxy-a
(trifuoromethyl)phenylacetic acid ((R)MPTA) and (,S~-a-methoxy-a
(trifouromethyl)phenylacetic acid ((.S~MPTA) were from Aldrich,
trifluoroacetic acid (TFA)
20 Biograde from Halocarbon, phenol from Fisher, and ninhydrin from Pierce.
All reagents were
used without further purification.
Quik-Sep polypropylene disposable filters were purchased from Isolab Inc. ~H
NMR
spectra were recorded on a General Electric-QE NMR spectrometer at 300 MHz
with chemical
25 shifts reported in parts per million relative to residual solvent. UV
spectra were measured in
water on a Hewlett-Packard Model 8452A diode array spectrophotometer. Optical
rotations
were recorded on a JASCO Dip 1000 Digital Polarimeter. Matrix-assisted, laser
desorption/ionization time of flight mass spectrometry (MALDI-TOF) was
performed at the
Protein and Peptide Microanalytical Facility at the California Institute of
Technology. HPLC
3o analysis was performed on either a HP 1090M analytical HPLC or a Beckman
Gold system
using a RAINEN C,g, Microsorb MV, Spm, 300 x 4.6 mm reversed phase column in
0.1%
(wtJv) TFA with acetonitrile as eluent and a flow rate of 1.0 mL/min, gradient
elution 1.25%
acetonitrile/min. Preparatory reverse phase HPLC was performed on a Beckman
HPLC with a
Waters DeltaPak 25 x 100 mm, 100 ltm C 18 column equipped with a guard, 0.1 %
(wt/v) TFA,
3s 0.25% acetonitrile/min. 18MS2 water was obtained from a Millipore MilliQ
water purification
system, and all buffers were 0.2 pm filtered.
z1
CA 02281947 1999-08-17
WO 98/37066 PCT/US98/01006
Activation of Boc-3-methoxypyrrole acid. The amino acid (0.5 mmol) was
dissolved in 2 mL
DMF. HBTU ( 190 mg, 0.5 mmol) was added followed by DIEA ( 1 mL) and the
resulting
mixture was shaken for 5 min.
Activation of Imidazole-2-carboxylic acid, y-aminobntyric acid, Boc-glycine,
and Boc-~i-
alanine. The appropriate amino acid or acid (2 mmol) was dissolved in 2 mL
DMF. HBTU
(720 mg, 1.9 mmol) was added followed by DIEA (1 mL) and the solution shaken
for at least 5
min.
to Activation of Boc-Imidazole acid. Boc imidazole acid (257 mg, 1 mmol) and
HOBt (135 mg,
1 mmol) were dissolved in 2 mL DMF, DCC (202 mg, 1 mmol) is then added and the
solution
allowed to stand for at least S min.
Acetylation Mix. 2 mL DMF, DIEA (710 ~L, 4.0 mmol), and acetic anhydride (380
~L, 4.0
mmol) were combined immediately before use.
Manual Synthesis Protocol. Boc-13-alanine-Pam-Resin ( 1.25 g, 0.25 mmol) is
placed in a 20
mL glass reaction vessel, shaken in DMF for 5 min and the reaction vessel
drained. The resin
was washed with DCM (2 x 30 s.) and the Boc group removed with 80%
TFA/DCM/O.SM
2o PhSH, 1 x 30s., 1 x 20 min The resin was washed with DCM {2 x 30 s.)
followed by DMF ( 1 x
30 s.) A resin sample (5 - 10 mg) was taken for analysis. The vessel was
drained completely and
activated monomer added, followed by DIEA if necessary. The reaction vessel
was shaken
vigorously to make a slung. The coupling was allowed to proceed for 90 min,
and a resin
sample taken. Acetic anhydride ( 1 mL) was added and the reaction shaken for 5
min. The
zs reaction vessel was then washed with DMF, followed by DCM.
Machine-Assisted Protocols. Machine-assisted synthesis was performed on a ABI
430A
synthesizer on a 0.18 mmol scale (900 mg resin; 0.2 mmol/gram). Each cycle of
amino acid
addition involved: deprotection with approximately 80% TFA/DCM/0.4M PhSH for 3
minutes,
3o draining the reaction vessel, and then deprotection for 17 minutes; 2
dichloromethane flow
washes; an NMP flow wash; draining the reaction vessel; coupling for 1 hour
with in situ
neutralization, addition of dimethyl sulfoxide {DMSO)/NMP, coupling for 30
minutes, addition
of DIEA, coupling for 30 minutes; draining the reaction vessel; washing with
DCM, taking a
resin sample for evaluation of the progress of the synthesis by HPLC analysis;
capping with
35 acetic anhydride/DIEA in DCM for 6 minutes; and washing with DCM. A double
couple cycle
is employed when coupling aliphatic amino acids to imidazole, all other
couplings are
performed with single couple cycles.
22
CA 02281947 1999-08-17
W0 98/37066 PCT/US98/01006
The ABI 430A synthesizer was left in the standard hardware configuration for
NMP-
HOBt protocols. Reagent positions 1 and 7 were DIEA, reagent position 2 was
TFA/O.SM
thiophenol, reagent position 3 was 70% ethanolamine/methanol, reagent position
4 was acetic
anhydride, reagent position S was DMSO/NMP, reagent position 6 was methanol,
and reagent
position 8 was DMF: New activator functions were written, one for direct
transfer of the
cartridge contents to the concentrator (switch list 21, 25, 26, 35, 37, 44),
and a second for
transfer of reagent position 8 directly to the cartridge (switch list 37, 39,
45, 46).
Boc-Py-OBt ester (357 mg, 1 mmol) was dissolved in 2 ml DMF and filtered into
a
synthesis cartridge. Boc-Im acid monomer was activated (DCC/HOBt), filtered,
and placed in a
synthesis cartridge. Imidazole-2-carboxylic acid was added manually. At the
initiation of the
coupling cycle the synthesis was interrupted, the reaction vessel vented and
the activated
monomer added directly to the reaction vessel through the resin sampling loop
via syringe.
When manual addition was necessary an empty synthesis cartridge was used.
Aliphatic amino
~5 acids (2 mmol) and HBTU (1.9 mmol) were placed in a synthesis cartridge. 3
ml of DMF was
added using a calibrated delivery loop from reagent bottle 8, followed by
calibrated delivery of
1 ml DIEA from reagent bottle 7, and a 3 minute mixing of the cartridge.
The activator cycle was written to transfer activated monomer directly from
the cartridge to
2o the concentrator vessel, bypassing the activator vessel. After transfer, 1
ml of DIEA was
measured into the cartridge using a calibrated delivery loop, and the DIEA
solution combined
with the activated monomer solution in the concentrator vessel. The activated
ester in 2:1
DMF/DIEA was then transferred to the reaction vessel. All lines were emptied
with argon
before and after solution transfers.
ImImOpPy-y-ImPyPyPy-~i-Dp. ImImOpPy-y-ImPyPyPy-~i-Pam-Resin was synthesized
in a stepwise fashion by machine-assisted solid phase methods from Boc-~3-Pam-
Resin (0.66
mmol/g). Baird, E. E. & Dervan, P. B. describes the solid phase synthesis of
polyamides
containing imidazole and pyrrole amino acids. J. Am. Chem. Soc. 118, 6141-6146
(1996); also
3o see PCT US 97/003332. 3-hydroxypyrrole-Boc-amino acid (0.7 mmol) was
incorporated by
placing the amino acid (0.5 mmol) and HBTU (0.5 mmol) in a machine synthesis
cartridge.
Upon automated delivery of DMF (2 mL) and DIEA ( 1 mL) activation occurs. A
sample of
ImImOpPy-y-ImPyPyPy-(i-Pam-Resin (400 mg, 0.40 mmol/gram) was placed in a
glass 20 mL
peptide synthesis vessel and treated with neat dimethylaminopropylamine (2 mL)
and heated
(55 °C) with periodic agitation for 16 h. The reaction mixture was then
filtered to remove resin,
0.1% (wtJv) TFA added (6 mL) and the resulting solution purified by reversed
phase HPLC.
ImImOpPy-Y-ImPyPyPy-~i-Dp is recovered upon lyophilization of the appropriate
fractions as a
white powder (97 mg, 49% recovery). UV (Hz0) 7~",~X 246, 316 (66,000); ~H NMR
(DMSO-d6)
23
CA 02281947 1999-08-17
W0 98/37066 PCT1US98/01006
8 10.24 (s, 1 H), 10.14 {s, 1 H), 9.99 (s, 1 H), 9.94 (s, 1 H), 9.88 (s, 1 H),
9.4 (br s, 1 H), 9.25 (s,
1 H), 9.11 (s, 1 H), 8.05 (m, 3 H), 7.60 (s, 1 H), 7.46 (s, 1 H), 7.41 (s, 1
H), 7.23 (d, 1 ), 7.21 (d,
1 H), 7.19 (d, 1 H), 7.13 (m, 2 H), 7.11 (m, 2 H), 7.02 (d, 1 H), 6.83 (m, 2
H), 3.96 (s, 6 H),
3.90 (s, 3 H), 3.81 (m, 6 H), 3.79 (s, 3 H), 3.75 (d, 9 H), 3.33 (q, 2 H, J=
S.4 Hz), 3.15 (q, 2 H,
J = S.S Hz), 3.08 (q, 2 H, J = 6.0 Hz), 2.96 (quintet, 2 H, J = 5.6 Hz), 2.70
(d, 6 H, J = 4.5 Hz),
2.32 (m, 4 H), 1.71 (m, 4 H); MALDI-TOF-MS (monoisotopic), 1253.5 (1253.b
calc. for
CssHrzNzzO~ i )~
ImImHpPy-y-ImPyPyPy. In order to remove the methoxy protecting group, a sample
of
to ImlmOpPy-Y-ImPyPyPy-(3-Dp (S mg, 3.9 pmol) was treated with sodium
thiophenoxide at 100
°C for 2 h. DMF (1000 pL) and thiophenol (500 pL) were placed in a (13
x 100 mm) disposable
Pyrex screw cap culture tube. A 60 % dispersion of sodium hydride in mineral
oil (100 mg) was
slowlv added. Upon completion of the addition of the sodium hydride, ImImOpPy-
y-ImPyPyPy-
~3-Dp (S mg) dissolved in DMF (500 pL) was added. The solution was agitated,
and placed in a
100 °C heat block, and deprotected for 2 h. Upon compietion of the
reaction the culture tube
was cooled to 0°C, and 7 ml of a 20 % (wt/v) solution of
trifluoroacetic acid added. The
aqueous Layer is separated from the resulting biphasic solution and purified
by reversed phase
HPLC. ImImHpPy-Y-ImPyPyPy-~3-Dp is recovered as a white powder upon
lyophilization of
the appropriate fractions (3.8 mg, 77 % recovery). UV (H20) 7~,~x 246, 312
(66,000); ~ H NMR
(DMSO-d6) 8 10.34 (s, 1 H), 10.24 (s, 1 H), 10.00 (s, 2 H), 9.93 (s, 1 H),
9.87 (s, 1 H), 9.83 (s,
1 H), 9.4 (br s, 1 H), 9.04 (s, 1 H), 8.03 (m, 3 H), 7.58 (s, 1 H), 7.44 (s, 1
H), 7.42 (s, 1 H), 7.23
(s, 1 H), 7.20 (m, 3 H), 7.12 (m, 2 H), 7.05 (d, 1 H), 7.02 (d, 1 H), 6.83 (s,
1 H), 6.79 (s, 1 H),
3.96 (s, 6 H), 3.90 (s, 3 H), 3.81 (s, 6 H), 3.79 (s, 3 H), 3.75 (d, 6 H),
3.33 (q, 2 H, J = 5.4 Hz),
3.14 (q, 2 H, J = 5.4 Hz), 3.08 (q, 2 H, J = 6. I Hz), 2.99 (quintet, 2 H, J =
5.4 Hz), 2.69 (d, 6 H,
2s J = 4.2 Hz), 2.31 (m, 4 H), 1.72 (m, 4 H); MALDI-TOF-MS (monoisotopic),
1239.6 (1239.6
calc. for CS~H"NzzO").
