Thermolabile phosphorus protecting groups, associated intermediates and methods of use Number:6,762,298 from the United States Patent and Trademark Office (PTO) owispatent The invention provides a method of thermally deprotecting the internucleosidic phosphorus linkage of an oligonucleotide, which method comprises heating a protected oligonucleotide in a fluid medium at a substantially neutral pH, so as to deprotect the oligonucleotide.The present invention further provides a method of synthesizing an oligonucleotide using the thermal deprotection method described above, and novel oligonucleotides and intermediates that incorporate the thermolabile protecting group used in accordance with the present invention.<H intermediates, Thermolabile, Thermolabile, ph, 6762298.html, Patent, pto, 6762298

Title: Thermolabile phosphorus protecting groups, associated intermediates and methods of use

Abstract: The invention provides a method of thermally deprotecting the internucleosidic phosphorus linkage of an oligonucleotide, which method comprises heating a protected oligonucleotide in a fluid medium at a substantially neutral pH, so as to deprotect the oligonucleotide.The present invention further provides a method of synthesizing an oligonucleotide using the thermal deprotection method described above, and novel oligonucleotides and intermediates that incorporate the thermolabile protecting group used in accordance with the present invention.

Patent Number: 6,762,298 Issued on 07/13/2004 to Beaucage, et al.

Inventors: Beaucage; Serge L. (Silver Spring, MD), Wilk; Andrzej (Bethesda, MD), Grajkowski; Andrzej (Bethesda, MD)
Assignee: The United States of America as represented by the Department of Health and Human Services (Washington, DC)

Appl. No.: 09/792,799

Filed: February 23, 2001

Current U.S. Class: 536/25.31 ; 536/25.3; 536/25.33; 536/25.34

Current International Class: C07H 19/00 (20060101); C07H 19/10 (20060101); C07H 21/00 (20060101)

Field of Search: 536/25.33,26.7,26.8,27.62,27.81,28.51,28.53,25.34,26.74,27.8,25.3,25.31

References Cited [Referenced By]

U.S. Patent Documents


3534017	October 1970	Fujimoto et al.
4415732	November 1983	Caruthers et al.
4417046	November 1983	Hsiung
4458066	July 1984	Caruthers et al.
4663446	May 1987	Wright
4668777	May 1987	Caruthers et al.
4725677	February 1988	Koster et al.
4739044	April 1988	Stabinsky
4757141	July 1988	Fung et al.
4808708	February 1989	Yoshida et al.
4816569	March 1989	Miyoshi
4816570	March 1989	Farquhar
4845205	July 1989	Huynh Dinh et al.
4849513	July 1989	Smith et al.
4950745	August 1990	Ishido et al.
4973679	November 1990	Caruthers et al.
4980460	December 1990	Molko et al.
5026838	June 1991	Nojiri et al.
5039796	August 1991	Engels et al.
5071974	December 1991	Groody
5091519	February 1992	Cruickshank
5134228	July 1992	Takaku
RE34069	September 1992	Koster et al.
5166330	November 1992	Engels et al.
5212304	May 1993	Fung et al.
5214135	May 1993	Srivastava et al.
5252760	October 1993	Urdea et al.
5258538	November 1993	Fung et al.
5324831	June 1994	Marquez et al.
5332845	July 1994	Ureda et al.
5348868	September 1994	Reddy et al.
5428148	June 1995	Reddy et al.
5430138	July 1995	Urdea et al.
5449602	September 1995	Royer et al.
5506351	April 1996	McGee
5510476	April 1996	Ravikumar et al.
5518651	May 1996	Reddy et al.
5519126	May 1996	Hecht
5525719	June 1996	Srivastava et al.
5556961	September 1996	Foote et al.
5571902	November 1996	Ravikumar et al.
5574146	November 1996	Reddy et al.
5614622	March 1997	Iyer et al.
5616700	April 1997	Reddy et al.
5623068	April 1997	Reddy et al.
5639867	June 1997	Brill
5645985	July 1997	Froehler et al.
5646265	July 1997	McGee
5652358	July 1997	Pfleiderer et al.
5670489	September 1997	Baxter et al.
5681940	October 1997	Wang et al.
5700919	December 1997	Seliger et al.
5703218	December 1997	Urdea et al.
5703223	December 1997	Wickstrom et al.
5705621	January 1998	Ravikumar
5712378	January 1998	Wang
5714597	February 1998	Ravikumar et al.
5731429	March 1998	Reddy et al.
5763599	June 1998	Pfleiderer et al.
5866700	February 1999	Pfleiderer et al.
5889165	March 1999	Fodor et al.
5908926	June 1999	Pirrung et al.
5959099	September 1999	Cheruvallath et al.
6001982	December 1999	Ravikumar et al.
6022963	February 2000	McGall et al.
6043060	March 2000	Imanishi
2001/0044529	November 2001	Beaucage et al.

Foreign Patent Documents


0 006 220	Jan., 1980	EP
0 090 789	Oct., 1983	EP
0 196 101	Oct., 1986	EP
0 219 342	Apr., 1987	EP
0 241 363	Oct., 1987	EP
0 323 152	Jul., 1989	EP
2 153 356	Aug., 1985	GB
WO 88/02004	Mar., 1988	WO
WO 93/12132	Jun., 1993	WO
WO 00/56749	Sep., 2000	WO

