US 5,773,571 A · Peptide nucleic acids

Abstract

A novel class of compounds, known as peptide nucleic acids, bind complementary ssDNA and RNA strands more strongly than a corresponding DNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker.

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Claims11

Dependent claims

A compound having the formula:

##STR6## wherein:
n is at least 2,
each of L.sup.1 -L.sup.n is independently selected from the group consisting of hydrogen, hydroxy, (C.sub.1 -C.sub.4)alkanoyl, naturally occurring nucleobases, non-naturally occurring nucleobases, aromatic moieties, DNA intercalators, nucleobase-binding groups, heterocyclic moieties, and reporter ligands, at least one of L.sup.1 -L.sup.n being a naturally occurring nucleobase, a non-naturally occurring nucleobase, a DNA intercalator, or a nucleobase-binding group;
each of T.sup.1 -T.sup.n is (CR.sup.6 R.sup.7)y where R.sup.6 is hydrogen and R.sup.7 is selected from the group consisting of the side chains of naturally occurring alpha amino acids, or R.sup.6 and R.sup.7 are independently selected from the group consisting of hydrogen, (C.sub.2 -C.sub.6)alkyl, aryl, aralkyl, heteroaryl, hydroxy, (C.sub.1 -C.sub.6)alkoxy, (C.sub.1 -C.sub.6)alkylthio, NR.sup.3 R.sup.4 and SR.sup.5 or R.sup.6 and R.sup.7 taken together complete an alicyclic or heterocyclic system;
each R.sup.3 and R.sup.4 is independently selected from the group consisting of hydrogen, (C.sub.1 -C.sub.4)alkyl, hydroxy- or alkoxy- or alkylthio-substituted (C.sub.1 -C.sub.4)alkyl, hydroxy, alkoxy, alkylthio and amino;
R.sup.5 is hydrogen, (C.sub.1 -C.sub.6)alkyl, hydroxy-, alkoxy-, or alkylthio- substituted (C.sub.1 -C.sub.6)alkyl;
each of D.sup.1 -D.sup.n is (CR.sup.6 R.sup.7), where R.sup.6 and R.sup.7 are as defined above;
each of y and z is zero or an integer from 1 to 10, the sum y+z being greater than 2 but not more than 10;
each of G.sup.1 -G.sup.n-1 is --NR.sup.3 CO-- in either orientation, where R.sup.3 is as defined above;
each of A.sup.1 -A.sup.n and J.sup.1 -J.sup.n are selected such that:
(a) A is a group of formula (IIa), (IIb) or (IIc), and J is N or R.sup.3 N.sup.+, provided that at least one A is a group of formula (IIc) wherein S is an interger from 1 to 5;
or
(b) A is a group of formula (IId) and J is CH;
or
(c) A is a group of formula (IIa) or (IIb) and J is N or R.sup.3 N.sup.+, provided at least one of y or z is not 1 or 2;
##STR7## wherein:
X is O, S, Se, NR.sup.3, CH.sub.2 or C(CH.sub.3).sub.2 where R.sup.3 is as defined above;
Y is a single bond, O, S or NR.sup.4 where R.sup.4 is as defined above;
each of r and s is zero or an integer from 1 to 5;
each R.sup.1 and R.sup.2 is independently selected from the group consisting of hydrogen, (C.sub.1 -C.sub.4)alkyl which may be hydroxy- or alkoxy- or alkylthio-substituted, hydroxy, alkoxy, alkylthio, amino and halogen;
Q is --CO.sub.2 H, --CONR.sup.8 R.sup.9, --SO.sub.3 H or --SO.sub.2 NR.sup.8 R.sup.9 or an activated derivative of --CO.sub.2 H or --SO.sub.3 H;
and
Z is --NHR.sup.10 R.sup.11 or --NR.sup.10 C(O)R.sup.11, where R.sup.8, R.sup.9, R.sup.10 and R.sup.11 are independently selected from the group consisting of hydrogen, alkyl, amino protecting groups, reporter ligands, intercalators, chelators, peptides, proteins, carbohydrates, lipids, steroids, oligonucleotides and soluble and non-soluble polymers.

20 more elements

A process for preparing a compound according to claim 28, comprising the steps of:

A) providing a polymer substrate, said polymer being functionalized with a chemical group capable of forming an anchoring linkage with an amino acid;
B) coupling said polymer with a first amino acid through said anchoring linkage, said first amino acid having formula (IV):
##STR8## wherein:
L is selected from the group consisting of naturally occurring nucleobases, non-naturally occurring nucleobases, aromatic moieties, DNA intercalators, nucleobase-binding groups, heterocyclic moieties, and reporter ligands, wherein amino groups are, optionally, protected by amino protecting groups;
each T is (CR.sup.6 R.sup.7)y where R.sup.6 is hydrogen and R.sup.7 is selected from the group consisting of the side chains of naturally occurring alpha amino acids, or R.sup.6 and R.sup.7 are independently selected from the group consisting of hydrogen, (C.sub.2 -C.sub.6)alkyl, aryl, aralkyl, heteroaryl, hydroxy, (C.sub.1 -C.sub.6)alkoxy, (C.sub.1 -C.sub.6)alkylthio, NR.sup.3 R.sup.4 and SR.sup.5 or R.sup.6 and R.sup.7 taken together complete an alicyclic or heterocyclic system;
each R.sup.3 and R.sup.4 is independently selected from the group consisting of hydrogen, (C.sub.1 -C.sub.4)alkyl, hydroxy- or alkoxy- or alkylthio-substituted (C.sub.1 -C.sub.4)alkyl, hydroxy, alkoxy, alkylthio and amino;
R.sup.5 is hydrogen, (C.sub.1 -C.sub.6)alkyl, hydroxy-, alkoxy-, or alkylthio-substituted (C.sub.1 -C.sub.6)alkyl;
each D is (CR.sup.6 R.sup.7).sub.z where R.sup.6 and R.sup.7 are as defined above;
each of y and z is zero or an integer from 1 to 10, the sum y+z being greater than 2 but not more than 10;
A and J are selected such that:
(a) A is a group of formula (IIc) and J is N or R.sup.3 N.sup.+ ;
or
(b) A is a group of formula (IId) and J is CH;
or
(c) A is a group of formula (IIa) or (IIb) and J is N or R.sup.3 N.sup.+, provided at least one of y or z is not 1 or 2;
##STR9## wherein:
X is O, S, Se, NR.sup.3, CH.sub.2 or C(CH.sub.3).sub.2 where R.sup.3 is as defined above;
Y is a single bond, O, S or NR.sup.4 where R.sup.4 is as defined above;
each of r and s is zero or an integer from 1 to 5;
each R.sup.1 and R.sup.2 is independently selected from the group consisting of hydrogen, (C.sub.1 -C.sub.4)alkyl which may be hydroxy- or alkoxy- or alkylthio-substituted, hydroxy, alkoxy, alkylthio, amino and halogen;
and
each E is COOH or an activated or protected derivative thereof;
and
each M is NHR.sup.3 or NR.sup.3 R.sub.12, where R.sup.3 is as defined above, and R.sup.12 is an amino protecting group;
C) removing said amino protecting group from said coupled first amino acid to generate a free amino group;
and
D) reacting said free amino group with a second amino acid having formula (IV) to form a peptide chain.

24 more elements

Dependencies are inferred from the claim text.

Description16,556 words

Summary3,627 words

#This invention is directed to compounds that are not polynucleotides yet which bind to complementary DNA and RNA strands. In particular, the invention concerns compounds wherein naturally-occurring nucleobases or other nucleobase-binding moieties are covalently bound to a polyamide backbone.

#Oligodeoxyribonucleotides as long as 100 base pairs (bp) are routinely synthesized by solid phase methods using commercially available, fully automatic synthesis machines. The chemical synthesis of oligoribonucleotides, however, is far less routine. Oligoribonucleotides also are much less stable than oligodeoxyribonucleotides, a fact which has contributed to the more prevalent use of oligodeoxyribonucleotides in medical and biological research directed to, for example, gene therapy or the regulation of transcription or translation.

#The function of a gene starts by transcription of its information to a messenger RNA (mRNA) which, by interaction with the ribosomal complex, directs the synthesis of a protein coded for by its sequence. The synthetic process is known as translation. Translation requires the presence of various co-factors and building blocks, the amino acids, and their transfer RNAs (tRNA), all of which are present in normal cells.

Detailed description12,929 words

#Additional objects, advantages, and novel features of this invention will become apparent to those skilled in the art upon examination of the following examples thereof, which are not intended to be limiting.

#Glycinamide hydrochloride (1, 11.0 g, 0.10 mol) is suspended in 500 mL of dioxane and diisopropylethylamine (12.9 g, 0.10 mol) is added and the mixture cooled to 0.degree. C. With stirring ethyl acrylate (10.0 g, 0.10 mol) is added dropwise over 15 minutes. After the addition is complete the reaction is allowed to warm to room temperature and stir for 12 hours. The reaction mixture is diluted with water 1.5 L and the pH adjusted to 4. The solution is extracted with diethyl ether (3.times.300 mL) . The aqueous layer is neutralized with sodium hydroxide and extracted 5 times with dichloromethane. The dichloromethane extracts are combined, dried (Na.sub.2 SO.sub.4), and the solvent removed to give a solid.

#The product from Example 1, 2, is dissolved in dichloromethane (500 mL) and to this is added 1-carboxymethyl thymine (3, 15.5 g, 0.1 mol), hydroxybenzotriazole (13.5 g, 0.1 mol) and the solution is cooled to 0.degree. C. in an ice bath. Diisopropylcarbodiimide (12.6 g, 0.1 mol) dissolved in So mL of dichloromethane is added in one portion and the reaction is stirred for 12 hours. The suspended solids are removed by filtration and washed with dichloromethane. The solution is evaporated to a solid and the desired product, 4, is obtained after chromatography on silica gel using dichloromethane/ethanol as eluent.

Facts

Application

08/595,387Filed 1 Feb 1996 · division

Priority

26 Apr 1993

Granted

30 Jun 1998

Term ended

26 Apr 2013

Estimated · how

20 years from earliest known nonprovisional domestic filing. Maintenance-fee lapses, terminal disclaimers and statutory extensions have not been checked. The continuity chain may be incomplete; this is not an enforceability determination. Patent term adjustment is unavailable; no adjustment was added.

Classification23 codes

C12N 15/113
DNA or RNA fragments; Modified forms thereof ; Non-coding nucleic acids having a biological activity: non-coding nucleic acids modulating the…
A61P 31/00
Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
A61P 31/12
Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics: antivirals
A61P 35/00
Antineoplastic agents

Peptide nucleic acids

Life of this patent

Peptide nucleic acids

Life of this patent

Life of this patent

Abstract

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Claims11

Description16,556 words

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Classification23 codes

Life of this patent

Description16,556 words