PROCESS FOR THE PREPARATION OF GALNAC PHOSPHORAMIDITE EPIMERS

Abstract

The invention comprises a process for the preparation of a GalNAc phosphoramidite epimer of the formula (I), wherein R.sup.1 is a hydroxy protecting group, n is an integer from 0 to 10 and m is an integer from 0 to 20, corresponding enantiomers and/or optical isomers thereof and the use of the process for the preparation of GalNAc-cluster oligonucleotide conjugates.

##STR00001##

Claims

1. Process for the preparation of epimerically pure GalNAc phosphoramidite epimers of the formula I, ##STR00031## wherein R.sup.1 is a hydroxy protecting group, n is an integer from 0 to 10 and m is an integer from 0 to 20, corresponding enantiomers and/or optical isomers thereof comprising a) coupling a compound of formula II, ##STR00032## wherein R.sup.3 is a hydroxy protecting groups and m is as above, or a salt thereof with a GalNAc moiety of the formula III ##STR00033## wherein R.sup.1 and n is as above to form the GalNAc amide of formula IV ##STR00034## wherein R.sup.1, R.sup.3, n and m are as above; and b) removing the hydroxyl protecting group R.sup.3 to form the free alcohol of the GalNAc amide of formula IV and c) reacting the free alcohol of the GalNAc amide of formula IV with a phosphoroamidating agent to form the GalNAc phosphoramidite epimer of the formula I.

2. Process of claim 1, wherein R.sup.1 is an acyl group.

3. Process of claim 1 or 2, wherein R.sup.1 is a C.sub.1-6-alkylcarbonyl group which is optionally substituted by C.sub.1-6-alkyl or phenyl.

4. Process of any one of claims 1 to 3, wherein n is an integer from 0 to 5 and m is an integer from 0 to 10.

5. Process of any one of claims 1 to 4, wherein R.sup.1 is acetyl, n is 2 and m is 5.

6. Process of anyone of claims 1 to 5, wherein R.sup.3 is benzyl.

7. Process of any one of claims 1 to 6, wherein the formula I comprises GalNAc phosphoramidite epimers of the formulas Ib to Ie. ##STR00035## ##STR00036##

8. Process of any one of claims 1 to 7, wherein the compound of formula II is prepared by a1) coupling a lysine compound of formula V ##STR00037## wherein R.sup.2 and R.sup.4 are amino protecting groups with an amine of the formula VI ##STR00038## wherein R.sup.3 and m are as above to form the carboxamide of formula VII; ##STR00039## wherein R.sup.2, R.sup.3, R.sup.4 and m are as above and b1) removing the amino protecting R.sup.4 to form the amine of formula VIII ##STR00040## wherein R.sup.2, R.sup.3 and m are as above; cl) coupling the amine of formula VIII with an amino group protected lysine to form the dipeptide of formula IX ##STR00041## wherein R.sup.2 and R.sup.3 and m are as above; and d1) removing the amino protecting groups R.sup.2 to form the compound of formula II.

9. Process of claim 8, wherein the coupling steps a1) and c1) are performed in the presence of a peptide coupling agent, an amine base and an organic solvent.

10. Process of claim 8 or 9, wherein the peptide coupling agent is n-propylphosphonic acid anhydride, the amine base is a tertiary amine, the organic solvent is a polar aprotic solvent and the reaction temperature is selected from 20° C. to 70° C.

11. Process of claims 8 to 10, wherein R.sup.4 is an amino protecting group cleavable under basic conditions, preferably FMOC.

12. Process of claim 11, wherein the basic conditions involve the treatment with a secondary aliphatic amine, preferably diethyl amine in the presence of an organic solvent.

13. Process of anyone of claims 8 to 12, wherein the amino protecting group R.sup.2 is tert-butyloxycarbonyl.

14. Process of claim 8, wherein in step d1) the amino protecting group R.sup.2 is removed under acidic conditions and a tri-ammonium salt of the dipeptide of formula IX with the respective acid is formed.

15. Process of claim 14, wherein the acid is a sulfonic acid, preferably methanesulfonic acid.

16. Process of claims 1 to 7, wherein the coupling of the compound of formula II with the GalNAc moiety of the formula III in step a) is performed in the presence of a peptide coupling agent, an amine base and an organic solvent.

17. Process of claim 16, wherein the peptide coupling agent is n-propylphosphonic acid anhydride, the amine base is a tertiary amine, the organic solvent is a polar aprotic solvent and the reaction temperature is selected from 20° C. to 70° C.

18. Process of claims 1 to 7, wherein the removing the hydroxyl protecting group R.sup.3 to form the free alcohol in step b) is performed by way of a catalytic hydrogenation with hydrogen in the presence of a hydrogenation catalyst.