ImImPyPy-y-ImOpPyPy-(3-Dp. ImhrtPyPy-y-ImOpPyPy-(3-Pam-Resin was synthesized
in a stepwise fashion by machine-assisted solid phase methods from Boc-(3-Pam-
Resin (0.66
3o mmol/g) as described for ImImOpPy-y-ImPyPyPy-(i-Dp. A sample of ImImPyPy-y-
ImOpPyPy-
(3-Pam-Resin (400 mg, 0.40 mmol/gram) was placed in a glass 20 mL peptide
synthesis vessel
and treated with neat dimethylaminopropylamine (2 mL) and heated (SS
°C) with periodic
agitation for 16 h. The reaction mixture was then filtered to remove resin,
0.1 % (wt/v) TFA
added (6 n1L) and the resulting solution purified by reversed phase HPLC.
ImImPyPy-y-
35 ImOpPyPy-~3-Dp is recovered upon lyophilization of the appropriate
fractions as a white
powder ( 1 O 1 mg, SO% recovery). UV (Hz0) ~ 246, 316 (66,000); MALDI-TOF-MS
(monoisotopic), 1253.6 ( 1253.6 calc. for CSgH~zNzzO").
24
CA 02281947 1999-08-17
WO 98/37066 PCT/US98/01006
ImImPyPy-y-ImHpPyPy. A sample of ImImpypy-y_ImOpPyPy-~-Dp (5 mg, 3.9 pmol)
was treated with sodium thiophenoxide and purified by reversed phase HPLC as
described for
ppY-Y-~YPYPY-~-Dp. ~ypy-y-ImHpPyPy-(3-Dp is recovered upon lyophilization
of the appropriate fractions as a white powder (3.2 mg, 66 % recovery). W
(H20) 7~",~,~ 246,
312 (b6,000); MALDI-TOF-MS (monoisotopic), 1239.6 (i239.6 calc. for
CS~H~iNZ20~,).
ImPyPy-y-OpPyPy-~i-Dp. ImPyPy-y-OpPypy_~i-pam_Resin was synthesized in a
stepwise fashion by machine-assisted solid phase methods from Boc-~3-Pam-Resin
(0.66
mmol/g). Baird, E. E. & Dervan, P. B. describes the solid phase synthesis of
polyamides
to containing imidazole and pyrrole amino acids. J. Am. Chem. Soc. 118, 6141-
6146 (1996); also
see PCT US 97/003332. 3-hydroxypyrrole-Boc-amino acid (0.7 mmol) was
incorporated by
placing the amino acid (0.5 mmol) and HBTU (0.5 mmol) in a machine synthesis
cartridge.
Upon automated delivery of DMF (2 mL) and DIEA ( 1 mL) activation occurs. A
sample of
I~YPY-Y-OPPYPY-~-P~-Resin (400 mg, 0.45 mmoUgram) was placed in a glass 20 mL
peptide synthesis vessel and treated with neat dimethylaminopropylamine (2 mL)
and heated
(55 °C) with periodic agitation for 16 h. The reaction mixture was then
filtered to remove resin,
0.1% (wt/v) TFA added (6 mL) and the resulting solution purified by reversed
phase HPLC.
ImPyPy-y-OpPyPy-j3-Dp is recovered upon lyophilization of the appropriate
fractions as a
white powder (45 mg, 25% recovery). UV (H20) ?"",ax 246, 310 (50,000); ~H NMR
(DMSO-d6)
8 10.45 (s, 1 H), 9.90 (s, 1 H), 9.82 (s, 1 H), 9.5 (br s, 1 H), 9.38 (s, 1
H), 9.04 (s, 1 H), 8.02 (m,
3 H), 7.37 (s, 1 H), 7.25 (m, 2 H), 7.15 (d, 1 H, J= 1.6 Hz), 7.11 (m, 2 H),
7.09 (d, 1 H), 7.03
(d, 1 H), 6.99 (d, 1 H), 6.87 (d, 1 H), 6.84 (d, 1 H), 3.96 (s, 3 H), 3.81 (s,
6 H), 3.77 (s, 6 H),
3.76 (s, 3 H), 3.74 (s, 1 H), 3.34 (q, 2 H, J= 5.6 Hz), 3.20 (q, 2 H, J= 5.8
Hz), 3.09 (q, 2 H, J=
6.1 Hz), 2.97 (quintet, 2 H, J = 5.3 Hz), 2.70 (d, 6 H, J = 3.9 Hz), 2.34 (m,
4 H), 1.73 (m, 4 H);
MALDI-TOF-MS (monoisotopic), 1007.6 (1007.5 calc. for C48H6;N,6O9).
ImPyPy-y-HpPyPy. In order to remove the methoxy protecting group, a sample of
ImPypy-y-OpPyPy-~i-Dp (5 mg, 4.8 ltmol) was treated with sodium thiophenoxide
at 100 °C
for 2 h. DMF (1000 pL) and thiophenol (500 pL) were placed in a (13 x 100 mm)
disposable
3o Pyrex screw cap culture tube. A 60 % dispersion of sodium hydride in
mineral oil (100 mg) was
slowly added. Upon completion of the addition of the sodium hydride, ImImPyPy-
y-ImOpPYPy
(3-Dp (S mg) dissolved in DMF (500 uL) was added. The solution was agitated,
and placed in a
100 °C heat block, and deprotected for 2 h. Upon completion of the
reaction the culture tube
was cooled to 0°C, and 7 ml of a 20 % (wt/v) solution of
trifluoroacetic acid added. The
aqueous layer is separated from the resulting biphasic solution and purified
by reversed phase
HPLC. ImImHpPy-y-ImHpPYPy-(3-Dp is recovered as a white powder upon
lyophilization of
the appropriate fractions (2.5 mg, 52 % recovery). LJV (HZO) 7~.r"aK 246, 310
(50,000); ~H NMR
(DMSO-d6) 8 10.44 (s, 1 H), 10.16 (s, 1 H), 9.90 (s, 1 H), 9.77 (s, 1 H), 9.5
(br s, 1 H), 9.00 (s,
CA 02281947 1999-08-17
WO 98/37066 PCT/US98/01006
1 H), 8.03 (m, 3 H), 7.37 (s, 1 H), 7.26 (m, 2 H), 7.14 (d, 1 H, J = 1.7 Hz),
7.12 (m, 2 H), 7.02
(d, 1 H), 6.93 (d, 1 H), 6.88 (d, 1 H), 6.82 (d, 1 H), 6.72 (d, 1 H), 3.96 (s,
3 H), 3.81 (s, 6 H),
3.77 (s, 3 H), 3.76 (s, 3 H), 3.74 (s, 1 H), 3.36 (q, 2 H, J = 5.4 Hz), 3.22
(q, 2 H, J = 5.9 Hz),
3.09 (q, 2 H, J = 5.5 Hz), 2.98 (quintet, 2 H, J = 5.3 Hz), 2.70 (d, 6 H, J =
4.3 Hz), 2.34 (m, 4
H), 1.78 (m, 4 H); MALDI-TOF-MS (monoisotopic), 994.2 (993.5 calc. for
C47H6,N~6Og).
Table 5. Mass spectral characterization of Op and Hp polyamides, synthesized
and purif ed as
described for ImImOpPy-Y-ImPyPyPy-(3-Dp and ImIm.HpPv-y-ImPvPvPv-Q-Dn.