Other References

Lefebvre et al. "Mononucleoside Phosphotriester Derivatives with S-Acyl-2-thioethyl Bioreversible Phosphate-Protecting Groups: Intracellular Delivery of 3'-Azido-2', 3'-dideoxythymidine 5'-Monophosphate" J. Med. Chem., 1995, 38 (20), 3941-3950.* .
Wang et al. "A Stereoselective Synthesis of Dinucleotide Phosphorothioates, Using Chiral Indol-oxazaphosphorine Intermediates" Tetrahedron Letters, 1997, 38 (22), 3797-3800.* .
Barone et al., Nucl. Acids Res., 12(10), 4051-4061 (1984). .
Beaucage et al., Ann. New York Acad. Sci., 616, 483-485 (1990). .
Beaucage et al., Current Protocols in Nucleic Acid Chemistry, vol. 1 (Beaucage S.L., Bergstrom, D.E., Glick, G.D., Jones, R.A. eds), John Wiley and Sons: New York (2000) pp. 3.3.1-3.3.20. .
Beaucage et al., Tetrahedron, 48(12), 2223-2311 (1992). .
Beaucage et al., Tetrahedron, 49(28), 6123-6194 (1993). .
Beaucage, Methods in Molecular Biology, vol. 20: Protocols for Oligonucleotides and Analogs, (S. Agrawal, ed.), Humana Press: Totowa, NJ (1993) pp. 33-61. .
Bigg et al., Synthesis, 277-278 (Mar. 1992). .
Boal et al., Nucl. Acids Res., 24(15), 3115-3117 (1996). .
Brown et al., J. Chem. Soc. Chem. Commun., 891-893 (1989). .
Gardrat et al., J. Heterocyclic Chem., 27, 811-812 (1990). .
Iyer et al., J. Org. Chem., 55(15), 4693-4699 (1990). .
Iyer, Current Protocols in Nucleic Acid Chemistry, vol. 1 (Beaucage S.L., Bergstrom, D.E., Glick, G.D. Jones R.A. eds); John Wiley and Sons: New York, (2000) pp. 2.1.1-2.1.17. .
Martin, Helv. Chim. Acta., 78, 486-504 (1995). .
McBride et al., J. Am. Chem. Soc., 108, 2040-2048 (1986). .
Mizrakh et al., Chemical Abstracts, 83(23), 454 (1975). .
Prakash et al., Org. Lett., 2(25), 3995-3998 (2000). .
Probst et al., Makromol. Chem., 177, 2681-2695 (1976). .
Pudovik et al., Chemical Abstracts, 79(11), 441 (1973). .
Pudovik et al., Chemical Abstracts, 81(11), 484 (1974). .
Regan et al., Org. Prep. Proc. Int., 24(4), 488-492 (1992). .
Saegusa et al., Makromol. Chem., 177, 2271-2283 (1976). .
Shibanuma et al., Chem Pharm. Bull., 28(9), 2609-2613 (1980). .
Smith et al., Nucleosides & Nucleotides, 15(10), 1581-1594 (1996). .
Waldner et al., Bioorg. Med. Chem. Letters, 6(19), 2363-2366 (1996). .
Wilk et al., J. org. Chem., 62(20), 6712-6713 (1997). .
Wilk et al., J. Org. Chem., 64(20), 7515-7522 (1999). .
Wincott, Current Protocols in Nucleic Acid Chemistry, vol. 1 (Beaucage S.L., Bergstrom, D.E., Glick, G.D, Jones, R.A. eds); John Wiley and Sons: New York, (2000) pp. 3.5.1-3.5.12. .
Cao et al.; Tetrahedron Letters, 24(10), 1019-1020 (1983). .
Grajkowski et al.; Organic Letters, 3(9), 1287-1290 (2001). .
Gray et al.; J. Am. Chem. Soc., 81, 4351-4355 (1959). .
Guzaev et al.; Tetrahedron Letters, 41, 5623-5626 (2000). .
Iyer et al.; J. Org. Chem., 60, 5388-5389 (1995). .
Iyer et al.; Tetrahedron: Asymmetry, 6 (5), 1051-1054 (1995). .
Kawanobe et al.; Chemistry Letters, Chem. Soc. of Japan, 825-828 (1982). .
Murphy et al.; Tetrahedron, 47(24), 4077-4088 (1991). .
Tsuruoka et al.; Tetrahedron Letters, 40, 8411-8414 (1999). .
Wilk et al.; J. Am. Chem. Soc., 122, 2149-2156 (2000). .
Wilk et al.; Tetrahedron Letters, 42, 5635-5639 (2001). .
Wilk et al.; J. Org. Chem., 67, 6430-6438 (2002). .
Yang et al.; Chem. Abs., 111, 97382x (1989). .
Zhang et al.; Chem. Abs., 126(2). 18939t (1997). .
Finger et al., J. Am. Chem. Soc., 81 (10), 2674-2675 (Jun. 2, 1959). .
Mizrakh et al., Zh. Obs. Khim., 45 (3), 549-552 (Mar., 1975). .
Mizrakh et al., Zh. Obs. Khim., 45 (7). 1469-1473 (Jul., 1975). .
Mizrakh et al., Zh. Obs. Khim., 45 (10), 2343-2344 (Oct., 1975). .
Scremin et al., J. Org. Chem., 59 (8), 1963-1966 (1994). .
Somei et al., Chem. Pharm. Bull., 28 (8), 2515-2518 (1980). .
Stec et al., Nucleic Acids Res., 19 (21), 5883-5888 (1991). .
Weiner et al., J. Org. Chem., 14, 868-872, (1949)..

Primary Examiner: Wilson; James O.
Assistant Examiner: Lewis; Patrick
Attorney, Agent or Firm: Leydig, Voit & Mayer, Ltd.

Parent Case Text

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This patent application is a continuation-in-part of copending international patent application No. PCT/US00/04032, filed Feb. 16, 2000, pending, which claims priority to U.S. provisional patent application No. 60/125,867, filed Mar. 24, 1999.

