Nucleoside derivatives

• US 5,552,540 A
• Filed: 05/27/1993
• Issued: 09/03/1996
• Est. Priority Date: 06/24/1987
• Status: Expired due to Term

• Current Assignee: Howard Florey Institute of Experimental Physiology and Medicine
• Original Assignee: Howard Florey Institute of Experimental Physiology and Medicine
• Inventors: Haralambidis, Jim
• Primary Examiner(s): Robinson, Douglas W.
• Assistant Examiner(s): Wilson, James O.
• Application Number:
  US08/068,604
• Time in Patent Office: 1,195 Days
• Field of Search: 536/26.7, 536/25.34, 536/28.53, 536/28.54, 536/28.55, 536/26.8, 536/26.6, 536/27.11
• US Class Current: 536/25.34
• 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: Y02P 20/55 Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

A nucleoside derivative of formula (1), characterized in that: Y is H or OH or a protected hydroxy group; X is H, a phosphonate group or a phosphoramidite group of formula (II), where R¹ and R² are the same or different, and are selected from alkyl and substituted alkyl, which may be branched or unbranched; and Q is a phosphate protecting group; Z is H, a phosphate or triphosphate group or hydroxy protecting group; X'"'"' is a C_1-15 alkyl group which may be branched or unbranched; R is an amino protecting group or a fluorophore, or other non-radioactive detectable marker; or the group Y'"'"'NHA, where Y'"'"' is an alkyl (C_1-40) carbonyl group which may be branched or unbranched, and A is an amino protecting group or a fluorophore or other non-radioactive detectable marker. Methods for the synthesis and sequencing of polynucleotides utilizing compounds of formula (I).

Claims

1. A nucleoside of the formula (I):

##STR18## wherein (A) Y is H, OH or a protected hydroxy group;

(B) X is H, a phosphonate group or a phosphoramidite group of the formula ##STR19## wherein (i) R¹ and R² are the same or different, and are selected from branched or unbranched C_1-30 alkyl or substituted C_1-30 alkyl having substituents selected from the group consisting of phenyl, benzyl, acyl, methyl, propyl, ethyl, isopropyl and butyl; and

(ii) Q is a phosphate protecting group;

(C) Z is H, a phosphate group, a triphosphate group or a hydroxy-protecting group;

(D) X'"'"' is a branched or unbranched C_1-15 alkyl group;

(E) R is an acid labile amino protecting group, an amino reactive detectable marker, or the group

space="preserve" listing-type="equation">Y'"'"'NHA,

wherein

(i) Y'"'"' is a branched or unbranched C_1-40 alkyl carbonyl group; and

(ii) A is an acid labile amino protecting group, or an amino reactive detectable marker.

2. A nucleoside as claimed in claim 1, wherein R^` and R² are both isopropyl groups.

3. A nucleoside as claimed in claim 1 wherein X'"'"' is methylene and R is

4. A nucleoside as claimed in claim 1, wherein

Y is H, OH or a protected hydroxy group;

X is ##STR20## Z is dimethoxytrityl;

X'"'"' is CH₂ ; and

R is CH₂ CO(CH₂)₅ NHA, wherein A is an acid labile amino protecting group, or an amino reactive detectable marker.

5. A nucleoside as claimed in claim 1, wherein

Y is H, OH or a protected hydroxy group;

X is H;

Z is triphosphate;

X'"'"' is CH₂ ; and

R is CO(CH₂)₅ NHA, wherein A is an acid labile amino protecting group, or an amino reactive detectable marker.

6. A nucleoside as claimed in any one of claims 1 to 5, wherein Q is selected from the group consisting of methyl, phenyl, substituted phenyl having substituents selected from the group consisting of halogen, hydroxy and NO₂ groups, benzyl, or cyanoethyl.

7. A nucleoside as claimed in any one of claims 1 to 5, wherein said non-radioactive detectable marker is selected from the group consisting of biotin, avidin, colloidal gold, colloidal silver and ferritin.

8. A nucleoside as claimed in any one of claims 1 to 5 wherein the amino reactive detectable marker is fluorescein.

9. A nucleoside as claimed in any one of claims 1 to 5, wherein the amino reactive detectable marker is a fluorophore precursor which contains one or more groups which suppress fluorescence, but which is capable of fluorescence once these groups are removed.

10. A nucleoside as claimed in claim 9, having the formula:

##STR21##