Nucleic acid probe containing a terminal carbamyl linking non-radioactive labeling and preparating processes

• US 5,218,102 A
• Filed: 02/23/1989
• Issued: 06/08/1993
• Est. Priority Date: 02/24/1988
• Status: Expired due to Fees

• Current Assignee: La Region Wallonne An Autonomous Public Body Attached To Belgium
• Original Assignee: Improbio
• Inventors: de Vos-Pierreux, Marie-Joelle, Cravador, Alfredo, Bollen, Alex
• Primary Examiner(s): Yarbrough, Amelia Burgess
• Assistant Examiner(s): Marschel, Ardin H.
• Application Number:
  US07/315,348
• Time in Patent Office: 1,566 Days
• Field of Search: 435/6, 435/91, 435/810, 436/501, 536/27, 536/26, 536/28, 536/29, 935/78, 935/88
• US Class Current: 536/24.3
• 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 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/708 for papilloma
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

The subject of the present invention is a nucleic acid probe containing a nucleic acid sequence, wherein the said sequence is linked at its 5'"'"' end, via a divalent bifunctional chemical "arm" L, to a "labeling component" M, M being a synthetic or natural molecule which is directly or indirectly detectable in a non-isotopic manner, according to the formula I: ##STR1## in which J=H or OH

n denotes the number of nucleotides from 1 to 100,000

B is a purine or pyrimidine nucleic acid base, which varies according to the nucleotide, as appropriate.

The subject of the invention is also a process for preparing such probes, employing an intermediate compound consisting of a nucleotide synthon of formula IV ##STR2## in which J, B, L and R₁ have the meanings given above, B optionally being protected,

R₂ denotes H or any phosphorylated group, optionally protected, suited to the introduction of the compound of formula IV at the 5'"'"' end of another nucleotide, for a given type of internucleotide-assembling synthesis.

Claims

1. A nucleotide or nucleic acid of the formula ##STR25## wherein:

J=H or OH;

n=1 to 100,000;

B=a purine or pyrimidine nucleic acid base;

##STR26## where Alk is a straight alkyl chain of from 2 to 20 carbon atoms; and

M=a non-radioactive detectable label.

5. A nucleotide or nucleic acid according to claim 1 wherein Alk is hexamethylene.

6. A nucleotide or nucleic acid according to claim 1 wherein M is an enzyme which catalyzes a reaction resulting in the loss or formation of a chromogenic product;

a fluorescer having at least one fluorophore;

or a molecule capable of specifically binding to another molecule to which a detectable molecule is bound.

7. A nucleotide or nucleic acid according to claim 6, wherein said enzyme is microperoxidase or alkaline phosphatase.

8. A nucleotide or nucleic acid according to claim 6, wherein said fluorescer is fluorescein.

9. A nucleotide or nucleic acid according to claim 6, wherein said specifically binding molecule is biotin.

10. A nucleotide or nucleic acid according to claim 1, wherein M is a synthetic polymer comprising a plurality of groups capable of directly or indirectly producing a detectable signal.

2. A method of preparing a nucleic acid probe comprising:

(a) synthesizing a nucleic acid of formula Ia;

##STR27## wherein;

J=H or OH;

n=an integer of up to 100,000 and n-1 is at least 1;

B=a purine or pyrimidine nucleic acid base;

(b) coupling said nucleic acid at its free 5'"'"'--(OH) end, to the 3'"'"'--(OH) of a nucleotide synthon which synthon is (1) protected at the 3'"'"' position by a phosphoryl group for linking to said 5'"'"' end of said sequence, (2) bonded at the 5'"'"' position to a linking group L terminating in a protective group R₁ capable of being activated for linking to a detectable label and (3) of the formula IV ##STR28## in which J, B, and R₁ have the meanings given above, B optionally being protected, ##STR29## where Alk is a straight alkyl chain of from 2 to 20 carbon atoms, R₂ is any phosphorylated group for joining to the 5'"'"' end of another nucleotide to give the compound of formula Ib ##STR30## (c) activating R₁ and reacting activated R₁ with said detectable label to produce said probe.

11. A method according to claim 2, wherein at least said synthesizing is carried out on a solid support.

12. A method according to claim 2, wherein said detectable label comprises a macromolecule to which the groups capable of being detected either directly or indirectly are bonded prior to or after reacting with R₁.

3. A compound of the formula:

##STR31## wherein;

J=H or OH;

B=a purine or pyrimidine nucleic acid base;

##STR32## where Alk is a straight alkyl chain of from 2 to 20 carbon atoms;

R₁ =a protective group capable of being activated for linking to a detectable label; and

R₂ =H or a phosphorylated group for joining to the 5'"'"' end of a nucleotide, with the proviso that B may be substituted with protective groups.

4. A process for preparing a compound according to claim 3, said process comprising:

selectively activating the 5'"'"'OH of a 3'"'"'OH protected and base protected nucleotide;

coupling at the 5'"'"' position a singly protected linking group of the formula R₁ --L--, wherein;

##STR33## where Alk is a straight alkyl chain of from 2 to 20 carbon atoms and R₁ is a protective group capable of being activated for linking to a detectable label;

deprotecting said 3'"'"'--OH position; and

optionally, phosphorylating said 3'"'"'--OH with a phosphoryl group capable of coupling to the 5'"'"'--OH of a nucleoside or nucleotide.

13. A compound according to claim 3, wherein R₂ is a cyanoethyldiisopropylphosphoramidite group.

14. A compound according to claim 3, wherein L is of the formula ##STR34## where L'"'"' is a diaminated residue and R₁ is a diphenylisopropyloxycarbonyl group.

15. An intermediate compound according to claim 14 of the formula:

##STR35##

16. A process according to claim 4, wherein ##STR36## the selective activation of the 5'"'"'--OH is with a carbonyldiimidazole, the coupling reaction is with the diaminated residue singly protected on a terminal amine, wherein R₁ is a di-phenylisopropyloxycarbonyl group.

17. A process according to claim 4, wherein said 3'"'"'--OH is protected with a dimethyl-tert-butylsilyl group.

18. A process according to claim 17, wherein said deprotection employs tetrabutylammonium fluoride.

19. A process according to claim 4, wherein said 3'"'"'--OH position is phosphorylated with diisopropylaminocyanoethoxychlorophosphine.

20. A process according to claim 4, wherein said nucleoside is selectively protected at the 5'"'"'--OH with an acid labile group;

the 3'"'"'--OH is protected with a mild or neutral condition liable group; and

said selective activation of said 5'"'"'--OH is by treatment under mild acid conditions.

21. A process according to claim 20, wherein said acid labile group is dimethoxytrityl and said mild or neutral condition labile group is dimethyl-tert-butylsilyl.