Note: Descriptions are shown in the official language in which they were submitted.
2144475
Hoechst Aktiengesellschaft HOE 94/F 057 Dr. WI/pp
Description
Polyamide-oligonucleotide derivatives, their preparation
and use
The present invention relates to novel polyamide-oligonu-
cleotide derivatives with valuable physical, biological
and pharmacological properties. Their application relates
to use as inhibitors of gene expression (antisense
oligonucleotides, ribozymes, sense oligonucleotides and
triplex forming oligonucleotides), as probes for detec-
ting nucleic acids and as aids in molecular biology.
Oligonucleotides are finding increasing application as
inhibitors of gene expression (G. Zon, Pharmaceutical
Research 5, 539 (1988); J.S. Cohen, Topics in Molecular
and Structural Biology 12 (1989) Macmillan Press;
C. Helene and J.J. Toulme, Biochimica et Biophysica Acta
1049, 99 (1990); E. Uhlmann and A. Peyman, Chemical
Reviews 90, 543 (1990)). Antisense oligonucleotides are
nucleic acid fragments whose base sequence is complemen-
tary to that of an mRNA to be inhibited. This target mRNA
can be of cellular, viral or other pathogenic origin.
Suitable cellular target sequences are, for example,
those of receptors, cell-adhesion proteins, enzymes,
immunomodulators, cytokines, growth factors, ion channels
or oncogenes. Inhibition of virus replication with the
aid of antisense oligonucleotides has been described, for
example, for HBV (hepatitis B virus), HSV-1 and -2
(herpes simplex virus type I and II), HIV (human immuno-
deficiency virus) and influenza viruses. This entails use
of oligonucleotides which are complementary to the viral
nucleic acid. Sense oligonucleotides are, by contrast,
designed to have a sequence such that, for example, they
bind ("trap") nucleic acid-binding proteins or nucleic
acid-processing enzymes and thus inhibit their biological
activity (C. Helene and J.J. Toulme, Biochimica et
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Biophysica Acta 1049, 99 (1990)). Viral targets which may
be mentioned here are, for example, reverse
transcriptase, DNA polymerase and transactivator
proteins. Triplex-forming oligonucleotides generally have
the DNA as target and, after binding thereto, form a
triple helix structure. Whereas in general the processing
(splicing etc.) of the mRNA or translation thereof into
protein is inhibited by antisense oligonucleotides, the
transcription or replication of the DNA is inhibited by
triplex-forming oligonucleotides (C. Helene and
J.J. Toulme, Biochim. Biophys. Acta 1049 (1990) 99-125;
E. Uhlmann and A. Peyman, Chemical Reviews 90, 543
(1990)). However, it is also possible to bind single-
stranded nucleic acids in a first hybridization with an
antisense oligonucleotide to form a double strand, which
then in a second hybridization with a triplex-forming
oligonucleotide forms a triplex structure. The antisense
and triplex binding regions may in this case be accommo-
dated either in two separate oligonucleotides or else in
one oligonucleotide. Another application of synthetic
oligonucleotides comprises so-called ribozymes which
destroy the target RNA as a consequence of their
ribonuclease activity (J.J. Rossi and N. Sarver, TIBTECH
(1990) 8, 179; Castanetto et al., Critical Rev. Eukar.
Gene Expr. (1992) 2, 331).
The compounds according to the invention can also be used
in therapy in the sense of aptamers. Aptamers are oligo-
meric nucleic acids or analogs thereof which bind with
high affinity to proteins. The aptamers are found by in
vitro selection from a random mixture (Famulok and
Szostak (1992) Angew. Chem. 104, 1001-1011) and this has
been carried out successfully for a thrombin-binding
aptamer (Bock et al. (1992) Nature 355, 564-566). The
procedure for this can be such that the base sequence of
the aptamer is determined by screening an oligonucleotide
mixture, and this base sequence is then transferred to
polyamide-oligonucleotide analogs. Another possibility
comprises encoding the binding region of the aptamer, to
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facilitate identification, by a separate non-binding part
of the molecule (Brenner and Lerner (1992) PNAS 89,
5381-5383).
In DNA diagnosis, nucleic acid fragments with suitable
labeling are used as so-called DNA probes for specific
hybridization onto a nucleic acid to be detected. The
specific formation of the new double strand is in this
case followed with the aid of the labeling, which is
preferably non-radioactive. It is possible in this way to
detect genetic, malignant or viral diseases or diseases
caused by other pathogens.
Oligonucleotides in their naturally occurring form have
little or no suitability for most of the said applica-
tions. They have to be chemically modified so that they
satisfy the specific requirements. For oligonucleotides
to be employable in biological systems, for example for
inhibition of virus replication, they must meet the
following requirements:
1. They must have sufficiently high stability under in
vivo conditions, that is to say both in serum and
intracellularly.
2. Their properties must be such that they can pass
through the cell membrane and nuclear membrane.
3. Under physiological conditions they must bind in a
base-specific manner to their target nucleic acid in
order to display the inhibitory effect.
Points 1 to 3 are not a requirement for DNA probes;
however, these oligonucleotides must be derivatized so
that detection is possible, for example by fluorescence,
chemiluminescence, colorimetry or specific staining (Beck
and Koster, Anal. Chem. 62, 2258 (1990)). The chemical
modification of the oligonucleotides usually takes place
by appropriate modification of the phosphate backbone,
ribose unit or the nucleotide bases (J.S. Cohen, Topics
in Molecular and Structural Biology 12 (1989) Macmillan
Press; E. Uhlmann and A. Peyman, Chemical Reviews 90,
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543 (1990)). Another frequently used method is to prepare
oligonucleotide 5' conjugates by reaction of the
5'-hydroxyl group with appropriate phosphorylation
reagents. If, on the other hand, all the internucleotide
phosphate residues are modified there is often a drastic
change in the properties of the oligonucleotides. For
example, the solubility of methyl phosphonates in aqueous
medium is greatly reduced, while all-phosphorothioate
oligonucleotides often act in a non-sequence-specific
manner.
There have recently been descriptions of polyamide-
nucleic acid derivatives (Michael Egholm, Peter
E. Nielsen, Rolf H. Berg and Ole Buchardt, Science 1991,
254, 1497-1500; WO 92/20702; M. Egholm et al. Nature
(1993) 365, 566-568; P. Nielsen, (1994) Bioconjugate
Chem. 5, 3-7) which bind to complementary target
sequences (DNA or RNA) with higher affinity than natural
oligonucleotides. These so-called peptide or polyamide
nucleic acids (PNA) are DNA-analogous compounds in which
the deoxyribose phosphate skeleton has been replaced by
a polyamide oligomer. These compounds have the advantage
compared with natural oligonucleotides that they are very
stable in serum. However, on the other hand, they have
the following disadvantageous properties:
(1) The amount taken up in cells is zero or undetectable.
However, since antisense or triplex-forming oligonucleo-
tides are able to display their activity only in the
cell, the PNAs as such are unsuitable for inhibition of
gene expression in vivo.
(2) The PNAs tend to aggregate in aqueous solution, that
is to say also under physiological conditions. Their
solubility in aqueous buffer is therefore low and they
are unavailable for hybridization to complementary
sequences.
(3) The PNAs additionally have high affinity for various
materials such as Sephadex (from Pharmacia) or Bond
Elut (from Varian) used to purify the oligomers, so that
the PNAs can often be isolated only in poor yields.
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(4) Another serious disadvantage of the PNAs is that they
do not bind in an unambiguous orientation to
complementary nucleic acids. The sequence specificity is
therefore reduced by comparison with natural oligonucleo-
tides. Whereas natural nucleic acids generally hybridize
to complementary nucleic acids in the antiparallel
orientation, PNAs may bind both in the antiparallel and
in the parallel orientation.
(5) WO 92/20702 mentions an oligonucleotide-PNA conjugate
(T) 7 (5'-L-N)(t)6-Ala (Fig. 25; substitute sheet), where
(T)7 is a natural heptathymidylate oligonucleotide which
is linked via its 5'-O-phosphate and 4-hydroxybutyric
acid (L) to the primary amino group (N) of a PNA-hexa-
thymidylate (t)7 and alanine (Ala). Neither the synthesis
of this compound nor any properties have been described.
(6) PNAs show highly cytotoxic properties in the molar
range in cell culture experiments.
The orientation of the base-pairing nucleic acid strands
is defined as follows: (cf. Egholm et al.; Nature 365
(1993) 566-568).
A)
5' ---------- 3' DNA ap Duplex ap = antiparallel
3' ---------- 5' DNA
B)
5' ---------- 3' DNA p Duplex p = parallel
5' ---------- 3' DNA
C)
5' ---------- 3' DNA ap Duplex (DNA=PNA)
C ----------- N PNA
D)
5' ---------- 3' DNA p Duplex (DNA=PNA)
N ----------- C PNA
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E)
C ----------- N PNA
5' ---------- 3' DNA (Pu) ap=ap triplex (DNA=DNA=PNA)
3' ---------- 5' DNA Pu = purine-rich strand
F)
N ----------- C PNA
5' ---------- 3' DNA (Pu) ap=p triplex (DNA=DNA=PNA)
3' ---------- 5' DNA
G)
N ----------- C PNA
5' ---------- 3' DNA (Pu) ap=p triplex (PNA=DNA=PNA)
C ----------- N PNA
H)
C ----------- N PNA
5' ---------- 3' DNA (Pu) ap=ap triplex (PNA=DNA=PNA)
C ----------- N' PNA
I)
N ----------- C PNA
5' ---------- 3' DNA (Pu) p=p triplex (DNA=DNA=PNA)
N ----------- C' PNA
K)
C ----------- N PNA
5' ---------- 3' DNA (Pu) p=ap triplex (DNA=DNA=PNA)
N ----------- C PNA
where 5' means the 5' end of an oligonucleotide,
3' means the 3' end of an oligonucleotide,
N means the amino terminus of a PNA
C means the carboxyl terminus of a PNA.
Cases A)-D) are examples of the types of orientation
which are possible in principle for the antisense oligo-
mers. Cases E)-F) show possibilities for triplex forma-
tion on single-stranded or double-stranded nucleic acids.
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It is moreover possible for two of the PNA or DNA single
strands to be linked together. For example, in E) the
N terminus of the PNA can be linked to the 5' end of the
DNA, or in F) the C terminus of the PNA can be linked to
the 5' end of the DNA.
The object of the invention therefore was to prepare
polyamide-oligonucleotide derivatives in which the
abovementioned disadvantages are eliminated.
The invention relates to polyamide-oligonucleotide
derivatives of the formula I
F[(DNA-Li)q(PNA-Li)r(DNA-Li)S(PNA)t],F' (I)
wherein
q, r, s, t are, independently of one another, zero or 1,
where the total of two or more adjacent q, r, s and
t > 2;
x is 1 to 20, preferably 1 to 5, particularly prefer-
ably 1;
DNA is a nucleic acid such as DNA or RNA or a known
derivative thereof;
Li is a covalent linkage between DNA and PNA, where the
covalent linkage comprises a bond or an organic
radical with at least one atom from the series
consisting of C, N, 0 or S;
PNA is a polyamide structure which contains at least one
nucleotide base which is different from thymine; and
F and F' are end groups and/or are linked together by a
covalent bond (cyclic compounds),
and the physiologically tolerated salts thereof.
Particular mention may furthermore be made of polyamide-
oligonucleotide derivatives of the formula I in which x
is 1 and, at the same time,
q = r = 1 and s = t= zero or
r = s = 1 and q = t = zero or
q = r = s = 1 and t = zero or
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r = s= t = 1 and q = zero.
