Reagents useful for synthesizing rhodamine-labeled oligonucleotides

1. A method of synthesizing an oligonucleotide labeled with an energy transfer dye pair that comprises an N-protected NH-rhodamine dye moiety, comprising the steps of:

i. coupling a donor or an acceptor dye phosphoramidite reagent to a support-bound protected oligonucleotide;

ii. optionally coupling one or more linker phosphoramidite reagents to the oxidized product of step i; and

iii. coupling an acceptor or a donor dye phosphoramidite reagent to the oxidized product of step ii,

wherein the acceptor dye phosphoramidite reagent is an N-protected NH-rhodamine phosphoramidite reagent, and wherein the N-protected NH-rhodamine dye moiety comprises a structure selected from:

wherein LM represents a label moiety comprising the N-protected NH-rhodamine dye, PEP represents the phosphate ester precursor group, B represents a suitably protected nucleobase, L 2 represents a linker linking label moiety LM to nucleobase B, and further wherein the N-protected NH-rhodamine dye moiety of the labeling moiety comprises a structure chosen from structural formulae (IIIa):

wherein:

R′ is selected from R 3′ and hydrogen;

R″ is selected from R 6′ and hydrogen;

R 9 is an acyl protecting group;

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, chosen 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 chosen 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(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, chosen from lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5-14 membered heteroaryl and 6-20 membered heteroarylalkyl, and each R c 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

R 3′ and R 6′ , when taken alone, are each, independently of one another, chosen from lower alkyl, (C6-C14) aryl, (C7-C20) arylalkyl, 5-14 membered heteroaryl and 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 , 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),

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) comprises a group of the formula —Y—, wherein —Y— is chosen from —C(O)—, —S(O) 2 —, —S—, and —NH.

2. The method of claim 1 in which the donor dye phosphoramidite reagent is an O-protected fluorecein phosphoramidite reagent.

3. The method of claim 1 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 each Ar represents, independently of the others, an optionally substituted monocyclic or polycyclic cycloalkylene, cycloheteroalkynene, arylene or heteroarylene group.

4. The method of claim 1 , in which at least one 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 is chosen from —R b and —(CH 2 ) x —R b .

5. The method of claim 1 , in which the N-protected NH-rhodamine dye moiety comprises a structure selected from structural formulae (IIIa.1), (IIIa.2):

wherein R′, R″, R 2′ , R 2″ , R 3′ , R 4′ , R 4″ , R 5′ , R 5″ , R 6′ , R 7′ , R 7″ , R 8′ , R 4 , R 5 , R 6 , R 7 , R 9 and Y are as previously defined in claim 1 .

6. The method of claim 1 in which the N-protected NH-rhodamine dye moiety has one or more applicable features chosen from:

R 4 and R 7 are each chloro;

(ii) R 1′ and R 8′ are each hydrogen;

(iii) R 1′ and R 2′ or R 7′ and R 8′ are taken together to form a benzo group;

(iv) R 2′ and R 7′ are each hydrogen or lower alkyl;

(v) R′ is R 3′ and R″ is R 6′ ; and

(vi) R′ is R 3′ , R″ is R 6′ , and R 3′ and R 6′ are taken together with a substituent group on an adjacent carbon atom to form a group selected from —CH 2 CH 2 —, —CH 2 CH 2 CH 2 —, —C(CH 3 ) 2 CH═C(CH 3 )—, —C(CH 3 ) 2 CH═CH—, —CH 2 —C(CH 3 ) 2 — and

7. The method of claim 1 in which the energy transfer dye label moiety is linked to the 3′- or 5′-hydroxyl of the oligonucleotide.

8. The method of claim 1 in which the energy transfer dye label moiety is linked to a nucleobase of the oligonucleotide.

9. The method of claim 1 in which the oligonucleotide labeled with an energy transfer dye is further labeled with a donor and/or acceptor moiety for the N-protected NH-rhodamine moiety.

10. The method of claim 9 in which the energy transfer dye label moiety comprises structural formula (VI):

A-Z 1 -Sp-Z 2 -D  (VI)

wherein A represents the N-protected NH-rhodamine moiety, D represents the donor moiety, Z 1 and Z 2 , which may be the same or different, represent a portion of a linkage contributed by a functional group F z , and Sp represents a spacing moiety.

11. The method of claim 10 in which A is selected from structural formula A.1 and D is selected from structural formula D.1, or A is selected from structural formula A.7 and D is selected from structural formula D.7;

wherein:

E 1 is selected from —NHR 9 , —NR 3′ R 9 and —OR 9b ;

E 2 is selected from —NHR 9 , —NR 6′ R 9 and —OR 9b ;

R 9b is R 9 ;

Y 1a , Y 1b , Y 2a , Y 2b , Y 3a and Y 3b are each, independently of one another, selected from —O—, —S—, —NH—, —C(O—) and —S(O) 2 —; and

R′, R″, R 2′ , R 2″ , R 3′ , R 4′ , R 4″ , R 5′ , R 5″ , R 6′ , R 7′ , R 7″ , R 8′ , R 4 , R 5 , R 6 , R 7 , and R 9 are as previously defined in claim 1 , with the proviso that when E 1 and E 2 are —OR 9b , then R 1′ and R 2′ and/or R 7′ and R 8′ may only be taken together with the carbon atoms to which they are bound to form an optionally substituted (C6-C14) aryl bridge.

12. The method of claim 11 in which A is selected from structural formula A.1.

13. The method of claim 1 in which the oligonucleotide labeled with an energy transfer dye is further labeled with a quencher moiety.

14. The method of claim 1 in which the oligonucleotide labeled with an energy transfer dye is further labeled with a minor groove binding moiety.

15. The method of claim 1 which further comprises the steps of deprotecting and cleaving the oligonucleotide labeled with an energy transfer dye from the support.