Coumarin-based compounds and related methods
The present invention generally relates to coumarin-based compounds that can be used for the labeling of biological molecules, as well as related synthetic and testing methods. The coumarin-based compounds typically survive conditions necessary for automated synthesis of nucleic acids without undergoing any substantial degree of degradation or alteration.
1. A compound of Structure 15 , 16 or 17 :
where
m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
n is 1,2 or 3;
o is 0, 1, 2, or 3;
p is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
R 2 is selected from a group consisting of —H or —CH 3 ;
X is —OH, —NH 2 , —SH, —OR 11 —NHR 12 —NR 13 R 14 —OC(O)R 15 —NHC(O)R 16 , SC(O)R 17 , —C(O)OH, —C(O)OR 18 , —C(O)NHR 19 , —C(O)NR 20 R 21 , —N 3 ;
R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , and R 21 , are independently selected from (C 1 -C 8 )alkyl, (C 1 -C 8 )substituted alkyl, —CH 2 CH(OH)CH 2 (OH), —CH(CH 2 OH) 2 , —CH 2 CCH, —(CH 2 CH 2 O)a —CH 2 CH 2 OH where a is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, —P(NR 22 R 23 )OCH 2 CH 2 -EWG, —P(O)(O − )-Oligonucleotide, —(CH 2 ) b CC-Nucleoside, —(CH 2 ) b CC-[Nucleoside Triphosphate], —(CH 2 ) b -Nucleoside, —(CH 2 ) b -[Nucleoside Triphosphate], —(CH 2 ) b NH-Nucleoside, —(CH 2 ) b NH-[Nucleoside Triphosphate], —(CH 2 ) b NHC(O)(CH 2 ) b -Nucleoside, and —(CH 2 ) b NHC(O)(CH 2 ) b -[Nucleoside Triphosphate], where b is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
EWG is an electron withdrawing group; and
R 22 and R 23 are independently selected from —CH 3 , —CH 2 CH 3 , —CH(CH 3 ) 2 or R 22 and R 23 together form a heterocycle; and
in the compound of Structure 15, R x and R y are independently H or CH 3 ,
in the compound of Structure 16, R 7 and R 8 are independently selected from H, (C 1 -C 8 ) alkyl, (C 1 -C 8 )substituted alkyl, or R 7 and R 8 together form a five-, six-, or seven-membered heterocycle; and
in the compound of Structure 17, R 24 is (C 1 -C 8 )alkyl or (C 1 -C 8 )substituted alkyl;
R 25 is (C 1 -C 8 )alkyl, (C 1 -C 8 )substituted alkyl or CH 2 SO 3 H; and
the dashed line represents an optional double bond.
2. The compound according to claim 1 , wherein m is 1, 2 or 3, n is 1 or 2, o is 1 or 2, and p is 0, 1, 2 or 3.
3. The compound according to claim 1 , wherein X is —OH, —OR 11 , or OC(O)R 15 .
4. A compound of Structure 14:
where
m is 0;
n is 1, 2 or 3;
o is 1, 2, or 3;
p is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
R 2 is selected from a group consisting of —H, or —CH 3 ;
R 3 is selected from a group consisting of —H, -halogen, —CF 3 , —OH, —NH 2 , —CCH, —CN, —NO 2 , (C 1 -C 8 )alkyl, (C 1 -C 8 )substituted alkyl, (C 1 -C 8 )alkylthio, (C 1 -C 8 ) substituted alkylthio, (C 1 -C 8 )alkoxy, (C 1 -C 8 )substituted alkoxy, —NR 7 R 8 where R 7 and R 8 are independently selected from H, (C 1 -C 8 )alkyl or R 7 and R 8 together form a four-, five-, six-, or seven-membered heterocycle, (C 1 -C 8 )heteroalkyl, (C 1 -C 8 )substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, or R 3 together with R 4 form a five- or six-membered carbocyclic or heterocyclic ring or substituted heterocyclic ring;