Preferred compounds have the formulae Ia and Ib
( (NU)n
0 a CN2
R2 C=0 0
il
(Lt~J (D). - CH2-CN2-N - CH= - C - (Li2J
T
(Nu)e
0 8 ~
CHI
R' C=0 0 (la)
II
(Li~) (D)o - CHZ-CHZ-N - CHZ - C P'
t ~
t
F CH=-CHI-N -H2C - C - (0')p L(
II
0=C 0 2
CH7
0
(Nu)e
1 t
(Li~)-CNI-CN=-N -NtC - C - (D')~ --{li
11 i
0=C 0
0
N= (Ib)
(Nu)~ I'
r Q
in which
x is 1 to 20, where
when x > 1 r = s = 1 and, at the same time,
q= t = zero and o = n= zero to 5;
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q, r, s, t are, independently of one another, zero or 1,
where the total of two or more adjacent q, r, s and
t z 2;
R2 is hydrogen, hydroxyl, C1-C18-alkoxy, preferably
C1-C6-alkoxy, halogen such as F or Cl, preferably F,
azido or amino;
B is, independently of one another, a base customary
in nucleotide chemistry, for example natural bases
such as adenine, cytosine, thymine, guanine, uracil,
inosine or unnatural bases such as, for example,
purine, 2,6-diaminopurine, 7-deazaadenine, 7-deaza-
guanine, N4,N4-ethanocytosine, N6,N6-ethano-2,6-di-
aminopurine, pseudoisocytosine, 5-methylcytosine,
5-fluorouracil, 5-(C3-C6)-alkynyluracil, 5-(C3-C6)-
alkynylcytosine or the prodrug forms thereof,
and the "curved bracket" indicates that R2 and the
adjacent substituent can be in the 2' position and
3' position or else conversely in the 3' position
and 2' position;
Nu is a radical of the formulae IIa or IIb
5' 51
0 8 0
FR RZ
Y Y
I I
U P V U P V
II II
W yy
(Ila) (Ilb)
in which
R2 and B are as defined above;
U is hydroxyl, mercapto, C1-C18-alkyl, preferably
C1-C8-alkyl, C1-C16-alkoxy, preferably C1-C8-
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alkoxy, C6-C20-aryl, preferably C6-C12-aryl,
C6-C14-aryl-C1-C8-alkyl, preferably C6-aryl-
C1-Cq-alkyl, NHR3 or NR3R4, and
R3 is C1-C18-alkyl or C1-Cq-alkoxy-C1-C4-alkyl, prefer-
ably C1-C8-alkyl or C1-C4-alkoxy-C1-C4-alkyl, par-
ticularly preferably C1-C4-alkyl or methoxyethyl and
R4 is C1-C18-alkyl, preferably C1-C8-alkyl and particu-
larly preferably C1-C4-alkyl, or
R3 and R4 is, together with the nitrogen atom carrying
them, a 5-6-membered heterocyclic ring which can
additionally contain another hetero atom from the
series consisting of 0, S, N, such as, for example,
morpholine;
V is oxy, thio or imino;
W is oxo or thioxo;
Y is oxy, thio, methylene or imino;
m is zero to 20;
o is zero to 20;
D is a radical of the formula III
B
C H 2
(
C-0
0 (III)
I II
CH2-CH2-N - CHZ - C - N
H
in which B is as defined above;
D' is a radical of the formula IV
- N - CH2-CH2-N - H2C - C
H I I
0
0-C (IV)
C H 2
B
in which B is as defined above;
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n is zero to 20;
p is zero to 20;
Lil, Li2, Li3 and Li4 are each, independently of one
another, a structure of the formula V
[(V')-(G)-(G')]E (V)
where, independently of one another,
E is 1 to 5, preferably 1-2,
V' is oxygen, NH, a bond or a radical of the formula
VI
U
I
Y P V (VI)
II
w
in which
U, V, W and Y are as defined above;
G can be C1-C12-alkanediyl, preferably C1-C6-alkane-
diyl, where alkanediyl can optionally be substituted
by halogen, preferably F or chlorine, amino,
hydroxyl, C1-C18-alkyl, preferably C1-C6-alkyl,
C1-C18-alkoxy, preferably C1-C6-alkoxy, C6-C14-aryl,
preferably C6-aryl, or C6-C14-aryl-C1-C18-alkyl,
preferably C6-aryl-C1-C4-alkyl; C6-C14-aryl-di-C1-
C12-alkanediyl, preferably C6-aryl-di-C1-C4-alkane-
diyl, or a group of the formula (CH2CH2O)SCH2CH2 in
which b can be 1 to 11, preferably 1 to 7; or a
bond; and
G' is oxy, thio, imino, -C(O)-, -C(O)NH-, a bond or a
radical of the formula VI in which U, V, W and Y are
as defined above; and
F and F' are linked by a bond (cyclic compounds) and/or
F is R 0 - (A)Wk V and
F' in formula Ia is -(Q) 1- R1 and in formula Ib is
V1 - (A)1 - R1,
where
R is hydrogen, C1-C18-alkanoyl, preferably C8-C18-
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alkanoyl, C1-C18-alkoxycarbonyl, C3-C8-cycloalkanoyl,
C7-C15-aroyl, C3-C13-heteroaroyl or a group which
favors intracellular uptake of the oligomer or
serves as labeling of a DNA probe or, in the
hybridization of the oligomer onto the target
nucleic acid, attacks the latter with binding,
crosslinking or cleavage; or
if k is zero, R is hydrogen or together with V is
a radical of the formula VII
V
I
Z P Z(vii)
(I
w
in which
Z and Z' are, independently of one another,
hydroxyl, mercapto, C1-C22-alkoxy, preferably
C12-C18-alkoxy, C1-C18-alkyl, preferably C12-C18-
alkyl, C6-C20-aryl, preferably C6-C16-aryl, C6-C14-
aryl-C1-C1e-alkyl, preferably C6-aryl-Cl-C4-alkyl,
C1-C22-alkylthio, preferably C12-C18-alkylthio, NHR3,
NR3R4, or a group which favors intracellular uptake
of the oligomer or serves as labeling of a DNA probe
or, in the hybridization of the oligomer onto the
target nucleic acid, attacks the latter with
binding, crosslinking or cleavage, and in which
R3, R4, V and W are as defined above;
R1 is hydrogen or Q
where R1 is always only hydrogen when at the
same time 1 is zero and in formula Ia t is zero
and s is 1 and Lil is a structure of the for-
mula V with V' = bond, G bond, E= 1 and
G' = oxy, thio, imino or a radical of the
formula VI with U = Z
or
in formula Ib q is 1 or q r = zero and in
F' = V1 -(A)1 - R1 with V1 = V,
A and Q are, independently of one another, the
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residue of a natural or unnatural amino acid, pre-
ferably from the series consisting of glycine,
leucine, histidine, phenylalanine, cysteine, lysine,
arginine, aspartic acid, glutamic acid, proline,
tetrahydroisoquinoline-3-carboxylic acid, octahydro-
indole-2-carboxylic acid, N-(2-aminoethyl)glycine;
Q is hydroxyl, OR', NH2, NHR" with
R' = C1-C1e-alkyl, preferably C12-C18-alkyl and
R" = C1-C18-alkyl, preferably C12-C18-alkyl,
C1-C1e-aminoalkyl, preferably C12-C18-amino-
alkyl, C1-C18-hydroxyalkyl, preferably C12-C18-
hydroxyalkyl;
V is as defined above;
V1 is a bond or V, where in F' only in formula Ib
with q = zero and r = 1 V1 is always a bond;
k is zero to 10;
1 is zero to 10;
with the proviso that
a) if in the compound of the formula Ia t is zero and
s is 1, and Lil is (V') - (G) -(G') with V' = a
compound of the formula VI, G = C2-C12-alkylene and
G' = CO, in F' _-(Q)1 - R1 1 is zero to 10 and R1
is Q ;
b) if in the compound of the formula Ia s = t = zero,
Li2 is a bond;
c) if in the compound of the formula Ib t is zero and
s is 1, Li3 is a bond;
d) if in the compound of the formula Ib s = t = zero,
Li4 is a bond;
where each nucleotide can be in its D or L configuration,
and the base can be in the a or Q position.
Particularly preferred compounds of the formula Ia and Ib
are those in which the base is located on the sugar in
the (3 position,
x is 1 and
q = r = 1, s= t = zero or
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r = s= 1, q = t = zero or
q = r = s= 1, t = zero or
r = s = t = 1, q = zero.
Especially preferred oligomers have the formulae Ia and
Ib in which V', V, Y and W have the meaning of thio, oxy,
oxo or hydroxyl; these are very particularly preferred
if, in addition, R2 is hydrogen.
Also especially preferred are oligomers of the formulae
Ia and Ib with E= 1, in which
Lil, Li4 are
a) a compound of the formula V in which
V' = oxygen or compound of the formula VI,
G = C1-Clp-alkylene, G' = -CONH-
b) a compound of the formula V in which G, V.
is a bond and G' is a compound of the formula
VI with, preferably, U = V = W = Y = oxygen or
U = W = Y = oxygen and V = imino
Li2, Li3 are
a) a compound of the formula V with V' = imino,
G = C1-C10-alkylene and G' = compound of the
formula VI
b) a compound of the formula V with V' = imino,
G and G' = bond
c) a compound of the formula V with V' = imino,
G = C1-Clo-alkylene and G' = V with, prefer-
ably, U = V = W = Y = oxygen.
Very particularly preferred oligomers have the formulae
Ia and Ib in which V' , V, Y and W have the meaning of
thio, oxy, oxo or hydroxyl, R2 is hydrogen, Lil has the
meaning of -V'-[CH2]nC(O)NH- with V' = compound of the
formula VI with U = V = W = Y = oxygen or Li2 has the
meaning of -HN-[CH2]n(G')-, where n is 2 to 5 and G' has
the formula VI with U, V, W and Y = oxygen.
Additionally preferred oligomers of the formulae Ia and
Ib are those in which V', V, Y and W have the meaning of
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thio, oxy, oxo or hydroxyl, R2 is hydrogen, Lil has the
meaning of -O-[CH2]õC(O)NH- or Li2 has the meaning of
-HN-[CH2]n(G')-, where n is 2 to 5 and G' has the formula
VI with U, V, W and Y = oxygen, and q = zero and
r= s= t= 1.
Additionally preferred are oligomers of the formulae Ia
and Ib in which the curved bracket means that R2 is in
the 3' position (see formula Iib). The preferred base in
this case is adenine.
The invention is not confined to a- and R-D- and L-ribo-
furanosides, a- and Q-D- and L-deoxyribofuranosides and
corresponding carbocyclic five-membered ring analogs but
also applies to oligonucleotide analogs which are com-
posed of different sugar building blocks, for example
ring-expanded and ring-contracted sugars, acyclic, ring-
bridged or other suitable types of sugar derivatives. The
invention is furthermore not confined to the derivatives,
indicated by way of example in formula I, of the phos-
phate residue but also relates to known dephospho
derivatives.
The oligonucleotide part (DNA in formula I) can therefore
be modified from the natural structure in a wide variety
of ways. Examples of such modifications, which are
introduced by methods known per se, are:
a) Modifications of the phosphate bridge
Examples which may be mentioned are: phosphorothioates,
phosphorodithioates, methylphosphonates, phosphor-
amidates, boranophosphates, phosphate methyl esters,
phosphate ethyl esters, phenylphosphonates. Preferred
modifications of the phosphate bridge are phosphorothio-
ates, phosphorodithioates and methylphosphonates.
b) Replacement of the phosphate bridge
Examples which may be mentioned are: replacement by
formacetal, 3'-thioformacetal, methylhydroxylamine,
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oxime, methylenedimethylhydrazo, dimethylene sulfone,
silyl groups. Replacement by formacetals and 3'-thio-
formacetals is preferred.
c) Modifications of the sugar
Examples which may be mentioned are: a-anomeric sugars,
2'-O-methylribose, 2'-O-butylribose, 2'-O-allylribose,
2'-fluoro-2'-deoxyribose, 2'-amino-2'-deoxyribose,
a-arabinofuranose, carbocyclic sugar analogs. The pre-
ferred modification is that by 2'-O-methylribose and
2'-O-n-butylribose.
d) Modifications of the bases with do not alter the
specificity of the Watson-Crick base pairing
Examples which may be mentioned are: 5-propynyl-2'-deoxy-
uridine, 5-propynyl-2'-deoxycytidine, 5-hexynyl-2'-deoxy-
uridine, 5-hexynyl-2'-deoxycytidine, 5-fluoro-2'-deoxy-
cytidine, 5-fluoro-2'-deoxyuridine, 5-hydroxymethyl-
2'-deoxyuridine, 5-methyl-2'-deoxycytidine, 5-bromo-
2'-deoxycytidine. Preferred modifications are 5-propynyl-
2'-deoxyuridine, 5-hexynyl-2'-deoxyuridine, 5-hexynyl-
2'-deoxycytidine and 5-propynyl-2'-deoxycytidine.
e) 3'-3' and 5'-5' inversions [for example M. Koga et
al., J. Org. Chem. 56 (1991) 3757]
f) 5'- and 3'-phosphates, and 5'- and 3'-thiophosphates.
Examples of groups which favor intracellular uptake are
various lipophilic radicals such as -O-(CH2),-CH3 in which
x is an integer from 6 to 18, -0-(CH2)n-CH=CH-(CH2)m CH3
in which n and m are, independently of one another, an
integer from 6 to 12, -O-(CH2CH2O)q-(CH2)9-CHg,
-O-(CH2CH2O)8-(CH2)13-CH3 and -O-(CH2CH2O)7-(CH2)15-CH3, but
also steroid residues such as cholesteryl or vitamin
residues such as vitamin E, vitamin A or vitamin D and
other conjugates which utilize natural carrier systems
such as bile acid, folic acid, 2-(N-alkyl-N-alkoxyamino)-
anthraquinone and conjugates of mannose and peptides of
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the appropriate receptors which lead to receptor-mediated
endocytosis of the oligonucleotides, such as EGF
(Epidermal Growth Factor), bradykinin and PDGF (Platelet
Derived Growth Factor). By labeling groups are meant
fluorescent groups, for example of dansyl (= 1-dimethyl-
aminonaphthalene-5-sulfonyl), fluorescein or coumarin
derivatives or chemiluminescent groups, for example of
acridine derivatives, and the digoxigenin system detect-
able by ELISA, the biotin group detectable by the
biotin/avidin system or else linker arms with functional
groups which permit subsequent derivatization with
detectable reporter groups, for example an aminoalkyl
linker which is reacted with an acridinium active ester
to give the chemiluminescence probe. Typical labeling
groups are:
0 01
H COOH
I
NH /CH0
~0
Fluorescein derivative
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CHs
N( '
0 0
0 N-(CH=)1-N-
H H
Acridinium ester
0 0-(CHI)=-0-
.
\ \ I ~
C 0 0 R x- 2-18 Drelerabiy 4
R = H or C,-C4-aikyl
i
(= 'rluorescein' for x 4 and R = CH3 )
Fluorescein derivative
0 R = H or amino protective group
R - N N H
H
0-
Is
0
Biotin conjugate (_ "Biotin" for R = Fmoc )
2144475
- 19 -
0
HO 0
\
OH
0 0
-
N 0
H
Digoxigenin conjugate
Oligonucleotide analogs which bind to or intercalate
and/or cleave or crosslink nucleic acids contain, for
example, acridine, psoralen, phenanthridine, naphtho-
quinone, daunomycin or chloroethylaminoaryl conjugates.
Typical intercalating and crosslinking radicals are:
-0-(CH2 ), iN
Acridine derivative x = 2-12, preferably 4
OCH~
/ \
-S - (CHZ)-NH N
CI
x = 2-12, preferably 4
2144475
- 20 -
0
CH 11
CH2X-(CH2)2-NH-C-
~
~
11 0 0 C H X - -NH or -0-
0 3
CH~
Trimethylpsorolene conjugate (- "psoralene" tor z-0)
0
NH
0
N
N
Phenonihroline conjugote
0-
HN
0 00
O
Psoralen conjugate
0
NH,,~/0-
~
CI
0
Nophthoquinone conjugote
2144475
- 21 -
0 ON 0
/ CN3
OH
N.
0CH3 0 OH 0
0
CHj
N0
NH
~
Daunomycin derivative
C I -CH2CH2\
N ( CHZ ) x-0-
H3C/
X
:= 1-18, X- Alkyl, Holoyen, NO2, CN, -C-R
0
C I -CHZCHZ\
/ N (CHZ),-0-
CI-CH2CHz
x
x- I-18, X- Alkyl, Holoflen, NOq, CN, -C-R
I )
0
Examples which may be mentioned of NR3R4 groups in which
R3 and R4 form, together with the nitrogen atom carrying
them, a 5- to 6-membered heterocyclic ring which addi-
tionally contains another hetero atom are the morpholinyl
and the imidazolidinyl radical.