Claims

What is claimed is:

1. A method of deprotecting an oligonucleotide, which method comprises heating an oligonucleotide of the formula: ##STR40##

in a fluid medium, at a substantially neutral pH, at a temperature up to about 100.degree. C. to produce an oligonucleotide of the formula: ##STR41##

wherein: R is a thermolabile protecting group of the formula: ##STR42## R.sup.1 is H, R.sup.1a, OR.sup.1a, SR.sup.1a or NR.sup.1a R.sup.1a', wherein R.sup.1a and R.sup.1a' are the same or different and each is H, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, an aryl, or an aralkyl; or, when R.sup.1 is NR.sup.1a R.sup.1a', R.sup.1a and R.sup.1a', together with the nitrogen atom to which they are bonded, comprise a heterocycle containing from 3 to about 7 atoms in the ring skeleton thereof; X.sup.1 is O, S or Se; X is O or S; Z is O, S, NR.sup.2a, CR.sup.2a R.sup.2a' or CR.sup.2a R.sup.2a CR.sup.2b R.sup.2b', wherein R.sup.2a, R.sup.2a', R.sup.2b and R.sup.2b' are the same or different and each is H, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, an aryl, or an aralkyl; or R.sup.1a or R.sup.1a', in combination with any of R.sup.2a, R.sup.2a', R.sup.2b or R.sup.2b', together with C.dbd.X of the protecting group to which they are bonded, comprise a ring containing from 3 to about 7 atoms in the skeleton thereof; provided that R.sup.1 is not R.sup.1a when Z is S, Z is not CR.sup.2a R.sup.2a' or CR.sup.2a R.sup.2a' CR.sup.2b R.sup.2b' when R.sup.1 is SR.sup.1a, and Z is not O or S when R.sup.1 is H; R.sup.2, R.sup.2', R.sup.3 and are the same or different and each is H, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, an aryl, or an aralkyl, or R.sup.2 or R.sup.2', in combination wit R.sup.3 or R.sup.3', together with the carbon atoms to which they are bonded, comprise a cyclic substituent of the formula: ##STR43## wherein p is an integer from 0-6 and a-d are the same or different and each is selected from the group consisting of H, an alkyl, a nitro, a dialkylamino, an alkoxy, an alkylthio, a cyano and a halogen, provided that the aromatic ring, which bears substituents a-d, is one carbon removed from the phosphate oxygen of formula (IIIa), wherein R.sup.1, R.sup.2a, R.sup.2a', R.sup.2b, R.sup.2b', R.sup.2, R.sup.2', R.sup.3 or R.sup.3' is unsubstituted or substituted with one or more substituents, which are the same or different, selected from the group consisting of OR.sup.8, CN, NO.sub.2, N.sub.3, and a halogen, wherein R.sup.8 is H or an alkyl; R.sup.4 and R.sup.15 are the same or different and each is H, a hydroxyl protecting group, or a solid support; Q.sup.1 is a nucleoside, an oligonucleotide or an oligomer comprising an oligonucleotide; n is an integer from 1 to about 300; and Q is a nucleoside, an oligonucleotide or an oligomer comprising an oligonucleotide and, when n is greater than 1, each Q is independently selected, provided that the deprotection is not by an enzyme.

2. The method of claim 1, wherein Q or Q.sup.1 comprises a nucleoside of the formula: ##STR44##

wherein: B is a labeling group, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, an aryl, a heteroaryl, a heterocycloalkyl, an aralkyl, an amino, an alkylamino, a dialkylamino, a purine, a pyrimidine, adenine, guanine, cytosine, uracil, or thymine, wherein B is unsubstituted or substituted with one or more substituants, which are the same or different, selected from the group consisting of a nucleobase protecting group, R.sup.11, OR.sup.11, NHR.sup.11, NR.sup.11 R.sup.12, N.dbd.C--NR.sup.11' R.sup.12', CN, NO.sub.2, N.sub.3, and a halogen, wherein R.sup.11 and R.sup.12 are the same or different and each is H, an alkyl or an acyl, and R.sup.11 and R.sup.12' are the same or different and each is an alkyl or R.sup.11' and R.sup.12', together with the nitrogen atom to which they are bonded, form a heterocycle containing 3 to about 7 atoms in the ring skeleton thereof; and E is H, a halogen, OR.sup.13, NHR.sup.13, or NR.sup.13 R.sup.14, wherein R.sup.13 and R.sup.14 are the same or different and each is H, a protecting group, an alkyl, or an acyl.

3. The method of claim 1, wherein R.sup.1 is H, an alkyl or NR.sup.1a R.sup.1a', wherein R.sup.1a and R.sup.1a', together with the nitrogen atom to which they are bonded, comprise a heterocycle containing from 3 to about 7 atoms in the ring skeleton thereof.

4. The method of claim 1, wherein X.sup.1 is S.

5. The method of claim 1, wherein Z is CR.sup.2a R.sup.2a' or CR.sup.2a R.sup.2a' CR.sup.2b R.sup.2b' and R.sup.2a, R.sup.2a', R.sup.2b and R.sup.2b' are the same or different and each is H or an alkyl.

