CA2628883C - Novel 3'-modified oligonucleotide derivatives - Google Patents

Novel 3'-modified oligonucleotide derivatives Download PDF

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CA2628883C
CA2628883C CA 2628883 CA2628883A CA2628883C CA 2628883 C CA2628883 C CA 2628883C CA 2628883 CA2628883 CA 2628883 CA 2628883 A CA2628883 A CA 2628883A CA 2628883 C CA2628883 C CA 2628883C
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formula
alkyl
compound
aryl
alkoxy
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CA2628883A1 (en
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Anuschirwan Peyman
Eugen Uhlmann
Carolin Carolus
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Hoechst AG
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Hoechst AG
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Priority claimed from DE4424263A external-priority patent/DE4424263A1/en
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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07HSUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12Antivirals
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07CACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/34Esters of acyclic saturated polycarboxylic acids having an esterified carboxyl group bound to an acyclic carbon atom
    • C07C69/40Succinic acid esters
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

Abstract

Novel oligonucleotide analogs of the formulae I and II (see Formula I) (see Formula II) in which A, B, D, R1, R2, T, U, V, W, X, Y, Z, a, b, m, m', n and n' have the meanings stated in the description, with valuable physical, biological and pharmacological properties, and a process for the preparation thereof are described. Application thereof relates to the use as inhibitors of gene expression (antisense oligonucleotides, ribozymes, sense oligonucleotides and triplex forming oligonucleotides), as probes for detecting nucleic acids and as aids in molecular biology.

Description

Hoechst Aktiengesellschaft HOE 94/F 161K Dr.WI
Novel 3'-modified oligonucleotide derivatives The present invention relates to novel oligonucleotide analogs with valuable physical, biological and pharmaco-logical properties and to a process for the preparation thereof. Application thereof relates to the use as inhibitors of gene expression (antisense oligonucleo-tides, ribozymes, sense oligonucleotides and triplex forming oligonucleotides), as probes for detecting nucleic acids and as aids in molecular biology.

Oligonucleotides are increasingly being used as inhibitors of gene expression (J. F. Milligan, M. D.
Matteucci and J. C. Martin, J. Med. Chem. 36 (1993) 1923;
E. Uhlmann and A. Peyman, Chemical Reviews 90 (1990) 543).

Antisense oligonucleotides are nucleic acid fragments whose base sequence is complementary to a mRNA to be inhibited. This target mRNA may be of cellular, viral or other pathogenic origin. Examples of appropriate cellular target sequences are those of receptors, enzymes, growth factors, immunomodulators, ion channels or oncogenes.
Inhibition of virus replication using antisense oligo-nucleotides has been described, for example, for RSV
(Rous sarcoma virus), HSV-1 and -2 (herpes simplex virus type I and II), HIV (human immunodeficiency virus) and influenza viruses. This entails use of oligonucleotides which are complementary to the viral nucleic acid.

Sense oligonucleotides are, by contrast, designed in their sequence so that they bind ("trap"), for example, nucleic acid-binding proteins or nucleic acid-processing enzymes and thus inhibit the biological activity thereof (C. Helene and J. J. Toulme, Biochim. Biophys. Acta 1049 (1990) 99). Examples of viral targets which may be mentioned in this connection are reverse transcriptase, DNA polymerase and transactivator proteins. Triplex forming oligonucleotides generally have DNA as target and, after binding thereto, form a triple helix struc-ture.

Whereas antisense oligonucleotides are used in general to inhibit the processing (splicing etc.) of the mRNA or the translation thereof into protein, triplex forming oligo-nucleotides inhibit the transcription or replication of DNA (N. T. Thuong and C. Helene, Angew. Chem. 105 (1993) 697; Uhlmann and Peyman, Chemical Reviews 90 (1990) 543).
However, it is also possible to bind single-stranded nucleic acids in a first hybridization with an antisense oligonucleotide to form a double strand which then, in a second hybridization with a triplex-forming oligonucleo-tide, forms a triplex structure. The antisense and triplex binding regions can moreover be located either in two separate oligonucleotides or else in one oligonucleo-tide.

A further application of synthetic oligonucleotides is in so-called ribozymes which destroy the target RNA as a consequence of their ribonuclease activity (D. Casta-notto, J. J. Rossi, J. 0. Deshler, Critical Rev. Eukar.
Gene Expr. 2 (1992) 331).

Nucleic acid fragments with suitable labeling are used in DNA diagnosis as so-called DNA probes for specific hybridization onto a nucleic acid which is to be detected. The specific formation of the new double strand is in this case followed by means of the labeling, which is preferably not radioactive. it is possible in this way to detect genetic, malignant or viral diseases or dis-eases caused by other pathogens.

For most of the said applications, oligonucleotides in their naturally occurring form are of little suitability or completely unsuitable. They must be chemically modi-fied so that they meet specific requirements. For oligo-nucleotides to be usable in biological systems, for example inhibiting virus replication, they must comply with the following conditions:
1. They must have a sufficiently high stability under in vivo conditions, that is to say both in serum and inside cells.
2. Their properties must be such that they can pass through the plasma membrane and nuclear membrane.
3. They must under physiological conditions bind in a base-specific manner to their target nucleic acid in order to display the inhibitory effect.

These conditions are not indispensable for DNA probes;
however, these oligonucleotides must be derivatized in such a way that detection, for example, by fluorescence, chemiluminescence, colorimetry or specific staining, is possible (Beck and Roster, Anal. Chem. 62 (1990) 2258).
Chemical modification of oligonucleotides usually takes place by appropriate modification of the phosphate backbone, ribose unit or the nucleotide bases (Uhlmann and Peyman, Chemical Reviews 90 (1990) 543). Another frequently used method is to prepare oligonucleotide 5'-conjugates by reacting the 5'-hydroxyl group with appropriate phosphorylation reagents. Oligonucleotides modified only at the 5' end have the disadvantage that they are broken down in serum. If, on the other hand, all the internucleotide phosphate residues are modified there are often drastic alterations in the properties of the oligonucleotides. For example, the solubility of methyl-phosphonate oligonucleotides in aqueous medium is dim-inished and the hybridization capacity is reduced.
Phosphorothioate oligonucleotides have non-specific effects so that, for example, even homooligomers (Uhlmann and Peyman, Chemical Reviews 90 (1990) 543) are active against viruses.

The breakdown of oligonucleotides by 3'-nucleolytic activity is generally regarded as the predominant breakdown by nucleases in serum. The object therefore is to provid4 3'-derivatized oligonucleotide analogs with specific activity, increased serum stability and good solubility.

This invention therefore relates to compounds of the formula I and formula II

Y
U-P V-CH X

W (C H 2 )b /C H 2-V-P=W
H1 (CH2)1 Z

U-V
=
W L
Y R
I

W (C"24 C H 2-V-P =W HC (C =H ), Z

I
T
I

W m CH2) HC-T-H
(CH2).
CHI
V
I
t-P-X
II
W
R'' V B
A

Y
U-P V-CH2 x W (CH2)e CH2-V-P=W ( I I ) I

HC (CHI )D Z
T

U-P
A
W M~
R=
U_P V
II A
W

and the physiologically tolerated salts thereof, in which a is a number from zero to 20, preferably from zero to 10, particularly preferably from zero to 6, very particu-larly preferably from zero to 4;

b is a number from zero to 20, preferably from zero to 10, particularly preferably from zero to 4, very particu-larly preferably of zero;

R1 is hydrogen, C1-C18-alkyl, preferably Cl-C6-alkyl, in particular methyl, C2-C18-alkynyl, C3-C18-alkynyl, C1-C18-alkylcarbonyl, C2-C19-alkenylcarbonyl, C3-C19-alkynyl-carbonyl, C6-C20-aryl, C6-C14-aryl-C1-C8-alkyl, or a radical of the formula III

I
Z-P-Z' w (rig) preferably hydrogen or a radical of the formula III, very particularly preferably hydrogen;

R2 is hydrogen, hydroxyl, Ci-C18-alkoxy, halogen, azido or NH2, preferably hydrogen, hydroxyl, Ci-C4-alkoxy, fluorine or NH2, particularly preferably hydrogen or hydroxyl, very particularly preferably hydrogen;

D is hydroxyl, O-PO32-, very particularly preferably hydroxyl;

B is a base customary in nucleotide chemistry, for example natural bases such as adenine, cytosine, guanine, uracil and thymine or unnatural bases such as, for example, purine, 2,6-diaminopurine, 7-deazaadenine, 7-deazaguanine, N4,N4-ethanocytosine, N6,N6-ethano-2,6-diaminopurine, pseudoisocytosine, 5-propinuracil, 5-prop-incytosine, 5-fluorocytosine, 5-fluorouracil, 5-hydroxy-methyluracil and 5-bromocytosine and very particularly preferably adenine, cytosine, guanine, uracil, thymine, 5-propinuracil and 5-propincytosine;

n is an integer from 1 to 100, preferably 5 to 40, particularly preferably 6 to 30, very particularly preferably 7 to 25;

n' is an integer from zero to 50, preferably zero to 40, particularly preferably zero to 30, very particularly preferably zero to 25;

m is an integer from zero to 5, very particularly preferably zero;

m' in formula I is an integer from zero to 5, very particularly preferably zero or 1;

m' in formula II is an integer from 1 to 5, very particularly preferably 1;

A is oxy, thioxy or methylene, preferably oxy;

W is oxo, thioxo or selenoxo, preferably oxo or thioxo, particularly preferably oxo;

V is oxy or thio, very particularly preferably oxy;

T is oxy, thio or imino, very particularly preferably oxy;

Y is oxy, thio, imino or methylene, very particularly preferably oxy;

X is hydroxyl or mercapto;

U is hydroxyl, mercapto, BH3, SeH, Cl-C18-alkoxy, preferably Ci-C6-alkoxy, C1-C18-alkyl, preferably C1-C6-alkyl, C6-C20-aryl, C6-C14-aryl-C1-C8-alkyl, NHR3, NR3R4 or a radical of the formula IV

(OCH2CH2).pO (CH2) gCH2R5 (IV) preferably hydroxyl, mercapto, C1-C6-alkoxy, C1-C6-alkyl, NR3R4 or NHR3 and particularly preferably hydroxyl or C1-C6-alkyl, in which R3 is C1-C18-alkyl, preferably C1-C8-alkyl, C6-C20-aryl, C6-C14-aryl-C1-C8-alkyl, - (CH2) C- INH (CH2) c) d-NR6R6, in which c is an integer from 2 to 6 and d is an integer from zero to 6, and R6 is, independently of one another, hydrogen, C1-C6-alkyl or C1-C4-alkoxy-C1-C6-alkyl, prefer-ably methoxyethyl, preferably C1-C8-alkyl, in particular C1-C4-alkyl;

R4 is C1-C18-alkyl, C6-C20-aryl or C6-C10-aryl-C1-C8-alkyl, preferably C1-C8-alkyl, in particular C1-C4-alkyl, C6-C20-aryl or C6-C10-aryl-C1-C8-alkyl, or, in the case of NR3R4, is, together with R3 and the nitrogen atom carrying them, a 5-6-membered heterocyclic ring which can additionally contain another hetero atom from the series consisting of 0, S and N;
P is an integer from 1 to 100, preferably 3 to 20 and particularly preferably 3 to 8;

q is an integer from zero to 22, preferably zero to 15;

R5 is hydrogen or a functional group such as hydroxyl, amino, NHR7, COON, CONH21 COORS or halogen, in which R7 is C1-C6-alkyl and R8 is Cl-C4-alkyl, preferably methyl;

Z and Z' are, independently of one another, hydroxyl, mercapto, SeH, C1-C22-alkoxy, preferably C6-C18-alkoxy, -O-(CH2)b-NR7R8, in which b is an integer from 1 to 6, and R7 is C1-C6-alkyl and RS is C1-C4-alkyl, or R7 and R8 form, together with the nitrogen atom carrying them, a 3-6-membered ring; C1-C18-alkyl, preferably C1-C8-alkyl, C6-C20-aryl, C6-C14-aryl-C1-C8-alkyl, preferably C6-C10-aryl-C1-C4-alkyl, C6-C14-aryl-C1-C8-alkoxy, preferably C6-C10-aryl-C1-C4-alkoxy, where aryl also means heteroaryl and aryl is optionally substituted by 1, 2 or 3 identical or different radicals from the series consisting of carboxyl, amino, nitro, Ci-C4-alkylamino, C1-C6-alkoxy, hydroxyl, halogen and cyano, or C1-C18-alkylmercapto, NHR3, NR3R4, in which R3 and R4 are as defined above, or a group which favors intracellular uptake or acts as labeling of a DNA probe or, on hybridization of the oligonucleotide analog onto to the target nucleic acid, interacts with the latter by binding, crosslinking or cleavage, or a nucleoside or oligonucleotide linked via the 5' or 3' ends; and the curved parenthesis indicates that R2 and the adjacent phosphoryl radical can be located in the 2' and 3' positions or else conversely in the 3' and 2' positions, it being possible for each nucleotide to be in its D or L configuration and for the base B to be located in the a or P position.
Oligonucleotide analogs of the formula I and the physio-logically tolerated salts thereof in which the base B is located in the P position, the nucleotides are in the D
configuration and R2 is located in the 2' position are preferred.

Oligonucleotide analogs of the formula I in which V and Y are oxy are particularly preferred.
Also particularly preferred are oligonucleotide analogs of the formula I in which V, Y and W are oxy and oxo respectively.
Oligonucleotide analogs of the formula I in which V, Y, W and Y are oxy, oxo and hydroxyl, respectively, are very particularly preferred.
Oligonucleotide analogs of the formula I in which R1 is hydrogen are furthermore preferred.
Oligonucleotide analogs of the formula I in which U, V, W, X and Y are oxy, oxo and hydroxyl, respectively, and R1 is hydrogen, are particularly preferred.

The radicals which occur repeatedly, such as R2, B, A, W, V, Y, U, R3, R4, T, a, b, p, q and Z can have meanings which are identical or different independently of one another, i.e., for example, V is, independently of one another, oxy, thio or imino.

Halogen is preferably fluorine, chlorine or bromine.