19. Process of claims 1 to 7, wherein the phosphoroamidating agent in step c) is selected from 2-cyanoethyl-N,N-di-(2-propyl)chlorophosphoroamidite or from 2-Cyanoethyl-N,N,N′,N′-tetra (2-propyl)phosphorodiamidite.

20. Process of claim 19, wherein the reaction in step c) is performed with 2-Cyanoethyl-N,N,N′,N′-tetra (2-propyl)phosphorodiamidite in the presence of an acidic ammonium salt of a secondary amine and a polar aprotic solvent at a reaction temperature between −20° C. and 50° C.

21. Use of the process of claims 1 to 20 in a process for the preparation of GalNAc-cluster oligonucleotide conjugates comprising the GalNAc moiety as single epimer.

22. Use of claim 21, wherein the preparation of GalNAc-cluster oligonucleotide conjugates comprises a) the preparation of the GalNAc phosphoramidite epimer of the formula I according to claims 1 to 20; b) the employment of the GalNAc phosphoramidite epimer of the formula I in a solid phase oligonucleotide synthesis together with a desired nucleoside building block in a desired sequence to form a desired GalNAc-cluster oligonucleotide conjugate bound to the solid support and finally the c) cleavage of the GalNAc-cluster_oligonucleotide conjugate from the solid phase support and full deprotection and purification

Description

EXAMPLES

Abbreviations:

[0089] DIPEA diisopropylethyl amine [0090] DMAP 4-(dimethylamino)-pyridine [0091] ESI Electron Spry Ionization [0092] EtOAc ethylacetate [0093] EtOH ethanol [0094] HRMS High-resolution mass spectrometry [0095] HSQC-NMR Heteronuclear Single Quantum Coherence-Nuclear Magnetic Resonance [0096] MeOH methanol [0097] MS Molecular sieves [0098] MsOH methanesulfonic acid [0099] rt room temperature (20-25° C.) [0100] SPOS solid phase oligonucleotide synthesis [0101] T3P n-propylphosphonic acid anhydride [0102] THF tetrahydrofuran [0103] TBME methyl tert-butyl ether

Process Scheme:

[0104] ##STR00018##

Example 1a

(2S)-6-(tert-butoxycarbonylamino)-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoic acid

[0105] ##STR00019##

[0106] To a solution of Fmoc-L-Lys(Boc)-OH (54 g, 115 mmol), 6-benzyloxyhexyl-1-amine hydrochloride (prepared in accordance with WO2017084987A1) (29.5 g, 121 mmol) and N-ethyldiisopropylamine (78.4 ml, 461 mmol) in THF (540 ml) was added n-propylphosphonic acid anhydride (cyclic trimer 50% in EtOAc, 122 ml, 207 mmol,) over 30 s at 20-25° C. The resulting light yellow solution at pH 7-8 was stirred at 20-25° C. for 1 h. Water (540 ml), TBME (135 ml) and n-heptane (540 ml) were added sequentially to the reaction mixture and the biphasic mixture was extracted. The organic layer was concentrated and concentrated in vacuo to afford the target (S)-amide (79 g) which was used without further purification. HRMS (ESI): calc. for C.sub.39H.sub.51N.sub.3O.sub.6 (MH.sup.+): 657.3778; found: 657.3781.

Example 1b

(2R)-6-(tert-butoxycarbonylamino)-2-(9H-fluoren-9-ylmethoxycarbonylamino)hexanoic acid

[0107] ##STR00020##

[0108] In the same fashion as for Example 1a, the crude (R)-amide was obtained as a white solid and used without further purification. HRMS (ESI): calc. for C.sub.39H.sub.51N.sub.3O.sub.6 (MH.sup.+): 657.3778; found: 657.3789.

Example 2a

tert-butyl N-[(5S)-5-amino-6-(6-benzyloxyhexylamino)-6-oxo-hexyl]carbamate

[0109] ##STR00021##

[0110] To a solution of the above crude amide (79 g, 120 mmol) in THF (237 ml) was added diethylamine (251 ml, 2.4 mol) and the colorless solution was stirred for 1.5 h at 20-25° C. Then the reaction mixture was concentrated and dried in vacuo to afford a light yellow oil, which was redissolved in TBME (521 ml) and water (521 ml). Methanesulfonic acid (7.02 ml, 108 mmol) was added to pH 4 and the layers were separated. The aqueous layer was reextracted with TBME (521 ml) and then basified with sodium hydroxide (32% in water, 12.9 ml, 139 mmol) to pH 14. The aqueous phase was extracted with TBME (521 ml), the organic layer was separated, dried over sodium sulfate, filtered, concentrated and dried in vacuo to afford (S)-B as a colorless oil (48.5g, 97% yield over 2 steps). HRMS (ESI): calc. for C.sub.24H.sub.41N.sub.3O.sub.6 (MH.sup.+): 435.3097; found: 435.3121.

