Chiral phosphoramidite auxiliaries and methods of their use

Abstract

Disclosed are chiral phosphoramidite auxiliary compositions for diastereoselective synthesis of stereochemically enriched oligonucleotides, and nucleoside phosphoramidite compounds comprising the same. Exemplary structures of the chiral phosphoramidite auxiliary compositions include Formulas (IA) and (IB). ##STR00001##

Claims

1. A compound of formula (IIIA) or (IIIB): ##STR00074## wherein is a single carbon-carbon bond or a double carbon-carbon bond; A is an optionally substituted C.sub.1-12 alkyl, optionally substituted C.sub.3-10 cycloalkyl, optionally substituted C.sub.3-10 cycloalkyl-C.sub.1-6-alkyl, optionally substituted C.sub.1-9 heterocyclyl, optionally substituted C.sub.1-9 heterocyclyl-C.sub.1-6-alkyl, sugar analogue, nucleoside, nucleotide, or oligonucleotide; each of R.sup.1 and R.sup.2 is independently an optionally substituted C.sub.1-6 alkyl or optionally substituted C.sub.6-10 aryl, or R.sup.1 and R.sup.2, together with the atoms to which each is attached, combine to form an optionally substituted 5- to 8-membered ring; and each of R.sup.3 and R.sup.4 is independently H, optionally substituted C.sub.1-6 alkyl, or optionally substituted C.sub.6-10 aryl.

2. The compound of claim 1, wherein R.sup.1 and R.sup.2, together with the atoms to which each is attached, combine to form an optionally substituted 5- to 8-membered ring.

3. The compound of claim 1, wherein the compound is of the following structure: ##STR00075##

4. A nucleoside phosphoramidite comprising a sugar bonded to a nucleobase and to a phosphoramidite of the following structure: ##STR00076## wherein is a single carbon-carbon bond or a double carbon-carbon bond; each of R.sup.1 and R.sup.2 is independently an optionally substituted C.sub.1-6 alkyl or optionally substituted C.sub.6-10 aryl, or R.sup.1 and R.sup.2, together with the atoms to which each is attached, combine to form an optionally substituted 5- to 8-membered ring; and each of R.sup.3 and R.sup.4 is independently H, optionally substituted C.sub.1-6 alkyl, or optionally substituted C.sub.6-10 aryl.

5. The nucleoside phosphoramidite of claim 4, wherein the nucleoside phosphoramidite is of the following structure: ##STR00077## wherein B.sup.1 is a nucleobase; Y.sup.1 is H or C.sub.1-6 alkyl; R.sup.5 is H, O-protected hydroxyl, optionally substituted C.sub.1-6 alkoxy, or halogen; and R.sup.6 is a hydroxyl protecting group.

6. The nucleoside phosphoramidite of any one of claims 5, wherein R.sup.6 is dimethoxytrityl.

7. The nucleoside phosphoramidite of claim 4, wherein R.sup.1 and R.sup.2, together with the atoms to which each is attached, combine to form an optionally substituted 5- to 8-membered ring.

8. The nucleoside phosphoramidite of claim 4, wherein the phosphoramidite is of the following structure: ##STR00078##

9. A method of preparing a composition comprising an oligonucleotide comprising a stereochemically enriched internucleoside phosphorothioate, the method comprising (i) reacting the nucleoside phosphoramidite of claim 4 with a coupling activator and a nucleoside comprising a 5′-hydroxyl or an oligonucleotide comprising a 5′-hydroxyl, (ii) reacting with an electrophilic source of acyl, and (iii) reacting with a sulfurizing agent to produce the oligonucleotide comprising a stereochemically enriched internucleoside phosphorothioate triester.

10. A method of preparing the nucleoside phosphoramidite comprising a sugar bonded to a nucleobase and phosphoramidite of the following structure: ##STR00079## wherein is a single carbon-carbon bond or a double carbon-carbon bond; each of R.sup.1 and R.sup.2 is independently an optionally substituted C.sub.1-6 alkyl or optionally substituted C.sub.6-10 aryl, or R.sup.1 and R.sup.2, together with the atoms to which each is attached, combine to form an optionally substituted 5- to 8-membered ring; and each of R.sup.3 and R.sup.4 is independently H, optionally substituted C.sub.1-6 alkyl, or optionally substituted C.sub.6-10 aryl; the method comprising reacting a sugar bonded to a nucleobase with a compound of formula (VIIIA) or (VIIIB): ##STR00080## wherein X is a halogen or pseudohalogen.

11. An oligonucleotide comprising one or more internucleoside groups independently selected from the group consisting of linkers of formulas (XIA), (XIB), (XIIA) and (XIIB): ##STR00081## where is a single carbon-carbon bond or a double carbon-carbon bond; each of R.sup.1 and R.sup.2 is independently an optionally substituted C.sub.1-6 alkyl or optionally substituted C.sub.6-10 aryl, or R.sup.1 and R.sup.2, together with the atoms to which each is attached, combine to form an optionally substituted 5- to 8-membered ring; each of R.sup.3 and R.sup.4 is independently H, optionally substituted C.sub.1-6 alkyl, or optionally substituted C.sub.6-10 aryl; and R.sup.7 is acyl.

12. The compound of claim 1, wherein each of R.sup.3 and R.sup.4 is independently H.

13. The compound of claim 2, wherein the optionally substituted 5- to 8-membered ring is an optionally substituted 6-membered carbocyclic ring.

14. The nucleoside phosphoramidite of claim 4, wherein each of R.sup.3 and R.sup.4 is independently H.

15. The nucleoside phosphoramidite of claim 7, wherein the optionally substituted 5- to 8-membered ring is an optionally substituted 6-membered carbocyclic ring.

16. The method of claim 10, wherein the optionally substituted 5- to 8-membered ring is an optionally substituted 6-membered carbocyclic ring.

17. The method of claim 10, wherein each of R.sup.3 and R.sup.4 is independently H.

18. The oligonucleotide of claim 11, wherein the substituted 5- to 8-membered ring is substituted 6-membered carbocyclic ring.

