Nucleic acid-binding oligomers for therapy and diagnosis

The present invention relates to compounds of the general formula (I), ##STR1## in which the radicals have the meaning given in the description, to processes for their preparation and to their use as medicaments.

The specific switching-off of gene expression by complementary nucleic 
acids, so-called antisense oligonucleotides, represents a new approach to 
therapy. Possible applications extend from the treatment of viral 
infections through to cancer therapy (S. Agrawal, Tibtech 10, 152 (1992); 
W. James, Antiviral Chemistry & Chemotherapy 2, 191 (1991); B. Calabretta, 
Cancer Research 51, 4504 (1991)). Gene expression is controlled at the DNA 
and RNA level and is achieved even using unmodified oligonucleotides (C. 
Helene, Anti-Cancer Drug Design 6, 569 (1991); E. Uhlmann, A. Peymann, 
Chemical Reviews 90, (1990)). However, the latter are not suitable for 
therapeutic applications because of their lack of stability towards 
enzymes and because they are not taken up to a sufficient extent by 
cellular systems. Therapeutic applications require chemically modified 
antisense oligonucleotides. 
Apart from the antisense strategy, the sense strategy can also be used to 
inhibit gene expression. In this case, the sense oligonucleotides compete 
specifically with DNA binding proteins such as transcription factors (M. 
Blumengeld, Nucleic Acids Research 21, 3405 (1993)). 
Oligonucleotides having a modified internucleotide phosphate or a 
phosphate-free internucleotide linkage have been investigated 
systematically in many studies; however, their synthesis was found to be 
very elaborate and their reported therapeutic effects were not adequate 
(E. Uhlmann, A. Peyman, Chemical Reviews 90, 543 (1990)). 
An alternative to modifying or substituting the phosphate group in nucleic 
acids is completely to replace ribose and phosphate by other backbones. 
This concept was first realized by Pitha et al., who replaced ribose 
phosphate by poly-N-vinyl derivatives, resulting in so-called "plastic 
DNA" (J. Pitha, P.O.P. Ts'O, J. Org. Chem. 33, 1341 (1968); J. Pitha, J. 
Adv. Polym. Sci. 50, 1 (1983)). However, this does not permit the specific 
construction of defined sequences. 
Synthesis of defined sequences is achieved if, for example, a polyamide 
backbone, which is constructed stepwise in analogy with conventional 
peptide synthesis (M. Bodanszky, Principles of Peptide Synthesis, 
Springer, Berlin 1984), is used instead of sugar phosphate. This concept 
has been realized in different ways by different research groups (J. E. 
Summerton et al., WO 86/05518; R. S. Varma et al., WO 92/18518; O. 
Buchardt et al., WO 92/20702; H. Wang, D. D. Weller, Tetrahedron Letters 
32, 7385 (1991); P. Garner, J. U. Yoo, Tetrahedron Letters 34; 1275 
(1993); S.-B. Huang, J. S. Nelson and D. D. Weller; J. Org. Chem. 56; 6007 
(1991)). 
Polyamide nucleic acids are also suitable for use in diagnostic and 
molecular biological applications (Buchardt et al., WO 92/20703 and Glaxo, 
WO 93/12129). 
During the processing of this type of structure, success was achieved in 
synthesizing novel N-branched oligomeric nucleic acids. The latter were 
found to bind surprisingly well to DNA and RNA. The substances are 
suitable for controlling gene expression and exhibit antiviral properties. 
Furthermore, substances of this nature can be used in diagnostics and 
molecular biology for isolating, identifying and quantifying nucleic 
acids. 
The invention relates to compounds of the general formula (I), 
##STR2## 
in which A represents --(CH.sub.2).sub.n -- or --CO--, 
B represents all natural or unnatural nucleotide bases, such as, for 
example, thymine, uracil, cytosine, adenine, guanine or hypoxanthine, or 
derivatives derived therefrom by means of chemical modification, or 
halogenated precursors thereof, which are optionally substituted on the 
amino groups by protective groups such as acetyl, trifluoroacetyl, 
trichloroacetyl, benzoyl, phenylacetyl, benzyloxycarbonyl, 
tert-butyloxycarbonyl, allyloxycarbonyl, (9-fluorenyl)methoxycarbonyl or 
other protective groups which are customary in peptide and nucleic acid 
chemistry, or which have free amino groups, 
D represents --(CO).sub.p --, 
E and G, independently of each other, represent --CHR--, where 
R represents H or a residue of a natural or unnatural amino acid, for 
example from glycine, alanine, valine, leucine, isoleucine, serine, 
threonine, cysteine, methionine, phenylalanine, tyrosine, histidine, 
tryptophan, lysine, ornithine, asparagine, aspartic acid, glutamine, 
glutamic acid, arginine, proline, hydroxyproline, sarcosine, dehydroamino 
acids, such as, for example, dehydroalanine or 
dehydro-.alpha.-aminobutyric acid, or other unnatural amino acids, such as 
phenylglycine, 4-nitrophenylalanine, 3-nitrophenylalanine, 
