Hoechst Aktiengesellschaft

501 Hoechst Aktiengesellschaft

Joachim Engels

Eugen Uhlmann

Matthias Mag

Mathias Herrlein

Renate Konrad

Wolfgang Konig

Waldemar Wetekam

Paul Habermann

Dominique Tripier

Matthias Scharf

Hubert Mullner

Peter Crause

Michael Leineweber

Wolfgang Ulmer

Arnold Ganser

Martin Kramer

Alfred Jager

Michaela Scherr

Enzymatic RNA molecules have been designed that cleave mutant N-ras mRNA, preferably at a NUX cleavage site (N=any base, X=A, C or U). Preferred ribozymes have nucleotide sequences 5'-CCAACACCUGAUGAGCGUUAGCGAAACCUGCU-3' or 5'-UCCCAACCUGAUGAGCGUUAGCGAAACACCUG-3' (SEQ ID NOS:1 and 2), and derivatives thereof. The present invention also provides pharmaceuticals containing such molecules and the use of such molecules for the preparation of pharmaceuticals for the treatment of diseases involving abnormal cell growth and/or differentiation.

Enzymatic RNA molecules that cleave mutant N-RAS

3'-(2')-Amino- or thiol-modified, fluorescent dye-coupled nucleosides, nucleotides and oligonucleotides, and a process for the preparation and the use thereof The OH group located in the 3' and/or 2' position of a nucleoside, nucleotide or oligonucleotide is derivatized to an amino or thiol group and subsequently a fluorescent dye is coupled thereto. The resulting 3'- and/or 2'-amino- and thiol-modified nucleosides, nucleotides and oligonucleotides can then be used for the synthesis of complementary strands in the presence of a template strand or of oligonucleotides and for the detection of genetic material. They have the advantage that the fluorescent label need no longer be attached to the 5' end of the oligonucleotide or to the nucleobase, and thus need not be introduced during the chemical synthesis as in labeling techniques hitherto known, while the known and conventional methods have the disadvantage that only a few polymerases can be employed for the synthesis, the acceptance of the triphosphates by the polymerases diminishes and, moreover, a large substrate excess is necessary.

3'(2')-amino- or thiol-modified, fluorescent dye-coupled nucleosides,

3'-(2')-Amino- or thiol-modified, fluorescent dye-coupled nucleosides, nucleotides and oligonucleotides, and a process for the preparation and the use thereof. The OH group located in the 3' and/or 2' position of a nucleoside, nucleotide or oligonucleotide is derivatized to an amino or thiol group and subsequently a fluorescent dye is coupled thereto. The resulting 3'- and/or 2'-amino- and thiol-modified nucleosides, nucleotides and oligonucleotides can then be used for the synthesis of complementary strands in the presence of a template strand or of oligonucleotides and for the detection of genetic material. They have the advantage that the fluorescent label need no longer be attached to the 5' end of the oligonucleotide or to the nucleobase, and thus need not be introduced during the chemical synthesis as in labeling techniques hitherto known, while the known and conventional methods have the disadvantage that only a few polymerases can be employed for the synthesis, the acceptance of the triphosphates by the polymerases diminishes and, moreover, a large substrate excess is necessary.

3'-(2')-amimo- or thiol-modified, fluorescent dye-coupled nucleosides,

The OH group located in the 3' and/or 2' position of a nucleoside, nucleotide or oligonucleotide is derivatized to an amino or thiol group and subsequently a fluorescent dye is coupled thereto. The resulting 3'- and/or 2'-amino- and thiol-modified nucleosides, nucleotides and oligonucleotides can then be used for the synthesis of complementary strands in the presence of a template strand or of oligonucleotides and for the detection of genetic material. They have the advantage that the fluorescent label need no longer be attached to the 5' end of the oligonucleotide or to the nucleobase, and thus need not be introduced during the chemical synthesis as in labeling techniques hitherto known, while the known and conventional methods have the disadvantage that only a few polymerases can be employed for the synthesis, the acceptance of the triphosphates by the polymerases diminishes and, moreover, a large substrate excess is necessary.

3'-(2')-amino or thiol-modified, fluorescent dye-coupled

Polypeptides having a carboxamide carboxyl terminal end and a methionine residue, which is optionally bonded to a bacterial protein, on the amino end can be prepared by synthesis by genetic engineering methods of the corresponding polypeptide having at the C terminal end a glycine residue, and conversion of the latter enzymatically into the amino group of the desired carboxamide group. Peptides which have the aminoacid sequence of growth hormone releasing factor, part sequences thereof, or modifications of these peptides, are readily accessible by this means. The synthesis by genetic engineering methods is advantageously carried out via two gene fragments which are synthesized chemically from smaller, single-stranded structural units. The two gene fragments are then linked enzymatically to give the complete gene, which is incorporated into a suitable vector, amplified there, and the peptide is isolated directly or as a fused protein, and is converted enzymatically into the desired amide.

Preparation of polypeptides having an amide carboxyl terminal end

Thrombin inhibitors from the hirudin family which differ from the previously known hirudins by mutation in the protein chain, and a process for their preparation are described. The novel hirudins are distinguished by a high specific activity, high stability and good pharmacokinetics.

Isohirudins

Deoxyribonucleoside phosphonates, thiophosphonates and selenophosphonates are obtained by condensation of a difunctional phosphonylating reagent of the formula

Process for the preparation of nucleoside alkyl-aralkyl- and

Compounds of the formula ##STR1## are phosphorylating reagents which react with acylatable hydrogen atoms with elimination of the amine HNR.sup.3 R.sup.4. The resulting compounds are oxidized to give the corresponding phosphate, thiophosphate or selenophosphate derivatives, and the radicals R.sup.1 and R.sup.2 are then split off by means of bases. The chemical nature of the radicals R.sup.1, R.sup.2, R.sup.3 and R.sup.4 accordingly depends on the requirements for these cleavage reactions.

Process for 5'-phosphorylation of nucleic acids

Partial sequences of human-.gamma.-interferon, comprising aminoacid sequences 5 to 127, 1 to 127 and 5 to 146, having biological activity. These partial sequences can be obtained by a genetic engineering process, for which purpose the appropriate DNA sequences are chemically synthesized. The DNA sequences are incorporated in hybrid plasmids, and the latter are introduced into host organisms and their expression is induced there. The biologically active polypeptides are suitable, as is human-.gamma.-interferon, for medicaments.

Preparation of polypeptides having human .gamma.-interferon activity

Secretin, which cannot be prepared directly by genetic engineering because of its carboxylic acid carboxyl-terminus, can be obtained by preparing secretylglycine by genetic engineering and then obtaining secretin therefrom by enzymatic conversion of the terminal glycine radical. The gene for the secretylglycine is synthesized chemically from smaller single-stranded units which are linked enzymatically to give the complete gene, incorporated into a suitable vector and amplified therein, after which the peptide is isolated directly or as a fusion protein and, after cyanogen bromide cleavage, is converted enzymatically into secretin.

Preparation of secretin

Steroid-21-phosphoric acids and pharmaceutically usable salts thereof of the formula III ##STR1## (in which U=H or CH.sub.3, V=H, OH, O or Hal; W=H or OH and Y=H or F) are obtained in a very pure state by reacting I ##STR2## (in which X=OH or Hal) with an organic phosphoric acid ester of the formula IVa or IVb ##STR3## (in which Z=optionally substituted alkyl and R=alkyl). The compounds II ##STR4## obtained thereby is saponified to give III and the latter, if appropriate, is neutralized to give the salt. Compounds II are new.

Growing community of inventors

Kronberg/Taunus, Germany

Joachim Engels