Note: Descriptions are shown in the official language in which they were submitted.
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WO 98/53801 PCT/US98/10804
CONJUGATED PEPTIDE NUCLEIC ACIDS HAVING ENHANCED
CELLULAR UPTAKE
Field of the Invention
The present invention is directed to compositions comprising a peptide nucleic
acid
(PNA) which is conjugated to a lipophilic group and incorporated into
liposomes. The PNA
is composed of naturally-occurring nucleobases or non-naturally-occurring
nucleobases which
are covalently bound to a polyamide backbone. The PNA compositions of the
present
invention may further comprise a PNA modified by an amino acid side chain. The
PNA
compositions of the present invention exhibit enhanced cellular uptake and
distribution. PNA
compositions which were incorporated into liposomes demonstrated increased
cellular uptake
and more diffuse distribution than PNA compositions without liposomes.
Background of the Invention
The function of a gene starts by transcription of its information to a
messenger RNA
(mRNA). By interacting with the ribosomal complex, mRNA directs synthesis of
proteins.
This protein synthesis process is known as translation. Translation requires
the presence of
various cofactors, building blocks, amino acids and transfer RNAs (tRNAs), all
of which are
present in normal cells.
Most conventional drugs exert their effect by interacting with and modulating
one
or more targeted endogenous proteins, e.g., enzymes. Typically, however, such
drugs are not
2 0 specific for targeted proteins but interact with other proteins as well.
Thus, use of a relatively
large dose of drug is necessary to effectively modulate the action of a
particular protein. If
the modulation of a protein activity could be achieved by interaction with or
inactivation of
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mRNA, a dramatic reduction in the amount of drug necessary and in the side-
effects of the
drug could be achieved. Further reductions in the amount of drug necessary and
the side-
effects could be obtained if such interaction is site-specific. Since a
functioning gene
continually produces mRNA, it would be even more advantageous if gene
transcription could
be arrested in its entirety. Oligonucleotides and their analogs have been
developed and
used as diagnostics, therapeutics and research reagents. One example of a
modification to
oligonucleotides is labeling with non-isotopic labels, e.g., fluorescein,
biotin, digoxigenin,
alkaline phosphatase, or other reporter molecules. Other modifications have
been made to the
ribose phosphate backbone to increase the resistance to nucleases. These
modifications
1 o include use of linkages such as methyl phosphonates, phosphorothioates and
phosphoro-
dithioates, and 2'-O-methyl ribose sugar moieties. Other oligonucleotide
modifications
include those made to modulate uptake and cellular distribution.
Phosphorothioate
oligonucleotides are presently being used as antisense agents in human
clinical trials for the
treatment of various disease states. Although some improvements in diagnostic
and
therapeutic uses have been realized with these oligonucleotide modifications,
there exists an
ongoing demand for improved oligonucleotide analogs.
There are several known nucleic acid analogs having nucleobases bound to
backbones other than the naturally-occurring ribonucleic acids or
deoxyribonucleic acids.
These nucleic acid analogs have the ability to bind to nucleic acids with
complementary
2 o nucleobase sequences. Among these, the peptide nucleic acids (PNAs), as
described. for
example, in WO 92/20702, have been shown to be useful as therapeutic and
diagnostic
reagents. This may be due to their generally higher affinity for complementary
nucleobase
sequence than the corresponding wild-type nucleic acids.
PNAs are useful surrogates for oligonucleotides in binding to DNA and RNA.
Egholm et al., Nature, 1993, 365, 566, and references cited therein. The
current literature
reflects the various applications of PNAs. Hyrup et al., Bioorganic do Med.
Chem., 1996, 4,
5; and Nielsen, Perspectives Drug Disc. Des., 1996, 4, 76.
PNAs are compounds that are analogous to oligonucleotides, but differ in
composition. In PNAs, the deoxyribose backbone of oligonucleotide is replaced
by a peptide
3 o backbone. Each subunit of the peptide backbone is attached to a naturally-
occurring or non
naturally-occurring nucleobase. One such peptide backbone is constructed of
repeating units
of N-(2-aminoethyl)glycine linked through amide bonds. The synthesis of PNAs
via
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preformed monomers was previously described in WO 92/20702 and WO 92/20703,
the
contents of which are herein incorporated by reference. More recent advances
in the structure
and synthesis of PNAs are illustrated in WO 93/12129 and U.S. Patent
5,539,082, issued July
23, 1996, the contents of both being herein incorporated by reference.
Further, the literature
is replete with publications describing synthetic procedures, biological
properties and uses of
PNAs. For example, PNAs possess the ability to effect strand displacement of
double-
stranded DNA. Patel, Nature,1993, 365, 490. Improved synthetic procedures for
PNAs have
also been described. Nielsen et al., Science,1991, 254, 1497; and Egholm, J.
Am. Chem. Soc.,
1992, 114, 1895. PNAs form duplexes and triplexes with complementary DNA or
RNA.
l0 Knudson et al., Nucleic Acids Research, 1996, 24, 494; Nielsen et al., J.
Am. Chem. Soc.,
1996,118, 2287; Egholm et al., Science,1991, 254, 1497; Egholm et al., J. Am.
Chem. Soc.,
1992,114, 1895; and Egholm et al., J. Am. Chem. Soc.,1992,114, 9677.
PNAs bind to both DNA and RNA and form PNA/DNA or PNA/RNA duplexes.
The resulting PNA/DNA or PNA/RNA duplexes are bound tighter than corresponding
DNA/DNA or DNA/RNA duplexes as evidenced by their higher melting temperatures
(Tm).
This high thermal stability of PNA/DNA(RNA) duplexes has been attributed to
the neutrality
of the PNA backbone, which results elimination of charge repulsion that is
present in
DNA/DNA or RNA/RNA duplexes. Another advantage of PNA/DNA(RNA) duplexes is
that
Tm is practically independent of salt concentration. DNA/DNA duplexes, on the
other hand,
2 0 are highly dependent on the ionic strength.
Triplex formation by oligonucleotides has been an area of intense
investigation since
sequence-specific cleavage of double-stranded deoxyribonucleic acid (DNA) was
demonstrated. Moser et al., Science, 1987, 238, 645. The potential use of
triplex-forming
oligonucleotides in gene therapy, diagnostic probing, and other biomedical
applications has
generated considerable interest. Uhlmann et al., Chemical Reviews, 1990, 90,
543.
Pyrimidine oligonucleotides have been shown to form triple helix structures
through binding
to homopurine targets in double-stranded DNA. In these structures the new
pyrimidine strand
is oriented parallel to the purine Watson-Crick strand in the major groove of
the DNA and
binds through sequence-specific Hoogsteen hydrogen bonding. The sequence
specificity is
3 0 derived from thymine recognizing adenine (T:A-T) and protonated cytosine
recognizing
guanine (C':G-C). Best et al., J. Am. Chem. Soc., 1995, 117, 1187. In a less
well-studied
triplex motif purine-rich oligonucleotides bind to purine targets of double-
stranded DNA.
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The orientation of the third strand in this motif is anti-parallel to the
purine Watson-Crick
strand, and the specificity is derived from guanine recognizing guanine (G:G-
C) and thymine
or adenine recognizing adenine (A:A-T or T:A-T). Greenberg et al., J. Am.
Chem. Soc.,1995,
117, 5016.
Homopyrimidine PNAs have been shown to bind complementary DNA or RNA
fomling (PNA)z/DNA(RNA) triplexes of high thermal stability. Egholm et al.,
Science,1991,
254, 1497; Egholm et al., J. Am. Chem. Soc.,1992, 114, 1895; Egholrn et al.,
J. Am. Chem.
Soc., 1992, 114, 9677. The formation of triplexes involving two PNA strands
and one
nucleotide strand has been reported in U.S. Patent Application Serial No.
08/088,661, filed
July 2, 1993, the contents of which are incorporated herein by reference. The
formation of
triplexes in which the Hoogsteen strand is parallel to the DNA purine target
strand is preferred
to formation of anti-parallel complexes. This allows for the use of bis-PNAs
to obtain triple
helix structures with increased pH-independent thermal stability using
pseudoisocytosine
instead of cytosine in the Hoogsteen strand. Egholm et al., J. Am. Chem. Soc.,
1992, 114,
1895. Further, see WO 96/02558, the contents of which are incorporated herein
by reference.
Peptide nucleic acids have been shown to have higher binding affinities (as
determined by their Tm's) for both DNA and RNA than that of DNA or RNA to
either DNA
or RNA. This increase in binding amity makes these peptide nucleic acid
oligomers
especially useful as molecular probes and diagnostic agents for nucleic acid
species.
2 0 In addition to increased affinity, PNAs have increased specificity for DNA
binding.
Thus, a PNA/DNA duplex mismatch show 8 to 20 °C drop in the T,"
relative to the DNA/DNA
duplex. This decrease in Tm is not observed with the corresponding DNA/DNA
duplex
mismatch. Egholm et al., Nature 1993, 365, 566.
A further advantage of PNAs, compared to oligonucleotides, is that the
polyamide
2 5 backbone of PNAs is resistant to degradation by enzymes.
Considerable research is being directed to the application of oligonucleotides
and
oligonucleotide analogs that bind to complementary DNA and RNA strands for use
as
diagnostics, research reagents and potential therapeutics. For many
applications, the
oligonucleotides and oligonucleotide analogs must be transported across cell
membranes or
3 0 taken up by cells to express their activity.
PCT/EP/01219 describes novel PNAs which bind to complementary DNA and RNA
more tightly than the corresponding DNA. It is desirable to append groups to
these PNAs
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_ 5 _ -
which will modulate their activity, modify their membrane permeability or
increase their
cellular uptake property. One method for increasing amount of cellular uptake
property of
PNAs is to attach a lipophilic group. United States Application Serial No.
117,363, filed Sept.
3, 1993, describes several alkylamino functionalities and their use in the
attachment of such
pendant groups to oligonucleosides.
United States Application Serial No. 07/943,516, filed Sept. 11, 1992, and its
corresponding published PCT application WO 94/06815, describe other novel
amine-
containing compounds and their incorporation into oligonucleotides for, inter
alia, the
purposes of enhancing cellular uptake, increasing lipophilicity, causing
greater cellular
retention and increasing the distribution of the compound within the cell.
United States Application Serial No. 08/116,801, filed Sept. 3, 1993,
describes
nucleosides and oligonucleosides derivatized to include a thiolalkyl
functionality, through
which pendant groups are attached.
Recently, liposomal drug-delivery systems incorporating various biomolecules
and
drugs have been studied and found to exhibit reduced toxicities and increased
efficacy due
to enhanced cellular uptake and distribution. Chonn and Cullis, Current
Opinion in
Biotechnolo~,1995, 6, 698; Mannino et al., Biotechniques, 1988, 6, 682; Blume
and Cevc,
Biochem et Biophys. Acta, 1990, 1029, 91; and Lappalainen et al., Antiviral
Res., 1994, 23,
119. Liposomes are microscopic spheres composed of an aqueous core and a lipid
bilayer
2 0 enveloping the core. Procedures for preparation of liposomes are available
in the literature.
G. Gregoridadis in "Liposome Technology," volume 2, G. Gregoridadis (ed.), CRC
Press,
1993, p.l; Watwe and Bellare, Curr. Sci., 1995, 68, 715. Several liposomal
drugs are
currently on the market or under development. Chonn and Cullis, Current
Opinion in
Biotechnology,1995, 6, 698.
WO 96/10391, published April 11, 1995, describes polyethylene glycol-modified
ceramide lipids which are used to form liposomes, and the use of these
liposomes as drug-
delivery vehicles.
WO 96/24334, published August 15, 1996, describes lipid constructs having an
aminomannose-derivatized crolesterol moiety for the delivery of drugs to the
cytoplasm of
3 o cells, particularly to vascular smooth muscle tissues.
WO 96/40627, published December 19, 1996, describes cationic lipid-containing
Iiposome formulations which are useful in the delivery of biomolecules such as
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oligonucleotides, nucleic acids, peptides and other agents.
Despite recent advances, there remains a need for stable compositions with
enhanced
cellular uptake and distribution.
Summary of the Invention
The present invention provides peptide nucleic acids (PNAs) conjugated to a
lipophilic group and having a modified backbone wherein an amino acid side
chain is attached
to the backbone. The present invention also provides liposomal compositions
comprising a
peptide nucleic acid (PNA) conjugated to a lipophilic group which is
incorporated into
liposomes. The PNAs of the present invention comprise nucleobases covalently
bound to a
polyamide backbone. Representative nucleobases include the four major
naturally-occurring
DNA nucleobases (i.e., thymine, cytosine, adenine and guanine), other
naturally-occurring
nucleobases (e.g. inosine, uracil, 5-methylcytosine, thiouracil and 2,6-
diaminopurine) and
artificial nucleobases (e.g., bromothymine, azaadenines and azaguanines).
These nucleobases
are attached to a polyamide backbone through a suitable linker.
Preferred peptide nucleic acids of the invention have the general formula (I):
L L
O p
O O
Rh N~N N~N/Ri\Ri
R7. H R7. H
n
wherein:
2 o each L is, independently, a naturally-occurring nucleobase or a non-
naturally-
occurring nucleobase;
each R'' is hydrogen or the side chain of a naturally-occurring or non-
naturally-
occurring amino acid, at least one R'~ being the side chain of an amino acid;
R'' is OH, NHz, or NHLysNH,;
2 5 each of R' and R' is, independently, a lipophilic group or an amino acid
labeled with
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a fluorescent group; or R' and R', together, are a lipophilic group;
n is an integer from 1 to 30.
PNAs having formula ()] wherein R' is D-lysine labeled with a fluorescent
group and
R' is an adamantoyl group are preferred. Even more preferred are PNAs of
formula (I)
wherein R' is D-lysine labeled with fluorescein and R' is an adamantoyl group.
Also preferred
are PNAs having formula (I) wherein R' and R', together, are an adamantoyl
group. Further
preferred are PNAs of formula (I) wherein at /east one of said R'' is the side
chain of D-lysine.
Preferably, the carbon atom to which substituent R'' is attached is
stereochemically
enriched. Hereinafter, "stereochemically enriched" means that one stereoisomer
predominates
over the other stereoisomer in a sufficient amount as to provide a beneficial
effect.
Preferably, one stereoisomer predominates by more than 50%. More preferably,
one
stereoisomer predominates by more than 80%. Even more preferably, one
stereoisomer
predominates by more than 90%. Still more preferably, one stereoisomer
predominates by
more than 95%. Even more preferably, one stereoisomer predominates by more
than 99%.
Still even more preferably, one stereoisomer is present substantially
quantitatively.
The present invention also provides liposomal compositions comprising a
peptide
nucleic acid incorporated in a liposome, said peptide nucleic acid having
formula (I) wherein:
each L is, independently, a naturally-occurring nucleobase or a non-naturally-
occurring nucleobase;
each R'' is hydrogen or the side chain of a naturally-occurnng or non-
naturally-
occurring amino acid;
R" is OH, NHz, or NHLysNH~;
each of R' and R' is, independently, a lipophilic group or an amino acid
labeled with
a fluorescent group; or R' and R', together, are a lipophilic group;
n is an integer from 1 to 30.