Heteroaryl means, in particular, radicals derived from phenyl or naphthyl in which one or more CH groups are replaced by N and/or in which at least two adjacent CH
groups are replaced by S, NH or 0 (to form a five-membered aromatic ring). Furthermore, one or both atoms at the point of fusion in bicyclic radicals (as indoliz-inyl) can be nitrogen atoms. Heteroaryl is, in particu-lar, furanyl, thienyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyrid-azinyl, indolyl, indazolyl, quinolyl, isoquinolyl, -phthalazinyl, quinoxalinyl, quinazolinyl, cinnolinyl.
Examples of groups which favor intracellular uptake are various lipophilic radicals such as -0- (CH2),-CH3 in which x is an integer from 6-18, -O- (CH2) e-CH=CH- (CH2) f-CH3 in 5 which e and f are, independently of one another, an integer from 6 to 12, -0-(CH2CH2O)4-(CH2)9-CH3, -0- (CH2CH2O) 8- (CH2)13-CH3 and -0- (CH2CH2O) 7- (CH2) 15-CH31 but also steroid residues such as cholesteryl and conjugates which utilize natural carrier systems, such as bile acid, 10 folic acid, 2-(N-alkyl-N-alkoxy)aminoanthraquinone and conjugates of mannose and peptides of the appropriate receptors which lead to receptor-mediated endocytosis of the oligonucleotides, such as EGF (epidermal growth factor), bradykinin and PDGF (platelet derived growth factor).. Labeling groups mean fluorescent groups, for example of dansyl (= N-dimethyl-1-aminonaphthyl-5-sul-fonyl), fluorescein or coumarin derivatives or chemiluminescent groups, for example of acridine deriva-tives, and the digoxigenin system detectable by ELISA, the biotin group detectable via the biotin/avidin system, or else linker arms with functional groups which permit subsequent derivatization with detectable reporter groups, for example an aminoalkyl linker, which is reacted with an acridinium active ester to give the chemiluminescent sample. Typical labeling groups are:
N(') 0 N-(CH2)x-N-H H
Acridinium eater 0 0 0-(CH2)1-0-COOK
Fluorescein derivative x = 2-18, preferably 4 R = H or C1-C4-alkyl "fluorescein" for x 4 and R = CH3 ) R = H or amino protective group RN/NH

H
N

Biotin conjugate (_ "biotin" for R = Fmoc) HO

OH
0\ 0 0_ \/\N
H
Digoxigenin conjugate Oligonucleotide analogs which bind or intercalate and/or cleave or crosslink to nucleic acids contain, for example, acridine, psoralen, phenanthroline, naphthoquin-one, daunomycin or chloroethylaminoaryl conjugates.
Typical intercalating and crosslinking radicals are:
-0-(CH2), N

\ /

Acridine derivative x = 2 - 12, preferably 4 -S-(CH2),-NH /N

CI
x = 2 - 12, preferably 4 CH(13.CH2X-(CH2)2_X_ I

X = -NH or -O-Trimethyipsoralen conjugate (= "psoralen" for X = 0) NH
S

N
N
Phenanthroline conjugate HN

Psoralen conjugate N H_--~O
CI

Naphthoquinone conjugate i I ( CH3 OH

NO
NH

Daunomycin derivative C I -CH2CHZNl N (CH2)x'0`
H3Cz -C-R
x = 1-18, X = alkyl, halogen, NO2, CN, to (CH2)x-0-X
x = 1-18, X = alkyl, halogen, NO2, CN, -C-R

The invention is not confined to a- and P-D-= or L-ribo-furanosides, a- and j5-D- or L-deoxyribofuranosides and corresponding carbocyclic five-membered ring analogs but also applies to oligonucleotide analogs which are assembled from different sugar building blocks, for example ring-expanded and ring-contracted sugars, acyclic or suitable other types of sugar derivatives. The inven-tion is furthermore not confined to the derivatives, listed by way of example in formula I and formula II, of the phosphate residue but also relates to the known dephospho derivatives (E. Uhlmann and A. Peyman in "Methods in Molecular Biology", Vol. 20, Protocols for Oligonucleotides and Analogs. S. Agarwal, Ed., Humana Press, Ottowa 1993). The invention also relates to other modifications familiar in the chemistry of oligonucleo-tide analogs, for example known conjugate modifications via phosphate residues, bases and at the 3' end in the case of formula II. The invention furthermore also relates to oligonucleotides in which the novel building blocks can additionally be present elsewhere in compounds of the formulae I and II.

Physiologically tolerated, salts of compounds of the formulae I and II mean both inorganic and organic salts as described in Remington's Pharmaceutical Sciences (Mack Publ. Co., Easton, PA, 17th edition (1985) 1418). Because of the physical and chemical stability, inter alia sodium, potassium, calcium and ammonium salts are pre-ferred for acidic groups.

Oligonucleotide analogs of the formula I and II are prepared by known methods analogous to the synthesis of biological oligonucleotides in solution or, preferably, on solid phase, where appropriate with the assistance of an automatic synthesizer.

There are various methods for introducing conjugate molecules at the 3' end of the oligonucleotides. However, these do not afford compounds of the formula I. A review of the prior art is given by: M. Manoharan in Antisense Research and Applications, Crooke and Lebleu, Eds., Chapter 17, pages 303 et seq., CRC Press Boca Raton, 1993, and EP-A 0 552 766 (HOE 92/F 012) and EP-A 0 552 767 (HOE 92/F 013). Whereas derivatization at the 5' end of an oligonucleotide is comparatively simple to bring about, for example by reaction with a phosphor-amidite of the appropriate conjugate molecule using the standard oligonucleotide synthesis cycle, there is no such universally applicable process for the 3' end. 3'-conjugation takes place either post-synthetically - that is to say after elimination from the support and after elimination of the protective groups - or via a support material which is to be prepared specifically for a specific conjugate molecule. P. S. Nelson et al. (Nucl.
Acids Res. 20 (1992) 6253) describe a 3' linker from which, after synthesis has taken place on the solid support, all protective groups are eliminated and then conjugate molecules are coupled onto the free amino group post-synthetically. Gamper et al. (Nucl. Acids Res. 21 (1993) 145) describe solid-phase synthesis using support material which has been derivatized with the conjugate molecule to be introduced. The support must be deriva-tized in an elaborate manner for every conjugate mole-cule. EP-A 0 552 766 and EP-A 0 552 767 describe a j5-eliminatable linker onto which are coupled nucleoside phosphoramidites which carry the appropriate conjugate molecule in place of the usual cyanoethyl protective group. The oligonucleotide synthesis then takes place.
This means that the conjugate molecule must not carry any acid-labile protective group, which would be eliminated during the synthesis cycle. In addition, the synthesis of the nucleoside conjugate monomer building blocks is very elaborate.

This invention therefore relates to a process which can be employed universally for the 3' modification of oligonucleotides on a solid support, which permits the introduction of a conjugate molecule by phosphoramidite chemistry during solid-phase synthesis. It is possible to employ for the conjugation the readily accessible conju-gate phosphoramidites which are familiar for 5' derivat-ization. The linker molecule with the appropriate protective group which is used for this purpose can be introduced not only at the 3' end of the oligonucleotide but also one or more times within the oligonucleotide using phosphoramidite chemistry.

The process for the preparation of the compounds of the formula I comprises a) reacting a compound of the formula V

I
(CH2)b HC-?-H
I (V) (IH2)0 in which a, b, V, T are defined as above in formula I and V' is V, and the functional groups V, V' and T can also be in temporarily protected form where appropriate (preferably, if V = V, = T = oxy and b = 0, as cyclic acetal which is obtained by reaction with acetone with Fell= catalysis and is eliminated again with acetic acid after introduction of the protective group Si), with a protective group Si which can be eliminated from an oligonucleotide which is still completely protected and linked to the support without cleaving other protec-tive groups or the linkage to the solid support, such as, for example, the levuloyl protective group, and ortho-, meta- or para-R-O-aryl, where R is C1-C20-alkyl, C2-C20-alkenyl, C3-C20-alkynyl, C6-C12-aryl-C1-C6-alkyl, prefer-ably the levuloyl protective group and the para-methoxy-phenyl protective group, and a protective group S2 which can be removed without cleaving the linker arm Li in formula VII and without cleaving the protective group Si, preferably dimethoxy-trityl, monomethoxytrityl, trityl, pixyl, 4-methoxytetra-hydropyranyl, particularly preferably monomethoxytrityl and dimethoxytrityl, by known processes (for example M. J. Gait, "Oligonucleo-tide Synthesis - a practical approach", IRL Press 1984), for example the para-methoxyphenyl group is introduced by reaction with para-methoxyphenol, diphenyl azodicarbox-ylate and triphenylphosphine in a suitable solvent, for example tetrahydrofuran (THF), under ref lux, then the acetal is eliminated again with acid, for example with acetic acid, and subsequently the monomethoxytrityl protective group is introduced by reaction with monometh-oxytrityl chloride in pyridine, to give a compound of the formula VI

I
(IH2)e HC-T-H
I (vi) (CH2)o NZ C
in which Si, S2, V, V', T, a and b are as defined above, b) subsequently reacting the compound of the formula VI
by known processes with 1 to 10 equivalents, preferably with 1 to 2 equivalents, of a linker Li such as, for example, succinic anhydride, in a suitable organic solvent such as, for example, methylene chloride, where appropriate after addition of a catalyst, for example 4-dimethylaminopyridine, to give a compound of the formula VII

(CH2)b Li-7-CH
I (V11) (CH2)e H2C -V'-S t in which Si, S2, V, V', T, a and b are as defined above, and Li is a linker arm which can attach the compound of the formula VI by chemical linkage (amide, ester, inter alia) to a solid support (Damka et al., Nucleic Acids Res. 18 (1990) 3813, Sonveaux (Bioorg. Chem. 14 (1986) 274), preferably a succinic acid residue (O-C (O) -CH2CH2-C (O) -) , an oxalic acid residue, (O-C(O)-C(O)-), an alkylamine, preferably LCAA (long chain alkylamine), or polyethylene glycol, particularly preferably a succinic acid residue, where in certain cases, for example in combination with substituents which do not withstand lengthy ammonia treatment, also more labile linkers such as the oxalyl linker are advantageous, and subsequently working up by known processes, such as, for example, extraction, crystallization, chromatography;

c) coupling the compound of the formula VII by known processes to a solid support SS such as, for example, aminopropyl-CPG (CPG = controlled pore glass) or Tentagel (from Rapp, Germany), for example by reaction with DCC and p-nitrophenol in a suitable solvent with O-(benzotriazol-l-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU) and a base such as, for example, N-ethylmorpholine, in a suitable solvent such as, for example, DMF (for example M. J. Gait, Oligonucleotide Synthesis - a practical approach, IRL Press, 1984) to obtain a compound of the formula VIII

I
(i H 2) b SS-L i-T-CH
I (VIII) (IH2)=
H2C-V$-SI
in which S1, S2, V, V', T, Li, a and b are as defined above, and SS is the solid support, for example of materials such as CPG (controlled pore glass), silica gel or an organic resin such as polystyrene (PS) or a graft copolymer of PS
and polyethylene glycol (POE), and is modified by func-tional groups such as hydroxyl, amino, halogen or COOH in the side chain:

d) eliminating the protective group S2 by known pro-cesses, for example by treatment with 1-4% dichloroacetic acid (DCA) in dichloromethane or chloroform, or alternatively previously eliminating the protective group Si by known processes, for example the levuloyl protec-tive group by treatment with hydrazine, carrying out reaction steps 1) and m), then reaction steps e) - i) and subsequently reaction step n), or alternatively after elimination of the protective group S2 carrying out reaction steps 1) and m), then elimina-ting the protective group S1 by known processes, for example the levuloyl protective group by treatment with hydrazine or the para-methoxyphenyl protective group by treatment with Ce1 , then carrying out reaction steps e) - i) and finally reaction step n);

e) subsequently, if m is 1 to 5, reacting the compound obtained in d) with a compound of the formula IX

R R10H (CH2)e P-T-CH
I (IX) R12 (CH2)e H2C _V'-S 1 in which Si, S2, V, V', T, a and b are as defined above, and R9 and R10 are identical or different and are C1-C8-alkyl, preferably isopropyl, or C5-C12-cycloalkyl, preferably up to C8, benzyl or phenyl or together with the nitrogen atom to which they are bonded a saturated or unsaturated heterocyclic ring, optionally with further hetero atoms, such as, for example, morpholine, and substituents such as OC(O)O-C1-C4-alkyl esters, R12 is OR13 or C1-C18-alkyl, C1-C18-alkoxy, C6-C20-aryl, C6-C14-aryl-C1-C8-alkyl, preferably OR13, C1-C6-alkyl, C1-C6-alkoxy, C6-C20-aryl, C6-C14-aryl-C1-C8-alkyl, parti-cularly preferably OR13 or C1-C6-alkyl, R13 is a group of the formulae -CH2-CH2 0 NO= -CH2-CH2-C=N, CI
or - CH3 or a benzyl group, which is not or is one to four times ring-substituted, preferably not substituted, where the substituent or substituents is, independently of one another, fluorine, chlorine, bromine, a C1-C4-alkyl, nitro, methoxy or carboxyl group, in the presence of a compound of the formula [HNR14R15R16] (+)E(-), where R14, R15 and R16 are identical to or different from one another and are a C1-C4-alkyl group and E is fluorine, chlorine, bromine, in particular chlorine, or in the presence of tetrazole or substituted tetrazole, such as, for example, 5-(4-nitrophenyl)-1H-tetrazole or 5-methylthio-1H-tetrazole or 5-ethylthio-lH-tetrazole, preferably in the presence of substituted tetrazole such as, for example, 5-(4-nitrophenyl)-1H-tetrazole or 5-methylthio-1H-tetrazole or 5-ethylthio-lH-tetrazole, particularly preferably in the presence of 5-methylthio-1H-tetrazole, in a suitable organic solvent, preferably acetonitrile, oxidizing the resulting compound by known processes, for example as described in reaction step m), carrying out a capping in the conventional way, eliminating the protective group S2 (for example Beaucage and Iyer Tetrahedron 49 (1993) 1925 & 2223 & 6123; E. Sonveaux, Bioorg. Chem. 14 (1986) 274; E. Uhlmann and A. Peyman, Chemical Reviews 90 (1990) 543) and, then repeating this reaction step (m-i) times where appropriate, resulting in a compound of the formula X