POLYAMIDE FORNIUL,A (M+IT)CALCD FOUND
to ImOpPy-Y-PyPyPy-~3-Dp CaaH63Nt609 1007.5 1007.5
~PPY-Y-PYPYPY-~-DP Ca~H6~N~609 993.5 993.2
ImPyOp-Y-PyPyPy-~3-Dp CasH63N, 1007.5 1007.5
609
ImPyHp-Y-PyPyPy-(i-Dp Ca~Hb t N 993.5 993.4
i 609
ImPyPy-Y-OpPyPy-(3-Dp CsaH63N~ 1007.5 1007.6
609
~5 ImPyPy-y-HpPyPy-~i-Dp C.,7H6,N~609993.5 993.2
~'YPY-Y-PYOPPY-~-DP CasH63Nib09 1007.5 1007.5
~YPY-Y-PYHPPY-~-Dp Ca7H6iNt609 993.5 993.4
ImOpOp-Y-PyPyPy-(3-Dp C49H65Ni6Oio1037.5 1037.5
ImHpHp-y-PyPyPy-(3-Dp Ca~H61 N 1009.5 1009.4
i 60, 0
2o ImImOpPy-Y-ImPyPyPy-~3-Dp CSgH~zNzzO" 1253.6 1253.5
ImImHpPy-Y-ImPyPyPy-(3-Dp CS~H~,NzzO" 1239.6 1239.6
ImImPyPy-Y-ImOpPyPy-(3-Dp CSgH~2Nzz0" 1253.6 1253.6
ImImPyPy-Y-ImHpPyPy-(3-Dp CS~H~~NzzO" 1239.6 1239.6
ImOpPyPy-Y-ImOpPyPy-(3-Dp C6oH~6Nz,O,z1282.6 1282.6
z5 ImHpPyPy-Y-ImHpPyPy-~-Dp CSgH~zNz~O,z1254.6 1254.6
ImImOpPy-Y-ImOpPyPy-~i-Dp CS9H~SNZZO,z1283.6 1283.6
ImImHpPy-Y-ImHpPyPy-~i-Dp CS~H~INzzOiz1255.6 1255.5
ImOpPyPy-Y-PyPyPyPy-~i-Dp C6oH~5Nzo0, 1251.6 1251.5
~
ImPyPyPy-Y-PyPyOpPy-~i-Dp C6oH~5Nzo0" 1251.6 1251.5
3o ImImPyPy-Y-ImPyOpPy-(3-Dp CSBH~zNzzO, 1253.6 1253.7
~
ImOpPyPyPy-Y-ImOpPyPyPy-(3-Dp C~zHggN250,41526.7 1526.6
~PPYPYPY-Y-I~PPYPYPY-~-Dp C~oHsaNzs0~41498.7 1498.0
ImImPyPyPy-Y-ImOpOpPyPy-~-Dp C~~H8~NZ60,41527.7 1527.7
26
CA 02281947 1999-08-17
WO 98/37066 PCT/US98/01006
EXAMPLE 3:
DETERMINATION OF POLYAMIDE
BINDING AFFINITY AND SEQUENCE SPECIFICITY.
s Representative-footprint titration experiments are shown in Figures 3 and
10. A 252-by
DNA fragment which is typically used for the footprint titration experiments
provides 247
possible 6-by binding sites for an eight-ring hairpin polyamide. Thus, in
addition to providing
DNA binding affinities, the footprint titration experiments also reveal DNA
binding sequence-
specificity. The DNA binding sequence specificity of polyamides which differ
by a single
to Py/Py, Hp/Py, or Py/Hp pair for sites which differ by a single A~T or T~A
base pair are
described in Tables 1, 6, and 7.
Quantitative DNaSe I Footprint Titrations All reactions were executed in a
total volume
of 400 pL (Brenowitz, M. et al., 1986). A polyamide stock solution or H,O (for
reference
is lanes) was added to an assay buffer containing 3'-3zP radiolabeled
restriction fragment (20,000
cpm), affording final solution conditions of 10 mM Tris~HC1, 10 mM KC1, 10 mM
MgClz, 5
mM CaClz, pH 7.0, and either (i) a suitable concentration range of polyamide,
or (ii) no
polyamide (for reference lanes). The solutions were allowed to equilibrate for
24 hours at 22°C.
Footprinting reactions were initiated by the addition of 10 pL of a stock
solution of DNase I (at
20 the appropriate concentration to give ~55% intact DNA) containing 1 mM
dithiothreitol and
allowed to proceed for 7 minutes a1 22°C. The reactions were stopped by
the addition of 50 pL
of a solution containing 2.25 M NaCI, 150 mM EDTA, 23 uM base pair calf thymus
DNA, and
0.6 mg/ml glycogen, and ethanol precipitated. The reactions were resuspended
in 1 x TBE/
80% fotmamide loading buffer, denatured by heating at 85°C for 15
minutes, and placed on ice.
25 The reaction products were separated by electrophoresis on an 8%
polyacrylamide gel (5%
crosslinking, 7 M urea) in 1 x TBE at 2000 V for 1.5 h. Gels were dried on a
slab dryer and
then exposed to a storage phosphor screen at 22°C.
Photostimuable storage phosphor imaging plates (Kodak Storage Phosphor Screen
30 50230 obtained from Molecular Dynamics) were pressed flat against dried gel
samples and
exposed in the dark at 22°C for 12-24 hours. A Molecular Dynamics 400S
PhosphorImager
was used to obtain ail data from the storage screens (3ohnston et al., 1990).
The data were
analyzed by performing volume integration of the target site and reference
blocks using the
ImageQuant v. 3.3 software running on a Compaq Pentium 80.
3s
Quantitative DNase I Footprint Titration Data Analysis was performed by taking
a
background-corrected volume integration of rectangles encompassing the
footprint sites and a
reference site at which DNase I reactivity was invariant across the titration
generated values for
27
CA 02281947 1999-08-17
WO 98/37066 PCTNS98/01006
the site intensities (Is;t~) and the reference intensity (I~r). The apparent
fractional occupancy
(6app) of the sites were calculated using the equation:
L~.I L.~
0'°° = 1 - ~ 1 )
I.de°llr.f°
where Is;te° and Iref are the site and reference intensities,
respectively, from a DNase I control
lane to which no polyamide was added.
The ([L]tot, eapp) data were fit to a Langmuir binding isotherm (eq. 2, n=1)
by
minimizing the difference between 6app and AF,, using the modified Hill
equation:
Ka" (L]" ~o~
e~;~ = e~, + Amax - 9min ) (2,)
1 + Ka" (L]" ~o~
where [L~o,] is the total polyamide concentration, Ka is the equilibrium
association constant, and
emm ~d emax ~e the experimentally determined site saturation values when the
site is
unoccupied or saturated, respectively. The data were fit using a nonlinear
least-squares fitting
procedure of KaleidaGraph software (v. 3Ø1, Abelbeck Software) with Ka,
Ar,,ax, and 9m;n as the
adjustable parameters. The goodness of fit of the binding curve to the data
points is evaluated
by the correlation coefficient, with R > 0.97 as the criterion for an
acceptable fit. Four sets of
acceptable data were used in determining each association constant. All lanes
from a gel were
used unless a visual inspection revealed a data point to be obviously flawed
relative to
neighboring points. The data were normalized using the following equation:
espp - emm
a nom, _
Bmau - emm
TABLE 6 Discrimination of 5'-TGTAA-3' and 5'-TGTTA-3"
Pair1 5'-TGTAA-3' S'-TGTTA-3' KrciT
5'-T G T A A-3' S'-T G T '1' A-3'
Py/Py + 2.0
3'-A C A T T-5' 3'-A C A 11 T-5'
Kd = 0.014 ~cM Kd = 0.007 pM
5'-T G T A A-3' S'-T G T Z A-3'
N +
p 3'-A C A T T-5' 3'-A C A Z T-5' 036
Kd = 0.20 pM Kd = 0.56 ttM
5'-T G T 11 A-3' S'-T G T T A-3'
N
HplPy + +14
3'-A C A T T-S' 3'-A C A Z T-5'
Kd = 4.0 pM - Kd = 0.28 wM
"1'he reported equilibrium dissociation constants are the mean values
obtained from two DNase 1 footprint titration experiments on a 3''~P
labeled 370-by pDEHl EcoIZI/PvuII DNA restriction fragmentl3. The
assays were carried out at 22 °C, pH 7.0 in the presence of 10 mM
Tris~HCI, 10 mM KCI, 10 mM MgClz and 5 mM CaCIZ.
tRing pairing opposite T~A and A~T in the third position
$Calculated as K~(5'-TGTAA-3'1/K~(S'-TGTTA-3'1.
28
CA 02281947 1999-08-17
WO 98/37066 PCT/US98/01006
TABLE 7 Discrimination of 5'-TGTTT-3' and 5'-TGATT-3'~
Pain 5'-TGATT-3' S'-TGTTT-3' K,~~
5'-T G ~~ T T-3' S'-T G T T T-3'
PY~Y + + v 5.2
3'-A C ." A ~-5' 3'-A C 1l A A-5'
Kd = 0.026 ~M Kd = 0.005 pIN
5'-T G ' T T-3' S'-T G T T T-3'
Hp/Py +~ 66
3'-A C . A A-5' 3'-A C A A A-5'
Kd = 0.53 ~M Kd = 0.008 liM
5'-T G A T T-3' S'-T G T T T-3'
+ H + " 0.56
3'-A C T A A-5' 3'-A C 7~ A A-5'
K~ = 0.33 pM K,, = 0.59 wM
'The reported equihbnum d~ssoc~ahon constants are the mean values
obtained from two DNase I footprint titration experiments. The assays
were carried out at 22 °C, pH 7.0 in the presence of 10 mM Tris~HCI,
mM KCI, 10 mM MgCl2, and 5 mM CaCI
tRing pairing opposite T~A and A~T in the third position.
Calculated as Kd(S'-TGATT-3')/Kd(5'-TGTTT-3').
EXAMPLE 5:
5 PREPARAT10N OF A BIFUNCTIONAL Hp-Py-Im-POLYAMIDE.
ImImOpPy-y-ImPvPvPv-ø-Dp-NH2. ImImOpPy-y-ImPyPyPy-ø-Pam-Resin was
synthesized in a stepwise fashion by machine-assisted solid phase methods from
Boc-ø-Pam
Resin (0.66 mmol/g). Baird, E. E. & Dervan, P. B. describes the solid phase
synthesis of
l0 polyamides containing imidazole and pyrrole amino acids. J. Am. Chem. Soc.