2-nitrophenylalanine, 2-, 3- or 4-aminophenylalanine, 
3,4-dichlorophenylalanine, 4-iodophenylalanine, 4-methoxyphenylalanine, 
1-triazolylalanine, 2-pyridylalanine, 3-pyridylalanine, 4-pyridylalanine, 
1-naphthylalanine or 2-naphthylalanine, optionally having protective 
groups, in their D or L form, or, where appropriate, 
E and G are linked to each other by way of a --(CHR').sub.q -- chain, 
K represents --CO--, --SO.sub.2 -- or --CH.sub.2 --, 
L can be a carrier system, a reporter ligand, a solubility-mediating group 
or OH, 
M can, independently of L, be a carrier system, a reporter ligand, a 
solubility-mediating group or hydrogen, 
Q represents NH, O, S or NR", 
R' can be selected, independently of each other, from a group consisting of 
H, OH, SH, NH.sub.2, NHR", N.sub.3, alkyl (where alkyl can be methyl, 
ethyl, n-propyl, n-butyl, iso-butyl, tert-butyl or longer-chain, branched 
or unbranched, saturated or unsaturated alkyl chains), aryl (where aryl 
can be phenyl, 2-pyridyl or 4-pyridyl) or aralkyl (where aralkyl can be 
benzyl, naphthylmethyl or .beta.-naphthylmethyl), 
R" represents protective groups such as, for example, Boc, Fmoc, Z, Pyoc, 
Alloc or other protective groups which are customary in peptide chemistry, 
or else represents alkyl substitution (where alkyl can be methyl, ethyl, 
n-propyl, n-butyl, iso-butyl, tert-butyl or longer-chain, branched or 
unbranched, saturated or unsaturated alkyl chains), aryl (where aryl can 
be phenyl, 2-pyridyl or 4-pyridyl) or aralkyl (where aralkyl can be 
benzyl, naphthylmethyl or .beta.-naphthylmethyl), 
m can be 0, 1, 2 or 3, 
n can be 0, 1, 2, 3 or 4, 
p can be 0, 1 or 2, 
q can be 0, 1 or 2, and 
r can be 0 or 1, and 
s can assume values of between 1 and 30. 
When r=1 in all the monomers, this structural component then occurs 
alternately and represents 50% of the total molecule. When r is 0 (zero) 
in individual monomers, the proportion of this structural component is 
correspondingly reduced to, for example, 40, 30 or 20%. This structural 
component should occur at least once in the total molecule. 
Compounds of the general formula (I) are preferred 
in which 
A represents --(CH.sub.2).sub.n -- or --CO--, 
B represents all natural nucleotide bases, such as, for example, thymine, 
uracil, cytosine, adenine, guanine or hypoxanthine, or halogenated 
precursors thereof, which are optionally substituted on the amino groups 
by protective groups such as acetyl, trifluoroacetyl, trichloroacetyl, 
benzoyl, phenylacetyl, benzyloxycarbonyl, tert-butyloxycarbonyl, 
allyloxycarbonyl, (9-fluorenyl)methoxycarbonyl or other protective groups 
which are customary in peptide and nucleic acid chemistry, or which have a 
free amino group, 
D represents --(CO).sub.p --, 
E and G, independently of each other, represent --CHR--, where 
R represents H or a residue of a natural or unnatural amino acid, for 
example from glycine, alanine, valine, leucine, isoleucine, serine, 
threonine, cysteine, methionine, phenylalanine, tyrosine, histidine, 
tryptophane, lysine, ornithine, asparagine, aspartic acid, glutamine, 
glutamic acid, arginine, proline, hydroxypyroline, sarcosine, dehydroamino 
acids, such as, for example, dehydroalanine or 
dehydro-.alpha.-aminobutyric acid, or other unnatural amino acids, such as 
phenylglycine, 2-pyridylalanine, 3-pyridylalanine, 4-pyridylalanine, 
1-naphthylalanine or 2-naphthylalanine, optionally having protective 
groups, in their D or L form, or, where appropriate, 
E and G are linked to each other by way of a --(CHR').sub.q -- chain, 
K can be --CO--, --SO.sub.2 -- or --CH.sub.2 --, 
L can be a carrier system, a reporter ligand, a solubility-mediating group 
or hydrogen, 
M can, independently of L, be a carrier system, a reporter ligand, a 
solubility-mediating group or hydrogen, 
Q represents NH, O or NR", 
R' can be selected, independently of each other, from a group consisting of 
H, OH, SH, NH.sub.2, NHR", N.sub.3, alkyl (where alkyl can be methyl, 
ethyl, n-propyl, n-butyl, iso-butyl, tert-butyl or longer-chain, branched 
or unbranched, saturated or unsaturated alkyl chains), aryl (where aryl 
can be phenyl, 2-pyridyl or 4-pyridyl) or aralkyl (where aralkyl can be 
benzyl, naphthylmethyl or .beta.-naphthylmethyl), 
R" represents protective groups such as, for example, Boc, Fmoc, Z, Pyoc, 
Alloc or other protective groups which are customary in peptide chemistry, 
or else represents alkyl substitution (where alkyl can be methyl, ethyl, 
n-propyl, n-butyl, iso-butyl, tert-butyl or longer-chain, branched or 
unbranched, saturated or unsaturated alkyl chains), aryl (where aryl can 
be phenyl, 2-pyridyl or 4-pyridyl) or aralkyl (where aralkyl can be 
benzyl, naphthylmethyl or .beta.-naphthylmethyl), 
m can be 0, 1, 2 or 3, 
n can be 0, 1, 2 or 3, 
p can be 0 or 1, 
q can be 0, 1 or 2, 
r can be 0 or 1, and 
s can assume values of between 3 and 20. 
