Polynucleotide reagents containing modified deoxyribose moieties and associated methods of synthesis and use

• US 5,594,117 A
• Filed: 04/26/1995
• Issued: 01/14/1997
• Est. Priority Date: 08/25/1994
• Status: Expired due to Fees

• Current Assignee: Chiron Corporation (Bayer AG)
• Original Assignee: Chiron Corporation (Bayer AG)
• Inventors: Urdea, Michael S., Horn, Thomas
• Primary Examiner(s): Zitomer, Stephanie W.
• Assistant Examiner(s): Atzel, Amy
• Application Number:
  US08/429,197
• Time in Patent Office: 629 Days
• Field of Search: 435/6, 536/24.3, 536/22.1, 536/23.1
• US Class Current: 536/23.1
• CPC Class Codes: C07F 9/65515 the oxygen atom being part of a five-membered ring
• CPC Class Codes: C07H 15/04 attached to an oxygen atom of the saccharide radical
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6823 Release of bound markers

Abstract

Methods and reagents are provided for synthesizing polynucleotides containing modified deoxyribose residues. Monomeric reagents having the structural formula (I) ##STR1## wherein R¹, R², R³, R⁴ and R⁵ are as defined herein, are used to create polynucleotides having nonnucleotidic moieties --A--Z--(R⁹)_n at the 1 position of selected deoxyribose units. The polynucleotides so provided are useful in a variety of hybridization assay formats.

Claims

1. A polynucleotide reagent having the structural formula:

##STR12## wherein;

DNA₁ is a first segment of DNA;

DNA₂ is a second segment of DNA;

R³ is selected from the group consisting of hydrogen, hydroxyl, sulfhydryl, halogeno, amino, alkyl, allyl, --OR⁶ wherein R⁶ is alkyl, allyl, silyl or phosphate;

R⁴ is either hydrogen or --(CH₂)_m OR⁷ wherein R⁷ is alkyl or --(CO)R⁸, R⁸ is alkyl, and m is an integer in the range of 0 to 12 inclusive;

R⁵ is --A--Z--X(R⁹)_n ;

A is oxygen, sulfur or methylene;

Z is arylene, C₆ -C₁₈ aralkylene or C₁ -C₁₂ alkylene containing 0 to 6 heteroatoms selected from the group consisting of O, S, N, Si and Se and 0 to 6 linkages selected from the group consisting of --CO--, --COO--, --CONH--, --NHCO--, --S--S--, --SO₂ --, --CH(OH)--CH(OH)--, --CH(OR¹⁵)--CH(OR¹⁵)--, --O--PO(O^--)--O--, --O--PO(R¹⁵)--, --O--PO(OR¹⁵)--O--, --O--PO(OR¹⁵)--R¹⁶ -- and --PO(OR¹⁵)--O--R¹⁶ -- in which R¹⁵ is lower alkyl and R¹⁶ is lower alkylene, and, if Z is aralkylene or alkylene, containing 0 to 3 unsaturated bonds;

X is selected from the group consisting of --NH--, --CONH--, --NHCO--, --CO--, --S-- and --Si≡

;

R⁹ is hydrogen, a protecting group, or a detectable label; and

n is 1 when X is --NH--, --CONH--, --NHCO--, --CO--, or --S--, and is 3 when X is --Si≡

.

2. A polynucleotide reagent having the structural formula ##STR13## wherein:

DNA ₁ is a first segment of DNA;

DNA ₂ is a second segment of DNA;

R³ is selected from the group consisting of hydrogen, hydroxyl, sulfhydryl, halogeno, amino, alkyl, allyl, --OR⁶ wherein R⁶ is alkyl, allyl, silyl or phosphate;

R⁴ is either hydrogen or --(CH₂)_m OR⁷ wherein R⁷ is alkyl or --(CO)R⁸, R⁸ is alkyl and m is an integer in the range of 0 to 12 inclusive;