The polyamide part (PNA in formula I) is composed of
amide structures which contain at least one nucleotide
2144475
- 22 -
base which is different from thymine. Polyamide struc-
tures of this type are composed, for example, of the
following building blocks a) to h), preferably a), in
which f is 1 to 4, preferably 1 or 2 and g is zero to 3,
preferably zero to 2:
a)
B
CHI
C=0
NH-(CH2)f-CH2-N-(CH1)f-CO
Hyrup et al.; J. Chem. Soc. Chem. Comm. 1993, 519
b)
B
(CHz)~
I
NH-CH-CO-NH-CH2-C0
De Konig et al. (1971) Rec. Trav. Chim. 91, 1069
c)
B
I
C Hz)r
(i
NH-CH-CHi-CH2-CHi-CO
Huang et al. (1991) J. Org. Chem. 56, 6007
d)
B
I
(CH2 ) f
N H- C H z-C O-N-C H z-C O
Almarsson et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90,
7518
2144475
- 23 -
e)
B
C H 2
C=0
NH-CH1- CH2- CH-CH2 -CO
Froehler et al. (1991) WO 93/10820
f)
B
0 1
C H
NH-CH2-CH2-N_,,~ CO
Froehler et al. (1991) WO 93/10820
g)
B
( i H 2 ) i
NH-CH-CO-N~
CO
Lewis (1993) Tetrahedron Lett. 34, 5697.
h)
0
~N,r( C H 2 cO
( C H 2 ) f
NH-(CH2)q
End groups for PNAs are described in the applications,
filed simultaneously, with the titles "PNA synthesis
using an amino protective group which is labile to weak
acids" (HOE 94/F 060, DE-P 44 08 531.1) and "PNA syn-
thesis using a base-labile amino protective group"
(HOE 94/F 059, DE-P 44 08 533.8).
2144475
- 24 -
Preferred polyamide structures are composed of structures
according to a). The latter are particularly preferred
when f is 1.
The preparation of polyamide-oligonucleotide derivatives
of the formula I takes place similarly to the synthesis
of oligonucleotides in solution or, preferably, on solid
phase, where appropriate with the assistance of an
automatic synthesizer. The oligomer of the formula I can
be assembled stepwise by successive condensation of one
PNA unit or DNA unit with in each case one nucleotide
base onto an appropriately derivatized support or onto a
growing oligomer chain. However, the assembly can also
take place in fragment fashion, in which case the frag-
ments are first synthesized as polyamide or oligonucleo-
tide structures which are then linked to give the
polyamide-oligonucleotide of the formula I. However, it
is also possible to use building blocks composed of PNA
and nucleotide, preferably dimers, which are then
assembled by the methods of nucleotide chemistry or
peptide chemistry to give polyamide-oligonucleotide
derivatives.
The assembly of the oligonucleotide part takes place by
processes known to the skilled worker, such as the
triester method, the H-phosphonate method or phosphor-
amidite method, preferably by the standard phosphor-
amidite chemistry of Caruthers (M.D. Matteucci and
M.H. Caruthers, J. Am. Chem. Soc. 103, 3185 (1981)). The
polyamide part can be synthesized by the methods of
peptide chemistry known to the skilled worker. If the
oligonucleotide part and polyamide part are not sepa-
rately synthesized and subsequently linked, the processes
used to assemble the oligonucleotide structure and
polyamide structure must be mutually compatible, in which
connection a preferred embodiment of the synthesis of the
polyamide part is described in the simultaneously filed
application with the title "PNA synthesis using an amino
2144475
- 25 -
protective group which is labile to weak acids"
(HOE 94/F 060, DE-P 44 08 531.1).
Depending on whether q, r, s and t are 1 or zero, the
synthesis starts with the oligonucleotide part or with
the polyamide part. The synthesis of compounds of the
formula I whose oligonucleotide part is modified at the
3' and/or at the 5' end takes place in respect of these
modifications by the processes described in
EP-A 0 552 766 (HOE 92/F 012) (compare synthetic scheme
for DNA). The synthesis of compounds of the formula I
takes place in respect of the polyamide part by the
process described in the simultaneously filed application
with the title "PNA synthesis using an amino protective
group which is labile to weak acids (HOE 94/F 060, DE-P
44 08 531.1) (compare synthetic scheme for PNA).
Synthetic scheme for DNA
[anchor group]-[polymer]
1. 1 coupling on of PG-(Nu')-active
PG-(Nu')-[anchor group]-[polymer]
2. 1 elimination of protective group PG
H-(Nu')-[anchor group]-[polymer]
3. 1 repetition of steps 1 and 2(n-1) times
H-(Nu')n-[anchor group]-[polymer]
4. 1 coupling on of R -V-active
R0-V-(Nu')n-[anchor group]-[polymer]
5. 1 elimination of polymer and protective groups
RO-V-(Nu)n
Synthetic scheme for PNA
[anchor group]-[polymer]
1. 1 coupling on of PG-(Q')-OH
PG-(Q')-[anchor group]-[polymer]
2. 1 elimination of protective group PG
H-(Q')-[anchor group]-[polymer]
3. 1 repetition of steps 1 and 2 (1-1) times
H-(Q')1-[anchor group]-[polymer]
4. 1 coupling on of PG-[B'/X]-OH
2144475
- 26 -
PG-[B'/X]-(Q')1-[anchor group]-[polymer]
5. 1 elimination of protective group PG
H-[B'/X]-(Q')1-[anchor group]-[polymer]
6. 1 repetition of steps 4 and 5(n-1) times
H-[B'/X]n-(Q')1-[anchor group]-[polymer]
7. 1 coupling on of PG-(A')-OH
PG-(A')-[B'/X]n-(Q')1-[anchor group]-[polymer]
8. 1 elimination of protective group PG
H-(A')-[B'/X]n-(Q')1-[anchor group]-[polymer]
9. 1 repetition of steps 7 and 8(k-1) times
H-(A')k-[B'/X]n-(Q')1-[anchor group]-[polymer]
10. 1 coupling on of the group R
R -(A')k-[B'/X]n-(Q')1-[anchor group]-[Polymer]
11. 1 elimination of polymer and protective groups
R0-(A)k-[B/X]n-(Q)1-Qo
The meanings in this are:
PG protective group, preferably a protective group labile
to weak acid;
Nu' nucleotide unit whose exocyclic amino group is pro-
tected by a suitable protective group;
Nu'-active an activated derivative customary in
nucleotide chemistry, such as, for example, of a phos-
phoramidite, a phosphodiester or an H-phosphonate;
A', B' and Q' are the forms of A, B and Q which are
protected where appropriate.
Synthetic scheme for PNA/DNA hybrids of the formula I
F[(DNA-Li)q(PNA-Li)=(DNA-Li)5(PNA)t],F' (I)
For q = r = s = t = 1 and x = 1, the following outline of
the synthesis applies:
1. 1 synthesis of the end group F'; where appro-
priate conjugation to polymer
PG-F'
2. 1 elimination of protective group PG
H-F'
2144475
- 27 -
3. 1 conjugation of the polyamide structure
PNA-F'
4. 1 coupling on of a linker
Li-PNA-F'
5. 1 conjugation of the nucleotide structure
DNA-Li-PNA-F'
6. 1 coupling on of a linker
Li-DNA-Li-PNA-F'
7. 1 repetition of steps 3 to 5
DNA-Li-PNA-Li-DNA-Li-PNA-F'
8. 1 coupling on of the end group F
F-DNA-Li-PNA-Li-DNA-Li-PNA-F'
The coupling on of the linker building block can be
omitted if appropriate junctions are present in the PNA
or DNA building blocks.
For clarification, a synthetic scheme for PNA/DNA
hybrids of the formula I is shown and explains by way
of example the preparation of a hybrid oligomer in which
q = r = s = t = 1 and x = 1. Initially, the end group F
is synthesized by known processes and, in the case of
solid-phase synthesis, coupled to a polymeric support
(step 1). After elimination of the protective group PG
(step 2), which preferably takes place in weakly acidic
medium, the polyamide building blocks are coupled on to
the desired length of the PNA part (step 3). As junction
to the DNA part it is now possible to attach a linker
unit (step 4). The conjugation of the nucleotide struc-
ture then takes place by successive condensation on of
the nucleotide building blocks (step 5), preferably by
the known phosphoramidite method. After a linker which
makes it possible to join DNA to PNA has been condensed
on (step 6), in turn a polyamide structure is assembled.
Introduction of a linker which makes it possible to join
PNA to DNA, conjugation of another DNA structure (step 7)
and final coupling on of the end group F (step 8) result
in the hybrid molecule [F-DNA-Li-PNA-Li-DNA-Li-PNA-F'].
The linker building blocks can in this case also
2144475
- 28 -
contain nucleotide bases. To synthesize a hybrid
F-DNA-Li-PNA-Li-F' (q = r = 1, s = t = zero), for example
first steps 1-5 are carried out and then the synthesis
is completed with step 8.
To synthesize a hybrid F-PNA-Li-DNA-F' (r = s = 1,
q = t = zero), for example first steps 1-2 are carried
out, then steps 5-6 follow, followed by step 3 and
completion of the synthesis with step 8.
To synthesize a hybrid F-PNA-Li-DNA-Li-PNA-F'
(r = s = t = 1, q = zero), the synthesis starts with
steps 1-6. After repetition of step 3, the synthesis is
completed with step 8.
If x in formula I is > 1, then steps 2-7 must be repeated
where appropriate. After assembly of the polymeric
chains, the PNA/DNA hybrids must in the case of solid-
phase synthesis be cleaved off the support and, where
appropriate, the protective groups on the bases, amino-
acid side chains and end groups must be eliminated.
However, the PNA part and DNA part can also be
synthesized separately by known methods and subsequently
coupled together via appropriate activation of at least
one component. Activation of the PNA part preferably
takes place via the carboxylic acid group, for example as
active ester or isothiocyanate, which are then reacted
with reactive groups in the DNA part, preferably an amino
group. Activation of the DNA part takes place, for
example, in the form of a cyanogen bromide condensation
known per se, in which the activated phosphate
functionality is reacted with a reactive group in the PNA
part, preferably an amino group.
It has been found, surprisingly, that the oligomers of
the formula Ia and Ib have a greatly increased cellular
uptake by comparison with pure PNAs. This improved
cellular uptake is very crucial because antisense- or
2144475
- 29 -
triplex-forming oligomers are able to act only if they
are efficiently taken up by cells. Their hybridization
behavior is likewise more favorable than in the case of
pure PNAs because they preferentially lead to anti-
parallel duplex formation. Compared with normal
oligonucleotides, they have an improved nuclease stabi-
lity, which is expressed by an increased biological
activity. The binding affinity to complementary nucleic
acids is better than the other nuclease-stable oligo-
nucleotides such as, for example, phosphorothioates or
methylphosphonates. The binding affinity of the compounds
according to the invention is at least equally good, but
usually better, by comparison with natural oligonucleo-
tides, which are rapidly degraded under serum conditions.
The increase in the binding affinity depends on the
length of the PNA part. Pure PNAs showed a potent
cytotoxic effect at concentrations > 5 M in cell-culture
experiments, whereas the compounds according to the
invention did not damage the cells. It has furthermore
been found that compounds of the formula I inhibit,
depending on the base sequence of the PNA part and DNA
part, the expression of specific genes, for example of
enzymes, receptors or growth factors, in cell culture and
in selected examples in animal models.
Further advantages of the PNA/DNA oligomers and PNA/RNA
oligomers comprise the possibility of stimulating
cellular endonucleases such as, for example, RNase H and
RNase L. In contrast to PNAs, the PNA-DNA chimeras
according to the invention which have some deoxyribo-
nucleotide units are able, after binding to the comple-
mentary target RNA, to cleave the latter in a sequence-
specific manner owing to induction of cellular RNase H.
A particular embodiment of the oligomers according to the
invention furthermore comprises those which are composed
of PNA part and a 2',5'-linked oligoadenylate part,
preferably tetraadenylate or its cordycepin analog, and
which activate cellular RNase L.
2144475
- 30 -
The present invention extends very generally to the use
of compounds of the formula I as therapeutically active
ingredients of a pharmaceutical. By therapeutically
active polyamide-oligonucleotide derivatives is meant in
general antisense oligonucleotides, triple helix-forming
oligonucleotides, aptamers or ribozymes, especially
antisense oligonucleotides.
The pharmaceuticals of the present invention can be used,
for example, to treat diseases caused by viruses, for
example by HIV, HSV-1, HSV-2, influenza, VSV, hepatitis
B or papilloma viruses.
Antisense polyamide-oligonucleotide derivatives according
to the invention which are active against such targets
have, for example, the following base sequence. The
length and position of the PNA part and DNA part in these
sequences can be altered appropriately to achieve optimal
properties.
a) against HIV, for example
5'-A C A C C C A A T T C T G A A A A T G G-3 ' or
(I)
5'-A G G T C C C T G T T C G G G C G C C A-3' or
(II)
5'-G T C G A C A C C C A A T T C T G A A A A T G G A T A A-3 ' or
(III)
5'-G C T A T G T C G A C A C C C A A T T C T G A A A-3' or
(IV)
5'-T C G T C G C T G T C T C C G C T T C T T C T T C C T G C C A or
(VI)
b) against HSV-1, for example
2144475
- 31 -
5'-G C G G G G C T C C A T G G G G G T C G-3 '
(VII)
The pharmaceuticals of the present invention are also
suitable, for example, for the treatment of cancer. In
this connection, it is possible to use, for example,
polyamide-oligonucleotide sequences which are directed
against targets which are responsible for the development
of cancer or the growth of cancers. Examples of such
targets are:
1) nuclear oncoproteins such as, for example, c-myc,
N-myc, c-myb, c-fos, c-fos/jun, PCNA, p120
2) cytoplasmic/membrane-associated oncoproteins such as,
for example, EJ-ras, c-Ha-ras, N-ras, rrg, bcl-2, cdc-2,
c-raf-1, c-mos, c-src, c-abl
3) cellular receptors such as, for example, the EGF
receptor, c-erbA, retinoid receptors, protein kinase
regulatory subunit, c-fms
4) cytokines, growth factors, extracellular matrix such
as, for example, CSF-1, IL-6, IL-la, IL-lb, IL-2, IL-4,
bFGF, myeloblastin, fibronectin.
