HK1038568B - Polyamide-oligonucleotide derivatives, their production and use - Google Patents
Polyamide-oligonucleotide derivatives, their production and use Download PDFInfo
- Publication number
- HK1038568B HK1038568B HK01109203.3A HK01109203A HK1038568B HK 1038568 B HK1038568 B HK 1038568B HK 01109203 A HK01109203 A HK 01109203A HK 1038568 B HK1038568 B HK 1038568B
- Authority
- HK
- Hong Kong
- Prior art keywords
- methyl
- dna
- formula
- zero
- alkyl
- Prior art date
Links
Description
The present invention relates to new polyamide oligonucleotide derivatives with valuable physical, biological and pharmacological properties, and their application relates to use as inhibitors of gene expression (antisense oligonucleotides, ribozymes, sense oligonucleotides and triplex forming oligonucleotides), probes for the detection of nucleic acids and as aids in molecular biology.
Oligonucleotides are increasingly used 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 complementary to an inhibitory mRNA. This target mRNA may be of cellular, viral or other pathogenic origin.Err1:Expecting ',' delimiter: line 1 column 416 (char 415)While antisense oligonucleotides generally inhibit splicing of mRNA or its translation into protein, triplex forming oligonucleotides inhibit DNA transcription or replication (C. Helene and J.J. Toulme; Biochem. Biophysics. 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 by forming a double strand, which then forms a triplex structure in a second hybridization with a triplex forming oligonucleotide.Another use of synthetic oligonucleotides is in the so-called ribozymes, which destroy the target RNA as a result 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 of the present invention can also be used in the sense of aptamers in therapy. Aptamers are oligomeric nucleic acids or their analogues which bind with high affinity to proteins. The aptamers are found by in vitro selection from a random mixture (Famulok and Szostak (1992) Appendix 104, 1001-1011) and have been successfully performed for a thrombin-binding aptamer (Bock et al. (1992) Nature 355, 564-566).Another possibility is that the binding region of the aptamer is encoded by a separate nonbinding part of the molecule to facilitate identification (Brenner and Lerner (1992) PNAS 89, 5381-5383).
In DNA diagnostics, DNA probes or DNA probes are used to identify specifically hybridized nucleic acid fragments with appropriate marking, and the specific formation of the new double strand is tracked using the marking, preferably non-radioactive, to detect genetic, malignant, viral or other pathogenic diseases.
For most of the above uses, oligonucleotides in their naturally occurring form are unsuitable or unsuitable; they must be chemically modified to meet the specific requirements; for oligonucleotides to be used in biological systems, for example inhibition of viral replication, they must meet the following conditions:
They must be sufficiently stable under in vivo conditions, i.e. both in serum and intracellular. They must be designed to pass through the cell and nucleus membranes. They must bind to their target nucleic acid in a base-specific manner under physiological conditions to produce the inhibitory effect.
For DNA probes, the requirements of points 1 to 3 are not necessary; however, these oligonucleotides must be derived in such a way that detection, for example by fluorescence, chemiluminescence, colorimetry or specific staining, is possible (Beck and Köster, Anal. Chem. 62, 2258 (1990)).
The chemical modification of oligonucleotides usually takes place by changing the phosphate backbone, ribose unit or nucleobases accordingly (J. S. Cohen, Topics in Molecular and Structural Biology 12 (1989) Macmillan Press; E. Uhlmann and A. Peyman, Chemical Reviews 90, 543 (1990)). Another commonly used method is the production of oligonucleotide-5'-conjugates by translating the 5'-hydroxy group with corresponding phosphorylation reactions.
Polyamide nucleic acid derivatives have recently been described (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 greater affinity than natural oligonucleotides. These so-called peptides or polyamide nucleic acids (PNA) are DNA-analog compounds in which the deoxyribose phosphate structure has been replaced by a polyamide oligomer. These compounds have the advantage over natural oligonucleotides that they are very stable, but on the other hand they have the following properties in serum:
(1) They are not or only in undetectable quantities taken up into cells.However, since antisense or triplex-forming oligonucleotides can only be active in the cell, PNAs are not suitable as such for inhibition of gene expression in vivo. (2) PNAs tend to aggregation in aqueous solution, i.e. under physiological conditions. They are therefore soluble in aqueous buffer and are not available for hybridization to complementary sequences. (3) PNAs also have a high affinity for various materials such as Sephadex (Fa. Pharmacia) or Bond Elut (Fa. Varian) used in the purification of oligomers, so that PNAs are often poorly isolated. (4) Another serious drawback of PNAs is that they are not very effective in cleaning the oligomers.While natural nucleic acids to complementary nucleic acids generally hybridise in antiparallel orientation, PNAs can bind in both antiparallel and parallel orientation.(5) In WO 92/20702, an oligonucleotide PNA conjugate (T) 7'-L-N) t) 6-alagat (Fig. 25; substitute sheet), wherein (T) 76) a natural heptamydyl substitute Oxyde Oligonucleotide, which means 5'-O-Phosphat and 4-Hydroxybutters (L) is strongly bound to the primary function (N) of an amino acid (PNA) and does not yet exhibit any properties similar to those of an amino acid (PNA) in this area. (Alagat) 7 has not been described in any of the cytotoxic properties of this compound.
The orientation of base-pairing nucleic acid strands is defined as follows: (see Egholm et al.; Nature 365 (1993) 566-56
Other
8), and
wherein
5'the 5' end of an oligonucleotide, 3'the 3' end of an oligonucleotide, N the aminoterminus of a PNA'sCden the carboxyterminus of a PNA's.
Cases A) - D) are examples of the orientation types of antisense oligomers that are in principle possible.Cases E) - F) show possibilities for triplex formation on single-stranded or double-stranded nucleic acids.Two of the PNA or DNA single strands may be linked together.For example, in E) the N-terminus of the PNA may be linked to the 5' end of the DNA or in F) the C-terminus of the PNA may be linked to the 5' end of the DNA.
The purpose of the invention was therefore to produce polyamide oligonucleotide derivatives in which the above disadvantages are eliminated.
Err1:Expecting ',' delimiter: line 1 column 836 (char 835)other than thymine; stands for a residue of formulae IIa or llb
In which
Other
R2 and B as defined above;
Hydroxy, Mercapto, C1-C18-alkyl, preferably C1-C8-alkyl, C1-C18-alkoxy, preferably C1-C8-alkoxy, C6-C20-aryl, preferably C6-C12-aryl, C6-C14-aryl-C1-C8-alkyl, preferably C6-aryl-C1-C4-alkyl, NHR3 or NR3R4 means andR3C1-C18-alkyl or C1-C4-alkoxy-C1-C4-alkyl, preferably C1-C8-alkyl or C1-C4-alkoxy-C1-CYO4-alkyl, preferably C1-C4-alkoxy or methoxy-methyl and C6-C18-aryl-C1-C1-C8-alkyl, preferably C6-C12-aryl-C1-C4-alkyl, preferably C6-C1-C1-C1-C4-alkyl, preferably C1-C1-C1-C4-C4-alkyl, preferably C4-C4-C4-C4-C4-C4-C4-C4-R4 or R4-R4 means andR3C1-C1-C1-C1-C1-C1-C1-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C4-C
Other
wherein B is defined as above;n = 0 to 20;p = 0 to 20;
Li3 and Li4 independently each form a structure of formula V
Other
The following shall be reported in the table:
Other
iswherein the two are independent of each other
Other
ε = 1 to 5, preferably 1 - 2 is
V' oxygen, NH, a bond or residue of formula VI
Other
means:
wherein U, V, W and Y are as defined above;GC1-C12-alcandyyl, preferably C1-C6-alcandyyl, wherein alcandyyl may be substituted by halogens, preferably F or chlorine, amino, hydroxy, C1-C18-alkyl, preferably C1-C6-alkyl, C1-C18-alkoxy, preferably C1-C6-alcoxy, C6-C14-aryl, preferably C6-aryl, or C6-C14-aryl-C1-C18-alkyl, preferably C6-aryl-C1-C4-alcandyyl;C6-C14-aryl-di-C1-C12-alcandyyl, preferably C6-aryl-di-C1-C4-alcandyyl, or the group (CH2CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-CH2-where U, V, W and Y are as defined above; andF and F' are linked by a bond (cyclic compounds) and/orF for R0 - (A) k - V - andF'in formula Ib for V1 - (A), - R1,
wherein
R0 means hydrogen, C1-C18-alkanoyl, preferably C8-C18-alkanoyl, C1-C18-alkoxycarbonyl, C3-C8-cycloalkanoyl, C7-C15-aroyl, C3-C13-heteroaroyl or a group that facilitates the intracellular uptake of the oligomer or acts as a marker for a DNA probe or attaches the oligomer to the target nucleic acid by binding, cross-linking or splitting, or
if k = zero, R0 is hydrogen or together with V for a residue of formula VII
It says in
Z and Z' independently of each other hydroxy,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-C18-alkyl, preferably C6-aryl-C1-C4-alkyl, C1-C22-alkylthio, preferably C12-C18-alkylthio, NHR3, NR3R4, or a group that favors the intracellular uptake of the oligomer or is used as a marker for a DNA probe or in the hybridisation of the oligomer to the target nucleic acid by binding, cross-linking or splitting it, and
R3, R4, V and W are as defined above;
R1Hydrogen or Q0
where R1 is always hydrogen,
if simultaneously I = zero and
in formula Ib q = 1 or q = r = zero and in F' = V1 - (A), - R1 with V1 = V means,A residue of a natural or unnatural amino acid,Err1:Expecting ',' delimiter: line 1 column 295 (char 294)Li4 is for bonding;
wherein each nucleotide may be present in its D or L configuration and the base may be in the α or β position.
