Phosphoramidite derivatives, their preparation and the use thereof in the incorporation of reporter groups on synthetic oligonucleotides

• US 5,567,811 A
• Filed: 11/03/1992
• Issued: 10/22/1996
• Est. Priority Date: 05/03/1990
• Status: Expired due to Fees

• Current Assignee: Amersham International Plc (General Electric Company)
• Original Assignee: Amersham International Plc (General Electric Company)
• Inventors: Gait, Michael, Misiura, Konrad
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): HOUTTEMAN, SCOTT W
• Application Number:
  US07/946,477
• Time in Patent Office: 1,449 Days
• Field of Search: 536/23.1, 536/25.3, 536/25.31, 536/25.32, 536/25.34, 435/6, 435/7.5
• US Class Current: 536/25.34
• CPC Class Codes: C07F 9/2408 of hydroxyalkyl compounds
• CPC Class Codes: C07F 9/6561 containing systems of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring or ring system, with or without other non-condensed hetero rings
• 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: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: Y02P 20/55 Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

Phosphoramidite derivatives of formula (V), ##STR1## wherein X is biotin and Y is a protecting group. There may be a linker arm, of variable length, between X and the rest of the molecule. Examples of the protecting group Y include 4,4'"'"'-dimethoxytrityl, trifluoroacetyl and fluorenylmethoxycarbonyl (Fmoc). The phosphoramidite derivatives are useful in single or multiple labelling of synthetic oligonucleotides. Process for the preparation of these phosphoramidite derivatives are also disclosed.

Claims

1. A phosphoramidite derivative of the following formula:

##STR15## wherein X=biotin, and

Y=a protecting group.

2. A phosphoramidite derivative as claimed in claim 1, wherein there is a linker arm of variable length between biotin and the rest of the molecule.

3. A phosphoramidite derivative as claimed in claim 1, wherein Y is 4,4'"'"'-dimethoxytrityl.

5. A method for the single or multiple labelling of synthetic oligonucleotides and which comprises the use of a phosphoramidite ligand as claimed in claim 1.

6. A method for the single or multiple labeling of synthetic oligonucleotides and which comprises the use of a phosphoramidite ligand as claimed in claim 1, wherein the labeling with said phosphoramidite ligand occurs at the 5'"'"' end or the 3'"'"' end of the oligonucleotide or at any internal position along the chain.

4. A method for the production of a phosphoramidite derivative of the following formula:

##STR16## and which comprises;

i) Reaction of solketal with acrylonitrile to form the addition product, 2-cyanoethyl solketal (I);

##STR17## ii) Reduction of the resultant compound (I) to form 3-aminopropyl solketal (II);

##STR18## iii) Reaction of the resultant compound (II) with biotin N-hydroxysuccinimide to form N-biotinyl-3-aminopropyl solketal (III);

##STR19## iv) Reaction of the resultant compound (III) with 4,4'"'"'-dimethoxytrityl chloride to form 1-0-(4, 4'"'"'-dimethoxytrityl)-3-0-(N-biotinyl-3-aminopropyl) glycerol (IV);

##STR20## v) Phosphitylation of the resultant compound (IV) to produce the desired biotinyl phosphoramidite derivative (V);

##STR21## wherein DMTr=4,4'"'"'-dimethoxytrityl.