6. The method of claim 1, wherein R.sup.2 or R.sup.2' is H or an alkyl.

7. The method of claim 1, wherein R.sup.3 or R.sup.3' is H, an alkyl or an

8. The method of claim 1, wherein R is a protecting group of the formula: ##STR45##

9. The method of claim 1, wherein the temperature is from about 50.degree. C. to about 90.degree. C.

10. The method of claim 1, wherein the deprotection is carried out at about pH 7.

11. The method of claim 1, wherein the fluid medium contains water.

12. A method of producing an oligonucleotide, which method comprises (a) reacting a nucleophile of the formula:

with an electrophile of the formula: ##STR46##

wherein W is a dialkylamino group that is displaced by the nucleophile, under conditions to displace W and produce an adduct comprising a tricoordinated phosphorus atom; (b) reacting the product obtained in step (a) with a reagent selected from the group consisting of oxidizing agents, sulfurizing agents, and selenizing agents to produce a protected oligonucleotide of the formula: ##STR47## (c) cleaving R.sup.15 from the protected oligonucleotide from step (b) to produce a nucleophile; (d) optionally repeating steps (a)-(c) until an oligomer of a specified length is obtained; and (e) heating the product from step (c) or (d) in a fluid medium, at a substantially neutral pH, at a temperature up to about 100.degree. C. to produce a deprotected oligonucleotide of the formula: ##STR48## wherein R is a thermolabile protecting group of the formula: ##STR49## R.sup.1 is H, R.sup.1a, OR.sup.1a, SR.sup.1a or NR.sup.1a R.sup.1a', wherein R.sup.1a and R.sup.1a' are the same or different and each is H, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, an aryl, or an aralkyl; or, when R.sup.1 is NR.sup.1a R.sup.1a', R.sup.1a and R.sup.1a', together with the nitrogen atom to which they are bonded, comprise a heterocycle containing from 3 to about 7 atoms in the ring skeleton thereof; X.sup.1 is O, S or Se; X is O or S; Z is O, NR.sup.2a, CR.sup.2a B.sup.2a' or CR.sup.2a R.sup.2a' CR.sup.2b R.sup.2b', wherein R.sup.2a, R.sup.2a', R.sup.2b and R.sup.2b' are the same or different and each is H, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, an aryl, or an aralkyl; or R.sup.1a or R.sup.1a', in combination with any of R.sup.2a, R.sup.2a', R.sup.2b or R.sup.2b', together with C.dbd.X of the protecting group to which they are bonded, comprise a ring containing from 3 to about 7 atoms in the skeleton thereof; provided that R.sup.1 is not R.sup.1a when Z is S, Z is not CR.sup.2a R.sup.2a' or CR.sup.2a R.sup.2a' CR.sup.2b CR.sup.2b' when R.sup.1 is SR.sup.1a, and Z is not O or S when R.sup.1 is H; R.sup.2, R.sup.2', R.sup.3 and R.sup.3' are the same or different and each is H, an alkyl, an alkenyl, alkynyl, a cycloalkyl, an aryl, or an aralkyl, or R.sup.2 or R.sup.2', in combination R.sup.3 or R.sup.3', together with the carbon atoms to which they are bonded, comprise a cyclic substituent of the formula: ##STR50## wherein p is an integer from 0-6 and a-d are the same or different and each is selected from the group consisting of H, an alkyl, a nitro, an amino, a hydroxyl, a thio, a cyano and a halogen, provided that the aromatic ring, which bears the substituents a-d, is one carbon removed from the phosphate oxygen of formula (IIIa), wherein R.sup.1, R.sup.2a, R.sup.2a', R.sup.2b, R.sup.2b' R.sup.2, R.sup.2', R.sup.3 or R.sup.3' is unsubstituted or substituted with one or more substituents, which are the same or different, selected from the group consisting of OR.sup.8, CN, NO.sub.2, N.sub.3, and a halogen, wherein R.sup.8 is H or an alkyl; R.sup.4 is H, a hydroxyl protecting group, or a solid support; R.sup.15 is a hydroxyl protecting group or a solid support; Q.sup.1 is a nucleoside, an oligonucleotide or an oligomer comprising an oligonucleotide; n is an integer from 1 to about 300; and Q is a nucleoside, an oligonueleotide or an oligomer comprising an oligonucleotide and, when a is greater than 1, each Q is independently selected, provided that the deprotection is not by an enzyme.

Description

FIELD OF THE INVENTION

This invention pertains to thermolabile phosphate protecting groups, intermediates therefor and methods of using them in oligonucleotide synthesis.

BACKGROUND OF THE INVENTION

There are significant potential therapeutic applications for oligonucleotides. The therapeutic application of oligonucleotides is based on the selective formation of hybrids between antisense oligonucleotides and complementary nucleic acids, such as messenger RNAs (mRNAs). Such hybrids inhibit gene expression by preventing protein translation. Nuclease-resistant oligonucleotides are highly desirable in this regard. Nucleosides bearing phosphorothioate internucleotide linkages are well-known for such nuclease resistance and, thus, are undergoing rapid development.

In view of their significant potential therapeutic application, there is a high demand for improved methods of preparing oligonucleotides and analogues thereof. A number of methods for synthesizing oligonucleotides have been developed. The most commonly used synthetic method for the synthesis of thioated oligonucleotides is the phosphoramidite method with stepwise sulfurization (see, e.g., U.S. Pat. Nos. 4,415,732, 4,668,777, 4,973,679, 4,845,205, and 5,525,719). Essentially, a phosphate precursor is sulfurized such that a sulfur atom is substituted for one of the non-bridging oxygen atoms normally present in phosphodiesters. This method uses tricoordinated phosphorus precursors that normally produce products containing a mixture of different thioated oligonucleotide stereoisomers, primarily due to the use of non-stereoselective and non-stereospecific acid-catalyzed nucleophilic substitution reactions.

Protecting groups for internucleosidic phosphorus linkages and associated deprotection methods are well-known in the art, and have been described, for example, in U.S. Pat. Nos. 4,417,046, 5,705,621, 5,571,902 and 5,959,099. However, the methods presently used for removing internucleosidic phosphorus protecting groups are disadvantageous in that they employ harsh reagents, such as bases (e.g., ammonium hydroxide) and acids (e.g., trichloroacetic acid). Under these deprotection conditions, there is a greater risk of problems, such as by-product formation and degradation of the desired oligonucleotide, which make oligonucleotide purification more difficult and increase the overall cost, particularly in large-scale production processes. Moreover, the range of structural analogs that one can prepare is limited to those that are stable under the acidic and/or basic deprotection conditions that are commonly employed in the art.