A carrier system or reporter ligand is intended to mean a cell-specific 
binding and recognition agent which binds specifically to the cell surface 
and which brings about internalization of the nucleic acid-binding 
oligomers on which the invention is based. The internalization can take 
place in different ways, for example by endocytosis or active transport 
mechanisms. 
The cell surface can be constructed from a protein, polypeptide, 
carbohydrate, lipid or a combination thereof. Uptake into a cell is 
typically brought about by surface receptors. For this reason, the binding 
and recognition agent can be a natural or synthetic ligand of a receptor. 
The ligand can be a protein, polypeptide, carbohydrate, lipid, steroid or a 
combination thereof, which is provided with functional groups which are so 
arranged that they can be recognized by the cell surface structure. The 
ligand can also be a component, or the entirety, of a biological organism, 
for example of a virus or a cell, or be an artificial transport system, 
for example liposomes. Furthermore, the ligand can be an antibody or an 
analogue of an antibody. 
Different ligands must be employed for directing the oligomers to different 
cells. 
Carbohydrates, such as, for example, mannose, polycations, such as, for 
example, polylysines, polyarginines or polyornithines, basic proteins, 
such as, for example, avidin, and also glycopeptides, steroids, peptides 
or lipopeptides are preferably used as ligands for directing the oligomers 
to macrophages (G. Y. Chu et al., WO 9304701). 
Solubility-mediating groups are intended to mean functional groups which 
mediate solubility in water. These groups can be, for example, esters or 
amides of amino acids, hydroxycarboxylic acids, aminosulphonic acids, 
hydroxysulphonic acids or diamines. Amides of diaminocarboxylic acids, 
such as ornithine, lysine or 2,4-diaminobutyric acid, are preferred. 
In the present application, nucleic acid-binding oligomers are described in 
which the great variability of the structural components from DE 4 331 
012.5 has been combined with the properties of the aminoethylglycine 
structural components (WO 92/20703 and WO 93/12129, obtainable 
commercially from Millipore). The structural components which are employed 
for the oligomerization have been described in WO 92/20703 and WO 
93/12129. Derivatives of the structural components which are described can 
be prepared by means of reaction steps which are known from the 
literature. 
DESCRIPTION OF THE EXPERIMENTS 
Investigations on the hybridization properties and also on stability 
towards nucleases and proteases were carried out in analogy with the 
experiments in DE 4 331 012.5. Capillary electrophoresis measurements were 
carried out as an additional investigative method for examining the 
hybridization properties. 
General Section 
Oligomerization 
While the linking of the structural components to form oligomers can take 
place in solution, it is preferably carried out by means of solid phase 
synthesis (see: Merrifield, R. B., J. Am. Chem. Soc., 85, (1963, 2149). A 
peptide synthesizer, in particular the 431-A model from Applied 
Biosystems, is preferably employed for this purpose. Various commercially 
available resins are available for use as polymeric supports; the PAM, 
MBHA and HMP resins from Applied Biosystems are preferably used. The 
structural components are linked, in analogy with conventional peptide 
synthesis, by selective use of a protective group strategy at the N 
terminus, preferably employing the Fmoc method or the Boc method. 
Activation is as a rule effected in N-methyl-2-pyrrolidone (NMP) by 
reacting with hydroxybenzotriazole/dicyclohexylcarbodiimide, or else using 
other known activation methods from peptide chemistry (for example uronium 
salts, such as TBTU, HBTU, BOP, PYBOP, etc., in NMP or other solvents, 
such as DMF, DMSO or DCM). Subsequent to the oligomerization, the solid 
phase-bound compounds are separated off using special cleavage reagents 
such as HF or trifluoromethanesulphonic acid (Boc method; PAM or MBHA 
resin), or using trifluoroacetic acid (Fmoc method; HMP resin), and 
removed from the polymeric support by filtration. Examples of well known 
reviews containing detailed descriptions of the method employed are a) 
Barany, G., Kneib-Cordonier, N., Mullen, D. G., Int. J. Pept. Protein 
Res., 30, 1987, 705ff and b) Fields, G. B., Noble, R. C., Int. J. Pept. 
Protein Res., 35, 1990, 161-214. The reaction products are isolated by 
preparative HPLC, in particular by means of the reversed phase method 
using RP 8 columns and employing a solvent mixture such as an ascending 
gradient of trifluoroacetic acid in acetonitrile or acetonitrile/water. 
The compounds are characterized by mass spectroscopy, in particular.

EXAMPLE 1 
Solid phase Synthesis of NH.sub.2 --T.sub.1 --T.sub.2 --T.sub.1 --T.sub.2 
--T.sub.1 --T.sub.2 --T.sub.1 --T.sub.2 --Lys--NH.sub.2 
T.sub.1 =Aminoethylglycine-thymine structural component, in accordance with 
WO 92/20703 
T.sub.2 =L-trans-4-Amino-N-(thymin-1-yl)-acetyl!-proline structural 
component, in accordance with DE 43 31 012.5 
The oligomerization is effected using the programme, which exists in the 
ABI 431-A peptide synthesizer, for Boc small-scale reactions. 