(CN2)b CH=-V'-SI
HC (CH=)1 (x) U- P V - CH, w (CH3)b N C-T-L I -SS

(CN2).
CH2-V'-SI

in which Li, Si, SS, T, U, V, VI, W, a, b and m are as defined above;

f) if m is 0, reacting the compound obtained in d) by the phosphoramidite method (E. Sonveaux, Bioorg. Chem. 14 (1986) 274) with a nucleoside phosphoramidite of the formula XI

S2-V B.
(XI) R' Y R2.
N-P -R' 2 in which B' is defined as B and R21 is defined as R2, and these can also be in protected form where appropriate, for example R2 can be hydroxyl protected by tert-butyldimethylsilyl, and R9, R10, R12, S2 and V are as defined above, oxidizing the resulting compound by known, processes, carrying out a capping in the conventional way, eliminating the protective group 02, preferably di-methoxytrityl or monomethoxytrityl, by known processes (for example Beaucage and Iyer, Tetrahedron 49 (1993) 1925 & 2223 & 6123; E. Sonveaux, Bioorg. Chem. 14 (1986) 274; E. Uhlmann and A. Peyman, Chemical Reviews 90 (1990) 543), and then repeating this reaction step (n-1) times where appropriate, resulting in a compound of the formula XII

H V 6' (XII) A

R
Y
U-P V- i H2 W n ( CH2) CHi V-S i HC (CH2)I
T

W m (~ H2)b HC-T-LI-SS
I
(CH2)e CH2-V'-S t in which A, B', Li, R2', Si, SS, T, U, V, V', W, Y, a, b, m and n are as defined above;

g) if m' is 1 to 5, carrying out reaction step e), which is repeated (m'-1) times where appropriate, resulting in the compound of the formula XIII

I
(i H2)` /CH2 V'-S 1 HC (CH2(X11 I) T
I 8' U-P V A
u W m' Y R

II
W (CN2 )- /CH2 V'-S t Hi (CH2), T
I

W m (CH2)1 HC-T-L I-SS
(cH2)e CH2-V'-S 1 in which A, B', Li, R2Si, SS, T, U, V, V', W, Y, a, b, m, m' and n are as defined above;

h) if m' is 0 and n' is 1-50, carrying out reaction step f), which is then repeated (n'-1) times where approp-riate, resulting in the compounds of the formula XIV

H -V

W-R, W (iH2)b /Ckj V'-SI
H (CH
I 2)1 (XIV) T

n U_ 1 V

Y R

u W ( CH2 )b CH2 V'-S 1 I
Hj (CHI), T

W m (CH2)b HC-T-L i-SS
(~H2)a CH2-V'-S I
in which A, B', Li, R21, S1, SS, T, U, V, V', W, Y, at b, m, m', n and n' are as defined above;

i) where appropriate if R1 s H in formula I, introducing the radical R1 by known processes into the compound obtained in f), g) or h), preferably by appropriate reaction analogous to reaction steps 1) and m), where R1 are C1-C18-alkyl, preferably C1-C6-alkyl, in particular methyl, C2-C18-alkenyl, C3-C18-alkynyl, C1-C16-alkyl-carbonyl, C2-C19-alkenylcarbonyl, C3-C19-alkynylcarbonyl, C6-C20-aryl, C6-C14-aryl-C1-C8-alkyl, or a radical of the formula III

Z-P-Z' II
W (III) in which W, Z and Z' are as defined above, preferably a radical of the formula III;

j) if R1 = H in formula I, capping by known methods, for example by reaction with acetic anhydride and N-methyl-imidazole;

f) if R1 = H in formula II, capping by known methods, for example by reaction with acetic anhydride and N-methyl-imidazole;

k) subsequently eliminating the protective group S1 by known processes (for example Greene, Wuts, "Protective Groups in Organic Synthesis", J. Wiley, New York 1991) from the oligonucleotides which are obtained in this way and are still linked to the support and protected, so that the linker to the solid support and the other protective groups present in the molecule are retained, for example for Si = levuloyl by treatment with hydrazine and for S1 = para-methoxyphenyl preferably by treatment with the Ce1", for example with a 0.05-1 M solution of Ce"v(NH4) 2 (NO3) 6 in acetonitrile/H20 at -10 to 100 C for 0.2 to 500 minutes, preferably with a 0.05 to 0.5 M, in particular 0.1 M, solution of Ce1 (NH4)2(NO3)6 in acetonitrile/H20 (2:1 to 8:1, in particular 4:1) at 0-50 C, in particular 20-30 C, for 1-30 min, in particu-lar for 2 to 10 min;

1) and reacting the compound obtained in this.way with a compound of the formula XV

R'R' N
iP-Z (XV) in which R9, R10, R12 have the abovementioned meanings, and Z" has the meaning of Z as defined above or else is Z
protected by known processes, the protective groups which are preferably used being those eliminated under con-ditions used for the elimination of protective groups in the oligonucleotide synthesis, examples which may be mentioned being hydroxyl, mercapto and SeH, which must be in the form of protected derivatives, for example as O-CH2-CH2-CN, O-CH3, S-CH2-CH2-CN or ci ti in the presence of a. compound of the formula Elm14R15R16] (+)E(-), where R14, R15, R16 and E are as defined above, or in the presence of tetrazole or substi-tuted tetrazole, such as, for example, 5- (4-nitrophenyl) -1H-tetrazole or 5-methylthio-1H-tetrazole or 5-ethylthio-1H-tetrazole, preferably in the presence of substituted tetrazole, such as, for example, 5-(4-nitrophenyl)-1H-tetrazole or 5-methylthio-1H-tetrazole or 5-ethylthio-lH-tetrazole, particularly preferably in the presence of 5-methylthio-1H-tetrazole, in a suitable organic solvent, preferably acetonitrile;

m) oxidizing the resulting compound by known processes, for example by reaction with iodine in the presence of aqueous pyridine, lutidine or collidine, where approp-riate also in-the presence of other organic solvents such as, for example, tetrahydrofuran, or, for example, by reaction with N,N,N',N'-tetraethylthiuram disulfide in acetonitrile, or, for example, by reaction with iodine in the presence of alkylamine or arylamine, the various oxidation processes which are known to the skilled worker and are used to prepare natural and modified oligonucleo-tides being summarized, for example, in Beaucage and Iyer, Tetrahedron 49 (1993) 1925 & 2223 & 6123; E.
Sonveaux, Bioorg. Chem. 14 (1986) 274 and E. Uhlmann and A. Peyman, Chemical Reviews 90 (1990) 543, and the oxidation preferably being carried out by reaction with iodine in the presence of aqueous pyridine, lutidine or collidine, where appropriate also in the presence of other organic solvents such as tetrahydrofuran;

n) eliminating the oligonucleotide from the support by known processes, for example with NH3 at 50-60 C, and eliminating the remaining protective groups on the phosphate and nucleotide bases likewise by known pro-cesses.

The process for the preparation of compounds of the formula II comprises a) eliminating in a compound of the formula XVI
S2-V 8.
(XVI) Li SS

in which A, B', Li, R21, S2, SS and V are as defined above, and Li can additionally be a linker which permits introduction of 3'-phosphate residue (see, for example, EP-A 0 552 766, Beaucage and Iyer, Tetrahedron 49 (1993) 2223 & 6123), the protective group S2 by known processes, for example by treatment with 1-4% dichloroacetic acid (DCA) in dichloromethane or chloroform;

b) subsequently reacting the resulting compound by the phosphoramidite method (E. Sonveaux, Bioorg. Chem. 14 (1986) 274) with a nucleoside phosphoramidite of the formula XI

S2-V VAB- (XI) Y R2.

's R
Rio/ N-P-R
in which B' is defined as B and R2' is defined as R2, and these can also be in protected form where appropriate, for example R2 can be hydroxyl protected by tert-butyldimethylsilyl, and R9, R10, R12, S2 and V are as defined above, oxidizing the resulting compound by known processes, carrying out a capping in the conventional way, eliminat-ing the protective group S2, preferably dimethoxytrityl or monomethoxytrityl, by known processes (for example Beaucage and Iyer, Tetrahedron 49 (1993) 1925 & 2223 &
6123; E. Sonveaux, Bioorg. Chem. 14 (1986) 274; E.
Uhlmann and A. Peyman, Chemical Reviews 90 (1990) 543), and then repeating this reaction step (n-1) times where appropriate, resulting in a compound of the formula XVII
H V BA

(XVI1) Y R=I B.
U-P V A

W

LI

SS
in which A, B', Li, R21, SS, U, V, W, Y and n are as defined above;

c) subsequently reacting the resulting compound with a compound of the formula IX

ORION ( H2)-P-T-CH
/
12 1 (IX) R iH2 H2C --V'-S 1 in which Si, S2, V, V', T, a and b are as defined above and R9 and R10 are identical or different and are C1-C8-alkyl, preferably isopropyl, or C5-C12-cycloalkyl, preferably up to C8, benzyl or phenyl or together with the nitrogen atom to which they are bonded a saturated or unsaturated heterocyclic ring, optionally with further hetero atoms, such as, for example, morpholine, and substituents such as 0C(O)0-C1-C4-alkyl esters, R12 is OR13 or C1-C18-alkyl, Ci-Ci8-alkoxy, C6-C20-aryl, C6-C14-aryl-C,-C8-alkyl, preferably 0R13, C1-C6-alkyl, C1-C6-alkoxy, C6-C20-aryl, C6-C14-aryl-C1-C8-alkyl, partic-ularly preferably OR13 or C1-C6-alkyl, R13 is a group of the formula -CH2-CH2 /^\ N02 C H 2 - C H 2 - C ~ N
CI
CI or --CH3 or a benzyl group, which is not or is one to four times ring-substituted, preferably not substituted, where the substituent or substituents is, independently of one another, fluorine, chlorine, bromine, a C1-C4-alkyl, nitro, methoxy or carboxyl group, in the presence of a compound of the formula (BM 14R15R16] (+)E(-), where R14, R15 and R16 are identical to or different from one another and are a C1-C4-alkyl group and E is fluorine, chlorine, bromine, in particular chlorine, or in the presence of tetrazole or substituted tetrazole, such as, for example, 5-(4-nitrophenyl)-1H-tetrazole or 5-methylthio-1H-tetrazole or 5-ethylthio-IH-tetrazole, preferably in the presence of substituted tetrazole such as, for example, 5-(4-nitrophenyl)-lH-tetrazole or 5-methylthio-1H-tetrazole or 5-ethylthio-lH-tetrazole, particularly preferably in the presence of 5-methylthio-lH-tetrazole, in a suitable organic solvent, preferably acetonitrile, oxidizing the resulting compound by known processes, for example as described in reaction step m), carrying out a capping in the conventional way, eliminating the protec-tive group S2 (for example Beaucage and Iyer Tetrahedron 49 (1993) 1925 & 2223 & 6123; E. Sonveaux, Bioorg. Chem.
14 (1986) 274; E. Uhlmann and A. Peyman, Chemical Reviews 90 (1990) 543) and, where appropriate, then repeating this reaction step (m'-1) times, resulting in a compound of the formula XVIII

HV-CH!
(i H= ) /CHI- V'-S I

Hi ( C H 2 (XVI I I) T
U-P V A B
11.
W

I BUP V A
W In Li SS
in which A, B', Li, R21, S1, SS, U, V, V', W, Y, a, b, m' and n are as defined above;

d) if n' is 1-50, carrying out reaction step b), which is repeated (n'-l) times where appropriate, resulting in the compound of the formula XIX

H V B.
A
R z' Y
U-P V-CH=
II I
W --fn' ( C H2 )` CH= V'-S 1 HC (CH=)a T (XIX) U-P V A
W ii m= I
Y R_ U-P V B' n A

Li SS

in which A, B', Li, R', S1, SS, U, V, V', W, Y, a, b, m', n and n' are as defined above;

e) where appropriate if R1 s H in formula II, introducing the radical R1 by known processes into the compound obtained in c) or d), preferably by appropriate reaction analogous to reaction steps h) and i), where R1 is C1-C18-alkyl, preferably C1-C6-alkyl, in particular methyl, C2-C18-alkenyl, C3-C18-alkynyl, C1-C18-alkyl-carbonyl, C2-C19-alkenylcarbonyl, C3-C19-alkynylcarbonyl, C6-C20-aryl, C6-C14-aryl-C1-C8-alkyl, or a radical of the formula III

2 -P- z , W
in which W, Z and Z' are as defined above, preferably a radical of the formula III;

f) if R1 = H in formula II, capping by known methods, for example by reaction with acetic anhydride and N-methyl-imidazole;

g) subsequently eliminating the protective group Si by known processes (for example Greene, Wuts, "Protective Groups in Organic Synthesis", J. Wiley, New York 1991) from the oligonucleotides which are obtained in this way and are still linked to the support and protected, so that the linker to the solid support and the other protective groups present in the molecule are retained, for example for Si = levuloyl by treatment with hydrazine and for S1 =.para-methoxyphenyl preferably by treatment with the Celt', for example with a 0.05-i M solution of Ce1 (NH4) 2 (N03) 6 in acetonitrile/H20 at -10 to 100 C for 0.2 to 500 minutes, preferably with a 0.05 to 0.5 M, in particular 0.1 M, solution of CeIv(NH4)2(N03)6 in acetonitrile/H20 (2:1 to 8:1, in particular 4:1) at 0-50 C, in particular 20-30 C, for 1-30 min, in particu-lar for 2 to 10 min;

h) and reacting the compound obtained in this way with a compound of the formula XV

R'R'0N
P-Z" (XV) RI=

in which R9, R10, R12 have the abovementioned meanings, and Z" has the meaning of Z as defined above or else is Z
protected by known processes, protective groups which are preferably used being those eliminated under conditions used for the elimination of protective groups in the oligonucleotide synthesis, examples which may be men-tioned being hydroxyl, mercapto and SeH, which must be in the form of protected derivatives, for example as O-CH2-CH2-CN, O-CH3, S-CH2-CH2-CN or :ixY' in the presence of a compound of the formula [HNR14R15R16 ] (+)E(-), where R14 , R15, R16 and E are as defined above, or in the presence of tetrazole or substi-tuted tetrazole, such as, for example, 5- (4-nitrophenyl)-1H-tetrazole or 5-methylthio-1H-tetrazole or 5-ethylthio-1H-tetrazole, preferably in the presence of tetrazole or substituted tetrazole, such as, for example, 5-(4-nitro-phenyl)-1H-tetrazole or 5-methylthio-lH-tetrazole or 5-ethylthio-lH-tetrazole, particularly preferably in the presence of 5-methylthio-1H-tetrazole, in a suitable organic solvent, preferably acetonitrile;

i) oxidizing the resulting compound by known processes, for example by reaction with iodine in the presence of aqueous pyridine, lutidine or collidine, where approp-riate also in the presence of other organic solvents such as, for example, tetrahydrofuran, or, for example, by reaction with N,N,N',N'-tetraethylthiuram disulfide in acetonitrile, or, for example, by reaction with iodine in the presence of alkylamine or arylamine, the various oxidation processes which are known to the skilled worker and are used to prepare natural and modified oligonucleo-tides being summarized, for example, in Beaucage and Iyer, Tetrahedron 49 (1993) 1925 & 2223 & 6123; E.
Sonveaux, Bioorg. Chem. 14 (1986) 274 and E. Uhlmann and A. Peyman, Chemical Reviews 90 (1990) 543, and the oxidation preferably being carried out by reaction with iodine in the presence of aqueous pyridine, lutidine or collidine, where appropriate also in the presence of other organic solvents such as tetrahydrofuran;

j) eliminating the oligonucleotide from the support by known processes, for example with NH3 at 50-60 C, and eliminating the remaining protective groups on the phosphate and nucleotide bases likewise by known pro-cesses.