PNAs having formula ()) wherein R' is D-lysine labeled with a fluorescent
group and
R' is an adamantoyl group are preferred. Even more preferred are PNAs of
formula (I)
wherein R' is D-lysine labeled with fluorescein and R' is an adamantoyl group.
Also preferred
are PNAs having formula (I) wherein R' and R', together, are an adamantoyl
group. Further
3 o preferred are PNAs of formula ()) wherein at least one of said R'~ is the
side chain of D-lysine.
Preferably, the carbon atom to which substituent R'' is attached is
stereochemically
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_ g _
enriched.
The PNAs of the present invention are synthesized by adaptation of standard
peptide
synthesis procedures, either in solution or on a solid phase.
The present invention further provides methods for enhancing the cellular
uptake and
distribution of peptide nucleic acids by incorporation of amino acid side
chains into PNA
backbones, conjugating lipophilic groups with PNAs and introducing PNAs into
liposomes.
Brief Description of the Drawing
Figure I shows structures of some lipophilic groups.
Detailed Description of the Invention
l0 In accordance with the present invention, peptide nucleic acids and
liposomal
compositions exhibiting enhanced cellular uptake and distribution are
provided. The peptide
nucleic acids (PNAs) of the invention are assembled from a plurality of
nucleobases which
are attached to a polyamide backbone by a suitable linker. In one preferred
embodiment of
the present invention, the PNAs are conjugated to a lipophilic group. As used
herein,
"conjugating" refers to attaching a lipophilic group to a PNA of the
invention. In another
preferred embodiment, the polyamide backbone of PNAs of the invention is
derivatized. As
used herein, "derivatizing" refers to modifying the backbone of a PNA by
attaching the side
chain of at least one naturally-occurring or non-naturally-occurnng amino acid
to the
polyamide backbone. The liposomal compositions of the present invention
comprise peptide
2 0 nucleic acids of the invention that are incorporated into liposomes. Thus,
the liposomal
compositions of the present invention comprise PNAs which are encapsulated by
liposomes.
The PNAs and liposomai compositions of the present invention exhibit enhanced
cellular
uptake and distribution.
The PNAs of the present invention have the formula (I) wherein nucleobase L is
a
2 5 naturally-occurring nucleobase attached at the position found in nature,
i. e., position 9 for
adenine or guanine, and position 1 for thymine or cytosine, a non-naturally-
occurring
nucleobase (nucleobase analog) or a nucleobase-binding moiety. Representative
nucleobases
include the four major naturally-occurring DNA nucleobases (i. e., thymine,
cytosine, adenine
and guanine), other naturally-occurnng nucleobases (e.g. inosine, uracil, 5-
methylcytosine,
3 o thiouracil and 2,6-diaminopurine) and artificial nucleobases (e.g.,
bromothymine, azaadenines
and azaguanines). These nucleobases are attached to a polyamide backbone
through a
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- 9 _ -.
suitable linker.
The PNAs of formula (I) include one or more amino acid moieties within their
structure. These amino acids may be naturally-occurring or non-naturally-
occurring.
Naturally-occurring amino acids include a-amino acids where the chiral center
has a D-
configuration. Such naturally-occurnng amino-acids may be either essential or
non-essential
amino acids. Non-naturally-occurring amino acids used in the PNAs of the
present invention
of formula (I) include a-amino acids with chiral centers bearing an L-
configuration. Non-
nat<ually-occurring amino acids also include amino acids bearing unusual side
chains that do
not exist in nature and are prepared synthetically, such as halo- and cyano-
substituted benzyl,
tetrahydroisoquinolylmethyl, cyclohexylmethyl, and pyridylmethyl. Other
synthetic amino-
acids include (3-amino acids.
The amino acids may be introduced into the PNAs of formula (I) either as part
of the
monomer used or at the terminal ends of the PNA. Any of the abovementioned
amino acids
could be incorporated into the monomeric building blocks used in PNA
synthesis. Preferably
the amino acid used is glycine, where R'' is H. R'~ can also be methyl, ethyl,
benzyl,
isopropyl, p-hydroxybenzyl, halobenzyl, carboxymethyl,
tetrahydroisoquinolinylmethyl, or
aminohexanoyl. Amino acids may also be attached at the C-terminus of PNAs such
that the
terminal R"-CO- group represents an amino acyl group derived from any
naturally- or non-
naturally-occurring amino acid, a- or ~3- amino acid, and with a D- or L-
configuration at the
2 o a-chiral center. Preferably the C-terminal amino acid is lysine. Amino
acids may also be
incorporated at the N-terminal end of the PNA of structure (I) where each of
R' and R' may,
independently, be an amino acyl group derived from any naturally- or non-
naturally-occurring
amino acid, a- or Vii- amino acid, and with a D- or L-configuration at the a-
chiral center.
Preferably the N-terminal amino acid is lysine.
2 5 Lipophilic groups attached to PNA's of formula (I) of the present
invention, include
natural and synthetic fatty acids, fatty alcohol derivatives and
diacylglycerol derivatives such
as adipic acid, palmitic acid, decanoic acid, octadecanoic acid, oleic acid,
elaidic acid,
linoleic acid, bile acids, heptylsuccinic acid, palmitylsuccinic acid,
polyglycolic acid,
dioctadecylglycerol phosphatidic acid, dioleoylglycerol phosphatidic acid,
adamantoyl,
3 0 octadecyloxycarbonyl, and decalinoyl. These lipophilic groups may be
attached at any
suitable location in the PNA molecule of formula (I). Preferably, the
lipophilic group is
attached to the N-terminus of the PNA of the invention wherein each of R' and
R' may,
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independently, be a lipophilic group. More preferably, R' and R', together,
are an adamantoyl
group.
The PNAs of the present invention have the formula (I) wherein labels, such as
fluorescent groups, are incorporated so as to allow a convenient means by
which to detect the
PNA. Fluorescent groups include, but are not limited to, dyes such as
fluorescein, rhodamine,
pyrenyl, cyanine dyes, CyS~ (Biological Detection Systems, Inc., Pittsburgh,
PA), and
derivatives of such dyes. These may be incorporated into the PNA of formula
(I) at any
suitable position in the PNA. Preferably, each of R' is a chemical moiety to
which is attached
a fluorescent group. It is more preferred that R' is an amino acid that has
been derivatized
1 o with a fluorescent group. It is further more preferred that R' is a lysine
with an s-fluoresceinyl
group.
Liposomal compositions of the invention comprise PNAs of the invention which
are
incorporated into liposomes. The liposomal compositions exhibit enhanced
cellular uptake
and distribution. Liposomes are a colloidal dispersion system, and constitute
a stable
delivery system which protects the incorporated PNA from the environment while
being
transported to target areas. Liposomes represent a stable delivery vehicle to
enhance the in
vitro and in vivo stability of the PNAs of the invention. The liposomal
compositions of the
present invention, comprising PNAs of the invention incorporated into
liposomes, can be
formulated as pharmaceutical compositions according to standard techniques
known by the
2 o art-skilled using suitable and acceptable carriers and adjuvants.
Liposomes that may be used include small unilamellar vesicles (SUVs), large
unilamellar vesicles (LUVs) and multilamellar vesicles (MLVs). It has been
shown that
LUVs, which range in size from 0.2-0.4 pm, can encapsulate a substantial
percentage of an
aqueous buffer containing large macromolecules (e.g., RNA, DNA and intact
virions can be
2 5 encapsulated within the aqueous interior and delivered to brain cells in a
biologically active
form: Fraley et al., Trends Biochem. Sci.,1981, 6, 77). The composition of the
liposome is
usually a combination of lipids, particularly phospholipids, in particular,
high phase transition
temperature phospholipids, usually in combination with one or more steroids,
particularly
cholesterol. Examples of lipids useful in liposome production include
phosphatidyl
3 0 compounds, such as phosphatidylglycerol, phosphatidylcholine,
phosphatidylserine,
phosphatidylethanolamine, sphingolipids, cerebrosides and gangliosides.
Particularly useful
are diacyl phosphatidylglycerols, where the lipid moiety contains from 14-18
carbon atoms,
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- 11 -
particularly from 16-18 carbon atoms, and is saturated (lacking double bonds
within the 14-18
carbon atom chain). Illustrative phospholipids include phosphatidylcholine,
dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.
The targeting of liposomes can be either passive or active. Passive targeting
utilizes
the natural tendency of liposomes to distribute to cells of the
reticuloendothelial system in
organs that contain sinusoidal capillaries. Active targeting, by contrast,
involves modification
of the liposome by coupling thereto a specific ligand such as a viral protein
coat (Morishita
et al., Proc. Natl. Acad. Sci. U.S.A., 1993, 90, 8474), monoclonal antibody
(or a suitable
binding portion thereof), sugar, glycolipid or protein (or a suitable
oligopeptide fragment
1 o thereof), or by changing the composition and/or size of the liposome in
order to achieve
distribution to organs and cell types other than the naturally occurring sites
of localization.
The surface of the targeted colloidal dispersion system can be modified in a
variety of ways.
In the case of a liposomal targeted delivery system, lipid groups can be
incorporated into the
lipid bilayer of the liposome in order to maintain the targeting ligand in
close association with
the lipid bilayer. Various linking groups can be used for joining the lipid
chains to the
targeting ligand. The targeting ligand, which binds a specific cell surface
molecule found
predominantly on cells to which delivery of the oligonucleotides of the
invention is desired,
may be, for example, (1 ) a hormone, growth factor or a suitable oligopeptide
fragment thereof
which is bound by a specific cellular receptor predominantly expressed by
cells to which
2 0 delivery is desired; or (2) a polyclonal or monoclonal antibody, or a
suitable fragment thereof
(e.g., Fab; F(ab')z) which specifically binds an antigenic epitope found
predominantly on
targeted cells. Two or more bioactive agents (e.g., a PNA and a conventional
drug, or two
PNAs) can be combined within, and delivered by, a single liposome. It is also
possible to add
agents to colloidal dispersion systems which enhance the intercellular
stability and/or
2 5 targeting of the contents thereof.
The PNAs of the present invention may be used for gene modulation (e.g., gene
targeted drugs}, diagnostics, biotechnology and other research purposes. The
PNAs may also
be used to target RNA and single-stranded DNA (ssDNA) to produce both
antisense-type
gene regulating moieties and as hybridization probes, e.g., for the
identification and
3 o purification of nucleic acids. Furthermore, the PNAs may be modified in
such a way that they
form triple helices with double stranded DNA (dsDNA). Compounds that bind
sequence-
specifically to dsDNA have applications as gene targeted drugs. These
compounds are
CA 02291839 1999-11-26
WO 98153801 PCT/US98/10804
- 12 -
extremely useful drugs for treating various diseases, including cancer,
acquired immune
deficiency syndrome (AIDS) and other virus infections and genetic disorders.
Furthermore,
these compounds may be used in research, diagnostics and for detection and
isolation of
specific nucleic acids.
Gene-targeted drugs are designed with a nucleobase sequence (preferably
containing
10-20 units) complementary to the regulatory region (the promoter) of the
target gene.
Therefore, upon administration, the gene-targeted drugs bind to the promoter
and prevent
RNA polymerase from accessing the promoter. Consequently, no mRNA, and thus no
gene
product (protein), is produced. If the target is within a vital gene for a
virus, no viable virus
1 o particles will be produced. Alternatively, the target region could be
downstream from the
promoter, causing the RNA polymerase to terminate at this position, thus
forming a truncated
mRNA/protein which is nonfunctional.
Synthesis of PNA Oligomers.
The principle of anchoring molecules during a reaction onto a solid matrix is
known
as Solid Phase Synthesis or Merrifield Synthesis. Merrifield, J. Am. Chem.
Soc., 1963, 85,
2149; and Science, 1986, 232, 341. Established methods for the stepwise or
fragment-wise
solid phase assembly of amino acids into peptides normally employ a beaded
matrix of cross
linked styrene-divinylbenzene copolymer. The cross-linked copolymer is formed
by the pearl
polymerization of styrene monomer to which is added a mixture of
divinylbenzenes. Usually,
1-2% cross-linking is employed. Such a matrix may be used in solid phase PNA
synthesis
of the present invention.
More than fifty methods for initial functionalization of the solid phase have
been
described in connection with traditional solid phase peptide synthesis. Barany
and Merrifield
in "The Peptides," Vol. 2, Academic Press, New York, 1979, pp. 1; and Stewart
and Young
2 5 in "Solid Phase Peptide Synthesis," 2nd ed., Pierce Chemical Company,
Illinois, 1984.
Regardless of its nature, the purpose of introducing a functionality on the
solid phase is to
form an anchoring linkage between the copolymer solid support and the C-
terminus of the
first amino acid to be coupled to the solid support. As will be recognized,
anchoring linkages
may also be formed between the solid support and the amino acid N-terminus.
The
3 0 "concentration" of a functional group present in the solid phase is
generally expressed in
millimoles per gram (mmol/g). All of these established methods are, in
principle, useful
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within the context of the present invention.
A preferred method for PNA synthesis employs aminomethyl as the initial
functionality. Aminomethyl is particularly advantageous as a "spacer" or
"handle" group
because it forms amide bonds with a carboxylic acid group in nearly
quantitative amounts.
A vast number of relevant spacer- or handle-forming bifunctional reagents have
been
described. Barany et al., Int. J. Peptide Protein Res., 1987, 30, 705. Certain
functionalities
(e.g., benzhydrylamino, 4-methylbenzhydrylamino and 4-methoxybenzhydrylamino)
which
may be incorporated for the purpose of cleavage of a synthesized PNA chain
from the solid
support such that the C-terminal of the PNA chain is released as an amide,
require no
introduction of a spacer group. Any such functionality may advantageously be
employed in
the context of the present invention.
Exemplary N-protecting groups are tent-butyloxycarbonyl (BOC) (Carpino, J. Am.
Chem. Soc.,1957, 79, 4427; McKay, et al., J. Am. Chem. Soc.,1957, 79, 4686;
and Anderson
et al., J. Am. Chem. Soc.,1957, 79, 6180), 9-fluorenylmethyloxycarbonyl (FMOC)
(Carpino
et al., J. Am. Chem. Soc.,1970, 92, 5748 and J. Org. Chem.,1972, 37, 3404),
Adoc (Hass et
al.,J. Am. Chem. Soc.,1966, 88,1988), Bpoc (SieberHelv. Chem. Acta.,1968, Sl,
614), Mcb
(Brady et al., J. Org. Chem., 1977, 42, 143), Bic (Kemp et al.,
Tetrahedron,1975, 4624), o-
nitrophenylsulfenyl (Nps) (Zervas et al., J. Am. Chem. Soc., 1963, 85, 3660)
and
dithiasuccinoyl (Dts) (Barany et al., J. Am. Chem. Soc., 1977, 99, 7363), as
well as other
2 o groups which are known to those skilled in the art. These amino-protecting
groups,
particularly those based on the widely-used urethane functionality, prohibit
racemization
(mediated by tautomerization of the readily formed oxazolinone (azlactone)
intermediates
(Goodman et al., J. Am. Chem. Soc., 1964, 86, 2918)) during the coupling of
most a-amino
acids. In addition to such amino-protecting groups, nonurethane-type of amino-
protecting
2 5 groups are also applicable when assembling PNA molecules.
