Diaminophenothiazinium derivatives for labelling biomolecules, method and substrate for labelling oligonucleotides, and oligonucleotides obtained

Patent metadata

Field	Value
Publication number	US-9540405-B2
Application number	US-201314379417-A
Country	US
Kind code	B2
Filing date	Feb 21, 2013
Priority date	Feb 27, 2012
Publication date	Jan 10, 2017
Grant date	Jan 10, 2017

Abstract

The present invention relates to diaminophenothiazinium derivatives of formula (I); in which R 1 , R 2 , R 3 , R 4 , R 5 , R 6 and X − are as defined in Claim 1 , and also the methods for labelling oligonucleotides using such a derivative, labeling substrates and the oligonucleotides which can be obtained by means of such methods or from such labelling substrates.

First claim

The invention claimed is:

1. Diaminophenothiazinium derivatives of formula (I):

in which:
one of the R 1 , R 2 , R 3 and R 4 groups represents -A 1 -OR 7 , with A 1 representing a linear or branched alkylene chain comprising from 2 to 12 carbons, the oxygen and nitrogen atoms being separated by at least two consecutive carbon atoms, and R 7 representing a group forming, with OA 1 to which it is bonded, a phosphoramidite, phosphodiester, or a hydrogen phosphonate group,
the other R 1 , R 2 , R 3 and R 4 groups, which may be identical or different, represent, independently of one another:
an -A 2 -OR 8 group, with A 2 representing a linear or branched alkylene chain comprising from 2 to 12 carbons, the oxygen and nitrogen atoms being separated by at least two consecutive carbon atoms, and R 8 representing a member selected from the group consisting of trityl, 4-O-monomethoxytrityl, 4,4′-O-dimethoxytrityl, tert-butyldimethylsilyl, acetyl, trifluoroacetyl, 9-phenylxanthen-9-yl, and fluorenylmethyloxycarbonyl,
an alkyl group having from 2 to 12 carbon atoms,
or, R 1 and R 2 or R 3 and R 4 are bonded to one another to form, with the nitrogen atom to which they are bonded, a piperidinyl or pyrrolidinyl group,
R 5 and R 6 , which may be identical or different, represent, independently of one another, a hydrogen, chlorine, bromine, iodine or fluorine atom, or an alkyl group having from 1 to 12 carbon atoms, an alkenyl group having from 2 to 12 carbon atoms, an alkynyl group having from 2 to 12 carbon atoms, an acyl group or a phenyl group, and
X − represents an anion.

2. The diaminophenothiazinium derivatives as claimed in claim 1 , characterized in that A 1 and A 2 are linear or branched alkylene chains in which from 2 to 6 consecutive carbon atoms separate the oxygen and nitrogen atoms.

3. The diaminophenothiazinium derivatives as claimed in claim 1 , characterized in that at least one of the R 1 , R 2 , R 3 and R 4 groups does not represent an -A 2 -OR 8 group, as defined in claim 1 , and said R 1 , R 2 , R 3 , or R 4 group(s) are different than -A 1 -OR 1 and wherein -A 2 -OR 8 represent(s) an alkyl group having from 2 to 12 carbon atoms.

4. The diaminophenothiazinium derivatives as claimed in claim 1 , wherein R 5 ═R 6 ═H.

5. The diaminophenothiazinium derivatives as claimed in claim 1 , wherein R 7 represents a —P{N[(C 2 -C 12 )alkyl] 2 }(OCH 2 CH 2 C≡N) group.

6. The diaminophenothiazinium derivatives as claimed in claim 5 , wherein R 7 represents the —P[N ( i Pr) 2 ](OCH 2 CH 2 C≡N) group.

7. The diaminophenothiazinium derivatives as claimed in claim 1 , of formula (Ia):

in which:
A 1 , A 2 , R 5 , R 6 , R 7 , R 8 and X − are as defined in claim 1 ,
R 1 and R 2 , which may be identical or different, represent, independently of one another, an alkyl group having from 2 to 12 carbon atoms, or R 1 and R 2 are bonded to one another to form, with the nitrogen atom to which they are bonded, a piperidinyl or pyrrolidinyl group.