Accordingly, there is a need for internucleosidic phosphorus protecting groups that can be removed under milder conditions and methods of making and using such protecting groups. Removal of such protecting groups should be fast and should be carried out under conditions that minimize the possibility for degradation of the desired oligonucleotide. In addition, the intermediates that introduce such protecting groups should be easy to synthesize inexpensively on a large scale. It is, therefore, of prime importance to develop low-cost, protected intermediates for oligonucleotide synthesis which are easy to synthesize, couple efficiently during stepwise synthesis, and are deprotected quickly in high yield under mild conditions.

The invention provides such protecting groups and methods. These and other objects and advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.

BRIEF SUMMARY OF THE INVENTION

The invention provides a method of thermally deprotecting an oligonucleotide. The method comprises heating an oligonucleotide of the formula: ##STR1##

in a fluid medium, at a substantially neutral pH, at a temperature up to about 100.degree. C. to produce an oligonucleotide of the formula: ##STR2##

wherein R is a thermolabile protecting group of the formula: ##STR3##

R.sup.1 is H, R.sup.1a, OR.sup.1a, SR.sup.1a or NR.sup.1a R.sup.1a', wherein R.sup.1a and R.sup.1a' are the same or different and each is H, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, an aryl, or an aralkyl. Alternatively, when R.sup.1 is NR.sup.1a R.sup.1a', R.sup.1a and R.sup.1a', together with the nitrogen atom to which they are bonded, comprise a heterocycle. Substituent X.sup.1 is O, S or Se and substituent X is O or S. Substituent Z is O, S, NR.sup.2a, CR.sup.2a R.sup.2a' or CR.sup.2a R.sup.2a' CR.sup.2b R.sup.2b', wherein R.sup.2a, R.sup.2a', R.sup.2b and R.sup.2b' are the same or different and each is H, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, an aryl, or an aralkyl. Alternatively, R.sup.1a or R.sup.1a', in combination with any of R.sup.2a, R.sup.2a', R.sup.2b or R.sup.2b', together with C.dbd.X of the protecting group to which they are bonded, comprise a ring containing from 3 to about 7 atoms in the skeleton thereof. R.sup.1 is not R.sup.1a when Z is S, Z is not CR.sup.2a R.sup.2a' or CR.sup.2a R.sup.2a' CR.sup.2b R.sup.2b' when R.sup.1 is SR.sup.1a, and Z is not O or S when R.sup.1 is H.

Substituents R.sup.2, R.sup.2', R.sup.3 and R.sup.3' are the same or different and each is H, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, an aryl, or an aralkyl. Alternatively, R.sup.2 or R.sup.2', in combination with R.sup.3 or R.sup.3', together with the carbon atoms to which they are bonded, comprise a cyclic substituent of the formulae: ##STR4## wherein p is an integer from 0-6 and a-d are the same or different and each is selected from the group consisting of H, an alkyl, a nitro, a dialkylamino, an alkoxy, an alkylthio, a cyano and a halogen, provided that the aromatic ring, which bears substituents a-d, is one carbon removed from the phosphate oxygen of formula (IIIa).

Substituents R.sup.1, R.sup.2a, R.sup.2b, R.sup.2b', R.sup.2, R.sup.2', R.sup.3 or R.sup.3' can be unsubstituted substituted, as further described herein. Substituents R.sup.4 and R.sup.15 are the same or different and each is H, a hydroxyl protecting group, or a solid support.

Q and Q.sup.1 are the same or different and each is a nucleoside, an oligonucleotide or an oligomer comprising an oligonucleotide. Variable n represents an integer from 1 to about 300. When n is greater than 1, each Q is independently selected.

The present invention further provides a novel compound selected from the group consisting of compounds of the formulae: ##STR5##

wherein R is a thermolabile protecting group as defined herein, R.sup.4, R.sup.15 and X.sup.1 are as defined herein, and W is a dialkylamino group.

The present invention further provides method of producing an oligonucleotide. The method comprises: (a) reacting a nucleophile of the formula:

with an electrophile of the formula: ##STR6##

wherein R, R.sup.4, Q, Q.sup.1 and W are as defined herein, and R.sup.15 is a protecting group, under conditions to displace W and produce an adduct comprising a tricoordinated phosphorus atom; (b) reacting the product obtained in step (a) with a reagent selected from the group consisting of oxidizing agents, sulfurizing agents, and selenizing agents to produce a protected oligonucleotide of the formula: ##STR7##

wherein n=1; (c) cleaving R.sup.15 from the protected oligonucleotide from step (b) to produce a nucleophile; (d) optionally repeating steps (a)-(c) until an oligomer of a specified length is obtained; and (e) thermally deprotecting the thermolabile protecting group R in accordance with the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A generally illustrates the thermal deprotection of a tetracoordinated phosphorus internucleosidic linkage.

FIG. 1B illustrates the thermal deprotection of a phosphate/thiophosphate internucleosidic linkage.

FIG. 1C illustrates the thermal deprotection of the phosphate/thiophosphate internucleosidic linkage of an oligonucleotide prepared from a phosphoramidite precursor.

FIG. 1D illustrates the thermal deprotection of various thermolabile phosphate/thiophosphate protecting groups.

FIG. 2A illustrates the synthesis of various phosphoramidite precursors.

FIG. 2B illustrates the structures of various phosphoramidite precursors.

FIG. 3 illustrates the synthesis of various N-acylphosphoramidite of various.

FIG. 4 illustrates the solid phase synthesis of an oligonucleotide using an N-acylphosphoramidite precursor.

FIG. 5 illustrates the solid phase stereocontrolled synthesis of phosphorothioate oligonucleotides using an N-acylphosphoramidite precursor.

FIG. 6 illustrates the synthesis of various N-acylphosphoramidites.

FIG. 7 illustrates the preparation of acyclic N-acylphosphoramidites and their application in solid phase synthesis.

FIG. 8 illustrates the preparation of cyclic and acyclic N-acylphosphoramidites.

FIG. 9 illustrates the preparation of an oligonucleotide using either cyclic or acyclic N-acylphosphoramidite precursors.