32.5 mg (0.025 mol) of MBHA resin are initially introduced into a reaction 
vessel. The support is neutralized with diisopropylethylamine and washed 
with DCM. The activation of 1 mmol of Boc-Lys(2-chloro-Z)-OH (0.41 g) and, 
at any one time, either 50 mg of the T.sub.2 structural component or 48 mg 
of the T.sub.1 structural component is effected by reacting them with 135 
mg (1.0 mmol) of hydroxybenzotriazole and 206 mg (1.0 mmol) of 
dicyclohexylcarbodiimide in N-methyl-2-pyrrolidone (NMP). To improve the 
solubility of the structural components, the hydroxybenzotriazole is 
introduced, together with NMP, into the cartridge containing the amino 
acid. Prior to each coupling step, the tert-butyloxycarbonyl protective 
group of the support-bound intermediate is cleaved off by treating with 
trifluoroacetic acid, and the support is then neutralized with 
diisopropylethylamine and washed with DCM. Stepwise coupling to the 
polymeric support then follows. After the final coupling, the Boc 
protective group is removed by treating with trifluoroacetic acid. 
Weighing the dried support indicates an increase in weight of 56.1 mg. The 
oligomer is cleaved off the support by treating the polymer for 2 hours 
with 4.5 ml of HF and 0.5 ml of anisole at 0.degree. C. in a Teflon flask. 
After effecting the HF-mediated cleavage, the residue is stirred 4 times 
with 15 ml of absolute diethyl ether on each occasion in order to dissolve 
out adhering anisole. The ether is carefully separated off after 15 
minutes in each case. The oligomer is now extracted with 60 ml of 30% 
acetic acid (4.times.15 ml, for 15 minutes in each case), and the solution 
is separated off from the polymer by filtering through a D3 frit, and the 
filtrate is lyophilized. 
46.5 mg of crude product are obtained, which crude product is purified by 
RP-HPLC. A Eurosil Bioselect 300 A (5 .mu.m) column is used as the 
stationary separation medium while employing the following elution system: 
Eluent A: 0.1% TFA in water, 
Eluent B: 0.1% TFA in water/acetonitrile (3/7) 
The gradient is designed as follows: 
______________________________________ 
Gradient (min) 
Eluent A (%) 
Eluent B (%) 
______________________________________ 
0.00 95 05 
60.00 40 60 
40.00 20 80 
45.00 20 80 
50.00 95 05 
______________________________________ 
Detection is at 260 nm using a UV detector. The oligomers in Examples 2 to 
7 were purified in an analogous manner to that used in Example 1. The 
retention time of the target substance is 15.2 minutes. 
Following the HPLC, the purified oligomer is lyophilized. A yield of highly 
pure product of 35.7 mg (0.0153 mmol, 61.2% based on the theoretically 
possible quantity; 63.6% based on the actual quantity bound to the resin) 
is obtained. The oligomer is characterized by mass spectroscopy (LDI 
method). The theoretical molecular weight is 2323 g/mol, with 2324.0 g/mol 
being measured. 
EXAMPLE 2 
Solid Phase Synthesis of NH.sub.2 --T.sub.1 --T.sub.3 --T.sub.1 --T.sub.3 
--T.sub.1 --T.sub.3 --T.sub.1 --T.sub.3 --Lys--NH.sub.2 
T.sub.1 =Aminoethylglycine-thymine structural component, in accordance with 
WO 92/20703 
T.sub.3 =L-cis-4-Amino-N-(thymin-1-yl)-acetyl!-proline structural 
component, in accordance with DE 43 31 012.5 
The oligomerization was carried out in an analogous manner to that in 
Example 1. 70.0 mg of crude product are obtained, which crude product is 
purified by RP-HPLC. 
The retention time of the target substance is 16.3 minutes. 
Following the HPLC, the purified oligomer is lyophilized. A yield of highly 
pure product of 20.4 mg (0.0087 mmol, 34.9% based on the theoretically 
possible quantity; 36.8% based on the actual quantity bound to the resin) 
is obtained. The oligomer is characterized by mass spectroscopy (LDI 
method). The theoretical molecular weight is 2323.3 g/mol, with 2325.6 
g/mol being measured. 
EXAMPLE 3 
Solid Phase Synthesis of NH.sub.2 --(T.sub.1 --T.sub.2 --T.sub.2 
--T.sub.2).sub.3 --Lys--NH.sub.2 SEQ ID NO: 1 
T.sub.1 =Aminoethylglycine-thymine structural component, in accordance with 
WO 92/20703 
T.sub.2 =L-trans-4-Amino-N-(thymin-1-yl)-acetyl!proline, in accordance 
with DE 43 31 012.5 
The oligomerization was carried out in an analogous manner to that in 
Example 1. 
65.2 mg of crude product are obtained, which crude product is purified by 
RP-HPLC. 