The nature of the amino protective groups on the bases and the properties of the linker Li depend in the individual case on the nature of the substituent Z
because it must be possible to eliminate the latter without problems after the synthesis is complete. For example, in the preparation of an isopropyl oligonucleo-tide-3'-phosphate (Z = O-i-C3H7) it is possible to use as protective groups benzoyl (Bz) for B = Ade and Cyt and isobutyryl (i-Bu) for B = Gua. On the other hand, to synthesize an oligonucleotide-3'-methylphosphonate (Z =
CH3) or ethyl ester (Z = O-C2H5) the protective groups used are preferably the more labile phenoxyacetyl (PAC) for B = Ade and Gua and isobutyryl for B = Cyt.

The compounds of the formula IX (pages 20 and 30) R'R'0N (CH2)b P-T-CH
~ I (IX) R' _ (?H2 H2C -V'-S 1 in which Si, S2, V, V', T, a and b are as defined above, and R9 and R10 are identical or different and are C1-C8-alkyl, preferably isopropyl, or C5-C12-cycloalkyl, preferably up to C8, benzyl or phenyl or together with the nitrogen atom to which they are bonded a saturated or unsaturated heterocyclic ring, optionally with further hetero atoms, such as, for example, morpholine, and substituents such as OC(O)O-C1-C4-alky1 esters, R12 is OR13 or C1-C18-alkyl, C1-C18-alkoxy, C6-C20-aryl, C6-C14-aryl-C1-C8-alkyl, preferably OR13, C1-C6-alkyl, C1-C6-alkoxy, C6-C20-aryl, C6-C14-aryl-C1-C8-alkyl, partic-ularly preferably OR13 or C1-C6-alkyl, R13 is a group of the formula -C H 2 - C H 2 0 N 0 2 , - CH2 - CH2 - CEN, CI
or --CH3 or a benzyl group, which is not or is one to four times ring-substituted, preferably not substituted, where the substituent or substituents is, independently of one another, fluorine, chlorine, bromine, a C1-C4-alkyl, nitro, methoxy or carboxyl group, can be obtained by reacting a compound of the formula VI

(iH2)b H-T-CH
(Vi) (CH2)a H2C -V'-S I
with a compound of the formula XX
R'R'ON P/ (XX) 'R12 in which R9, R10 and R12 are as defined above, and R11 is chlorine or bromine or a radical of the formula NR9R10, where R9 and R10 are as defined above;

in the presence of a base, preferably pyridine, or of a mixture of tetrahydrofuran (THF), dioxane, dichloro-methane (DCM), chloroform and/or acetonitrile with a C1-C4-trialkylamine, preferably trimethyl-, triethyl- or diisopropylethylamine, or, if R11 is a radical of the formula NR9R10, then in the presence of a compound of the formula (HNR14R15R16] (+)E(") where R14, R15, R16 are identi-cal to or different from one another and are a C1-C4-alkyl group and E is fluorine, chlorine, bromine, in particular chlorine, or in the presence of tetrazole or substituted tetrazole such as, for example, 5-(4-nitro-phenyl)-1R-tetrazole or 5-methylthio-1H-tetrazole or 5-ethylthio-1H-tetrazole, preferably in the presence of tetrazole.

in place of the phosphoramidite method, it is also possible to obtain the compounds of the formulae I and II
by solid-phase synthesis by the H-phosphonate method or the phosphotriester method (E. Uhlmann and A. Peyman, Chemical Reviews 90 (1990) 543).

When the H-phosphonate method is used, the compound of the formula VI obtained after reaction step a) (prepara-tion of compounds of the formula I) is converted by known processes (for example B. Froehler, Tetrahedron Lett. 27 (1986) 5575) into a compound of the formula XXI

I
0_ (CH2)b H-P-T-CH
11 1 (XXI) W (CH2)0 H2)0 H2C -V'-S 1 in which V, V', T, a, b and W have the abovementioned meaning. An example which may be mentioned is the reac-tion with N, N P

in a suitable organic solvent, for example dichioro-methane, and subsequent hydrolysis. On introduction of the group Z (reaction step i) for compounds of the formula I and reaction step e) for compounds of the formula II) in the H-phosphonate method there is reaction with a compound of the formula XXII

H-P--Z
11 (XXiI) W

in which Z" and W have the abovementioned meanings, in the presence of a condensing agent such as pivaloyl or adamantoyl chloride, and of a base such as pyridine. The H-phosphonate diester which is formed is then subjected to an oxidative phosphoramidation (B. Froehler, Tetrahedron Lett. 27, (1986) 5575) or an oxidation with iodine water, sulfur or selenium. it is possible in this way, for example, to prepare an oligonucleotide with a 3'-terminal cholesteryl group using cholesteryloxy-carbonylaminoalkylamine in the presence of tetrachloro-methane. Oxidative amidation with 2-methoxyethylamine results, for example, in oligonucleotides with a 3'-0-(2-methoxyethyl)phosphoramidate residue.

In the triester method, the compound of the formula VI
obtained after reaction step a) (preparation of compounds of the formula I) is converted by known processes (for example Sonveaux, Bioorg. Chem. 14 (1986) 274) into a compound of the formula XXIII

i I (IH2)b R '7-0-P -T - C H
II 1 (XXIII) W (I H2 ).

H2C-V'-S1 in which V, V', T, a, b and W have the abovementioned meaning, and R17 is one of the protective groups used in the triester process and known to the skilled worker, for example 2,4-dichlorophenyl (E. Sonveaux, Bioorg. Chem. 14 (1986) 274). On introduction of the group Z (reaction step i) for compounds of the formula I and reaction step e) for compounds of the formula II) by the triester method there is reaction with a compound of the formula XXIV

yl W (XXIV) in which Z, W and R17 are as defined above, in the pres-ence of a condensing agent. Preferred condensing reagents are arylsulfonyl chlorides such as mesitylene-, 2,4,6-triisopropylbenzene- or 8-quinolinesulfonyl chlor-ide in the presence of nucleophilic catalysts such as imidazole, triazole or tetrazole or substituted deri-vatives thereof, such as N-methylimidazole, 3-nitro-triazole or 5-(p-nitrophenyl)tetrazole. Particularly preferred condensing agents are 4-substituted derivatives of pyridine N-oxide or quinoline N-oxide (Efimov et al., Nucleic Acids Research 13 (1985) 3651).

Oligonucleotide analogs of the formula I or of the formula II are used as inhibitors of gene expression.
The compounds of the present invention can be used, for example, as pharmaceuticals for the treatment of diseases caused by viruses (HIV, HSV-1, HSV-2, influenza, VSV, hepatitis B or papilloma viruses).

Antisense oligonucleotide sequences modified according to the invention and effective against such targets are, for example:

a) against HIV, for example 5'-ACACCCAATTCTGAAAATGG-3' (I) or 5'-AGGTCCCTGTTCGGGCGCCA-3' (II) or 5'-GTCGACACCCAATTCTGAAAATGGATAA-3' (III) or 5'-GCTATGTCGACACCCAATTCTGAAA-3' (IV) or 5'-TCGTCGCTGTCTCCGCTTCTTCTTCCTGCCA (V) or 5'- CTGTCTCCGCTTCTTCTTCCTGCCATAGGAG-3' (VI) or b) against HSV-1, for example 5'-GCGGGGCTCCATGGGGGTCG-3' (VII) The compounds of the present invention are also suitable, for example, for the treatment of cancer. Examples of oligonucleotide sequences which can be used for this purpose are those directed against targets which are responsible for the development of cancer or growth of cancer. Pharmaceuticals of the present invention are furthermore suitable, for example, also for preventing restenosis. Examples of oligonucleotide sequences which can be used for this purpose are those directed against targets which are responsible for proliferation or migration. Examples of such targets are:

1) nuclear oncoproteins such as, for example, c-myc, N-myc, c-myb, c-fos, c-fos/jun, PCNA, p120 2) cytoplasmic/membrane-associated oncoproteins such as, for example, EJ-ras, c-Ha-ras, N-ras, rrg, bcl-2, cdc-2, c-raf-1, c-mos, c-src, c-abl 3) cellular receptors, such as, for example, EGF
receptor, FGF receptor, c-erbA, retinoid receptors, protein kinase regulatory subunit, c-fms, cdc2 kinase, 4) cytokines, growth factors, extraceliular matrix, such as, for example, CSF-1, IL-6, IL-la, IL-lb, IL-2, IL-4, bFGF, IGF, myeloblastin, fibronectin.

Antisense oligonucleotide sequences modified according to the invention and active against such targets are, for example a) against c-Ha-ras, for example 5'-CAGCTGCAACCCAGC-3' (VIII) or c) c-myc, for example 5'-GGCTGCTGGAGCGGGGCACAC-3' (IX) or 5'-AACGTTGAGGGGCAT-3' (X) or d) c-myb, for example 5'-GTGCCGGGGTCTTCGGGC-3' (XI) or e) c-fos, for example 5'-GGAGAACATCATGGTCGAAAG-3' (XII) or 5'-CCCGAGAACATCATGGTCGAAG-3' (XIII) or 5'-GGGGAAAGCCCGGCAAGGGG-3' (XIV) or f) p120, for example 5'-CACCCGCCTTGGCCTCCCAC-3' (XV) or g) EGF receptors, for example 5'-GGGACTCCGGCGCAGCGC-3' (XVI) or 5'-GGCAAACTTTCTTTTCCTCC-3' (XVII) or h) p53 tumor suppressor, for example 5'-GGGAAGGAGGAGGATGAGG-3' (XVIII) or 5'-GGCAGTCATCCAGCTTCGGAG-3' (XIX).
The compounds of the present invention are furthermore suitable, for example, for the treatment of disorders which are influenced by integrins or cell-cell adhesion receptors, for example by VLA-4, VLA-2, ICAM or ELAM.
Antisense oligonucleotide sequences modified according to the invention and active against such targets are, for example a) VLA-4, for example 5'-GCAGTAAGCATCCATATC-3' (XX) or b) ICAM, for example 5'-CCCCCACCACTTCCCCTCTC-3' (XXI) or 5'-CTCCCCCACCACTTCCCCTC-3' (XXII) or 5'-GCTGGGAGCCATAGCGAGG-3' (XXIII) or c) ELAM-1, for example 5'-ACTGCTGCCTCTTGTCTCAGG-3' (XXIV).
The oligonucleotide analogs of the formula I or of the formula II can furthermore be used as probe for detecting nucleic acids or as aids in molecular biology.

The invention furthermore relates to pharmaceutical compositions containing one or more oligonucleotide analogs of the formula I or II, where appropriate together with physiologically tolerated ancillary sub-stances and/or vehicles and/or together with other known active substances, and to processes for the preparation thereof.

Examples 1) Synthesis of 4-methoxyphenyl 6-O-(4-methoxytri-phenylmethyl)-S-O-succinylhexyl ether la) 2,2-Dimethyl-4-hydroxybutyl-1,3-dioxolane 15 g (112 mmol) of 1,2,6-hexanetriol were dissolved together with 0.5 g of FeC13 in 1 1 of acetone and boiled under ref lux for 7 h. The mixture was filtered, and excess acetone was removed by distillation, resulting in the product in pure form.

Yield: 18.8 g (96%);
1H-NMR (200 MHz, CDC13/TMS): d = 1.35 (s, 3H, CH3); 1.40 (a, 3H, CH3); 1.30-1.40 (m, 6H, -(CH2)3-); 3.52 (t, iH, C4-H); 3.68 (t, 2H, CH2-OH); 4.00-4.20 (m, 2H, -C5H2-);
MS (EI) : m/e = 175 (M + H+, 50%) ; 159 (30%) ib) 4-Methoxyphenyl 4-(2,2-dimethyl-l,3-dioxolan-4-yl)-butyl ether 1.74 g (10 mmol) of 2,2-dimethyl-4-hydroxybutyl-l,3-di-oxolane from Example la, 3.41 g (13 amiol) of triphenyl-phosphine, 2.26 g (13 mmol) of diethyl azodicarboxylate and 3.72 g (30 mmol) of 4-methoxyphenol were dissolved in 30 ml of absolute tetrahydrofuran (THF) and boiled under reflux for 1 h. The solvent was removed by distillation, and the residue was chromatographed on silica gel using ethyl acetate (EA)/n-heptane (1:4).