FIG. 10A illustrates the structure of a P-chiral (S.sub.P) N-acylphosphoramidite.

FIG. 10B illustrates the structure of a P-chiral (R.sub.P) N-acylphosphoramidite.

FIG. 11 illustrates the structure of a P-diastereomeric (R.sub.P, S.sub.P) N-acylphosphoramidite.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated, at least in part, on the surprising and unexpected discovery of a method for thermally deprotecting the internucleosidic phosphorus linkage of an oligonucleotide, new thermolabile protecting groups that can be removed under such conditions and intermediates that incorporate them. The methods and protecting groups of the present invention simplify, and improve the efficiency and cost-effectiveness effectiveness of, oligonucleotide synthesis by avoiding the use of harsh reagents, such as alkaline or acidic reagents. In one embodiment, the present invention provides a method of deprotecting an oligonucleotide, which method comprises heating an oligonucleotide of the formula: ##STR8##

in a fluid medium, at a substantially neutral pH, at a temperature up to about 100.degree. C. to produce an oligonucleotide of the formula: ##STR9##

wherein:

R is a thermolabile protecting group of the formula: ##STR10##

The deprotection method of the present invention can be performed in any suitable fluid medium. Suitable fluid media include, for example, liquid media and gaseous media. A preferred fluid medium comprises or contains water. Liquid media include, for example, solvents, preferably solvents that are liquid at room temperature. Suitable solvents include organic solvents and inorganic solvents.

Organic solvents preferably include those that are easily removed by evaporation. Preferably, the organic solvent is a polar organic solvent. Preferred polar organic solvents include, for example, acetonitrile; cyclic ethers such as, for example, dioxane and tetrahydrofuran; alcohols such as, for example, methanol, ethanol and isopropanol; mixtures thereof; and the like. Non-polar organic solvents such as, for example, hydrocarbons, e.g., hexane, cyclohexane and heptane; aromatic hydrocarbons, e.g., toluene and benzene; mixtures thereof, and the like can be included in the fluid medium, for example, as co-solvents.

Inorganic solvents include, for example, water. In a particularly preferred embodiment, the solvent is water or a mixture of one or more organic solvents and water.

The liquid medium can be a homogeneous solution or heterogeneous mixture, but is preferably a homogeneous solution. Most preferably, the liquid medium is a homogeneous solution that contains water as a co-solvent.

Suitable solvents include, for example, acetonitrile/water mixtures ranging from about 10:1 (v/v) to about 1:10 (v/v) acetonitrile/water. Suitable acetonitrile/water mixtures include, for example, about 9:1 (v/v) acetonitrile/water, about 5:1 (v/v) acetonitrile/water, about 2:1 (v/v) acetonitrile/water, about 1:1 (v/v) acetonitrile/water, about 1:2 (v/v) acetonitrile/water, about 1:5 (v/v) acetonitrile/water, and about 1:9 (v/v) acetonitrile/water.

Suitable solvents also include, for example, dioxane/water mixtures ranging from about 10:1 (v/v) to about 1:10 (v/v) dioxane/water. Suitable dioxane/water mixtures include, for example, about 9:1 (v/v) dioxane/water, about 5:1 (v/v) dioxane/water, about 2:1 (v/v) dioxane/water, about 1:1 (v/v) dioxane/water, about 1:2 (v/v) dioxane/water, about 1:5 (v/v) dioxane/water, and about 1:9 (v/v) dioxane/water.

Suitable solvents also include other organic solvent/water mixtures, e.g., using the ratios described herein. Other suitable solvents include organic solvents, such as, for example, acetonitrile, dioxane, mixtures thereof, and the like, that contain a trace amount of water (e.g., from about 0.05-2 wt. %, from about 0.1-2 wt. %, from about 0.5-2 wt. %, from about 1-2 wt. %, and the like).

The method of the present invention can be performed in a gaseous medium, most preferably a gaseous medium that contains water in a gaseous or fluid state (e.g., steam, hot water mist or vapor, or the like). The gaseous medium also can include the gaseous phases of any of the organic solvents or solvent mixtures described herein. In a particularly preferred embodiment, the method of the present invention includes contacting the oligonucleotide of formula (IIIa) (e.g., bound to a solid support) with steam.

As indicated above, the method of the present invention is carried at a substantially neutral pH. As utilized herein, the term "substantially neutral pH" means a pH in the range from about 5.5-7.5, preferably from about 6-7.5, most preferably about 7 (e.g., about 7.0-7.4). Optionally, a buffer can be added to the solvent system to maintain a substantially neutral pH throughout the course of the deprotection reaction. Suitable buffers include, for example, phosphate buffers, trialkylammonium acetate buffers (e.g., 0.1 M triethylammonium acetate), and the like.

The deprotection method of the present invention is preferably performed at a temperature that is sufficient to remove the protecting group at a rate that is practical for commercial scale production (e.g., about 3 hours or less), but should be low enough to avoid thermal degradation of the desired oligonucleotide. Typically, the deprotection is performed at a temperature up to about 100.degree. C. (at about 100.degree. C. or less), e.g., from above about ambient temperature (e.g., above about 20-25.degree. C.) to about 100.degree. C. Preferably, the deprotection is performed at a temperature from about 50-100.degree. C., more preferably from about 60-100.degree. C., still more preferably from about 70-100.degree. C., most preferably from about 80-100.degree. C. About 90.degree. C. or about 100.degree. C. is especially preferred. When a liquid solvent medium is used, the deprotection is preferably performed from about 50-90.degree. C., more preferably from about 60-90.degree. C., still more preferably from about 70-90.degree. C., even still more preferably from about 80-90.degree. C., and most preferably at about 90.degree. C. However, in some circumstances, it may be desirable to carry out the deprotection at somewhat higher temperatures (e.g., up to about 110.degree. C., e.g., from about 100-105.degree. C.).