The retention time of the target substance is 20.5 minutes. Following the 
HPLC, the purified oligomer is lyophilized. A yield of highly pure product 
of 15.9 mg (0.0046 mmol; 18.4% based on the theoretically possible 
quantity; 21.0% based on the actual quantity bound to the resin) is 
obtained. The oligomer is characterized by mass spectroscopy (LDI method, 
measurement carried out without adding any mass standard). The theoretical 
molecular weight is 3448.3 g/mol, with 3449 g/mol being measured. 
EXAMPLE 4 
Solid phase synthesis of NH.sub.2 --(T.sub.1 --T.sub.2 --T.sub.2).sub.4 
--Lys--NH.sub.2 SEQ ID NO: 2 
T.sub.1 =Aminoethylglycine-thymine structural component, in accordance with 
WO 92/20703 
T.sub.2 =L-trans-4-Amino-N-(thymin-1-yl)-acetyl!proline, in accordance 
with DE 43 31 012.5 
The oligomerization was carried out in an analogous manner to that in 
Example 1. 
60.2 mg of crude product are obtained, which crude product is purified by 
RP-HPLC. 
The retention time of the target substance is 19.6 minutes. Following the 
HPLC, the purified oligomer is lyophilized. A yield of highly pure product 
of 11.8 mg (0.0034 mmol; 13.7% based on the theoretically possible 
quantity; 15.7% based on the actual quantity bound to the resin) is 
obtained. The oligomer is characterized by mass spectroscopy (LDI method, 
no internal mass standard is added). The theoretical molecular weight is 
3436.3 g/mol, with 3442 g/mol being measured. 
EXAMPLE 5 
Solid Phase Synthesis of NH.sub.2 --(T.sub.2 --T.sub.1).sub.4 
--Lys--NH.sub.2 
T.sub.1 =Aminoethylglycine-thymine structural component, in accordance with 
WO 92/20703 
T.sub.2 =L-trans-4-Amino-N-(thymin-1-yl)-acetyl!proline, in accordance 
with DE 43 31 012.5 
The oligomerization was carried out in an analogous manner to that in 
Example 1. 
59.3 mg of crude product are obtained, which crude product is purified by 
RP-HPLC. 
The retention time of the target substance is 17.4 minutes. Following the 
HPLC, the purified oligomer is lyophilized. A yield of highly pure product 
of 44.5 mg (0.0192 mmol; 76.6% based on the theoretically possible 
quantity; 66.0% based on the actual quantity bound to the resin) is 
obtained. The oligomer is characterized by mass spectroscopy (LDI method, 
no internal mass standard is added). The theoretical molecular weight is 
2323.2 g/mol, with 2325 g/mol being measured. 
EXAMPLE 6 
Solid Phase Synthesis of NH.sub.2 --(T.sub.1 --T.sub.4).sub.6 
--Lys--NH.sub.2 SEQ ID NO: 3 
T.sub.1 =Aminoethylglycine-thymine structural component, in accordance with 
WO 92/20703 
T.sub.4 =D-trans-4-Amino-N-(thymin-1-yl)-acetyl!proline, in accordance 
with DE 43 31 012.5 
The oligomerization was carried out in an analogous manner to that in 
Example 1. 
89 mg of crude product are obtained, which crude product is purified by 
RP-HPLC. 
The retention time of the target substance is 23.0 minutes. Following the 
HPLC, the purified oligomer is lyophilized. A yield of highly pure product 
of 78 mg (0.022 mmol; 91% based on the theoretically possible quantity; 
90% based on the actual quantity bound to the resin) is obtained. The 
oligomer is characterized by mass spectroscopy (LDI method, no internal 
mass standard is added). The theoretical molecular weight is 3412.3 g/mol, 
with 3417 g/mol being measured. 
EXAMPLE 7 
Solid Phase Synthesis of NH.sub.2 --(T.sub.1 --T.sub.4 --T.sub.4).sub.4 
--Lys--NH.sub.2 SEQ ID NO: 4 
T.sub.1 =Aminoethylglycine-thymine structural component, in accordance with 
WO 92/20703 
T.sub.4 =D-trans-4-Amino-N-(thymin-1-yl)-acetyl!proline, in accordance 
with DE 43 31 012.5 
The oligomerization was carried out in an analogous manner to that in 
Example 1. 
81.0 mg of crude product are obtained, which crude product is purified by 
RP-HPLC. 
The retention time of the target substance is 22.0 minutes. Following the 
HPLC, the purified oligomer is lyophilized. A yield of highly pure product 
of 70.7 mg (0.02 mmol; 81.5% based on the theoretically possible quantity; 
63.1% based on the actual quantity bound to the resin) is obtained. The 
oligomer is characterized by mass spectroscopy (LDI method, no internal 
mass standard is added). The theoretical molecular weight is 3436.3 g/mol, 
with 3462 g/mol being measured. 
EXAMPLE 8 
Solid Phase Synthesis of NH.sub.2 --(T.sub.1 --T.sub.4 --T.sub.2).sub.4 
--Lys--NH.sub.2 SEQ ID NO: 5 
T.sub.1 =Aminoethylglycine-thymine structural component, in accordance with 
WO 92/20703 
T.sub.2 =L-trans-4-Amino-N-(thymin-1-yl)-acetyl!proline, in accordance 
with DE 43 31 012.5 
T.sub.4 =D-trans-4-Amino-N-(thymin-1-yl)-acetyl!proline, in accordance 
with DE 43 31 012.5 
The oligomerization was carried out in an analogous manner to that in 
Example 1. 