Compounds of formula lb in which the base is in the β position on the sugar are particularly preferred.
x = 1 is andq=r=1, s=t=zero orr=s=1, q=t=zero orq=r=s=1, t=zero orr=s=t=1, q=zero.
In particular, oligomers of formula Ib, where V', V, Y and W have the meanings of thio, oxy, oxo or hydroxy, are preferred; these are particularly preferred if R2 is hydrogen in addition.
In particular, oligomers of formula Ib with ε = 1 are preferred, where
(a) a compound of formula V in which V' = oxygen or a compound of formula VI, G = C1-C10 alkyl, G' =-CONH-b) a compound of formula V in which G, V' a bond and G' a compound of formula VI is preferred with
(a) a compound of formula V with V' = imino, G = C1-C10 alkyl and G' = compound of formula VIb) a compound of formula V with V' = imino, G and G' = bonding c) a compound of formula V with V' = imino, G = C1-C10 alkyl and G' = V with preferably U = V = W = Y = oxygen.
Particularly preferred are oligomers of formula Ib, where V', V, Y and W have the meaning of thio, oxy, oxo or hydroxy, R2 is equal to hydrogen, Li1 the meaning of -V'-[CH2]nC(O)NH- with V' = connection of formula Vl with U =V=W=Y=oxygen or Li2 have the meaning of -HN-[CH2]n(G') - where n = 2 to 5 and G' is equal to formula VI with U, V, W and Y = oxygen.
In addition, oligomers of formula Ib are preferred, wherein V', V, Y and W have the meaning of Thio, Oxy, Oxo or Hydroxy, R2 is equal to hydrogen, Li1, the meaning of -O-[CH2]nC(O)NH- or Li2 have the meaning of -HN-[CH2]n(G')-, wherein n=2 to 5 and G' is equal to formula VI with U,V,W and Y=oxygen and q=zero and r=s=t=1.
The preferred base is adenine, where the bracket means that R2 is in the 3' position (see formula llb).
The invention is not limited to α- and β-D- or L-ribofuranosides, α- and β-D- or L-deoxyribofuranosides and their corresponding carbocyclic pentacyclic analogues, but also applies to oligonucleotide analogues made from other sugar building blocks, such as ring-extended and ring-narrowed sugars, acyclic, ring-broken or other suitable sugar derivatives.
The oligonucleotide part (DNA in formula l) can therefore be modified from the natural structure in a variety of ways.
The following are examples: phosphorothioates, phosphorodithioates, methylphosphonates, phosphoramidates, borano-phosphates, phosphate methyl esters, phosphate ethyl esters, phenyl phosphonates.
For example, substitution by formacetal, 3'-thioformacetal, methyl hydroxylamine, oxime, methyldimethyl hydrazone, dimethyl sulfone, silyl groups, and the preferred substitution by formacetals and 3'-thioformacetals.
The following are examples: α-anomic sugars, 2'-O-methylribose, 2'-O-butylribose, 2'-O-allylribose, 2'-fluoro-2'-deoxyribose, 2'-amino-2'-deoxyribose, α-arabinofuranose, carbocyclic sugar analogues.
Base pairing not changed
The following are examples: 5-propinyl-2'-deoxyurin, 5-propinyl-2'-deoxycytidine, 5-hexinyl-2'-deoxyurin, 5-hexinyl-2'-deoxyurin, 5-fluoro-2'-deoxyurin, 5-fluor-2'-deoxyurin, 5-hydroxymethyl-2'-deoxyurin, 5-methyl-2'-deoxyurin, 5-bromo-2'-deoxyurin. The preferred modifications are 5-propinyl-2'-deoxyurin, 5-hinyl-2'-deoxyurin, 5-hexinyl-2'-deoxyurin and 5-propyl-2'-deoxyurin.
Examples of groups that favor intracellular uptake are various lipophilic residues such as -O-(CH2)x-CH3, where x means an integer from 6 to 18, -O-(CH2)n-CH=CH-(CH2)m-CH3, where n and m independently mean an integer from 6 to 12, -O-(CH2CH2O)4-(CH2)9-CH3, -O-(CH2CH2O) (CH2) 13-CH3 and -O-(CH2CH2O) 7-(CH2) 15-CH3, but also steroid residues such as cholesterol groups or vitamin A derivatives such as vitamin A, vitamin E, vitamin A, or vitamin D, and other transporters, which are natural carriers of fluorescent acids such as fluorescent acids, fluorescent flavonoids, 2-nitrogen, 2-nitrogen, such as N-methyl-Diphenyl-Diphenyl-Diphenyl-Diphenyl-Diphenyl-Diphenyl-Diphenyl-Diphenyl-Diphenyl-Diphenyl-Diphenyl-Diphenyl-Diphenyl-Diphenyl-Diphenyl-Diphenyl-Diphenyl-Diphenyl-Diphenyl-Diphenyl-Diphenyl-Diphenyl-Diphenyl-Diphenyl-Diphenyl-Diphenyl-Diphenyl-Diphenyl-Diphenyl-Diphenyl-Diphenyl-Diphenyl-Diphenyl-Diphenyl-Diphenyl-Diphenyl-Diphen-Diphenyl-Diphen-Diphen-Diphen-Diphen-Diphen-Diphen-Diphen-DiphenDiphenDiphenDiphenDiphenDiphenDiphenDiphenDiphenDiphenDiphenDiphenDiphenDiphenDiphenDiphenDiphenDiphenDiphenDiphenDiphenDiphenDiphenDiphenDiphenDiphenDiphenDiphenDiphenDiphenDip
Other
Oligonucleotide analogues that bind to or intercalate and/or cleavage or cross-linking nucleic acids contain, for example, acridine, psoralen, phenanthridine, naphtoquinone, daunomycin or chlorethylaminoaryl conjugates.
Other
Examples of groups NR3R4 in which R3 and R4 together with their carrier nitrogen atom form a 5- to 6-membered heterocyclic ring containing an additional heteroatom are the morpholinyl and imidazolidinyl residues.
The polyamide component (PNA in formula I) consists of amide structures containing at least one nucleobase other than thymine, such polyamide structures being, for example, composed of the following components (a) to h, preferably a), where f is 1 to 4, preferably 1 or 2, and g is 0 to 3, preferably 0 to 2:
(a)
Other
The following table shows the results of the analysis:
Other
The following are the main characteristics of the product:
Other
The use of the term 'chemical' in this context is not excluded.
Other
The use of the term 'science' in the definition of the term 'science' is not excluded.
Other
The Commission has not yet adopted a decision on the application of Article 93 (2) of the EC Treaty.
Other
The Commission has not yet adopted a decision on the application of Article 93 (2) of the EC Treaty.
Other
The following is a list of the main components of the system:
Other
Err1:Expecting ',' delimiter: line 1 column 130 (char 129)
The preferred polyamide structures are those constructed from structures as defined in (a) and the latter are particularly preferred if f = 1.
The synthesis of formula 1 polyamide oligonucleotide derivatives is similar to the synthesis of oligonucleotides in solution or preferably in the solid phase, where appropriate using an automatic synthesis device. The construction of the formula 1 oligomer can be done in stages by condensing a PNA unit or DNA unit with one nucleobase each to a correspondingly derived carrier or to a growing oligomer chain. However, the construction can also be fragmented, with the fragments first synthesized as polyamide or oligonucleotide structures, which are then formed into polynucleotide-dimethyl-knitriol-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl-methyl
Err1:Expecting ',' delimiter: line 1 column 803 (char 802)
Err1:Expecting ',' delimiter: line 1 column 579 (char 578)
PG protective group, prefers a weak acid-labile protective group;Nu' nucleotide unit, whose exocyclic amino group is protected by an appropriate protective group;Nu'-active a commonly activated derivative in nucleotide chemistry, such as a phosphoramide, phosphor ester or H-phosphonate; R', B' and Q' stand for the possibly protected forms of A, B and Q.