The choice of side chain protecting groups, in general, depends on the choice
of the
. amino-protecting group, because the side chain protecting group must
withstand the
conditions of the repeated amino deprotection cycles. This is true whether the
overall strategy
for chemically assembling PrdA molecules relies on, for example, different
acid stability of
3 o amino and side chain protecting groups (such as is the case for the above-
mentioned "BOC-
benzyl" approach) or employs an orthogonal, that is, chemoselective,
protection scheme (such
as is the case for the above-mentioned "FMOC-t-Bu" approach).
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Following coupling of the first amino acid, the next stage of solid phase
synthesis
is the systematic elaboration of the desired PNA chain. This elaboration
involves repeated
deprotection/coupling cycles. A temporary protecting group, such as BOC or
FMOC, on the
last coupled amino acid is quantitatively removed by a suitable treatment, for
example, by
acidolysis, such as with trifluoroacetic acid in the case of BOC, or by base
treatment, such as
with piperidine in the case of FMOC, so as to liberate the N-terminal amine
function.
The next desired N-protected amino acid is then coupled to the N-terminal of
the last
coupled amino acid. This coupling of the C-terminal of an amino acid with the
N-terminal
of the last coupled amino acid can be achieved in several ways. For example,
it can be
achieved by providing the incoming amino acid in a form with the carboxyl
group activated
by any of several methods, including the initial formation of an active ester
derivative such
as a phthalimido ester (Neflcens et al., J. Am. Chem. Soc., 1961, 83, 1263), a
pentafluoro-
phenyl ester {Kovacs et al., J. Am. Ckem. Soc., 1963, 85, 183), an imidazole
ester (Li et al.,
J .. Am. Chem. Soc., 1970, 92, 7608), and a 3-hydroxy-4-oxo-3,4-
dihydroquinazoline (Dhbt-
OH) ester (Konig et al., Chem. Ber., 1973, 103, 2024 and 2034), or the initial
formation of
an anhydride such as a symmetrical anhydride (Wieland et al., Angew. Chem.,
Int. Ed. Engl.,
1971, 10, 336). Alternatively, the carboxyl group of the incoming amino acid
can be reacted
directly with the N-terminal of the last coupled amino acid with the
assistance of a
condensation reagent such as, for example, dicyclohexylcarbodiimide (Sheehan
et al., J. Am.
2 0 Chem. Soc., 1955, 77, 1067) or derivatives thereof. Benzotriazolyl N-oxy-
trisdimethylaminophosphonium hexafluorophosphate (BOP), "Castro's reagent"
(see Rivaille
et al., Tetrahedron, 1980, 36, 3413), is recommended when assembling PNA
molecules
containing secondary amino groups.
Following the assembly of the desired PNA chain, including protecting groups,
the
2 5 next step will normally be deprotection of the amino acid moieties of the
PNA chain and
cleavage of the synthesized PNA from the solid support. These processes can
take place
substantially simultaneously, thereby providing the free PNA molecule in the
desired form.
In the above-mentioned "BOC-benzyl" protection scheme, the final deprotection
of
side chains and release of the PNA molecule from the solid support is most
often carried out
30 by the use of strong acids such as anhydrous HF (Sakakibara et al., Bull.
Chem. Soc. Jpn.,
1965, 38, 4921), boron tris (trifluoroacetate) (Pless et al., Helv. Chim.
Acta,1973, 46, 1609)
and sulfonic acids, such as trifluoromethanesulfonic acid and methanesulfonic
acid (Yajima
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et al., J. Chem. Soc., Chem. Comm., 1974, 107). A strong acid (e.g., anhydrous
HF)
deprotection method may produce very reactive carbocations that may lead to
alkylation and
acylation of sensitive residues in the PNA chain. Such side reactions are only
partly avoided
by the presence of scavengers such as anisole, phenol, dimethyl sulfide, and
mercaptoethanol.
Thus, the sulfide-assisted acidolytic SN2 deprotection method (Tam et al., J.
Am. Chem. Soc.,
1983,105, 6442 and J. Am. Chem. Soc.,1986,108, 5242), the so-called "low"
method, which
removes the precursors of harmful carbocations to form inert sulfonium salts,
is frequently
employed in peptide and PNA synthesis. Other methods for deprotection and/or
final
cleavage of the PNA-solid support bond may include base-catalyzed alcoholysis
(Barton et
al., J. Am. Chem. Soc., 1973, 95, 4501), ammonolysis, hydrazinolysis
(Bodanszky et al.,
Chem. Ind., 1964 1423), hydrogenolysis (Jones, Tetrahedron Lett. 1977 2853 and
Schlatter
et al., Tetrahedron Lett. 1977 2861 )) and photolysis (Rich and Gurwara, J.
Am. Chem. Soc.,
1975 97, 1575)).
Finally, in contrast with the chemical synthesis of conventional peptides,
stepwise
chain building of achiral PNAs such as those based on aminoethylglycyl
backbone units can
start either from the N-terminus or the C-terminus. Those skilled in the art
will recognize that
synthesis commencing at the C-terminus typically employ protected amine groups
and free
or activated acid groups, and syntheses commencing at the N-terminus typically
employ
protected acid groups and free or activated amine groups.
Likely therapeutic and prophylactic targets include herpes simplex virus
(HSV),
human papillomavirus (HPV), human immunodeficiency virus (HIV), candida
albicans,
influenza virus, cytomegalovirus (CMV), intercellular adhesion molecules
(ICAM), 5-
lipoxygenase (5-LO), phospholipase AZ (PLAz), protein kinase C (PKC), and the
ras
oncogene. Potential treatment of such targeting include ocular, labial,
genital, and systemic
herpes simplex I and II infections; genital warts; cervical cancer; common
warts; Kaposi's
sarcoma; AIDS; skin and systemic fungal infections; flu; pneumonia; retinitis
and
pneumonitis in immunosuppressed patients; mononucleosis; ocular, skin and
systemic
inflammation; cardiovascular disease; cancer; asthma; psoriasis;
cardiovascular collapse;
cardiac infarction; gastrointestinal disease; kidney disease; rheumatoid
arthritis; osteoarthritis;
3 0 acute pancreatitis; septic shock; and Crohn's disease.
In general, for therapeutic or prophylactic treatment, a patient suspected of
requiring
such therapy is administered a PNA or liposomal composition of the present
invention,
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commonly in a pharmaceutically acceptable carrier, in amounts and for periods
of time which
will vary depending upon the nature of the particular disease, its severity
and the patient's
overall condition. The PNAs and liposomal compositions of the invention can be
formulated
in a pharmaceutical composition, which may include carriers, thickeners,
diluents, buffers,
preservatives, surface active agents and the like. Pharmaceutical compositions
may also
include one or more active ingredients such as antimicrobial agents, anti-
inflammatory agents,
anesthetics and the like, in addition to the peptide nucleic acids.
The pharmaceutical composition may be administered in a number of ways
depending upon whether local or systemic treatment is desired, and upon the
area to be
treated. Administration may be topical (including ophthalmic, vaginal, rectal,
intranasal,
transdermal), oral or parenteral, for example, by intravenous drip,
subcutaneous,
intraperitoneal or intramuscular injection or intrathecal or intraventricular
administration.
Formulations for topical administration may include transdermal patches,
ointments,
lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
Conventional
pharmaceutical carriers, nucleic acid carriers, aqueous, powder or oily bases,
thickeners and
the like may be necessary or desirable in certain circumstances. Coated
condoms, gloves and
the like may also be useful. Topical administration also includes delivery of
the PNAs and
liposomal compositions of the invention into the epidermis of an animal by
electroporation.
Zewart et al., WO 96/39531, published December 12, 1996.
2 0 Compositions for oral administration include powders or granules,
suspensions or
solutions in aqueous or non-aqueous media, capsules, sachets, or tablets.
Thickeners,
flavorings, diluents, emulsifiers, dispersing aids or binders may be
desirable.
Intravitreal injection, for direct delivery of the PNAs and liposomal
compositions of
the invention to the vitreous humor of the eye of an animal is described in
U.S. Patent
5,595,978, issued January 21, 1997, the contents of which are herein
incorporated by
reference.
Intraluminal administration, for direct delivery of PNAs and liposomal
compositions
of the invention to an isolated portion of a tubular organ or tissue (e.g.,
artery, vein, ureter or
urethra) may be desired for the treatment of patients with diseases or
conditions afflicting the
3 0 lumen of such organs or tissues. To effect this mode of administration, a
catheter or cannula
is surgically introduced by appropriate means. After isolation of the portion
of the tubular
organ or tissue for which treatment is sought, the PNA or liposomal
composition of the
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invention is infused through the catheter or cannula. The infusion catheter or
cannula is then
removed, and flow within the tubular organ or tissue is restored by removal of
the ligatures
which effected the isolation of a segment thereof. Morishita et al., Proc.
Natl. Acad. Sci.,
U.S.A.,1993, 90, 8474.
Intraventricular administration, for direct delivery of PNAs or liposomal
compositions of the invention to the brain of a patient, may be desired for
the treatment of
patients with diseases or conditions afflicting the brain. To effect this mode
of administration,
a silicon catheter is surgically introduced into a ventricle of the brain, and
is connected to a
subcutaneous infusion pump (Medtronic, Inc., Minneapolis, MN) that has been
surgically
implanted in the abdominal region. Zimm et a1, Cancer Research,1984, 44, 1698;
and Shaw,
Cancer, 1993, 72(11 Suppl.), 3416. The pump is used to inject the PNA or
liposomal
composition, and allows precise dosage adjustments and variation in dosage
schedules with
the aid of an exten~al programming device. The reservoir capacity of the pump
is 18-20 mL,
and infusion rates may range from 0.1 mL/hour to 1 mL/hour. Depending on the
frequency
of administration, ranging from daily to monthly, and the dose to be
administered, ranging
from 0.01 p.g to 100 g per kg of body weight, the pump reservoir may be
refilled at 3-10 week
intervals. Refilling of the pump is accomplished by percutaneous puncture of
the self sealing
septum of the pump. Compositions for intraventricular administration may
include sterile
aqueous solutions which may also contain buffers, diluents and other suitable
additives.
2 o Intrathecal administration, for the direct delivery of PNAs or liposomal
compositions
of the invention into the spinal column of a patient, may be desired for the
treatment of
patients with diseases of the central nervous system. To effect this route of
administration,
a silicon catheter is surgically implanted into the L3-4 lumbar spinal
interspace of the patient,
and is connected to a subcutaneous infusion pump which has been surgically
implanted in the
2 5 upper abdominal region. Luer and Hatton, The Annals of
Pharmacotherapy,1993, 2 7, 912;
Ettinger et al., Cancer, 1978, 41, 1270; and Yaida et al., Regul. Pept., 1995,
59, 193. The
pump is used to inject the PNA or liposomal composition, and allows precise
dosage
adjustments and variations in dose schedules with the aid of an external
programming device.
The reservoir capacity of the pump is 18-20 mL, and infusion rates may vary
from 0.1
3 0 mL/hour to 1 mL/hour. Depending on the frequency of administration,
ranging from daily
to monthly, and dosage to be administered, ranging from 0.01 pg to 100 g per
kg of body
weight, the pump reservoir may be refilled at 3-10 week intervals. Refilling
of the pump is
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accomplished by a single percutaneous puncture to the self sealing septum of
the pump.
Compositions for intrathecal administration may include sterile aqueous
solutions which may
also contain buffers, diluents and other suitable additives.
To effect delivery to areas other than the brain or spinal column via this
method, the
silicon catheter may be configured to connect the subcutaneous infusion pump
to, e.g., the
hepatic artery, for delivery to the liver. Kemeny et al., Cancer, 1993, 71,
1964. Infusion
pumps may also be used to effect systemic delivery. Ewel et al., Cancer
Research,1992, 52,
3005; and Rubenstein et al., J. Surg. Oncol.,1996, 62, 194.
Compositions for parenteral, intrathecal or intraventricular administration,
or
liposomal systems, may include sterile aqueous solutions which may also
contain buffers,
diluents and other suitable additives. Dosing is dependent on severity and
responsiveness of
the disease state to be treated, with the course of treatment lasting from
several days to several
months, or until a cure is effected or a diminution of the disease state is
achieved. Optimal
dosing schedules can be calculated from measurements of drug accumulation in
the body of
the patient. Persons of ordinary skill can easily determine optimum dosages,
dosing
methodologies and repetition rates. Optimum dosages may vary depending on the
relative
potency of individual PNAs, and can generally be estimated based on ECSOs
found to be
effective in in vitro and in vivo animal models. In general, dosage is from
0.01 ~g to 100 g
per kg of body weight, and may be given once or more daily, weekly, monthly or
yearly, or
2 o even once every 2 to 20 years.
Synthesis of monomer subunits.
The monomer subunits preferably are synthesized by a general scheme that
commences with the preparation of either the methyl or ethyl ester of (BOC-
aminoethyl)glycine, via a protection/deprotection procedure, as described in
Examples 1 and
2 5 2. The synthesis of thymine monomer is described in Examples 4 and 5, and
the synthesis of
protected cytosine monomer is described in Example 6.
The synthesis of a protected adenine monomer involves alkylation of adenine
with
ethyl bromoacetate (Example 7) and verification of the position of
substitution (i. e. position
9) by X-ray crystallography. The N6-amino group is then protected with the
benzyloxy-
3 0 carbonyl group by the use of the reagent N-ethyl-
benzyloxycarbonylimidazole
tetrafluoroborate (Example 8). Simple hydrolysis of the product ester (Example
9) gave N6-
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benzyloxycarbonyl-9-carboxymethyl adenine (Examples 10 and 11), which was used
in the
standard PNA oligomer synthesis.
For the synthesis of the protected G-monomer, the starting material, 2-amino-6-
chloropurine, was alkylated with bromoacetic acid (Example 12), and the 6-
chloro group was
then substituted with a benzyloxy group (Example 13). The resulting acid was
coupled to the
(BOC-aminoethyl)glycine methyl ester (from Example 2) with agent PyBropTM
being used
as a coupling agent, and the resulting ester was hydrolyzed (Example 14) to
afford the
protected G monomer. The O6-benzyl group was removed in the final HF-cleavage
step
following synthesis of the PNA-oligomer.
l0 Additional objects, advantages, and novel features of the present invention
will
become apparent to those skilled in the art upon examination of the following
examples
thereof. The following examples illustrate the invention and are not intended
to limit the
same. Those skilled in the art will recognize, or be able to ascertain through
routine
experimentation, numerous equivalents to the specific substances,
compositions, and
procedures described herein. Such equivalents are considered to be within the
scope of the
present invention.
General Remarks.