8. The diaminophenothiazinium derivatives as claimed in claim 1 , of formula (Ib):

in which:
A 1 , A 2 , R 5 , R 6 , R 7 , R 8 and X − are as defined in claim 1 ,
R 2 and R 4 , which may be identical or different, represent, independently of one another, an alkyl group having from 2 to 12 carbon atoms.

9. The diaminophenothiazinium derivatives as claimed in claim 1 , of formula (Ic):

in which:
A 1 , R 5 , R 6 , R 7 and X − are as defined in claim 1 ,
R 1 and R 2 , which may be identical or different, represent, independently of one another, an alkyl group having from 2 to 12 carbon atoms, or R 1 and R 2 are bonded to one another to form, with the nitrogen atom to which they are bonded, a piperidinyl or pyrrolidinyl group,
R 3 represents an alkyl group having from 2 to 12 carbon atoms.

10. The diaminophenothiazinium derivatives as claimed in claim 1 , wherein X − is selected from the group consisting of Cl − , I − , ClO 4 − , and NO 3 − .

11. A method for labeling an oligonucleotide with a diaminophenothiazinium derivative as claimed in claim 1 , which comprises the growth of an oligonucleotide grafted onto a solid substrate, and the replacement of one or more of the nucleotides of which it is formed with one or more of said diaminophenothiazinium derivatives, before the oligonucleotide is detached from the solid substrate.

12. The labeling method as claimed in claim 11 , wherein at least one replacement with a diaminophenothiazinium derivative is carried out before the end of the growth of the oligonucleotide.

13. The labeling method as claimed in claim 11 , wherein at least one substitution with a diaminophenothiazinium derivative is carried out in the 3′ or 5′ positions, on the first or the last nucleotide, respectively.

14. The labeling method as claimed in claim 11 , comprising a final step of treatment in a basic medium, either using a basic solution of sodium hydroxide, of aqueous ammonia, or of potassium carbonate, or a solution of methylamine and of aqueous ammonia, or a solution of diisopropylamine and of β-mercaptoethanol, or using ammonia or methylamine in the gas phase.

15. Labeled oligonucleotides obtained by the method of claim 11 .

16. An oligonucleotide synthesis substrate comprising at least one diaminophenothiazinium derivative covalently grafted at the surface according to the series:

in which:
R 5 , R 6 , which may be identical or different, represent, independently of one another, a hydrogen, chlorine, bromine, iodine, or fluorine atom, or an alkyl group having from 1 to 12 carbon atoms, an alkenyl group having from 2 to 12 carbon atoms, an alkynly group having from 2 to 12 carbon atoms, an acyl group, or a phenyl group,
X − represents an anion,
R′ 1 and R′ 2 , identical or different, represent, independently of one another, an alkyl group having from 2 to 12 carbon atoms, or R 1 and R 2 are bonded to one another to form, with the nitrogen atom to which they are bonded, a piperidinyl or pyrrolidinyl group,
A 1 ′ and A 1 ″, which may be identical or different, represent, independently of one another, a linear or branched alkylene chain comprising from 2 to 12 carbons, and
R 9 represents a member selected from the group consisting of trityl, 4-O-monomethoxytrityl, 4, 4′-O-dimethoxytrityl, tert-butyldimethylsilyl, acetyl, trifluoroacetyl, 9-phenylxanthen-9-yl, and fluorenylmethyloxycarbonyl; and
R 10 represents —CO-A 3 -CO—NH-Substrate, with A 3 representing a linear or branched alkylene chain comprising from 1 to 6 carbon atoms; or

in which:
R 5 , R 6 , which may be identical or different, represent, independently of one another, a hydrogen, chlorine, bromine, iodine or fluorine atom, or an alkyl group having from 1 to 12 carbon atoms, alkenyl group having from 2 to 1

Assignees

• Univ Claude Bernard Lyon
• Centre Nat Rech Scient

Inventors

• Chaix Carole
• De Crozals Gabriel

Classifications

• C07H1/00

  Processes for the preparation of sugar derivatives · CPC title

• C09B21/00

  Thiazine dyes · CPC title

• C07F9/6547 Primary

  condensed with carbocyclic rings or {carbocyclic} ring systems · CPC title

• C07H21/00

  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 title

• C07D279/18 Primary

  [b, e]-condensed with two six-membered rings · CPC title