Yield: 2.1 g (74%);
1H-NMR (200 MHz, CDC13/TMS) : d = 1.35 (s, 3H, CH3) ; 1.41 (s, 3H, CH3) ; 1.45-1.90 (m, 6H, - (CH2) 3-) ; 3.53 (t, 1H, C4. -H) ; 3.77 (s, 3H, O-CH3) ; 3.92 (t, 2H, CH2-OAr) ;
3.99-4.21 (m, 2H, -C5' H2-); 6.84 (a, 4H, Ar-H);
MS (EI) : m/e = 280 (M + H+, 90%); 265 (50%); 223 (100%) ic) 4-Methoxyphenyl 5,6-dihydroxyhexyl ether 2.08 g of 4-methoxyphenyl 4-(2,2-dimethyldioxolan-4-yl)butyl ethyer from Example lb were dissolved in 165 ml of 80% acetic acid and stirred at room temperature for 4 h. The acetic acid was separated off in vacuo, and the mixture was then coevaporated with toluene/methanol twice. This resulted in a crystalline product.

Yield: 1.15 g (65%), mp: 69 C
1H-NMR (200 MHz, CDC13/TMS): d = 1.40-1.91 (m, 6H, -(CH2)3-); 3.39-3.52 (m, 1H, C5-H); 3.42-3.74 (m, 2H, C6H2) ; 3.77 (s, 3H, O-CH3) ; 3.93 (t, 2H, CH2-OAr) ; 6.82 (s, 4H, Ar-H);
MS (EI) : m/e = 241 (M + H+, 60%) ; 240 (M+, 100%) ; 223 (30%), 205 (30%) id) 4-Methoxyphenyl 6-0-(4-methoxytriphenylmethyl)-5-hydroxyhexyl ether 1.96 g (8.2 mmol) of 4-methoxyphenyl 5,6-dihydroxyhexyl ether from Example lc and 2.78 g (9.0 mmol) of 4-methoxy-triphenylmethyl chloride were dissolved in 30 ml of absolute pyridine and stirred at room temperature for 3 h. The pyridine was evaporated off in vacuo, the residue was taken up in 40 ml of dichloromethane (DCM) and extracted first with 40 ml of 5% NaHCO3 solution and then with 40 ml of saturated NaCl solution and washed twice with water. The solution was dried over sodium sulfate, the solvent was removed by distillation, and the residue was chromatographed on silica gel using EA/n-heptane (1:2).

Yield: 3.10 g (74%);
1H-NMR (200 MHz, CDC13/TMS): d = 1.37-1.80 (m, 6H, - (CH2) 3-) ; 2.30 (d, J =5Hz, 1H, C5-H) ; 3.00-3.23 (m, 2H, CH2-OMMTr) ; 3.73 (s, 3H, O-CH3) ; 3.80 (s, 3H, O-CH3) ; 3.88 (t, 2H, CH2-OAr); 6.80 (s, 4H, Ar-H); 7.15-7.47 (m, 14H, Ar-H);
MS (ES+, + LiCl): m/e = 519 (M + Li+, 100%) le) 4-Methoxyphenyl 6-0-(4-methoxytriphenylmethyl)-5-0-succinylhexyl ether 3.1 g (6.05 mmol) of 4-methoxyphenyl 6-0-(4-methoxytri-phenylmethyl)-5-hydroxyhexyl ether from Example Id were dissolved together with 0.85 g (8.47 mmol) of succinic anhydride and 1.04 g (8.47 mmol) of N,N-dimethylamino-pyridine (DMAP) in 20 ml of absolute pyridine and stirred at room temperature for 19 h. The solvent was evaporated off in vacuo. it was then coevaporated twice with toluene/methanol, the residue was taken up in 280 ml of DCM and washed with 140 ml of 10% citric acid and twice with water and dried over sodium sulfate. The solvent was removed by distillation, and the residue was chromato-graphed on silica gel using EA/n-heptane 2:1.

Yield: 2.55 g (69%);
1H-NMR (200 MHz, CDC13/TMS) : d = 1.27-1.49 (m, 2H, C2H2);
1.60-1.82 (m, 4H, C1H2 & C3H2); 2.66 (s, 4H, CO-(CH2)2-CO); 3.15 (d, 2H, CH2-O14MTr); 3.75 (s, 3H, O-CH3); 3.78 (s, 3H, O-CH3); 3.89 (t, 2H, CH2-OAr); 5.12 (dt, 1H, CH-Osucc); 6.80 (s, 4H, Ar-H); 7.11-7.52 (m, 14H, Ar-H);
MS (FAB + LiCl) : m/e = 625.3 (M + 2Li+-H+, 100%) ; 619.2 (M + Li+, 70%) ; 612.2 (M+, 100%) 2) Synthesis of 4-methoxyphenyl 6-O-(4-methoxytri-phenylmethyl)-5-O-diisopropylamino-p-cyanoethoxy-phosphinohexyl ether 512 mg (1.0 umol) of 4-methoxyphenyl 6-0-(4-methoxytri-phenylmethyl)-5-hydroxyhexyl ether from Example ld were coevaporated together with 390 mg (3.0 mmol) of diiso-propylethylamine with absolute acetonitrile and then dissolved in 4 ml of absolute THF. Under protective gas, 330 mg (1.4 mmol) of cyanoethyl N,N-diisopropylchloro-phosphoramidite were slowly added dropwise. The mixture was stirred at room temperature for 2 h. The solvent was evaporated off, and the residue was taken up in 20 ml of EA and extracted with 40 ml of saturated NaCl solution.
The organic phase was then washed twice with water and subsequently dried over sodium sulfate. The solvent was removed by distillation, and the residue was chromato-graphed on silica gel using DCM/ethanol/triethylamine (TEA) (100:4:2).

Yield: 520 mg;
1H-NMR (200 MHz, CDC13/TMS): d = 1.00-1.93 (m, 18H, -(CH 2)3- & 4 x CH3) ; 2.38 & 2.57 (each: t, 1H, CH2-CN) ;
2.92-3.26 (m, 2H, P-O-CH2); 3.45-4.20 (m, 13H, 2 x OCH3 &

2 x CH(CH3)2 & CH2-OAr & CH2-O-MMTr & C5H); 6.70-6.87 (s, 4H, Ar-H); 7.14-7.32 (m, 14H, Ar-H);
MS (FAB, LiCl; NBA): m/e = 735.5 (M + Na+, 100%); 719.5 (M + Li+, 50%) 3) Synthesis of 4-methoxyphenyl 3-0-(4-methoxytri-phenylmethyl)-2-0-succinylpropyl ether 3a) 4-Methoxyphenyl (2,2-dimethyl-1,3-dioxolan-4-yl)-methyl ether Synthesis took place in analogy to Example lb from 2,2-dimethyl-4-hydroxymethyl-1,3-dioxolane.

Yield: 56%;
1H-NMR (200 MHz, CDC13/TMS) : d = 1.40 (s, 3H, CH3) ; 1.44 (s, 3H, CH3); 3.78 (a, 3H, O-CH3); 3.89 (dd, 2H, CH2-OAr);
3.97-4.21 (m, 2H, -C3H2-); 4.45 (dt, 1H, C2H); 6.83 (a, 4H, Ar-H);
MS (EI) : m/e = 239 (M + H+, 40%); 238 (M+, 50%) 3b) 4-Methoxyphenyl 2,3-dihydroxypropyl ether Synthesis took place in analogy to Example lc from 4-methoxyphenyl (2,2-dimethyl-1,3-dioxolan-4-yl)methyl ether (Example 3a).

Yield: 98%;
MS (EI) : m/e = 199 (M' + H+, 1000 ; 198 (M+, 80%) ; 181 (40%) ; 163 (70%) ;

3c) 4-Methoxyphenyl 3-0-(4-methoxytriphenylmethyl)-2-hydroxypropyl ether Synthesis took place in analogy to Example id from 4-methoxyphenyl 2,3-dihydroxypropyl ether (Example 3b).
Yield: 46%;
1H-NMR (200 MHz, CDC13/TMS): d = 3.3.31 (d, 2H, CH2-OMMTr) ; 3.77 (s, 3H, O-CH3) ; 3.79 (s, 3H, O-CH3);
3.96-4.20 (m, 3H, O-CH2-CH); 6.76-6.90 (m, 4H, Ar-H);
7.15-7.55 (m, 14H, Ar-H);
MS (FAB + LiCl) : m/e = 477.2 (M + Li+, 20%) ; 470.2 (M+, 10%) ;

3d) 4-Methoxyphenyl 3-0-(4-methoxytriphenylmethyl)-2-0-succinylpropyl ether Synthesis took place in analogy to Example le from 4-methoxyphenyl 3-0-(4-methoxytriphenylmethyl)-2-hydroxy-propyl ether (Example 3c).

Yield: 98%;
1H-NMR (200 MHz, CDC13/TMS): d = 2.63 (s, 4H, CO-(CH2)2-CO); 3.31-3.40 (m, 2H, CH2-OMMTr); 3.76 (s, 3H, O-CH3) ; 3.79 (s, 3H, O-CH3) ; 4.04-4.10 (m, 2H, CH2-0-MOP) ;
5.35 (dt, 1H, CH-Osucc); 6.79 (a, 4H, Ar-H); 7.15-7.47 (m, 14H, Ar-H);
MS (FAB + LiCl) : m/e = 583.3 (M + 2Li+-H+, 40%) ; 577.3 (M + Li+, 100%).

4) Synthesis of 6-0-(4-methoxytriphenylmethyl)-5-0-succinylhexyl levulinate 4a) 4-(2,2-Dimethyl-1,3-dioxolan-4-yl)butyl levulinate 0.81 g (5 mmol) of 2,2-dimethyl-4-hydroxybutyl-1,3-di-oxolane from la were coevaporated twice with absolute acetonitrile, then dissolved together with 1.5 g (7 mmol) of levulinic anhydride and 0.86 g (7 mmol) of dimethyl-aminopyridine (DMAP) in absolute pyridine and stirred at room temperature for 15 h. The solvent was evaporated off in vacuo, and then three coevaporations with toluene were carried out. The residue was taken up in EA, and the organic phase was washed with saturated NaCl solution and with water and then dried over sodium sulfate. The solvent was evaporated off, and the residue was chromato-graphed on silica gel using EA.

Yield: 0.65 g (48%);
1H-NMR (200 MHz, CDC13/TMS) : d = 1.37 (s, 3H, CH3) ; 1.41 (a, 3H, CH3); 1.42-1.75 (m, 6H, -(CH2)3-); 2.19 (s, 3H, CH3-CO); 2.49-2.82 (m, 4H, COCH2CH2CO); 3.45-3.58 (m, 1H, C4'-H) ; 3.97-4.16 (m, 4H, -C5' H2- & CH2-OCO) ;
MS (EI): m/e = 273 (M + H+, 45%); 257 (35%);
4b) 5,6-Dihydroxyhexyl levulinate Synthesis took place in analogy to Example is from 4-(2,2-dimethyl-1,3-dioxolan-4-yl)butyl levulinate (Example 4a).

Yield: 90%;
1H-NMR (200 MHz, CDC13/TMS): d = 1.37-1.75 ((m, 6H, -(CH2)3-); 2.20 (s, 3H, CH3-CO); 2.47-2.82 (m, 4H, COCH2CH2CO); 3.39-3.52 (dd, 1H, CH-OH); 3.60-3.79 (m, 2H, CH2-OH); 4.11 (t, 2H, CH2-OLev);
MS (El): m/e = 233 (M + H+, 20%); 215 (15%);

4c) 6-0-(4-Methoxytriphenylmethyl)-5-hydroxyhexyl levulinate Synthesis took place in analogy to Example ld from 5,6-dihydroxyhexyl levulinate (Example 4b).

Yield: 40%;
1H-NMR (200 MHz, CDC13/TMS): d = 1.22-1.70 ((m, 6H, -(CH2)3-); 2.19 (s, 3H, CH3-CO); 2.48-2.79 (m, 4H, COCH2CH2CO); 2.97-3.21 (m, 2H, CH2-OMMTr); 3.79 (a, 3H, OCH3); 3.68-3.82 (m, 1H, CH-OH); 4.03 (t, 2H, CH2-OLev);
6.80-7.48 (m, Ar-H, 14H);
MS (ES+ + LiCl): m/e = 511 (M + Li+, 100%);

4d) 6-0-(4-methoxytriphenylmethyl)-5-O-succinylhexyl levulinate Synthesis took place in analogy to Example le from 6-0- (4-methoxytriphenylmethyl) -5-hydroxyhexyl levulinate (Example 4c).

Yield: 80%;
1H-NMR (200 MHz, CDC13/TMS): d = 1.20-1.72 (m, 6H, -(CH2)3-); 2.19 (s, 3H, CH3-CO); 2.49-2.80 (m, 8H, 2 x COCH2CH2CO); 3.15 (d, 2H, CH2-OMMTr) ; 3.79 (s, 3H, OCH3); 4.03 (t, 2H, CH2-OLev); 5.15 (m, 1H, CH-OSucc);
6.79-7.50 (m, Ar-H, 14H);
MS (ES+ + LiCl): m/e = 627 (M + Na+, 20%); 611 (M + Li+, 50%).

5) Preparation of a support of the formula VIII-1 by loading aminopropyl-CPG with 4-methoxyphenyl 6-0- (4-methoxytriphenylmethyl) -5-0-succinylhexyl ether 123 mg (20 mmol) of 4-methoxyphenyl 6-0-(4-methoxytri-phenylmethyl)-5-0-succinylhexyl ether (from Example 1) were coevaporated twice with absolute acetonitrile and then dissolved together with 7.1 mg (22 mmol) of 0-(1-benzotriazolyl)-N,N,N',N'-tetramethyluronium tetra-fluoroborate (TBTU) and 3.2 mg (28 mmol) of N-ethylmor-pholine in 0.75 ml of absolute dimethylformamide (DMF).
100 mg of aminopropyl-CPG (0.1 mmol/g, 550A) supplied by Fluka were added to this solution, and the suspension was shaken at room temperature for 7 h. The derivatized support was filtered off with suction, washed with methanol, DMF, THF, acetonitrile, again with methanol and with methylene chloride and dried at 40 C in vacuo for 1 h. The loading of the support with monomethoxytrityl-containing component was 12.2 mmol/g. Reactive groups are capped in a DNA synthesizer using capping reagent (acetic anhydride/2,6-lutidine/1-methylimidazole; 0.25 M each in THF), followed by washing with acetonitrile.