Example 2b

tert-butyl N-[(5R)-5-amino-6-(6-benzyloxyhexylamino)-6-oxo-hexyl]carbamate

[0111] ##STR00022##

[0112] In the same fashion as for Example 2a, (R)-B was obtained as a light yellow oil (923 mg, 85% over two steps). HRMS (ESI): calc. for C.sub.24H.sub.41N.sub.3O.sub.6 (MH.sup.+): 435.3097; found: 435.3113.

Example 3a

tert-butyl N-[(5S)-6-(6-benzyloxyhexylamino)-6-oxo-5-[[(2S)-2,6-bis(tert-butoxycarbonylamino)hexanoyl]amino]hexyl]carbamate

[0113] ##STR00023##

[0114] To a solution of (S)-B (48 g, 110 mmol), DIPEA (75 ml, 441 mmol) and Boc-L-Lys(Boc)-OH (45.8 g, 132 mmol) in THF (480 ml) was added T3P (50% in ethyl acetate, 97.4 ml, 165 mmol) over 30 s at 20-25° C. and the colorless solution was stirred for 45 min. Water (480 ml) was then added and the biphasic mixture was stirred for 5 min. n-heptane (480 ml) was added and the layers were separated. The organic layer was washed with 0.5M HCl in water (230 ml), 0.5M NaOH (230 ml), dried over sodium sulfate, filtered and concentrated. The crude product was dissolved in EtOH (45 ml) and n-heptane (428 ml) was added. The resulting white suspension was stirred for 16 h at 20-25° C. The suspension was filtered, the cake washed with EtOH/n-heptane (0.5/9.5, 50 ml) and the white solid dried in vacuo to afford (S,S)-C (63.3 g, 75% yield) as a white crystalline solid. HRMS (ESI): calc. for C.sub.40H.sub.69N.sub.5O.sub.9 (MH.sup.+): 763.5095; found: 763.5098.

Example 3b

tert-butyl N-[(5R)-6-(6-benzyloxyhexylamino)-6-oxo-5-[[(2S)-2,6-bis(tert-butoxycarbonylamino)hexanoyl]amino]hexyl]carbamate

[0115] ##STR00024##

[0116] In the same fashion as for Example 3a, (R,S)-C was obtained as a white solid (16.4 g, 71%). HRMS (ESI): calc. for C.sub.40H.sub.69N.sub.5O.sub.9 (MH.sup.+): 763.5095; found: 763.5076.

Example 4a

(2S)-2,6-bis[[2-[2-[2-[2-[(2R,3R,4S,5R,6R)-3-acetamido-6-ethyl-4,5-dimethyl-tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetyl]amino]-N-[(1S)-1-(6-benzyloxyhexylcarbamoyl)-5-[[2-[2-[2-[2-[(2R,3R,4S,5R,6R)-3-acetamido-6-ethyl-4,5-dimethyl-tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetyl]amino]pentyl]hexanamide

[0117] ##STR00025##

[0118] (S,S)-C (36.1 g, 47.3 mmol) was suspended in acetonitrile (366 ml) and methanesulfonic acid (15.4 ml, 237 mmol) was added. The resulting yellowish, cloudy solution was heated to 55-60° C. After 20 min, additional acetonitrile (366 ml) was added to enable stirring. After 2h the oil bath was removed and the white slurry was used for the coupling. DIPEA (137 ml, 804 mmol) and a solution of F (which has been prepared in accordance with WO2017084987A (7.6% w/w, 1.35 kg, 191 mmol) were added to the above reaction mixture and the light yellow solution was warmed to 40-45° C. Then, T3P (50% in ethyl acetate, 139 ml, 237 mmol) was added over 5 min and the colorless solution was stirred at 40-45° C. After 30 min the reaction mixture was cooled to 20-25° C. and concentrated in vacuo to roughly 500 g. This crude solution was dissolved in 1M sodium bicarbonate (236 ml, 236 mmol) and was purified by reverse phase chromatography in 4 portions (Redisep R.sub.f C18, 360 g, H.sub.2O/acetonitrile 100:0 to 70:30 to 60:40 to 10:90). The product-containing fractions were concentrated in vacuo to remove acetonitrile and then lyophilized to obtain a white foam which was azeotroped with acetonitrile (2×) to afford partly deacetylated (S,S)-G (77.1 g) as a white foam.