The following abbreviations are used in the experimental examples: DMF, N,N-
dimethylfomnamide; Tyr, tyrosine; Lys, lysine; DCC, N,N-dicyclohexyl-
carbodiimide; DCU,
2 0 N,N-dicyclohexylurea; THF, tetrahydrofuran; aeg, N-acetyl-N-(2'-
aminoethyl)glycine; aek,
N-acetyl-N-(2'- aminoethyl)lysine; Pfp, pentafluorophenyl; BOC, tert-
butoxycarbonyl; Z,
benzyloxycarbonyl; NMR, nuclear magnetic resonance; s, singiet; d, doublet;
dd, doublet of
doublets; t; triplet; q, quartet; m, multiplet; b, broad; b, chemical shift;
ppm, parts per million
(chemical shift).
NMR spectra were recorded on JEOL FX 90Q spectrometer or a Bruker 250 MHz
with tetramethylsilane as an internal standard. Mass spectrometry was
performed on a
MassLab VG 12-250 quadropole instrument fitted with a VG FAB source and probe.
Melting
points were recorded on a Buchi melting point apparatus and are uncorrected.
N,N-
Dimethylformamide was dried over 4 ~ molecular sieves, distilled and stored
over 4 ~
3 0 molecular sieves. Pyridine (HPLC quality) was dried and stored over 4 t~
molecular sieves.
Other solvents used were either the highest quality obtainable or were
distilled prior to use.
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Dioxane was passed through basic alumina prior to use. BOC-anhydride, 4-
nitrophenol,
methyl bromoacetate, benzyloxycarbonyl chloride, pentafluorophenol were all
obtained from
Aldrich Chemical Company. Thymine, cytosine, adenine were all obtained from
Sigma.
Thin layer chromatography (tlc} was performed using the following solvent
systems:
{1) chloroformariethyl amine:methanol, 7:1:2; (2) methylene chloride:methanol,
9:1; (3)
chloroform:methanol:acetic acid 85:10:5. Spots were visualized by UV (254 nm)
and/or
spraying with a ninhydrin solution {3 g ninhydrin in 1000 mL of 1-butanol and
30 mL of
acetic acid), after heating at 120°C for 5 minutes and, after spraying,
heating again.
The carboxyl terminal {C terminus) end of PNA oligomers can be substituted
with
l0 a variety of functional groups. One way this is performed is through the
use of different
resins. The amino terminal (N terminus) end of PNA oligomers can also be
capped with a
carboxylic acid-based capping reagent for the final PNA monomer in the final
coupling step,
or substituted with a variety of conjugate groups. Representative examples of
the types of C
and N terminal groups are shown below.
Resin Employed aeg-PNA/aeg-PNA Derivative Prepared
(Capping Reagent = Acetyl)
Merrifield CH3CONH-(PNA)-COOH
HZN-(PNA)-COOH
Lys Substituted Merifield HZN-(PNA)-Lys-COON
2 o Merrifield HzN-(PNA)-CONH,
Lys Substituted MBHA HZN-(PNA)-Lys-CONHZ
Lys Substituted Merrifield CH3CONH-(PNA)-Lys-COOH
HzN-(PNA)-COON
Lys Substituted Merrif eld HzN-{PNA)-Lys-COOH
2 5 Mernfield HzN-{PNA)-CONHZ
MBHA HEN-(PNA)-CONHz
Lys Substituted MBHA HEN-(PNA)-Lys-CONHZ
MBHA CH3CONH-(PNA)-CONH
H,N-(PNA}-CONHZ
3 o Lys Substituted MBHA CH3CONH-(PNA)-Lys-CONHz
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(Capping Reagent = N-Boc glycine)
Mernfield BocGly-(PNA)-COON
Lys Substituted Merrifield BocGly-(PNA)-Lys-COOH
MBHA BocGly-(PNAj-CONHZ
Lys Substituted MBHA BocGly-(PNA)-Lys-CONHZ
(Capping Reagent = 1. Glycine; 2. Cholic Acid (Chol))
Mernfield Chol-Gly-(PNA)-COOH
Lys Substituted Merrifield Chol-Gly-(PNA)-Lys-COOH
1 o MBHA Chol-Gly-(PNA)-CONHZ
Lys Substituted MBHA Chol-Gly-(PNA)-Lys-CONHZ
Further examples are found in United States Application Serial No. 08/275,951,
filed
July 15, 1994, and incorporated herein by reference.
Example 1
Synthesis of N-benzyloxycarbonyl-N-'(BOC-aminoethyl)glycine.
Aminoethyl glycine (52.86 g, 0.447 moi) was dissolved in water (900 mL) and
dioxane (900 mL) was added. The pH was adjusted to 11.2 with 2N NaOH. While
the pH
was kept at 11.2, tert-butyl p-nitrophenyl carbonate (128.4 g, 0.537 mol) was
dissolved in
dioxane (720 mL) and added dropwise over the course of 2 hours. The pH was
kept at 11.2
2 0 for at least three more hours and then allowed to stand overnight, with
stirring. The yellow
solution was cooled to 0°C and the pH was adjusted to 3.5 with 2 N HCI.
The mixture was
washed with chloroform (4x100 mL), and the pH of the aqueous phase was
readjusted to 9.5
with 2 N NaOH at 0 °C. Benzyloxycarbonyl chloride (73.5 mL, 0.51 S mol)
was added over
half an hour, while the pH was kept at 9.5 with 2 N NaOH. The pH was adjusted
frequently
2 5 over the next 4 hours, and the solution was allowed to stand overnight,
with stirring. On the
following day the solution was washed with ether (3x600 mL) and the pH of the
solution was
afterwards adjusted to 1.5 with 2 N HCl at 0°C. The title compound was
isolated by
extraction with ethyl acetate (5x1000 mL). The ethyl acetate solution was
dried over
magnesium sulfate and evaporated to dryness, in vacuo. This afforded 138 g of
the product,
3 0 which was dissolved in ether (300 mL) and precipitated by the addition of
petroleum ether
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(1800 mL). Yield 124.7 g (79%). M.p. 64.5-85 °C. Anal. for C~~H24NZO6
found(calc.) C:
58.40(57.94); H: 7.02(6.86); N: 7.94(7.95). 'H-NMR (250 MHz, CDC13) 7.33 &
7.32 (5H,
Ph); 5.15 & 5.12 {2H, PhCH2); 4.03 & 4.01 (2H, NCHZCOZH); 3.46 {b, 2H, BOC-
NHCHZCH~; 3.28 (b, 2H, BOC-NHCHZCH~; 1.43 & 1.40 (9H, t-Bu). HPLC (260 nm)
20.71
min. (80.2%) and 21.57 min. (19.8%). The UV-spectra (200 nm - 300 nm) are
identical,
indicating that the minor peak consists of Bis-Z-AEG.
Example 2
Synthesis of N'-BOC-aminoethylglycine esters.
(a) Ethyl ester: N-Benzyloxycarbonyl-N'-(BOC-aminoethyl)glycine (60 g, 0.170
mol) and N,N-dimethyl-4-aminopyridine (6 g) were dissolved in absolute ethanol
(500 mL),
and cooled to 0°C before the addition of DCC (42.2 g, 0.204 mol). The
ice bath was removed
after 5 minutes and stirring was continued for 2 more hours. The precipitated
DCU (32.5 g,
dried) was removed by filtration and washed with ether (3x100 mL). The
combined filtrate
was washed successively with diluted potassium hydrogen sulfate (2x400 mL),
diluted
sodium hydrogencarbonate (2x400 mL) and saturated sodium chloride (1x400 mL).
The
organic phase was filtered, then dried over magnesium sulfate, and evaporated
to dryness, in
vacuo, which yielded 66.1 g of an oily substance which contained some DCU.
The oil was dissolved in absolute ethanol (600 mL) and was added 10% palladium
on carbon (6.6 g) was added. The solution was hydrogenated at atmospheric
pressure. After
2 0 4 hours, 3.3 L was consumed out of the theoretical 4.2 L. The reaction
mixture was filtered
through celite and evaporated to dryness, in vacuo, affording 39.5 g (94%) of
an oily
substance. A 13 g portion of the oily substance was purified by silica gel
(Si02, 600 g)
chromatography. After elution with 300 mL of 20% petroleum ether in methylene
chloride,
the title compound was eluted with 1700 mL of 5% methanol in methylene
chloride. The
2 5 solvent was removed from the fractions, in vacuo, to yield 8.49 g product
of satisfactory
purity. Alternatively 10 g of the crude material was purified by Kugelrohr
distillation. 'H-
NMR (250 MHz, CD30D); 4.77 (b. s, NH); 4.18 (q, 2H, MeCH,-); 3.38 (s, 2H,
NCH~CO,Et);
3.16 (t, 2H, BOC-NHCH~CHZ); 2.68 (t, 2H, BOC-NHCHZCH~); 1.43 (s, 9H, t-Bu} and
I.26
(t, 3H, CH3) '3C-NMR 171.4 (COEt); 156.6 (CO); 78.3 ((CH3)3C); 59.9 (CHI; 49.0
(CHI;
3 0 48.1 (CHZ); 39.0 (CHZ); 26.9 (CH,) and 12.6 (CH3).
(b) Meth 1~: The above procedure for the ethyl ester was used, with methanol
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- 23 -
being substituted for ethanol. The final product was purified by column
chromatography.
Example 3
Alternate Large-scale Synthesis of (N'-BOC-aminoethyl)glycine ethyl ester.
(a) Preparation of BOC-aminoacetaldehyde.
3-Amino-1,2-propanediol (80 g, 0.88 mol) was dissolved in water (1500 mL) and
the
solution was cooled to 4°C, after which BOC-anhydride (230 g, 1.05 mol)
was added in one
portion. The solution was gently heated to room temperature in a water bath.
The pH was
maintained at 10.5 by the dropwise addition of sodium hydroxide. Over the
course of the
reaction, a total of 70.2 g of NaOH, dissolved in 480 rnL of water, was added.
After stirring
overnight, ethyl acetate {1000 mL) was added, the mixture cooled to 0°C
and the pH adjusted
to 2.5 by the addition of 4 M hydrochloric acid. The ethyl acetate layer was
removed and the
acidic aqueous solution was extracted with more ethyl acetate (8x500 mL). The
combined
ethyl acetate solution was reduced to a volume of 1500 mL using a rotary
evaporator. The
resulting solution was washed with half saturated potassium hydrogen sulphate
(1500 mL)
and then with saturated sodium chloride. It then was dried over magnesium
sulphate and
evaporated to dryness, in vacuo. Yield: 145.3 g (86%).
3-BOC-amino-1,2-propanediol (144.7 g, 0.757 mol) was suspended in water (750
mL) and potassium periodate (191.5 g, 0.833 mol) was added. The mixture was
stirred under
nitrogen for 2.5 h and the precipitated potassium iodate was removed by
filtration and washed
2 0 once with water (100 mL). The aqueous phase was extracted with chloroform
{6x400 mL).
The chloroform extracts were dried and evaporated to dryness, in vacuo. Yield:
102 g (93%)
of an oil. BOC-aminoacetaldehyde was purified by kugelrohr distillation at
84°C and 0.3 mm
Hg, in two portions. Yield: 79 g (77%) as a colorless oil.
(b) Preparation of (N'-BOC-aminoethyl)glycine methyl ester.
2 5 Palladium on carbon ( 10%, 2.00 g) was added to a solution of BOC-
aminoacetaldehyde (10 g, 68.9 mmol) in methanol {150 mL) at 0°C. Sodium
acetate (11.3
g, 138 mmol) in methanol (150 mL), and glycine methyl ester hydrochlon~ide
(8.65 g; 68.9
mmol) in methanol (75 mL) were added. The mixture was hydrogenated at
atmospheric
pressure for 2.5 h, then filtered through celite and evaporated to dryness, in
vacuo. The
30 material was redissolved in water (150 mL) and the pH adjusted to 8 with
0.5 N NaOH. The
aqueous solution was extracted with methylene chloride (Sx 150 mL). The
combined extracts
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were dried over sodium sulphate and evaporated to dryness, in vacuo. This
resulted in 14.1
g (88%) yield of (N'-BOC-aminoethyl)glycine methyl ester. The crude material
was purified
by kugelrohr destination at 120°C and 0.5 mm Hg to give 11.3 g (70%) of
a colorless oil. The
product had a purity that was higher than the material produced in example 26
according to
tlc analysis ( 10% methanol in methylene chloride).
Alternatively, sodium cyanoborohydride can be used as reducing agent instead
of
hydrogen (with Pd(C) as catalyst), although the yield (42%) was lower.
(c) Preparation of (N'-BOC-aminoethyl)glycine ethyl ester.
The title compound was prepared by the above procedure with glycine ethyl
ester
1 o hydrochloride substituted for glycine methyl ester hydrochloride. Also,
the solvent used was
ethanol. The yield was 78%.
Example 4
Synthesis of 1-(BOC-aeg)thymine ethyl ester.
N'-BOC-aminoethylglycine ethyl ester (13.5 g, 54.8 mmol), DhbtOH (9.84 g, 60.3
mmol) and 1-carboxymethyl thymine (11.1 g, 60.3 mmol) were dissolved in DMF
(210 mL).
Methylene chloride (210 mL) was added. The solution was cooled to 0°C
in an ethanol/ice
bath and DCC (13.6 g, 65.8 mmol) was added. The ice bath was removed after 1
hour and
stirring was continued for another 2 hours at ambient temperature. The
precipitated DCU was
removed by filtration and washed twice with methylene chloride (2x75 mL). To
the
2 0 combined filtrate was added more methylene chloride (650 mL). The solution
was washed
successively with diluted sodium hydrogen carbonate (3x500 mL), diluted
potassium
hydrogen sulfate (2x500 mL), and saturated sodium chloride ( 1 x500 mL). Some
of the
precipitate was removed from the organic phase by filtration, The organic
phase was dried
over magnesium sulfate and evaporated to dryness, in vacuo. The oily residue
was dissolved
2 5 in methylene chloride ( 1 SO mL), filtered, and the title compound was
precipitated by the
addition of petroleum ether (350 mL} at 0°C. The methylene
chloride/petroleum ether
procedure was repeated once. This afforded 16 g (71 %) of a material which was
more than
99% pure by HPLC.
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Example 5
Synthesis of f-(BOC-aeg)thymine.
- The material from Example 4 was suspended in THF (194 mL, gives a 0.2 M
solution), and 1 M aqueous lithium hydroxide (116 mL) was added. The mixture
was stirred
for 45 minutes at ambient temperature and then filtered to remove residual
DCU. Water (40
mL) was added to the solution which was then washed with methylene chloride
(300 mL).
Additional water (30 mL) was added, and the alkaline solution was washed once
more with
methylene chloride (i50 mL). The aqueous solution was cooled to 0°C and
the pH was
adjusted to 2 by the dropwise addition of 1 N HCl (approx. 110 mL). The title
compound
was extracted with ethyl acetate (9x200 mL), the combined extracts were dried
over
magnesium sulfate and were evaporated to dryness, in vacuo. The residue was
evaporated
once from methanol, which after drying overnight afforded a colorless glassy
solid. Yield:
9.57 g (64 %). HPLC > 98% RT 14.8 minutes. Anal. for C,6H24N40~~0.25 H20 Found
(calc.)
