Reagents useful for synthesizing rhodamine-labeled oligonucleotides

Patent metadata

Field	Value
Publication number	US-9783560-B2
Application number	US-201514702499-A
Country	US
Kind code	B2
Filing date	May 1, 2015
Priority date	Mar 31, 2006
Publication date	Oct 10, 2017
Grant date	Oct 10, 2017

Abstract

The present disclosure provides reagents that can be used to label synthetic oligonucleotides with rhodamine dyes or dye networks that contain rhodamine dyes.

First claim

What is claimed is:

1. A reagent useful for labeling an oligonucleotide, which is a compound according to structural formula (VII):
LM-L-PEP  (VII)

wherein LM represents a label moiety that comprises an N-protected NH-rhodamine moiety, PEP represents a phosphate ester precursor group which comprises a phosphoramidite group or an H-phosphonate group, and L represents an optional linker linking the label moiety to the phosphate ester precursor group, and
further wherein the in which the N-protected NH-rhodamine moiety comprises a structure selected from structural formulae (IIIc), (IIIb) and (IIIc):

wherein:
R′ is selected from R 3′ and hydrogen;
R″ is selected from R 6′ and hydrogen;
R 9 is a protecting group having a structure of the formula —C(O)R 10 , wherein R 10 is selected from hydrogen, a lower alkyl, —CX 3 , —CHX 2 , —CH 2 X, —CH 2 —OR d , and phenyl optionally monosubstituted with a lower alkyl, —X, —OR d , cyano or nitro group, wherein R d is selected from a lower alkyl, phenyl and pyridyl, and each X is a halo group;
each of R 1′ , R 2′ , R 2″ , R 4′ , R 4″ , R 5′ , R 5″ , R 7′ , R 7″ , R 8′ , R 4 , R 5 , R 6 , and R 7 , when taken alone, are each, independently of one another, selected from hydrogen, lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5-14 membered heteroaryl, 6-20 membered heteroarylalkyl, —R b and —(CH 2 ) x —R b , where x is an integer ranging from 1 to 10 and R b is selected from —X, —OH, —OR a , —SH, —SR a , —NH 2 , —NHR a , —NR c R c , —N + R c R c R c , perhalo lower alkyl, trihalomethyl, trifluoromethyl, —B(OH) 3 , —B(OR a ) 3 , —B(OH)O − , —B(OR a ) 2 O − , —B(OH)(O − ) 2 , —B(OR a )(O − ) 2 , —P(OH) 2 , —P(OH)O − , —P(OR a ) 2 , —P(OR a )O − , —P(O)(OH) 2 , —P(O)(OH)O − , —P(O)(O − ) 2 , —P(O)(OR a ) 2 , —P(O)(OR a )O − , —P(O)(OH)(OR a ), —OP(OH) 2 , —OP(OH)O − , —OP(OR a ) 2 , —OP(OR a )O − , —OP(O)(OH) 2 , —OP(O)(OH)O − , —OP(O)(O − ) 2 , —OP(O)(OR a ) 2 , —OP(O)(OR a )O − , —OP(O)(OR a )(OH), —S(O) 2 O − , —S(O) 2 OH, —S(O) 2 R a , —C(O)H, —C(O)R a , —C(S)X, —C(O)O − , —C(O)OH, —C(O)NH 2 , —C(O)NHR a , —C(O)NR c R c , —C(S)NH 2 , —C(O)NHR a , —C(O)NR c R c , —C(NH)NH 2 , —C(NH)NHR a , and —C(NH)NR c R c , where X is halo, each R a is, independently of the others, selected from lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5-14 membered heteroaryl and 6-20 membered heteroarylalkyl, and each R e is, independently of the others, an R a , or, alternatively, two R c bonded to the same nitrogen atom may be taken together with that nitrogen atom to form a 5- to 8-membered saturated or unsaturated ring that may optionally include one or more of the same or different ring heteroatoms, which are typically selected from O, N and S, or, alternatively, R 1′ and R 2′ or R 7′ and R 8′ are taken together with the carbon atoms to which they are bonded to form an optionally substituted (C6-C14) aryl bridge and/or R 4′ and R 4″ and/or R 5′ and R 5″ are taken together with the carbon atoms to which they are bonded to form a benzo group; and

each of R 3′ and R 6′ , when taken alone, are each, independently of one another, selected from lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5-14 membered heteroaryl and 6-20 membered heteroarylalkyl, or alternatively, R 3′ and R 2′ or R 4′ and/or R 6′ and R 5′ or R 7′ in the compounds of structural formula (IIIa), R 3′ and R 2′ or R 4′ and/or R 6′ and R 5′ or R 7″ in the compounds of structural formula (III6), or R 3′ and R 2″ or R 4′ and/or R 6′ and R 5′ or R 7″ in the compounds of structural formula (IIIc) are taken together with the atoms to which they are bonded to form a 5- or 6-membered saturated or unsaturated ring which is optionally substituted with one or more of the same or different lower alkyl, benzo or pyrido groups,
with the proviso that at least one of R 2′ , R 4′ , R 5′ , R 7′ , R 5 or R 6 in the compounds structural formula (IIIa), at least one of R 2′ , R 4′ , R 5′ , R 7″ , R 5 or R 6 in the compounds of structural formula (IIIb) and at least one of R 2″ , R 4′ , R 5′ , R 7″ , R 5 or R 6 in the compounds of structural formula (IIIc) comprises a group of the formula —Y—, where Y is chosen from —C(O)—, —S(O) 2 —, —S—, and —NH.