Antisense polyamide-oligonucleotides of the formula I
according to the invention which are active against such
targets have, for example, the following base sequence:
a) against c-Ha-ras, for example
5'-C A G C T G C A A C C C A G C-3'
(VIII)
c) c-myc, for example
5'-G G C T G C T G G A G C G G G G C A C A C-3'
(IX)
2144475
- 32 -
5'-A A C G T T G A G G G G C A T-3'
(X)
d) c-myb, for example
5'-G T G C C G G G G T C T T C G G G C-3 '
(XI)
e) c-fos, for example
5'-G G A G A A C A T C A T G G T C G A A A G-3 '
(XII)
5'-C C C G A G A A C A T C A T G G T C G A A G-3'
(XIII)
5'-G G G G A A A G C C C G G C A A G G G G-3
(XIV)
f) p120, for example
5'-C A C C C G C C T T G G C C T C C C A C-3'
(XV)
g) EGF receptor, for example
5'-G G G A C T C C G G C G C A G C G C-3 '
(XVI)
5'-G G C A A A C T T T C T T T T C C T C C-3
(XVII)
h) p53 tumor suppressor, for example
5'-G G G A A G G A G G A G G A T G A G G-3'
(XVIII)
5'-G G C A G T C A T C C A G C T T C G G A G-3'r
(XIX)
2144475
- 33 -
The pharmaceuticals of the present invention are further-
more suitable, for example, for the treatment of diseases
which are influenced by integrins or cell-cell adhesion
receptors, for example by VLA-4, VLA-2, ICAM, VCAM or
ELAM.
Antisense polyamide-oligonucleotide derivatives according
to the invention which are active against such targets
have, for example, the following base sequence:
a) VLA-4, for example
5'-G C A G T A A G C A T C C A T A T C-3' or
(XX)
b) ICAM, for example
5'-C C C C C A C C A C T T C C C C T C T C-3'
(XXI)
5'-C T C C C C C A C C A C T T C C C C T C-3 '
(XXII)
5'-G C T G G G A G C C A T A G C G A G G-3'
(XXIII)
c) ELAM-1, for example
5'-A C T G C T G C C T C T T G T C T C A G G-3'
(XXIV)
5'-C A A T C A A T G A C T T C A A G A G T T C-3'
(XXV)
The pharmaceuticals of the present invention are also
suitable, for example, for preventing restenosis. In this
connection, examples of polyamide-oligonucleotide
sequences which can be used are those directed against
targets which are responsible for proliferation or
migration. Examples of such targets are:
2144475
- 34 -
1) nuclear transactivator proteins and cyclins such as,
for example, c-myc, c-myb, c-fos, c-fos/jun, cyclins and
cdc2 kinase
2) mitogens or growth factors such as, for example, PDGF,
bFGF, EGF, HB-EGF and TGF-R
3) cellular receptors such as, for example, bFGF recep-
tor, EGF receptor and PDGF receptor.
Antisense polyamide-oligonucleotides according to the
invention of the formula I which are active against such
targets have, for example, the following base sequence:
a) c-myb
5'-G T G T C G G G G T C T C C G G G C-3'
(XXVI)
b) c-myc
5'-C A C G T T G A G G G G C A T-3'
(XXVII)
c) cdc2 kinase
5'-G T C T T C C A T A G T T A C T C A-3 '
(XXVIII)
d) PCNA (proliferating cell nuclear antigen of rat)
5'-G A T C A G G C G T G C C T C A A A-3 '
(XXIX)
The pharmaceuticals can be used, for example, in the form
of pharmaceutical products which can be administered
orally, for example in the form of tablets, coated
tablets, hard or soft gelatin capsules, solutions, emul-
sions or suspensions. Inclusion of the pharmaceuticals in
liposomes, which optionally contain further components
21444 75
- 35 -
such as proteins, is a likewise suitable administration
form. They can also be administered rectally, for example
in the form of suppositories, or parenterally, for
example in the form of injection solutions. To produce
pharmaceutical products, these compounds can be processed
in therapeutically inert organic and inorganic exci-
pients. Examples of such excipients for tablets, coated
tablets and hard gelatin capsules are lactose, corn
starch or derivatives thereof, talc and stearic acid or
salts thereof. Suitable excipients for producing
solutions are water, polyols, sucrose, invert sugar and
glucose. Suitable excipients for injection solutions are
water, alcohols, polyols, glycerol and vegetable oils.
Suitable excipients for suppositories are vegetable and
hardened oils, waxes, fats and semiliquid polyols. The
pharmaceutical products may also contain preservatives,
solvents, stabilizers, wetting agents, emulsifiers,
sweeteners, colorants, flavorings, salts to alter the
osmotic pressure, buffers, coating agents, antioxidants
and, where appropriate, other therapeutic active
substances.
Preferred administration forms are topical applications,
local applications such as, for example, with the aid
of a catheter or else injections. For injection, the
antisense polyamide-oligonucleotide derivatives are
formulated in a liquid solution, preferably in a physio-
logically acceptable buffer such as, for example, Hank's
solution or Ringer's solution. The antisense polyamide-
oligonucleotides can, however, also be formulated in
solid form and be dissolved or suspended before use. The
dosages preferred for systemic administration are about
0.01 mg/kg to about 50 mg/kg of body weight and day.
The invention extends very generally to the use of
compounds of the formula I as DNA probes or primers in
DNA diagnosis, in particular in the sense of the gene
probes mentioned in HOE 92/F 406 (EP-A 0 602 524), and
generally as aids in molecular biology.
2144475
- 36 -
Gene probes, also called DNA probes or hybridization
probes, play a large part in DNA diagnosis for sequence-
specific detection of particular genes. A gene probe is
generally composed of a recognition sequence and of a
suitable labeling group (label). The specificity of the
determination of a target sequence in an analytical
sample by hybridization with a complementary gene probe
is determined by the recognition sequence and its chemi-
cal structure. The PNAs have the advantage, compared with
oligonucleotides of natural structure, that they have a
higher affinity for the target sequence. However, the
specificity of the hybridization is reduced because PNAs,
in contrast to natural DNA, are able to bind both in
parallel and in antiparallel orientation to single-
stranded nucleic acids. The PNA/DNA oligomers according
to the invention likewise show an increased binding
affinity but very preferentially bind in the desired
antiparallel orientation.
It is moreover possible, by appropriate selection of the
PNA part and DNA part in a gene probe, to have a bene-
ficial effect on the differentiation capacity because
base mispairing in the PNA part leads to a greater
depression of the melting temperature of a hybrid than
does a base mispairing in the DNA part. This is particu-
larly important with regard to differentiation in the
case of point mutations as occur, for example, in the
transition from protooncogenes into the corresponding
oncogenes (pathogenic state). The advantage of the better
discrimination between pathogenic and non-pathogenic
state can also be utilized in the form of the primer pro-
perty of the PNA/DNA oligomers according to the invention
as long as these have a free 3'-hydroxyl group in the DNA
part. PNAs as such have no primer function for poly-
merases. It has been found, surprisingly, that even one
nucleoside unit at the end of a PNA/DNA oligomer is
sufficient to initiate the DNA polymerase reaction, for
example with DNA polymerase (Klenow fragment). Various
polymerases can be employed depending on the characteris-
2144475
- 37 -
tics of the PNA/DNA primer and the nature of the template
onto which the primer hybridizes in a sequence-specific
manner. These polymerases are generally commercially
available, such as, for example, Taq polymerase, Rlenow
polymerase or reverse transcriptase.
Another advantage by comparison with the use of natural
oligonucleotide primers is that the nucleic acid strand
which is copied with the aid of the PNA/DNA primer and
which contains the PNA part at the 5' end is stable to
5'-exonucleases. It is thus possible to degrade all
natural DNA or RNA sequences in the reaction mixture by
5'-exonucleases without attack on the PNA-containing
strand.
Another advantage of the PNA/DNA oligomers is that they
can also be used to carry out other biochemical reactions
on the DNA part which are impossible with PNAs them-
selves. Examples of such reactions are the 3'-tailing
with 3'-terminal transferase, the digestion with restric-
tion enzymes in the DNA double-stranded region, and
ligase reactions. For example, a (PNA)-(DNA)-OH oligomer
with free 3'-hydroxyl group can be linked to a second
p-(DNA)-(PNA) oligomer which contains a nucleoside
5'-phosphate at the 5' end after hybridization to a
complementary DNA auxiliary sequence of natural origin in
the presence of a DNA ligase.
(DNA)-(PNA)-(DNA) oligomers can furthermore be incorpora-
ted into genes, which is not at present possible with
PNAs.
The linkage of labeling groups onto PNA/DNA oligomers
takes place by methods known per se, as described for
oligonucleotides or peptides. The nature of the labeling
group can vary within wide limits and depends essentially
on the type of assay used. Known embodiments of gene
probe assays are the hybridization protection assay, the
energy transfer assay and the kissing probes assay.
2144475
- 38 -
PNA/DNA oligomers are additionally particularly suitable
for a strand displacement assay. In many cases it is
advantageous to remove the hybrid which is formed from
excess gene probe with the aid of magnetic particles. The
stability of the PNA/DNA gene probes according to the
invention is higher than that of conventional DNA probes.
Polymerase chain reaction (PCR) and ligase chain reaction
(LCR) are techniques for target amplification in which
the oligomers according to the invention can likewise be
used as primers. The PNA/DNA oligomers can be used par-
ticularly advantageously as gene probes on the Christmas
tree principle because in this case the PNA/DNA probes
can be shorter than corresponding DNA probes.
Examples:
The abbreviations used for amino acids correspond to the
three-letter code customary in peptide chemistry, as
described in Europ. J. Biochem. 138, 9 (1984). Other
abbreviations used are listed below.
Aeg N-(2-Aminoethyl)glycyl,
-NH-CH2-CH2-NH-CH2-CO-
Aeg (aMeosz ) N- (2-Aminoethyl) -N- (N6- ( 4-methoxybenzoyl ) -
9-adenosylacetyl)-glycyl
Aeg(cBz) N-(2-Aminoethyl)-N-(N4-benzoyl-l-cytosyl-
acetyl)-glycyl
Aeg(cMeosz) N-(2-Aminoethyl)-N-(N4-(4-methoxybenzoyl)-
1-cytosylacetyl)-glycyl
Aeg ( ctB"Bz ) N- ( 2 -Aminoethyl ) -N- ( N4 - ( 4 -tert . butyl -
benzoyl)-1-cytosylacetyl)-glycyl
Aeg(gl$u) N- ( 2-Aminoethyl ) -N- (N2 -isobutanoyl-
9-guanosylacetyl)-glycyl
Aeg (g2-Ac, 4-np ) N- (2-Aminoethyl) -N- (N2-acetyl-04-diphenyl-
carbamoyl-9-guanosyl)glycyl
Aeg(t) N-(2-Aminoethyl)-N-((1-thyminyl)acetyl)-
glycyl
Bnpeoc 2,2-[bis(4-Nitrophenyl)]-ethoxycarbonyl)
Boc tert.-butyloxycarbonyl
2144475
- 39 -
BOI 2-(Benzotriazol-l-yloxy)-1,3-dimethyl-
imidazolidinium hexafluorophosphate
BOP Benzotriazolyl-l-oxy-tris(dimethylamino)-
phosphonium hexafluorophosphate
BroP Bromo-tris(dimethylamino)phosphonium
hexafluorophosphate
BSA N,O-bis(Trimethylsilyl)-acetamide
But tert.-butyl
Bz Benzoyl
Bzl Benzyl
C1-Z 4-Chloro-benzyloxycarbonyl
CPG Controlled pore glass
DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene
DCM Dichloromethane
Ddz 2-(3,5-Dimethoxyphenyl)-2-propyloxycar -
bonyl
DMF Dimethylformamide
Dmt di-(4-Methoxyphenyl)phenylmethyl
Dnpeoc 2-(2,4-Dinitrophenyl)-ethoxycarbonyl
Dpc Diphenylcarbamoyl
FAM Fluorescein.residue
Fm 9-Fluorenylmethyl
Fmoc 9-Fluorenylmethyloxycarbonyl
H-Aeg-OH N-(2-Aminoethyl)glycine
HAPyU O-(7-Azabenzotriazol-l-yl)-1,1,3,3-bis -
(tetramethylene)uronium hexafluorophos-
phate
HATU O-(7-Azabenzotriazol-1-yl)-1,1,3,3-tetra-
methyluronium hexafluorophosphate
HBTU O-(Benzotriazol-1-yl)-1,1,3,3-tetra-
methyluronium hexafluorophosphate
HOBt 1-Hydroxybenzotriazole
HONSu N-Hydroxysuccinimide
HOObt 3-Hydroxy-4-oxo-3,4-dihydrobenzotriazine
iBu Isobutanoyl
MeOBz 4-Methoxybenzoyl
Mmt 4-Methoxytriphenylmethyl
Moz 4-Methoxybenzyloxycarbonyl
2144475
- 40 -
MSNT 2-Mesitylenesulfonyl-3-nitro-1,2,4-tria-
zole
Mtt (4-Methylphenyl)diphenylmethyl
NBA Nitrobenzyl alcohol
NMP N-Methylpyrrolidine
Obg N-(4-Oxybutyl)glycyl, -O-(CH2)4-NH-CH2-CO-
Obg(t) N-(4-Oxybutyl)-N-((1-thyminyl)acetyl)-
glycyl
Oeg N-(2-Oxyethyl)glycyl,
-O-CH2-CH2-NH-CH2-CO-
oeg(t) N-(2-Oxyethyl)-N-((i-thyminyl)acetyl)-
glycyl
Opeg N-(5-Oxypentyl)glycyl,
-O-(CH2)5-NH-CH2-CO-
opeg(t) N-(5-Oxypentyl)-N-((1-thyminyl)acetyl)-
glycyl
Oprg N-(3-Oxypropyl)glycyl,
-0-(CH2)3-NH-CH2-CO-
Oprg(t) N-(3-Oxypropyl)-N-((1-thyminyl)acetyl) -
glycyl
Pixyl 9-(9-Phenyl)xanthenyl
PyBOP Benzotriazolyl-l-oxytripyrrolidinophos -
phonium hexafluorophosphate
PyBroP Bromotripyrrolidinophosphonium hexa-
fluorophosphate
TAPipU 0-(7-Azabenzotriazol-1-yl)-1,1,3,3-bis-
(pentamethylene)uronium tetrafluoroborate
TBTU 0-(Benzotriazol-1-yl)-1,1,3,3-tetra-
methyluronium tetrafluoroborate
tBu tert.-Butyl
tBuBz 4-tert.Butylbenzoyl
TDBTU O-(3,4-Dihydro-4-oxo-1,2,3-benzotriazin-
3-yl)-1,1,3,3-tetramethyluronium tetra-
fluoroborate
TDO 2,5-Diphenyl-2,3-dihydro-3-oxo-4-hydroxy-
thiophene dioxide
Teg N-(2-Thioethyl)glycyl,
-S-CH2-CH2-NH-CH2-CO-
2144475
- 41 -
Teg(t) N-(2-Thioethyl)-N-((1-thyminyl)acetyl) -
glycyl
TFA Trifluoroacetic acid
THF Tetrahydrofuran
TNTU O-(5-Norbornene-2,3-dicarboximido)-
1,1,3,3-tetramethyluronium tetrafluoro-
borate
TOTU O-[(Cyano(ethoxycarbonyl)methylene)-
amino]-1,1,3,3-tetramethyluronium tetra-
fluoroborate
TPTU O-(1,2-Dihydro-2-oxo-l-pyridyl)-1,1,3,3-
tetramethyluronium tetrafluoroborate
Trt Trityl
TSTU O-(N-Succinimidyl)-1,1,3,3-tetramethyl-
uronium tetrafluoroborate
Z Benzyloxycarbonyl
MS(ES+) Electrospray mass spectrum (positive ion)
MS(ES ) Electrospray mass spectrum (negative ion)
MS(DCI) Desorption chemical ionization mass
spectrum
MS(FAB) Fast atom bombardment mass spectrum
Example 1
1-Hydroxy-6-((4-methoxyphenyl)-diphenylmethylamino)-
hexane
Mmt-hex
6-Amino-1-hexanol (1 g; 8.55 mmol) is dissolved in
anhydrous pyridine (7 ml), and triethylamine (0.2 ml) is
added. To this solution is added over the course of
45 minutes a solution of (4-methoxyphenyl)diphenylmethyl
chloride (2.5 g; 8.12 mmol) in anhydrous pyridine (9 ml).