6) Preparation of a support of the formula VIII-2 by loading aminopropyl-CPG with 4-methoxyphenyl (2,2-dimethyl-1,3-dioxolan-4-yl)methyl ether Preparation in analogy to Example 5 using 4-methoxyphenyl (2,2-dimethyl-1,3-dioxolan-4-yl)methyl ether (from Example 3). The loading of the support with monomethoxy-trityl-containing component was 36.7 amnol/g.

7) Preparation of a support of the formula VIII-3 by loading aminopropyl-CPG with 6-0-(4-methoxyphenyl-methyl)-5-0-succinylhexyl levulinate Preparation in analogy to Example 5 using 6-0- (4-methoxy-triphenylmethyl)-5-0-succinylhexyl levulinate (from Example 4). The loading of the support with monomethoxy-trityl-containing component was 14.3 mmol/g.

8) Preparation of a support of the formula VIII-4 by loading Tentagel with 4-methoxyphenyl 6-0-(4-meth-oxytriphenylmethyl)-5-0-succinylhexyl ether 306 mg (0.5 mmol) of 4-methoxyphenyl 6-O-(4-methoxytri-phenylmethyl)-5-0-succinylhexyl ether (from Example 1) were coevaporated twice with absolute acetonitrile and dissolved in a mixture of 1.25 ml of absolute THE and 65 ml of absolute pyridine. Then a. solution of 70 mg (0.5 mmol) of 4-nitrophenol and 115 mg (0.55 mmol) of dicyclohexylcarbodiimide (DCC) in 0.35 ml of absolute THE
was added, and the mixture was stirred at room temper-ature for 2 h. After the reaction was complete, the precipitated dicyclohexylurea was removed by centrifugation. The sediment was resuspended in 1 ml of ether and again centrifuged. 200 mg of Tentagel resin (PS/POE copolymer with 175 mmol/g amino functionality) were suspended in a mixture of 0.7 ml of absolute DMF and 0.14 ml of TEA, and the 4-nitrophenyl succinate solution obtained above was added, and the mixture was shaken at room temperature for 17 h. Filtration with suction was followed by working up as described in Example 5. The loading of the support with monomethoxytrityl-containing component was 28.7 mmol/g.

Oligonucleotide synthesis: the oligonucleotides are initially purified by butanol precipitation (Sawadogo, Van Dyke, Nucl. Acids Res. 19 (1991) 674). The sodium salt is then obtained by precipitation from a 0.5 M NaCl solution with 2.5 parts by volume of ethanol.

The oligonucleotides are analyzed by a) analytical gel electrophoresis in 20% acrylamide, 8 M urea, 454 M trio-borate buffer, pH 7.0 and/or b) HPLC analysis: Waters GenPak FAX, gradient CH3CN
(400 ml) H2O (1.6 1), NaH2PO4 (3.1 g), NaCl (11.7 g) pH 6.8 (0.1 M in NaCl) to CH3CN (400 ml). H2O
(1.6 1), NaH2PO4 (3.1g), NaCl (175.3 g), pH 6.8 (1.5 M in NaCl) and/or c) capillary gel electrophoresis, Beckmann eCAPTM
capillary, U100P gel. column, 65 cm length, 100 mm I.D., window 15 cm from one end, buffer 140 AM trio, 360 mM boric acid, 7 M urea and/or d) electrospray mass spectroscopy.

9) Preparation of oligonucleotides of the formula I:
TpTpTpTpTpTpTpTp-CH2-CH(OH)CH2)4-(O-methoxyphenyl) The monomer is in each case a P-D-deoxyribonucleoside;
R1 = R2 = H; Z = O-(4-methoxyphenyl); n = 8, m = m' = n' = b = 0; A = V = W = U = X = Y = T = oxy;
a = 3;

a) 0.2 mol of the support VIII-4 from Example 8 is treated successively with the following reagents:

1. absolute acetonitrile 2. 3% trichloroacetic acid in dichioromethane 3. absolute acetonitrile 4. 4 pmol of 5'-O-dimethoxytritylthymidine-3'-phos-phorous acid P-cyanoethyl ester diisopropylamide and 25 mol of tetrazole in 0.15 ml of absolute acetonitrile 5. acetonitrile 6. 20% acetic anhydride in THE with 40% lutidine and 10% dime thylaminopyridine 7. acetonitrile 8. iodine (0.1 M 12 in THE/water/pyridine; 70:20:5 =
v:v:v) Steps 1 to 8, called one reaction cycle hereinafter, are repeated seven times to assemble the octathymidylate derivative.

b) After the synthesis is complete, the dimethoxytrityl group is eliminated as described in steps 1 to 3.

c) Treatment with ammonia for 1.5 hours cleaves the oligonucleotide off the support and simultaneously eliminates the P-cyanoethyl groups. Since the oligo-nucleotide contains no amino protective groups, no further ammonia treatment is necessary.

10) Preparation of oligonucleotides of the formula I:
TpTpTpTpTpTpTpTp-CH2-CH(OH)CH2)4-(OH) The monomer is in each case a P-D-deoxyribonucleoside;
R1 = R2 = H; Z = OH; n = 8, m = m' = n' = b = 0;
A V = W = U = X = Y= T = Oxy; a = 3;

a) Preparation takes place in analogy to Example 9a;
b) After the synthesis is complete, the dimethoxytrityl group (DMTr group) is eliminated as described in steps 1 to 3. Subsequently the 4-methoxyphenyl group (MOP group) is eliminated by treatment with 0.1 M Celv(NH4)2(NO3)6 in acetonitrile/H20 4:1 at room temperature for 5 min.

c) Treatment with ammonia for 1.5 hours cleaves the oligonucleotide off the support and simultaneously eliminates the P-cyanoethyl groups.

11) Preparation of oligonucleotides of the formula I:
TpTpTpTpTpTpTpTp-CH2-CH(OH) CH2) 4- (O- (CH2) 4-pyrene) starting from support VIII-4 The monomer is in each case a P-D-deoxyribonucleoside;
R1 = R2 = H; Z = O-(-(CH2)4-pyrene); n = 8, m = m' = n' = b = 0; A = V = W = U = X = Y = T = Oxy;
a = 3;

a) Preparation takes place in analogy to Example 9a;
b) After the synthesis is complete, the dimethoxytrityl group is eliminated as described in steps 1 to 3.
Subsequently the resulting free 5'-hydroxyl group is capped as described in steps 6 and 7. The 4-methoxyphenyl group is subsequently eliminated by treatment with 0.1 M
Ce1V (NH4) 2 (NO3) 6 in acetonitrile/H20 4:1 at room temperature for 5 min.

c) Introduction of the 4-(1-pyrenyl)butyl phosphodi-ester at the 5' end takes place as described in J.S. Mann et al. Bioconj. Chem. 3 (1992) 554 by treatment with 4 mol of 4-(1-pyrenyl)butyl 2-cyanoethyl N,N-diisopro-pylphosphoramidite and 25 mol of methylthio-lH-tetrazole in 0.15 ml of absolute acetonitrile and subsequent washing with acetonitrile.

d) Oxidation with 0.1 M 12 in THE/water/pyridine;
70:20:5 = v:v:v.

e) Treatment with ammonia for 1.5 hours cleaves the oligonucleotide off the support and simultaneously eliminates the P-cyanoethyl groups.

12) Preparation of oligonucleotides of the formula I:

TpTpTpTpTpTpTpTp-CH2-CH(OH)CH2)4-(0-(CH2)4-pyrene) starting from support VIII-1 The monomer is in each case a j3-D-deoxyribonucleoside;
R1 = R2 = H; Z = 0- (- (CH2) 4-pyrene) ; n = 8, m=m' = n' = b = 0; A = V = W = U = X = Y = T = Oxy;
a = 3;

Preparation takes place in analogy to Example 9a but using support VIII-1.

13) Preparation of oligonucleotides of the formula I:
TpTpTpTpTpTpTpTp-CH2-CH (OH) CH2) 4- (0- (CH2) 11CH3 ) starting from support VIII-4 The monomer is in each case a Q-D-deoxyribonucleoside;
R1 = R2 = H; Z = 0 - (CH2) 11CH3) ; n = 8, m = m' = n' = b = 0 ; A = V = W = U = X = Y = T = Oxy;
a = 3;

a) Preparation takes place in analogy to Example 9a;
b) Elimination of the DMTr group, capping and elimina-tion of the MOP group as described in Example llb;

c) Treatment with 4 mol of dodecyl 2-cyanoethyl N,N-diisopropylphosphoramidite and 25 mol of methylthio-1H-tetrazole in 0.15 ml of absolute acetonitrile and subsequent washing with acetonitrile.

d) Oxidation with 0.1 M 12 in THE/water/pyridine;
70:20:5 = v:v:v.

e) Treatment with ammonia for 1.5 hours cleaves the oligonucleotide off the support and simultaneously eliminates the P-cyanoethyl groups.

14) Preparation of oligonucleotides of the formula I:
TpTpTpTpTpTpTpTp-CH2-CH(OH) CH2) 4- (0- (CH2) 13CH3) starting from support VIII-1 The monomer is in each case a 16-D-deoxyribonucleoside;
R1 = R2 = H; Z = 0- (CH2) 13CH3) ; n = 8, m = m' = n' = b = 0 ; A = V = W = U = X = Y = T = oxy;
a = 3;

a) Preparation takes place in analogy to Example 9a;
b) Elimination of the DMTr group, capping and elimina-tion of the MOP group as described in Example lib;

c) Treatment with 4 mol of tetradecyl 2-cyanoethyl N,N-diisopropylphosphoramidite and 25 mol of methylthio-1H-tetrazole in 0.15 ml of absolute acetonitrile and subsequent washing with acetonitrile.

d) Oxidation with 0.1 M 12 in THE/water/pyridine;
70:20:5 = v:v:v.

e) Treatment with ammonia for 1.5 hours cleaves the oligonucleotide off the support and simultaneously eliminates the P-cyanoethyl groups.

15) Preparation of oligonucleotides of the formula I:
TpTpTpTpTpTpTpTp-CH2-CH(OH)CH2)-(O-3'-T-ODMtr) starting from support VIII-2 The monomer is in each case a P-D-deoxyribonucleoside;
Ri = R2 = H; Z = O-3'-T-ODMTr; n = 8, m = m' = n' = b = 0; A = V = W = U = X = Y = T = oxy;
a = 0;

a) Preparation takes place in analogy to Example 9a;
b) Elimination of the DMTr group, capping and elimina-tion of the MOP group as described in Example lib;

c) Treatment with 4 FLmol of 5'-O-dimethoxytritylthymi-dine-3'-phosphorous acid P-cyanoethyl ester diisopropyl-amide and 25 mol of methylthio-lH-tetrazole in 0.15 ml of absolute acetonitrile and subsequent washing with acetonitrile.

d) Oxidation with 0.1 M 12 in THE/water/pyridine;
70:20:5 = v:v:v.

e) Treatment with ammonia for 1.5 hours cleaves the oligonucleotide off the support and simultaneously eliminates the P-cyanoethyl groups.

16) Preparation of oligonucleotides of the formula I:
TpTpTpTpTpTpTpTp- (CH2) 4-CH (OH) CH2) - (0- (CH2) 13CH3) starting from support VIII-4 The monomer is in each case a P-D-deoxyribonucleoside;
R1 = R2 = H; Z = 0-(4-methoxyphenyl); n = 8, m=m' = n' = a = 0; A = V = W = U = X = Y = T = oxy;
b = 3;

a) 0.2 mol of support VIII-4 from Example 8 are treated successively with the following reagents:

1. absolute acetonitrile 2. 3% trichloroacetic acid in dichioromethane 3. absolute acetonitrile 4. 4 mol of tetradecyl 2-cyanoethyl N,N-diiso-propylphosphoramidite and 25 mol of methyl-thio-1H-tetrazole in 0.15 ml of absolute acetonitrile 5. acetonitrile 6. 0.1 M CeI (NH4) 2 (NO3) 6 in acetonitrile/H2O 4:1 at room temperature for 5 min 7. acetonitrile S. 4 mol of 5'-O-dimethoxytritylthymidine-3'-phosphorous acid P-cyanoethyl ester diiso-propylamide and 25 mol of tetrazole in 0.15 ml of absolute acetonitrile 9. acetonitrile 10. 20% acetic anhydride in THE with 40% lutidine and 10% dimethylaminopyridine 11. acetonitrile 12. iodine (1.3 g in THE/water/pyridine; 70:20:5 =
v:v:v) 13. acetonitrile 14. 3% trichloroacetic acid in dichloromethane.
Steps 7-14, called one reaction cycle hereinafter, are repeated 7 times to assemble the octathymidylate derivative.

b) Treatment with ammonia at 60 C for 12 hours cleaves the oligonucleotide off the support and simultaneously eliminates the P-cyanoethyl groups.
17) Preparation of oligonucleotides of the formula I:
GpGpApCpCpGpApApGpGp- (CH2) 4-CH (OH) -CH2- (O- (CH2) 13CH3) starting from support VIII-4 The monomer is in each case a P-D-deoxyribonucleoside;
R1 = R2 = H; Z = O-(4-methoxyphenyl); n = 10, m = m' = n' = a = 0; A = V = W = U = X = Y = T = oxy;
b = 3;

Synthesis takes place in analogy to Example 16 but the relevant 3'-phosphorous acid P-cyanoethyl ester diiso-propylamide of the appropriate base is used in step 8.
18) Preparation of oligonucleotides of the formula I:
TpTpTpTpTpTpTpTp - (CH2) 4 - CH (OH) CH2) - (O - (CH2) 13 CH3 ) starting from support VIII-3 The monomer is in each case a P-D-deoxyribonucleoside;
R1 = R2 = H; Z = O- (4-methoxyphenyl) ; n = 8, m m' = n' = a = 0; A = V = W = U = X = Y = T = oxy;
b = 3;

