Transfection reagents
View Patent ↗Disclosed are compounds capable of facilitating transport of biologically active agents or substances into cells having the general structure: wherein Q is selected from the group consisting of N, O and S; L is any bivalent organic radical capable of linking each Q, such as C, CH, (CH 2 )l, or {(CH 2 )i-Y—(CH 2 )j}k, wherein Y is selected from the group consisting of CH 2 , an ether, a polyether, an amide, a polyamide, an ester, a sulfide, a urea, a thiourea, a guanidyl, a carbamoyl, a carbonate, a phosphate, a sulfate, a sulfoxide, an imine, a carbonyl, and a secondary amino group and wherein Y is optionally substituted by —X 1 -L′-X 2 -Z or -Z; R 1 -R 6 , independently of one another, are selected from the group consisting of H, —(CH 2 )p-D-Z, an alkyl, an alkenyl, an aryl, and an alkyl or alkyl ether optionally substituted by one or more of an alcohol, an aminoalcohol, an amine, an amide, an ether, a polyether, a polyamide, an ester, a mercaptan, an alkylthio, a urea, a thiourea, a guanidyl, or a carbamoyl group, and wherein at least one of R 1 , R 3 , R 4 and R 6 is a straight chain or branched, cyclic, alkyl, alkenyl, alkynyl or aryl group; and anyone of R 1 , R 3 , R 4 and/or R 6 may optionally be covalently linked with each other, with Y or with L when L is C or CH to form a cyclic moiety; Z is selected from the group consisting of amine, spermiyl, carboxyspermiyl, guanidyl, spermidinyl, putricinyl, diaminoalkyl, pyridyl, piperidinyl, pyrrolidinyl, polyamine, amino acid, peptide, and protein; X 1 and X 2 , independently of one another, are selected from the group consisting of NH, O, S, alkylene, and arylene; L′ is selected from the group consisting of alkylene, alkenylene, alkynylene, arylene, alkylene ether, and polyether; D is Q or a bond; A 1 and A 2 , independently of one another, are selected from the group consisting of CH 2 O, CH 2 S, CH 2 NH, C(O), C{NH), C(S) and (CH 2 )t; X is a physiologically acceptable anion; m, n, r, s, u, v, w and y are 0 or 1, with the proviso that when both m and n are 0 at least one of r, s, u and y is other than 0; i, j, k, l, p and are integers from 0 to about 100; q is an integer from 1 to about 1000; and a is the number of positive charge divided by the valence of the anion.
1. A method for introducing a nucleic acid into a cell, comprising the steps of contacting the nucleic acid with a polycation, thereby forming a complex;
and incubating the complex with the cell, wherein the polycation has the formula:
where:
Q is N;
L is [(CH 2 ) i —Y—(CH 2 ) j ] k , where Y is a CH 2 , an ether, an amide, a thiourea, an imine, a carbonyl, or a secondary amino group;
R 1 , R 3 , R 4 , and R 6 , independently of one another, are selected from H, alkyl, alkenyl, or alkynyl;
R 2 and R 5 , independently of one another, are —(CH 2 ) p -D-Z, wherein (CH 2 ) p is optionally substituted with —OH;
D is a bond or Q;
Z is an amine, a polyamine or a diaminoalkyl optionally substituted with —OH;
X − is a physiologically acceptable anion;
a is the number of positive charges divided by the valence of the anion;
m and n are 1;
r, s, u, and y are 1;
i and j are independently an integer from 0 to about 100;
k is an integer from 1 to about 100;
p is an integer from 1 to about 100;
q is an integer from 1 to about 100, and wherein,
i) R 1 , R 3 , R 4 , and R 6 are optionally substituted by one or more of an alcohol, an amino alcohol, or an amine; and
ii) any two or more of R 1 , R 3 , R 4 and R 6 are covalently linked with each other thereby forming a cyclic moiety.
2. The method of claim 1 , wherein Y is CH 2 .
3. The method of claim 1 , wherein D is a bond.
4. The method of claim 1 , wherein p and k independently are integers from 1 to 25, and i and j independently are integers from 0 to 25.
5. The method of claim 1 , wherein p and k independently are integers from 1 to 10, and i and j independently are integers from 0 to 10.
6. The method of claim 1 , wherein p and k independently are integers from 1 to 4, i and j independently are integers from 0 to 4, and q is 1.
7. The method of claim 2 , wherein R 2 and R 5 are (CH 2 ) p -D-Z, wherein (CH 2 ) ) p is substituted with an —OH.
8. The method of claim 7 , wherein p and k independently are integers from 1 to 4, i and j independently are integers from 0 to 4, and q is 1.
9. The method of claim 7 , wherein Z is a diaminoalkyl optionally substituted with —OH.
10. The method of claim 7 , wherein D is a bond.
11. The method of claim 1 , wherein R 1 , R 3 , R 1 , and R 6 , independent of one another, are selected from H or an alkyl group.
12. The method of claim 1 , wherein R 2 and R 5 are an amino alcohol group.
13. The method of claim 1 , wherein R 2 and R 5 are independently an aminoalcohol selected from aminoethanol, aminopropanol, or aminobutanol.