19. The oligonucleotide of claim 11, comprising one or more internucleoside groups independently selected from the group consisting of linkers of formulas (XIIA) and (XIIB): ##STR00082##

20. The oligonucleotide of claim 11, comprising one or more internucleoside groups independently selected from the group consisting of linkers of formulas (XIA) and (XIB): ##STR00083##

Description

DETAILED DESCRIPTION

(1) The invention provides P-stereogenic groups for diastereoselective synthesis of stereochemically enriched P-stereogenic compounds. P-stereogenic groups of the invention can be used in highly diastereoselective synthesis of P-stereogenic phosphorothioates (e.g., with dr of 90:10 or greater (e.g., 95:5 or greater or 98:2 or greater)). Advantageously, P-stereogenic groups (e.g., those having R.sup.3 and R.sup.4 be H)) can be readily accessed through a short (e.g., a two-step synthesis) from commercially available materials. A P-stereogenic group of the invention is a group of formula (IA), (IB), (IC), or (ID):

(2) ##STR00022## where is a single carbon-carbon bond or a double carbon-carbon bond; each of R.sup.1 and R.sup.2 is independently an optionally substituted C.sub.1-6 alkyl or optionally substituted C.sub.6-10 aryl, or R.sup.1 and R.sup.2, together with the atoms to which each is attached, combine to form an optionally substituted 5- to 8-membered ring; and each of R.sup.3 and R.sup.4 is independently H, optionally substituted C.sub.1-6 alkyl, or optionally substituted C.sub.6-10 aryl.

(3) In some embodiments, the P-stereogenic group is a group of formula (IA) or (IB).

(4) In certain embodiments, the P-stereogenic group is of the following structure:

(5) ##STR00023## ##STR00024##

(6) In particular embodiments, R.sup.3 and R.sup.4 are each H. In some embodiments, the P-stereogenic group is a group of formula (IIA), (IIB), (IIA′), (IIB′), (IIA″), or (IIB″).

(7) The P-stereogenic groups of the invention may be provided in a compound of formula (IIIA), (IIIB), (IIIC), or (IIID):

(8) ##STR00025## wherein is a single carbon-carbon bond or a double carbon-carbon bond; A is an optionally substituted C.sub.1-12 alkyl, optionally substituted C.sub.3-10 cycloalkyl, optionally substituted C.sub.3-10 cycloalkyl-C.sub.1-6-alkyl, optionally substituted C.sub.1-9 heterocyclyl, optionally substituted C.sub.1-9 heterocyclyl-C.sub.1-6-alkyl, sugar analogue, nucleoside, nucleotide, or oligonucleotide; each of R.sup.1 and R.sup.2 is independently an optionally substituted C.sub.1-6 alkyl or optionally substituted C.sub.6-10 aryl, or R.sup.1 and R.sup.2, together with the atoms to which each is attached, combine to form an optionally substituted 5- to 8-membered ring; and each of R.sup.3 and R.sup.4 is independently H, optionally substituted C.sub.1-6 alkyl, or optionally substituted C.sub.6-10 aryl.

(9) In certain embodiments, the P-stereogenic group is of formula (IIIC′) or (IIID′):

(10) ##STR00026##

(11) In certain embodiments, the compound is of the following structure:

(12) ##STR00027## ##STR00028##

(13) In particular embodiments, A is an optionally substituted C.sub.1-12 alkyl, optionally substituted C.sub.3-10 cycloalkyl, optionally substituted C.sub.3-10 cycloalkyl-C.sub.1-6-alkyl, optionally substituted C.sub.1-9 heterocyclyl, optionally substituted C.sub.1-9 heterocyclyl-C.sub.1-6-alkyl, or sugar analogue.

(14) In further embodiments, A is a group of formula (X):

(15) ##STR00029## where each of R.sup.A1, R.sup.A2, and R.sup.A3 is independently H, —OR.sup.A4, or —N(R.sup.A4)(R.sup.A5); where R.sup.A4 is optionally substituted C.sub.1-16 alkyl, optionally substituted C.sub.2-16 heteroalkyl, or a protecting group, and R.sup.A5 is H optionally substituted C.sub.1-16 alkyl, optionally substituted C.sub.2-16 heteroalkyl, or a protecting group; and each of m1, m2, m3, and m4 is independently an integer from 0 to 11, provided that the quaternary carbon in the structure above is bonded to 0 or 1 atoms other than carbon and hydrogen, and provided that the sum of m1, m2, m3 and m4 is 11 or less.

(16) In some embodiments, the compound is of formula (IVA), (IVB), (IVA′), (IVB′), (IVA″), or (IVB″). In other embodiments, the phosphoramidite is of formula (IVE), (IVF), (IVE′), (IVF′), (IVE″), or (IVF″):

(17) ##STR00030##

(18) In certain embodiments, P-stereogenic groups may be provided in nucleoside phosphoramidites. The nucleoside phosphoramidites of the invention can be used to prepare oligonucleotides having P-stereogenic phosphorothioates with high diastereoselectivity (e.g., with dr of 90:10 or greater (e.g., 95:5 or greater or 98:2 or greater)). Advantageously, nucleoside phosphoramidites of the invention (e.g., those having R.sup.3 and R.sup.4 be H) can be readily accessed through a short synthesis (e.g., a two-step synthesis) from commercially available materials. Accordingly, the nucleoside phosphoramidites of the invention are a practical solution for high-yield synthesis of oligonucleotides having stereochemically enriched P-stereogenic phosphorothioates.

(19) The nucleoside phosphoramidites of the invention include a sugar bonded to a nucleobase and to a phosphoramidite of the following structure:

(20) ##STR00031## where is a single carbon-carbon bond or a double carbon-carbon bond; each of R.sup.1 and R.sup.2 is independently an optionally substituted C.sub.1-6 alkyl or optionally substituted C.sub.6-10 aryl, or R.sup.1 and R.sup.2, together with the atoms to which each is attached, combine to form an optionally substituted 5- to 8-membered ring; and each of R.sup.3 and R.sup.4 is independently H, optionally substituted C.sub.1-6 alkyl, or optionally substituted C.sub.6-10 aryl.