59.0 mg of crude product are obtained, which crude product is purified by 
RP-HPLC. 
The retention time of the target substance is 20.0 minutes. Following the 
HPLC, the purified oligomer is lyophilized. A yield of highly pure product 
of 52.2 mg (0.015 mmol; 60.8% based on the theoretically possible 
quantity; 61.3% based on the actual quantity bound to the resin) is 
obtained. The oligomer is characterized by mass spectroscopy (LDI method). 
The theoretical molecular weight is 3436.3 g/mol, with 3440 g/mol being 
measured. 
Test For Biological Stability Towards Proteases and Nucleases 
EXAMPLE 9 
Stability Towards Proteinase K 
In each case, 20 .mu.l of 1M Tris/HCl (pH 7.5), 80 .mu.l of a 50 mM 
solution of calcium chloride, and 1 U of proteinase K (Serva) were added 
to 75 .mu.g each of the compounds from Example 1 and Example 2, each of 
which was in 75 .mu.l of double distilled water, and the mixtures were 
incubated at 37.degree. C. for 3 hours. Each of the reaction mixtures was 
then investigated by HPLC (reversed phase, Eurosil-Bioselect, eluent: 
5-70% 0.1% trifluoroacetic acid in water/acetonitrile (3/7) against 0.1% 
trifluoroacetic acid in water). In neither case was it possible to detect 
any newly formed degradation product, whereas the signals of the compounds 
from Example 1 and Example 2 were present as before. Consequently, the 
compounds are stable towards proteinase K. 
EXAMPLE 10 
Stability Towards S1 Nuclease 
In each case, 20 .mu.l of nuclease buffer (Promega) and 4 .mu.l of S1 
nuclease (Promega, 50 U/ml) were added to 75 .mu.g each of the compounds 
from Example 1 and Example 2, each of which was in 75 .mu.l of double 
distilled water, and the mixtures were incubated at 37.degree. C. for 3 
hours. Each of the reaction mixtures was then investigated by HPLC 
(reversed phase, Eurosil-Bioselect, eluent: 5-70% 0.1% trifluoroacetic 
acid in water/acetonitrile (3/7) against 0.1% trifluoroacetic acid in 
water). In neither case was it possible to detect a newly formed 
degradation product, whereas the signals of the compounds from Example 1 
and Example 2 were present as before. Consequently, the compounds are 
stable towards S1 nuclease. 
EXAMPLE 11 
Determination of the Temperature at Which Selected Oligomers Anneal to an 
A.sub.8 DNA Strand or an A.sub.12 DNA Strand. 
The corresponding DNA strands were prepared on an "ABI 380B" Applied 
Biosystems DNA synthesizer using the phosphoramidite method in accordance 
with the manufacturer's (Applied Biosystems) small-scale cycle. 
The annealing temperature was determined using a Perkin Elmer "Lambda Bio" 
UV-Vis spectrometer and employing the "PE-TEMP" method specified by the 
manufacturer. 
For this purpose, sufficient PNA is dissolved in 700 .mu.l of water to give 
an absorbtion of 0.3. The same is done with the corresponding DNA strand. 
The two strands are then combined and the volume of water is increased to 
1.5 ml. The combined strands are next heated at 95.degree. C. for 5 
minutes and then cooled slowly overnight in a Styropor vessel. 
The absorbtion of the double strands in the temperature range of from 
20.degree. C. to 80.degree. C. is then investigated using the "PE-temp" 
method. The turning point (maximum of the 1st derivative) of the resulting 
absorption curve then corresponds to the annealing temperature which is 
measured on the temperature scale. 
The following oligomers were measured: 
______________________________________ 
Oligomers from Example 
DNA strand Annealing temp. in .degree. C. 
______________________________________ 
1 A.sub.8 47.0 
2 A.sub.8 27.0 
3 A.sub.12 34.4 
4 A.sub.12 48.8 
5 A.sub.8 46.0 
6 A.sub.12 27.0 
______________________________________ 
the oligomer A.sub..sub.12 is designated SEQ ID NO:6. 
EXAMPLE 12 
Demonstration of Strand Displacement by Nucleic Acid-Binding Oligomers in 
Double-Stranded Plasmid DNA 
In that which follows, the test is described for experimentally 
demonstrating DNA double strand displacement by the nucleic acid-binding 
oligomers. This ability to displace DNA double strands cannot be achieved 
using a ribose phosphate backbone, a ribose methylphosphonate backbone, a 
ribose phosphorothioate backbone, or other nucleic acid-like backbone 
types. 
The plasmid DNA employed in the example is a model substrate for 
demonstrating DNA double strand displacement. Other plasmids which contain 
appropriate target sequences having a base sequence which is complementary 
to the nucleic acid-binding oligomers to be tested can also be used for 
the test in the same way. 
In the test described here, use is made of double-stranded, circular 
plasmid DNA which is 4880 base pairs in length and which contains, 1150 
base pairs apart, two regions of polyadenine sequence containing at least 
nine consecutive adenine nucleotides. 