A is oxygen, sulfur or methylene; and

Z is arylene, C₆ -C₁₈ aralkylene or C₁ -C₁₂ alkylene containing 0 to 6 heteroatoms selected from the group consisting of O, S, N, Si and Se and 0 to 6 linkages selected from the group consisting of --CO--, --COO--, --CONH--, --NHCO--, --S--S--, --SO₂ --, --CH(OH)--CH(OH)--, --CH(OR¹⁵)--CH(OR¹⁵)--, --O--PO(O^--)--O--, --O--PO(R¹⁵)--, --O--PO(OR¹⁵)--O--, --O--PO(OR¹⁵)--R¹⁶ -- and --PO(OR¹⁵)--O--R¹⁶ -- in which R¹⁵ is lower alkyl and R¹⁶ is lower alkylene, and, if Z is aralkylene or alkylene, containing 0 to 3 unsaturated bonds.

3. A polynucleotide reagent having the structural formula ##STR14## wherein:

DNA₁ is a first segment of DNA;

DNA₂ is a second segment of DNA;

R³ is selected from the group consisting of hydrogen, hydroxyl, sulfhydryl, halogeno, amino, alkyl, allyl, --OR⁶ wherein R⁶ is alkyl, allyl, silyl or phosphate;

R⁴ is either hydrogen or --(CH₂)_m OR⁷ wherein R⁷ is alkyl or --(CO)R⁸, R⁸ is alkyl and m is an integer in the range of 0 to 12 inclusive;

A is oxygen, sulfur or methylene; and

Z is arylene, C₆ -C₁₈ aralkylene or C₁ -C₁₂ alkylene containing 0 to 6 heteroatoms selected from the group consisting of O, S, N, Si and Se and 0 to 6 linkages selected from the group consisting of --CO--, --COO--, --CONH--, --NHCO--, --S--S--, --SO₂ --, --CH(OH)--CH(OH)--, --CH(OR¹⁵)--CH(OR¹⁵)--, --O--PO(O^--)--O--, --O--PO(R¹⁵)--, --O--PO(OR¹⁵)--O--, --O--PO(OR¹⁵)--R¹⁶ -- and --PO(OR¹⁵)--O--R¹⁶ -- in which R¹⁵ is lower alkyl and R¹⁶ is lower alkylene, and, if Z is aralkylene or alkylene, containing 0 to 3 unsaturated bonds.

4. A branched polynucleotide reagent having the structural formula ##STR15## wherein:

DNA₁ is a first segment of DNA;

DNA₂ is a second segment of DNA;

DNA₃ is a third segment of DNA;

R³ is selected from the group consisting of hydrogen, hydroxyl, sulfhydryl, halogeno, amino, alkyl, allyl, --OR⁶ wherein R⁶ is alkyl, allyl, silyl or phosphate;

R⁴ is either hydrogen or --(CH₂)_m OR⁷ wherein R⁷ is alkyl or --(CO)R⁸, R⁸ is alkyl, and m is an integer in the range of 0 to 12 inclusive;

A is oxygen, sulfur or methylene; and

Z is arylene, C₆ -C₁₈ aralkylene or C₁ -C₁₂ alkylene containing 0 to 6 heteroatoms selected from the group consisting of O, S, N, Si and Se and 0 to 6 linkages selected from the group consisting of --CO--, --COO--, --CONH--, --NHCO--, --S--S--, --SO₂ --, --CH(OH)--CH(OH)--, --CH(OR¹⁵)--CH(OR¹⁵)--, --O--PO(O^--)--O--, --O--PO(R¹⁵)--, --O--PO(OR¹⁵)--O--, --O--PO(OR¹⁵)--R¹⁶ -- and --PO(OR¹⁵)--O--R¹⁶ -- in which R¹⁵ is lower alkyl and R¹⁶ is lower alkylene, and, if

Z is aralkylene or alkylene, containing 0 to 3 unsaturated bonds.

5. In a method for making a polynucleotide reagent comprising sequentially coupling nucleotidic monomers to a growing oligonucleotide chain, the improvement which comprises introducing an abasic site into the polynucleotide reagent by replacing a fraction of the nucleotidic monomers with the reagent of claim 4.