14. The method of claim 13 , wherein the amino alcohol group has the structure
15. The method of claim 1 , wherein the nucleic acid is DNA.
16. The method of claim 1 , wherein the nucleic acid is RNA.
17. The method of claim 1 , wherein the nucleic acid inhibits expression of nucleic acids in the cell.
18. The method of claim 1 , wherein the nucleic acid catalyzes reactions.
19. The method of claim 1 , wherein the complex formed is a liposome.
20. The method of claim 1 , wherein the complex is a liposome and wherein the method further comprises the step of forming liposomes after contacting the nucleic acid with the polycation.
21. The method of claim 1 , wherein a mixture of the polycation and a neutral lipid is formed and the nucleic acid is contacted with the mixture to form the complex.
22. The method of claim 1 , wherein a mixture of the polycation and a neutral lipid selected from DOPE, DOPC or cholesterol is formed and the nucleic acid is contacted with the mixture to form the complex.
23. The method of claim 1 , wherein a mixture of the nucleic acid and a peptide or a protein is formed and the polycation is contacted with the mixture to form the complex.
24. A method of making a compound having the structure
wherein L is [(CH 2 ) i —Y—(CH 2 ) j ] k , where Y is a CH 2 , an ether, an amide, a carbonyl, or a secondary amino group; i and j are independently an integer from 0 to about 100; k is an integer from 1 to about 100and R 1 and R 4 are independently C 1 to C 8 alkyl covalently linked with each other thereby forming a cyclic moiety;
wherein the method comprises alkylating a cyclic diamine having the structure
with an epoxyalkyl phthalimide reagent, followed by admixing the resulting compound with a reducing agent.
25. The method of claim 24 . wherein epoxyalkyl phthalimide reagent is epoxypropyl phthalimide and the reducing agent is hydrazine.
26. A compound for introducing a nucleic acid into a cell having the formula
where:
Q is N;
L is [(CH 2 ) i —Y—(CH 2 ) j ] k , where Y is a CH 2, an ether, an amide, a thiourea, an imine, a carbonyl, or a secondary amino group;
R 1 , R 3 , R 4 , and R 6 , independently of one another, are selected from H, alkyl, alkenyl, or alkynyl;
R 2 and R 5 , independently of one another, are —(CH 2 ) p -D-Z, wherein (CH 2 ) p is optionally substituted with OH;
D is a bond or Q;
Z is an amine, a polyamine or a diaminoalkyl optionally substituted with —OH;
X − is a physiologically acceptable anion;
a is the number of positive charges divided by the valence of the anion;
m and n are 1;
r, s, u, andy are 1;
i and j are independently an integer from 0 to about 100;
k is an integer from 1 to about 100;
p is an integer from 1 about 100;
q is an integer from 1 to about 100, and wherein,
i) R 1 , R 3 , R 4 , and R 6 are optionally substituted by one or more of an alcohol, an amino alcohol, or an amine; and
ii) any two or more of R 1 , R 3 , R 4 and R 6 are covalently linked with each other thereby forming a cyclic moiety,
provided that when two of R 1 , R 3 , R 4 , and R 6 are covalently linked with each other thereby forming a cyclic moiety, at least one of R 1 , R 3 , R 4 , and R 6 not forming the cyclic moiety is not alkyl.
27. The compound of claim 26 , wherein Y is CH 2 .
28. The compound of claim 26 , wherein D is a bond.
29. The compound of claim 26 , wherein p and k independently are integers from 1 to 4, i and j independently are integers from 0 to 4, and q is 1.
30. The compound of claim 27 , wherein R 2 and R 5 are (CH 2 ) p -D-Z, wherein (CH 2 ) p is substituted with an —OH.
31. The compound of claim 30 , wherein p and k independently are integers from 1 to 4, i and j independently are integers from 0 to 4, and q is 1.
32. The compound of claim 30 , wherein Z is a diaminoalkyl optionally substituted with —OH.
33. The compound of claim 30 , wherein D is a bond.
34. The compound of claim 26 , wherein R 1 , R 3 , R 4 , and R 6 , independent of one another, are selected from H or an alkyl group.
35. The compound of claim 26 , wherein R 2 and R 5 are an amino alcohol group.
36. The compound of claim 35 , wherein R 2 and R 5 are independently an aminoalcohol group selected from aminoethanol, aminopropanol, or aminobutanol.
37. The compound of claim 35 , wherein the amino alcohol group has the structure
38. The method of claim 8 , wherein Z is a diaminoalkyl optionally substituted with —OH.
39. The compound of claim 31 , wherein Z is a diaminoalkyl optionally substituted with —OH.
40. The method of claim 8 , wherein i is 0 and j is 1.
41. The method of claim 40 , wherein k is 1.
42. The method of claim 41 , wherein p is 3.
43. The compound of claim 31 , wherein i is 0 and j is 1.
44. The compound of claim 43 , wherein k is 1.
45. The compound of claim 44 , wherein p is 3.
46. The method of claim 6 , wherein k is 1.
47. The compound of claim 29 , wherein k is 1.