Synthesis takes place in analogy to Example 16 but step 6 is replaced by treatment with 0.5 M hydrazine hydrate in acetic acid/pyridine 2:3 for 30 min.
19) Preparation of oligonucleotides of the formula I:.
TpTPTPTPTPTPTpTP-CH2-CH(OH)CH2)4-(O-acridin) start-ing from support VIII-4 The monomer is in each case a P-D-deoxyribonucleoside;
R1 = R2 = H; Z = acridin; where acridin is 6-(2-methoxy-6-chloro-9-acridinylamino)-2-hydroxymethyihexoxy; n = 8, m = m' = n' = b = 0; A = V = W = U = X = Y = T = oxy;
a = 3;

a) Preparation takes place in analogy to Example 9a;
b) Elimination of the DMTr group, capping and elimina-tion of the MOP group as described in lib;

c) Treatment with 4 mol of 6-(2-methoxy-6-chloro-9-acridinylamino)-2-dimethoxytrityloxymethyl-l-(2-cyano-ethoxy-N,N-diisopropylaminophosphino)hexane (from Glen Research) and 25 mol of methylthio-lH-tetrazole in 0.15 ml of absolute acetonitrile and subsequent washing with acetonitrile.

d) Oxidation with 0.1 M 12 in THE/water/pyridine;
70:20:5 = v:v:v and washing with acetonitrile e) Elimination of the DMTr group f) Treatment with ammonia for 1.5 hours cleaves the oligonucleotide off the support and simultaneously eliminates the P-cyanoethyl groups.
20) Preparation of oligonucleotides of the formula I:
TpTpTpTpTpTpTpTp-CH2-CH(OR)CH2)4-(0-biotin) starting from support VIII-4 The monomer is in each case a P-D-deoxyribonucleoside;
R1 = R2 = H; Z = biotin; where biotin is 6-biotinamido-5-hydroxymethylhexoxy; n = 8, m = m' = n' = b = 0;
A = V = W= U= X = Y = T oxy; a = 3;

a) Preparation takes place in analogy to Example 9a;
b) Elimination of the DMTr group, capping and elimina-tion of the MOP group as described in lib;

c) Treatment with 4 mol of 6-biotinamido-5-dimethoxy-trityloxymethylhexyl 2-cyanoethyl N, N-diisopropylphos-phoramidite (from Glen Research) and 25 mol of methyl-thio-1H-tetrazole in 0.15 ml of absolute acetonitrile and subsequent washing with acetonitrile.

d) Oxidation with 0.1 M 12 in THE/water/pyridine;
70:20:5 = v:v:v and washing with acetonitrile e) Elimination of the DMTr group f) Treatment with ammonia for 1.5 hours cleaves the oligonucleotide off the support and simultaneously eliminates the 5-cyanoethyl groups.
21) Preparation of oligonucleotides of the formula I:
TpTpTpTpTpTpTpTp-CH2-CH(OH)CH2)4-(O-TEGBiotin) starting from support VIII-4 The monomer is in each case a Q-D-deoxyribonucleoside;
R1 = R2 = H; Z = TEGBiotin; where TEGBiotin is 16-biotin-amido-4,7,10,13-tetraoxy-l-hydroxy-2-hexadecoxy; n = 8, m = m' = n' = b = 0; A = V = W = U = X = Y = T = oxy;
a = 3;

a) Preparation takes place in analogy to Example 9a;
b) Elimination of the DMTr group, capping and elimina-tion of the MOP group as described in lib;

c) Treatment with 4 mol of 16-biotinamido-4,7,10,13-tetraoxy-1-dimethyltrityloxy-2-hexadecyl 2-cyanoethyl N,N-diisopropylphosphoramidite (from Glen Research) and 25 mol of methylthio-1H-tetrazole in 0.15 ml of absolute acetonitrile and subsequent washing with acetonitrile.
d) Oxidation with 0.1 M 12 in THE/water/pyridine;
70:20:5 = v:v:v and washing with acetonitrile e) Elimination of the DMTr group f) Treatment with ammonia for 1.5 hours cleaves the oligonucleotide off the support and simultaneously eliminates the P-cyanoethyl groups.
22) Preparation of oligonucleotides of the formula I:
TpTpTpTpTpTpTpTp-CE2-CH(OH)CH2)4-(O-cholesterol) starting from support VIII-4 The monomer is in each case a P-D-deoxyribonucleoside;
R1 = R2 = H; Z = cholesterol; where cholesterol is 16-cholesterylamino-4,7,10,13-tetraoxy-l-hydroxy-2-hexa-decoxy; n = 8, m = m' = n' = b = 0;
A= V= W= U= X= Y= T- oxy; a= 3;

a) Preparation takes place in analogy to Example 9a;
b) Elimination of the DMTr group, capping and elimina-tion of the MOP group as described in lib;

c) Treatment with 4 gmol of 16-cholesterylamino-4,7,10,13-tetraoxy-l-dimethoxytrityloxy-2-hexadecyl 2-cyanoethyl N,N-diisopropylphosphoramidite (from Glen Research) and 25 mol of methylthio-1H-tetrazole in 0.15 ml of absolute acetonitrile and subsequent washing with acetonitrile.

d) Oxidation with 0.1 M 12 in THE/water/pyridine;
70:20:5 = v:v:v and washing with acetonitrile CA 02628883 2010-05-05 QQtt'y~ J

e) Elimination of the DMTr group f) Treatment with ammonia for 1.5 hours cleaves the oligonucleotide off the support and simultaneously eliminates the fl-cyanoethyl groups.
23) Preparation of oligonucleotides of the formula I:
TpTpTpTpTpTpTpTp-CH2-CH(OH)CH2)4-(O-psoralen)start-ing from support VIII-4 The monomer is in each case a P-D-deoxyribonucleoside;
R1 = R2 = H; Z = psoralen; where psoralen is 2-[4-'(hy-droxymethyl)-4,5',8-trimethylpsoralen]ethyl; n = 8;
m m' = n' = b = 0; A = V = W = U = X = Y = T = oxy;
a = 3;

a) Preparation takes place in analogy to Example 9a;
b) Elimination of the DMTr group, capping and elimina-tion of the MOP group as described in lib;

c) Treatment with 4 mol of 2-[4-'(hydroxymethyl)-4,5', 8-trimethylpsoralen]ethyl 2-cyanoethyl N,N-diiso-propylphosphoramidite (from Glen Research) and 25 mol of methylthio-lH-tetrazole in 0.15 ml of absolute aceto-nitrile and subsequent washing with acetonitrile.

d) Oxidation with 0.1 M 12 in THE/water/pyridine;
70:20:5 = v:v:v and washing with acetonitrile e) Treatment with ammonia for 1.5 hours cleaves the oligonucleotide off the support and simultaneously eliminates the P-cyanoethyl groups.
24) Preparation of oligonucleotides of the formula I:
TpTpTpTpTpTpTpTp-CH2-CH (OH) CH2) - (O- (CH2)13CH3) start-ing from support VIII-2 The monomer is in each case a )4-D-deoxyribonucleoside;

R1 = R2 = H; Z = O- (CH2)13CH3; n = 8, m = m' = n' = b = 0;
A = V = W = U = X = Y = T = oxy; a = 0;

a) 0.2 mol of support VIII-2 from Example 6 are treated successively with:

1. absolute acetonitrile 2. 3% trichloroacetic acid in dichloromethane 3. absolute acetonitrile 4. 4 mol of tetradecyl 2-cyanoethyl N,N-diiso-propylphosphoramidite and 25 mol of methyl-thin-1H-tetrazole in 0.15 ml of absolute acetonitrile 5. acetonitrile 6. 20% acetic anhydride in THE with 40% lutidine and 10% dimethylaminopyridine 7. acetonitrile 8. iodine (0.1 M I2 in THE/water/pyridine; 70:20:5 v:v:v) 9. 0.1 M Celv(NH4) 2 (NO3) 6 in acetonitrile/H2O 4:1 (see also Example 11b).
10. acetonitrile b) and subsequently treated with 1. 4 mol of 5'-O-dimethoxytritylthymidine-3-phos-phorous acid p-cyanoethyl ester diisopropylamide and 25 mol of tetrazole in 0.15 ml of absolute acetonitrile 2. acetonitrile 3. 20% acetic anhydride in THE with 40% lutiine and 10% dimethylaminopyridine 4. acetonitrile S. iodine (0.1 M 12 in THE/water/pyridine; 70:20:5 v:v:v) 6. absolute acetonitrile 7. 3% trichloroacetic acid in dichloromethane 8. absolute acetonitrile.

Steps 1 to 8, hereinafter called one reaction cycle, are repeated 7 times to assemble the octathymidylate derivative.

c) Treatment with ammonia for 1.5 hours cleaves the oligonucleotide off the support and simultaneously eliminates the P-cyanoethyl groups. Since the oligo-nucleotide contains no amino protective groups, no further ammonia treatment is necessary.
25) Preparation of oligonucleotides of the formula I:
CpApCpGpTpTpGpApGpGpGpGpCpApTp-CH2-CH(OH)(CH2)-(0- (CH2) 13CH3) starting from support VIII-2 The monomer is in each case a P-D-deoxyribonucleoside;
R1 = R2 = H; Z = O-(CH2)13CH3; n = 15, m = m' = n' = b = 0; A = V = W = U = X = Y = T = oxy;
a = 0;

Synthesis in analogy to Example 24 but using the appro-priate standard 5'-O-dimethoxytritylthymidine-protected 3'-(2-cyanoethyl)-N,N-diisopropylphosphoramidite nucleo-sides in step bl. Treatment with ammonia for 1.5 hours cleaves the oligonucleotide off the support, and depro-tection took place by treatment with ammonia at 60 C for 16 h.
26) Preparation of oligonucleotides of the formula I:
CpApCpGpTpTpGpApGpGpGpGpCpApTp-CH2-CH(OH)(CH2)-(0-vitamin E) starting from support VIII-2 The monomer is in each case a P-D-deoxyribonucleoside;
R1 = R2 = H; Z = O-vitamin E; n = 15, m=m' = n' =b= 0; A=V=W=U=X=Y=T=oxy;
a = 0;

Synthesis in analogy to Example 24 but using the vitamin E 2-cyanoethyl N,N-diisopropylphosphoramidite in step a4.
27) Synthesis of3-O-(4-methoxytriphenylmethyl)-2-0-suc-cinylpropyl levulinate 27a) (2,2-Dimethyl-l,3-dioxolan-4-yl)methyl levulinate Synthesis in analogy to Example 4a from 2,2-dimethyl-4-hydroxymethyl-1,3-dioxolane.
Yield: 71%.

1H-NMR (200 MHz, CDC13/TMS): 6 = 1.38 (s, 3H, CH3); 1.42 (s, 3H, CH3); 2.19 (s, 3H, CH3-CO); 2.51-2.82 (m, 4H, COCH2CH2CO); 3.75 (dd, 1H, C4'-H); 4.01-4.39 (m, 4H, -C51H2- & CH2-OCO);

27b) 2,3-Dihydroxypropyl levulinate Synthesis in analogy to lc from (2,2-dimethyl-1,3-dioxo-lan-4-yl)methyl levulinate (27a);
Yield: 90%;

1H-NMR (200 MHz, CDC13/TMS) : 6 = 2.20 (a, 3H, CH3-CO) ;
2.60, 2.80 (each t, 4H, COCH2CH2CO); 3.54-3.80 (m, 2H, CH2-OH); 3.80 (t, 1H, OH); 3.95 (m, 1H, CH-OH); 4.21 (d, 2H, CH2-OLev);

27c) 3-0-(4-Methoxytriphenylmethyl)-2-hydroxypropyl levulinate Synthesis in analogy to ld from 2,3-dihydroxypropyl levulinate (24b);
Yield: 20%;

27d) 3-0-(4-Methoxytriphenylmethyl)-2-0-succinylpropyl levulinate Synthesis in analogy to 1e from 3-0-(4-methoxytriphenyl-methyl)-2-hydroxypropyl levulinate (27c);
Yield: 51%;

MS (FAB/LiCl) : m/e = 599.3 (M + Li+);
28) Preparation of a support of the formula VIII-5 by loading aminopropyl-CPG with 3-0-(4-methoxytri-phenylmethyl)-2-0-succinylpropyl levulinate Preparation in analogy to Example 5 using 3-0-(4-methoxy-triphenylmethyl)-2-0-succinylpropyl levulinate (from Example 27). The loading of the support with monomethoxy-trityl-containing component was 24.7 mol/g.