C: 49.29{49.42); H: 6.52(6.35); N: 14.11(14.41). Due to the limited rotation
around the
secondary amide, several of the signals were doubled in the ratio 2:1
(indicated in the list by
mj. for major and mi. for minor). 'H-NMR (250 MHz, DMSO-db) 8: 12.75 (bs, 1H,
CO,H);
11.28 (s, 1H, mj, imide NH); 11.26 (s, 1H, mi, imide NH); 7.30 (s, 1H, mj, T H-
6); 7.26 (s,
1 H, mi, T H-6); 6.92 (bt, 1 H, mj, BOC-NH); 6.73 (bt, 1 H, mi, BOC-NH); 4.64
(s, 2H. mj,
CHZCON); 4.46 (s, 2H, mj, CHZCON); 4.19 (s, 2H, mi, CH~COzH); 3.97 (s, 2H, mj,
2 o CHzCOzH); 3.63-3.01 (unresolved m, includes water, CHzCH2); 1.75 (s, 3H,
CH3) and 1.38
(s, 9H, t-Bu).
Example 6
Synthesis of N'-benzyloxycarbonyl-1-(BOC-aeg)cytosine.
N'-BOC-aminoethyl glycine ethyl ester (5 g, 20.3 mmol), DhbtOH (3.64 g, 22.3
mmol) and N4-benzyloxycarbonyl-1-carboxymethyl cytosine (6.77 g, 22.3 mmol)
were
suspended in DMF ( 100 mL). Methylene chloride ( 100 mL) was added. The
solution was
cooled to 0°C and DCC {5.03 g, 24.4 mmol) was added. The ice bath was
removed after 2
h and stirnng was continued for another hour at ambient temperature. The
reaction mixture
then was evaporated to dryness, in vacuo. The residue was suspended in ether
(100 mL) and
3 0 stirred vigorously for 30 minutes. The solid material was isolated by
filtration and the ether
wash procedure was repeated twice. The material was then stirred vigorously
for 15 minutes
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with dilute sodium hydrogencarbonate (approx. 4% solution, 100 mL), filtered
and washed
with water. This procedure was then repeated once, which after drying left 17
g of yellowish
solid material. The solid was then refluxed with dioxane (200 mL) and filtered
while hot.
After cooling, water {200 mL) was added. The precipitated material was
isolated by filtration,
washed with water, and dried. According to HPLC (observing at 260 nm) this
material has
a purity higher than 99%, besides the DCU. The ester was then suspended in THF
( 100 mL),
cooled to 0°C, and 1 N LiOH (61 mL) was added. After stirring for 15
minutes, the mixture
was filtered and the filtrate was washed with methylene chloride (2x150 mL).
The alkaline
solution then was cooled to 0°C and the pH was adjusted to 2.0 with 1 N
HCI. The title
compound was isolated by filtration and was washed once with water, leaving
11.3 g of a
white powder after drying. The material was suspended in methylene chloride
(300 mL) and
petroleum ether (300 mL) was added. Filtration and wash afforded 7.1 g (69%)
after drying.
HPLC showed a purity of 99% RT 19.5 minutes, and a minor impurity at 12.6
minutes
(approx. 1%) most likely the Z-deprotected monomer. Anal. for C23HZ9N508
found(calc.) C:
54.16(54.87); H: 5.76(5.81 ) and N: 13.65(13.91 ). 'H-NMR (250 MHZ, DMSO-db).
10.78 (bs,
1H, COZH); 7.88 (2 overlapping doublets, 1H, Cyt H-5); 7.41-7.32 (m, SH, Ph);
7.01 (2
overlapping doublets,1H, Cyt H-6); 6.94 & 6.78 (unresolved triplets, 1H, BOC-
NH); 5.19 (s,
2H, PhCHz); 4.81 & 4.62 (s, 2H, CH,CON); 4.17 & 3.98 (s, 2H, CH~COzH); 3.42-
3.03 (m,
includes water, CH CHZ) and 1.38 & 1.37 (s, 9H, '-Bu). '3C-NMR. 150.88;
128.52; 128.18;
127.96; 93.90; 66.53; 49.58 and 28.22. IR: Frequency in cm'. 3423, 3035, 2978,
1736, 1658,
1563, 1501 and 1456.
Example 7
Synthesis of 9-carboxymethyladenine ethyl ester.
Adenine (10 g, 74 mmol) and potassium carbonate (10.29 g, 74 mmol) were
2 5 suspended in DMF and ethyl bromoacetate (8.24 mL, 74 mmol) was added. The
suspension
was stirred for 2.5 h under nitrogen at room temperature and then filtered.
The solid residue
was washed three times with DMF (10 mL). The combined filtrate was evaporated
to
dryness, in vacuo. Water (200 mL) was added to the yellowish-orange solid
material and the
pH adjusted to 6 with 4 N HCI. After stirring at 0°C for 10 minutes,
the solid was filtered off,
3 0 washed with water, and recrystallized from 96% ethanol (150 mL). The title
compound was
isolated by filtration and washed thoroughly with ether. Yield: 3.4 g (20%).
M.p. 215.5-
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220 ° C. Anal. for C9H"N502 found(calc.): C: 48.86(48.65); H: 5.01
(4.91 ); N: 3 I .66(3 I .42).
'H-NMR (250 MHZ; DMSO-db): 7.25 (bs, 2H, NHZ), 5.06 (s, 2H, NCHZ), 4.17 (q,
2H, J=7.1 I
Hz, OCHZ) and 1.21 (t, 3H, J=7.13 Hz, NCHZ). '3C-NMR. 152.70, 141.30, 61.41,
43.97 and
14.07. FAB-MS. 222 (MH+), IR: Frequency in crri '. 3855, 3274, 3246, 3117,
2989, 2940,
2876, 2753, 2346, 2106, 1899, 1762, 1742, 1742, 1671, 1644, 1606, 1582, 1522,
1477, 1445
and 1422. The position of allcylation was verified by X-ray crystallography on
crystals, which
were obtained by recrystallization from 96% ethanol.
Alternatively, 9-carboxymethyladenine ethyl ester can be prepared by the
following
procedure. To a suspension of adenine (50 g, 0.37 mol) in DMF (1100 mL) in 2 L
three-
necked flask equipped with a nitrogen inlet, a mechanical stirrer and a
dropping funnel, was
added 16.4 g (0.407 mol) of hexane-washed sodium hydride-mineral oil
dispersion. The
mixture was stirred vigorously for 2 hours, after which ethyl bromoacetate (75
mL, 0.67 mol)
was added dropwise over the course of 3 hours. The mixture was stirred for one
additional
hour, after which tlc indicated complete conversion of adenine. The mixture
was evaporated
to dryness at 1 mm Hg and water (500 mL) was added to the oily residue which
caused
crystallization of the title compound. The solid was recrystallised from 96%
ethanol (600
mL). Yield (after drying): 53.7 g (65.6%). HPLC (215 nm) purity > 99.5%.
Example 8
Synthesis of N~'benzyloxycarbonyl-9-carboxymethyiadenine ethyl ester.
2 0 9-Carboxymethyiadenine ethyl ester (3.4 g, 15.4 mmol) was dissolved in dry
DMF
(SO mL) by gentle heating, cooled to 20°C, and added to a solution of N-
ethyl-ben-
zyloxycarbonylimidazole tetrafluoroborate (62 mmol) in methylene chloride (50
mL) over a
period of 15 minutes in an ice bath. Some precipitation was observed. The ice
bath was
removed and the solution was stirred overnight. The reaction mixture was
treated with
2 5 saturated sodium hydrogen carbonate ( 100 mL). After stirring for 10
minutes, the phases
were separated and the organic phase was washed successively with one volume
of water,
dilute potassium hydrogen sulfate (twice), and with saturated sodium chloride.
The solution
was dried over magnesium sulfate and evaporated to dryness, in vacuo, which
afforded 11 g
of an oily material. The material was dissolved in methylene chloride (25 mL),
cooled to
3 0 0°C, and precipitated with petroleum ether (50 mL). This procedure
was repeated once to
give 3.45 g (63%) of the title compound. M.p. 132-35°C. Analysis for
C"H"N504 found
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(calc.): C: 56.95{57.46); H: 4.71(4.82); N: 19.35(19.71). 'H-NMR (250 MHZ;
CDC13): 8.77
(s, 1H, H-2 or H-8); 7.99 (s, 1H, H-2 or H-8); 7.45-7.26 (m, 5H, Ph); 5.31 (s,
2H, N-CHZ);
4.96 (s, 2H, Ph-CHZ); 4.27 (q, 2H, J=7.15 Hz, CHZCH3) and 1.30 (t, 3H, J=7.15
Hz, CHzCH ).
'3C-NMR: 153.09; 143.11; 128.66; 67.84; 62.51; 44.24 and 14.09. FAB-MS: 356
(MH+) and
312 (MH+-COZ). IR: frequency in cm': 3423; 3182; 3115; 3031; 298I; 1747; 1617;
15.87;
1552; 1511; 1492; 1465 and 1413.
Example 9
Synthesis of N~benzyloxycarbonyl-9-carboxymethyladenine.
N6-Benzyloxycarbonyl-9-carboxymethyladenine ethyl ester (3.2 g, 9.01 mmol) was
to mixed with methanol (50 mL) cooled to 0°C. Sodium hydroxide solution
(2 N, 50 mL) was
added, whereby the material quickly dissolved. After 30 minutes at 0 °
C, the alkaline solution
was washed with methylene chloride (2x50 mL). The pH of the aqueous solution
was
adjusted to 1 with 4 N HCl at 0 °C, whereby the title compound
precipitated. The yield after
filtration, washing with water, and drying was 3.08 g (104%). The product
contained salt, and
the elemental analysis reflected that. Anal. for C,SH,3N504 found(calc.): C:
46.32(55.05); H:
4.24(4.00); N: 18.10(21.40) and C/N: 2.57(2.56). 'H-NMR(250 MHZ; DMSO-db):
8.70 (s,
2H, H-2 and H-8); 7.50-7.35 (m, 5H, Ph); 5.27 (s, 2H, N-CHZ); and 5.15 (s, 2H,
Ph-CHz).
'3C-NMR. 168.77, 152.54, 151.36,148.75,145.13, 128.51, 128.17,127.98, 66.76
and 44.67.IR
(KBr) 3484; 3109; 3087; 2966; 2927; 2383; 1960;1739;1688; 1655; 1594; 1560;
1530; 1499;
1475; 1455; 1429 and 1411. FAB-MS: 328 (MH+) and 284 (MH+-COz). HPLC (2I5 nm,
260 nm) in system 1: 15.18 min, minor impurities all less than 2%.
Example 10
Synthesis of N6-benzyloxycarbonyl-1-(BOC-aeg)adenine ethyl ester.
N'-BOC-aminoethylglycine ethyl ester (2 g, 8.12 mmol), DhbtOH (1.46 g, 8.93
2 5 mmol) and N6-benzyloxycarbonyl-9-carboxymethyl adenine (2.92 g, 8.93 mmol)
were
dissolved in DMF (15 mL). Methylene chloride (15 mL) was then added. The
solution was
cooled to 0°C in an ethanol/ice bath. DCC (2.01 g, 9.74 mmol) was
added. The ice bath was
removed after 2.5 h and stirring was continued for another 1.5 hour at ambient
temperature.
The precipitated DCU was removed by filtration and washed once with DMF (15
mL), and
twice with methylene chloride (2x15 mL). To the combined filtrate was added
more
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methylene chloride (100 mL). The solution was washed successively with dilute
sodium
hydrogen carbonate (2x100 mL), dilute potassium hydrogen sulfate (2x100 mL),
and saturated
sodium chloride ( 1 x 100 mL). The organic phase was evaporated to dryness, in
vacuo, which
afforded 3.28 g (73%) of a yellowish oily substance. HPLC of the raw product
showed a
purity of only 66% with several impurities, both more and less polar than the
main peak. The
oil was dissolved in absolute ethanol (SO mL) and activated carbon was added.
After stirring
for 5 minutes, the solution was filtered. The filtrate was mixed with water
(30 mL) and was
allowed to stir overnight. The next day, the white precipitate was removed by
filtration,
washed with water, and dried, affording 1.16 g (26%) of a material with a
purity higher than
98% by HPLC. Addition of water to the mother liquor afforded another 0.53 g of
the product
with a purity of approx. 95%. Anal. for CZ6H33N,O,oH,O found(calc.) C: 55.01
(54.44; H:
6.85(6.15) and N: 16.47(17.09). 'H-NMR (250 MHZ, CDC13) 8.74 (s, 1H, Ade H-2);
8.18 (b.
s, 1H, ZNH); 8.10 & 8.04 (s, 1H, H-8); 7.46-7.34 (m, SH, Ph); 5.63 (unres. t,
1H, BOC-NH);
5.30 (s, 2H, PhCH2); 5.16 & 5.00 (s, 2H, CHZCON); 4.29 & 4.06 (s, 2H,
CHZCOZH); 4.20 (q,
2H, OCHzCH3); 3.67-3.29 (m, 4H, CH,CH~; 1.42 (s, 9H, t-Bu) and 1.27 (t, 3H,
OCHZCH3).
The spectrum shows traces of ethanol and DCU.
Example 11
Synthesis of Nb-benzyloxycarbonyl-1-(BOC-aeg)adenine.
N6-Benzyloxycarbonyl-1-(BOC-aeg)adenine ethyl ester (1.48 g, 2.66 mmol) was
suspended in THF (13 mL) and the mixture was cooled to 0°C. Lithium
hydroxide (8 mL,
1 N) was added. After 15 minutes of stirring, the reaction mixture was
filtered, extra water
(25 mL) was added, and the solution was washed with methylene chloride (2x25
mL). The
pH of the aqueous solution was adjusted to 2 with 1 N HCI. The precipitate was
isolated by
filtration, washed with water, and dried, affording 0.82 g (58%) of the
product. The product
2 5 was additionally precipitated twice from methylene chloride/petroleum
ether. Yield (after
drying): 0.77 g (55%). M.p. 119°C (decomp.). Anal. for C24H~9N,O~oH20
found(calc.) C:
53.32(52.84); H: 5.71(5.73); N: 17.68(17.97). FAB-MS. 528.5 (MH+). 'H-NMR (250
MHZ,
DMSO-db). 12.75 (very b, 1H, CO,H); 10.65 (bs, 1H, ZNH); 8.59 (d, 1H, J= 2.14
Hz, Ade
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H-2); 8.31 (s, 1 H, Ade H-8); 7.49-7.31 (m, SH, Ph); 7.03 & 6.75 (unresol. t,
1 H, BOC-NH);
5.33 & 5.16 (s, 2H, CH2CON); 5.22 (s, 2H, PhCH2); 4.34-3.99 (s, 2H, CHZCOZH);
3.54-3.03
(multiplets, includes water, CHZCHZ) and 1.39 & 1.37 (s, 9H, t-Bu). '3C-NMR.
170.4; 166.6;
152.3; 151.5; 149.5; 145.2; 128.5; 128.0; 127.9; 66.32; 47.63; 47.03; 43.87
and 28.24.
Example 12
Synthesis of 2-amino-6-chloro-9-carboxymethylpurine.