Other
Other
The following information shall be provided for the purpose of the calculation of the value of the product:
Other
For q=r=s=t=1 and x=1, the following synthesis sequence applies:
Other
The linker may be removed if there are transitions in the PNA or DNA.
For illustration, a synthesis scheme for PNA/DNA hybrids of formula I is shown, illustrating the production of a hybrid oligomer in which q=r=s=t=1 and x=1. First, the end group F is synthesized according to known methods and, in the case of solid-phase synthesis, coupled to a polymer carrier (step 1). After splitting the protective group PG (step 2), which preferably takes place in the weakly acidic medium, the polyamide bactinium blocks are heated up to the desired deletion of the PNA part (step 3).After condensation of a linker (step 6) that allows the transition from DNA to PNA, a polyamide structure is again built. Introduction of a linker that allows the transition from PNA to DNA, conjugation of another DNA structure (step 7) and final coupling of the end group F (step 8) yield the hybrid molecule [F-DNA-Li-PNA-Li-DNA-Li-PNA-F']. The linker blocks may also contain nucleobases. For example, to synthesize a hybrid F-DNA-Li-PNA-Li-F' (qr=1, s=t=Null) base, steps 1-5 are performed and the synthesis is completed only with step 8.
For example, for the synthesis of a hybrid F-PNA-Li-DNA-F' (r=s=1, q=t=zero), steps 1-2 are first performed, followed by steps 5-6, followed by step 3 and the completion of the synthesis with step 8.For the synthesis of a hybrid F-PNA-Li-DNA-Li-PNA-F' (r=s=t=1, q = zero), the synthesis begins with steps 1 to 6, and after repeating step 3, the synthesis is completed with step 8.
If x is in formula I > 1, then steps 2-7 must be repeated if necessary. After the polymer chains are formed, the PNA/DNA hybrids must be separated from the carrier in the case of solid-phase synthesis and, if not, the protective groups at the bases, amino acid side chains and end groups must be separated.
However, the PNA and DNA components can be synthesized separately by known methods and then coupled with at least one component by corresponding activation.The activation of the DNA part is for example in the form of a known bromcyan condensation, in which the activated phosphate function is reacted with a reactive group in the PNA part, preferably an amino function.
Surprisingly, the oligomers of formula la and lb were found to have a greatly increased cellular uptake compared to pure PNAs. This improved uptake is crucial because antisense or triplex-forming oligomers can only act if they are effectively absorbed into cells. Their hybridization behavior is also more favorable than that of pure PNAs, as they preferentially lead to antiparallel duplex formation. Compared to normal DNA examples, they have a greatly improved nuclear stability, which is manifested in an increased biological activity.
Other advantages of PNA/DNA oligomers or PNA/RNA oligomers are the ability to stimulate cellular endonucleases such as RNase H and RNase L. Unlike PNAs, PNA DNA chimeras, which contain some deoxyribonucleotide units, can, when attached to the complementary target RNA, split them in a sequence-specific manner by induction of the cellular RNase H. A special embodiment of the acenylate oxides of the invention is furthermore those composed of PNA and a 2'5' tetradalgoadlate-linked oligoadlate-part, its analogue cordarlat or ancepylamine-activated RNA, which are activated and activated by the L-terminal nuclease.
In general, the present invention covers the use of compounds of formula 1 as therapeutically active components of a medicinal product. Therapeutically active polyamide oligonucleotide derivatives are generally understood to be antisense oligonucleotides, triple helix-forming oligonucleotides, aptamers or ribozymes, especially antisense oligonucleotides.
The medicinal products of the present invention may be used, for example, to treat diseases caused by viruses, such as HIV, HSV-1, HSV-2, influenza, VSV, hepatitis B or papilloma viruses.
For example, the antisense polyamide oligonucleotide derivatives of the invention that are effective against such targets have the following base sequence, in which the length and position of the PNA and DNA portion can be varied accordingly to obtain optimal properties.
(a) against HIV, for example.
The following shall be indicated in the column 'D' of the table:
The following shall be reported for the following categories of vehicles:
The following shall be indicated in the table:
The following shall be indicated in the table:
The test chemical is used to test the antibody against the antibody in the test chemical.
The following shall be added to the list of products:
The drugs of the present invention are, for example, also suitable for the treatment of cancer, for example by using polyamide oligonucleotide sequences directed against targets responsible for the development or growth of cancer, such targets being:
1) Nuclear oncoproteins such as c-myc, N-myc, c-myb, c-fos, c-fos/jun, PCNA, p1202) Cytoplasmic/membrane-associated oncoproteins such as EJras, c-Ha-ras, N-ras, rrg, bcl-2, cdc-2, c-raf-1, c-mos, c-src, c-abl3) Cellular receptors such as EGF receptor, c-erbA, retinoid receptors, protein kinase regulatory subunit, c-fms4) Cytokines, growth factors, extracellular matrix such as CSF-6, IL-1a, IL-1b, IL-2, IL-4, bF, fibroblastin, myc,
For example, antisense polyamide oligonucleotides of formula 1 according to the invention that are effective against such targets have the following base sequence:
(a) against c-ha-ras, for example.
The following shall be added to the list of substances:
(VIII) (b) the FIFG, for example
The following shall be reported in the table:
(XXX) c) c-myc, for example
The following shall be reported in the table:
(IX)
The following shall be added to the list of products:
(X) (d) c-myb, for example
The following shall be reported in the table:
(XI) e) c-fos, e.g.
The following shall be added to the list of products:
(XII)
The following shall be added to the list of products:
(XIII)
The following shall be added to the list of products:
(XIV) (f) p 120, for example
The following shall be added to the list of products:
(XV) (g) EGF receptor, e.g. the following:
The following shall be reported in the table:
(XVI)
The following shall be reported in the table:
(XVII) h) p53 tumor suppressor, such as
The following shall be added to the list of products:
(XVIII)
The following shall be reported in the table:
(XIX)
For example, the drugs of the present invention are also suitable for the treatment of diseases affected by integrins or cell-cell adhesion receptors, such as VLA-4, VLA-2, ICAM, VCAM or ELAM.
The method of analysis is based on the following equation:
The following shall be indicated in the column 'D' of the table:
(XX) (b) ICAM, for example
The following shall be reported in the table:
(XXI)
The following shall be added to the list of substances:
(XXII)
The following shall be added to the list of products:
(XXIII) (c) ELAM-1, for example
The following shall be reported in the table:
(XXIV)
The following shall be added to the list of substances:
(XXV)
The drugs of the present invention are also suitable, for example, for the prevention of restenosis.
The Commission shall, by means of implementing acts, adopt implementing acts laying down the rules for the application of this Regulation and laying down the procedures for implementing them.
1) Nuclear transactivator proteins and cyclines such as c-myc, c-myb, c-fos, c-fos/jun, cycline and cdc2-kinase2) Mitogens or growth factors such as PDGF, bFGF, EGF, HB-EGF and TGF-β.3) Cellular receptors such as bFGF receptor, EGF receptor and PDGF receptor.
For example, antisense polyamide oligonucleotides of formula 1 according to the invention that are effective against such targets have the following base sequence:
(a) c-myb
The following shall be reported in the table:
(XXVI) (b) c-myc
The following shall be added to the list of products:
(XXVII) c) cdc2-kinase
The following shall be reported in the table:
(XXVIII) (d) PCNA (proliferating cell nuclear antigen of rat)
The following shall be reported in the table:
(XXIX)
The use of medicinal products in liposomes, which may contain other components such as proteins, is also a suitable form of application. They may also be administered rectally, e.g. in the form of suppositories, or parenterally, e.g. in the form of solutions for injection. For the manufacture of pharmaceutical products, these compounds may be processed in a network of organised and inorganic vectors. Examples of such vectors are tablet-specific polyolefin, hard wax and emulsifying oils, anti-oxidants for the production of oil, gas, salts, salts, salts, salts, salts, salts, salts, salts, salts, salts and other therapeutic agents, and other products for the manufacture of polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyolefins, polyo
The preferred forms of administration are topical application, topical application such as by means of a catheter or injections. For injection, the antisense polyamide oligonucleotide derivatives are formulated in a liquid solution, preferably in a physiologically acceptable buffer, such as Hank's solution or Ringer's solution.
In general, the invention covers the use of compounds of formula I as DNA probes or primers in DNA diagnostics, in particular as defined in HOE 92/F 406 (EP-A 0 602 524) and as aids in molecular biology in general.