The reaction solution is stirred further at 22 C for
30 minutes and stopped by adding methanol (3 ml). The
solution is concentrated in a rotary evaporator, and the
resulting residue is coevaporated with toluene three
times to remove the pyridine. The resulting residue is
dissolved in ethyl acetate, and this solution is washed
successively with a saturated sodium bicarbonate solu-
2144475
- 42 -
tion, with water and with a saturated potassium chloride
solution. The organic phase is dried (Na2SO4), filtered
and concentrated in vacuo. The crude product is purified
by silica gel chromatography using heptane:ethyl acetate:
triethylamine/49.5:49.5:1.
Yield: 1.64 g
MS (FAB,NBA/LiCl) 396.3 (M + Li)+, 390.3 (M + H)+, 273.2
(Mmt)+
Rf 0.44 (heptane:ethyl acetate = 1:1)
Example 2
6-((4-Methoxyphenyl)diphenylmethylamino)-1-hexyl hemi-
succinate
Mmt-hex-succ
1-Hydroxy-6-((4-methoxyphenyl)diphenylmethylamino)hexane
(1.00 g; 2.57 mmol) is dissolved in anhydrous pyridine
(10 ml). To this solution are added succinic anhydride
(0.257 g; 2.57 mmol) and 4-dimethylaminopyridine
(31.3 mg; 0.257 mmol). After stirring at 22 C for
3 hours, further succinic anhydride (25.7 mg; 0.257 mmol)
and 4-dimethylaminopyridine (62.6 mg; 0.56 mmol) are
added, and this solution is heated at 50 C for 6 hours.
After a further 16 hours at 22 C, the mixture is concen-
trated, the residue is taken up in ethyl acetate, and the
resulting solution is washed with ice-cold 5% strength
aqueous citric acid. After the org. phase has been dried
(Na2SO4)1 the solution is concentrated in a rotary
evaporator. Purification of the residue by silica gel
chromatography using 50% CH2C12/1% triethylamine in ethyl
acetate and then using 5% methanol/1% triethylamine in
dichloromethane affords the required compound as color-
less oil.
MS (ES ) 978.0 (2M-H) , 488.3 (M-H)
Rf 0.30 (CH2C12:ethyl acetate = 1:1).
2144475
- 43 -
Example 3
6-((4-Methoxyphenyl)diphenylmethylamino)-1-hexylsuccinyl-
amido-Tentagel
(Mmt-hex-succ-Tentagel)
The amino form of TentagelR (Rapp polymers) (0.5 g;
0.11 mmol amino groups) is left to swell in 4-ethylmor-
pholine (0.1 ml) and DMF (5 ml) for 10 minutes. A solu-
tion of 6-((4-methoxyphenyl)diphenylmethylamino)-1-hexyl
hemisuccinate (97.4 mg; 0.165 mmol), 4-ethylmorpholine
(15.9 mg; 0.138 mmol; 17.4 ml) and TBTU (52.9 mg;
0.165 mmol) in DMF (3 ml) is then added, and the suspen-
sion is shaken at 22 C for 16 hours. The derivatized
Tentagel support is filtered off and washed successively
with DMF (3 x 3 ml), CH2C12 (3 x 1 ml) and diethyl ether
(3 x 1 ml) and dried. Unreacted amino groups are blocked
by treatment with acetic anhydride/lutidine/1-methyl-
imidazole in THF (1 ml) for 1 hour. The completed support
is washed with CH2C12 (3 x 1 ml) and diethyl ether
(3 x 1 ml) and dried in vacuo. Based on the monomethoxy-
trityl group introduced, the loading is 168 mmolg-1.
Example 4
6-((4-Methoxyphenyl)diphenylmethylamino)-1-hexylsuccinyl-
amidopropyl-controlled pore glass.
(Mmt-hex-succ-CPG)
The preparation takes place in analogy to the description
in Example 3 starting from aminopropyl-CPG (supplied by
Fluka) (550 Angstrom; 1.0 g) and 6-((4-methoxyphenyl)-
diphenylmethylamino)-1-hexyl hemisuccinate (48.7 mg;
0.082 mmol), 4-ethylmorpholine (7.6 ml) and TBTU
(26.4 mg; 0.082 mmol) in DMF (3 ml). The loading of the
Mmt-hex-succCPG is 91 mmolg-1.
2144475
- 44 -
Example 5
N-((4-Methoxyphenyl)diphenylmethylamino)ethyl-
N-(N4-(4-tert-butylbenzoyl)-1-cytosylacetyl)glycine
( Mmt-Aeg ( ctBuBz ) -OH)
1.63 g (2.28 mmol) of N-((4-methoxyphenyl)diphenylmethyl-
amino)ethyl-N-(N4-(4-tert-butylbenzoyl)-1-cytosylacetyl)-
glycine methyl ester were dissolved in a mixture of 10 ml
of dioxane and 1 ml of water and, while stirring at 0 C,
4.56 ml of 1 N NaOH were added dropwise. After 2 h, the
pH was adjusted to 5 by dropwise addition of 1 N RHSOQ,
and precipitated salts were filtered off and washed with
a little dioxane. The combined filtrates were evaporated
in vacuo, and the residue was coevaporated twice with
methanol and dichloromethane. The crude product obtained
in this way was purified by chromatography on silica gel
using a gradient of 2-10% methanol and 1% triethylamine
in dichloromethane. The fractions containing the product
were combined and concentrated in vacuo. Excess triethyl-
amine still present was removed by coevaporation with
pyridine and then toluene. 0.831 g of product was
obtained as an almost white foam.
Electrospray MS (negative ion) 700.7 (M-H) .
Rf 0.28 (CH2C12:MeOH/9:1), 0.63 (CH2C12:MeOH/7:3).
Example 6
N-((4-Methoxyphenyl)diphenylmethylamino)ethyl-N-((1-thy-
minyl)acetyl)glycine
(Mmt-Aeg(t))-OH
The product from the above reaction was dissolved in a
mixture of 10 ml of dioxane and 2 ml of water, the
solution was cooled to 0 C, and 1 N sodium hydroxide
solution was added dropwise until the pH reached 11.
After a reaction time of 2 h, the reaction was complete
and the solution was adjusted to pH 5 by cautious addi-
tion of 2 N RHSO4 solution. The solution was extracted
three times with ethyl acetate, and the combined organic
phases were dried over sodium sulfate and concentrated in
2144475
- 45 -
vacuo. The crude product obtained in this way was puri-
fied by chromatography on silica gel using a gradient of
5-10% methanol and 1% triethylamine in dichloromethane.
The fractions containing the product were combined and
concentrated in vacuo. Excess triethylamine still present
was removed by coevaporation with pyridine and then
toluene. 1.065 g of product were obtained as a colorless
foam.
Electrospray MS (negative ion) 1112.0 (2M-H) , 555.3
(M-H)
Rf 0.28 (CH2C12:MeOH/8:2).
Example 7
N-((4-Methoxyphenyl)diphenylmethylamino)ethyl-
N-(N2-isobutanoyl-9-guanosylacetyl)glycine
(Mmt-Aeg (g1B71) -OH
N-((4-Methoxyphenyl)diphenylmethylamino)ethyl-N-(NZ-iso-
butanoyl-9-guanosylacetyl)glycine methyl ester (1.15 g;
1.72 mmol) is dissolved in dioxane (10 ml) and, at 0 C,
1 M aqueous sodium hydroxide solution (10.32 ml) is added
dropwise in 5 portions over a period of 2.5 h. After a
further reaction time of 2 h at room temperature, the
solution is adjusted to pH 5 by dropwise addition of 2 M
aqueous potassium bisulfate solution. The precipitated
salts are filtered off and washed with a little dioxane.
The combined filtrates are evaporated to dryness in
vacuo, and the residue is coevaporated twice each with
ethanol and 1/1 dichloromethane:methanol. Purification
takes place by column chromatography on silica gel by
elution with a gradient of 10-20% methanol in dichloro-
methane (with 1% triethylamine). The product is obtained
as a white foam.
Yield: 1.229 g
ESMS (negative ion): 650.3 (M-H)
Rf 0.25 (dichloromethane:methanol/8:2)
2144475
- 46 -
Example 8
N-((4-Methoxyphenyl)diphenylmethylamino)ethyl-
N-(N6-(4-methoxybenzoyl)-9-adenosylacetyl)glycine
(Mmt-Aeg ( aMeoBZ ) -OH)
N-((4-Methoxyphenyl)diphenylmethylamino)ethyl-
N-(N6-(4-methoxybenzoyl)-9-adenosylacetyl)glycine methyl
ester (1.70 g; 2.38 mmol) is dissolved in dioxane (10 ml)
and, at 0 C, 1 M aqueous sodium hydroxide solution
(10.32 ml) is added dropwise in 5 portions over a period
of 2.5 h. After a further reaction time of 2 h at room
temperature, the solution is adjusted to pH 5 by dropwise
addition of 2 M aqueous potassium bisulfate solution. The
precipitated salts are filtered off and washed with a
little dioxane. The combined filtrates are evaporated to
dryness in vacuo, and the residue is coevaporated twice
each with ethanol and 1/1 dichloromethane:methanol.
Purification takes place by column chromatography on
silica gel by elution with a gradient of 10-20% methanol
in dichloromethane (with 1% triethylamine). The product
is obtained as a white foam.
Yield: 1.619 g
ESMS (negative ion): 698.3 (M-H)
Rf 0.10 (dichloromethane:methanol/8:2)
Example 9
N-((4-Methoxyphenyl)diphenylmethyloxy)ethyl-N-((1-thy-
minyl)acetyl)glycine
(Mmt-Oeg(t)-OH)
0.5 g (1.28 mmol) of N-((4-methoxyphenyl)diphenylmethyl-
oxy)ethylglycine was suspended in 10 ml of DMF, and
0.47 ml (1.92 mmol) of BSA was added dropwise. Then,
0.7 ml (5.1 mmol) of triethylamine and 0.26 g (1.28 mmol)
of chlorocarboxymethylthymine were successively added.
The reaction mixture was stirred at room temperature for
4 h and then a further 65 mg (0.32 mmol) of chlorocar-
boxymethylthymine were added, and the mixture was stirred
for 16 h. The solvent was then stripped off in vacuo, and
2144475
- 47 -
the crude product was purified on a silica gel column
using a gradient of 5-15% methanol and 1% triethylamine
in dichloromethane. The fractions containing the product
were combined and concentrated in vacuo. The resulting
brownish oil was dissolved in a little dichloromethane;
and the product was precipitated by adding diethyl ether.
The product was obtained as an almost white powder.
Yield: 0.219 g
Electrospray MS (negative ion) 556.3 (M-H) .
Rf 0.54 (CH2C12:MeOH/8:2).
Example 10
4-Nitrophenyl 4-(4,41-dimethoxytrityloxy)butyrate
Dmt-but-NPE
The sodium salt of 4-hydroxybutyric acid (1.26 g;
10 mmol) is dissolved in anhydrous pyridine (30 ml), and
4,4'-dimethoxytrityl chloride (3.39 g; 3.05 mmol) is
added. After 16 hours, 4-nitrophenol (1.39 g; 10 mmol)
and N,N'-dicyclohexylcarbodiimide (2.06 g; 10 mmol) are
added, and the mixture is stirred at 22 C for a further
48 hours. The precipitated dicyclohexylurea is filtered
off and washed with dichloromethane. The filtrate is
concentrated and the resulting residue is coevaporated
twice with toluene. The residue is purified on a silica
gel column (10-50% ethyl acetate and 1% triethylamine in
petroleum ether). The desired compound is obtained in the
form of a pale yellowish-colored oil.
Yield: 2.694 g
MS (FAB, MeOH/NBA/LiCl) 534.2 (M + Li)+, 527.2 M+.
Rf 0.34 (petroleum ether:ethyl acetate = 75:25)
Example 11
H-Oprg(t) -OH
3.68 g of thyminylacetic acid are dissolved in 20 ml of
dry DMF, and 6.65 g of TOTU and 2.77 ml of triethylamine
are added. The mixture is stirred at room temperature for
30 min and then slowly added dropwise to a solution
2144475
- 48 -
composed of 5.32 g of (3-hydroxypropyl)glycine, 20 ml of
water, 20 ml of DMF and 5.54 ml of triethylamine. The
mixture is stirred at room temperature for 1 h and then
concentrated in a rotary evaporator in vacuo. The residue
is taken up in water, adjusted to pH 1.5 with 1 N hydro-
chloric acid and extracted with ethyl acetate. The
aqueous phase is adjusted to pH 5 with saturated sodium
bicarbonate solution and concentrated in a rotary evapor-
ator. The residue is mixed with 250 ml of ethanol, and
the sodium chloride precipitated thereby is filtered off
with suction. The filtrate is concentrated and the crude
product is purified by chromatography on silica gel using
dichloromethane/methanol/ethyl acetate 10:2:1 with the
addition of 1% triethylamine followed by dichloromethane/
methanol/ethyl acetate 10:4:1 with the addition of 1%
triethylamine. The fractions containing the product are
combined and concentrated in a rotary evaporator in
vacuo.