[0119] Reacetylation:

[0120] The above obtained (S,S)-G (77.1 g) were taken up in acetonitrile (231 ml) and treated with DMAP (465 mg, 3.81 mmol), DIPEA (4.86 ml, 28.6 mmol) and acetic anhydride (2.51 ml, 26.7 mmol) at 20-25° C. for 1 h. After dilution with water (1.01), the solution was purified again by reverse phase chromatography in 5 portions (Redisep R.sub.f C18, 360 g, H.sub.2O/acetonitrile 100:0 to 70:30 to 65:35 to 0:100). The product-containing fractions were concentrated in vacuo to obtain a white foam which was azeotroped with acetonitrile to afford (S,S)-G (67.0 g, 87%) as a white foam. HRMS (ESI): calc. for C.sub.91H.sub.144N.sub.8O.sub.42 ((M+2H)/2.sup.2+): 1011.4762; found: 1011.4761.

Example 4b

(2S)-2,6-bis[[2-[2-[2-[2-[(2R,3R,4S,5R,6R)-3 -acetamido-6-ethyl-4,5-dimethyl-tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetyl]amino]-N-[(1R)-1-(6-benzyloxyhexylcarbamoyl)-5-[[2-[2-[2-[2-[(2R,3R,4S, 5R,6R)-3-acetamido-6-ethyl-4,5-dimethyl-tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetyl]amino]pentyl]hexanamide

[0121] ##STR00026##

[0122] In the same fashion as for Example 4a but without the re-acetylation procedure, (R,S)-G was obtained as a white foam (29.1 g, 66%). HRMS (EI): calc. for C.sub.91H.sub.144N.sub.8O.sub.41 (M.sup.+): 2020.9378; found: 2020.9365.

Example 5a

(2S)-2,6-bis[[2-[2-[2-[2-[(2R,3R,4S,5R,6R)-3-acetamido-6-ethyl-4,5-dimethyl-tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetyl]amino]-N-[(1S)-1-(6-hydroxyhexylcarbamoyl)-5-[[2-[2-[2-[2-[(2R,3R,4S,5R,6R)-3-acetamido-6-ethyl-4,5-dimethyl-tetrahydropyran-2-yl]oxyethoxy]-ethoxy]ethoxy]acetyl]amino]pentyl]hexanamide

[0123] ##STR00027##

[0124] (S,S)-G (67.0 g, 33.1 mmol) was dissolved in 2-propanol (670 ml) and palladium on carbon 10% (3.8 g, 3.57 mmol) was added. The mixture was hydrogenated in a pressurized reactor at 20° C. under 60 bar of H.sub.2 for 2 h. The suspension was filtered and the filter was washed with 2-propanol (150 ml). The resulting colorless solution was concentrated in vacuo and the residue azeotroped with acetonitrile (3×500 ml) to afford crude (S,S)-H (61.1 g, 95%) as a white foam which was used without further purification and stored at −20° C. HRMS (ESI): calc. for C.sub.84H.sub.139N.sub.8O.sub.42 (MH.sup.+): 1930.8908; found: 1931.9004.

Example 5b

(2S)-2,6-bis[[2-[2-[2-[2-[(2R,3R,4S,5R,6R)-3-acetamido-6-ethyl-4,5-dimethyl-tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetyl]amino]-N-[(1R)-1-(6-hydroxyhexylcarbamoyl)-5-[[2-[2-[2-[2-[(2R,3R,4S,5R,6R)-3-acetamido-6-ethyl-4,5-dimethyl-tetrahydropyran-2-yl]oxyethoxy]-ethoxy]ethoxy]acetyl]amino]pentyl]hexanamide

[0125] ##STR00028##

[0126] In the same fashion as for Example 5a, crude (R,S)-H was obtained as a white foam (29.1 g, quant.). LC-MS (ESI): calc. for C.sub.84H.sub.139N.sub.8O.sub.42 (MH.sup.+): 1931.9; found: 1931.5.

Example 6a

(2S)-2,6-bis[[2-[2-[2-[2-[rac-(2R,3R,4S,5R,6R)-3-acetamido-6-ethyl-4,5-dimethyl-tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetyl]amino]-N-[(1S)-1-[6-[2-cyanoethoxy-(diisopropylamino)phosphanyl]oxyhexylcarbamoyl]-5-[[2-[2-[2-[2-[(2R,3R,4S,5R,6R)-3-acetamido-6-ethyl-4,5-dimethyl-tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetyl]amino]pentyl]hexanamide

[0127] ##STR00029##

[0128] To a solution of (S,S)-H (3.4 g, 1.76 mmol) in anhydrous dichloromethane (20 ml) was added 3-((bis(diisopropylamino)phosphino)oxy)propanenitrile (902 mg, 2.99 mmol,) and Diisopropyl-ammonium tetrazolide (151 mg, 0.88 mmol). The light yellow solution was stirred at 20-25° C. for 1 h. The reaction mixture was diluted with TBME and purified directly by preparative chromatography (Redisep R.sub.f Gold Cyano, 275 g, TBME (containing 1% v/v NEt.sub.3)/acetonitrile 90:10 to 70:30). The product containing fractions were concentrated in vacuo to afford (S,S)-I (3.0 g, 80%) as a white foam. .sup.31P NMR (162 MHz, DMSO-d6): ppm 146.32; HRMS (Nanospray from anhydrous CHCl.sub.3): calc. for C.sub.91H.sub.144N.sub.8O.sub.42 ((M+2H)/2.sup.2+): 1066.5066; found: 1066.5078.