In DNA diagnostics, gene probes, also called DNA probes or hybridization probes, play a major role in sequence-specific detection of certain genes. A gene probe generally consists of a detection sequence and a suitable marking group (label). The specificity of determining a target sequence in an analytical sample by hybridization with a complementary gene probe is determined by the detection sequence and its chemical structure. PNAs have the advantage over natural-structure oligonucleotides that they have a higher affinity for the target sequence.
The advantage of better discrimination between pathogenic and non-pathogenic DNA can also be used in the form of a hybrid primer with the help of the PNA/DNA oligomers of the invention, provided that these are free-splitting 3'-polymerase polymers that have a specific function. For example, a primer such as PNA/PNA/PNA-E has been found to be more accessible at the end of a DNA-transferase, which is a type of DNA-transferase, and a primer such as PNA/PNA-E can be found at the end of a DNA-transferase, which is a type of DNA-transferase that is not available for any type of DNA-transferase.
Another advantage over the use of natural oligonucleotide primers is that the nucleic acid strand, which contains the PNA portion at the 5' end, is stable against 5' exonucleases, and thus all natural DNA or RNA sequences in the reaction mixture can be degraded by 5' exonucleases without attacking the PNA-containing strand.
Err1:Expecting ',' delimiter: line 1 column 267 (char 266)
Furthermore, (DNA) - (PNA) - (DNA) oligomers can be embedded in genes, which is not currently possible with PNAs.
Err1:Expecting ',' delimiter: line 1 column 382 (char 381)
Err1:Expecting ',' delimiter: line 1 column 43 (char 42)
The abbreviations used for amino acids correspond to the three-letter code commonly used in peptide chemistry as described in Europ. J. Biochem. 138, 9 (1984).
The following substances, which are not classified in the same heading as the active substance, are to be classified in the same heading as the active substance: sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric acid, sulphuric
6-aminohexan-1-ol (1g; 8.55mmol) is dissolved in anhydrous pyridine (7ml) and mixed with triethylamine (0.2ml). To make this solution, a solution of (4-methoxyphenyl) diphenylmethyl chloride (2.5g; 8.12mmol) is added to anhydrous pyridine (9ml) for 45 minutes. The reaction solution is agitated for 30 minutes at 22°C and stopped by adding methanol (3ml). The solution is diluted in a rotary steam engine, the resulting residue is co-evaporated with thymol to remove the pyridine. The resulting residue is in ethyl acetate and this solution is successively dissolved in a dilute solution of sodium tricarbonate, water and potassium trichloride. The product is concentrated in a vacuum filter (NaOH:NaOH:NaOH/49.5:19.54) and purified in a vacuum filter.
Production: 1.64 g
The following substances are to be classified as 'methanol' and 'methanol' in the Annex to Regulation (EC) No 1272/2008 of the European Parliament and of the Council:
Rf 0.44 (heptane:ethyl acetate = 1:1),
After stirring for 3 hours at 22°C, add further 1-hydroxy-6- ((((4-methoxyphenyl) diphenylmethylamino) hexane (1.00g; 2.57mmol) to anhydrous pyridine (10ml) and heat this solution for 6 hours at 50°C. After another 16 hours at 22°C, mix the residue in ethyl acetate and obtain the solution with 5% ethyl acetate.
MS (ES-) 978.0 (2M-H) and 488.3 (M-H)
The test chemical is used to determine the concentration of the test chemical in the test medium.
The aminoform of TentagelR (0.5 g; 0.11 mmol of Rapp polymers) is boiled for 10 minutes in 4-ethylmorpholine (0.1 ml) and DMF (5 ml) and then given a solution of 6- ((((4-methoxyphenyl) -diphenylmethylamino) -hex-1-yl 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) and shaken the suspension for 16 hours at 22°C. The derived Tentagel carrier is filtered and mixed successively with DMF (33 ml), CH3 (32 ml) and CH2 (1 ml) of diethylmethylamine (31 ml) into a non-reactive solution.
The manufacture is carried out in analogy with that described in example 3 from aminopropyl-CPG (Fluka) (550Å;1.0g) and 6- ((((4-Methoxyphenyl) diphenylmethylamino) -hex-1-yl hemisuccinate (48.7 mg; 0.082 mmol), 4-ethylmorpholine (7.6 ml) and TBTU (26.4 mg; 0.082 mmol) in DMF (3 ml).
After 2 h, the pH was brought to 5 by adding 1N KHSO4 by drip, filtered from waste salts and filtered with a small amount of dioxin. The combined esters were evaporated in a vacuum and the residue was co-evaporated with methanol and dichloromethane. The product was then mixed with a 0.31 to 0.85% triethylamine triethylamine (Methyl methanol) in a semi-transparent solution containing 0.31 to 0.8 percent triethylamine (Methyl methanol) and purified in a vacuum. The product was combined with pyridine and chloropyridine.
The test chemical is a chemical that is used to treat the effects of the chemical on the body.
The product from the above reaction was dissolved in a mixture of 10 ml dioxane and 2 ml water. The solution was cooled to 0 °C and 1 N of sodium salts were added by dripping until a pH of 11 was reached. After 2 h of reaction time, the reaction was completed and the solution was brought to pH 5 by gently adding 2 N of KHSO4 solution. The solution was extracted three times with ethyl acetate, the combined organic phases dried with sodium sulphate and pressed in the electrum. The resulting raw product was chromatographically purified in 5-10 MS gel with a gradient of 1.0% methanol and 1 % triethylamine in dichloromethane. The remaining pyrethyl fractions were combined and the product was vacuumed.
The following formulae are used:
N- ((((4-methoxyphenyl) diphenylmethylamino) ethyl-N-(9-(N2- (((isobutanoyl) guanosyl) acetyl) glycine methyl ester (1.15 g; 1.72 mmol) is dissolved in dioxin (10 ml) and divided into 5 parts by 1 M of aqueous sodium salt (10.32 ml) at 0°C over a period of 2.5 h. After a further 2 h reaction time at room temperature, the solution is brought to pH5 by adding 2 M of aqueous potassium hydrogen sulphate solution by 1 drop. The resulting salts are filtered and rinsed with a small amount of dioxin.
The combined filtrate is evaporated in a vacuum until dry and the residue is co-evaporated twice with ethanol and dichloromethane:methanol 1/1. The purification is carried out by column chromatography on silica gel by elution with a gradient of 10-20% methanol in dichloromethane (with 1% triethylamine).
The yield is 1.229 g.
The following information is provided for the purpose of the ESMS:
The following shall be reported for the product concerned:
N- ((((4-methoxyphenyl) diphenylmethylamino) ethyl-N-(9-(N6-(4-methoxybenzoyl) adenosyl) acetyl) glycine methyl ester (1.70 g; 2.38 mmol) is dissolved in dioxan (10 ml) and divided into 5 parts by 1 M of aqueous sodium solution (10.32 ml) at 0°C for 2.5 h. After a further 2 h reaction time at room temperature, the solution is brought to pH5 by adding 2 M of aqueous hydrogen sulphate solution by drops. The resulting salts are filtered and washed with a small amount of potassium dioxide.
The combined filtrate is evaporated in a vacuum until dry and the residue is co-evaporated twice with ethanol and dichloromethane:methanol 1/1. The purification is carried out by column chromatography on silica gel by elution with a gradient of 10-20% in methanol.
Production: 1.619 g
The ESMS (negative-lon): 698.3 ((M-H)
The following shall be reported for the product concerned:
0.5 g (1.28 mMol) N-di-methylmethyloxy-methylglycine was suspended in 10 ml DMF and 0.47 ml (1.92 mMol) BSA was administered by drip. 0.7 ml (5.1 mMol) triethylamine and 0.26 g (1.28 mMol) chlorcarboxymethylthymine were added successively. The reaction mixture was stirred at room temperature for 4 h, then another 65 mg (0.32 mMol) chlorcarboxymethylthymine was added and stirred for 16 h. The solvent was then removed in a vacuum and the raw product was purified in a silica gel with a grade of 5-15% methanol and 1 % triethylthymine. The product containing diethyl methyl methyl was obtained by a vacuum dissolving process and was almost completely dissolved in white oil and diethyl methanol.
The yield is 0.219 g.
Electrospray MS (negative) 556.3 (M-H)
The test chemical is a chemical that is used in the manufacture of a mixture of chemicals.
The sodium salt of 4-hydroxybutyric acid (1.26 g; 10 mmol) is dissolved in anhydrous pyridine (30 ml) and mixed with 4,4'-dimethoxytrithyl chloride (3.39 g; 3.05 mmol). After 16 hours, 4-nitrophenol (1.39 g; 10 mmol) and N,N'-dicyclohexylcarbodiimide (2.06 g; 10 mmol) are added and stirred at 22 °C for another 48 hours. The separated diclohexyl urine is filtered and washed with dichloromethane. The filtrate is concentrated and the resulting residue is co-evaporated twice with toluene. The residue is cleaned over a silica column (10-50% ethyl acetate and 1% triethylamine).