Seven samples, which were set up in parallel and which were designated 
(1-7), each contained 1.0 .mu.g of uncut plasmid DNA in 14 .mu.l of 
H.sub.2 O. 1 .mu.l of solution containing 0.0001 .mu.g, 0.001 .mu.g, 0.01 
.mu.g, 0.1 .mu.g and 1.0 .mu.g, respectively, of nucleic acid-binding 
oligomer from one of Examples 1 to 5 was added to samples 3 to 7, 
respectively, and the mixtures were incubated in sealed Eppendorf tubes at 
37.degree. C. for 45 min. 4 .mu.l of buffer (250 mM Na acetate, 1M NaCl, 
2.5% glycerol, 5 mM ZnCl.sub.2, pH 4.4) were then added to all the samples 
while 1 .mu.l of Aspergillus oryzae S1 nuclease (from Boehringer 
Mannheim), having an activity of 10 U/.mu.l, was also added to each of 
samples 2 to 7. After these samples had been incubated at 30.degree. C. 
for 15 minutes, they were placed on ice and 1 .mu.l of 0.5M EDTA and 3 
.mu.l of loading buffer (50% glycerol, 0.25% bromophenol blue in 40 mM 
Tris-HCl, 20 mM sodium acetate, 1 mM EDTA, pH 7.2) were added; the samples 
were then immediately subjected to electrophoretic separation on 1.2% 
agarose gels and, after staining with ethidium bromide, the sizes of the 
resulting plasmid fragments in the gel were determined on a UV light 
transilluminator at 254 nm by comparing with a molecular weight standard 
(1 kb ladder, from Gibco-BRL, D-7514 Eggenstein). 
It was found that, in the samples (samples 5-7) containing a concentration 
of the oligomer from Example 1&gt;0.001 .mu.g (.apprxeq.4.3.10.sup.-7 M), the 
S1 nuclease reaction produced DNA fragments of 4880 base pairs (plasmid 
linearization) and of 3270, 2570 and 1150 base pairs. These fragments 
demonstrate that the octamer from Example 1 bound in a sequence-selective 
manner to the double-stranded DNA leading to strand displacement and 
subsequent cleavage by S1 nuclease. 
DNA fragments of 3730, 4480, 2570 and 1150 base pairs in length, which 
confirm the sequence-selective double strand displacement, were likewise 
detected in samples 5-7 using a modified test mixture in which a plasmid 
DNA which had been linearized by restriction endonuclease digestion in the 
immediate vicinity of one of the two regions of polyadenine sequence was 
added to the samples in place of the circular, uncut plasmid DNA. 
Furthermore, DNA double strand displacement was also detected in the 
double-stranded plasmid DNA when the octamer from Example 1 was employed 
at higher salt concentrations, using 5 mM Tris HCl, 1 mM Mg Cl.sub.2, 10 
mM NaCl, pH 7.0, in place of water. 
In addition, a comparable DNA double strand displacement was demonstrated 
in this test using the oligomers from Examples 3 to 5 by way of example. 
The oligomers from Examples 3 to 5 also gave rise in the test, at 0.01 
.mu.g to 0.001 .mu.g (approximately 5.multidot.10.sup.-6 M to 
5.multidot.10.sup.-7 M), to a sequence-selective cleavage of 
double-stranded DNA by Aspergillus oryzae S1 nuclease. 
This series of tests rendered it possible to demonstrate the 
concentration-dependent and sequence-selective binding of the oligomers 
from Examples 1 to 5 to double-stranded DNA and to demonstrate the 
presence of the single-stranded DNA, which arose as a result, by means of 
digestion with S1 nuclease (at high salt concentrations to ensure the 
single-strand specificity of the S1 nuclease). 
EXAMPLE 13 
Gel Shift Analyses 
Gel shift analyses can verify, qualitatively and quantitatively, the 
potential ability of nucleic acid-binding oligomers to hybridize to normal 
diester oligomers. For this purpose, single-stranded DNA of appropriate 
base sequence is incubated with the oligomer to be investigated and the 
mixture is then fractionated by gel electrophoresis. As compared with free 
DNA, hybridized DNA exhibits a clear shift in the gel. By varying the 
concentration of the nucleic acid-binding oligomers, it becomes possible 
to make quantitative statements about the extent of the hybridization. 
Implementation of the Test 
1 .mu.g of diester oligonucleotide of appropriate base sequence is labelled 
at the 5' end, in the current manner and in a volume of 10 .mu.l, using 
polynucleotide kinase and .gamma.-ATP (Sambrook, Fritsch, Maniatis: 
Molecular Cloning, A Laboratory Manual, Cold Spring Harbor, 1989). After 
the labelling, the sample is heated at 70.degree. C. for 10 min to 
denature the enzyme and is then mixed with 9 .mu.g of unlabelled oligomer. 
1 .mu.l of this mixture is then treated with a desired quantity of the 
nucleic acid-binding oligomer to be tested (1-10 .mu.g) and the whole is 
incubated (hybridization) in a volume of 20 .mu.l at 22.degree. C. (room 
temperature) for 30 min. After that, the sample is placed on ice for 30 
min. An unhybridized, labelled oligomer is treated in the same way and 
serves as the control. The samples are loaded onto a 15% polyacrylamide 
gel in 1.times.Tris-borate-EDTA buffer. The gel and the buffer have been 
precooled in a refrigerator (8.degree. C.), and the electrophoresis is 
left to run overnight at 55 V in a refrigerator. Following the 
electrophoresis, an autoradiogram is prepared on AGFA film (exposure times 
1 to 16 hours). 