11$, 6141-6146
(1996); also see PCT US 97/003332. 3-hydroxypyrrole-Boc-amino acid (0.7 mmol)
was
incorporated by placing the amino acid (0.5 mmol) and HBTU (0.5 mmol) in a
machine
synthesis cartridge. Upon automated delivery of DMF (2 mL) and DIEA ( 1 mL)
activation
occurs. A sample of ImImOpPy-y-ImPyPyPy-ø-pam-Resin (400 mg, 0.40 mmol/gram)
was
placed in a glass 20 mL peptide synthesis vessel and treated with neat 3,3'-
diamino-N
methyldipropylamine (2 mL) and heated (55 °C) with 'periodic agitation
for 16 h. The reaction
mixture was then filtered to remove resin, 0.1 % (wt/v) TFA added (6 mL) and
the resulting
solution purified by reversed phase HPLC. ImImOpPy-y-Impypypy_~3_Dp-~2 is
recovered
upon lyophilization of the appropriate fractions as a white powder (93 mg, 46%
recovery). UV
(H20) ~,m~ 246, 316 (66,000); 1 H NMR (DMSO-d6) 8 10.34 (s, 1 H), 10.30 (br s,
1 H), 10.25
(s, 1 H), 9.96 (s, 1 H), 9.95 (s, 1 H), 9.89 (s, 1 H), 9.24 (s, I H), 9.11 (s,
1 H), 8.08 {t, 1 H, J=
5.6 Hz), 8.0 (m, 5 H), 7.62 (s, 1 H), 7.53 (s, 1 H), 7.42 (s, 1 H), 7.23 (d, 1
H, J = 1.2 Hz), 7.21
(m, 2 H), 7.1 S (m, 2 H), 7.13 (d, I H), 7.11 (m, 2 H), 7.04 (d, 1 H), 6.84
(m, 3 H), 3.98 (s, 3 H),
3.97 (s, 3 H), 3.92 (s, 3 H), 3.82 (m, 6 H), 3.80 (s, 3 H), 3.77 (d, 6 H),
3.35 (q, 2 H, J= 5.8 Hz)
3.0-3.3 (m, 8 H), 2.86 (q, 2 H, J = 5.4 Hz), 2.66 (d, 3 H, J = 4.5 Hz), 2.31
(m, 4 H), 1.94
29
CA 02281947 1999-08-17
WO -98/37066 PCT/US98/01006
(quintet, 2 H, J = 6.2 Hz), 1.74 (m, 4 H); MALDI-TOF-MS (monoisotopic), 1296.0
( 1296.6
calc. for C6pH78N23O11)~
ImImOpPy-y-ImPyPyPy-~3-Dp-EDTA. Excess EDTA-dianhydride (50 mg) was dissolved
in DMSO/hIMP (1 mL) and DIEA (1 mL) by heating at 55 °C for 5 min. The
dianhydride
solution was added to ImImOpPy-y-ImPyPyPy-(3-NH2 (13 mg, 10 ~cmol) dissolved
in DMSO
(750 pL). The mixture was heated (55 °C, 25 min.) and the remaining
EDTA-anhydride
hydrolyzed (O.1M NaOH, 3 mL, 55 °C, 10 min). Aqueous TFA (0.1% wtlv)
was added to
adjust the total volume to 8 mL and the solution purified directly by reversed
phase HPLC to
l0 provide ImImOpPy-y-ImPyPyPy-(3-Dp-EDTA as a white powder upon
lyophilization of the
appropriate fractions (5.5 mg, 40% recovery}. MALDI-TOF-MS (monoisotopic),
1570.9
(1570.7 calc. for C70H92N25O18).
ImImHpPy-y-ImPvPyPy-(3-Dp-EDTA. In order to remove the methoxy protecting
group,
a sample of ImImOpPy-y-ImPyPyPy-~3-Dp-EDTA (5 mg, 3.1 hmol) was treated with
sodium
thiophenoxide at 100 °C for 2 h. DMF ( 1000 ~L) and thiophenol (500
1tL) were placed in a ( 13
x 100 mm) disposable Pyrex screw cap culture tube. A 60 % dispersion of sodium
hydride in
mineral oil (100 mg) was slowly added. Upon completion of the addition of the
sodium hydride,
ImImOpPy-y-ImPyPyPy-(3-Dp-EDTA (5 mg) dissolved in DMF (500 ~L) was added. The
2o solution was agitated, and placed in a 100 °C heat block, and
deprotected for 2 h. Upon
completion of the reaction the culture tube was cooled to 0°C, and 7 ml
of a 20 % (wt/v)
solution of trifluoroacetic acid added. The aqueous layer is separated from
the resulting biphasic
solution and purified by reversed phase HPLC. ImImHpPy-y-ImPyPypy-~_Dp-EDTA is
recovered as a white powder upon lyophilization of the appropriate fractions
(3.2 mg, 72
recovery). UV (H20) 7~m~ 246, 312 (66,000); MALDI-TOF-MS (monoisotopic),
1556.6
(1556.7 calc. for C69H90N25018)~
ImImPyPy-y-ImOpPyPy-~3-Dp-NH2. ImImPyPy-y-ImOpPyPy-~i-Pam-Resin was
synthesized in a stepwise fashion by machine-assisted solid phase methods from
Boc-~i-Pam-
3o Resin (0.66 mmol/g). Baird, E. E. & Dervan, P. B. describes the solid phase
synthesis of
polyamides containing imidazole and pyrrole amino acids. J. Am. Chem. Soc.
118, 6141-6146
(i996); also see PCT US 97/003332. 3-hydroxypyrrole-Boc-amino acid (0.7 mmol)
was
incorporated by placing the amino acid (0.5 mmol) and HBTU (0.5 mmol) in a
machine
synthesis cartridge. Upon automated delivery of DMF (2 mL) and DIEA (1 mL)
activation
occurs. A sample of ImImPyPy-y-ImOpPyPy-~i-Pam-Resin (400 mg, 0.40 mmol/gram)
was
placed in a glass 20 mL peptide synthesis vessel and treated with neat 3,3'-
diamino-N
methyldipropylanvne (2 mL) and heated (55 °C) with periodic agitation
for 16 h. The reaction
mixture was then filtered to remove resin, 0.1 % (wtlv) TFA added (6 mL) and
the resulting
CA 02281947 1999-08-17
W0 98/37066 PCT/US98/01006
solution purified by reversed phase HPLC. ImImPyPy-y-Im,Oppypy_(3-Dp-~2 is
recovered
upon lyophilization of the appropriate fractions as a white powder (104 mg,
54% recovery). UV
(H20) ~,m~ 246, 316 (66,000); MALDI-TOF-MS (monoisotopic), 1296.6 (1296.6
calc. for
C60H78N23411 )~
-
ImImPyPy-y-ImOpPyPy-(3-Dp-EDTA. Excess EDTA-dianhydride (SO mg) was dissolved
in DMSO/NMP (1 mL) and DIEA (1 mL) by heating at 55 °C for 5 min. The
dianhydride
solution was added to ImImPyPy-y-ImOpPyPy-~i-NH2 (13 mg, 10 pmol) dissolved in
DMSO
(750 pL). The mixture was heated (55 °C, 25 min.) and the remaining
EDTA-anhydride
to hydrolyzed (0.1 M NaOH, 3 mL, 55 °C, 10 min). Aqueous TFA (0.1 %
wt/v) was added to
adjust the total volume to 8 mL and the solution purified directly by reversed
phase HPLC to
provide ImImPyPy-y-ImOpPyPy-~i-Dp-EDTA as a white powder upon lyophilization
of the
appropriate fractions (5.9 mg, 42% recovery). MALDI-TOF-MS (monoisotopic),
1570.8
( 1570.7 calc. for C7pH92N25018)~
ImImPvPy-y-ImHpPvPv-(3-Dp-EDT.4. In order to remove the methoxy protecting
group,
a sample of ImImPyPy-y-ImOppypy_(3_Dp-EDTA (5 mg, 3.1 umol) was treated with
sodium
thiophenoxide at 100 °C for 2 h. DMF ( 1000 pL) and thiophenol (500 pL)
were piaced in a ( 13
x 100 mm) disposable Pyrex screw cap culture tube. A 60 % dispersion of sodium
hydride in
2o mineral oil (100 mg) was slowly added. Upon completion of the addition of
the sodium hydride,
ImImPyPy-y-ImOpPyPy-~-Dp-EDTA (5 mg) dissolved in DMF (500 pL) was added. The
solution was agitated, and placed in a 100 °C heat block, and
deprotected for 2 h. Upon
completion of the reaction the culture tube was cooled to 0°C, and 7 ml
of a 20 % (wt/v)
solution of trifluoroacetic acid added. The aqueous layer is separated from
the resulting biphasic
solution and purified by reversed phase HPLC. ImImPyPy-y-ImHpPyPy-(3-Dp-EDTA
is
recovered as a white powder upon lyophilization of the appropriate fractions
(3.2 mg, 72
recovery). UV (H20) ~,m~ 246, 312 (66,000); MALDI-TOF-MS (monoisotopic),
1555.9
(1556.7 calc. for C69H90N25018)~
3o EXAMPLE 6:
DETERMINATION OF POLYAMIDE BINDING ORIENTATION
Affinity cleavage experiments using hairpin polyamides modified with
EDTA~Fe(II) at
either the C-terminus or on the y-turn, were used to determine polyamide
binding orientation
and stoichiometry. The results of affinity cleavage experiments are consistent
only with
recognition of 6-by by an 8-ring hairpin complex and rule out any extended 1:1
or overlapped
complex formation. In addition, affinity cleavage experiments reveal hairpin
formation
31
CA 02281947 1999-08-17
WO 98/37066 PCT/US98/01006
supporting the claim that it is the Hp/Py and Py/Hp pairing which form at both
match and
mismatch sites to discriminate A~T from T~A.
Affinity cleavage reactions were executed in a total volume of 40 ~L. A stock
solution of
polyamide or Hz0 was added to a solution containing labeled restriction
fragment (20,000
cpm), affording final solution conditions of 25 mM Tris-Acetate, 20 mM NaCI,
100 p.Mlbp calf
thymus DNA, and pH 7Ø Solutions were incubated for a minimum of 4 hours at
22°C.
Subsequently, 4 pL of freshly prepared 100 pM Fe(IVH~)2(S04)Z was added and
the solution
allowed to equilibrate for 20 min. Cleavage reactions were initiated by the
addition of 4 ~L of
to 100 mM dithiothreitol, allowed to proceed for 30 min at 22 °C, then
stopped by the addition of
~L of a solution containing 1.5 M NaOAc (pH 5.5), 0.28 mg/mL glycogen, and 14
~M base
pairs calf thymus DNA, and ethanol precipitated. The reactions were
resuspended in lx
TBE/80% formamide loading buffer, denatured by heating at 85 °C for 15
min, and placed on
ice. The reaction products were separated by electrophoresis on an 8%
polyacrylamide geI
(5% cross-Link, 7 M urea) in lx TBE at 2000 V for 1.5 hours. Gels were dried
and exposed to a
storage phosphor screen. Relative cleavage intensities were determined by
volume integration
of individual cleavage bands using ImageQuant software.