Harvested 2,694 g.
The following substances are to be classified as MS (FAB, MeOH/NBA/LiCI) 534.2 (M+Li) +; 527.2 M+.
The following table shows the results of the analysis:
The mixture is stirred at room temperature for 30 min and then slowly dripped to a solution of 5.32 g (3-hydroxypropyl) glycine, 20 ml water, 20 ml DMF and 5.54 ml triethylamine. Stir for 1 h at room temperature and then press tightly in the rotary evaporator in a vacuum. The residue is placed in water, taken with 1 N of hydrochloric acid at pH 1.5 and extracted with ethyl methacrylate. The wet phase is combined with saturated sodium trihydroxycarbonate solution at pH 5 and roasted at the rotary lamination stage. The back is then pressed with 250 ml of ethyl methacrylate, which is then removed from the vacuum and the product is dissolved in 10% of the raw ethyl methacrylate and 10% of the chlorine methacrylate.
The yield is 3.2 g.
The following substances are to be classified in the same group as the active substance:
The following information is provided for the purpose of the calculation of the maximum amount of the premium:
3.2 g H-Oprg (t) -OH are dissolved in 40 ml DMF, 5.93 ml triethylamine are added and a solution of 7.25 g Dmt-CI is dripped at 0°C into 40 ml dichloromethane for 20 min. Stir for 2 h at room temperature, filter the failed triethylamine hydrochloride and compress the filtrate in the rotary evaporator in the vacuum. The residue is absorbed in the dichloromethane, extracted with water, the organic phase dried with sodium sulphate and compressed in the vacuum in the rotary evaporator. The raw product is purified in pebbles with an addition of dichloromethane/ethanol/ethylacyl gel of 1% triethylamine. The product containing the red evaporator is combined in the vacuum with dissolving agents and pressed in the vacuum.
Production: 3.46 g
The following substances are to be classified in the same category as the active substance:
The following shall be added to the list of products:
The mixture is stirred at room temperature for 30 minutes and then slowly dripped to a solution of 4.41 g (4-hydroxybutyl) glycine, 10 ml water, 10 ml DMF and 4.16 ml triethylamine. Stir and narrow for 3 h at room temperature. Mix at the rotary evaporator in a vacuum. The retrieved product is placed in red water, with 1 N of hydrochloric acid at pH 1.5 and extracted with ethyl acetate. The water phase is mixed with a dilute sodium hydrocarbonate solution at pH 5 and at the rotation product. The product is mixed with a dilute thiocyanate/methanol/ethyl methanol solution containing 10% of the product and purified in a vacuum with a 10% dilution solution of triethylamine.
The yield is 3.7 g.
The following shall be reported for the product concerned:
The following shall be added to the list of products:
After stirring for another 2 h at room temperature, add another 40 ml of dichloromethane, filter the failed triethylamine hydrochloride and compress the filtrate in the vacuum evaporator. The residue is absorbed in the dichloromethane, extracted with water, the organic phase dried with sodium sulphate and compressed in the vacuum evaporator. The raw product is purified in a pebble gel with an addition of ethylene/methanol/ethyl chloride of 15:1:1 under the 1% triethylamine product.
The yield is 3.45 g.
The following substances are to be classified in the same category as the active substance:
The following shall be added to the list of substances:
The mixture is stirred at room temperature for 30 minutes and then slowly dripped to a solution of 4.83 g (5-hydroxypentyl) glycine, 10 ml water, 10 ml DMF and 4.16 ml triethylamine. The mixture is stirred for 3 h at room temperature and pressed in the rotary evaporator stand in a vacuum. The retention is placed in red water, with 1 N of hydrochloric acid at pH 1.5 and extracted with ethyl acetate. The water phase is mixed with a dilute sodium hydrocarbonate solution at pH 5 and the rotation product. The product is mixed with a dilute petroleum cyanide/methanol/ethyl methyl methyl chloride solution containing 10% of the product and purified in a vacuum.
Production: 3.34 g
The following shall be reported for the product concerned:
The following shall be added to the list of products:
3.2 g H-Opegt-OH are dissolved in 40 ml DMF, 6.77 ml triethylamine is added and a solution of 9.94 g Dmt-CI is dripped at 0°C into 40 ml dichloromethane for 15 min. Stir for 2 h at room temperature, add another 40 ml dichloromethane, filter the failed triethylamine hydrochloride and compress the filtrate in the rotary evaporator in the vacuum. The residue is absorbed in the dichloromethane, extracted with water, the organic phase dried with sodium sulphate and compressed in the vacuum by the rotary evaporator. The raw product is then purified by dipping a pebble gel with a 15% dilution of methanol/ethyl ethanol/ethyl ether.
The yield is 3.6 g.
The following substances are to be classified in the same category as the active substance:
The Commission shall be assisted by the Member States in the preparation of the annual report.
The DNA sequence is shown in upper case, the PNA sequence in lower case (example of structure type Xlla in Figure 1).
For example, the synthesis of PNAs is carried out on an Ecosyn D-300 DNA synthesizer (Fa. Eppendorf/Biotronik, Maintal) or an ABI 380B DNA synthesizer (Fa. Applied Biosystems, Weiterstadt). The synthesis of the DNA part is in principle carried out according to the standard phosphoramide chemistry and the commercially available synthesis cycles.
Other
with n = 1 - 8, preferably 1 - 5,
Other
3 μmol of the Mmt-hex-succ loaded CPG carrier (load 91 μmol/g) from example 4 are treated successively with the following reagents:
The following is the synthesis of the PNA part (agtc-hex):
The test chemical is a solution of 4-ethylmorpholine in acetonitrile (neutralization) 5. 0.4 M solution of (Mmt-Aeg ((ctBuBz) -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 (10 minutes coupling time).6.
Steps 1 to 7, hereinafter referred to as a PNA reaction cycle, are repeated 3 times to build up the PNA part,wherein in step 5 the sequence-required monomer building block from examples 5 to 8 is used in each case.Conjugation of the linker (agtc-hex ---> (but) -agtc-hex ):
Repeat steps 1 to 4 from above9. 4-Nitrophenyl-4- (((4, 4'-dimethoxytrityloxy) butyrate (105 mg) from example 10 and hydroxybenzotriazole (27 mg) in 2 ml NEM in DMF for 15 hours10. Wash with DMF11. Wash with acetonitrile12.
The following information shall be provided for the purpose of the calculation of the maximum number of vehicles:
The following are the active substances which may be used in the manufacture of the product: acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonitrile, acetonit, acetonit
| V | ||||
| VIII a | H | O | ||
| VIII b | H | O | ||
| VIII c | H | O | ||
| VIII d | -N-pyrrolidin-1-yl | H | O | |
| VIII e | O | |||
| VIII f | H | NH |
Steps 13 to 20, hereinafter referred to as a DNA reaction cycle, are repeated 10 times to build up the nucleotide portion, with step 16 using the sequence corresponding 5'-0-dimethoxytrityl ((nucleobase) -3-phosphoric acid-β-cyanoethylester-diisopropylamide.
After completion of synthesis, the dimethoxytrithyl group is separated as described in steps 1 to 3. 1.5 hours of treatment with ammonia separates the oligomer from the carrier while eliminating the β-cyanethyl groups. To separate the exocyclic amino protective groups, the ammonia solution is kept at 55°C for 5 hours. The resulting raw product (325 OD260) contains 5'-ATC GTC GTA TT-but-but) hexaglyclohex which is purified by 180 OD280 polyalamide gel electrophoresis.
The synthesis is performed in the same way as in example 17, except that in step 9 the linker Mmt-Oeg-t-OH from example 9 is coupled under the conditions described in step 5 instead of the Dmt-butyric acid-p-nitrophenyl ester. 135 OD260 is purified from the resulting raw product (235 OD260) to 5'-ATC GTC GTA TT-Oeg-t-agtc-hex by polyacrylamide gel chlorase.
The synthesis is initiated by a 5'-Dmtthymidine carrier which is bound by its 3'-end. The DNA component is first synthesised as described in Example 17 (steps 13 to 20), with oxidation with tetraethylthiuramine disulfide (TETD; User Bulletin No. 65 of Applied Biosystems Inc.) in step 20 in the case of phosphorothioate bridges (steps 13 to 20), and a Dmt-protected 5'-Amino-5'-Desoxycytilate-3'-phosphorothrin protease of the Vlll-Bactyl group is selected as the linker. The PNA-tropine analogues are then completed in Step 17 by steps 1 to 7. The polymerization is then completed in Step 1.