Claims (17)

1. A compound of the formula II:

and the physiologically tolerated salts thereof, in which a is an integer from zero to 20;

b is an integer from zero to 20;

R1 is hydrogen, C1-C18-alkyl, C2-C18-alkenyl, C3-C18-alkynyl, C1-C18-alkylcarbonyl, C2-C19-alkenylcarbonyl, C3-C19-alkynylcarbonyl, C6-C20-aryl, C6-C14-aryl-C1-C8-alkyl, or a radical of the formula III

R2 is hydrogen, hydroxyl, C1-C18-alkoxy, halogen, azido or NH2;

D is hydroxyl or O-PO3 2-;

B is a natural or unnatural base customary in nucleotide chemistry wherein the natural base is adenine, cystosine, guanine, uracil or thymine, and the unnatural base is purine, 2,6-diaminopurine, 7-deazaadenine, 7-deazaguanine, N4, N4-ethanocytosine, N6, N6-ethano-2, 6-diaminopurine, psuedoisocytosine, 5-propinuracil, 5-propincytosine, 5-fluorocytosine, 5-fluorouracil, 5-hydroxymethyluracil, or 5-bromocytosine;

n is an integer from 7 to 25;

n' is an integer from zero to 50;
m' in is an integer from 1 to 5;
A is oxy, thioxy or methylene;

W is oxo, thioxo or selenoxo;
V is oxo or thio;

T is oxy, thio or imino;

Y is oxy, thio, imino or methylene;
X is hydroxyl or mercapto;

U is hydroxyl, mercapto, BH3r SeH, C1-C18-alkoxy, C1-C18-alkyl, C6-C20-aryl, C6-C14-aryl-C1-C8-alkyl, NHR3, NR3R4 or a radical of the formula IV

(OCH2CH2)p O(CH2)q CH2R5 (IV) in which R3 is C1-C18-alkyl, C6-C20-aryl, C6-C14-aryl-C1-C8-alkyl or -(CH2)c-[NH(CH2)c]d-NR6R6, in which c is an integer from 2 to 6 and d is an integer from zero to 6, and R6 is independently of one another, hydrogen, C1-C6-alkyl or C1-C4-alkoxy-C1-C6-alkyl;

R4 is C1-C18-alkyl, C6-C20-aryl or C6-C10-aryl-C1-C8-alkyl, or, in the case of NR3R4, together with R3 and the nitrogen atom carrying them is a 5-6-membered heterocyclic ring which can additionally contain another heteroatom selected from the series consisting of O, S
and N;

p is an integer from 1 to 100;
q is an integer from zero to 22;

R5 is hydrogen, hydroxyl, amino, NHR7, COOH, CONH2, COOR8 or halogen, in which R7 is C1-C6-alkyl and R8 is C1-C4-alkyl;

Z, Z' are, independently of one another, hydroxyl;
mercapto; SeH; C1-C22-alkoxy; -O-(CH2)b'-NR7R8, in which b' is an integer from 1 to 6, and R7 is C1-C6-alkyl and R8 is C1-C4-alkyl, or R7 and R8 form, together with the nitrogen atom carrying them, a 3-6-membered ring; C1-C18-alkyl; C6-C20-aryl, C6-C14-aryl-C1-C8-alkyl, C6-C14-aryl-C1-C8-alkoxy, C6-C20-heteroaryl, C6-C14-heteroaryl-C1-C8-alkyl, or C6-C14-heteroaryl-C1-C8-alkoxy in which aryl and heteroaryl are unsubstituted or substituted by 1, 2 or 3 identical or different radicals selected from the series consisting of carboxyl, amino, nitro, C1-C4-alkylamino, C1-C6-alkoxy, hydroxyl, halogen and cyano; C1-C18-alkylmercapto; NHR3 or NR3R4 in which R3 and R 4 are as defined above; or a group a) which favours intracellular uptake selected from -O-(CH2)x-CH3, in which x is an integer from 8 to 18;
-O-(CH2)e-CH=CH-(CH2)f-CH3, in which e and f independently of one another are an integer from 6 to 12;
-O-(CH2CH2O)4-(CH2)9-CH3; -O-(CH2CH2O)8-(CH2)13-CH3; and -O-(CH2CH2O)7-(CH2)15-CH3; steroid residues and conjugates which utilize natural carrier systems; conjugates of mannose; and conjugates of peptides of the appropriate receptors which lead to receptor mediated endocytosis of the compounds of formula II wherein the conjugates which utilize natural carrier systems are bile acid, folic acid, 2-(N-alkyl-N-alkoxy)-aminoanthraquinone or conjugates of mannose, and wherein the conjugates of peptides of the appropriate receptors which lead to receptor mediated endocytosis of the compounds of formula II are epidermal growth factor (EGF), bradykinin or platelet derived growth factor (PDGF); or b)which acts as a labelling group selected from fluorescent groups; chemiluminescent groups; and linker groups having functional groups which permit subsequent derivatization with detectable reporter groups wherein the functional groups which permit subsequent derivitization with detectable reporter groups is an aminoalkyl linker, which is reacted to an acridinium active ester to give the chemiluminescent sample; or c)which on hybridization of the compound of formula II
onto a target nucleic acid, interacts with the target nucleic acid by binding, crosslinking or cleavage; or d) which is a nucleoside or oligonucleotide linked via the 5' or 3' end;

and the curved parenthesis indicates that R2 and the adjacent phosphoryl radical can be located in the 2' and 3' positions or else conversely in the 3' and 2' positions, each nucleotide is in its D or L configuration and the base B is located in the .alpha. or .beta. position.
2. The compound of the formula II as claimed in claim 1, wherein the natural base is adenine, cytosine, guanine, uracil or thymine, and the unnaturual base is 5-propinuracil or 5-propincytosine.
3. The compound of the formula II as claimed in claim 1 or claim 2, and the physiologically tolerated salts thereof, wherein the base B is located in the .beta. position, the nucleotides are in the D configuration, and R2 is located in position 2'.
4. The compound of the formula II as claimed in any one of claims 1 to 3, and the physiologically tolerated salts thereof, wherein a is an integer from zero to 10;
b is an integer from zero to 10;

R1 is hydrogen or a radical of the formula III
R2 is hydrogen or hydroxyl;

n' is an integer from zero to 30;
A is oxy;

W is oxo or thioxo;

U is hydroxyl, mercapto, C1-C6-alkoxy, C1-C6-alkyl, NR3R4 or NHR3, in which R3 is C1-C18-alkyl; and R4 is C1-C8-alkyl, C6-C20-aryl or C6-C10-aryl-C1-C8-alkyl, or, in the case of NR3R4, together with R3 and the nitrogen atom carrying them is a 5-6-membered heterocyclic ring which can additionally contain another heteroatom selected from the series consisting of O, S
and N.
5. The compound of the formula II as claimed in any one of claims 1 to 4, and the physiologically tolerated salts thereof, wherein a is an integer from zero to 4;
b is zero;

R1 is hydrogen;
R2 is hydrogen;
D is hydroxyl;

B is adenine, cytosine, guanine, uracil, thymine, 5-propinuracil or 5-propincytosine;

n' is an integer from zero to 25;
m' is 1;

T is oxy;

Y is oxy; and U is hydroxyl or C1-C6-alkyl.
6. The compound of the formula II as claimed in any one of claims 1 to 5, and the physiologically tolerated salts thereof, wherein Z, Z' are, independently of one another, hydroxyl;
mercapto; SeH; C1-C20-alkoxy; -O-(CH2)b'-NR7R8, in which b' is an integer from 1 to 6, and R7 is C1-C6-alkyl and R8 is C1-C4-alkyl, or R7 and R8 form, together with the nitrogen atom carrying them, a 3-6-membered ring; C1-C8-alkyl; C6-C20-aryl, C6-C10-aryl-C1-C4-alkyl, C6-C10-aryl-C1-C4-alkoxy, C6-C20-heteroaryl, C6-C10-heteroaryl-C1-C4-alkyl, or C6-C10-heteroaryl-C1-C4-alkoxy in which aryl and heteroaryl are unsubstituted or substituted by 1, 2 or 3 identical or different radicals selected from the series consisting of carboxyl, amino, nitro, C1-C4-alkylamino, C1-C6-alkoxy, hydroxyl, halogen and cyano; C1-C18-alkylmercapto; NHR3 or NR3R4 in which R3 and R4 are as defined in claim 1; or a group a) which favours intracellular uptake selected from -O-(CH2)x-CH3, in which x is an integer from 8 to 18;
-O-(CH2)e-CH=CH-(CH2)f-CH3, in which e and f independently of one another are an integer from 6 to 12;
-O-(CH2CH2O)4-(CH2)9-CH3; -O-(CH2CH2O)8-(CH)13-CH3; and -O-(CH2CH2O)7-(CH2)15-CH3; steroid residues and conjugates which utilize natural carrier systems; conjugates of mannose; and conjugates of peptides of the appropriate receptors which lead to receptor mediated endocytosis of the compounds of formula II wherein the conjugates which utilize natural carrier systems are bile acid, folic acid, 2-(N-alkyl-N-alkoxy)-aminoanthraquinone or conjugates of mannose, and wherein the conjugates of peptides of the appropriate receptors which lead to receptor mediated endocytosis of the compounds of fomrula II are epidermal growth factor (EGF), bradykinin or platelet derived growth factor (PDGF); or b)which acts as a labelling group selected from fluorescent groups; chemiluminescent groups; and linker groups having functional groups which permit subsequent derivatization with detectable reporter groups wherein the functional groups which permit subsequent derivitization with detectable reporter groups is an aminoalkyl linker, which is reacted to an acridinium active ester to give the chemiluminescent sample; or c)which on hybridization of the compound of formula II
onto a target nucleic acid, interacts with the target nucleic acid by binding, crosslinking or cleavage; or d) which is a nucleoside or oligonucleotide linked via the 5' or 3' end.
7. The compound of the formula II as claimed in any one of claims 1 to 6, and the physiologically tolerated salts thereof, wherein the steroid residue is cholesterol.
8. The compound of the formula II as claimed in any one of claims 1 to 6, and the physiologically tolerated salts thereof, wherein the conjugates which utilize natural carrier systems are selected from bile acid, folic acid and 2-(N-alkyl-N-alkoxy)aminoanthraquinone.
9. The compound of the formula II as claimed in any one of claims 1 to 6, and the physiologically tolerated salts thereof, wherein the peptides of the appropriate receptors which lead to receptor-mediated endocyctosis of the compounds of formula II are selected from epidermal growth factor, bradykinin and platelet derived growth factor.
10. A compound of the formula II as claimed in any one of claims 1 to 6, and the physiologically tolerated salts thereof, wherein the fluorescent groups are selected from N-dimethyl-l-aminonaphthyl-5-sulfonyl (dansyl), fluorescein and coumarin derviatives.
11. A compound of the formula II as claimed in any one of claims 1 to 6, and the physiologically tolerated salts thereof, wherein the chemiluminescent groups are selected from acridine derivatives, the digitoxin system and the biotin/avidin system.
12. A process for the preparation of compounds of the formula II as claimed in any one of claims 1-11, which comprises a) eliminating from a compound of the formula XVI
in which B' is B, R2' is R2, S2 is a suitable protective group, SS
is a solid support, Li is a linker arm, and A, B, R2, Y and V are as defined in claim 1 and Li is absent or is a linker which permits introduction of a 3' phosphate residue, the protective group S2;

b) subsequently reacting the resulting compound by the phosphoramidate method with a nucleoside phosphoramidite of the formula XI

in which A, B, R2', S2, Y and V are as defined in step a) , and B
is in unprotected or protected form, R9 and R10 are identical or different and are C1-C8-alkyl or C5-C12-cycloalkyl, benzyl or phenyl or together with the nitrogen atom to which they are bonded form a saturated or unsaturated heterocyclic ring, with or without further heteroatoms selected from N, O and S and a substituent wherein the substituent is OC(O)-C1-C4-alkyl esters, R12 is OR13 or C1-C18-alkyl, C1-C18-alkoxy, C6-C20-aryl or C6-C14-aryl-C1-C8-al kyl, R13 is a group of the formula or a benzyl group, which is unsubstituted or is one to four times ring-substituted, where the substituent or substituents are, independently of one another, fluorine, chlorine, bromine, a C1-C4-alkyl, nitro, methoxy or carboxyl group, oxidizing the resulting compound, carrying out a capping, eliminating the protective group S2 and then repeating this reaction step (n - 1) times, resulting in a compound of the formula XVII

in which A, B', Li, R2', SS, Y and V are as defined in step a) and U, W and n are as defined in claim 1;

c) subsequently reacting the resulting compound with a compound of the formula IX

in which S1 is a suitable protective group, V' is V, R9, R10, R12, V and S2 are as defined in step b), and T, a and b are as defined in claim 1, in the presence of a compound of the formula [HNR14R15R16](+)E(-), where R14, R15 and R16 are identical to or different from one another and are a C1-C4-alkyl group and E is fluorine, chlorine, bromine, or in the presence of tetrazole or substituted tetrazole in a suitable organic solvent, wherein the substituents on the tetrazole are C1-C4-alkoxy, C1-C4-thio-alkoxy or nitrophenyl substituents, oxidizing the resulting compound, carrying out a capping, eliminating the protective group S2, and then repeating this reaction step (m' - 1) times, resulting in a compound of the formula XVIII

in which A, B', Li, R2', SS, U, V, V', W, Y and n are as defined in step b), S1 is a suitable protective group, m' is m and m, a and b are defined in claim 1;

d) if n' is 1-50, carrying out reaction step b), which is repeated (n' - 1) times, resulting in a compound of the formula XIX

in which A, B', Li, R2', S1, SS, U, V, V', W, Y, a, b, m' and n are as defined in step c) and n' is n;

e) if R1 .noteq. H in formula II, introducing the radical R1' into the compound obtained in c) or d), where R1 is C1-C18-alkyl, C2-C18-alkenyl, C3-C18-alkynyl, C1-C18-alkylcarbonyl, C2-C1g-alkenylcarbonyl, C3-C19-alkynylcarbonyl, C6-C20-aryl, C6-C19-aryl-C1-C8-alkyl, or a radical of the formula III

in which W is as defined in step b) and Z and Z' are as defined in claim 1;

f) if R1 = H in formula II, capping the compound obtained in c) or d) ;

g) subsequently eliminating the protective group S1 so that the linker to the solid support and the other protective groups present in the molecule are retained;

h) and reacting the compound obtained in this way with a compound of the formula XV

in which R9, R10 and R12 are as defined in step b) , and Z" has the meaning of Z as defined in step e) or else Z is protected, in the presence of a compound of the formula [HNR19R15R16] (+)E(-), where R14, R15, R16 and E are as defined in step c), or in the presence of tetrazole or substituted tetrazole in a suitable organic solvent, wherein the substituents on the tetrazole are C1-C4-alkoxy, C1-C4-thio-alkoxy or nitrophenyl substituents;

i) oxidizing the compound obtained in step h);

j) eliminating the compound obtained in step i) from the support, and eliminating the remaining protective groups on the phosphate and nucleotide bases.
13. The process according to claim 12, wherein the protective group S2 is dimethoxytrityl or monomethoxytrityl.
14. The process as claimed in claim 12 or 13, wherein the linker arm Li is an aminoalkyl linker.
15. The process as claimed in claim 14 wherein the aminoalkyl linker is derivatized by reaction with an acridinium active ester.
16. A use of the compounds of the formula II as claimed in any one of claims 1 to 11 as a probe for detecting nucleic acids.
17. A pharmaceutical composition comprising a compound of the formula II as claimed in any one of claims 1 to 11 and a physiologically tolerated ancillary substance and/or vehicle.
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DE4420737A DE4420737A1 (en) 1994-06-15 1994-06-15 New 3'-derivatised oligo:nucleotide analogues
DE4424263A DE4424263A1 (en) 1994-07-09 1994-07-09 New 3'-derivatised oligo:nucleotide analogues
DEP4424263.8 1994-07-09
CA 2151801 CA2151801C (en) 1994-06-15 1995-06-14 3'-modified oligonucleotide derivatives

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US5696248A (en) 1997-12-09
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ATE312840T1 (en) 2005-12-15

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