To a suspension of 2-amino-6-chloropurine (5.02 g, 29.6 mmol) and potassium
carbonate (12.91 g, 93.5 mmol) in DMF (50 mL) was added bromoacetic acid (4.7
g, 22.8
mmol). The mixture was stirred vigorously for 20 h under nitrogen. Water (150
mL) was
l0 added and the solution was filtered through celite to give a clear yellow
solution. The
solution was acidified to a pH of 3 with 4 N hydrochloric acid. The
precipitate was filtered
and dried, in vacuo, over sicapent. Yield: 3.02 g (44.8%). 'H-NMR(DMSO-db) b:
4.88 ppm
(s, 2H); 6.95 (s, 2H); 8.10 (s, 1 H).
Example 13
Synthesis of 2-amino-6-benzyloxy-9-carboxymethylpurine.
Sodium (2 g 87 mmol) was dissolved in benzyl alcohol (20 mL) and heated to
130°C
for 2 h. After cooling to 0 ° C, a solution of 2-amino-6-chloro-9-
carboxymethylpurine {4.05
g, 18 mmol) in DMF (85 mL) was slowly added, and the resulting suspension
stirred
overnight at 20°C. Sodium hydroxide solution (1 N, 100 mL) was added
and the clear
2 0 solution was washed with ethyl acetate (3x100 mL). The water phase was
then acidified to
a pH of 3 with 4 N hydrochloric acid. The precipitate was taken up in ethyl
acetate (200 mL),
and the water phase was extracted with ethyl acetate (2x 100 mL). The combined
organic
phases were washed with saturated sodium chloride solution (2x75 mL), dried
with anhydrous
sodium sulfate, and evaporated to dryness, in vacuo. The residue was
recrystallized from
2 5 ethanol (300 mL). Yield, after drying in vacuo, over sicapent: 2.76 g
(52%). M.p. 159-65 °C.
Anal. (calc.; found): C{56.18; 55.97), H(4.38; 4.32), N{23.4; 23.10). 'H-NMR
(DMSO-db)
8: 4.82 (s, 2H); 5.51 (s, 2H); 6.45 (s, 2H); 7.45 (m, SH); 7.82 (s, 1 H).
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Example 14
Synthesis of N-([2-amino-6-benzyloxy-purine-9-yl]-acetyl)-N-(2-BOC-
aminoethyl)glycine
(BOC-Gaeg-OH monomer].
2-Amino-6-benzyloxy-9-carboxyrnethyl-purine (0.5 g, 1.67 mmol), methyl-N(2-
[tert-butoxycarbonylamino]ethyl)glycinate (0.65 g, 2.8 mmol), diisopropylethyl
amine (0.54
g, 4.19 mmol), and bromo-tris-pyrrolidino-phosphonium-hexafluoro-phosphate
(PyBroP~)
(0.798 g, 1.71 mmol) were stirred in DMF (2 mL) for 4 h. The clear solution
was poured into
an ice-cooled solution of sodium hydrogen carbonate ( 1 N, 40 mL) and
extracted with ethyl
acetate (3x40 mL). The organic layer was washed with potassium hydrogen
sulfate solution
l0 (1 N, 2x40 mL), sodium hydrogen carbonate (1 N, 1x40 mL) and saturated
sodium chloride
solution (60 mL). After drying with anhydrous sodium sulfate and evaporation
in vacuo, the
solid residue was recrystallized from 2:1 ethyl acetate/hexane (20 mL) to give
the methyl ester
in 63% yield. (MS-FAB 514 (M+1 ). Hydrolysis was accomplished by dissolving
the ester
in 1:2 ethanol/water (30 mL) containing concentrated sodium hydroxide (1 mL).
After
stirring for 2 h, the solution was filtered and acidified to a pH of 3, by the
addition of 4 N
hydrochloric acid. The title compound was obtained by filtration. Yield: 370
mg (72% for
the hydrolysis). Purity by HPLC was more than 99%. Due to the limited rotation
around the
secondary amide several of the signals were doubled in the ratio 2:1
(indicated in the list by
mj for major and mi for minor). 'H-NMR{250, MHZ, DMSO-db) 8: 1.4 (s, 9H); 3.2
(m, 2H);
2 0 3.6 (m, 2H); 4.1 (s, mj, CONRCH,COOH); 4.4 {s, mi, CONRCHZCOOH); 5.0 (s,
mi, Gua-
CH,CO-); 5.2 (s, mj, Gua-CHZCO); 5.6 (s, 2H); 6.5 (s, 2H); 6.9 (m, mi, BOC-
NH); 7.1 (m,
mj, BOC-NH); 7.5 (m, 3H); 7.8 (s, 1 H); 12,8 (s, 1 H). "C-NMR. 170.95; 170.52;
167.29;
166.85; 160.03; 159.78; 155.84; 154.87; 140.63; 136.76; 128.49; 128.10;
113.04; 78.19;
77.86; 66.95; 49.22; 47.70; 46.94; 45.96; 43.62; 43.31 and 28.25.
2 5 Example 15
Synthesis of ethyl-N6-(benzyloxycarbonyl)-2,6-diaminopurin-9-yl-acetate.
To a suspension of 2,6-diaminopurine (3 g, 19.46 mmol) in dry DMF (90 mL) was
added NaH (60% in oil, 0.87 g, 21.75 mmol). After 1 hour ethyl bromoacetate
(4.23 g, 25.34
mmol) was added. The reaction mixture became homogenous in 30 minutes and was
allowed
3 0 to stir for an additional 90 minutes. The DMF was removed in vacuo
resulting in a tan
powder. The tan powder was then refluxed with 1,4-dioxane (200 mL) for 10
minutes and
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filtered through celite. The solution was concentrated to give a light yellow
powder. To the
light yellow powder (5.52 g) in 1,4-dioxane (150 mL) was added freshly
prepared N-
benzyloxycarbonyl-N'-methylimidazolium triflate (10.7 g, 29.2 mmol). The
reaction mixture
was stirred at room temperature for 16 h resulting in a reddish solution. The
dioxane was
removed in vacuo and the crude material was recrystallized from MeOH:diethyl
ether to give
4.56 g (63%) of the title compound as a cream-colored solid.
'H NMR (DMSO-db) 8: 10.12 (bs, 1H), 7.43 (m, SH), 6.40 (bs, 2H), 5.17 (s, 2H),
4.94 (s, 2H), 4.18 (q, J = 7.2, 3H), 1.21 (t, J = 7.2, 3H). '3C NMR (DMSO-db)
ppm: 167.81,
159.85,154.09, 152.07,149.77,140.62,136.42, 128.22, 127.74, 127.61, 166.71.
65.87, 61.21,
43.51, 13.91.
Example 16
Synthesis of N6-(benzyloxycarbonyl)-2,6-diaminopurin-9-yl-acetic acid.
Ethyl-N6-(benzyloxycarbonyl)-2,6-diaminopurin-9-yl-acetate (3 g, 8.1 mmol) was
dissolved in NaOH (2 N, 30 mL). After 1 h the solution was acidified to pH 2.5
with 2 M
HCI. The precipitate was filtered, washed with water, and dried to give 2.82 g
(98%) of the
title compound as a white solid.
IR (KBr): 3300, 3095, 1750, 1630, 1590, 1410. 'H NMR (DMSO-db) S: 10.11 (s,
1 H), 7.91 (s, 1 H), 7.45-7.33 (m, SH), 6.40 (s, 2H), 5.17 {s, 2H), 4.83 (s,
2H).
Example 17
2 0 Synthesis of BOC-aminoacetaldehyde.
The title compound was prepared according to a published literature procedure
(Dueholm et al., Organic Preparations and Procedures Intl., 1993, 25, 457).
Example 18
Synthesis of E-(2-chlorobenzyloxycarbonyl)-lysine allyl ester.
2 5 The title compound was prepared according to a published literature
procedure
(Waldmann and Horst, Liebigs Ann. Chem, 1983, 1712).
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Example 19
Synthesis of N-(BOC-aminoethyl)-s-(2-chlorobenzyloxycarbonyl)-lysine allyl
ester.
E-{2-chlorobenzyloxycarbonyl)-lysine allyl ester (from example 18) was
dissolved
in methanol (50 mL) and cooled to 0°C. To the resulting solution was
added sodium
cyanoborohydride (5.9 mmol) followed by acetic acid (0.75 mL). After 5 minutes
BOC-
aminoacetaldehyde (13.3 mmol) was added and the reaction mixture was stirred
for an
additional 1 h. The methanol was removed in vacuo and the oil was dissolved in
ethyl acetate
(40 mL), washed with saturated aqueous NaHC03, brine, dried over NazS04 and
concentrated
to give a clear colorless oil. This oil was dissolved in dry ether (80 mL),
cooled to -20°C, and
a molar equivalent of HCl in ether was added slowly. The resulting white solid
was collected
by filtration and air dried. Precipitation of the air-dried white solid from
dry ether gave
analytically pure title compound.
Example 20
Synthesis of N-(BOC-aminoethyl)-N-(N6-(benzyloxycarbonyl)-2,6-diaminopurin-9-
yl-
acetyl]-s-(2-chlorobenzyloxycarbonyl)-lysine allyl ester.
To N6-(benzyloxycarbonyl)-2,6-diaminopurin-9-yl-acetic acid (3.6 g, 10.5 mmol)
in DMF (150 mL) was added N,N-diisopropylethylamine (2.75 mL, 2I mmole), and N-
(BOC-
aminoethyl)-s-(2-chlorobenzyloxycarbonyl)-lysine allyl ester hydrochloride
(7.31 gm, 15.8
mmol). The reaction mixture was stirred under nitrogen for 20 minutes and
bromo-tris-
2 0 pyrrolidino-phosphonium hexafluorophosphate (PyBrop, 5.4 gm, 11.6 mmol)
was added. The
reaction mixture was stirred overnight at room temperature under an atmosphere
of nitrogen
gas. The resulting mixture was concentrated and dissolved in ethyl acetate.
The ethyl acetate
solution was washed with aqueous saturated sodium bicarbonate, separated and
concentrated.
The crude material was purified by silica gel flash column chromatography
using ethyl
acetate:hexane: methanol (6:3:1, v/v/v), as the eluent. Concentration and
drying of the
appropriate fractions gave 3.1 g (37%) of the title compound.
'Example 21
Synthesis of N-(BOC-aminoethyl)-N-[N6-(benzyloxycarbonyl)-2,6-diaminopurin-9-
yl-
acetyl)-E-(2-chlorobenzyloxycarbonyl)-lysine.
3 0 To N-(BOC-aminoethyl~N-[N6-(benzyloxycarbonyl)-2,6-diaminopurin-9-yl-
acetyl]-
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s-(2-chlorobenzyloxycarbonyl)-lysine allyl ester hydrochloride (3.1 gm, 3.93
mmol) was
added THF (100 mL) morpholine (3.5 mL, 39.3 mmol), and
tetrakis(triphenylphosphine)-
palladium(0) (0.45 gm, 0.393 mmol). The reaction mixture was stirred under an
atmosphere
of nitrogen for 2.5 h at room temperature. The resulting mixture was
concentrated and
dissolved in ethyl acetate. The ethyl acetate solution was washed with aqueous
saturated
potassium hydrogen sulfate (that was half diluted with water), separated and
concentrated.
The crude material was purified by silica gel flash column chromatography
using
chloroform:methanol (9:1, v/v), as the eluent. Concentration and drying of the
appropriate
fi~actions gave 1.25 g {42%) of the title compound.
Example 22
Preparation of Guanine Monomer (BOC-Gaek-OH).
To N6-benzyl-9-carboxymethylene-guanine (2.63 g, 8.78 mmol) was added DIEA
(2.6 mL, 20 mmol), DMF (30 mL), dichloromethane (70 mL), and N-(BOC-
aminoethyl)-s-(2-
chlorobenzyloxycarbonyl)-lysine allyl ester (3.7 g, 8.04 mmol). The reaction
mixture was
stirred under nitrogen for 20 minutes. PyBrop (4 g, 8.58 mmol) was added and
the reaction
mixture stirred for an additional 16 h. The reaction mixture was concentrated
and the residue
was purified by silica gel flash column chromatography using chloroform/
hexanes/methanol
(12:7:1, v/v/v) to give 4 g (60%) of the title compound as the allyl ester.
To the allyl ester (4 g, 5.37 mmol) was added THF (100 mL),
tetrakispalladium(0)
2 0 (0.18 g, 0.15 mmol), and morpholine (6.1 mL, 70 mmol). The reaction
mixture was stirred
under nitrogen for 2.5 h and concentrated. The residue was purified by silica
gel flash column
chromatography using chloroforn~/hexanes/methanol (11:8:1, v/v/v) to give 2.67
g (60%) of
the title compound.
Example 23
Preparation of Adenine Monomer (BOC-Aaek-OH).
The procedure used for the guanine monomer in Example 22 above was followed
for
the synthesis of the adenine monomer using N6-benzyl-9-carboxymethylene-
adenine.
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Example 24
Preparation of Cytosine Monomer (BOC-Caek-OH).
To N-(BOC-aminoethyl)-s-(2-chlorobenzyloxycarbonyl)-lysine allyl ester (8.21
g,
17.7 mmol), were added triethylamine (10 mL, 98 mmol) and dichloromethane (200
mL).
The solution was cooled to about 0°C in an ice bath under nitrogen. To
the cooled solution
was added chloroacetyl chloride (2.2 mL, 27.6 mmol) over 10 minutes and the
reaction
mixture stirred at room temperature for 16 h. The reaction mixture was
concentrated and 'the
residue was purified by silica gel flash column chromatography using ethyl
acetate/ hexanes
(1:1, v/v) to give 6.54 g (68%) of the N-acetylated lysine backbone.
1 o Cytosine was protected at the N4- position by treatment with benzyl
chloroformate
in pyridine at 0°C to give N4-benzyl-cytosine.
To N4-benzyl-cytosine ( 1.31 g, 5.34 mmoI) was added DMF (200 mL), and 60%
NaH in mineral oil (0.22 g, 5.4 mmol) and the resulting mixture was stirred
under nitrogen
for 30 minutes. To the resulting mixture was added the N-acetylated lysine
backbone (2.9 g,
5.34 mmol) in DMF (25 mL) and the mixture stirred for 16 h. The reaction
mixture was
concentrated and the residue dissolved in dichloromethane (250 mL). The
dichloromethane
phase was washed with water (200 mL) and concentrated. The resulting residue
was purified
by silica gel flash column chromatography using dichloromethane:hexanes:
methanol (8:2:1 )
to give 2.4 g (85%) of the cytosine attached to the aminoethyl-lysine backbone
as the allyl
2 0 ester.
The allyl ester is converted to the active monomer by deprotection using
palladiun
following the procedure used in Example 22 above to give 1.05 g (46%) of the
title
compound.
Example 25
2 5 Preparation of Tbymine Monomer (BOC-Taek-OH).
The thymine monomer was prepared following the procedure of Example 24 above.
Example 26
Solid Phase Synthesis of H-Taeg-Aaeg-[Taeg]8-Lys-NHZ.
(a) Stepwise Assembly of BOC-Taeg-A(Z)aeg-[Taeg]8-Lys(CIZ)-MBHA Resin.