(21) In certain embodiments, the phosphoramidite is of formula (VA) or (VB). In particular embodiments, the phosphoramidite is of formula (VC′) or (VD′):

(22) ##STR00032##

(23) In some embodiments, the nucleoside phosphoramidite is of the following structure:

(24) ##STR00033## where B.sup.1 is a nucleobase; Y.sup.1 is H or C.sub.1-6 alkyl (e.g., methyl); R.sup.5 is H, O-protected hydroxyl, optionally substituted C.sub.1-6 alkoxy, or halogen (e.g., F); and R.sup.6 is a hydroxyl protecting group; and the remaining variables are as defined for formulas (VA), (VB), (VC), and (VD).

(25) In particular embodiments, the nucleoside phosphoramidite is of formula (VIC′) or (VID′):

(26) ##STR00034##

(27) In certain embodiments, the phosphoramidite is of the following structure:

(28) ##STR00035## ##STR00036##

(29) In particular embodiments, R.sup.3 and R.sup.4 are each H. In some embodiments, the phosphoramidite is of formula (VIIA), (VIIB), (VIIA′), or (VIIB′). In other embodiments, the phosphoramidite is of formula (VIIE), (VIIF), (VIIE′), (VIIF′), (VIIE″), or (VIIF″):

(30) ##STR00037##
Diastereoselective Preparation of Oligonucleotides Containing Phosphorothioate Phosphodiester

(31) The nucleoside phosphoramidites of the invention may be used for the diastereoselective preparation of oligonucleotides containing a phosphorothioate phosphodiester using reaction conditions known in the art for the phosphoramidite route for oligonucleotide synthesis.

(32) Typically, a nucleoside phosphoramidite of formula (VA) produces an internucleoside (R.sub.P)-phosphorothioate, and a nucleoside phosphoramidite of formula (VB) produces an internucleoside (S.sub.P)-phosphorothioate.

(33) In a typical oligonucleotide chain growth step, a nucleoside phosphoramidite of the invention is coupled to a nucleoside having a 5′-hydroxyl (e.g., a nucleoside linked to a solid support) or an oligonucleotide having a 5′-hydroxyl (e.g., an oligonucleotide linked to a solid support) to produce a product oligonucleotide including an internucleoside phosphite substituted with a ring-opened chiral auxiliary. Typically, the coupling step is performed in the presence of a coupling activator. Coupling activators are known in the art; non-limiting examples of coupling activators are (benzylthio)-1H-tetrazole (BTT), N-phenylimidazolium trifluoromethanesulfonate (PhIMT), 1-(cyanomethyl)pyrrolidinium trifluoromethanesulfonate (CMPT), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 4,5-dicyanoimidazole (DCI), 4,5-dichloroimidazole, benzimidazolium trifluoromethanesulfonate (BIT), benzotriazole, 3-nitro-1,2,4-triazole (NT), tetrazole, (ethylthio)-1H-tetrazole (ETT), (4-nitrophenyl)-1H-tetrazole, 1-(cyanomethyl)piperidinium trifluoromethanesulfonate, and N-cyanomethyldimethylammonium trifluoromethanesulfonate. In certain embodiments (e.g., when the nucleoside phosphoramidite includes 2′-deoxyribose), the coupling activator is preferably CMPT. The product oligonucleotide may be an oligonucleotide (e.g., an oligonucleotide having a total of 2-100 nucleosides (e.g., 2 to 50 or 2 to 35) including one or more (e.g., 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1) internucleoside groups independently selected from the group consisting of linkers of formula (XIA) and (XIB):

(34) ##STR00038##
where is a single carbon-carbon bond or a double carbon-carbon bond; each of R.sup.1 and R.sup.2 is independently an optionally substituted C.sub.1-6 alkyl or optionally substituted C.sub.6-10 aryl, or R.sup.1 and R.sup.2, together with the atoms to which each is attached, combine to form an optionally substituted 5- to 8-membered ring; each of R.sup.3 and R.sup.4 is independently H, optionally substituted C.sub.1-6 alkyl, or optionally substituted C.sub.6-10 aryl; and R.sup.7 is acyl (e.g., alkanoyl).

(35) The oligonucleotides including one or more internucleoside groups of formula (XIA) and/or (XIB) may be intermediates in the synthesis of an oligonucleotide including at least one stereochemically enriched internucleoside phosphorothioate. For example, these oligonucleotides may be subjected to a sulfurization reaction with a sulfurizing agent (e.g., Beaucage reagent; 3-((N,N-dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-5-thione (DDTT); S.sub.8; or a compound of formula (XA) or (XB)) to produce an oligonucleotide (e.g., an oligonucleotide having a total of 2-100 nucleosides (e.g., 2 to 50 or 2 to 35) including one or more (e.g., 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1) internucleoside groups independently selected from the group consisting of linkers of formula (XIIA) and (XIIB):

(36) ##STR00039##
where the variables are as describe for formulae (XIA) and (XIB).

(37) Sulfurizing agents are known in the art; non-limiting examples of the sulfurizing agents are Beaucage reagent; 3-((N,N-dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-5-thione (DDTT); S.sub.8; and compounds of formula (XA) and (XB).

(38) The compound of formula (XA) is of the following structure:
R.sup.8—S—S—R.sup.8,   (XA) or a salt thereof, where each R.sup.8 is independently R.sup.9C(X.sup.1)—, (R.sup.10).sub.2P(X.sup.1) or R.sup.11S(O).sub.2—, where each R.sup.9 is independently alkylamino or dialkylamino; each R.sup.10 is independently alkoxy or aryloxy; each R.sup.11 is independently hydroxyl, alkyl, aryl, or heteroaryl; and X.sup.1 is ═O or ═S.

(39) The compound of formula (XB) is of the following structure:

(40) ##STR00040## where X.sup.2 is O or S; and R.sup.12 is aryl, amino, or alkoxy.

