Thermally-cleavable protecting and linker groups
The present invention relates to chemical linkers and protecting groups, compounds and compositions containing the chemical linkers or protecting groups, and intermediates and processes that can be used to prepare them. The chemical linkers and protecting groups are based on pyrrolidine and piperidine activating groups, which undergo intramolecular cyclisation upon heating with release of carbon dioxide, thereby releasing the organic compound from a substrate. In particular, those chemical linkers and protecting groups are useful in the solid phase synthesis of oligonucleotides according to the following representative schemes.
1. A compound of formula (I), or an acid addition salt thereof:
wherein: M is —W—R 6 , wherein W is —O—, and R 6 is an organic fragment;
X is hydrogen or hydrocarbyl;
Y is hydrocarbyl or
each of R 1 , R 2 , R 3 , R 4 , R 5 and R 7 are hydrogen;
PG is a cleavable protecting group for nitrogen;
n is (i) 0 or (ii) 1; and
ring A is a 5, 6, or 7-membered nitrogen-containing monocyclic heterocyclic group;
wherein PG and A at each occurrence, is the same or different.
2. The compound according to claim 1 , wherein at least one PG is cleavable under a first reaction condition to produce a compound of formula (I*):
wherein the compound of Formula (I*) undergoes intramolecular cyclisation and cleavage with release of carbon dioxide under a second, different, reaction condition, to produce a compound of formula (II):
thereby releasing M or an organic compound comprising M;
wherein:
PG′ is hydrogen or a cleavable protecting group for nitrogen, provided that at least one PG′ is hydrogen;
Y′ is hydrocarbyl, or
3. The compound according to claim 1 , having the formula (IA):
wherein:
Y is hydrocarbyl;
optionally wherein PG is cleavable under a first reaction condition to produce a compound of formula (IA′):
wherein the compound of Formula (IA*) undergoes intramolecular cyclisation and cleavage with release of carbon dioxide under a second, different reaction condition, to produce a compound of formula (IIA):
thereby releasing M or an organic compound comprising M.
4. The compound according to claim 1 , having the formula (IB):
optionally wherein PG and A at each occurrence in formula (IB), is the same.
5. The compound according to claim 4 , wherein at least one PG is cleavable under a first reaction condition to produce a compound of formula (IB*):
wherein
PG′ is hydrogen or a cleavable protecting group for nitrogen, provided that at least one PG′ is hydrogen; and
wherein the compound of Formula (IB*) undergoes intramolecular cyclisation and cleavage with release of carbon dioxide under a second, different reaction condition, to produce a compound of formula (IIB):
thereby releasing M or an organic compound comprising M; optionally wherein PG′ are both H.
6. The compound according to claim 1 , wherein ring A is a heterocycle that is piperidyl, morpholinyl, pyrrolidinyl, or thiomorpholinyl.
7. The compound according to claim 1 , wherein X is H or hydrocarbyl, wherein the hydrocarbyl is alkyl, aryl or arylalkyl.
8. The compound according to claim 1 , wherein the cleavage of at least one PG is activated by pH, temperature, a chemical activating agent, or by a combination thereof; optionally wherein at least one PG is (i) thermally cleavable in the presence of an activating agent; or (ii) not thermally cleavable in the absence of an activating agent.
9. The compound according to claim 8 , wherein the chemical activating agent is an acid or a base.
10. The compound according to claim 1 , wherein at least one PG is thermally cleavable in the presence of an acid, and the intramolecular cyclisation and cleavage of the linker is effected by heating in the presence of a base.
11. The compound according to claim 1 , wherein at least one PG is cleavable in the presence of an acid, and wherein PG is optionally tert-butyloxycarbonyl (Boc), trityl (Trt), benzyloxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl (Ddz), 2-(4-biphenyl)isopropoxycarbonyl (Bpoc), 2-nitrophenylsulfenyl (Nps), or tosyl (Ts).
12. The compound according to claim 1 , wherein PG is Alloc.
13. The compound according to claim 1 , wherein at least one Y group is hydrocarbyl.
14. The compound according to claim 1 , wherein at least one Y group is aralkyl which is substituted with an alkynyl group.
15. The compound according to claim 1 , wherein R 6 is a residue of a nucleotide, an oligonucleotide or a polynucleotide, which is optionally attached via an ether bond at the 3′ or 5′ position of a nucleotide.
16. The compound according to claim 1 , wherein:
M is —W—R 6 ,
W is —O—,
R 6 is an organic fragment which is covalently bound W;
X is hydrogen or phenyl;
Y is hydrocarbyl, or the group
each of R 1 , R 2 , R 3 , R 4 and R 5 is hydrogen;
R 7 is hydrogen;
PG is a cleavable protecting group for nitrogen;
n is (i) 0 or 1, or (ii) 1; and
ring A is a 5, 6, or 7-membered nitrogen-containing monocyclic heterocyclic group.