2. The reagent of claim 1 in which L is selected from —Z—(CH 2 ) 3-6 —O − , —Z—(CH 2 ) a —[(Ar) b —(CH 2 ) a ] c —O—, —Z—(CH 2 ) a —[C≡C—(CH 2 ) a ] c —O—, —Z—(CH 2 ) a —[C≡C—(Ar) b ] c —(CH 2 ) a —O—, —Z—(CH 2 ) d —NH—C(O)—[(CH 2 ) a —(Ar)—(CH 2 ) a —C(O)—NH] c —(CH 2 ) d —O—, and —Z—[CH 2 (CH 2 ) e O] f —CH 2 (CH 2 ) e O—, wherein:
each Z represents, independently of the others, a portion of a linkage contributed by a functional group F z ;
each a represents, independently of the others, an integer ranging from 0 to 4;
each b represents, independently of the others, an integer ranging from 1 to 2;
each c represents, independently of the others, an integer ranging from 1 to 5;
each d represents, independently of the others, an integer ranging from 1 to 10;
each e represents, independently of the others, an integer ranging from 1 to 4;
each f represents, independently of the others, an integer ranging from 1 to 10; and
each Ar represents, independently of the others, an optionally substituted monocyclic or polycyclic cycloalkylene, cycloheteroalkynene, arylene or heteroarylene group.

3. The reagent of claim 2 in which each Ar is, independently of the others, a group derived from cyclohexane, piperazine, benzene, napthalene, phenol, furan, pyridine, piperidine, imidazole, pyrrolidine or oxadizole.

4. The reagent of claim 1 which is a compound according to structural formula (VII.1):

wherein B represents a suitably protected nucleobase and L 2 represents a linker linking nucleobase B to label moiety LM.

5. The reagent of claim 4 in which the nucleobase is selected from adenine, 7-deazaguanine, guanine, 7-deazaguanine, cytosine, uracil, thymine, inosine, xanthene and hypoxanthene.

6. The reagent of claim 4 in which B is selected from A iBu , A Pac , C Ac , G iPr-Pac , T and U.

7. The reagent of claim 4 in which L 2 is selected from —C≡C—CH 2 —NH—, —C≡C—C(O)—, —CH═CH—NH—, —CH═CH—C(O)—, —C≡C—CH 2 —NH—C(O)—(CH 2 ) 1-6 —NH—, —CH═CH—C(O)—NH—(CH 2 ) 1-6 —NH—C(O)—, —C≡CH—CH 2 —O—CH 2 CH 2 —[O—CH 2 CH 2 ] 0-6 —NH—, —C≡C—C≡C—CH 2 —O—CH 2 CH 2 —[O—CH 2 CH 2 ] 0-6 —NH—, —C≡C—(Ar) 1-2 —C≡C—CH 2 —O—CH 2 CH 2 —[O—CH 2 CH 2 ] 0-6 —NH—, —C≡C—(Ar) 1-2 —O—CH 2 CH 2 —[O—CH 2 CH 2 ] 0-6 —NH— and —C≡C—(Ar) 1-2 —O—CH 2 CH 2 —[O—CH 2 CH 2 ] 0-6 —NH—, where Ar is as defined in claim 2 .

8. The reagent of claim 4 in which —B-L 2 - is selected from

9. The reagent of claim 1 further comprising a suitably protected synthesis handle.

10. The reagent of claim 9 which is a compound according to structural formula (VIII):
R e O-L-LM-L-PEP  (VIII)

wherein R e represents an acid-labile protecting group, each L represents, independently of the other, an optional linker, LM represents the label moiety and PEP represents the phosphate ester precursor group.

11. The reagent of claim 9 which is a compound according to structural formula (IX):

wherein LM represents the label moiety, L represents a linker, R e represents an acid-labile p

Assignees

• Applied Biosystems Llc

Inventors

• Benson Scott C
• Zou Ruiming N
• Upadhya Krishna G
• Kenney Paul M
• Cassel Jonathan M

Classifications

• C07F9/6561 Primary

  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 title

• C07H19/04

  Heterocyclic radicals containing only nitrogen atoms as ring hetero atom · CPC title

• C07H21/04

  with deoxyribosyl as saccharide radical · CPC title

• C07D311/96

  spiro-condensed with carbocyclic rings or ring systems · CPC title

• C09B11/24

  Phthaleins containing amino groups {; Phthalanes; Fluoranes; Phthalides; Rhodamine dyes; Phthaleins having heterocyclic aryl rings; Lactone or lactame forms of triarylmethane dyes} · CPC title