Example 6b

(2S)-2,6-bis[[2-[2-[2-[2-[(2R,3R,4S,5R,6R)-3-acetamido-6-ethyl-4,5-dimethyl-tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetyl]amino]-N-[(1R)-1-[6-[2-cyanoethoxy-(diisopropylamino)phosphanyl]oxyhexylcarbamoyl]-5-[[2-[2-[2-[2-[(2R,3R,4S,5R,6R)-3-acetamido-6-ethyl-4,5-dimethyl-tetrahydropyran-2-yl]oxyethoxy]ethoxy]ethoxy]acetyl]amino]pentyl]hexanamide

[0129] ##STR00030##

[0130] To a solution of (R,S)-H (2.5 g, 1.29 mmol) in acetonitrile (20 ml, dried over CaH.sub.2) was added 3-((bis(diisopropylamino)phosphino)oxy)propanenitrile (624 mg, 2.07 mmol,) and Diisopropyl-ammonium tetrazolide (44.3 mg, 0.26 mmol). The colorless solution was stirred at 20-25° C. for 1.5 h. The reaction mixture concentrated in vacuo to a volume of 12 ml and was applied preparative chromatography (Redisep R.sub.f Gold Cyano, 275 g, TBME/acetonitrile 95:5 to 75:25). The product containing fractions were concentrated in vacuo to afford (R,S)-I (2.1 g, 76%) as a colorless wax. .sup.31P NMR (162 MHz, DMSO-d6): ppm 146.83.

[0131] For the solid phase oligonucleotide synthesis (SPOS), either (S,S)-I and (R,S)-I was dissolved in anhydrous MeCN or CH.sub.2Cl.sub.2 to afford a 0.1-0.2M solution. This solution was dried for one hour over 4 Å MS, 3 Å MS, anhydrous K.sub.2CO.sub.3, basic activated alumina, CaCl.sub.2 or CaH.sub.2 and then used directly on an oligonucleotide synthesizer.

[0132] Solid-Phase Oligonucleotide Synthesis

[0133] GalNAc-cluster-modified LNA/DNA was produced by standard phosphoramidite chemistry (see WO2017084987A1 and WO2018215391A1) on solid phase at a scale of 1 or 20 μmol on a BioAutomation Mermade 12 or at a 0.2, 0.95 or 1.9 mmol scale using an AKTA Oligopilot 100 (GE Healthcare, Freiburg, Germany). Solid supports used include Primer Support 5G Unylinker 200 (GE Healthcare, Freiburg, Germany), Primer Support 5G Unylinker 350 (GE Healthcare, Freiburg, Germany) or Kinovate NittoPhase HL Unylinker 400. Oligonucleotides containing, 2-OCH.sub.2-4 bridged nucleotides (Sigma-Aldrich, SAFC, Hamburg, Germany) and DNA (Sigma-Aldrich, SAFC, Hamburg, Germany) were generated employing the corresponding phosphoramidites. The above prepared solutions of GalNAc-cluster phosphoramidite (S,S)-I and (R,S)-I in MeCN or CH.sub.2Cl.sub.2 were used with standard SPOS activators, such as 4,5-dicyanoimidazole (with and without N-methylimidazole) or tetrazole activators such as 5-(benzylthio)-1H-tetrazole or Activator 42, employing 1.5-4.0 equivalents of phosphoramidite, an amidite to activator ratio of 30/70-40/60 and coupling times of 10-30 min. Oxidation is performed by organic oxidants such as camphorsulfonyloxaziridine, cumene hydroperoxide, tert-butylhydrogenperoxide or iodine in pyridine/H.sub.2O (9:1). Thiolation can be effected by standard thiolation reagents used for SPOS, such as 3-amino-1,2,3-dithiazole-5-thione (xhanthane hydride), 3-dimethylamino-1,2,3-dithiazole-5-thione, 3-ethoxy-1,2,4-dithiazoline-5-one, Beaucage reagent or phenylacetic acid disulfide in their respective solvents. No capping step was employed for the coupling of GalNAc-cluster phosphoramidites. Cleavage and deprotection was achieved by methods known in the field (Wincott F. et al. Nucleic Acid Research, 1995, 23,14, 2677-84), such as concentrated NH.sub.4OH (28-33%) at temperatures between 25-55° C. The deprotected and dried crude GalNAc-clusters modified LNA as ammonium salt were characterized and the identity was confirmed with ion pair HPLC-MS.