R 4 is selected from a group consisting of —H, -halogen, —CF 3 , —OH, —NH 2 , —CCH, —CN, —NO 2 , (C 1 -C 8 )substituted alkyl, (C 1 -C 8 )alkylthio, (C 1 -C 8 ) substituted alkylthio, (C 1 -C 8 )alkoxy, (C 1 -C 8 )substituted alkoxy, —NR 7 R 8 where R 7 and R 8 are independently selected from H, (C 1 -C 8 )alkyl or R 7 and R 8 together form a four-, five-, six-, or seven-membered heterocycle, (C 1 -C 8 )heteroalkyl, (C 1 -C 8 )substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, or R 4 together with R 5 form a five- or six-membered carbocyclic or heterocyclic ring having nitrogen as a heteroatom or substituted heterocyclic ring having nitrogen as a heteroatom;
R 5 is selected from a group consisting of —H, -halogen, —CF 3 , —OH, —NH 2 ,—CCH, —CN, —NO 2 , (C 1 -C 8 )alkyl, (C 1 -C 8 )substituted alkyl, (C 1 -C 8 )alkylthio, (C 1 -C 8 ) substituted alkylthio, (C 1 -C 8 )alkoxy, (C 1 -C 8 )substituted alkoxy, —NR 7 R 8 where R 7 and R 8 are independently selected from H, (C 1 -C 8 )alkyl or R 7 and R 8 together form a four-, five-, six-, or seven-membered heterocycle, (C 1 -C 8 )heteroalkyl, (C 1 -C 8 )substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, or R 5 together with R 6 form a five- or six-membered carbocyclic or heterocyclic ring or substituted heterocyclic ring; or R 4 , R 5 and R 6 together form fused carbocyclic or heterocyclic ring systems or substituted heterocyclic ring systems having five- or six-membered rings;
R 6 is selected from a group consisting of —H, -halogen, —CF 3 , —OH, —NH 2 , —CCH, —CN, —NO 2 , (C 1 -C 8 )alkyl, (C 1 -C 8 )substituted alkyl, (C 1 -C 8 )alkylthio, (C 1 -C 8 ) substituted alkylthio, (C 1 -C 8 )alkoxy, (C 1 -C 8 )substituted alkoxy, —NR 7 R 8 where R 7 and R 8 are independently selected from H, (C 1 -C 8 )alkyl or R 7 and R 8 together form a four-, five-, six-, or seven-membered heterocycle, (C 1 -C 8 )heteroalkyl, (C 1 -C 8 )substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;
X is selected from —OH —NH 2 —SH —OR 11 —NHR 12 —NR 13 R 14 —OC(O)R 15 —NHC(O)R 16 —SC(O)R 17 , —C(O)OH, —C(O)OR 18 , —C(O)NHR 19 , —C(O)NR 20 R 21 , —N 3 ;
R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 , and R 21 , are independently selected from (C 1 -C 8 )alkyl, (C 1 -C 8 )substituted alkyl, —CH 2 CH(OH)CH 2 (OH), —CH(CH 2 OH) 2 , —CH 2 CCH, —(CH 2 CH 2 O)a—CH 2 CH 2 OH where a is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, —P(NR 22 R 23 )OCH 2 CH 2 -EWG, —P(O)(O − )-Oligonucleotide, —(CH 2 ) b CC-Nucleoside, —(CH 2 ) b CC -[Nucleoside Triphosphate], —(CH 2 ) b -Nucleoside, —(CH 2 ) b -[Nucleoside Triphosphate], —(CH 2 ) b NH-Nucleoside, —(CH 2 ) b NH-[Nucleoside Triphosphate], —(CH 2 ) b NHC(O)(CH 2 ) b -Nucleoside, —(CH 2 ) b NHC(O)(CH 2 ) b -[Nucleoside Triphosphate], where b is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
EWG is an electron withdrawing group; and
R 22 , and R 23 , are independently selected from —CH 3 , —CH 2 CH 3 , —CH(CH 3 ) 2 or R 22 and R 23 together form a heterocycle.
5. The compound according to claim 4 , wherein R 3 is —H or (C 1 -C 8 )alkyl.
6. The compound according to claim 5 , wherein X is —OH, —OR 11 , or OC(O)R 15 .
7. The compound according to claim 6 , wherein R 4 is —H or R 4 together with R 5 form a five- or six-membered carbocyclic or heterocyclic ring.