17. The compound according to claim 16 , wherein R 6 is a residue of a nucleotide, an oligonucleotide or a polynucleotide, which is optionally attached via an ether bond at the 3′ or 5′ position of a nucleotide.
18. The compound according to claim 16 , wherein at least one PG is: i) activated by pH, temperature, a chemical activating agent, or by a combination thereof, ii) thermally cleavable in the presence of an activating agent, or iii) not thermally cleavable in the absence of an activating agent.
19. The compound according to claim 16 , wherein Y is (i) benzyl; or (ii)
wherein each R 3 , R 4 and R 5 is hydrogen, both protecting groups PG are the same and both ring A are the same.
20. The compound according to claim 16 , wherein ring A is a heterocycle that is piperidyl, morpholinyl, pyrrolidinyl, or thiomorpholinyl.
21. The compound according to claim 16 , wherein at least one PG thermally cleavable in the presence of an acid or base.
22. The compound according to claim 16 , wherein at least one protecting group PG is thermally cleavable in the presence of an acid, and the intramolecular cyclisation and cleavage of the linker is effected by heating in the presence of a base.
23. The compound according to claim 16 , wherein at least one protecting group PG is cleavable in the presence of an acid.
24. A composition comprising a compound according to claim 1 , wherein the compound is (i) covalently bound to a substrate or (ii) covalently bound via a linker group to a substrate
25. The composition according to claim 24 , wherein the substrate is a solid support comprising particles that are gold or silicon.
26. A process for the preparation of one or more oligonucleotides or polynucleotides comprising:
(i) providing a composition comprising the compound claim 1 , wherein M is —W—R 6 , wherein R 6 is a first optionally protected nucleotide, wherein the compound is covalently bound to a solid support;
(ii) conducting solid phase synthesis by a phosphoramidite method to produce the oligonucleotide or polynucleotide; and
(iii) cleaving the oligonucleotide or polynucleotide from the solid support.
27. The process according to claim 26 , comprising:
(i) providing a composition comprising the compound of claim 1 , wherein the compound is covalently bound to a solid support;
(ii) removing the 5′-protecting group from the 5′-protected nucleoside;
(iii) coupling at the 5′-OH of the nucleoside with a nucleoside phosphoramidite monomer to form a support-bound phosphite triester;
(iv) optionally capping the 5′-hydroxyl groups on the unreacted nucleoside phosphoramidite monomer by acetylation;
(v) oxidising the phosphite triester to a phosphotriester;
(vi) repeating steps (ii) to (v) in order to produce the oligonucleotide or polynucleotide;
(vii) cleaving the oligonucleotide or polynucleotide from the solid support; and
(viii) optionally isolating the oligonucleotide; and
(ix) the process optionally further comprising:
a step of annealing a complementary oligonucleotide or a complementary polynucleotide before step (vii), and optionally isolating the double stranded oligonucleotide or double stranded polynucleotide; or
a step of annealing a complementary oligonucleotide or a complementary polynucleotide after step (vii) or step (viii), and optionally isolating the double stranded oligonucleotide or double stranded polynucleotide.
28. The process according to claim 27 , wherein step (vii) comprises:
(vii-a) heating in the presence of an activating agent to remove PG from the cleavable linker; and
(vii-b) heating, optionally in the presence of an activating agent, to effect intramolecular cyclisation and cleavage of the linker and solid substrate from the compound.
29. The process according to claim 28 , wherein (a) the activating agent in step (vii-a) is an acid, and step (vii-b) comprises heating in the presence of a base; or (b) the activating agent in step (vii-a) is an acid and after step (vii-a), the reaction mixture is neutralised, and step (vii-b) comprises heating to effect intramolecular cyclisation and cleavage of the linker and solid substrate from the compound.
30. The process according to claim 28 , wherein the activating agent in step (vii-a) is a base, and step (vii-b) comprises heating; optionally wherein step (vii-a) comprises heating to a first temperature in the presence of a base, and wherein the intramolecular cyclisation and cleavage in step (vii-b) is effected by heating to a second temperature, wherein the second temperature is higher than the first temperature.
31. The process according to claim 28 , wherein step (vii-a) or step (vii-b), or both step (vii-a) and step (vii-b), are carried out in a non-aqueous system, optionally wherein step (vii-a) or step (vii-b), or both steps (vii-a) and (vii-b), are carried out in an organic solvent, optionally wherein the organic solvent is acetonitrile, dimethylformamide, tetrahydrofuran, or dioxane.
32. The process according to claim 28 , wherein the step (vii-b) is carried out in the presence of (i) least one polar solvent, or (ii) a polar solvent that comprises acetonitrile and dimethylsulfoxide.