[0134] They can be purified by standard purification methods for oligonucleotides (see e.g. WO2018215391A1).

[0135] (Uppercase letters denote beta-D-oxy-LNA units; lowercase letters denote DNA units; the subscript “s” denotes a phosphorothioate linkage; superscript Me denotes a DNA or beta-D-oxy-LNA unit containing a 5-methylcytosine base and AM-C6 denote a 6-aminohexyl-1-phosphate linkage).

Example 7a:

Synthesis of 5′-(S,S)-GalNAc-C6-caG.SUB.s..SUP.Me.C.SUB.s.G.SUB.s.t.SUB.s.a.SUB.s.a.SUB.s.a.SUB.s.g.SUB.s.a.SUB.s.g.SUB.s.a.SUB.s.G.SUB.s.G-3′

[0136] According to the above standard SPOS conditions using (S,S)-I, the title product was synthesized on a 1.9 mmol scale on an AKTA Oligopilot 100 (GE Healthcare, Freiburg, Germany). The deprotected and concentrated crude oligonucleotide as ammonium salt was purified by reverse-phase HPLC and, after ultrafiltration/diafiltration and lyophilization, afforded the sodium salt of 5′-(S,S)-GalNAc-C6-caG.sub.s.sup.MeC.sub.sG.sub.st.sub.sa.sub.sa.sub.sa.sub.sg.sub.sa.sub.sg.sub.sa.sub.sG.sub.sG-3′ (1.7 g, 87.0 a% HPLC purity) as a white lyophilisate. IP-RP-HPLC-HRMS (ESI): calc. for C.sub.220H.sub.303N.sub.76O.sub.111P.sub.15S.sub.12 (M.sup.−): 6633.3115; found: 6633.3089. The epimeric purity was determined by .sup.1H-.sup.13C-HSQC-NMR to be >95% (LOD). Additionally, hydrolysis in 6M HCl, derivatization of the free amino acids and gas chromatographic separation of the lysine enantiomers on CHIRASIL VAL showed an epimeric purity of 99.8%.

Example 7b

Synthesis of 5′-(R,S)-GalNAc-C6-caG.SUB.s..SUP.Me.C.SUB.s.G.SUB.s.t.SUB.s.a.SUB.s.a.SUB.s.a.SUB.s.g.SUB.s.a.SUB.s.g.SUB.s.a.SUB.s.G.SUB.s.G-3′

[0137] According to the above standard SPOS conditions using (R,S)-I, the title product was synthesized on a 1.9 mmol scale on an AKTA Oligopilot 100 (GE Healthcare, Freiburg, Germany). The deprotected and concentrated crude oligonucleotide as ammonium salt was purified by reverse-phase HPLC and, after ultrafiltration/diafiltration and lyophilization, afforded the sodium salt of 5′-(R,S)-GalNAc-C6-caG.sub.s.sup.MeC.sub.sG.sub.st.sub.sa.sub.sa.sub.sa.sub.sg.sub.sa.sub.sg.sub.sa.sub.sG.sub.sG-3′ (1.6 g, 91.6 a% HPLC purity) as a white lyophilisate. IP-RP-HPLC-HRMS (ESI): calc. for C.sub.220H.sub.303N.sub.76O.sub.111P.sub.15S.sub.12 (M.sup.−): 6633.3115; found: 6633.3134. The epimeric purity was determined by .sup.1H-.sup.13C-HSQC-NMR to be >95% (LOD). Additionally, hydrolysis in 6M HCl, derivatization of the free amino acids and gas chromatographic separation of the lysine enantiomers on CHIRASIL VAL showed an epimeric purity of <95.6%.

Example 8a

Synthesis of 5′-(S,S)-GalNAc-C6-ca.SUP.Me.C.SUB.s..SUP.Me.C.SUB.s.t.SUB.s.a.SUB.s.t.SUB.s.t.SUB.s.t.SUB.s.a.SUB.s.a.SUB.s.c.SUB.s.a.SUB.s.t.SUB.s.c.SUB.s.A.SUB.s .G.SUB.s.A.SUB.s..SUP.Me.C-3′

[0138] According to the above standard SPOS conditions using (S,S)-I, the title product was synthesized on a 0.2 mmol scale on an AKTA Oligopilot 100 (GE Healthcare, Freiburg, Germany). The deprotected and concentrated crude oligonucleotide as ammonium salt was purified by IEX-MPLC and, after ultrafiltration/diafiltration and lyophilization, afforded the sodium salt of 5′-(S,S)-GalNAc-C6-ca.sup.MeC.sub.s.sup.MeC.sub.st.sub.sa.sub.st.sub.st.sub.st.sub.sa.sub.sa.sub.sc.sub.sa.sub.st.sub.sc.sub.sA.sub.sG.sub.sA.sub.s.sup.MeC-3′ (350 mg, 79.1 a% HPLC purity) as a white lyophilisate. IP-RP-HPLC-HRMS (ESI): calc. for C.sub.259H.sub.359N.sub.76O.sub.135P19S16 (M.sup.−): 7793.4109; found: 7793.4127. The epimeric purity was determined by .sup.1H-.sup.13C-HSQC-NMR to be >96% (LOD). Additionally, hydrolysis in 6M HCl, derivatization of the free amino acids and gas chromatographic separation of the lysine enantiomers on CHIRASIL VAL showed an epimeric purity of 99.8%.