(41) For example, the compound of formula (XB) can be:

(42) ##STR00041##

(43) The oligonucleotide including one or more internucleoside groups of formula (XIIA) and/or (XIIB) is then fed back into the synthesis, e.g., by deprotecting the 5′-protecting group and treating the resulting 5′-hydroxyl as described above or using a different nucleoside phosphoramidite (e.g., those known in the art). Alternatively, if the synthesis of the oligonucleotide chain is complete, the oligonucleotide may be subjected to further modifications (e.g., capping the 5′ end). If the oligonucleotide chain is linked through a linker to solid support, the linker may be cleaved using methods known in the art after the synthesis of the oligonucleotide chain is complete. The remainder of the ring-opened chiral auxiliaries of the invention may be removed from phosphotriesters through hydrolysis with aqueous ammonia (30% (w/w)) (e.g., by heating for 12-24 hours at, e.g., about 55° C.). The remainder of the ring-opened chiral auxiliaries of the invention may be removed before, after, or concomitantly with the oligonucleotide chain removal from the solid support.

(44) A non-limiting example of an oligonucleotide synthesis route is shown in Scheme 1.

(45) ##STR00042##

(46) As shown in Scheme 1, compound A, which is a protected nucleoside optionally linked to a solid support, may be subjected to a deprotection reaction to remove the 0-protecting group (e.g., DMT) at R.sup.6 and produce compound B. Compound B is then coupled to phosphoramidite C to produce phosphite D. In certain embodiments (e.g., when the nucleoside phosphoramidite includes 2′-deoxyribose, e.g., when R.sup.5 is H), the coupling activator is preferably CMPT.

(47) Compound D is oxidized using a sulfurizing agent to afford phosphorothioate E with retention of stereochemistry.

(48) Nucleoside phosphoramidites including phosphoramidites of formula (IA), (IB), (IC), and (ID) can be in the synthesis of oligonucleotides in accordance with the procedure described above using reaction conditions known in the art.

(49) Preparation of Nucleoside Phosphoramidites

(50) Phosphoramidite Precursors

(51) The nucleoside phosphoramidites of the invention may be prepared from a compound of formula:

(52) ##STR00043## where is a single carbon-carbon bond or a double carbon-carbon bond; X is a halogen (e.g., Cl or Br) or pseudohalogen; each of R.sup.1 and R.sup.2 is independently an optionally substituted C.sub.1-6 alkyl or optionally substituted C.sub.6-10 aryl, or R.sup.1 and R.sup.2, together with the atoms to which each is attached, combine to form an optionally substituted 5- to 8-membered ring; and each of R.sup.3 and R.sup.4 is independently H, optionally substituted C.sub.1-6 alkyl, or optionally substituted C.sub.6-10 aryl.

(53) In particular embodiments, the precursor to a nucleoside phosphoramidite may be a compound of formula:

(54) ##STR00044## ##STR00045## where each of R.sup.3 and R.sup.4 is independently H, optionally substituted C.sub.1-6 alkyl, or optionally substituted C.sub.6-10 aryl; and X is halogen (e.g., Cl or Br) or pseudohalogen.

(55) A non-limiting example of the preparation of a nucleoside phosphoramidite of the invention is shown in Scheme 2.

(56) ##STR00046##

(57) As shown in Scheme 2, aminoalcohol G can be converted to oxazaphospholane of formula (VIIIA) using an electrophilic source of phosphorus (III), e.g., phosphorus (III) halide (e.g., PCl.sub.3). An oxazaphospholane of formula (VIIIA) may be coupled to nucleoside H to give a nucleoside phosphoramidite of formula (VIA). The reaction conditions useful for this coupling are known in the art and typically involve the use of a sterically hindered organic base (e.g., N,N-diisopropylethylamine). Typically, the oxazaphospholane formation and phosphoramidite formation are performed in a one-pot transformation without isolation or purification of the oxazaphospholane of formula (VIIIA).

(58) Nucleoside phosphoramidites including phosphoramidites of formula (VA), (VB), (VC), and (VD) can be prepared according to the procedure described above using reaction conditions known in the art.

(59) Aminoalcohol G and its enantiomer can be prepared from the corresponding amino acid using methods and reactions known in the art. Aminoalcohol G can be used in the preparation of compounds containing a P-stereogenic group of formula (IA) or (IB) (e.g., compounds of formula (IIIA) or (IIIB)). Aminoalcohol I and its enantiomer for the preparation of phosphoramidites of formula (VC) and (VD) can be prepared from the corresponding amino acids using methods and reactions known in the art. Aminoalcohol I can be used in the preparation of compounds containing a P-stereogenic group of formula (IC) or (ID) (e.g., compounds of formula (IIIC) or (IIID)). Aminoalcohol I is a compound of the following structure:

(60) ##STR00047##
where each of R.sup.1 and R.sup.2 is independently optionally substituted C.sub.1-6 alkyl or optionally substituted C.sub.6-10 aryl, or R.sup.1 and R.sup.2, together with the atoms to which each is attached, combine to form an optionally substituted 5- to 8-membered ring.

(61) Advantageously, when R.sup.3 and R.sup.4 are each H, nucleoside phosphoramidites of the invention can be prepared through a short reaction sequence of only three steps, two of which can be carried out in one pot.

(62) The following examples are meant to illustrate the invention. They are not meant to limit the invention in any way.

EXAMPLES

(63) Chiral Auxiliaries and Phosphoramidites

(64) ##STR00048##

(65) To a solution of (2S)-dihydro-1H-indole-2-carboxylic acid 1 (1.63 g, 10.0 mmol) in ether (50 mL) was added a solution of LiAlH.sub.4 in THF (2M, 7.5 mL, 15.0 mmol) under argon, and the mixture was stirred overnight. After completion of the reaction, the mixture was quenched with Na.sub.2SO.sub.4.10H.sub.2O. The solid was filtered off and washed with ethyl acetate, and the filtrate was dried over anhydrous Na.sub.2SO.sub.4. The mixture was filtered, and the solvent evaporated to give a residue, which was subjected to flash silica gel column purification on an ISCO (hexane/ethyl acetate, 10-70%) to give 1.44 g (96%) of compound 2 as a gray solid. .sup.1H NMR (500 MHz, CDCl.sub.3; ppm): δ7.10 (1H, d, J 7.5 Hz), 7.04 (1H, t, J 7.5 Hz), 6.75 (1H, t, J 7.5 Hz), 6.69 (1H, d, J 7.5 Hz), 4.10-4.06 (2H, m), 3.75 (1H, dd, J 11.0, 4.0 Hz), 3.60 (1H, dd, J 11.0, 6.0 Hz), 3.12 (1H, dd, J 16.0, 9.0 Hz), 2.87 (1H, dd, J 16.0, 8.0 Hz); ESI MS for C.sub.9H.sub.11NO calculated 149.2, observed [M+H].sup.+ 150.1.