32
CA 02281947 1999-08-17
WO 98/37066 PCT/IJS98/01006
EXAMPLE 7:
IMPROVEMENT TO POLYAMIDE SEQUENCE SPECIFICITY.
s The polyamides of this invention provide improved specificity relative to
existing
polyamide technology. Turner, J. T., Baird, E. E., and Dervan, P.B. describe
the recognition of
seven base pair sequences in the minor groove of DNA by ten-ring pyrrole-
imidazole
polyamide hairpins J. Am. Chem. Soc. 1997 119, 7636. For example, quantitative
DNaseI
footprint titrations reveal that the 10-ring hairpin Impypypypy_Y_~pypypypy-~-
Dp binds a 5'-
to TGTAACA-3- sequence with an equlibrium dissociation constant of 0.083 nM,
and I8-fold
specificity versus a single base mismatch site. A number of other sites are
also bound on the
252-by DNA fragment used for the footprint titration experiments. (Figure 13).
Introduction of
a Hp/Py and Py/Hp pair in the 10-ring polyamide, ImHpPypypy_Y_l~ppypypy_~-Dp,
to
recognize a T~A and A~T within the 7-by target sequence, increases the
sequence-specificty. For
15 example, a single base mismatch site 5'-TGGAACA-3 is discriminated by >
5000-fold (Figure
i 3, Table 8). In fact all 245 7-by mismatch sites present on the restriction
fragment are
discriminated > 5000-fold by the polyamide ImHpPypypy_Y_IrnHppypypy_~3_Dp
(Figure i3).
For cases where three A,T base pairs are present in succession it is preferred
to substitute Py/Py
in place of at least one Hp/Py or Py/Hp to provide for recognition of A~T and
T~A at a single
2o position.
TABLE 8 Equilibrium dissociation constants'
Polyamidet 5'-TGGTCA-3' S'-TGGACA-3' K~~#
5'-T G T A S C A-3' S'-T G G T A C A-3'
18
3'-A C A T ? G T-5' 3'-A C C A T G T-5'
Kd = 0.083 nM Kd ~ 1.5 nM
5'-T G T. A A' C A-3' S'-T G O T A C A-3'
H H
HP~ + " + H >5000
3'-A C A T T G T-5' 3'-A C C A T G T-5'
Kd = 0.2 nM Kd > 1000 nM
'The reported dissociation constants are the average values obtained from
three
DNase I footprint titration experiments. The standard deviation for each data
set is
less than 15% of the reported number. Assays were carried out in the presence
of 10
mM Tris~HCI, 10 mM KCI, 10 mM MgCl2, and 5 mM CaCl2 at pH 7.0 and 22
°C.
tRing pairing opposite T~A and A~T in the fourth position.
#Calculated as Kd(5'-TGGTACA-3')/Kd(5'-TGTAACA-3').
2s EXAMPLE 8:
USE OF PAIRING CODE
There are 256 possible four base pair combinations of A, T, G, and C. Of
these, there are
a possible 240 four base pair sequences which contain at least 1 A~T or T~A
base pair and
33
CA 02281947 1999-08-17
WO 98/37066 PCT/US98/01006
therefore can advantageously use an Hp/Py, or Py/Hp carboxamide binding.
Polyamides
binding to any of these sequences can be designed in accordance with the code
of TABLE 2.
Table 9 lists the sixteen eight-ring hairpin polyamides (1-16) which recognize
the sixteen 5'-
WGTNIVW-3' sequences (W = A or T, X = A, G, C, or T). Table 10 lists the
sixteen eight-ring
hairpin polyamides ( 17-32) which recognize the sixteen 5'-WGAIVNW-3'
sequences ( 17-32).
Table 11 lists the twelve eight-ring hairpin polyamides (33-44) which
recognize twelve 5'-
WGGNNW-3' sequences which contain at least one A,T base pair. Table 11 lists
the four eight-
ring hairpin polyamides (G1-G4) which target the four 5'-WGGNNW-3' sequences
(G1-G4)
which contain exclusively G~C base pairs. Table 12 lists the twelve eight-ring
hairpin
to polyamides (45-56) which recognize twelve 5'-WGCNNW-3' sequences which
contain at least
one A,T base pair. Table 12 lists the four eight-ring hairpin polyamides (GS-
G8) which target
the four 5'-WGCNNW-3' sequences (GS-G8) which contain exclusively G~C base
pairs. Table
13 lists the sixteen eight-ring hairpin polyamides (57-72) which recognize the
sixteen 5'-
WTTNNW-3' sequences (57-72). Table 14 lists the sixteen eight-ring hairpin
polyamides (73-
88) which recognize the sixteen 5'-WTANNW-3' sequences (73-88). Table 15 lists
the sixteen
eight-ring hairpin polyamides (89-104) which recognize the sixteen 5'-WTGNNW-
3' sequences
(89-104). Table 16 lists the sixteen eight-ring hairpin polyamides (105-120)
which recognize
the sixteen 5'-WTCNNW-3' sequences ( 1 OS-120). Table 17 lists the sixteen
eight-ring hairpin
polyamides (121-136) which recognize the sixteen 5'-WATNNW-3' sequences (121-
136).
2o Table 18 lists the sixteen eight-ring hairpin polyamides (137-152) which
recognize the sixteen
5'-WAANNW-3' sequences (137-152). Table 19 lists the sixteen eight-ring
hairpin polyamides
(153-168) which recognize the sixteen 5'-WAGNNW-3' sequences (153-168). Table
20 lists
the sixteen eight-ring hairpin polyamides (169-184) which recognize the
sixteen 5'-WACNNW-
3' sequences ( 169-184). Table 21 lists the sixteen eight-ring hairpin
polyamides ( 185-200)
which recognize the sixteen 5'-WCTNNW-3' sequences ( 185-200). Table 22 lists
the sixteen
eight-ring hairpin polyamides (201-2I6) which recognize the sixteen 5'-WCANNW-
3'
sequences (201-216). Table 23 lists the twelve eight-ring hairpin polyamides
(217-228) which
recognize the twelve 5'-WCGNNW-3' sequences which contain at least one A,T
base pair.
Table 23 lists the four eight-ring hairpin polyamides (G9-G12) which target
the four 5'-
3o WCGNNW-3' sequences (G9-G12) which contain exclusively C~G base pairs.
Table 24 lists
the twelve eight-ring hairpin polyamides (229-240) which recognize the twelve
5'-WCCNNW-
3' sequences which contain at least one A,T base pair. Table 24 lists the four
eight-ring hairpin
polyamides (G13-G16) which target the four 5'-WCCNNW-3' sequences (G13-G16)
which
contain exclusively C~G base pairs.
34
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W0 98/37066 PCTNS98/01006
TABLE
9:
8-ring
Hairpin
Polyamides
for
recognition
of
6-by
5'-WGTNNW-3'
DNA aromatic amino acid sequence
sequence
1) 5' GT T T W-3' 1) ImHpHpHp-y-PyPypypy
-W
2 ) 5' GT T A W-3 2 ) ImHpHpPy-y-HpPyPyPy
-W '
3) 5'-W GT T G W-3' 3)ImFipHpIm-y-PyPyPyPy
4) 5' GT T C W-3' 4) ImHpHpPy-y-ImPyPyPy
-W
5) 5' GT A T W-3' S) ImHpPyHp-y-PyHpPyPy
-W
IS
6) 5' GT A A W-3' 6) ImHpPyPy-Y-HpHpPyPy
-W
7) 5' GT A G W-3' 7) ImHpPyIm-y-pyHpPyPy
-W
8) 5'-W GT A C W-3' B)ImHpPyPy-y-ImHppyPy
9) 5' GT G T W-3' 9> ImHpImHp-Y-pyPyPypy
-W
10) 5' GT G A W-3' 10) ImHpImPy-y-HpPyPyPy
-W
11) 5' GT G G W-3' 11) ImHpImIm-y-pypypypy
-W
12) 5' GT G C W-3' 12) ImI-IpImPy-y-ImPyPyPy
-W
13) 5' GT C T W-3' 13) ImHpPyHp-y-PyImPyPy
-W
14) 5'-W GT C A W-3' 14)ImHpPyPy-y-HpImPyPy
15) 5' GT C G W-3' 15) ImHpPyIm-y-PyImPyPy
-W
16 5 GT C C W-3 16 ) ImHpPyPy-y- ImImPyPy