3 0 About 0.3 g of wet BOC-[Taeg)g-Lys(C1Z)-MBHA resin was placed in a 3 mL
SPPS
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reaction vessel. BOC-Taeg-A(Z)aeg-[Taeg]8-Lys(C1Z)-MBHA resin was assembled by
in situ
DCC coupling (single) of the A(Z)aeg residue utilizing 0.19 M of BOC-A(Z)aeg-
OH together
with 0.15 M DCC in 2.5 mL of 50% DMF/CHzCl2 and a single coupling with 0.15 M
BOC-
Taeg-OPfp in neat CHZCl2 ("Synthetic Protocol I"). The synthesis was monitored
by the
quantitative ninhydrin reaction, which showed about 50% incorporation of
A(Z)aeg and about
96% incorporation of Taeg.
(b) Cleavage, Purification, and Identification of H-Taeg-Aaeg-[Taeg]8-Lys-NHZ.
The protected BOC-Taeg-A(Z)aeg-[Taeg]8-Lys(C1Z)-BHA resin was treated with
50% trifluoroacetic acid in methylene chloride to remove the N-terminal BOC
group (which
is a precursor of the potentially harmful tert-butyl cation) prior to the HF
cleavage. Following
neutralization and washing (performed in a way similar to those of steps 2-4
in "Synthetic
Protocol 1 "), and drying for 2 h in vacuum, the resulting 53.1 mg of H-
[Taeg]5-BHA resin was
cleaved with 5 mL of HF:anisoie (9:1, v/v) while stirring at 0°C for 60
minutes. After
removal of HF, the residue was stirred with dry diethyl ether (4x15 mL, 15
minutes each) to
remove anisole, filtered under gravity through a fritted glass funnel, and
dried. The PNA was
then extracted into a 60 mL (4x15 mL, stirnng 1 S minutes each) 10% aqueous
acetic acid
solution. Aliquots of this solution were analyzed by analytical reverse-phase
HPLC to
establish the purity of the crude PNA. The main peak at 13 minutes accounted
for about 93%
of the total absorbance. The remaining solution was frozen and lyophilized to
afford about
2 o 15.6 mg of crude material. The main peak at 14.4 minutes accounted for
less than 50% of the
total absorbance. A 0.5 mg portion of the crude product was purified to give
approximately
0.1 mg of H-Taeg-Aaeg-[Taeg]g-Lys-NHz. For (MH+)+ the calculated m/z value was
2816.16
and the measured m/z value was 2816.28.
(c) Synthetic Protocol I.
2 5 ( 1 ) BOC-deprotection with TFA/CHZCI ~ ( 1:1, v/v), 2.5 mL, 3x 1 minute
and 1 x30
minutes; (2) washing with CHZCIz, 2.5 mL, 6x1 minute; (3) neutralization with
DIEA/CHZCIz
( 1: 19, v/v), 2.5 mL, 3x2 minutes; (4) washing with CH~CIZ, 2.5 mL, 6x 1
minute, and drain
for 1 minute; (5) 2-5 mg sample of PNA-resin was removed and dried thoroughly
for a
quantitative ninhydrin analysis to determine the substitution; (6) addition of
0.47 mmol (0.25
3 o g) BOC-A(Z)aeg-OH dissolved in 1.25 mL of DMF followed by addition of 0.47
mmol (0.1
g) DCC in 1.25 mL of CHZCh or 0.36 mmol (0.2 g) BOC-Taeg-OPfp in 2.5 mL of
CHzCl2;
the coupling reaction was allowed to proceed for a total of 20-24 h while
shaking; (7) washing
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with DMF, 2.5 mL, 1x2 minutes; (8) washing with CHzCl3 2.5 mL, 4x1 minute; {9)
neutralization with DIEA/CHZCh (1: 19, v/v), 2.5 mL, 2x2 minutes; (10) washing
with
CHZCIz, 2.5 mL, 6x1 minute; (11) 2-5 mg sample ofprotected PNA-resin was
removed and
dried thoroughly for a quantitative ninhydrin analysis to determine the extent
of coupling;
(12) blocking of unreacted amino groups by acetylation with a 25 mL mixture of
acetic
anhydride/pyridine/CHZC12 ( 1:1:2, v/v/v) for 2 h (except after the last
cycle); and ( I 3 ) washing
with CHZC12, 2.5 mL, 6 x 1 minute; ( 14) 2x2-5 mg samples of protected PNA-
resin are
removed, neutralized with DIEA/CHZCIz (1: 19, v/v) and washed with CHzCl2 for
ninhydrin
analyses.
l0 Example 27
Solid Phase Synthesis of H-[Taeg]i-Aaeg-(Taeg]s-Lys-NHZ.
(a) Stepwise Assembly of BOC-[Taeg]2-A(Z)aeg-[Taeg]5-Lys(C1Z)-MBHA Resin.
About 0.5 g of wet BOC-[TaegJs-Lys(CIZ)-MBHA resin was placed in a 5 mL SPPS
reaction vessel. BOC-[Taeg]2-A(Z)aeg-[Taeg]5-Lys(CIZ)-MBHA resin was assembled
by in
situ DCC coupling of both the A(Z)aeg and the Taeg residues utilising 0.1 S M
to 0.2 M of
protected PNA monomer (free acid) together with an equivalent amount of DCC in
2 mL neat
CHZC12 ("Synthetic Protocol II"). The synthesis was monitored by the
quantitative ninhydrin
reaction which showed a total of about 82% incorporation of A(Z)aeg after
coupling three
times {the first coupling gave about 50% incorporation; a fourth HOBt-mediated
coupling in
2 0 50% DMF/CHZCIz did not increase the total coupling yield significantly)
and quantitative
incorporation (single couplings) of the Taeg residues.
(b) Cleavage, Purification, and Identification of H-[Taeg]Z-Aaeg-[Taeg]$-Lys-
NH2.
The protected BOC-[Taeg]2-A(Z)aeg-[Taeg] sLys(C1Z~BHA resin was treated as
described in Example 26(b) to yield about 16.2 mg of crude material upon HF
cleavage of
2 5 102.5 mg dry H-[Taeg]~-A(Z)aeg-[Taeg]5-Lys(CIZ)-BHA resin. A small portion
of the crude
product was purified. For (MH+)', the calculated m/z value was 2050.85 and the
measured
m/z value was 2050.90
(c) Synthetic Protocol I1.
(1) BOC-deprotection with TFA/CHZCIz (1:1, v/v), 2 mL, 3x1 minute and 1x30
3 o minutes; (2) washing with CHZCI,, 2 mL, 6x1 minute; (3) neutralization
with DIEA/CH,Ch
(1: 19, v/v), 2 mL, 3x2 minutes; (4) washing with CHZC12, 2 mL, 6x1 minute,
and drain for
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_ _
1 minute; (5) 2-5 mg sample of PNA-resin was removed and dried thoroughly for
a
quantitative ninhydrin analysis to determine the substitution; (6) addition of
0.44 mmol (0.23
g) BOC-A(Z~eg-OH dissolved in 1.5 mL of CHZC12 followed by addition of 0.44
mmol (0.09
g) DCC in 0.5 mL of CHZC12 or 0.33 mmol (0.13 g) BOC-Taeg-OH in 1.5 mL of CH
X12
followed by addition of 0.33 mmol (0.07 g) DCC in 0.5 mL of CHzCl2;; the
coupling reaction
was allowed to proceed for a total of 20-24 h with shaking; (7) washing with
DMF, 2 mL, 1 x2
minutes; (8) washing with CHZCIz, 2 mL, 4x 1 minute; (9) neutralization with
DIEA/CH,C12
( 1: 19, v/v), 2 mL, 2x2 minutes; ( 10) washing with CHZC12, 2 mL, 6x 1
minute; ( 11 ) 2-5 mg
sample of protected PNA-resin was removed and dried thoroughly for a
quantitative ninhydrin
analysis to determine the extent of coupling; (12) blocking of unreacted amino
groups by
acetylation with a 25 mL mixture of acetic anhydride/pyridine/CHzCl2 (l:l :2,
v/v/v) for 2 h
(except after the last cycle); (13) washing with CHZC12, 2 mL, 6x1 minute; and
(14) 2x2-5 mg
samples of protected PNA-resin were removed, neutralized with DIEA/CHZCI2 (1:
19, v/v)
and washed with CHZCh for ninhydrin analyses.
Example 28
Standard Protocol For PNA Synthesis and Characterization.
Instrument: PerSeptive Biosystems 8909 Expedite.
Synthesis Scate: 2 pmole.
Reagents:
2 o Wash A: 20% DMSO in NMP
Wash B: 2 M Collidine in 20% DMSO in NMP
Deblock: 5% m-Cresol, 95% TFA
Neutralizer: 1 ~I DIEA in 20% DMSO in NMP
Cap: 0.5 M Acetic Anhydride, 1.5 M Collidine in 20% DMSO in NMP
2 5 Activator: 0.2 M HATU in DMF
Monomers: 0.22 M in 2 M ColIidine (50% Pyridine in DMF)
Synthesis: The solid support (BOC-BHA-PEG-resin) is washed with 708 pl of Wash
A.
Deblock (177 pL) is passed through the column 3 times over 6.3 minutes. The
resin is then
washed with 1416 pL of Wash A. The free amine is neutralized with 1063 ~.L of
Neutralizer.
3 0 The resin is washed with 1062 L of Wash B. Monomer and Activator (141 ~L
each) are
slowly added to the column over 14 minutes. The resin is washed with 708 ~L of
Wash B and
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708 ~L of Wash A. Unreacted amine is capped with slow addition of 708 ~.L of
Cap solution
over 5 minutes. The resin is then washed 2124 pL of Wash A. The cycle is
repeated until
synthesis of the desired PNA sequence is completed.
Cleavage: The PNA-resin is washed with 5 mL of MeOH and dried under vacuum.
The
dried resin is emptied into a 1.5 mL Durapore ultrafree filter unit.
Thioanisole (25 ~L), 25
~,L of m-Cresol, 100 gL of TFA and 100 ~L of TFMSA is added to the resin,
vortexed for
about 30 seconds and allowed to stand for 2 h. The reaction mixture is then
centrifuged for
5 minutes at 10 K and the inner tube with resin is removed. Approximately 1.5
mL of ether
is added to the TFA solution to precipitate the product. The TFA solution is
vortexed,
followed by centrifugation at 10 K for 2 minutes. The ether is removed in
vacuo. Ether
precipitation and centrifugation are repeated an additional 2 times. The dry
pellet is heated
in a heat block (55°C) for 15 to 30 minutes to remove excess ether and
redissolved in 200 ~L
of H20. Solvent is added to 100 mg of Dowex Acetate Resin in a 1.5 mL Durapore
ultrafree
filter unit, vortexed, allowed to stand for 30 minutes and centrifuged at 10 K
for 2 minutes.
Characterization: The absorbance of a 1 gL sample in 1 mL of H20 is measured
at 260 nm.
Isopropanol (50%) in Hz0 with 1 % Acetic acid ( 100 pL) is added to 4 ~,L of
the sample. This
sample is characterized by electrospray mass spectrometry.
Common Abreviations
NMP: N-methyl pyrrolidinone
2 o TFA: Trifluoroacetic acid
DIEA: N,N-Diisopropylethylamine
HATU: O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate
TFMSA: Trifluormethanesulfonic Acid
Example 29
Synthesis and Cellular Uptake of Conjugated PNA Oligomers.
Using the procedures of Example 28, the aminoethylglycine PNA monomers of
examples 5 through 14, and monomers of Examples 1 S through 21, the following
PNA
oligomers were synthesized.
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PNA LiposomesCellular Uptake
Fl-GGT-GCT-CAC-GCT-GGC-Lys-NH2 x -
Fl-GGT-GCT-CAC-GCT-GGC-Lys-NH2 J -
Ada-Fl-GGT-GCT-CAC-TGC-GGC-Lys-NH2 x -
Ada-Fi-GGT-GCT-CAC-TGC-GGC-Lys-NH2 ,/ (+)
Fl-GGTk-GCTk-CAC-TkGC-GGC-Lys-NH2 x -
Fl-GGTk-GCTk-CAC-TkGC-GGC-Lys-NH2 ./ -
Ada-Fl-GGTk-GCTk-CAC-TkGC-GGC-Lys-NH2x +
Ada-Fl-GGTk-GCTk-CAC-TkGC-GGC-Lys-NH2,/ +
[(+) = pNA aggregates; - = no cellular uptake observed; + = cellular uptake
observed;
./ = presence of liposomes; x = absence of liposomes; Tk is thymine attached
to an
aminoethyl-lysine backbone; Lys is D-Lysine; Ada is adamantyl; and Fl is
fluoresceinyl
lysine.]
Example 30
Synthesis of Linolenyl-TAG-CAG-AGG-AGC-TC (SEQ ID NO:1)
Linolenic acid (40 p.moles) was dissolved in coupling solvent (100 p.L) (0.5 M
DIEA
in 20% DMSO/NMP),to which HATU (90 pL of 0.4 M) was added and the solution was
mixed. After a 2 minute activation period, the solution was mixed with
protected PNA resin
(15.4 mg, 2 pmoles). After 1 hour, the resin was washed with 20% DMSO/NMP,
CHzCI, and
2 0 MeOH (about 3 mL each). The resulting linolenyl-conjugated PNA was cleaved
from the
solid support and characterized according to the procedure described in
Example 28.
Example 31
Synthesis of Oleyl-TAG-CAG-AGG-AGC-TC (SEQ ID NO:1)
Oleic acid (40 p.moles) was dissolved in coupling solvent (100 ~L ) (0.5 M
DIEA
2 5 in 20% DMSO/NMP),to which HATU (90 pL of 0.4 M) was added and the solution
was
mixed. After a 2 minute activation period, the solution was mixed with
protected PNA resin
(5.4 mg, 2 pmoles). After 1 hour, the resin was washed with 20% DMSO/NMP,
CHZCI, and
MeOH (about 3 mL each). The resulting oleyl-conjugated PNA was cleaved from
the solid
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support and characterized according to the procedure described in Example 28.
Example 32
Synthesis of Caproyl-Gly-TAG-CAG-AGG-AGC-TC (SEQ ID NO:1)
Caproyl-gly (40 pmoles) was dissolved in coupling solvent (100 ~.L) (0.5 M
DIEA
in 20% DMSO/NMP), to which HATU (90 pL of 0.4 M) was added and the solution
was
mixed. After 2 minutes of activation, the solution was mixed with protected
PNA resin ( 15.4
mg, 2 pmoles). After 1 hour, the resin was washed with 20% DMSO/NMP, CH,Ch and
MeOH (about 3 mL each). The resulting PNA was cleaved from the solid support
and
characterized according to the procedure described in Example 28.
1 o Example 33
Synthesis of N-BOC-s-(Fluoresceinyl carbonyl)-D-lysine and its ethyl ester
a-BOC protected lysine ethyl ester was treated with excess fluorescein
isocyanate
in a mixture of THF and DMF at room temperature for several hours. The
reaction was
monitored by tlc for the disappearance of the starting amino acid. The
reaction was then
treated with equal volumes of water and chloroform and the phases separated.