The synthesis is carried out in the same way as described in example 18, but the corresponding methylphosphonate building blocks of formula Vlllb are used in the DNA reaction cycle to construct the methylphosphonate bridges Me.
The synthesis is carried out in the same way as described in example 17, but the Vllld component is used to install the dithiohydrate bridges and the oxidation (step 20) with TETD is carried out at these sites.
The synthesis follows the analogy described in example 18, whereby, after completion of DNA synthesis, a 5'-aminonucleotide is condensed in analogy to example 13, which allows the conjugation of the second PNA part. So, first, six PNA synthesis cycles are performed and then the linker block from example 9 is coupled.
The synthesis is similar to that described in example 19, but from a CPG carrier that holds the glycerol hexadecylether bound. After 12 DNA synthesis cycles, the linker Vlllf is condensed. After four PNA synthesis cycles and the final Mmt group is cleaved, the free amino function can be quantitatively realized with a 30-fold excess of fluorescein isothiocyanate (FITC).
The synthesis of the PNA part follows the same process as in example 17. After condensing six PNA units, the (Mmt-Oeg(t) -OH) from example 9 is coupled. As a linker, the tetraethylene glycol derivative of formula XV is first condensed twice before the synthesis of the DNA part is performed in reverse (from 5' to 3').
The synthesis is performed in a similar way to the one described in example 17 (DNA synthesis cycle), but starting from a CPG carrier that holds 3'-O-Dmt-deoxyguanosine bound via a 5'-O-succinate group. After six DNA units have been condensed with the help of the building blocks of formula XIV, the tetraethylene glycol derivative of formula XV is first condensed as a linker before the phosphoride of formula XVI is doubled to introduce a C6-link.
The synthesis is carried out in a similar way as described in Example 17: before the product is separated from the carrier and protected, one half of the carrier-bound DNA/PNA hybrid is removed for fluorescence marking (Example 27), and the other half is protected and processed as described in Example 17.
The carrier-bound DNA/PNA hybrid from example 26 is fluorescently labelled by performing steps 13 to 20 as described in example 17, using the fluorescein phosphoramidite from Fa. Applied Biosystems in step 16.
The synthesis is performed in a similar way as described in example 17. Before the product is split and insulated from the carrier, one half of the carrier-bound DNA/PNA hybrid is removed for fluorescence marking (example 29). The other half is insulated and processed as described in example 17. The title link binds as a triplex-forming oligonucleotide with high affinity to a DNA double strand containing the homopurin motif 5'-AAA AAA GGG GGG-3'.
The carrier-bound DNA/PNA hybrid from Example 28 is fluorescently labelled by performing steps 13 to 20 as described in Example 17, using the fluorescein phosphoramidite from Fa. Applied Biosystems in step 16.
Synthesis is carried out analogously to that described in example 17 from CPG loaded with vitamin E (MacKellar et al. (1992) Nucleic Acids Res, 20(13), 3411-17) and four-fold coupling of the Vllle component after the DNA synthesis cycle. Recoupling of the 5'-aminonucleotide component of the Vlllf formula follows, six PNA units are condensed after the PNA synthesis cycle. After neutralization, the phosphoramid is coupled to the amino function by a known method and the DNA synthesis cycle is described as the DNA phenyl group structure, with the DNA part repeated accordingly, with the bafosphate bafosphate sections of the Vllllllle group being inserted into the 16th and 16th step. Finally, at the end of the treatment, the forophosphoramine is removed with the oxygen group, as described in the example of Glenorhospiracetam, at a temperature of 22°C. The final step is the treatment with the forophosphoramine group, which is protected by the glyphosate oxide group at the end of the treatment.
The synthesis is performed in the same way as in example 18, first by five PNA synthesis cycles and then by coupling the linker Oeg ((t) from example 9, then by six DNA synthesis cycles, then by splitting from the carrier and further processing as in example 18.
The synthesis is performed in the same way as in example 18, first by five PNA synthesis cycles and then by coupling the linker Oeg ((t) from example 9, then by six DNA synthesis cycles, then by splitting from the carrier and further processing as in example 18.
This pure PNA oligomer was produced as a reference compound analogous to that in example 18, except that twelve PNA cycles were performed. The exocyclic amino protective groups were expectorated in ammonia solution (5 hours at 55°C) and the monomethoxytrithyl group was only then cleaved by a 2-hour treatment with 80% acetic acid at 22°C.
The synthesis is carried out in the same way as described in example 31, but the corresponding 5-hexinylpyrimidine nucleoside phosphoramidites are used in the condensation reaction instead of the normal pyrimidine phosphoramidites.
The synthesis of this PNA/DNA oligomer is analogous to that described in Example 27, but only two thymidylate units are condensed.
This PNA/DNA oligomer, which is composed of 15 PNA units and one nucleoside unit, has been synthesized as a primer for the DNA polymerase reaction. It is made from a solid-phase carrier (aminoalkyl-CPG) from which the 5'-monomethoxytritylamino-5'-deoxythymidine is bound to its 3'-hydroxy group as a succinate. After splitting the monomethoxytrityl group with 3% TCA in dichloromethane, 15 PNA cycles are performed as described in example 17. The exocyclic protective amino groups are defused in ammonia solution (5 hours at 55°C). First, the monomethoxytrymethymide group is obtained by a 2-hour treatment with 80% C-Kloromethane at 22°C (as a primer for DNA with 3'-hydroxy-DNA).
(ps means a 5' thiophophat; spacer means a triethylene glycol phosphate; rA is a riboadenylate; (2'5') means that the internucleotide bond is 2' to 5' in the ribose)
After introducing the linker Mmt-Oeg(t) -OH from example 9 under the conditions described in step 5, the M group is cleaved with 3% TCA and spacer is made with the commercially available Dmt-O-(CH2CH2O) 3-0-P-OCH2CH2CN) N(i-C3H7) 3 Spacer phosphorite (Eurogenetics; Brussels). The (2'5') linker is then cooled as described in example 17 with the help of N6-Benzo-5'-D'-D'-Oxyethyl-3'-Oxyethyl-3'-Oxyethyl-2-Oxyethyl-2-Oxyethyl-2-Oxyethyl-D-Oxyethyl-Oxyethyl-Oxyethyl-Oxyethyl-D-Oxyethyl-Oxyethyl-Oxyethyl. The last step is to add the 5-methyl-D-Oxyethyl-Oxyethyl group to the active substance, and the 5-methyl-D'-Oxyethyl-Oxyethyl-Oxyethyl-Oxyethyl-Oxyethyl-Oxyethyl-Oxyethyl-Oxyethyl-Oxyethyl-Oxyethyl-Oxyethyl-Oxyethyl-Oxyethyl-Oxyethyl (D-Oxyethyl-Oxyethyl-Oxyethyl-Oxyethyl-Oxyethyl-Oxyethyl-Oxyethyl) is added to the final product, and the 5-methyl-D-Oxyethyl-Oxyethyl-Oxyethyl-Oxyethyl-Oxyethyl-Oxyethyl-Oxyethyl-Oxyethyl-Oxyethyl-Oxyethyl-Oxyethyl-Oxyethyl-Oxyethyl-Oxyethyl-Oxy-Oxyethyl-Oxy-Oxy-Oxy-Oxy-Oxy-Oxy-Oxy-Oxy-Oxy-Oxy-Oxy-Oxy-Oxy-O-Oxy-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-O-
The synthesis is carried out in the same way as in example 37, but the corresponding N6-benzoyl-5'-O-Dmt-3'-O-tert-butyldimethylsilyl-adenosine-2'-O-cyanoethyl-di-isopropylamino phosphoramidites are replaced by the corresponding N6-benzoyl-5'-O-Dmt-cordycepin-2'-O-cyanoethyl-di-isopropylamino phosphoramide (Fa. chemogenoram, constant) and the fluoride treatment is eliminated.
The synthesis is performed analogously to that described in example 18, whereby, after nine PNA couplings, the linker Mmt-Oeg ((t) -OH from example 9 is condensed under the conditions described in step 5, allowing the subsequent condensation of eight guanylate residues. The resulting PNA/DNA oligomer binds with high affinity in antiparallel orientation as a triplex-forming oligonucleotide to double-stranded DNA having the sequence 5'..AAAAAAAAGGGGGG..3'.
The products are purified as described above and then show a uniform band at the PAGE (20% acrylamide, 2% acrylamide and 7 M urea). The HPLC is performed on Reverse Phase Columns RP-18 of the Merck Company (eluent A: Water with 0.1% TFA, B: Water/acetonitrile = 1:4; linear gradient) or on a PA-100 column of the Fa. Dionex Company (Eluent A: 20 mM NaOH and 20 mM NaCI; B: 20 mM NaOH and 1.5 mM Bisa; linear gradient NaCI). For the ESF-NA, the ammonium salts are converted into ammonium salts by ca. 1.5 μl of O2 or other ammonium salts. The efficiency of the OMS is approximately 1.5 ± 5 μl.