) ' '
-W
CA 02281947 1999-08-17
W0 98/37066 PCT/US98/01006
TABLE
10:
8-ring
Hairpin
Polyamides
for
recognition
of
6-by
5'-WGANNW-3'
DNA aromatic amino acid sequence
sequence
17) 5'-W G A TT W-3' 17)ImPyHpHp-y-PyPyHpPy
18) 5'-W G R TA W-3' 18)ImPyHpPy-y-HpPyHpPy
19) 5'-W G A TG W-3' 19)ImPyHpIm-y-PyPyHpPy
20) 5' G A TC W-3' 20) ImPyHpPy-y-ImPyHpPy
-W
21) 5' G A AT W-3' 21) ImPyPyHp-y-PyHpHpPy
-W
22) 5'-W G A AA W-3' 22)ImPyPyPy-y-HpHpHpPy
23) 5' G A AG W-3' 23) ImPyPyIm-y-PyHpHpPy
-W
24) 5'-W G A AC W-3' 24)ImPyPyPy-y-ImHpHpPy
25) 5' G A GT W-3' 25) ImPyImHp-y-PyPyHpPy
-W
26) 5' G A GA W-3' 26) ImPyImPy-y-HpPyHpPy
-W
27) 5'-W G A GG W-3' 27)ImPyImIm-~!-PyPyHpPy
28) 5' G A GC W-3' 28) ImPyImPy-y-ImPyHpPy
-W
29) 5'-W G A CT W-3' 29)ImPyPyHp-y-PyImHpPy
30) 5'-W G A CA W-3' 30)ImPyPyPy-y-HpImHpPy
31) 5' G A CG W-3' 31) ImPyPyIm-y-PyImHpPy
-W
32) 5' G A CC W-3' 32) ImPyPyPy--!-ImImHpPy
-W
36
CA 02281947 1999-08-17
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TABLE
11: 8-ring
Hairpin
Polyamides
for recognition
of 6-by
5'-WGGNNW-3'
DNA aromatic amino acid sequence
sequence
33) 5'-W GG T T W-3' 33)ImImHpHp-y-Pypypypy
34) 5'-W GG T A W-3' 34)ImImHpPy-y-HpPyPyPy
35) 5' GG T G W-3' 35) ImImHpIm-y-pypypypy
-W
36) 5' GG T C W-3' 36) ImImHpPy-y-ImpyPyPy
-W
37) 5'-W GG A T W-3' 37)ImImPyHp-y-pyHpPyPy
38) 5' GG A A W-3' 38) ImImPyPy-y-HpHpPypy
-W
39) 5'-W GG A G W-3' 39)ImImPyIm-y-pyHpPyPy
40) 5'-W GG A C W-3' 40)ImImpypy-y-ImHpPyPy
41) 5'-W GG G T W-3' 41)ImImImHp-y-Pypypypy
42} 5'-W GG G A W-3' 42)ImImImPy-y-HpPypyPy
43) 5'-W GG C T W-3' 43)ImImPyHp-y-pyImPyPy
44) 5'-W GG C A W-3' 44)ImImPyPy-y-HpImPypy
G1) 5'-W GG G G W-3' G1)ImImImIm-y-Pypypypy
G2 ) 5 GG G C W-3 G2 ) ImImImPy-y- ImPyPypy
' '
-W
G3) 5'-W GG C G W-3' G3)ImImPyIm-y-PyImPyPy
G4) 5'-W GG C C W-3' G4)ImImPyPy-y-ImImPyPy
37
CA 02281947 1999-08-17
WO 98/37066 PCT/US98/01006
TABLE
12:
8-ring
Hairpin
Polyamides
for
recognition
of 6-by
5'-WGCNNW-3'
DNA aromatic amino acid sequence
sequence
45) 5' G C TT W-3' 45) ImPyHpHp-y-PyPyImPy
-W
46) 5' G ~ TA W-3' 46) ImPyHpPy-y-HpPyImPy
-W
47 ) 5' G C TG W-3' 47) ImPyHpIm-y-PyPyImPy
-W
48) 5' G C TC W-3' 48) ImPyHpPy-y-ImPyImPy
-W
49) 5' G C AT W-3' 49) ImPyPyHp-y-PyHpImPy
-W
50) 5' G C AA W-3' S0) ImPyPyPy-y-HpHpImPy
-W
51) 5' G C AG W-3' S1) ImPyPyIm-y-PyHpImPy
-W
52) 5' G C AC W-3' S2) ImPyPyPy-y-ImHpImPy
-W
53 ) 5' G C GT W-3' S3 ) ImPyImHp-y-PyPyImPy
-W
54) 5' G C GA W-3' S4) ImPyImPy-y-HpPyImPy
-W
55) 5' G C CT W-3' S5) ImPyPyHp-y-PyImImPy
-W
56) 5' G C CA W-3' S6) ImPyPyPy-y-HpImImPy
-W
G5) 5' G C GG W-3' G5) ImPyImIm-y-PyPyImPy
-W
G6) 5' G C GC W-3' G6) ImPyImPy-y-ImPyImPy
-W
G7) 5' G C CG W-3' G7) ImPyPyIm-y-PyImimPy
-W
G8) 5' G C CC W-3' GB) ImPyPyPy-y-ImImImPy
-W
38
CA 02281947 1999-08-17
VVO 98/37066 PCT/US98/01006
TABLE
13:
8-ring
Hairpin
Polyamides
for
recognition
of
6-by
5'-WTTNNW-3'
DNA sequence aromatic amino acid sequence
57) 5'-W T T T T W-3' S7)HpHpHpHp-y-PyPyPyPy
58) 5'-W T T T A W-3' S8)HpHpHpPy-y-HpPyPyPy
59) 5'-W T T T G W-3' S9)HpHpHpIm-y-PyPypypy
60) 5'-W T T T C W-3' 60)HpHpHpPy-y-ImPyPyPy
61) 5' -W T T A T W-3' 61) HpHpPyHp-y-PyHpPyPy
62) 5'-W T T A A W-3' 62)HpHpPyPy-y-HpHpPyPy
63 ) 5' -W T T A G W-3' 63 ) HpHpPyIm-y-PyHpPyPy
64) 5'-W T T A C W-3' 64)HpHpPyPy-y-ImHpPyPy
65) 5'-W T T G T W-3' 65)HpHpImAp-y-PyPyPyPy
66) 5'-W T T G A W-3' 66)HpHpImPy-y-HpPyPyPy
67) 5'-W T T G G W-3' 67)HpHpImIm-y-PyPyPyPy
68) 5'-W T T G C W-3' 68)HpHpImPy-y-ImPyPyPy
69) 5'-W T T C T W-3' 69)HpHpPyHp-y-PyImPyPy
70) 5'-W T T C A W-3' 70)HpHpPyPy-y-HpImPyPy
71) 5'-W T T C G W-3' 71)HpHpPyIm-y-PyImPyPy
72) 5'-W T T C C W-3' 72)HpHpPyPy-y-ImImPyPy
39
CA 02281947 1999-08-17
WO 98/37066 PCT/US98/01006
TABLE
I4:
8-ring
Hairpin
Polyamides
for
recognition
of 6-by
5'-WTANNW-3'
DNA aromatic amino acid sequence
sequence
73 ) 5' TA T T W-3' 73 ) HpPylipHp-y-PyPyHpPy
-W
74) 5'-W T?r T A W-3' 74)HpPyHpPy-y-HpPyHpPy
75) 5'-W TA T G W-3' 75)HpPyHpIm-y-PyPyHpPy
76) 5'-W TA T C W-3' 76)HpPyHpPy-y-ImPyHpPy
77) 5'-W TA A T W-3' 77)HpPyPyHp-y-PyHpHpPy
78) 5'-W TA A A W-3' 78)HpPyPyPy-y-HpHpl3pPy
79) 5' TA A G W-3' 79)HpPyPyIm-y-PyHpHpPy
-W
80) 5'-W TA A C W-3' 80)HpPyPyPy-y-ImHpHpPy
81) 5'-W TA G T W-3' el)HpPyImHp-y-PyPyHpPy
82) 5'-W TA G A W-3' 82)HpPyImPy-y-HpPyHpPy
83) 5' TA G G W-3' 83 ) HpPyImIm-y-PyPyHpPy
-W
84) 5'-W TA G C W-3' 84)HpPyImPy-y-ImPyHpPy
85) 5'-W TA C T W-3' 85)HpPyPyHp-y-PyImHpPy
86) 5'-W TA C A W-3' 86)HpPyPyPy-y-HpImHpPy
87) 5'-W TA C G W-3' 87)HpPyPyIm-y-PyImIipPy
88) 5'-W TA C C W-3' BB)HpPyPyPy-y-ImImHpPy
CA 02281947 1999-08-17
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TABLE
15: 8-ring
Hairpin
Polyamides
for recognition
of 6-by
5'-WTGNNW-3'
DNA aromatic
sequence amino
acid
sequence
89) 5' T G T TW-3' 89) HpImHpHp-y-PyPyPyPy
-W
90) 5' T G T AW-3' 90) HpImHpPy-y-HpPyPyPy
-W
91) 5' T G T GW-3' 91) HpImHpIm-y-PyPyPyPy
-W
92) 5'-WT G T CW-3' 92) HpImHpPy-y-ImPyPyPy
93) 5'-WT G A TW-3' 93) HpImPyHp-y-pyHpPyPy
94) 5' T G A AW-3' 94) HpImPyPy-y-HpHpPyPy
-W
95) 5' T G A GW-3' 95) HpImPyIm-y-PyHpPyPy
-W
96) 5' T G A CW-3' 96) HpImPyPy-y-ImHpPyPy
-W
97) 5'-WT G G TW-3' 97) HpImImHp-y-pypypypy
98) 5'-WT G G AW-3' 98) HpImImPy-y-HppyPypy
99) 5' T G C TW-3' 99) HpImPyHp-y-pyImPyPy
-W
100) 5' T G C AW-3' 100) HpImPyPy-y-HpImPyPy
-W
101) 5' T G G GW-3' 101) HpImImIm-y-pyPypypy
-W
102) 5' T G G CW-3' 102) HpImImPy-y-ImPyPyPy
-W
103) 5'-WT G C GW-3' 103) HpImPyIm-y-PyImPyPy
104) 5'-WT G C CW-3' 104) HpImPyPy-y-ImImPyPy
41
CA 02281947 1999-08-17
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TABLE
16:
8-ring
Hairpin
Polyamides
for
recognition
of 6-by
5'-WTCNNW-3'
DNA aromatic amino acid sequence
sequence
105) 5' T CT T W-3' 105)HpPyHpHp-y-PyPyImPy
-W
106) 5'-W T CT A W-3' 106)HpPyHpPy-y-HpPyImPy
107) 5'-W T CT G W-3' 107)HpPyHpIm-y-PyPyImPy
108) 5'-W T CT C W-3' l08)HpPyHpPy-y-ImPyImPy
109) 5'-W T CA T W-3' 109)HpPyPyHp-y-PyHpImPy
110) 5'-W T CA A W-3' 110)HpPyPyPy-y-HpHpImPy
111) 5'-W T CA G W-3' 111)HpPyPylm-y-PyHpImPy
112) 5'-W T CA C W-3' 112)HpPyPyPy-y-ImHpImPy
113) 5'-W T CG T W-3' 113)HpPyImHp-y-PyPyImPy
114) 5'-W T CG A W-3' 114)HpPyImPy-y-HpPyImPy
115) 5'-W T CC T W-3' 115)HpPyPyHp-y-PyImImPy
I16) 5'-W T CC A W-3' 116)HpPyPyPy-y-HpImImPy
117) 5'-W T CG G W-3' 117)HpPyImIm-y-PyPyImPy
118) 5'-W T CG C W-3' 118)HpPyImPy-y-ImPyImPy
119) 5'-W T CC G W-3' 119)HpPyPyIm-y-PyImImPy
120) 5'-W T CC C W-3' 120)HpPyPyPy-y-ImImImPy
42
CA 02281947 1999-08-17
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TABLE