The aqueous
phase was extracted with more chloroform and the combined organic solutions so
obtained,
dried with magnesium sulfate. This solution was concentrated, in vacuo, and
the crude
product obtained was purified by column chromatography to afford the N-BOC-s-
(Fluoresceinyl carbonyl)-D-lysine ethyl ester.
The ethyl ester was hydrolyzed using 1M aqueous lithium hydroxide and
tetrahydrofuran as solvent. The progress of the reaction was followed by tlc
and upon
completion the reaction mixture was treated with water and then washed 2x with
dichloromethane. The basic solution was then cooled to <IOC, neutralized with
1N HCl to
a pH below 4 and the product extracted out using ethyl acetate. The organic
extract was dried
2 5 using magnesium sulfate, and concentrated in vacuo to afford the N-BOC-s-
(Fluoresceinyl
carbonyl)-D-lysine.
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Example 34
Coupling of N-BOC-e-(Fluoresceinyl carbonyl)-D-Lysine to PNA of sequence GGT-
GCT-CAC-TGC-GGC-Lys-NHZ (SEQ ID N0:2)
PNA of sequence GGT-GCT-CAC-TGC-GGC-Lys-NHZ was synthesized following
standard PNA synthesis protocols {as in examples 27 and 28) and commencing
with lysine
derivatized synthesis resin. The N-terminal BOC group of the PNA bound to
resin was
deprotected using:
1. 3 mL of 1:1 v/v TFA/DCM 1 x2 mins and then 1 x0.5 hours
2. Washing with 3 mL DCM, 4x20 seconds.
1 o Washing with 3 mL DMF, 2x20 seconds
Washing with 3 mL DCM, 2x20 seconds.
Draining for 30 seconds.
3. Neutralizing with 3 mL DIEA/DCM, 1:19 v/v, 2x3 minutes.
Coupling of the Fluoresceinyl lysine was then performed according to the
following steps:
1. Wash with DCM, 3 mL, 4x20 seconds.
Drain for 1 minute.
2. Addition of 4 equivalents of DIC, and 4 equivalents of N-BOC-e-
(Fluoresceinyl
carbonyl)-D-Lysine dissolved in 1:1 v/v DCM/DMF (final concentration of the
amino acid
being 0.1 M).
2 0 3. Coupling allowed to proceed for 0.5 hour with shaking at room
temperature.
4. Drain for 20 seconds.
Wash with 3 mL DMF, 2x20 seconds and 1 x2 minutes.
Wash with 3 mL DCM, 4x20 seconds.
5. Neutralize with 3 mL, DIEA/DCM, 1:19 v/v, 2x3 minutes.
2 5 Wash with 3 mL DCM, 4x20 seconds; Drain for 1 minute.
6. Perform a qualitative Kaiser test. A negative result indicates near 100%
coupling.
The BOC group was cleaved and then the PNA (Fl-GGT-GCT-CAC-TGC-GGC-Lys-NHz)
was cleaved and purified as in examples 27 and 28. Alternatively, the PNA is
left attached
to the resin and used for derivatization with a lipophilic group as in example
35.
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Example 35
Synthesis of Ada-Fl-GGT-GCT-CAC-TGC-GGC-Lys-NHz (SEQ ID N0:2)
The N-terminal BOC group of Fl-GGT-GCT-CAC-TGC-GGC-Lys-NHz PNA bound
to the resin was first cleaved (as in example 34) and the free amino terminus
then derivatized
' 5 as follows:
100 mole adamantoyl chloride was dissolved in 1:5 v/v DIEA/DMF was added to
2 mole resin bound PNA (that is completely protected except at the N-terminus
where the
BOC group has been cleaved). After 1 hour of reaction, the resin was washed
with 3 mL each
of 20% NMP/DMSO, DCM and methanol. The PNA was cleaved from the resin and
purified
following standard protocols as in examples 27 and 28.
Example 36
Synthesis of Adamantyl-Ahx-TAGCAG-AGG-AGC-TC (SEQ ID NO:1)
Adamantyl carbonyl chloride ( 100 p.moles) was dissolved in DMF { 1.0 mL) and
DIEA (200 p.moles). This solution was mixed with protected PNA resin ( 15.4
mg, 2 pmoles)
with an attached amino hexanoic acid group linking group. After 1 hour, the
resin was
washed with 20% DMSO/NMP, CHZCh and MeOH (about 3 mL each). The resulting PNA
was cleaved from the solid support following known methods and techniques.
Example 37
Preparation of PNA/Liposome.
Liposomes containing the PNA Adamantyl-(Fl*)-TTT AGC TTC AGC-LysNH,
(SEQ ID N0:3), where Fl* is a fluoresceinated PNA monomer, were prepared by a
modification of the ethanol injection method described by Campbell.
Biotechniques, 1995,
18, 1027. DOPE (dioleyl-L-a-phosphatidylethanolamine) (13.4 mmol) and DDAB
(dimethyldiocadecylammonium bromide) (6.6 mmol) were dissolved in 1 mL of 96%
ethanol.
2 5 A solution of PNA (10 mL, 2.5 mM) in DMSO was combined with the lipid
mixture (40 mL).
The resulting 50 mL of material was then rapidly added to sterile distilled
H20 (1 mL) while
vortexing. The resulting PNA concentration in the liposome mix was 25 mM. For
cell uptake
experiments, the PNA-liposome mix (40 mL) was added to OptiMEM~'~"'' ( 1 mL)
and fed to
cells. The final concentration of PNA was 1 mM.
3 0 Liposome transfection reagents: Four commercially available transfection
liposome reagents
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were employed: Lipofectin'~"'' (Gibco BRL), Lipofectamine (~ibco BRL), Tfx-50
(Promega) and DOTAP'~"'' (Boehringer Mannheim). Each liposome reagent was
mixed with
conjugated PNA I 118 to give a final PNA concentration of 1 mM in the culture
medium (I
mL). The optimal concentration of each liposome reagent in terms of PNA cell
uptake was
determined. The table below shows the amount of reagent used per mL of
OptiMEM.
LipofecNnTM LipofectamineTM Tfx-SOTM DOTAPTM
2 mL 4 mL 10 mL 10 mL
Cells: The human carcinoma cell line HeLa was grown in RPMI 1640 medium
containing
Glutamax~''', penicillin, streptomycin and fetal bovine serum. On the day
preceding the
l0 experiment, the cells were plated at a density of 2 x I05 cells per dish in
35 mm dishes
containing coverslips. The following day the cells were washed once with
OptiMEM, then
fed with 1 mL OptiMEM containing 1 ~M PNA or PS-ODN, either alone, mixed with
one of
the 4 liposome reagents or incorporated in DOPE/DDAB liposomes, as described
above.
After an overnight incubation, the PNA-treated cells were fixed in 3%
formaldehyde/0.2%
glutaraldehyde on ice. The coverslips were then mounted on objective glasses
and the cells
observed by fluorescence microscopy on a Leits Diaplan microscope. Micrographs
were taken
with Kodak Ektacrome 1600 ASA film.
Example 38
Cellular uptake of conjugated PNAs.
Four conjugated PNAs (Example 30-32 and 36) having the title formula were
prepared following the standard procedure illustrated in Example 28. Lysine
residues were
incorporated into PNA's by using a modified MBHA resin (Dueholm, J. Org.
Chem.,1994,
59, 5767) using a Boc-Lys- CICbz (ClCbz = 2-chlorobenzyloxycarbonyl). The PNA
oligomer
was then extended with a protected Lys group that was previously
fluoresceinated at the E-
amino group. Deprotection of the amino group followed by conjugation with a
lipophilic
group afforded the support bound conjugated PNA. Cleavage from the solid
support afforded
the free PNA conjugate having a fluorescent label. The lipophilic groups (R)
investigated
include adamantoyl, decanoyl, heptyl-succinyl and palmityl-succinyl groups (as
shown in
Figure 1 ).
3 0 Stock solutions of the four conjugated PNAs were prepared by dissolving
the PNAs
in DMSO. Dilutions of these stock solutions were made in either water or
OptiMEM (Gibco
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BRL). The human carcinoma cell line HeLa was grown in RPMI 1640 medium
containing
GlutamaxTM, penicillin, streptomycin and fetal calf serum (10% v/v). On the
day preceding
the experiment, the cells were plated at a density of 2x105 cells per dish in
35 mm dishes
containing coverslips. The next day the cells were rinsed once with OptiMEM,
then fed with
3 pM PNA in 1 mL of OptiMEM and further incubated. In order to visualize PNA
uptake,
the coverslips were washed twice with PBS and the cells were fixed for 15
minutes in 3%
formaldehyde/0.2% glutaraldehyde on ice. After washing twice with PBS, the
coverslips
were mounted on objective glasses using 90% glycerol in PBS, and the cells
were observed
by fluorescent microscopy on a Leitz Diaplan Microscope. Micrographs were
taken with
l0 Kodak Ektachrome 1600 ASA film.
The four conjugated PNAs were tested for uptake into human cells in culture.
The
PNAs were added directly to the cell culture medium. HeLa cells grown on
coverslips were
incubated with PNA (3 pm) in serum free medium overnight, then fixed and
examined by
fluorescence microscopy. Both the palmityl-succinyl and the heptyl-succinyl
conjugated
PNAs showed punctate and spotted fluorescence in ali cells. Generally, the
spots were evenly
distributed over the cell with a tendency of an enhanced staining at the edges
of the cells,
probably the cell membrane. The adamantoyl- and decanoyl-conjugated PNAs
showed much
less cell-associated fluorescence with large fluorescent aggregates seen
outside the cells.
The palmityl-succinyl PNA conjugate was further studied by confocal microscopy
2 0 to determine the exact location and distribution of the PNA conjugate
inside the cell. A cell
was selected from the above study and further scanned through 12 sections. The
images
confirm that the PNA conjugate was indeed taken up by the cells and
distributed in spots
throughout the cytoplasm. There was, apparently, no fluorescence in the
nucleus. This
pattern is indicative of the endocytotic pathway of uptake, implying that the
PNA conjugates
2 5 end up in endosomes.
The palmityl-succinyl PNA conjugate was also observed in a time course
experiment. Cells were incubated for different lengths of time in the presence
of 3 ~M of
PNA. The uptake of the PNA conjugate by the cells increased with time up until
24 hours of
incubation when the PNA-containing medium was replaced with fresh serum
containing
3 o medium. After 48 hours intracellular PNA was concentrated in compartments
of the cells,
probably secondary lysosomes. After 72 hours there was virtually no PNA left
inside the
cells.
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Example 39
In vitro translation assay using conjugated PNAs.
PNA having the sequence R-Lys(Fluorescein)-TTT-AGC-TTC-CTT-AGC-Lys-NH,
(SEQ ID N0:3) is complementary to the 15 nucleotides immediately 5' to the AUG
start
codon in CAT mRNA and the corresponding unconjugated PNA has previously been
shown
to be able to inhibit translation of CAT in vitro. The four conjugated PNAs of
Example 38
(SEQ ID N0:2) were tested in this assay. All four conjugated PNAs specifically
inhibited
CAT translation at similar concentrations as the unconjugated PNA.
Example 40
Preparation of liposome constructs using PNA conjugates (SEQ ID N0:2).
Liposome constructs were prepared using two of the conjugated PNAs having SEQ
ID N0:2. The adamantoyl- and decanoyl-conjugated PNAs were combined with
liposomes
by a modification of the ethanol injection method described by Campbell in
Biotechnigues,
1995, 18, 1027. Following this method, 13.4 mole of DOPE (dioleyl-L-a-
phosphatidyl-
ethanolamine) and 6.6 mole of DDAB (dimethyldioctadecylammonium bromide) were
dissolved in 1 mL of absolute ethanol. A solution of PNA (10 ~L, 3 mM
PNA/DMSO) was
combined with 40 ~L of the lipid mixture. The resulting 50 pL of reaction
mixture was then
rapidly added to 1 mL of sterile distilled H20 while vortex mixing. The PNA
concentration
in the liposome mixture was thus 30 ~M. For cell uptake experiments, 60 pL of
the PNA-
2 0 liposome mixture was added to 1 mL of OptiMEM and fed to the cells.
Incorporation of the conjugated PNAs into the liposome constructs was verified
by
fluorescent microscopy. The fluorescent micrographs showed spots of
fluorescence
associated with the cells as observed for the PNA conjugates. In addition, a
more diffuse
fluorescence was observed throughout the cells with fluorescence observed in
the nuclei of
2 5 some cells.
When other cell lines were used (COS-7, green monkey kidney derived cells; and
NIH 3T3, mouse fibroblast cells) identical uptake patterns were observed.
Example 41
Cellular Uptake of an Adamantyl-Conjugated PNA.
3 0 The cellular uptake of an adamantyl-PNA (prepared according to Examples 35
or 36)
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was determined, and was also compared to the uptake of a phosphorothioate
oligonucleotide.
The adamantyl-conjugated PNA and oligonucleotide were added directly to
subconfluent
HeLa cells at 1 ~M concentrations and left over night. The cells were next
fixed and uptake
visualized by fluorescence microscopy. The oligonucleotide exhibited fine
punctate
fluorescence, mainly confined to clusters in the cytoplasm of the cells and
absent from the
nuclei. With the PNA, punctate fluorescence was similarly observed. However,
the spots were
somewhat larger and present both in the cytoplasm and on the cell membrane.
In an attempt to improve uptake of the adamantyl-PNA, the PNA was combined
with
various commercially available cationic liposomes normally used for
transfection of DNA.
In addition, PNA-containing liposomes composed of the lipids DOPE and DDAB
were also
prepared. In theory, the hydrophobic adamantyl-group of the PNA should insert
into the lipid
layer of the liposomes and thus entrap the PNA. The liposomes were prepared by
a simple
ethanol injection technique, which was reported to be efficient for the
transport of plasmid
DNA into cells. The different PNA-liposome mixtures were fed to cells with a
final PNA
concentration of 1 p.M and incubated over night. The presence of either of the
liposome
reagents or the DOPE/DDAB liposomes resulted in a much more diffuse
fluorescence inside
the cells, compared to when the PNA was added alone. However, the fluorescence
was still
confined to the cytoplasm with no sign of nuclear uptake. In contrast, when
the
oligonucleotide was mixed with Lipofectamine~'~"'' and fed to the cells at a
concentration of 1
2 0 ~M, the majority of the cells had fluorescently stained nuclei.
In conclusion, adding adamantyl-conjugated PNA to cells resulted in an uptake
pattern reminiscent of an endocytotic pathway, where the PNA ends up in
endosomal or
lysosomal compartments of the cell. When PNA is pre-mixed with liposome
transfection
reagents or incorporated into DOPE/DDAB liposomes, it is distributed
throughout the cell
2 5 cytoplasm in a much more diffuse fashion.
Those skilled in the art will appreciate that numerous changes and
modifications may
be made to the preferred embodiments of the present invention and that such
changes and
modifications may be made without departing from the spirit of the invention.
It is therefore
intended that the appended claims cover all such equivalent variations as fall
within the true
3 o spirit and scope of the invention.