A commonly used 35S marking is that at least one oxidation in the DNA synthesis cycle (step 20 in example 17) is performed with elemental 35S in the synthesis of the 35S DNA part. PNA/DNA hybrids with a free 5'-hydroxy group can be marked with 32P or 35S by polynucleotide kinase using known methods. PNA/DNA hybrids with a free 3'-hydroxy group can be marked with 3'-terminal transferase by known methods.
Err1:Expecting ',' delimiter: line 1 column 472 (char 471)
| Inkubationszeit | Zellaufnahme | |
| in Stunden | DNA | |
| 1 | 0.25 | 0.36 |
| 7 | 0.54 | 0.57 |
| 24 | 0.75 | 0.78 |
Err1:Expecting ',' delimiter: line 1 column 355 (char 354)
The PNA/DNA oligomer from example 26 is 69% stable after 24 hours incubation; the DNA oligomer is 3% stable.
The PNA/DNA oligomer from example 31 has a half-life of 32 h under these conditions, while the corresponding DNA oligonucleotide has a half-life of approximately 2 h.
The COS cells are grown in 5 cm Petri dishes until they converge in Dulbecco's MEM, which has been supplemented with 10% FCS. The cells are washed twice with serum-free DMEM. A sterile needle is used to scrape an area of about 1 cm2 into the centre of the petri dish. The PNA/DNA oligomer solution (0.1 mM) to be examined is placed in this area. It is incubated at 37°C under CO2 atmosphere. After 2, 4 and 16 hours, the cells are examined by fluorescent microscopy. The cells are washed with four serum-free DMEM, covered with a glass tube and subjected to fluorescent heat or contrast.After two hours of incubation of the cells with this PNA, > 90% of the cells show signs of severe morphological changes and cell death. Most cells show strong vacuolization. The plasma membrane, the cytosol and the nucleus show no uptake of PNA. After another two hours of incubation with the pure PNA, all cells have died. The DNA/PNA oligomers of the invention are different.
The melting temperatures are determined by means of an HP 8452A diodenarray spectrophotometer, an HP 89090A Peltier element and HP Temperature Control Software Rev. B5.1 (Fa. Hewlett Packard). It is measured in 0.5°C/min steps in 10mM HEPES and 140 mM NaCl (pH 7.5) as a buffer. The oligomer concentration is 0.5 to 1 OD260 per ml.
The result for the product from example 17 and 18:
Other
| 5'-ATC GTC GTA T(Oeg(t)a gtc-hex | |
| 3'-TAG CAG CAT A A T CAG-5' | antiparallel |
| 5'-ATC GTC GTA T(Oeg(t)a gtc-hex | |
| 5'-TAG CAG CAT A A T CAG-3' | parallel |
| 5'-ATC GTC GTA TT(but)a gtc-hex | |
| 3'-TAG CAG CAT AA T CAG-5' | antiparallel |
| 5'-ATC GTC GTA TTA GTC-3' | |
| 3'-TAG CAG CAT AAT CAG-5' | DNA·DNA antiparallel |
| 5'-ATC GTC GTA TT(but) a gtc-hex | |
| 5'-TAG CAG CAT AA T CAG-3' | parallel |
| gegen DNA | gegen 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 |
| 5'- 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' | |||
| S-DNA ap | 46.7 °C | 43.8 °C | |
| 3'- TGT AGT ACC AGC -5' |
TGG TCG means a part of DNA in which all internucleotide bonds are present as a phosphorothioate.
The antiviral activity of the test substances against various human pathogenic herpes viruses is tested in the cell culture test system. For the test, monkey kidney cells (Vero, 2x105/ml) are sown in serum Dulbecco's MEM (5% fetal calf serum FCS) in 96-pot microtiter plates and incubated for 24 h at 37°C and 5% CO2. The serum medium is then sucked and the cells are rinsed twice with unfilled Dulbecco's MEM (-FCS). The test substances are pre-diluted in H2O to a concentration of 600 μM and preserved at -18°C. For further dilution, further steps are taken in Dulbecco's MEM (100 μM) to dilute the test medium with each of the individual Dulbecco's MEM (100 μM) at -18°C.After 3 h incubation at 37°C and 5% CO2, the cells are infected with herpes simplex virus type 1 (ATCC VR733, HSV-1 F strain) or herpes simplex virus type 2 (ATCC VR734, HSV-2 G strain) at concentrations where the cell wall is completely destroyed within 3 days. In HSV-1, the infection rate is 500 plaque-forming units (PFU) per nap, in HSV-2, 350 PFU/nap. The test pieces then contain test substance at concentrations of 80 μM to 0.04 μM in MEM, supplemented by 100 U/ml penicillin G and 100 mg/l streptomycin. All tests are performed as a double determination with eight controls, each with one exception.The test substances are incubated for 17 h at 37°C and 5% CO2. The cytotoxicity of the test substances is determined after 20 h total incubation time by microscopic examination of the cell cultures. The dose tolerata maxima (DTM) is the highest concentration of the drug that does not cause microscopically detectable cell damage under the specified test conditions. FCS is then added to a final concentration of 4% with further incubation for 55 h at 37°C and 5% CO2. The untreated infection controls then show a complete cytopath effect (ECP). After microscopic examination of the cell cultures, these are then neutralised according to the Vital Color Finterpretation (1966). The activity of the test substance is defined as inhibition of the anti-inflammatory (MCP) activity of the test substance.The activity of the PNA/DNA chimeras is always better than that of the corresponding DNA oligomers or PNA oligomers.
The determination is as described (Sandkühler et al. (1991) in: Proceedings of the Vlth World Congress on Pain, Charlton and Woolf, Editors; Elsevier, Amsterdam; page 313-318) by superfusion of the spinal cord. After laminctomy of a barbiturate-anesthetized Sprague-Dawley rat, a two-chamber silicone container is formed to accommodate the antisense oligomer. One chamber is filled with the antisense PNA/DNA derivative, while the other chamber is filled with the control oligomer (concentration 75 μM each). The superfossil is exchanged every hour for one hour. 6 After superfusion, the c-phosphate expression is stimulated by heat treatment at 33.2 °C (52 °C) to stimulate the corresponding HO. The corresponding example of the c-phosphate expression can be demonstrated by the corresponding example of the c-O-DNA enzyme and the corresponding example of the c-O-phosphate expression in the c-O-DNA enzyme.
To determine RNase H activity, dissolve 1.3 OD of the PNA/DNA oligomer to be tested with 0.5 OD of the complementary RNA sequence (TargetSequence) in 50 μl of autoclaved water treated with DEPC (diethylpyrocarbonate), heat for 5 minutes at 80 °C and then cool to 37 °C within 15 minutes, denaturing both oligomers to form a sequence-specific nucleic acid double strand after cooling.
The test is to incubate this RNA·PNA/DNA duplex with 10 μl of RNase H 10x buffer, 1 μl of Dithiothreitol (DTT) and 2 μl (respectively 10 u) of RNase H from USB. The incubation approach is completed with autoclaved DEPC-treated water to the desired total volume of 100 μl. The samples are incubated at 37 °C. For kinetic analysis, 20 μl of the solution were taken after 0.2 min, 10 min and 1 h, heated for 5 minutes at 95 °C and frozen at -70 °C. The analysis of the RNase H cleavage is carried out by Otrophor P. The analysis showed that RNA/NAH is broken down into DNA/NAH, the corresponding P-Oxybonucleotide, which is somewhat incomplete and is not compatible with the RNA/NAH.
To stimulate the activity of cellular endoribonuclease L by the 2'5'-tetraadenylate PNA/DNA conjugate, a HeLa cell extract has been prepared by adding 35 vials of 20 ml of medium with Dulbeccos MEM (minimum essential medium) and 10% FCS (foetal calf serum) to the cells to terminate the treatment. The cells can be harvested after treatment with trypsin. After centrifugation at 1000 rpm, 4 ml of cell grains are obtained. This is first filled with 4 ml and after 3 minutes, 4 ml of buffer A (5.48 HEPES; 15.5 KCI g; 2.488 mg g acetate; 1232 μl 2-mercaptoethanol ad 1 l) are added to the water to terminate the treatment. The solution is then centrifuged for 20 minutes at 30 °C (ultra-centigrade) and taken in a solution of 30,000 g of water at an ultra-centigrade temperature of 30 °C.