Example 8b

Synthesis of 5′-(R,S)-GalNAc-C6-ca.SUP.Me.C.SUB.s..SUP.Me.C.SUB.s.t.SUB.s.a.SUB.s.t.SUB.s.t.SUB.s.t.SUB.s.a.SUB.s.a.SUB.s.c.SUB.s.a.SUB.s.t.SUB.s.c.SUB.s.A.SUB.s.G.SUB.s .A.SUB.s..SUP.Me.C-3′

[0139] According to the above standard SPOS conditions using (R,S)-I, the title product was synthesized was produced on a 0.2 mmol scale on an AKTA Oligopilot 100 (GE Healthcare, Freiburg, Germany). The deprotected and concentrated crude oligonucleotide as ammonium salt was purified by IEX-MPLC and, after ultrafiltration/diafiltration and lyophilization, afforded the sodium salt of 5′-(R,S)-GalNAc-C6-ca.sup.MeC.sub.s.sup.MeC .sub.st.sub.sa.sub.st.sub.st.sub.st.sub.sa.sub.sa.sub.sc.sub.sa.sub.st.sub.sc.sub.sA.sub.sG.sub.sA.sub.s.sup.MeC-3′ (630 mg, 82.9 a% HPLC) as a white lyophilisate. IP-RP-HPLC-HRMS (ESI): calc. for C.sub.259H.sub.359N.sub.76 O.sub.135P.sub.19S.sub.16 (M.sup.−): 7793.4109; found: 7793.4127. The epimeric purity was determined by .sup.1H-.sup.13C-HSQC-NMR to be >96% (LOD). Additionally, hydrolysis in 6M HCl, derivatization of the free amino acids and gas chromatographic separation of the lysine enantiomers on CHIRASIL VAL showed an epimeric purity of >95.4%.

Example 9a

Synthesis of 5′-(S,S)-GalNAc-C6-caT.SUB.s..SUP.Me.C.SUB.s.A.SUB.s.a.SUB.s.c.SUB.s.t.SUB.s.t.SUB.s.t.SUB.s.c.SUB.s.a.SUB.s.c.SUB.s.t.SUB.s.t.SUB.s..SUP.Me.C.SUB.s.A.SUB.s.G.SUB.s.-3′

[0140] According to the above standard SPOS conditions using (S,S)-I, the title product was synthesized on 2*1.9 mmol scale on an AKTA Oligopilot 100 (GE Healthcare, Freiburg, Germany). The deprotected and concentrated crude oligonucleotide as ammonium salt was purified by IEX-MPLC, followed by reverse-phase HPLC and, after ultrafiltration/diafiltration and lyophilization, afforded the sodium salt of 5′-(S,S)-GalNAc-C6-caT.sub.s.sup.MeC.sub.sA.sub.sa.sub.sc.sub.st.sub.st.sub.st.sub.sc.sub.sa.sub.sc.sub.st.sub.st.sub.s.sup.MeC.sub.sA.sub.sG.sub.s-3′ (12.5 g, 92.7 a% HPLC purity) as a white lyophilisate. IP-RP-HPLC-HRMS (ESI): calc. for C.sub.248H.sub.346N.sub.68O.sub.133P.sub.18S.sub.15 (M.sup.−): 7441.3490; found: 7441.3730. The epimeric purity was determined by .sup.1H-.sup.13C-HSQC-NMR to be >98% (LOD). Additionally, hydrolysis in 6M HCl, derivatization of the free amino acids and gas chromatographic separation of the lysine enantiomers on CHIRASIL VAL showed an epimeric purity of 99.6%.