(66) ##STR00049##

(67) To a solution of compound 2 (1.0 g, 6.7 mmol) in anhydrous THF (5 mL) was added N,N-diisopropylethylamine (2.41 mL, 13.4 mmol) under argon. The resulting mixture was added dropwise to a solution of phosphorus trichloride (0.58 mL, 6.7 mmol) in anhydrous THF (8 mL) at 0° C. under argon. The mixture was warmed to room temp and stirred for 1.5 h. In a separate round bottom flask, a solution of 5′-O-(4,4′-dimethoxytrityl)-2′-methoxy-uridine (2.25 g, 4.0 mmol) and N,N-diisopropylethylamine (4.81 mL, 26.8 mmol) in THF (5 mL) under argon was cooled to −78° C., and the above mixture was slowly added. The mixture was warmed to room temp, stirred for 3 h, diluted with dichloromethane (30 mL), and washed with saturated aqueous sodium bicarbonate (20 mL). The organic layer dried over anhydrous sodium sulfate and filtered. The filtrate was evaporated to afford a residue, which was subjected to flash silica gel amine column purification on an ISCO (1-8% methanol in dichloromethane) to give 1.14 g (39%) of the title compound 3 as a white foam. ESI MS for C.sub.40H.sub.40N.sub.3O.sub.9P Calculated 737.7, Observed 738.2 (M+1); .sup.31 P NMR (202 MHz, CDCl.sub.3): δ141.2 (s).

(68) ##STR00050##

(69) Compound 4 was prepared by the same procedure as reported here for Compound 3 using 5′-O-(4,4′-dimethoxytrityl)-2′-fluoro-uridine as a starting material. Compound 4 was produced in 24% yield. ESI MS for C.sub.39H.sub.37FN.sub.3O.sub.8P Calculated 725.7, Observed 748.3 (M+Na); .sup.31 P NMR (202 MHz, CDCl.sub.3): δ141.0 (s).

(70) ##STR00051##

(71) To a solution of (2S,3aS,7aS)-octahydro-1H-indole-2-carboxylic acid 5 (1.69 g, 10.0 mmol) in ether (50 mL) was added a solution of LiAlH.sub.4 in THF (2M, 7.5 mL, 15 mmol) under argon, and the mixture was stirred overnight. After completion of the reaction, the mixture was quenched with Na.sub.2SO.sub.4.10H.sub.2O, and the solids were filtered off and washed with ethyl acetate. The filtrate was dried over anhydrous Na.sub.2SO.sub.4 and evaporated to give 1.23 g (79%) of the crude compound 6 as a colorless oil. .sup.1H NMR (500 MHz, CDCl.sub.3; ppm): δ3.70 (1H, dd, J 11.0, 3.5 Hz), 3.60 (1H, dd, J 11.0, 6.0 Hz), 3.50-3.40 (1H, m), 3.24 (1H, q, J 6.0 Hz), 2.13-2.08 (1H, m), 1.94-1.86 (1H, m), 1.75-1.65 (1H, m), 1.65-1.40 (6H, m), 1.35-1.23 (2H, m); ESI MS for C.sub.9H.sub.11NO calculated 155.2, observed [M+H].sup.+ 156.1.

(72) ##STR00052##

(73) Compound 7 was prepared by the same procedure as reported here for Compound 3 using 5′-O-(4,4′-dimethoxytrityl)-2′-fluoro-uridine as a starting material. ESI MS for C.sub.39H.sub.43FN.sub.3O.sub.8P calculated 731.7, observed 732.2 (M+1); .sup.31P NMR (202 MHz, CDCl.sub.3): δ140.7 (s).

(74) ##STR00053##

(75) Compound 8 was prepared by the same procedure as reported here for Compound 3 using 5′-O-(4,4′-dimethoxytrityl)-2′-methoxy-uridine as a starting material. ESI MS for C.sub.40H.sub.46N.sub.3O.sub.9P calculated 743.8, observed 742.5 (M−1); .sup.31 P NMR (202 MHz, CDCl.sub.3): δ140.0 (s).

(76) ##STR00054##

(77) To a solution of (2R)-dihydro-1H-indole-2-carboxylic acid 9 (4.90 g, 30.0 mmol) in ether (100 mL) was added a solution of LiAlH.sub.4 in THF (2M, 22.5 mL, 45 mmol) under argon, and the mixture was stirred overnight. After completion of the reaction, the reaction mixture was quenched with Na.sub.2SO.sub.4.10H.sub.2O, and the solids were filtered off and washed with ethyl acetate. The filtrate was dried over anhydrous Na.sub.2SO.sub.4, the mixture was filtered, and the solvent evaporated to give a residue, which was subjected to flash silica gel column purification on an ISCO (hexane/ethyl acetate, 10-70%) to give 3.68 g (82%) of compound 10 as a gray solid. .sup.1H NMR (500 MHz, CDCl.sub.3; ppm): δ7.10 (1H, d, J 7.5 Hz), 7.04 (1H, t, J 7.5 Hz), 6.75 (1H, t, J 7.5 Hz), 6.69 (1H, d, J 7.5 Hz), 4.10-4.06 (1H, m), 3.75 (1H, dd, J 11.0, 4.0 Hz), 3.60 (1H, dd, J 11.0, 6.0 Hz), 3.12 (1H, dd, J 16.0, 9.0 Hz), 2.87 (1H, dd, J 16.0, 8.0 Hz); ESI MS for C.sub.9H.sub.11NO calculated 149.2, observed [M+H].sup.+ 150.1.

(78) ##STR00055##

(79) Compound 11 was prepared by the same procedure as reported here for Compound 3 using 5′-O-(4,4′-dimethoxytrityl)-2′-methoxy-uridine as a starting material. Compound 11 was produced in 56% yield. ESI MS for C.sub.40H.sub.40N.sub.3O.sub.9P calculated 737.7, observed 738.2 (M+1); .sup.31 P NMR (202 MHz, CDCl.sub.3): δ141.3 (s).