Yield: 3.2 g
Rf 0.15 (dichloromethane/methanol/ethyl acetate 10:2:1
+ 1% triethylamine)
MS(ES+): 300.2 (M + H)
Example 12
Dmt-Oprg(t)-OH
3.2 q of H-Oprg(t)-OH are dissolved in 40 ml of DMF,
5.93 ml of triethylamine are added and, at 0 C, a solu-
tion of 7.25 g of Dmt-Cl in 40 ml of dichloromethane is
added dropwise over the course of 20 min. The mixture is
stirred at room temperature for 2 h, then the precipita-
ted triethylamine hydrochloride is filtered off, and the
filtrate is concentrated in a rotary evaporator in vacuo.
The residue is taken up in dichloromethane and extracted
with water, and the organic phase is dried with sodium
sulfate and concentrated in a rotary evaporator in vacuo.
The crude product is purified on silica gel using
dichloromethane/methanol/ethyl acetate 10:2:1 with the
addition of 1% triethylamine. The fractions containing
2144475
- 49 -
the product are combined and concentrated in a rotary
evaporator in vacuo.
Yield: 3.46 g
Rf 0.28 (dichloromethane/methanol/ethyl acetate 10:2:1
+ 1% triethylamine)
MS (ES+) 602.4 (M + H)'.
Example 13
H-Obg(t) OH
2.76 g of thyminylacetic acid are dissolved in 15 ml of
dry DMF, and 4.92 g of TOTU and 2.08 ml of triethylamine
are added. The mixture is stirred at room temperature for
30 min and then slowly added dropwise to a solution
composed of 4.41 g of (4-hydroxybutyl)glycine, 10 ml of
water, 10 ml of DMF and 4.16 ml of triethylamine. The
mixture is stirred at room temperature for 3 h and then
concentrated in a rotary evaporator in vacuo. The residue
is taken up in water, adjusted to pH 1.5 with 1 N hydro-
chloric acid and extracted with ethyl acetate. The
aqueous phase is adjusted to pH 5 with saturated sodium
bicarbonate solution and concentrated in a rotary
evaporator. The crude product is purified by chromato-
graphy on silica gel using dichloromethane/methanol/ethyl
acetate 10:2:1 with the addition of 1% triethylamine. The
fractions containing the product are combined and concen-
trated in a rotary evaporator in vacuo.
Yield: 3.7 g
Rf 0.11 (dichloromethane/methanol/ethyl acetate 10:2:1
+ 1% triethylamine)
MS (ES+) 314.2 (M + H)+.
Example 14
Dmt-Obg(t)-OH
3.6 g of H-Obg(t)-OH are dissolved in 40 ml of DMF,
9.5 ml of triethylamine are added and, at 0 C, a solution
of 15.4 g of Dmt-Cl in 40 ml of dichloromethane is added
dropwise over the course of 15 min. The mixture is
2144475
-50-
stirred at room temperature for 2 h, a further 40 ml of
dichloromethane are added, then the precipitated
triethylamine hydrochloride is filtered off, and the
filtrate is concentrated in a rotary evaporator in vacuo.
The residue is taken up in dichloromethane and extracted
with water, and the organic phase is dried with sodium
sulfate and concentrated in a rotary evaporator in vacuo.
The crude product is purified on silica gel using
dichloromethane/methanol/ethyl acetate 15:1:1 with the
addition of 1% triethylamine. The fractions containing
the product are combined and concentrated in a rotary
evaporator in vacuo.
Yield: 3.45 g
Rf 0.29 (dichloromethane/methanol/ethyl acetate 10:2:1
+ 1% triethylamine)
MS (ES+ + LiCl) 622.3 (M + Li)+.
Example 15
H-Opeg(t) OH
2.76 g of thyminylacetic acid are dissolved in 15 ml of
dry DMF, and 4.92 g of TOTU and 2.08 ml of triethylamine
are added. The mixture is stirred at room temperature for
min and then slowly added dropwise to a solution
composed of 4.83 g of (5-hydroxypentyl)glycine, 10 ml of
water, 10 ml of DMF and 4.16 ml of triethylamine. The
25 mixture is stirred at room temperature for 3 h and then
concentrated in a rotary evaporator in vacuo. The residue
is taken up in water, adjusted to pH 1.5 with 1 N hydro-
chloric acid and extracted with ethyl acetate. The
aqueous phase is adjusted to pH 5 with saturated sodium
30 bicarbonate solution and concentrated in a rotary
evaporator. The crude product is purified by chromato-
graphy on silica gel using dichloromethane/methanol/ethyl
acetate 10:2:1 with the addition of 1% triethylamine. The
fractions containing the product are combined and concen-
trated in a rotary evaporator in vacuo.
21~~473-
- 51 -
Yield: 3.34 g
Rf 0.19 (dichloromethane/methanol/ethyl acetate 10:2:1
+ 1% triethylamine)
MS (DC1) 328.2 (M + H)+.
Example 16
Dmt-Opeg(t)-OH
3.2 g of H-Opeg(t)-OH are dissolved in 40 ml of DMF,
6.77 ml of triethylamine are added and, at 0 C, a solu-
tion of 9.94 g of Dmt-Cl in 40 ml of dichloromethane is
added dropwise over the course of 15 min. The mixture is
stirred at room temperature for 2 h, a further 40 ml of
dichioromethane are added, then the precipitated
triethylamine hydrochloride is filtered off, and the
filtrate is concentrated in a rotary evaporator in vacuo.
The residue is taken up in dichloromethane and extracted
with water, and the organic phase is dried with sodium
sulfate and concentrated in a rotary evaporator in vacuo.
The crude product is purified on silica gel using
dichloromethane/methanol/ethyl acetate 15:1:1 with the
addition of 1% triethylamine. The fractions containing
the product are combined and concentrated in a rotary
evaporator in vacuo.
Yield: 3.6 g
Rf 0.27 (dichloromethane/methanol/ethyl acetate 10:2:1
+ 1% triethylamine)
MS (ES + + LiCl) 636.4 (M + Li)+.
Example 17
5'-ATC GTC GTA TT-(but)-agtc-hex
The DNA sequence is indicated in capital letters and the
PNA sequence is indicated in small letters (example of
the structural type XIIa in scheme 1). The PNAs are
synthesized, for example, in an Ecosyn D-300 DNA synthe-
sizer (from Eppendorf/Biotronik, Maintal) or an ABI 380B
DNA synthesizer (from Applied Biosystems, Weiterstadt).
The synthesis of the DNA part is carried out in principle
2144475
- 52 -
by standard phosphoramidite chemistry and commercially
obtainable synthesis cycles. For the synthesis of the PNA
part the methods of peptide synthesis are approximated to
the DNA synthesis cycles as explained hereinafter.
H1N`
MO
0 N
CH,
o~`q l o
Y l
U- P V N
1'Q`) Q
w r\r/~ LNI
H
o N
U-PY\ I (~n
0~
~ 0,-r, 0 B
U P V, 0(')
n 0
o q~ a Y
u-v- ve
N-~ M Q
CN,
Y
0 N\
H 1\ ~ V-P-V\
y lk\~I_'0'"'`/1
Schsme 1 CHt Q ON
ONA/PNA hy6riC moIACUIo s ON
0 M Farmula XI11
N (XIII a; n=1)
Formula X11
(XI I a, n=1)
2144475
- 53 -
H=N\ ~
B l`
H 0 N
CH: 0
o~`p B
Y
U -P- V N
ii ~ CH= 0
w
O~N\ B
Y H I
8 `
U P - V
r N0~ 0
0 X CN~
N BI
H~ ~oV
U -P -V B\Y/
W
N
0
CH U-P-V\ 8
2 W 11
}r OY`~
ON\ B
Y
H I\
N
I 0 O -P- V B
CH= jy
O1^1 N g
`iF
N 0
PNA/DN- hybrid Yolooulet CH2 OH
Sthem= 2 0 N Formula XI
H
Formula X (Xo, V-0; Xb, V-NH)
2144475
- 54 -
Dmt -V 80
0 R
I
R5-' p-, Rs
Formulo V I I I
R5 R6 R2 V
VIII a NC-CH CH -0- -N(i-C H )H 0
VIII b CH -N(i-C H )H 0
VIII c C H -N(i-C H )H 0
VIII d C H-C(O)-S(CH )-S -N-pyrrolidin-1 I H 0
VIII e NC-CH CH -0- -N(i-CH1 OCH 0
VIII f NC-CH CH -0- -N(i-C H )H NH
Mm t - V B '
( n
I I
N ~O
CHZ
I
0 H
Formulo IX (IXa, V=NH; IXb, V=O)
2144473-
- 55 -
with n 1-8, preferably 1-5,
y ,
0
\P B
0-Dmt
NC
Formulo XIV
y
N,,, - -~ p/OL~j~Of-,, Omi
0
f
NC
Formulo XV
~--r
N Wmt
I \P/0 N/
H
0
f
NC
Formulo XVI
2144475
- 56 -
3 mol of the CPG support loaded with Mmt-hex-succ
(loading 91 mol/g) from Example 4 are treated succes-
sively with the following reagents:
Synthesis of the PNA part (agtc-hex):
1. dichloromethane
2. 3% trichloroacetic acid in dichioromethane
3. acetonitrile abs.
4. 3.5 M solution of 4-ethylmorpholine in acetonitrile
(neutralization)
5. 0.4 M solution of (Mmt-Aeg(ctB'BZ)-OH) from Example 5
in acetonitrile:DMF = 9:1/0.9 M solution of ByBOP in
acetonitrile/3.5 M solution of 4-ethylmorpholine in
acetonitrile (coupling time of 10 minutes).
6. step 5 is repeated four times.
7. acetonitrile
Steps 1 to 7, called a PNA reaction cycle hereinafter,
are repeated 3 times to assemble the PNA part, using in
step 5 in each case the monomer building block, necessary
according to the sequence, from Examples 5 to S.
Conjugation of the linker (agtc-hex ---> (but)-agtc-hex):
8. repeat steps 1 to 4 from above
9. 4-nitrophenyl 4-(4,4'-dimethoxytrityloxy)butyrate
(105 mg) from Example 10 and hydroxybenzotriazole
(27 mg) in 2 ml of NEM in DMF for 15 hours
10. wash with DMF
11. wash with acetonitrile
12. dichloromethane
Synthesis of the DNA part
((but)-agtc-hex) --> 5'-ATC GTC GTA TT-(but)-agtc-hex):
13. acetonitrile abs.
14. 3% trichloroacetic acid in dichioromethane
15. acetonitrile abs.
16. 10 mol of R-cyanoethyl 5'-O-dimethoxytritylthymi-
dine 3'-diisopropylphosphoramidite and 50 mol of
tetrazole in 0.3 ml of acetonitrile abs.
2144475
- 57 -
17. acetonitrile
18. 20% acetic anhydride in THF with 40% lutidine and
10% dimethylaminopyridine
19. acetonitrile
20. iodine (1.3 g in THF/water/pyridine; 70:20:5
= v:v:v)
Steps 13 to 20, called a DNA reaction cycle hereinafter,
are repeated 10 times to assemble the nucleotide part,
using in step 16 in each case the P-cyanoethyl 5'-O-di-
methoxytrityl(nucleotide base) 3'-diisopropylphosphor-
amidite corresponding to the sequence.
After the synthesis is complete, the dimethoxytrityl
group is eliminated as described in steps 1 to 3. The
oligomer is cleaved off the support and, at the same
time, the (3-cyanoethyl groups are eliminated by treatment
with ammonia for 1.5 hours. To eliminate the exocyclic
amino protective groups, the ammoniacal solution is kept
at 55 C for 5 hours. 180 OD260 of the resulting crude
product (325 OD260) of 5' -ATC GTC GTA TT- (but) -agtc-hex
are purified by polyacrylamide gel electrophoresis.
Desalting on a BiogelR column (from Biorad) results in
50 OD260 of high-purity oligomer from this.
Example 18
5'-ATC GTC GTA TT-(Oeg(t))-agtc-hex
(Example of structural type Xa in scheme 2; see Example 9
for explanation of Oeg(t))
The synthesis takes place in analogy to the description
in Example 17 but in step 9 coupling the linker building
block Mmt-Oeg(t)-OH from Example 9, in place of the
p-nitrophenyl Dmt-butyrate, under the conditions
described in step S. 135 OD260 of the resulting crude
product (235 OD260) of 5'-ATC GTC GTA TT-(Oeg(t)-agtc-hex
are purified by polyacrylamide gel electrophoresis.
Desalting on a BiogelR column (from Biorad) results in
20 OD260 of high-purity oligomer from this.
2144475
- 58 -
Example 19
N-ggg g(5'NH-C)T CSCSAS TGG GGSGS T (sequence complemen-
tary to HSV-1)
(Example of structural type XI in scheme 2; s means a
phosphorothioate bridge; (5'NH-C) means a 5'-aminocytidy-
late residue; N equals amino terminus)
The synthesis takes place starting from a CPG support on
which 5'-Dmt-thymidine is bound via its 3' end. The
synthesis of the DNA part is first carried out as
described in Example 17 (steps 13 to 20), carrying out
the oxidation in step 20 in the case of the phosphoro-
thioate bridges (S) using tetraethylthiuram disulfide
(TETD; User Bulletin No. 65 of Applied Biosystems Inc.).
A Dmt-protected 5'-amino-5'-deoxycytidylate 3'-phosphor-
amidite building block of the formula VIIIf is used as
linker building block. The PNA building blocks are then
condensed on in analogy to steps 1 to 7 in Example 17.
After the synthesis is complete, the oligomer is cleaved
off the support and, at the same time, the R-cyanoethyl
groups are eliminated by treatment with ammonia for
1.5 hours. To eliminate the exocyclic amino protective
groups, the ammoniacal solution is kept at 55 C for
5 hours. Only then is the monomethoxytrityl group elimi-
nated by treatment with 80% strength acetic acid at 22 C
for 2 hours. The product is purified by polyacrylamide
gel electrophoresis and desalted on a BiogelR column
(from Biorad).
Example 20
5' -GmeGMeG GCT CCA (Oeg (t) ) gg ggg t-hex
(Example of structural type Xa in scheme 2; 2õte means a
methylphosphonate bridge; see Example 9 for explanation
of Oeg(t))
The synthesis takes place in analogy to the description
in Example 18 but using the appropriate methylphosphonate
building blocks of the formula VIIib in the DNA reaction
cycle to incorporate the methylphosphonate bridges Me.