8. A compound of Structure 14:
where
m is 0;
n is 0;
o is 0;
p is 3, 4, 5, 6, 7, 8, 9, or 10
R 2 is selected from a group consisting of —H, or —CH 3 ;
R 3 is selected from a group consisting of —H, -halogen;
R 4 is selected from a group consisting of —H, -halogen, —CF 3 , —OH, —NH 2 , —CCH, —CN, —NO 2 , (C 1 -C 8 )alkyl, (C 1 -C 8 )substituted alkyl, (C 1 C 8 )alkylthio, (C 1 -C 8 ) substituted alkylthio, (C 1 -C 8 )alkoxy, (C 1 C 8 )substituted alkoxy, -NR 7 R 8 where R 7 and R 8 are independently selected from H, (C 1 -C 8 )alkyl or R 7 and R 8 together form a four-, five-, six-, or seven-membered heterocycle, (C 1 -C 8 )heteroalkyl, (C 1 -C 8 )substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, or R 4 together with R 5 form a five- or six-membered carbocyclic or heterocyclic ring having nitrogen as a heteroatom or substituted heterocyclic ring having nitrogen as a heteroatom;
R 5 is selected from a group consisting of —H, -halogen, —CF 3 , —OH, —NH 2 , —CCH, —CN, —NO 2 , (C 1 -C 8 )alkyl, (C 1 -C 8 )substituted alkyl, (C 1 C 8 )alkylthio, (C 1 -C 8 ) substituted alkylthio, (C 1 -C 8 )alkoxy, (C 1 C 8 )substituted alkoxy, —NR 7 R 8 where R 7 and R 8 are independently selected from H, (C 1 -C 8 )alkyl or R 7 and R 8 together form a four-, five-, six-, or seven-membered heterocycle, (C 1 -C 8 )heteroalkyl, (C 1 -C 8 )substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, or R 5 together with R 6 form a five- or six-membered carbocyclic or heterocyclic ring or substituted heterocyclic ring; or R 4 , R 5 and R 6 together form fused carbocyclic or heterocyclic ring systems or substituted heterocyclic ring systems having five- or six-membered rings;
R 6 is selected from a group consisting of —H, -halogen, —CF 3 , —OH, —NH 2 ,—CCH, —CN, —NO 2 , (C 1 -C 8 )alkyl, (C 1 -C 8 )substituted alkyl, (C 1 -C 8 )alkylthio, (C 1 -C 8 ) substituted alkylthio, (C 1 -C 8 )alkoxy, (C 1 -C 8 )substituted alkoxy, —NR 7 R 8 where R 7 and R 8 are independently selected from H, (C 1 -C 8 )alkyl or R 7 and R 8 together form a four-, five-, six-, or seven-membered heterocycle, (C 1 -C 8 )heteroalkyl, (C 1 -C 8 )substituted heteroalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl;
X is selected from —OH, —OR 11 and —OC(O)R 15 —;
R 11 , and R 15 , are independently selected from (C 1 -C 8 )alkyl, (C 1 -C 8 )substituted alkyl, —CH 2 CH(OH)CH 2 (OH), —CH(CH 2 OH) 2 , —CH 2 CCH, —(CH 2 CH2O)a—CH 2 CH 2 OH where a is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 , —P(NR 22 R 23 )OCH 2 CH 2 -EWG, —P(O)(O − )-Oligonucleotide, —(CH 2 ) b CC-Nucleoside, —(CH 2 ) b CC -[Nucleoside Triphosphate], —(CH 2 ) b -Nucleoside, —(CH 2 ) b -[Nucleoside Triphosphate], —(CH 2 ) b NH-Nucleoside, —(CH 2 ) b NH-[Nucleoside Triphosphate], —(CH 2 ) b NHC(O)(CH 2 ) b -Nucleoside, —(CH 2 ) b NHC(O)(CH 2 ) b -[Nucleoside Triphosphate], where b is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
EWG is an electron withdrawing group; and
R 22 , and R 23 , are independently selected from —CH 3 , —CH 2 CH 3 , —CH(CH 3 ) 2 , or R 22 and R 23 together form a heterocycle.
9. The compound according to claim 8 , wherein R 4 is —H or R 4 together with R 5 form a five- or six-membered carbocyclic or heterocyclic ring.
10. The compound according to claim 1 , wherein X is —OR 11 and R 11 represents —P(NR 22 R 23 )OCH 2 CH 2 -EWG, wherein
EWG is an electron withdrawing group and R 22 , and R 23 are independently selected from —CH 3 , —CH 2 CH 3 , —CH(CH 3 ) 2 , or R 22 and R 23 together form a heterocycle.
11. The compound according to claim 1 , wherein the compound is of Structure 15.
12. The compound according to claim 1 , wherein the compound is of Structure 16.
13. The compound according to claim 1 , wherein the compound is of Structure 17.
14. The compound according to claim 1 , wherein the compounds of structure 15 or 16, X is —NH 2 , —NHR 12 , —NR 13 R 14 , —NHC(O)R 16 .
15. The compound according to claim 1 , wherein, in the compound of structure 16, R 7 and R 8 are independently selected from H or (C 1 -C 8 )alkyl.