The structure of the thermolabile protecting group (substituent R of formula IIIa) can vary considerably in terms of different combinations of R.sup.1, R.sup.2, R.sup.2', R.sup.3, R.sup.3', Z and X, while maintaining thermal lability. In other words, the bond linking protecting group R to the non-bridging phosphate, phosphorothioate or phosphoroselenoate oxygen can be thermally cleaved using different combinations of R.sup.1, R.sup.2 , R.sup.2', R.sup.3, R.sup.3', Z and X.

While R.sup.1 can be any suitable substituent, R.sup.1 preferably is H, R.sup.1a, OR.sup.1a, SR.sup.1a or NR.sup.1a R.sup.1a', wherein R.sup.1a and R.sup.1a' are the same or different and each is H, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, an aryl, or an aralkyl. Alternatively, when R.sup.1 is NR.sup.1a R.sup.1a', R.sup.1 and R.sup.1a', together with the nitrogen atom to which they are bonded, comprise a heterocycle containing from 3 to about 7 atoms in the ring skeleton thereof.

Preferably, X.sup.1 is O, S or Se; X is O or S; and Z is O, S, NR.sup.2a, CR.sup.2a R.sup.2a' or CR.sup.2a R.sup.2a' CR.sup.2b R.sup.2b', wherein R.sup.2a, R.sup.2a', R.sup.2b and R.sup.2b' are the same or different and each is H, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, an aryl, or an aralkyl. Alternatively, R.sup.1a or R.sup.1a', in combination with any of R.sup.2a, R.sup.2a', R.sup.2b or R.sup.2b', together with C.dbd.X of the protecting group to which they are bonded, comprise a ring containing from 3 to about 7 atoms in the skeleton thereof.

It is preferred that thioesters are not utilized in the methods or the protecting groups of the present invention as they are believed to have a tendency to hydrolyze rather easily in the presence of water. Thus, when Z is S, it is preferred that R.sup.1 is not R.sup.1a. Similarly, when R.sup.1 is SR.sup.1a, Z is not CR.sup.2a R.sup.2a' or CR.sup.2a R.sup.2a' C.sup.R2b R.sup.2b'. Further, it is preferred that formate esters or formate thioesters are not utilized in the methods or the protecting groups of the present invention as they also are believed to have a tendency to hydrolyze rather easily in the presence of water. Thus, when R.sup.1 is H, it is preferred that Z is not O or S.

While R.sup.2, R.sup.2', R.sup.3 and R.sup.3' can be any suitable substituent, R.sup.2, R.sup.2', R.sup.3 and R.sup.3' preferably are the same or different and each is H, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, an aryl, or an aralkyl. Alternatively, R.sup.2 or R.sup.2', in combination with R.sup.3 or R.sup.3', together with the carbon atoms to which they are bonded, can comprise a cyclic substituent of the formula: ##STR11##

wherein p is an integer from 0-6 and a-d are the same or different and each is selected from the group consisting of H, an alkyl, a nitro, a dialkylamino, an alkoxy, an alkylthio, a cyano and a halogen, provided that the aromatic ring, which bears substituents a-d, is one carbon removed from (i.e., is benzylic relative to) the phosphate oxygen of formula (IIIa).

The foregoing substituents can be unsubstituted or substituted. Preferably, R.sup.1, R.sup.2a, R.sup.2a', R.sup.2b, R.sup.2b', R.sup.2, R.sup.2', R.sup.3 or R.sup.3' is unsubstituted or substituted. Preferably, R.sup.1, substituents, which are the same or different, selected from the group consisting of OR.sup.8, CN, NO.sub.2, N.sub.3, and a halogen, wherein R.sup.8 is H or an alkyl.

Substituents R.sup.4 and R.sup.15 are the same or different and each is preferably H, a hydroxyl protecting group, or a solid support. Substituent Q.sup.1 represents a nucleoside, an oligonucleotide or an oligomer comprising an oligonucleotide. The variable n is an integer from 1 to about 300, preferably from about 3 to about 200, more preferably from about 10 to about 40, and most preferably from about 15 to about 25. Substituent Q represents a nucleoside, an oligonucleotide or an oligomer comprising an oligonucleotide. When n is an integer greater than 1, each Q is independently selected, i.e., each Q in each monomeric unit can be the same or different.

As utilized herein, the term "alkyl" means a straight-chain or branched-chain alkyl radical which, unless otherwise specified, contains from about 1 to about 20 carbon atoms, preferably from about 1 to about 10 carbon atoms, more preferably from about 1 to about 8 carbon atoms, and most preferably from about 1 to about 6 carbon atoms. Examples of such alkyl radicals include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isoamyl, hexyl, octyl, dodecanyl, and the like.

The term "alkenyl" means a straight-chain or branched-chain alkenyl radical, which has one or more double bonds and, unless otherwise specified, contains from about 2 to about 20 carbon atoms, preferably from about 2 to about 10 carbon atoms, more preferably from about 2 to about 8 carbon atoms, and most preferably from about 2 to about 6 carbon atoms. Examples of alkenyl radicals include vinyl, allyl, 1,4-butadienyl, isopropenyl, and the like.

The term "alkynyl" means a straight-chain or branched-chain alkynyl radical, which has one or more triple bonds and contains from about 2 to about 20 carbon atoms, preferably from about 2 to about 10 carbon atoms, more preferably from about 2 to about 8 carbon atoms, and most preferably from about 2 to about 6 carbon atoms. Examples of alkynyl radicals include ethynyl, propynyl (propargyl), butynyl, and the like.