Results 
The compounds from Examples 1, 3, 4 and 6 already exhibit clear gel shifts 
when they are present at a concentration which is equimolar to that of a 
diester, and these shifts are complete when the compounds are present in a 
5 to 10-fold excess; this verifies the very good hybridization properties 
of these compounds. In comparison, the compounds from Examples 5 and 7 
exhibit a somewhat lower hybridization potential. 
Legend to FIG. 1 
Agarose gel electrophoresis of the test samples from Example 5 for the 
purpose of demonstrating DNA double strand displacement following 
incubation of plasmid DNA with the DNA-binding octamer from Example 1 and 
subsequent reaction with S1 nuclease. The contents of the tracks of the 
ethidium bromide-stained gel are as follows: 
0=molecular weight standard for double-stranded fragments of DNA 
1=uncut plasmid DNA (identical substrate for each test sample) 
2=as 1, but containing 10 U of S1 nuclease 
3-7=as 2, but additionally containing 0.0001; 0.001, 0.01; 0.1 and 1.0 
.mu.g of DNA-binding octamer from Example 1 
DNA double strand displacement can be detected in the test when the 
concentration of the octamer from Example 1 is &gt;4.3.times.10.sup.-7 M. 
EXAMPLE 14 
Capillary Electrophoresis 
Introduction 
The hybridization properties of oligonucleotide analogues are frequently 
conveyed by way of the melting temperature, T.sub.M, of the complementary 
oligonucleotides. Very recently, capillary gel electrophoresis (CE using a 
solid gel in the capillary) has also been employed for determining PNA-DNA 
binding (Rose, D. J. Anal. Chem. (1993), 65, 3545-3549). Dynamic liquid 
gels have also been successfully employed for separating DNA fragments 
(Barron, A. E.; Soane, D. S. & Blanch, H. W. J. Chromatogr. (1993), 652, 
3-16). 
Dynamic gel capillary electrophoresis (DGCE) is particularly suitable for 
separating oligomeric DNA nucleotides due to its high degree of 
reproducibility and its availability. 
Experimental 
Capillary Electrophoresis (CE) 
The investigations were carried out on an ABI 270A-HT (Applied Biosystems, 
Weiterstadt); any other CE apparatus having a UV detector is also 
suitable. The conditions are given in Table 1. The data were transferred 
to a PC via an AD converter and recorded and evaluated using the HPCHEM 
software (Hewlett-Packard, Waldbronn). 
Hybridization 
The nucleic acid-binding octamer from Example 1, at a constant 
concentration of 45 .mu.M in Tris-HCl (5 mM, 0.1 ml), was hybridized, in 
differing ratios, to the complementary DNA octamer .delta.(A).sub.8. The 
ratios of 0.2/1, 0.25/1, 0.33/1, 0.5/1, 1/1, 2/1 and 3/1 were chosen. 
The samples were heated in parallel, and in accordance with the 
standardized method, at 93.degree. C. for 5 min and were then gradually 
cooled down to RT; the samples were then measured directly after they had 
been diluted 1:5 with water. 
TABLE 1 
______________________________________ 
Measurement parameters for determining hybridization using 
______________________________________ 
DGCE 
Buffer 100 nM Tris/borate + 
0.5% dextran, pH 8.5 
Capillary: Fused silica (ABI) 
Lengths: total: 
50 cm effective: 29 cm 
Internal diameter: 
50 .mu.m 
Measurement parameters: 
Vacuum injection: 
1.7 .multidot. 10.sup.4 Pa 
Injection time: 
3 sec 
Voltage: 25 kV 
Temperature: 30.degree. C. 
Run time: 10 min 
Detection: 257 nm 
Column conditioning: 
Washing vacuum: 
6.8 .multidot. 10.sup.4 Pa 
1st washing step: 
2 min, 0.1 N NaOH 
2nd washing step: 
6 min, buffer 
______________________________________ 
Results and Discussion 
The hybridization yield plotted against the concentration of complementary 
DNA octamer .delta.(A).sub.8 shows that the hybridization yield increases 
strongly up to a ratio of 1:0.5. After that, the quantity of hybridization 
product remains relatively constant despite increasing concentration of 
complementary .delta.(A).sub.8 octamer (FIG. 2). The ratio of 1:0.5 
indicates that the nucleic acid-binding octamer from Example 1 hybridizes 
to the complementary .delta.(A).sub.8 octamer in a ratio of 2:1. 
Legend to FIG. 2 
In the figure, the maximum peak height of the hybridization product formed 
from the nucleic acid-binding octamer from Example 1 and the complementary 
.delta.(A).sub.8 octamer (y axis) is plotted against the relative 
concentration of complementary .delta.(A).sub.8 octamer (x axis). Maximum 
hybridization is achieved at a 2:1 ratio of nucleic acid-binding octamer 
from Example 1 to complementary .delta.(A).sub.s octamer. 
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