(80) ##STR00056##

(81) Compound 12 was prepared by the same procedure as reported here for Compound 3 using 5′-O-(4,4′-dimethoxytrityl)-2′-fluoro-uridine as a starting material. Compound 12 was produced in 72% yield. ESI MS for C.sub.39H.sub.37FN.sub.3O.sub.8P Calculated 725.7, Observed 748.3 (M+Na); .sup.31P NMR (202 MHz, CDCl.sub.3): δ141.8 (s).

(82) ##STR00057##

(83) Compound 13 was prepared by the same procedure as reported here for Compound 3 using 5′-O-(4,4′-dimethoxytrityl)-2′-deoxy-cytidine (N-acetyl) as a starting material. Compound 13 was produced in 33% yield. ESI MS for C.sub.41H.sub.41N.sub.4O.sub.8P Calculated 748.8, Observed 747.4 (M-1); .sup.31P NMR (202 MHz, CDCl.sub.3): δ140.2 (s).

(84) ##STR00058##

(85) Compound 14 was prepared by the same procedure as reported here for Compound 3 using 5′-O-(4,4′-dimethoxytrityl)-2′-deoxy-cytosine (N-acetyl) as a starting material. Compound 14 was produced in 27% yield. ESI MS for C.sub.41H.sub.41N.sub.4O.sub.8P Calculated 748.8, Observed 747.4 (M-1); .sup.31P NMR (202 MHz, CDCl.sub.3): δ139.7 (s).

(86) ##STR00059##

(87) Compound 15 was prepared by the same procedure as reported here for Compound 3 using 5′-O-(4,4′-dimethoxytrityl)-2′-methoxy-cytosine (N-acetyl) as a starting material. Compound 15 was produced in 35% yield. ESI MS for C.sub.42H.sub.43N.sub.4O.sub.8P Calculated 778.8, Observed 779.3 (M); .sup.31 P NMR (202 MHz, CDCl.sub.3): δ141.0(s).

(88) ##STR00060##

(89) Compound 16 was prepared by the same procedure as reported here for Compound 3 using 5′-O-(4,4′-dimethoxytrityl)-2′-methoxy-adenosine (N-benzoyl) as a starting material. Compound 16 was produced in 48% yield. ESI MS for C.sub.48H.sub.45N.sub.6O.sub.8P Calculated 864.8, Observed 865.3 (M); .sup.31 P NMR (202 MHz, CDCl.sub.3): δ140.0 (s).

(90) ##STR00061##

(91) Compound 17 was prepared by the same procedure as reported here for compound 3 using 5′-O-(4,4′-dimethoxytrityl)-2′-methoxy-guanosine (N-i-butyryl) as a starting material. Compound 17 was produced in 10% yield. ESI MS for C.sub.45H.sub.47N.sub.6O.sub.9P Calculated 846.9, Observed 847.3 (M); .sup.31 P NMR (202 MHz, CDCl.sub.3): δ138.9 (s).

(92) Compounds listed in Table 1 were prepared by the same procedure as reported here for compound 3.

(93) TABLE-US-00001 TABLE 1 Chem- ical .sup.31P NMR Yield (202 Compound (%) MHz) 33 — 26 — 34 δ138.84 (s) 33 δ138.53 (s) 57 δ138.90 (s) 44 δ139.58 (s) 45 δ138.63 (s) 42 δ139.41 (s) 0 32 δ140.93 (s) 14 δ139.13 (s) *iBu in this table stands for isobutyryl.
Synthesis of the Polynucleotide Constructs

(94) ##STR00072##

(95) All the polynucleotide constructs synthesized were modified at the 2′-ribose sugar position with 2′-F, 2′-OMe, or 2′-deoxy modification. 0-protecting groups, such as 2′-OTBDMS, can also be used. Automated polynucleotide synthesis (1 μmol scale) was carried out with the following reagents/solvents: Solid support—CPG Glen Uny support Coupling agent—0.25 M BTT in acetonitrile Oxidizer—0.02 M 12 in THF/Pyridine/H.sub.2O (2×30 s oxidation per cycle) Deblock—3% Trichloroacetic Acid/DCM (2×40 s deblocks per cycle) Cap Mix A—THF/2, 6-Lutidine/Ac.sub.2O (2×30 s capping per cycle) Cap Mix B—16% Methyl imidazole in THF (2×30 s capping per cycle) Sulfurization—0.05 M sulfurizing reagent, 3-((N,N-dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-5-thione (DDTT), in 60% pyridine/40% acetonitrile (3×60 s sulfurization per cycle) Coupling—Phosphoramidites were suspended to a concentration of 100 mM in anhydrous acetonitrile prior to synthesis, phosphoramidite activation was performed with 2.5-fold molar excess of BTT, 0.25 M in acetonitrile. Activated phosphoramidites were coupled for 3×60 seconds per cycle
Polynucleotide Deprotection and Purification Protocol: When polynucleotides contain standard nucleobase protecting groups (such as A-Bz, C-Ac and G-iBu etc.), the following cleavage and deprotection conditions were used: polynucleotides were cleaved and deprotected in 1.0 mL of AMA (1:1 ratio of 36% aq. ammonia and 40% methylamine in methanol) for 2 h at room temperature followed by centrifugal evaporation. Crude polynucleotide pellets were re-suspended in 100 μL of 50% acetonitrile/water, briefly heated to 65° C., and vortexed thoroughly. Total 100 μL crude polynucleotide samples were injected onto reverse phase HPLC with the following buffers/gradient: Buffer A=50 mM aqueous triethylammonium acetate (TEAA) Buffer B=90% acetonitrile in water Flow Rate=1 mL/min Gradient: 0-2 min (100% Buffer A/0% Buffer B) 2-42 min (0% to 60% Buffer B) 42-55 min (60% to 100% Buffer B) Across the dominant reverse phase HPLC peaks, 0.5 mL fractions were collected and analyzed by MALDI-TOF mass spectrometry to confirm the presence of compounds with the desired mass peaks. Purified fractions containing compounds with the correct mass peaks were frozen and lyophilized. Once dry, fractions were re-suspended, combined with corresponding fractions, frozen, and lyophilized to give the final product.