The terms "alkylamino" and "dialkylamino" mean an alkyl or a dialkyl amine radical, wherein the term "alkyl" is defined as above. Examples of alkylamino radicals include methylamino (NHCH.sub.3), ethylamino (NHCH.sub.2 CH.sub.3), n-propylamino, isopropylamino, n-butylamino, isobutylamino, sec-butylamino, tert-butylamino, n-hexylamino, and the like. Examples of dialkylamino radicals include dimethylamino (N(CH.sub.3).sub.2), diethylamino (N(CH.sub.2 CH.sub.3).sub.2), di-n-propylamino, diisopropylamino, di-n-butylamino, diisobutylamino, di-sec-butylamino, di-tert-butylamino, di-n-hexylamino, and the like.

The term "cycloalkyl" means a monocyclic alkyl radical, or a polycyclic alkyl which comprises one or more alkyl carbocyclic rings, which can be the same or different when the polycyclic radical has 3 to about 10 carbon atoms in the carbocyclic skeleton of each ring. Preferably, the cycloalkyl has from about 4 to about 7 carbon atoms, more preferably from about 5 to about 6 carbons atoms. Examples of monocyclic cycloalkyl radicals include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclodecyl, and the like. Examples of polycyclic cycloalkyl radicals include decahydronaphthyl, bicyclo[5.4.0]undecyl, adamantyl, and the like.

The term "aryl" refers to an aromatic carbocyclic radical, as commonly understood in the art, and includes monocyclic and polycyclic aromatics such as, for example, phenyl and naphthyl radicals, which radicals are, unless indicated otherwise, unsubstituted or substituted with one or more substituents selected from the group consisting of a halogen, an alkyl, an alkoxy, an amino, a cyano, a nitro, and the like. Preferably, the aryl has one or more six-membered carbocyclic rings including, for example, phenyl, naphthyl, and biphenyl, and are unsubstituted or substituted as set forth herein.

The term "aralkyl" means alkyl as defined herein, wherein an alkyl hydrogen atom is replaced by an aryl as defined herein. Examples of aralkyl radicals include benzyl, phenethyl, 1-phenylpropyl, 2-phenylpropyl, 3-phenylpropyl, 1-naphthylpropyl, 2-naphthylpropyl, 3-naphthylpropyl, 3-naphthylbutyl, and the like.

The terms heterocycle and heterocyclic refer to both heterocycloalkyls and heteroaryls. The term "heterocycloalkyl" means a cycloalkyl radical as defined herein (including polycyclics), wherein at least one carbon of a carbocyclic ring is substituted with a heteroatom such as, for example, O, N, or S. The heterocycloalkyl optionally has one or more double bonds within a ring, and may be aromatic, but is not necessarily aromatic. The heterocycloalkyl preferably has 3 to about 10 atoms (members) in the skeleton of each ring, more preferably from about 3 to about 7 atoms, more preferably from about 5 to about 6 atoms. Examples of heterocycloalkyl radicals include epoxy, aziridyl, oxetanyl, tetrahydrofuranyl, ribose, dihydrofuranyl, piperidinyl, piperazinyl, pyranyl, morpholinyl, and the like.

The term "heteroaryl" means a radical defined by an aromatic heterocyclic ring as commonly understood in the art, including monocyclic radicals such as, for example, imidazole, thiazole, pyrazole, pyrrole, furane, pyrazoline, thiophene, oxazole, isoxazole, pyridine, pyridone, pyrimidine, cytosine, 5-methylcytosine, thymine, pyrazine, and triazine radicals, and polycyclics such as, for example, quinoline, isoquinoline, indole, purine, adenine, guanine, N6-methyladenine, and benzothiazole radicals, which heteroaryl radicals are unsubstituted or substituted with one or more substituents, which are the same or different, selected from the group consisting of a halogen, an alkyl, an alkoxy, an amino, a cyano, a nitro, and the like. The heteroaryl preferably has 3 to about 10 atoms (members) in the ring skeleton of each ring, more preferably from about 3 to about 7 atoms, more preferably from about 5 to about 6 atoms.

It will be appreciated that the heterocycloalkyl and the heteroaryl substituents can be coupled to the compounds of the present invention via a heteroatom, such as nitrogen (e.g., 1-imidazolyl), or via a carbon atom (e.g., 4-thiazolyl). It will also be appreciated that heteroaryls, as defined herein, are not necessarily "aromatic" in the same context as phenyl is aromatic, although heteroaryls nonetheless demonstrate physical and chemical properties associated with aromaticity, as the term is understood in the art.

The term "carboxyl" means any functional group with a carbonyl backbone, and includes functional groups such as, for example, a carboxylic acid, an esters (e.g., ethoxycarbonyl), and amides (e.g., benzamido).

The term "nucleoside" includes all modified and naturally occurring nucleosides, including all forms of furanosides found in nucleic acids. Naturally occurring nucleosides include, for example, adenosine, guanosine, cytidine, thymidine, and uridine.

Nucleoside "derivatives" or "analogs" include synthetic nucleosides as described herein. Nucleoside derivatives also include nucleosides having modified base moieties, with or without protecting groups. Such analogs include, for example, deoxyinosine, 2,6-diaminopurine-2'-deoxyriboside, 5-methyl-2'-deoxycytidine, and the like. The base rings most commonly found in naturally occurring nucleosides are purine and pyrimidine rings. Naturally occurring purine rings include, for example, adenine, guanine, and N.sup.6 -methyladenine. Naturally occurring purine rings include, for example, cytosine, thymine, and 5-methylcytosine. The compounds and methods of the present invention include such base rings and synthetic analogs thereof, as well as unnatural heterocycle-substituted base sugars, and even acyclic substituted base sugars. Moreover, nucleoside derivatives include other purine and pyrimidine derivatives, for example, halogen-substituted purines (e.g., 6-fluoropurine), halogen-substituted pyrimidines, N.sup.6 -ethyladenine, N.sup.6 -(alkyl)-cytosines, 5-ethylcytosine, and the like.

The term "oligonucleotide" as used herein includes linear oligomers of natural or modified nucleosides,