16. The compound according to claim 1 , wherein, in the compound of structure 17, R 24 and R 25 are (C 1 -C 8 )alkyl.
17. A compound of Structure 27, 28 or 29
where
m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10,
R 2 is selected from a group consisting of —H or -CH 3 ;
R 9 is —CH 3 , —CH 2 CH 3 , —CH(CH 3 ) 2 ;
X is —OH, —NH2, —SH, —OR 11 , —NHR 12 , —NR 13 R 14 , —OC(O)R 15 , —NHC(O)R 16 , —SC(O)R 17 , —C(O)OH, —C(O)OR 18 , —C(O)NHR 19 , —C(O)NR 20 R 21 , —N 3 ;
R 11 , R 12 , R 13 , R 14 , R 15 , R 16 , R 17 , R 18 , R 19 , R 20 and R 21 are independently selected from (C 1 -C 8 )alkyl, (C 1 -C 8 )substituted alkyl, —CH 2 CH(OH)CH 2 (OH), —CH(CH 2 OH) 2 , —CH 2 CCH, —(CH 2 CH 2 O) a -CH 2 CH 2 OH where a is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, —P(NR 22 R 23 )OCH 2 CH 2 -EWG, —P(O)(O—)-Oligonucleotide, —(CH 2 ) b CC-Nucleoside, —(CH 2 ) b CC-[Nucleoside Triphosphate], —(CH 2 ) b -Nucleoside, —(CH 2 ) b -[Nucleoside Triphosphate], —(CH 2 ) b NH-Nucleoside, —(CH 2 ) b NH-[Nucleoside Triphosphate], —(CH 2 ) b NHC(O)(CH2) b -Nucleoside, —(CH 2 ) b NHC(O)(CH 2 ) b -[Nucleoside Triphosphate], where b is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10,
EWG is an electron withdrawing group,
R x and R y are independently H and CH 3 ;
R 22 and R 23 are independently selected from —CH 3 , —CH 2 CH 3 , —CH(CH 3 ) 2 or R 22 and R 23 together form a heterocycle; and
in the compound of Structure 27, R x and R y are independently H and CH 3 ;
in the compound of Structure 28, R 7 and R 8 are independently selected from H, (C 1 - C 8 )alkyl, (C 1 -C 8 )substituted alkyl, or R 7 and R 8 together form a five-, six-, or seven-membered heterocycle; and
in the compound of Structure 29,
R 24 is (C 1 -C 8 )alkyl or (C 1 -C 8 )substituted alkyl;
R 25 is (C 1 -C 8 )alkyl, (C 1 -C 8 )substituted alkyl or CH 2 SO 3 H; and
the dashed line represents an optional double bond.
18. The compound according to claim 17 , wherein X is —OH, —OR 11 , or OC(O)R 15 .
19. The compound according to claim 17 , wherein, in the compound of Structure 28, R 7 and R 8 are independently selected from H or (C 1 -C 8 )alkyl.
20. The compound according to claim 17 , wherein, in the compound of Structure 29, R 24 and R 25 are (C 1 -C 8 )alkyl.
21. The compound according to claim 17 , wherein X is —OR 11 and R 11 represents —P(NR 22 R 23 )OCH 2 CH 2 -EWG, wherein
EWG is an electron withdrawing group and R 22 , and R 23 are independently selected from —CH 3 , —CH 2 CH 3 , —CH(CH 3 ) 2 , or R 22 and R 23 together form a heterocycle.
22. The compound according to claim 17 , wherein the compound is of Structure 27.
23. The compound according to claim 17 , wherein the compound is of Structure 28.
24. The compound according to claim 17 , wherein the compound is of Structure 29.
25. A coumarin-CPG compound of Structure 150, wherein the coumarin-CPG compound is of Structure 150,
wherein the coumarin moiety is of structure 153, 154 or 155,
wherein:
m is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
R 2 is H or CH 3 ;
R 9 is CH 3 , CH 2 CH 3 or CH(CH 3 ) 2 ;
in the moiety of Structure 153, R x and R y are independently H or CH 3 ;
in the moiety of Structure 154, R 7 and R 8 are independently selected from H, (C 1 -C 8 )alkyl, (C 1 -C 8 )substituted alkyl, or R 7 and R 8 together form a four-, five-, six-, or seven-membered heterocycle;
in the moiety of Structure 155, R 24 is (C 1 -C 8 )alkyl or (C 1 -C 8 )substituted alkyl, R 25 is (C 1 -C 8 )alkyl, (C 1 -C 8 )substituted alkyl or CH 2 SO 3 H and the dashed lines represents an optional double bond;
Z is (CH 2 ) x , wherein x is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;
Linker 1 is selected from OC(O)(CH 2 ) y O(CH 2 ) z (CO), NHC(O)(CH 2 ) y O(CH 2 ) z (CO), wherein y is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, and wherein z is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; and
Linker 2 is selected from NH(CH 2 ) a , wherein a is 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.