17:
8-ring
Hairpin
Polyamides
for
recognition
of
6-by
5'-WATNNW-3'
DNA aromatic amino acid sequence
sequence
121) 5'-W A T TT W-3' 121)PyHpHpHp-y-pypypyHp
122) 5'-W A T TA W-3' 122)PyHpHpPy-y-HpPyPyHp
123) 5'-W A T TG W-3' I23)PyHpHpIm-y-PypypyHp
124) 5'-W A T TC W-3' 124)PyHpHpPy-y-ImPyPyHp
125) 5'-W A T AT W-3' 125)PyHpPyHp-y-PyHpPyHp
126) 5'-W A T AA W-3' 126)PyHpPyPy-y-HpHpPyHp
127) 5'-W A T AG W-3' I27)pyHppyIm-y-PyHpPyHp
128) 5'-W A T AC W-3' 128)PyHpPyPy-y-ImHpPyHp
129) 5'-W A T GT W-3' 129)PyHpImHp-y-pypypyHp
130) 5'-W A T GA W-3' 130)PyHpImPy-y-HpPyPyHp
13 1) 5'-W A T GG W-3' 131)PyHpImIm-y-PypypyHp
132) 5'-W A T GC W-3' 132)PyHpImPy-y-ImPyPyHp
133) 5'-W A T CT W-3' 133)PyHpPyHp-y-PyImPyHp
134) 5'-W A T CA W-3' 134)PyHpPyPy-y-HpImPyFip
135) 5'-W A T CG W-3' 135)PyHpPyIm-y-PyImPyHp
136) 5'-W A T CC W-3' 136)PyHpPyPy-y-ImImPyHp
43
CA 02281947 1999-08-17
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TABLE
18: 8-ring
Hairpin
Polyamides
for recognition
of 6-by
5'-WA,ANNW-3'
DNA aromatic amino acid sequence
sequence
137 ) 5' AA T T W-3' 137 ) PyPyFipHp-y-PyPyHpHp
-W
138) 5'-W AA T A W-3' 138)PyPyHpPy-y-HpPyHpHp
139) 5'-W AA T G W-3' 139)PyPyHpIm-y-PyPyHpHp
140) 5'-W AA T C W-3' 140)PyPyHpPy-y-ImPyHpHp
141) 5'-W AA A T W-3' 141)PyPyPyHp-y-PyHpHpHp
142) 5'-W AA A A W-3' 142)PyPyPyPy-y-HpHpHpHp
143) 5'-W AA A G W-3' 143)PyPyPyIm-y-PyHpHpHp
144) 5'-W AA A C W-3' 144)PyPyPyPy-y-ImHpHpHp
145) 5'-W AA G T W-3' 145)PyPyImHp-y-PyPyHpHp
146) 5' AA G A W-3' 146) PyPyImPy-y-HpPyHpHp
-W
2~ 147) 5'-W AA G G W-3' 147)PyPyImIm-y-PyPyHpHp
148) 5'-W AA G C W-3' 148)PyPyImPy-y-ImPyHpHp
149) 5' AA C T W-3' 149) PyPyPyHp-y-PyImHpHp
-W
150) 5'-W AA C A W-3' 150)PyPyPyPy-y-HpImHpHp
151) 5'-W AA C G W-3' 151)PyPyPyIm-y-PyImHpHp
152) 5'-W AA C C W-3' 152)PyPyPyPy-y-=mImHpHp
44
CA 02281947 1999-08-17
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TABLE
19: 8-ring
Hairpin
Polyamides
for recognition
of 6-by
5'-WAGNNW-3'
DNA aromatic amino acid sequence
sequence
153 ) 5' -W G T T W-3' 153 ) PyImHpHp-y-Pypypygp
A
154) 5' -W G T A W-3' 154) PyImHppy-Y-HpPyPYHp
A
155) 5'-W G T G W-3' 155)PyImHpIm-y-pypypyHp
A
156) 5'-W G T C W-3' 156)PyImHpPy-Y-ImPyPYHp
A
157) 5'-W G A T W-3' 157)PyImPyHp-y-pyHpPYHp
A
158) 5'-W G A A W-3' 158)PyImpyPy-y-HpHpPYHp
A
159) 5' -W G A G W-3' 159) PyImPyIm-y-pyHpPyHp
A
160) 5'-W G A C W-3' 160)PyImPyPy-y-ImHpPyHp
A
161) 5'-W G G T W-3' 161)PYImImHp-Y-PypypyHp
A
162 ) 5' -W G G A W-3' 162 ) PyImImPy-~~-HpPyPyHp
A
163) 5' -W G C T W-3' 163) PyImPyHp-y-PyImPyHp
A
164) 5'-W C A W-3' 164)PyImPyPy-~-HpImPyHp
A G
165) 5' -W G G W-3' 165) PyImImIm-y-pypypyHp
A G
166) 5' -W G C W-3' 166) PYImImPy-Y-ImPyPyHp
A G
167 ) 5' -W C G W-3 167 ) PyImPyIm-~~-PyIrnPyHp
A G '
168) 5' -W C C W-3' 168) PyImPyPy-~~-ImImPyHp
A G
CA 02281947 1999-08-17
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TABLE
20: 8-ring
Hairpin
Polyamides
for recognition
of 6-by
5'-WACNNW-3'
DNA aromatic amino acid sequence
sequence
169) 5'-W A C TT W-3' 169)PyPyHpHp-y-PyPyImHp
170) 5' A C TA W-3' 170) PyPyHpPy-y-HpPyImAp
-W
171) 5' A C TG W-3' 171) PyPyHpIm-y-PyPyImHp
-W
172) 5' A C TC W-3' 172) PyPyHpPy-y-ImPyImHp
-W
173) 5'-W A C AT W-3' 173)PyPyPyHp-y-PyHpImHp
174) 5'-W A C AA W-3' 174)PyPyPyPy-y-HpHpImHp
175) 5'-W A C AG W-3' 175)PyPyPyIm-y-PyHpImHp
176) 5'-W A C AC W-3' 176)PyPyPyPy-y-ImHpImHp
177) 5'-W A C GT W-3' 177)PyPyImHp-y-PyPyImHp
178) 5' A C GA W-3' 178) PyPyImPy-y-HpPyImHp
-W
179) 5' A C CT W-3' 179) PyPyPyHp-y-PyImImHp
-W
180) 5'-W A C CA W-3' 180)PyPyPyPy-y-HpImImHp
181) 5'-W A C GG W-3' 181)PyPyImIm-y-PyPyImHp
182) 5'-W A C GC W-3' 182)PyPyImPy-y-imPyImHp
183) 5' A C CG W-3' 183) PyPyPyIm-y-PyImImHp
-W
184) 5'-W A C CC W-3' 184)PyPyPyPy-y-ImImImHp
46
CA 02281947 1999-08-17
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TABLE
21:
8-ring
Hairpin
Polyamides
for
recognition
of 6-by
5'-WCTNNW-3'
DNA aromatic amino acid sequence
sequence
185) 5'-WC T T TW-3' 185)PyHpHpHp-y-PyPypyIm
186) 5'-WC T T AW-3' 186)PyHpHpPy-y-HpPyPyIm
187) 5'-WC T T GW-3' 187)PyHpHpIm-y-PyPyPyIm
188) 5'-WC T T CW-3' 188)PyHpHpPy-y-ImPyPyIm
189) 5' C T A TW-3' 189) PyHpPyFip-y-pyHpPyIm
-W
190) 5' C T A AW-3' 190) PyHpPyPy-y-HpHpPyIm
-W
191) 5'-WC T A GW-3' 191)PyHpPyIm-y-PyHpPyIm
192) 5'-WC T A CW-3' 192)PyHpPyPy-y-ImHpPyIm
193 ) 5' C T G TW-3' 193 ) PyHpImHp-y-PyPyPyIm
-W
194 ) 5' C T G AW-3' 194 ) PyHpImPy-y-HpPyPyIm
-W
195) 5' C T G GW-3' 195) PyHpImIm-y-PyPyPyIm
-W
196) 5'-WC T G CW-3' 196)PyHpImPy-y-ImPyPylm
197) 5'-WC T C TW-3' 197)PyHpPyHp-y-PyImPyIm
198) 5'-WC T C AW-3' 198)PyHpPyPy-y-HpImPyIm
199) 5'-WC T C GW-3' 199)PyHpPyIm-y-PyImPyIm
200) 5'-WC T C CW-3' 200)PyHpPyPy-y-ImImPyIm
47
CA 02281947 1999-08-17
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TABLE
22: 8-ring
Hairpin
Polyamides
for recognition
of 6-by
5'-WCANNW-3'
DNA aromatic amino acid sequence
sequence
201) 5' CA T T W-3' 201) PyPyHpHp-y-PyPyHpIm
-W
202) 5'-W C-A T A W-3' 202)PyPyHpPy-y-HpPyHpIm
203) 5' CA T G W-3' 203 ) PyPyHpIm-y-PyPyHpIm
-W
204) 5' CA T C W-3' 204) PyPyHpPy-y-ImPyHpIm
-W
205) 5'-W CA A T W-3' 205)PyPyPyHp-y-PyHpHpIm
206) 5' CA A A W-3' 206) PyPyPyPy-y-HpHpHpIm
-W
207) 5'-W CA A G W-3' 207)PyPyPyIm-y-PyHpHpIm
208) 5'-W CA A C W-3' 208)PyPyPyPy-y-ImHpHpIm
209) 5' CA G T W-3' 209) PyPyImHp-y-PyPyHplm
-W
210) 5'-W CA G A W-3' 210)PyPyImPy-y-HpPyHpIm
211) 5' CA G G W-3' 211) PyPyImIm-y-PyPyHpIm
-W
212) 5'-W CA G C W-3' 212)PyPyImPy-y-ImPyHpIm
213) 5'-W CA C T W-3' 213)PyPyPyHp-y-PyImHpIm
214) 5'-W CA C A W-3' 214}PyPyPyPy-y-HpImHpIm
215) 5'-W CA C G W-3' 215}PyPyPyIm-y-PyImHpIm
216) 5'-W CA C C W-3' 216)PyPyPyPy-y-ImImHpIm
48
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TABLE
23: 8-ring
Hairpin
Polyarnides
for recognition
of 6-by
5'-WCGNNW-3'
DNA aromatic amino acid sequence
sequence
217) 5'-W C G T T W-3'217)PyImHpHp-y-PypyPylm
218) 5'-W C G T A W-3'218)PyImHpPy-y-HpPyPyIm
219) 5'-W C G T G W-3'219)PyImHpIm-y-PyPyPyIm
220) 5'-W C G T C W-3'220)PyImHpPy-y-ImPyPyIm
221) 5'-W C G A T W-3'221)PyImPyHp-y-PyHpPyIm
IS 222) 5'-W C G A A W-3'222)PyImPyPy-y-HpHpPyIm
223) 5'-W C G A G W-3'223)PyImPyIm-y-PyHpPyIm
224) 5'-W C G A C W-3'224)PyImPyPy-y-ImHpPyIm
225) 5'-W C G G T W-3'225)PyImImHp-y-PyPyPyIm
226) 5'-W C G G A W-3'226)PyImImPy-y-HpPyPyIm
227) 5'-W C G C T W-3'227)PyImPyHp-y-PyImPyIm
228) 5'-W G C A W-3' 22B)PyImPyPy-y-HpImPyIm
C
G9) 5'-W G G G W-3' G9) PyImImIm-y-PyPyPyIm
C
G10) 5'-W G G C W-3' G10)PyImImPy-y-ImPyPyIm
C
G11) 5'-W G C G W-3' G11)PyImPyIm-y-PyImPyIm