Example 9b

Synthesis of 5′-(R, S)-GalNAc-C6-caT.SUB.s..SUP.Me.C.SUB.s.A.SUB.s.a.SUB.s.c.SUB.s.t.SUB.s.t.SUB.s.t.SUB.s.c.SUB.s.a.SUB.s.c.SUB.s.t.SUB.s.t.SUB.s..SUP.Me.C.SUB.s.A.SUB.s.G.SUB.s.-3′

[0141] According to the above standard SPOS conditions using (R,S)-I, the title product was synthesized was produced on a 1.9 mmol scale on an AKTA Oligopilot 100 (GE Healthcare, Freiburg, Germany). The deprotected and concentrated crude oligonucleotide as ammonium salt was purified by reverse-phase HPLC and, after ultrafiltration/diafiltration and lyophilization, afforded the sodium salt of 5′-(R,S)-GalNAc-C6-caT.sub.s.sup.MeC.sub.sA.sub.sa.sub.sc.sub.st.sub.st.sub.st.sub.sc.sub.sa.sub.sc.sub.st.sub.st.sub.s.sup.MeC.sub.sA.sub.sG.sub.s-3′ (6.0 g, 82.0 a% HPLC) as a white lyophilisate. IP-RP-HPLC-HRMS (ESI): calc. for C.sub.248H.sub.346N.sub.68O.sub.133P.sub.18S.sub.15 (M.sup.−): 7441.3490; found: 7441.3508. The epimeric purity was determined by .sup.1H.sup.13C-HSQC-NMR to be >98% (LOD). Additionally, hydrolysis in 6M HCl, derivatization of the free amino acids and gas chromatographic separation of the lysine enantiomers on CHIRASIL VAL showed an epimeric purity of >97.0%.

Example 10a

Synthesis of 5′-(S,S)-GalNAc-C6.SUB.s.-A.SUB.s.A.SUB.s.T.SUB.s.g.SUB.s.c.SUB.s.t.SUB.s.a.SUB.s.c.SUB.s.a.SUB.s.a.SUB.s.a.SUB.s.a.SUB.s.c.SUB.s..SUP.Me.C.SUB.s..SUP.Me.C.SUB.s.A-3′

[0142] According to the above standard SPOS conditions using (S,S)-I, the title product was synthesized on 0.95 mmol scale on an AKTA Oligopilot 100 (GE Healthcare, Freiburg, Germany). The deprotected and concentrated crude oligonucleotide as ammonium salt was purified by reverse-phase HPLC and, after ultrafiltration/diafiltration and lyophilization, afforded the sodium salt of 5′-(S,S)-GalNAc-C6.sub.s-A.sub.sA.sub.sT.sub.sg.sub.sc.sub.st.sub.sa.sub.sc.sub.sa.sub.sa.sub.sa.sub.sa.sub.sc.sub.s.sup.MeC.sub.s.sup.MeC.sub.sA-3′ (1.6 g, 87.7 a% HPLC purity) as a white lyophilisate. IP-RP-HPLC-HRMS (ESI): calc. for C.sub.229H.sub.318N.sub.72O.sub.113P.sub.16S.sub.16 (M.sup.−): 6891.2684; found: 6891.2714. The epimeric purity was determined by .sup.1H-.sup.13C-HSQC-NMR to be >94% (LOD). Additionally, hydrolysis in 6M HCl, derivatization of the free amino acids and gas chromatographic separation of the lysine enantiomers on CHIRASIL VAL showed an epimeric purity of 99.6%.

Example 10b

Synthesis of 5′-(R,S)-GalNAc-C6.SUB.s.-A.SUB.s.A.SUB.s.T.SUB.s.g.SUB.s.c.SUB.s.t.SUB.s.a.SUB.s.c.SUB.s.a.SUB.s.a.SUB.s.a.SUB.s.a.SUB.s.c.SUB.s..SUP.Me.C.SUB.s..SUP.Me.C.SUB.s.A-3′

[0143] According to the above standard SPOS conditions using (R,S)-I, the title product was synthesized was produced on a 0.95 mmol scale on an AKTA Oligopilot 100 (GE Healthcare, Freiburg, Germany). The deprotected and concentrated crude oligonucleotide as ammonium salt was purified by reverse-phase HPLC and, after ultrafiltration/diafiltration and lyophilization, afforded the sodium salt of 5′-(R,S)-GalNAc-Ch.sub.s-A.sub.sA.sub.sT.sub.sg.sub.sc.sub.st.sub.sa.sub.sc.sub.sa.sub.sa.sub.sa.sub.sa.sub.sc.sub.s.sup.MeC.sub.s.sup.MeC.sub.sA-3′ (2.0 g, 88.0 a% HPLC) as a white lyophilisate. IP-RP-HPLC-HRMS (ESI): calc. for C.sub.229H.sub.318N.sub.72O.sub.113P.sub.16S.sub.16 (M.sup.−): 6891.2684; found: 6891.2715. The epimeric purity was determined by .sup.1H-.sup.13C-HSQC-NMR to be >95% (LOD). Additionally, hydrolysis in 6M HCl, derivatization of the free amino acids and gas chromatographic separation of the lysine enantiomers on CHIRASIL VAL showed an epimeric purity of 99.4%.