For the endonuclease L test, 0.3 OD of the target RNA sequence is first heated at 80 °C for 5 minutes with the respective PNA/DNA oligomers and then cooled to 37 °C for hybridisation. The duplex is mixed with 20 μl of the extract, 1.2 μl of glycerin and RNase L-buffer and incubated at 37 °C. The total volume is then 70 μl. For the kinetic tests, samples are pipetted at 0, 20 and 60 minutes intervals and heated to 95 °C for 5 minutes for denaturation of the enzymes. The tetradenylates are stimulated in a speed lyopoly and by gel phosphatase. The L-2'NA5' tetracycline is stimulated by tetracycline and activates the RNase N-acetate while the L-N-acetate is not activated by the corresponding L-N-acetate compounds.
The following 81st oligodeoxynucleotide is used as the template for the DNA polymerase reaction:
Other
A corresponding oligodeoxynucleotide of sequence 5'- TAA TAC GAC TCA CTA TAG-3' is used as the control primer.
The primer (0.15 nmol) and template (0.15 nmol) are then diluted with 35 μl of water and hybridised by heating to 95°C and cooling. Then 10 μl of 2 μm dNTP mixture (nucleoside-5'-triphosphate) and 3 μl DNA polymerase (Klenow DNA fragment) are added to 5 μl of 10x PCR buffer (500 mM KCI, 100 mM Tris-HCI, pH9.1% Triton X-100, 15 mM MgCl2) and incubated for 0.5 hours at 22 °C and 37 °C. The reaction solution is then analysed on a 10% polyalamide gel (with 1 %) as marker pBR223/Hazel. The reaction with a double-strand test on the DNA strand shows a slightly faster change in the DNTP/Primor DNA strand than expected during the two-strand test.
Claims (11)
- A polyamide-oligonucleotide derivative of the formula Ib wherein x is 1 and q = r = 1 and s = t = zero or r = s = 1 and q = t = zero or q = r = s and t = zero;R2 is hydrogen, hydroxyl, C1-C18-alkoxy, halogen, azido or amino;B is, independently of one another, a base customary in nucleotide chemistry, 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, and in which the polyamide structure contains at least one nucleotide base which is different from thymine;Nu is a radical of the formulae IIa or IIb in whichR2 and B are as defined above;U is hydroxyl, mercapto, C1-C18-alkyl, C1-C18-alkoxy, C6-C20-aryl, C6-C14-aryl-C1-C8-alkyl, NHR3 or NR3R4, andR3 is C1-C18-alkyl or C1-C4-alkoxy-C1-C4-alkyl, andR4 is C1-C18-alkyl orR3 and R4 is, together with the nitrogen atom carrying them, a 5-6-membered heterocyclic ring which can additionally contain another heteroatom from the series consisting of 0, S, N;V is oxy, sulfanidyl or imino;W is oxo or thioxo;Y is oxy, sulfanidyl, methylene or imino;m is zero to 20;o is zero to 20;D' is a radical of the formula IVin which B is as defined above;n is zero to 20;p is zero to 20;Li3 and Li4 are each, independently of one another, a structure of the formula V [(V') - (G) - (G')]ε (V) where, independently of one another,ε is 1 to 5,V' is oxygen, NH, a bond or a radical of the formula VI in which U, V, W and Y are as defined above;G can be C1-C12-alkanediyl, where alkanediyl can optionally be substituted by halogen, amino, hydroxyl, C1-C18-alkyl, C1-C18-alkoxy, C6-C14-aryl, or C6-C14-aryl-C1-C18-alkyl C6-C14-aryl-di-C1-C12-alkanediyl, or a group of the formula (CH2CH2O)δCH2CH2 in which δ can be 1 to 11; or a bond; andG' is oxy, sulfanidyl, 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; andF and F' are linked to one another by a bond and/orF is R0 - (A)k - V - andF' in formula Ib is - V1 - (A)1 - R1 where R0 is hydrogen, C1-C18-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 bonding, crosslinking or cleavage; or if k is zero, R0 is hydrogen or together with V is a radical of the formula VII in which Z and Z' are, independently of one another, hydroxyl, mercapto, C1-C22-alkoxy, C1-C18-alkyl, C6-C20-aryl, C6-C14-aryl-C1-C18-alkyl, C1-C22-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 bonding, crosslinking or cleavage, and in which R3, R4, V and W are as defined above;R1 is hydrogen or Qo where R1 is always only hydrogen when at the same time 1 is zero and in formula Ib q = 1 or q = r = zero and in F' = V1 - (A) 1 - R1 with V1 - V,A is the residue of a natural or unnatural amino acid;Qo is hydroxyl, OR', NH2, NHR" with R' = C1-C18-alkyl and R" = C1-C18-alkyl, C1-C18-aminoalkyl, C1-C18-hydroxyalkyl;V is as defined above;V1 is a bond or is V, where V1 is always a bond in F' only in formula Ib with q = zero and r = 1;k is zero to 10;l is zero to 10;with the proviso thata) if in the compound of the formula Ib t is zero and s is 1, Li3 is a bond;b) if in the compound of the formula Ib s = t = zero, Li4 is a bond;where each nucleotide can be in its D configuration or L configuration, and the base can be in the α or β position.
- A peptide-oligonucleotide derivative of the formula Ib as claimed in claim 1, wherein the base is in the β position.
- A process for the preparation of peptide-oligonucleotide derivatives as claimed in either of claims 1 and 2, which comprises successive condensation of a PNA unit or DNA unit with, in each case, one nucleotide base onto an appropriately derivatized support or onto a growing oligomer chain.
- A peptide-oligonucleotide derivative as claimed in either of claims 1 and 2 for use as medicine.
- A peptide-oligonucleotide derivative as claimed in either of claims 1 and 2 for use as medicine for the treatment of diseases caused by viruses or of diseases influenced by integrins or cell-cell adhesion receptors, for the treatment of cancer or for preventing restenosis.
- A pharmaceutical containing a polyamide-oligonucleotide derivative as claimed in either of claims 1 and 2.
- A polyamide-oligonucleotide derivative as claimed in either of claims 1 and 2 for use as gene probe.
- A polyamide-oligonucleotide derivative as claimed in either of claims 1 and 2, wherein a nucleoside unit having a 3'-hydroxyl group is located on at least one end for use as primer.
- A gene probe assay for the determination of an oligo- or polynucleotide target (RNA or DNA), wherein a gene probe as claimed in claim 7 is used in a homogeneous or heterogeneous assay.
- A gene probe assay for the determination of an oligo- or polynucleotide target (RNA or DNA), wherein a primer as claimed in claim 8 is used.
- A gene probe assay as claimed in either of claims 9 and 10, wherein the target is determined by hybridization after target amplification.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| DE4408528A DE4408528A1 (en) | 1994-03-14 | 1994-03-14 | Peptide oligonucleotide derivatives, their preparation and use |
| DE4408528 | 1994-03-14 |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| HK98113225.3A Addition HK1012003B (en) | 1994-03-14 | 1998-12-11 | Polyamide-oligonucleotide derivatives, their production and use |
Related Child Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| HK98113225.3A Division HK1012003B (en) | 1994-03-14 | 1998-12-11 | Polyamide-oligonucleotide derivatives, their production and use |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| HK1038568A1 HK1038568A1 (en) | 2002-03-22 |
| HK1038568B true HK1038568B (en) | 2007-03-30 |
Family
ID=
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA2144475C (en) | Polyamide-oligonucleotide derivatives, their preparation and use | |
| JP3717081B2 (en) | Oligonucleotide analogues, their production and use | |
| AU5469399A (en) | 2'-o-aminoethyloxyethyl-modified oligonucleotides | |
| JP4908714B2 (en) | Polyamide nucleic acid derivative and drug, and preparation method thereof | |
| US6033909A (en) | Oligonucleotide analogs, their preparation and use | |
| SK1172002A3 (en) | Conjugates and methods for the production thereof, and their use for transporting molecules via biological membranes | |
| US7485421B2 (en) | Polyamide-oligonucleotide derivatives, their preparation and use | |
| AU2642595A (en) | Complementary dna and toxins | |
| JP5149475B2 (en) | Peptide nucleic acid derivative having negative charge, drug and method for producing the same | |
| US6136965A (en) | Deoxynucleic alkyl and alkoxy thiourea compounds | |
| HK1038568B (en) | Polyamide-oligonucleotide derivatives, their production and use | |
| HK1012003B (en) | Polyamide-oligonucleotide derivatives, their production and use | |
| WO1994015620A1 (en) | Novel oligonucleotides modified with non-nucleotide bridging groups | |
| WO1997014708A1 (en) | Oligonucleotides containing thiol-substituted nucleoside derivatives and methods of use thereof | |
| US6673912B1 (en) | 2′-O-aminoethyloxyethyl-modified oligonucleotides | |
| HK1012001B (en) | Modified oligonucleotides, their preparation and their use | |
| HK1012001A1 (en) | Modified oligonucleotides, their preparation and their use |