C
G12) 5'-W G C C W-3' G12)PyImPyPy-y-ImImPyIm
C
49
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TABLE 24: 8-ring Hairpin Polyamides for recognition of 6-by 5'-WCCNNW-3'
DNA sequence aromatic amino acid sequence
229) 5'-W C C T T W-3' 229)PyPyHpHp-y-PyPyImIm
230) 5'-W C-C T A W-3' 230)PyPyHpPy-y-HpPyImIm
231) 5'-W C C T G W-3' 231)PyPyHpIm-y-PyPyImIm
232) 5'-W C C T C W-3' 232)PyPyHpPy-y-ImPyImIm
233) 5'-W C C A T W-3' 233)PyPyPyHp-y-PyHpImIm
234) 5' -W C C A A W-3' 234 ) PyPyPyPy-y-HpHpImIm
235) 5'-W C C A G W-3' 235)PyPyPyIm-y-PyHpZmIm
236) 5' -W C C A C W-3' 236) PyPyPyPy-y-ImHpImIm
237) 5'-W C C G T W-3' 23~)PyPyImHp-y-PyPyImIm
238) 5' -W C C G A W-3' 238) PyPyImPy-y-HpPyImIm
239) 5' -W C C C T W-3' 239) PyPyPyHp-y-PyImImIm
240) 5' -W C C C A W-3' 240) PyPyPyPy-y-HpImImIm
G13) 5'-W C C G G W-3' G13)PyPyImIm-y-PyPyImIm
G14) 5'-W C C G C W-3' G14)PyPyImPy-y-ImPyImIm
G15) 5'-W C C C G W-3' G15)PyPyPyIm-y-PyImImIm
G16) 5'-W C C C C W-3' G16)PyPyPyPy-y-ImImImIm
EXAMPLE 9:
Aliphatic/Aromatic amino acid pairing for recognition of the DNA minor groove.
Selective placement of an aliphatic (I-alanine (~3) residue paired side-by-
side with either
a pyrrole {Py) or imidazole (Im) aromatic amino acid is found to compensate
for sequence
composition effects for recognition of the minor groove of DNA by hairpin
pyrrole-imidazole
polyamides. A series of polyamides were prepared which contain pyrrole and
imidazole
aromatic amino acids, as well as y-aminobutylic acid (y) "tum" and ~i-alanine
"spring" aliphatic
amino acid residues. The binding affinities and specificities of these
polyamides are regulated
by the placement of paired (3/(3 Py/~i and Im/(3 residues. Quantitative
footprint titrations
demonstrate that replacing two Py/Py pairings in a 12-ring hairpin (6-y-6)
with two Py/(3
CA 02281947 1999-08-17
WO 98/37066 PCT/US98/01006
pairings affords 10-fold enhanced affinity and similar sequence specificity
for an 8-by target
sequence.
Tabk 25 Equilibrium association constants (M-I ) for polyatnides a-~
Polyemlde 5'-TGTTAACA-3'S'-TGTGAACA-3'Specificityd
a~~~~~j~ 2.5 x 3.9 x 6
109 IOs
.d 1.3 x 2.0 x 7
109 IOa
~-c.~jp~= 1.7 x 2.7 x 6
IOta 109
~~p~~,~,~,~.~,1.2 x 2.2 x 55
IOrt l0
6.6x109 2.5xIpA 26
4.5xlOt~7.7x109 6
-f y
2.7xlOt~5.7x 5
109
s I x s 1 x I
IOe 108
°Values reported are the mean values obtained from three DNase 1
footprint utrauon cxpcrtments. b The assays were tamed out ac 22 °C at
pH 7.0 in the prcsence of 10 mM Tris-HCI, 10 mM KCI. 10 mM MgCl2,
and 5 mM CaCI, ' klatch sne associauon constants and spccificities
tugher than the parent hatrptn are shown tn boldtype. ~Spccificity is
calculated as Ka(match) / Ka(mismatch).
The 6-y-6 hairpin ImPyImPyPyPy-y-ImPyPyPyPyPy-~i-Dp, which contains six
consecutive amino acid pairings, is unable to discriminate a single-base-pair
mismatch site 5'-
TGTTAACA-3' from a 5'-TGTGAACA-3' match site. The hairpin polyamide Im-~i-
l~'YPYPY-Y-I~'YPYPY-~-Py-~-Dp binds to the 8-by match sequence 5'-TGTGAACA-3'
with
t o an equilibrium association constant of Ka = 2.4 x 1010 M 1 and > 48-fold
specificity versus the
5'-TGTTAACA-3' single-base-pair mismatch site.
51
CA 02281947 1999-08-17
WO 98/37066 PCT/LJS98/01006
Table26 Equilibrium association constants (M-I) for polyamides °-~
Polyamide 5'-TGTTAACA-3' S'-TGTGAACA-3' Specificityd
2.5 x 109 3-9 x IOs 6
6.6x10' ~.5x10g 26
x 109 5 x 109 1
s 5 x 108 2.4 x 10~s : 48
° Values reported for i. 5, and 10 are the mean values obtained from
three DNase I footprint titration experiments. 'The assays were carried
out at 22 °C at pH 7.0 in the presence of 10 tnM Tris-HCI, 10 mM KCI,
mM MgCl2, and 5 mM CaClz. 'Match site association constants
and specificities higher than parent hairpins are shown in
boldtype. dSpecificity is calculated as Ka(match) l Ka(mismatch).
Modeling indicates that the ~3-alanine residue relaxes ligand curvature,
providing for
optimal hydrogen bond formation between the floor of the minor groove and both
Im-residues
5 within the Im-~3-Im polyamide subunit. This observation provided the basis
for design of a
hairpin polyamide. Im-~3-ImPy-y-Im-(3-ImPy-(3-Dp, which incorporates Im/(3
pairings to
recognize a "problematic" 5'-GCGC-3' sequence at subnanomolar concentrations.
Table 27 Equilibrium association constamts (M-1) for polyamides.°-
b
Polyamide 5'-TGCGCA-3' Si'-TGGCCA-3' S'-TGGGGA-3'
3.7x10' <l0' <10'
3.7x109 1.4x108 l.lxlOR
Values reported are the mean wlues obttained from a minimum of three
DNase I footprint titration experiments. l6 The assays were carried out at
22 °C at pH 7.0 in the presence of 10 mM( Tris-HCI, 10 mM KCI, 10 mM
MgClz, and 5 mM CaClz.
to These results identify Im/~ and ~/Im pairings that respectively
discriminate G~C and
C~G from A~T/T~A as well as Py/~i and (3/Py pairings that discriminate A~T/T~A
from
G~C/C~G. These aliphatic/aromatic amino acid pairings will facilitate the
design of hairpin
polyamides which recognize both a larger binding site size as well as a more
diverse sequence
repertoire.
EXAMPLE 10:
POLYAMIDE BIOTIN CONJUGATES
Bifunctional conjugates prepared between sequence specific DNA binding
polyamides
and biotin are useful for a variety of applications. First, such compounds can
be readily attached
52
CA 02281947 1999-08-17
WO 98/37066 PCT/L1S98/01006
to a variety of matrices through the strong interaction of biotin with the
protein streptavidin.
Readily available strepdavidin-derivatized matrices include magnetic beads for
separations as
well as resins for chromatography.
A number of such polyamide-biotin conjugates have been synthesized by solid
phase
synthetic methods outlined in detail above. Following resin cleavage with a
variety of diamines,
the polyamides were reacted with various biotin carboxylic acid derivatives to
yield
bifunctional conjugates. The bifunctional conjugates were purified by HPLC and
characterized
by MALDI-TOF mass spectroscopy and'H NMR.
to
The scheme for the synthesis of an exemplary biotin-polyamide conjugate is
shown
below.
~i-Py-Py-Py-Im-y-Py-Py-Py-Im
Resin
HpN~N~NH2
O
HN~NH O
H H O
.,,~ N ~O-
O O
p DMF, DIEA 12h RT
HN~NH ~ p \ \
H 'SW.,~N~N~N~N~N w~ H \~~ HO \ I N~N
O H ~ H H . H N
~~H
N' ~' N
,, N HN
s \
1
The foregoing is intended to be illustrative of the present invention, but not
limiting.
Numerous variations and modifications of the present invention may be effected
without
departing from the true spirit and scope of the invention.
53