(96) Polynucleotides requiring additional deprotection were initially isolated as described above followed by the necessary secondary deprotection steps (see below):

(97) Secondary Deprotection of Polynucleotides Having TBDMS Protection:

(98) Reverse phase HPLC-purified polynucleotide products were re-suspended in 219 μL of anhydrous DMSO, heated briefly to 65° C., and vortexed thoroughly. To the DMSO solution, 31 μL of 6.1 M triethylamine trihydrofluoride (TEA.3HF) was added to give a final concentration of 0.75 M. The reaction was allowed to proceed at room temperature for ˜1 h per TBDMS-protected hydroxyl modification. Reaction was monitored by MALDI-TOF mass spectrometry to confirm complete deprotection. Once deprotection was complete, 35 μL of 3M sodium acetate and 1 mL of butanol were sequentially added. Samples were vortexed thoroughly and placed at −80° C. for 2 h. After 2 h, samples were centrifuged to pellet the polynucleotides. The butanol layer was removed, and the polynucleotide pellet was re-suspended in 1 mL of aqueous 20% acetonitrile. Samples were gel-filtered for isolation of the final polynucleotide construct.

(99) Synthesis of Polynucleotide Constructs with Stereochemically Enriched Internucleoside Phosphorothioates (PS):

(100) The following modified experimental conditions have been used for the synthesis of polynucleotide constructs including stereochemically enriched internucleoside phosphorothioates from chiral phosphoramidite monomers. Automated polynucleotide synthesis (1 μmol scale) was carried out with the following reagents/solvents: Solid support—CPG Glen Uny support Coupling agent—BTT/ETT/CMPT/phenyl imidazole as required Oxidizer—0.02 M 12 in THF/Pyridine/H.sub.2O (2×30 s oxidation per cycle) Deblock—3% dichloroacetic Acid/DCM (2×40 s deblocks per cycle) Cap Mix A—THF/2,6-lutidine/Ac.sub.2O (2×30 s capping per cycle) Cap Mix B—16% methyl imidazole in THF (2×30 s capping per cycle) Sulfurization—0.05 M sulfurizing reagent, 3-((N,N-dimethylaminomethylidene)amino)-3H-1,2,4-dithiazole-5-thione (DDTT), in 60% pyridine/40% acetonitrile (3×120 s sulfurization per cycle) Coupling—chiral phosphoramidites (e.g., compounds of formula (IIA) or (IIB)) were suspended to a concentration of 100 mM in anhydrous acetonitrile prior to synthesis, phosphoramidite activation was performed with 2.5-fold molar excess of respective activators as specified (BTT=0.25 M in acetonitrile, CMPT=0.5 M in acetonitrile, Ph-Imidazole=0.5 M in Acetonitrile). Activated chiral phosphoramidites were coupled for 3×200 seconds per cycle.
Chiral Polynucleotide Deprotection and Purification Protocol: Following automated polynucleotide synthesis, stereopure phosphorothioate containing polynucleotides with standard nucleobase protecting groups (such as A-Bz, C-Ac, and G-iBu etc.) and chiral auxiliary were deprotected and cleaved with the following conditions: DMT protected chiral phosphorothioate polynucleotides on solid support was suspended in 1.0 mL of aqueous ammonia (30 wt %) and heated at 55° C. for 12-24 h, followed by centrifugal evaporation. Crude chiral polynucleotide pellets were re-suspended in 100 μL of 50% acetonitrile, briefly heated to 65° C., and vortexed thoroughly. Total 100 μL crude polynucleotide samples were injected onto reverse phase HPLC with the following buffers/gradient: Buffer A=100 mM aqueous triethylammonium acetate (TEAA) Buffer B=90% acetonitrile in water Flow Rate=1 mL/min Gradient: 0-2 min (100% Buffer A/0% Buffer B) 2-50 min (0% to 45% Buffer B) 50-55 min (45% to 100% Buffer B) Across the dominant reverse phase HPLC peaks, 1.0 mL fractions were collected and analyzed by MALDI-TOF mass spectrometry to confirm the presence of compounds with the desired mass peaks. Purified fractions containing compounds with the correct mass peaks were frozen and lyophilized. Once dry, fractions were re-suspended, combined with corresponding fractions, frozen, and lyophilized to give the final product.
Analysis of Stereochemical Purity of Phosphorothioate Containing Polynucleotides:

(101) DMT protected oligonucleotides with stereochemically enriched phosphorothioates were analyzed by HPLC/UPLC to determine the diastereoselectivity of R.sub.P and S.sub.P isomers. The absolute stereochemical identity of the internucleoside phosphorothioate identified with an asterisk (*) was determined through comparison of the HPLC traces of the oligonucleotides of the invention to the HPLC traces of authentic racemic and diastereomerically enriched oligonucleotides that were prepared using methods known in the art. The HPLC conditions were as follows: Reverse Phase HPLC Column: AdvancedBio Oligonucleotide, 2.1×100 mm, 2.7 μm Mobile Phase A: 100 mM tetraethylammonium acetate in water Mobile Phase B: acetonitrile Gradient: 10-12% mobile phase B in 45 min Column Temperature: 60° C. Flow Rate: 0.35 mL/min Detection: 260 nm (UV)

(102) For comparison, reference standards of the same oligonucleotide with R.sub.P and S.sub.P isomers were prepared using literature methods as described elsewhere (Oka et al., Chem. Soc. Rev., 40:5829-5843, 2011; Oka et al., Org. Lett., 11:967-970, 2009; and U.S. pre-grant publication Nos. 2013/0184450 and 2015/0197540).

(103) Diastereomer ratios (S.sub.P:R.sub.P) have been established by integrating the product peaks in UPLC traces of the prepared oligonucleotides. Absolute stereochemical identity of the dominant diastereomer was determined by comparison to the reference standard. UPLC was performed as follows. Samples were dissolved in water, injected onto UPLC, and analyzed under the following conditions: Column: Xbridge C18, 4.6×150 mm, 5 μm Mobile phase A=50 mM aqueous triethylammonium acetate (TEAA) in water Mobile phase B=Acetonitrile in water Flow Rate=1 mL/min Column Temperature=50° C. Detection=260 nm Gradient: 0-1 min (90% mobile phase A/10% mobile phase B) 1-30 min (88.5% mobile phase A/11.5% mobile phase B)

(104) The stereochemical purity, stereochemical identity, and coupling activators used in the synthesis of the prepared oligonucleotides are shown in Table 2.