2144475
- 59 -
Example 21
5'-C$ SAS SC GTs ST GAG (but)Ggg cat-hex (c-myc antisense)
(Example of structural type XIIa in scheme 1; S s means a
phosphorodithioate bridge). ~
The synthesis takes place in analogy to the description
in Example 17 but the building block VIIid is used to
incorporate the dithioate bridges, and the oxidation at
these sites (step 20) is carried out with TETD.
Example 22
N-cga g(5'NH-A)A CAT CA (Oeg(t))ggt cg-hex (c-fos
antisense)
(5'NH-A means 5'-amino-5'-deoxyadenylate; see Example 9
for explanation of Oeg(t))
The synthesis takes place in analogy to the description
in Example 18 with, after completion of the DNA syn-
thesis, in analogy to Example 13 condensation on of a
5'-aminonucleotide which permits conjugation of the
second PNA part. Thus, firstly six PNA synthesis cycles
are carried out and then the linker building block from
Example 9 is coupled on. Then seven DNA synthesis cycles
are carried out, using the building block of the formula
VIIIf in the last cycle. After a further four PNA syn-
thesis cycles have been carried out, the elimination from
the support and further working up are carried out as
described in Example 19.
Example 23
F-cga g(5'NH-A)A CAT CAT GGT SCSG-O-CH2CH(OH)CH2-O-C16H33
(5'NH-A means 5'-amino-5'-deoxyadenylate; F a fluorescein
residue on the amino terminus of the PNA and S a
phosphorothioate bridge)
The synthesis takes place in analogy to the description
in Example 19 but starting from a CPG support onto which
the glycerol hexadecyl ether is bound. After 12 DNA
synthesis cycles have been carried out, the linker
2144475
- 60 -
building block VIIIf is condensed on. After four PNA
synthesis cycles have been carried out and the terminal
Mmt group has been eliminated, it is possible to react
the free amino group quantitatively with a 30-fold excess
of fluorescein isothiocyanate (FITC).
Example 24
3'-CCC TCT T-5'-(PEG)(PEG)-(Oeg(t))tg tgg g-hex
(PEG means a tetraethylene glycol phosphate residue)
The synthesis in respect of the PNA part takes place in
analogy to the description in Example 17. After six PNA
units have been condensed on, the (Mmt-Oeg(t)-OH) from
Example 9 is coupled on. Then as linker initially the
tetraethylene glycol derivative of the formula XV is
condensed on twice as described in the DNA synthesis
cycle before the synthesis of the DNA part with reversed
orientation (from 5' to 3') is carried out. For this
purpose, in place of the nucleoside 3'-phosphoramidites
in each case the corresponding nucleoside 5'-phosphor-
amidites of the formula XIV, which are commercially
available, are used in step 16 in the DNA synthesis
cycles. Further deprotection and working up take place as
described in Example 17.
Example 25
N-ccc tct t-(C6-link)(PEG)-3'-AAG AGG G-5'
(PEG means a tetraethylene glycol phosphate residue;
C6-link is a 6-aminohexanol phosphate residue)
The synthesis takes place in analogy to the description
in Example 17 (DNA synthesis cycle) but starting from a
CPG support to which 3'-O-Dmt-deoxyguanosine is bound via
a 5'-O-succinate group. After six DNA units have been
condensed on using the building blocks of the formula
XIV, initially the tetraethylene glycol derivative of the
formula XV is condensed on once as linker before coupling
the phosphoramidite of the formula XVI to introduce
C6-link. The PNA part is then synthesized on as in
2144475
- 61 -
Example 17 (PNA synthesis cycle). Further deprotection
and working up take place as described in Example 19.
Example 26
5'-TTT TTT TTT (but) ttt ttt-hex
The synthesis takes place in analogy to the description
in Example 17. Before the product is cleaved off the
support and deprotected, half the support-bound DNA/PNA
hybrid is taken for fluorescence labeling (Example 27).
The other half is deprotected and worked up as described
in Example 17.
Example 27
(FAM is fluorescein residue)
5'-FAM-TTT TTT TTT (but) ttt ttt-hex
The support-bound DNA/PNA hybrid from Example 26 is
fluorescence labeled by carrying out steps 13 to 20 as
described in Example 17 using the fluorescein phosphor-
amidite from Applied Biosystems in step 16.
Example 28
5'-GGG GGG GGG (but) ttt ttt-hex
The synthesis takes place in analogy to the description
in Example 17. Before the product is cleaved off the
support and deprotected, half the support-bound DNA/PNA
hybrid is taken for fluorescence labeling (Example 29).
The other half is deprotected and worked up as described
in Example 17. The title compound binds as triplex-
forming oligonucleotide with high affinity to a DNA
double strand which contains the homopurine motif
5'-AAA AAA GGG GGG GGG-3'.
2144475
- 62 -
Example 29
(FAM is fluorescein residue)
5'-FAM-GGG GGG GGG (but) ttt ttt-hex
The support-bound DNA/PNA hybrid from Example 28 is
fluorescence labeled by carrying out steps 13 to 20 as
described in Example 17 using the fluoresceine phosphor-
amidite from Applied Biosystems in step 16.
Example 30
Biotin-CpheGPheA GAA cat ca t(5' NH-G) G(Ome) U(Ome) C(Ome) -
G(Ome)-VitE (c-fos antisense)
(N(Ome) means a nucleotide unit N with a 2'-O-methoxy
group; Phe means a phenylphosphonate bridge; 5'NH-G means
5'-amino-5'-deoxyguanylate).
The synthesis takes place in analogy to the description
in Example 17 starting from CPG which is loaded with
vitamin E(MacRellar et al. (1992) Nucleic Acids Res,
20(13), 3411-17) and coupling the building block of the
formula VIIIe four times after the DNA synthesis cycle.
After the 5'-aminonucleotide building block of the
formula VIIIf has been coupled on, six PNA units are
condensed on after the PNA synthesis cycle. After neutra-
lization, the phosphoramidite is coupled to the amino
group by a known method, and the DNA synthesis cycle is
repeated appropriately to assemble the DNA part, using in
the case of the phenylphosphonate bridges the building
blocks of the formula VIIIc in step 16. Lastly the end
group is coupled on using the biotin phosphoramidite from
Glen Research. After the synthesis is complete, the
oligomer is deprotected as described in Example 19,
eliminating the dimethoxytrityl group at the end by
treatment with 80% strength acetic acid at 22 C for
2 hours.
2144475
- 63 -
Example 31
A CAT CA (Oeg(t)) ggt cg-hex (c-fos antisense)
(See Example 9 for explanation of Oeg(t))
The synthesis takes place in analogy to the description
in Example 18. In this case, firstly five PNA synthesis
cycles are carried out and then the linker building block
Oeg(t) from Example 9 is coupled on. Then six DNA syn-
thesis cycles are carried out. Subsequently, the elimi-
nation from the support and the further working up are
carried out as described in Example 18.
Example 32
A TAA TG (Oeg(t)) tct cg-hex (control oligomer for c-fos)
The synthesis takes place in analogy to the description
in Example 18. In this case, firstly five PNA synthesis
cycles are carried out and then the linker building block
Oeg(t) from Example 9 is coupled on. Then six DNA syn-
thesis cycles are carried out. Subsequently, the elimi-
nation from the support and the further working up are
carried out as described in Example 18.
Example 33
a cat cat ggt cg-hex (c-fos antisense)
This pure PNA oligomer was prepared as reference compound
in analogy to Example 18 but with the exception that
twelve PNA cycles were carried out. Deprotection of the
exocyclic amino protective groups is carried out in
ammoniacal solution (5 hours at 55 C). Only then is the
monomethoxytrityl group eliminated by treatment with 80%
strength acetic acid at 22 C for 2 hours.
Example 34
A (5-hexy-C)A(5-hexy-U) (5-hexy-C)A (Oeg(t)) ggt cg-hex
(c-fos antisense)
(See Example 9 for explanation of Oeg(t); 5-hexy-C means
5-hexynylcytidine, 5-hexy-U means 5-hexynyluridine)
2144475
- 64 -
The synthesis takes place in analogy to the description
in Example 31 but using in place of the normal pyrimidine
phosphoramidites the corresponding 5-hexynylpyrimidine
nucleoside phosphoramidites in the condensation reaction.
Example 35
(FAM is fluorescein residue)
5'-FAM-TT (but) ttt ttt-hex
The synthesis of this PNA/DNA oligomer takes place in
analogy to the description in Example 27 although only
two thymidylate units are condensed on.
Example 36
taa tac gac tca cta (5'HN-T)
(5'HN-T means 5'-amino-51-deoxythymidine)
This PNA/DNA oligomer which is composed of 15 PNA units
and one nucleoside unit was synthesized as primer for
the DNA polymerase reaction. This entails starting from
a solid phase support (aminoalkyl-CPG) to which the
5'-monomethoxytritylamino-5'-deoxythymidine is bound
via its 3'-hydroxyl group as succinate. After elimination
of the monomethoxytrityl group with 3% TCA in dichloro-
methane, 15 PNA cycles are carried out as described in
Example 17. Deprotection of the exocyclic amino protec-
tive groups is carried out in ammoniacal solution
(5 hours at 55 C). Only then is the monomethoxytrityl
group eliminated by treatment with 80% strength acetic
acid at 22 C for 2 hours. A PNA/DNA oligomer with a free
3'-hydroxyl group, which is used as primer for a DNA
polymerase (Klenow) is obtained.
Example 37
ps-rA(2'5')rA(2'5')rA(2'5')rA-spacer-(Oeg(t)tc ctc ctg
cgg-hex
(ps means a 5'-thiophosphate; spacer means a triethylene
glycol phosphate; rA is a riboadenylate; (2'5') means
that the internucleotide linkage is from 2' to 5' in the
2144475
- 65 -
ribose)
The synthesis of this compound takes place in analogy
to the description in Example 18 by initially condensing
on 14 PNA units. After the linker building block
Mmt-Oeg(t)-OH from Example 9 has been introduced under
the conditions described in step 5, the Mmt group is
eliminated with 3% TCA, and the spacer is introduced with
the aid of the commercially available Dmt-O-(CH2CH2O)3-
O-P(-OCH2CH2CN)N(i-C3H7)3 spacer phosphoramidite (from
Eurogentech; Brussels). The (2'5')-linked tetradenylate
is synthesized on as described in Example 17 using the
commercially available N6-benzoyl-5'-O-Dmt-3'-O-tert-
butyldimethylsilyladenosine 2'-O-cyanoethyl diisopropyl-
aminophosphoramidite (from Milligen, Bedford, USA),
extending the condensation time to 2 x 5 min. The
stronger activator 5-ethylthiotetrazole is used in place
of tetrazole in the coupling reaction. After elimination
of the last Dmt group, the oligomer is phosphitylated on
the 5'-OH group with bis(R-cyanoethyloxy)diisopropyl-
aminophosphine. Oxidation with TETD and deprotection with
ammonia and desilylation with fluoride result in the
title compound, which stimulates RNase L.
Example 38
ps-Co(2'S')Co(2'5')Co(2'5')Co-spacer-(Oeg(t))tc ctc ctg
cgg-hex
(ps means a 5'-thiophosphate; spacer means a triethylene
glycol phosphate; Co is cordycepin (3'deoxyadenosine);
(2'5') means that the internucleotide linkage is from 2'
to 5')
The synthesis is carried out in analogy to Example 37 but
in place of the N6-benzoyl-5'-O-Dmt-3'-O-tert-butyl-
dimethylsilyladenosine 2'-O-cyanoethyl diisopropylamino-
phosphoramidite, the corresponding N6-benzoyl-5'-O-Dmt-
cordycepin 2'-O-cyanoethyl diisopropylaminophosphorami-
dite (from Chemogen, Konstanz) is used, and the fluoride
treatment is omitted.
2144475
- 66 -
Example 39
5'-GG GGG GGG (Oeg(t)) ttt ttt ttt-hex
The synthesis takes place in analogy to the description
in Example 18, following nine PNA couplings by condensa-
tion on of the linker building block Mmt-Oeg(t)-OH from
Example 9 under the conditions described in step 5, which
permits subsequent condensation of eight guanylate
residues. The resulting PNA/DNA oligomer binds with high
affinity in the antiparallel orientation as triplex-
forming oligonucleotide to double-stranded DNA which has
the sequence 5'.. GGGGGGGG..3'.
Example 40
Characterization of the PNA/DNA hybrids
The characterization takes place with the aid of HPLC,
polyacrylamide gel electrophoresis (PAGE) and negative
ion electrospray mass spectrometry (ES-MS-). The products
are purified as described above and thereafter show in
the PAGE (20% acrylamide, 2% bisacrylamide and 7 M urea)
a single band. The HPLC takes place on RP-18 reversed
phase columns from Merck (eluent A: water with 0.1% TFA,
B: water/acetonitrile = 1:4; linear gradient) or on a
PA-100 column from Dionex (eluent A: 20 mM NaOH and 20 mM
NaCl; B: 20 mM NaOH and 1.5 M NaCl; linear gradient). For
the ES-MS , the PNA/DNA hybrids are converted by ammonium
acetate precipitation or other metathesis into the
ammonium salts. Sample introduction takes place from a
solution in acetonitrile/water (1:1) containing 5 OD260/ml
oligomer. The accuracy of the method is about
1.5 Dalton.
Example 41
Determination of cellular uptake and stability after
radioactive labeling
Radioactive labeling:
A generally applicable labeling with 35S comprises
2144475
- 67 -
carrying out at least one oxidation in the DNA synthesis
cycle (step 20 in Example 17) for the synthesis of the
DNA part using elemental sulfur-35. PNA/DNA hybrids which
have a free 5'-hydroxyl group can be labeled with 32P or
35S with the aid of polynucleotide kinase by methods
known per se. PNA/DNA hybrids which carry a free
3'-hydroxyl group can be labeled in a known manner with
3'-terminal transferase. As an example, the 5'-labeling
of the DNA part is described here: the PNA/DNA hybrid
with a free 5'-hydroxyl group (500 pmol) from Example 17,
18 or 26 is dissolved in 425 1 of water, and this
solution is heated to 90 C and rapidly cooled. Then 50 l
of 10 x kinase buffer and 50 l of 32P-gamma-ATP
(6,000 Ci/mmol) or 35S-gamma-ATP are added, and the
mixture is incubated at 37 C for 1 hour. The reaction is
stopped by adding 0.5 M EDTA solution. Desalting takes
place with the aid of an NAPR column from Pharmacia.
