Indocarbocyanine and benzindocarbocyanine phosphoramidites

• US 5,808,044 A
• Filed: 02/10/1997
• Issued: 09/15/1998
• Est. Priority Date: 01/22/1993
• Status: Expired due to Term

• Current Assignee: GE Healthcare Bio-Sciences Corporation (General Electric Company)
• Original Assignee: Pharmacia Biotech Inc. (General Electric Company)
• Inventors: Anderson, Eric Dean, Brush, Charles K.
• Primary Examiner(s): Wilson, James O.
• Application Number:
  US08/799,593
• Time in Patent Office: 582 Days
• Field of Search: 536/25.32, 536/241, 536/25.3, 536/25.31, 548/416, 548/455, 435/6
• US Class Current: 536/25.32
• 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 chemical compound of the following formula is disclosed: ##STR1## where R is selected from the group consisting of H, trityl, 4-O-monomethoxytrityl, 4,4'"'"'-O-dimethoxytrityl, and acyl groups, and whereby R can be used as a protecting group or is an H;

R'"'"' is a phosphoramidite;

R" is selected from the group consisting of H and lower alkyl groups;

R'"'"'" is selected from the group consisting of H and lower alkyl groups;

R⁴ is selected from the group consisting of H, lower alkyl, acyl, ##STR2## and (CH₂)_p COO(CH₂)_q CH₃ wherein p is an integer from 0 to 4 and q is an integer from 0 to 4;

R⁵ is selected from the group consisting of H, lower alkyl, acyl, ##STR3## and (CH₂)_p COO(CH₂)_q CH₃ wherein p is an integer from 0 to 4 and q is an integer from 0 to 4;

n is an integer from 0 to 10;

m is an integer from 0 to 10;

r is 1, 2, or 3; and

X^- is a negative ion.

Claims

1. A chemical compound of the following formula:

##STR14## wherein;

R is selected from the group consisting of H, trityl, 4-O-monomethoxytrityl, 4,4'"'"'-O-dimethoxytrityl, and acyl groups, ##STR15## and whereby R can be used as a protecting group or is an H;

R'"'"' is a phosphoramidite;

R" is selected from the group consisting of H and lower alkyl groups;

R"'"'"' is selected from the group consisting of H and lower alkyl groups;

R⁴ is selected from the group consisting of H, lower alkyl, acyl, ##STR16## and (CH₂)_p COO(CH₂)_q CH₃ wherein p is an integer from 0 to 4 and q is an integer from 0 to 4;

R⁵ is selected from the group consisting of H, lower alkyl, acyl, ##STR17## and (CH₂)_p COO(CH₂)_q CH₃ wherein p is an integer from 0 to 4 and q is an integer from 0 to 4;

n is an integer from 0 to 10;

m is an integer from 0 to 10;

r is 1, 2, or 3; and

X^- is a negative ion.

2. The chemical compound of claim 1 wherein:

R is 4-O-monomethoxytrityl;

R'"'"' is N,N-diisopropyl-O-β

-cyanoethyl phosphoramidite;

R" and R'"'"'" are H;

n is 1;

m is 1;

r is 2; and

R⁴ and R⁵ are 5,6-butadienyl.

3. The chemical compound of claim 1 wherein:

R is 4-O-monomethoxytrityl;

R'"'"' is N,N-diisopropyl-O-β

-cyanoethyl phosphoramidite;

R" and R'"'"'" are H;

n is 1;

m is 1;

r is 1; and

R⁴ and R⁵ are 5,6-butadienyl.

4. The chemical compound of claim 1 wherein:

R is acetyl;

R'"'"' is N,N-diisopropyl-O-β

-cyanoethyl phosphoramidite;

R" and R'"'"'" are H;

n is 1;

m is 1;

r is 2; and

R⁴ and R⁵ are 5,6-butadienyl.

7. A method of attaching a fluorescent label to an oligonucleotide, comprising:

reacting the compound of claim 1 with the oligonucleotide such that the label becomes linked to the oligonucleotide.

8. The method of claim 7 wherein the reacting step comprises linking a 5'"'"' end of the oligonucleotide to a phosphorus on the compound, oxidizing the linkage product, and removing the protecting group R from the oxidized linkage product.

9. The method of claim 8 wherein the linkage is done in the presence of tetrazole and acetonitrile.

5. A chemical compound of the following formula:

##STR18## wherein;

R is selected from the group consisting of aryl-group-containing moieties, wherein the moiety does not interfere with the attachment of an oligonucleotide at the R'"'"' position;

R'"'"' is a oligonucleotide;

R" is selected from the group consisting of H and lower alkyl groups;

R'"'"'" is selected from the group consisting of H and lower alkyl groups;

R⁴ is selected from the group consisting of H, lower alkyl, acyl, ##STR19## and (CH₂)_p COO(CH₂)_q CH₃ wherein p is an integer from 0 to 4 and q is an integer from 0 to 4;

R⁵ is selected from the group consisting of H, lower alkyl, acyl, ##STR20## and (CH₂)_p COO(CH₂)_q CH₃ wherein p is an integer from 0 to 4 and q is an integer from 0 to 4;

n is an integer from 0 to 10;

m is an integer from 0 to 10;

r is 1, 2, or 3; and

X^- is a negative ion.

6. A benzindocarbocyanine dye attached to a phosphoramidite, said dye comprising a benzindolinium ring and a benzindolenine ring in resonance

wherein said rings are connected by a carbon chain with conjugated double bonds,

wherein the rings each have a dimethyl substituent at the 3 position,

wherein said phosphoramidite is attached through a first linker at the nitrogen of either ring,

wherein a protecting group is attached through a second linker at the nitrogen of the other ring, and

wherein no glyceryl linker is interposed between (a) said protecting group and said phosphoramidite and (b) said benzindocarbocyanine dye.