(105) TABLE-US-00002 TABLE 2 Oligonu- En- cleotide Acti- try (5′-3′) vator PN (**) Sp:Rp 1 uUGAAGUAAA BTT Racemic Racemic Racemic 2 u*UGAAGUAAA BTT (R.sub.P)-OHI (S.sub.P) >99.0:<1.0 3 u*UGAAGUAAA BTT (R.sub.P)-DHI (S.sub.P) >99.0:<1.0 4 u*UGAAGUAAA PhIMT (R.sub.P)-OHI (S.sub.P) >99.0:<1.0 5 u*UGAAGUAAA PhIMT (R.sub.P)-DHI (S.sub.P) >99.0:<1.0 6 u*UGAAGUAAA CMPT (R.sub.P)-OHI (S.sub.P) >99.0:<1.0 7 u*UGAAGUAAA CMPT (R.sub.P)-DHI (S.sub.P) >99.0:<1.0 8 uUGAAGUAAA BTT Racemic Racemic Racemic 9 u*UGAAGUAAA BTT (R.sub.P)-OHI (S.sub.P) 91:11 10 u*UGAAGUAAA BTT (R.sub.P)-DHI (S.sub.P) >99.0:<1.0 11 u*UGAAGUAAA CMPT (R.sub.P)-OHI (S.sub.P) 95.0:5.0 12 u*UGAAGUAAA CMPT (R.sub.P)-DHI (S.sub.P) >99.0:<1.0 13 u*UGAAGUAAA PhIMT (R.sub.P)-OHI (S.sub.P) >99.0:<1.0 14 u*UGAAGUAAA PhIMT (R.sub.P)-DHI (S.sub.P) >99.0:<1.0 15 u*UGAAGUAAA BTT (S.sub.P)-DHI (R.sub.P) 18:82 16 u*UGAAGUAAA BTT (S.sub.P)-DHI (R.sub.P) <1:>99.0 17 u*UGAAGUAAA CMPT (S.sub.P)-DHI (R.sub.P) 1.7:98.3 18 u*UGAAGUAAA CMPT (S.sub.P)-DHI (R.sub.P) <1:>99.0 19 u*UGAAGUAAA PhIMT (S.sub.P)-DHI (R.sub.P) 4.2:95.8 20 u*UGAAGUAAA PhIMT (S.sub.P)-DHI (R.sub.P) 7.4:92.6 21 mNUAAGUAAA BTT Racemic Racemic Racemic 22 m*NUAAGUAAA BTT (S.sub.P)-DHI (R.sub.P) 16:84 23 m*NUAAGUAAA BTT (R.sub.P)-DHI (S.sub.P) 73.9:26.1 24 m*NUAAGUAAA CMPT (S.sub.P)-DHI (R.sub.P) <1.0:>99.0 25 m*NUAAGUAAA CMPT (R.sub.P)-DHI (S.sub.P) 96.3:3.7 26 m*NUAAGUAAA PhIMT (S.sub.P)-DHI (R.sub.P) 12.1:87.9 27 m*NUAAGUAAA PhIMT (R.sub.P)-DHI (S.sub.P) 74.4:25.6 28 aUGAAGUAAA BTT Racemic Racemic Racemic 29 a*UGAAGUAAA CMPT (S.sub.P)-DHI (R.sub.P) <1.0:>99.0 30 mUGAAGUAAA BTT Racemic Racemic Racemic 31 m*UGAAGUAAA CMPT (S.sub.P)-DHI (R.sub.P) <1.0:>99.0 30 gUGAAGUAAA BTT Racemic Racemic Racemic 31 g*UGAAGUAAA CMPT (S.sub.P)-DHI (R.sub.P) <1.0:>99.0 32 aNUAAGUAAA BTT Racemic Racemic Racemic 33 a*NUAAGUAAA CMPT (R.sub.P)-DHI (S.sub.P) 98.4:1.6 34 gNUAAGUAAA BTT Racemic Racemic Racemic 35 g*NUAAGUAAA CMPT (R.sub.P)-DHI (S.sub.P) 90.9:9.1 36 tNUAAGUAAA BTT Racemic Racemic Racemic 37 t*NUAAGUAAA CMPT (R.sub.P)-DHI (S.sub.P) 86.8:13.2

(106) In Table 2, lower case u is uridine having 2′-F and a 3′ position bonded to phosphorothioate; lower case bold u is uridine having 2′-OMe and a 3′ position bonded to phosphorothioate; lower case a is 2′-deoxyadenosine having a 3′ position bonded to phosphorothioate; lower case bold a is adenosine having a 2′-OMe and a 3′ position bonded to phosphorothioate; lower case g is 2′-deoxyguanosine having a 3′ position bonded to phosphorothioate; lower case bold g is guanosine having 2′-OMe and a 3′ position bonded to phosphorothioate; lower case m is 2′-deoxycytidine having a 3′ position bonded to phosphorothioate; lower case bold m is cytidine having 2′-OMe and a 3′ position bonded to phosphorothioate; lower case t is 2′-deoxythymidine having a 3′ position bonded to phosphorothioate; * indicates a stereochemically enriched internucleoside phosphorothioate; UPPER CASE LETTERS identify nucleosides having 2′-F and a 3′ position bonded to phosphate; UPPER CASE BOLD LETTERS identify nucleosides having 2′-OMe and a 3′ position bonded to phosphate; N is a 2′-deoxyguanosine having a 3′ position bonded to phosphate; PN means phosphoramidite; (**) provides stereochemical identity of the internucleoside phosphorothioate identified with * in the oligonucleotide column; and DHI and OHI represent the following structures:

(107) ##STR00073##
respectively.

OTHER EMBODIMENTS

(108) Various modifications and variations of the described invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention.

(109) Other embodiments are in the claims.