Determination of cellular uptake:
Vero cells are incubated in DMEM, 5% FCS, in 96-well
microtiter plates at 37 C for 24 hours. After removal of
the medium, the cells are washed twice with serum-free
DMEM. The radioactively labeled oligomer (106 cpm) is
diluted with unlabeled oligomer to a concentration of
10 M in serum, and the cells are incubated at 37 C
therewith. 150 l portions are removed after 1, 7 and
24 hours (called "supernatant 1"). The cells in the wells
of the microtiter plates are washed 7 times with 300 l
of fresh medium, and the combined washing media (called
"supernatant 2") are measured in a scintillation counter.
30- Then 100 l of trypsin solution are added, 30 seconds are
allowed to elapse, and the supernatant is aspirated off.
The cells are detached from the plate by incubating at
37 C for 3 min. The detached cells are transferred into
1.5 ml Eppendorf tubes and centrifuged at 2,000 rpm for
6 minutes ("supernatant 3"). Supernatants 1 (5 l), 2 and
5 (0.5 ml) are each measured separately in a scintilla-
tion counter. From this is calculated the uptake of
oligomer in pmol per 100,000 cells, with supernatant 3
representing the cell-bound oligomer fraction and the
2144475
- 68 -
total of supernatants 1 and 2 representing the non-cell-
bound oligomer fraction.
Results:
Incubation time Cellular uptake in pmol
in hours of oligomer/105 cells
PNA/DNA hybrid DNA
1 0.25 0.36
7 0.54 0.57
24 0.75 0.78
Investigation of the stability of the oligomer in medium
containing cells:
Supernatant 1 (10 l) is mixed with 5 l of 80% formamide
(with Xylenecyanol and bromphenolblue), heated to 95 C
(5 minutes) and loaded onto a polyacrylamide gel (20%
acrylamide, 7 M urea) . After development of the gel in
the electric field, the bands on the gel are assigned by
autoradiography to the "stable oligomer", and the missing
bands to the "degraded oligomer".
The PNA/DNA oligomer from Example 26 is 69% stable after
an incubation time of 24 hours; the DNA oligomer is 3%
stable.
The PNA/DNA oligomer from Example 31 has a half-life of
32 h under these conditions, whereas the corresponding
DNA oligonucleotide has a half-life of about 2 h.
Example 42
Determination of cellular uptake by fluorescence
labeling:
COS cells are allowed to grow to confluence in Dulbecco's
MEM supplemented with 10% FCS in 5 cm Petri dishes. The
cells are washed twice with serum-free DMEM. A sterile
needle is used to scratch an area of about 1 cm2 in the
214447a
- 69 -
middle of the Petri dish. The PNA/DNA oligomer solution
(0.1 mM) to be investigated is applied to this area.
Incubation is carried out at 37 C under a CO2 atmosphere.
The cells are investigated by fluorescence microscopy
after 2, 4 and 16 hours. For this, the cells are washed
four times with serum-free DMEM, covered with a glass
slide and assessed under a fluorescence microscope or by
phase contrast. A fluorescence-labeled PNA (without DNA
part) F-(but)-tttt ttt-hex was investigated as comparison
for the PNA/DNA hybrid molecules. After the cells had
been incubated with this PNA for two hours, > 90% of the
cells show signs of pronounced morphological changes and
cell death. Most of the cells exhibit pronounced vacuo-
lization. The plasma membrane, the cytosol and the
nucleus show no uptake of PNA. After incubation with the
pure PNA for a further two hours, all the cells have
died. The situation is different with the DNA/PNA oligo-
mers according to the invention. After incubation of the
cells with the DNA/PNA oligomers for only two hours the
cells show punctiform intracellular distribution of the
PNA/DNA oligomers. The cells suffer no cell death even
after prolonged incubation.
Example 43
Determination of the melting temperatures:
The melting temperatures are determined using an HP 8452A
diode array spectrophotometer, an HP 89090A Peltier
element and the HP temperature control software Rev.B5.1
(from Hewlett Packard). Measurements are carried out in
0.5 C/min steps in 10 mM HEPES and 140 mM NaCl (pH 7.5)
as buffer. The oligomer concentration is 0.5 to 1 OD260
per ml.
214447~
- 70 -
Result for the product from Example 17 or 18 (TM with
DNA)
5'-ATC GTC GTA T(Oeg(t))a gtc-hex TM = 51.5 C
3'-TAG CAG CAT A A T CAG-5' antiparallel
5'-ATC GTC GTA T(Oeg(t))a gtc-hex TM < 20 C
5'-TAG CAG CAT A A T CAG-3' parallel
5'-ATC GTC GTA TT(but)a gtc-hex TM = 51.0 C
3'-TAG CAG CAT AA T CAG-5' antiparallel
5'-ATC GTC GTA TTA GTC-3' TM = 50.5 C
3'-TAG CAG CAT AAT CAG-5' DNA=DNA antiparallel
5'-ATC GTC GTA TT(but)a gtc-hex TM < 20 C
5'-TAG CAG CAT AA T CAG-3' parallel
Sequence
T. with DNA TM with RNA
(T = U)
5'-ACA TCA TGG TCG-3' DNA ap 50.7 C 48.6 C
3'-TGT AGT ACC AGC-5'
5'-ACA TCA tgg tcg-3' (PNA/DNA) ap 54.5 C 54.7 C
3'-TGT AGT ACC AGC-5'
5'-ACA TCA tgg tcg-3' (PNA/DNA) p 20 C < 20 C
3'-TGT AGT ACC AGC-3'
5'-aca tca tgg tcg-3' PNA ap 58.8 C 66.6 C
3'-TGT AGT ACC AGC-5'
5'-aca tca tgg tcg-3' PNA p 46.3 C 44.8 C
5'-TGT AGT ACC AGC-3'
5'-ACA TCA TGG TCG-3' S-DNA ap 46.7 C 43.8 C
3'-TGT AGT ACC AGC-5'
TGG TCG means a DNA part in which all internucleotide
linkages are in phosphorothioate form. See page 5 for
2144475
- 71 -
definition of p and ap.
Example 44
Tests for antiviral activity:
The antiviral activity of the test substances on various
human-pathogenic Herpesviruses is investigated in a cell
culture test system. For the experiment, monkey kidney
cells (Vero, 2 x 105/ml) are inoculated in serum-contain-
ing Dulbecco's MEM (5% fetal calf serum FCS) in 96-well
microtiter plates and incubated at 37 C and 5% C02 for
24 h. The serum-containing medium is then aspirated off
and the cells are rinsed twice with serum-free Dulbecco's
MEM (-FCS). The test substances are prediluted in H20 to
a concentration of 600 M and stored at -18 C. Further
dilution steps in Dulbecco's minimal essential medium
(MEM) are carried out for the test. 100 l portions of
the individual test substance dilutions are added to-
gether with 100 l of serum-free Dulbecco's MEM (-FCS) to
the rinsed cells. After incubation at 37 C and 5% C02 for
3 h, the cells are infected with Herpes simplex virus
type 1 (ATCC VR733, HSV-1 F-strain) or with Herpes
simplex virus type 2 (ATCC VR734, HSV-2 G-strain) in
concentrations at which the cell lawn is completely
destroyed within 3 days. The infection concentration for
HSV-1 is 500 plaque-forming units (PFU) per well, and for
HSV-2 it is 350 PFU/well. The test mixtures then contain
test substance in concentrations of 80 M to 0.04 M in
MEM supplemented with 100 U/ml penicillin G and 100 mg/1
streptomycin. All the experiments are carried out as
duplicate determination with the exception of the con-
trols which are carried out eight times per plate. The
test mixtures are incubated at 37 C and 5% C02 for 17 h.
The cytotoxicity of the test substances is determined
after a total incubation time of 20 h by microscopic
inspection of the cell cultures. The maximum tolerated
dose (MTD) is defined as the highest concentration of
product which, under the stated test conditions, does not
yet cause microscopically detectable cell damage.
Subsequently FCS is added to a final concentration of 4%,
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and incubation is continued at 37 C and 5% C02 for 55 h.
The untreated infection controls then show a complete
cytopathic effect (CPE). After microscopic inspection of
the cell cultures they are then stained with neutral red
in accordance with the vital staining method of Finter
(1966). The antiviral activity of a test substance is
defined as the minimum inhibitory concentration (MIC)
which is needed to protect 30-60% of the cells from the
cytopathogenic effect caused by the virus. The activity
of the PNA/DNA chimeras is in each case better than that
of the corresponding DNA oligomers or PNA oligomers.
Example 45
Determination of the in vivo activity: inhibition of
c-Fos protein expression in the rat:
The determination takes place as described (SandkUhler et
al. (1991) in: Proceedings of the VIth World Congress on
Pain, Charlton and Woolf, Editors; Elsevier, Amsterdam;
pages 313-318) by superfusion of the spinal cord. After
laminectomy of a barbiturate-anesthetized Sprague-Dawley
rat, a two-chamber container is formed from silicone to
receive the antisense oligomer. One chamber is filled
with the antisense PNA/DNA derivative, while the other
chamber is filled with the control oligomer (concentra-
tion of each 75 M). The superfusate is exchanged in each
case after one hour. After superfusion for 6 hours, c-fos
expression is stimulated by heat treatment (52 C) of the
rear legs. Inhibition of c-fos expression can be demon-
strated immunohistochemically on appropriate tissue
section samples. The c-fos antisense oligonucleotide from
Example 31 brings about greater inhibition of c-fos
expression than does the corresponding DNA oligonucleo-
tide and the corresponding PNA oligomer from Example 33.
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Example 46
RNase H assay
To determine the RNase H activity, 1.3 OD of the PNA/DNA
oligomer to be investigated are heated with 0.5 OD of the
complementary RNA sequence (target sequence) dissolved in
50 l of autoclaved water, treated with DEPC (diethyl
pyrocarbonate), at 80 C for 5 minutes and subsequently
cooled to 37 C within 15 minutes. This results in initial
denaturation of both oligomers which, after cooling, form
a nucleic acid double strand in sequence-specific manner.
For the assay, this RNA-PNA/DNA duplex is incubated with
10 l of RNase H 10 x buffer, 1 l of dithiothreitol
(DTT) and 2 l (corresponding to 10 u) of RNase H
supplied by USB. The incubation mixture is made up with
autoclaved, DEPC-treated water to the required total
volume of 100 l. The samples are incubated as 37 C. For
the kinetic investigation, 20 l portions of the solution
were removed after 0, 2 min, 10 min and 1 h, heated at
95 C for 5 minutes and frozen at -70 C until analyzed.
The investigation of the RNase H cleavage of RNA takes
place by gel electrophoresis. It emerged that PNA/DNA
hybrids which contain deoxyribonucleotide building blocks
activate RNase H, with cleavage of the complementary RNA
strand whereas the PNA/DNA oligomer emerges undamaged
from the reaction. The cleavage reaction with the PNA/DNA
oligomer takes place somewhat more slowly than with a
corresponding oligodeoxyribonucleotide of equal length
and sequence.
Example 47
Preparation of an HeLa cell extract with RNase L activity
An HeLa cell extract was prepared in order to stimulate
the activity of cellular endoribonuclease L by the
2',5'-tetraadenylate-PNA/DNA conjugates. For this pur-
pose, 35 bottles were each charged with 20 ml of medium
containing Dulbecco's MEM (mimimal essential medium) and
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10% FCS (fetal calf serum) . The cells can be harvested
after trypsin treatment. 4 ml of cell harvest are
obtained after centrifugation at 1,000 rpm. This is
initially made up with 4 ml of water and, after
3 minutes, 4 ml of buffer A (5.48 g of HEPES; 15.5 g of
KC1; 2.488 g of Mg acetate; 1,232 l of 2-mercaptoethanol
ad 1 1 with water) are added in order to lyze the cells.
The solution is then centrifuged at 30,000 rpm (about
100,000 g) in an ultracentrifuge at 0 C for 30 minutes.
The supernatant from 8 ml of cell extract is removed and
stored at -20 C for the following investigations.
Example 48
Investigation of activiation of RNase L
For investigation of this extract for endonuclease L,
initially 0.3 OD of the RNA target sequence is heated
with the particular PNA/DNA oligomers at 80 C for
5 minutes and subsequently cooled to 37 C for the
hybridization. The duplex is mixed with 20 l of the
extract, 1.2 l of glycerol and RNase L buffer and
incubated at 37 C. The total volume is then 70 l. For
the kinetic investigations, samples are removed by
pipette at the times of 0, 20 and 60 minutes and heated
at 95 C for 5 minutes to denature the enzymes. The
samples are lyophilized in a Speedvac and analyzed by gel
electrophoresis. The PNA-2',5'-tetraadenylate conjugates
and tetracordycepin analogs activate cellular RNase L,
whereas corresponding compounds without the tetra-
adenylate part do not stimulate RNase L.
Example 49
DNA polymerase reaction
The following 81-mer oligodeoxynucleotide is used as
template for the DNA polymerase reaction:
5'-GCC CCA GGG AGA AGG CAA CTG GAC CGA AGG CGC TTG TGG AGA AGG AGT
TCA TAG CTG GGC TCC CTA TAG TGA GTC GTA TTA-3'
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The sequence of the PNA/DNA primer is:
H-taa tac gac tca cta (5NH-T)-OH 3'.
A corresponding oligodeoxynucleotide of the sequence
5'-TAA TAC GAC TCA CTA TAG-3' is used as control primer.
The primer (0.15 nmol) and the template (0.15 nmol) in
5 l of 10 x PCR buffer (500 m RC1, 100 mM tris-HC1,
pH 9, 1% Triton X-100, 15 mM MgC12) are diluted with
35 l of water and hybridized by heating to 95 C and
cooling. Then 10 l of 2 mM dNTP mixture (nucleoside
5'-triphosphates) and 3 l of DNA polymerase (Rlenow
fragment) are added, and the mixture is incubated at 22 C
and 37 C for 0.5 hour each. The reaction solution is then
analyzed on a 10% polyacrylamide gel (with 1% bis).
pBR322/HaeIII digestion is loaded as marker. The reaction
with the control primer shows a double-stranded DNA
fragment with the expected size relative to the marker,
whereas the product from the PNA/DNA primer migrates
somewhat more quickly. In both cases the double strand
migrates considerably faster than the template single
strand in the gel electrophoresis.
