HK40060361A - Process for the preparation of galnac phosphoramidite epimers - Google Patents

Description

Process for the preparation of GalNAc phosphoramidite epimers

The present invention relates to a novel process for the preparation of epimerically pure GalNAc phosphoramidite epimers of the formula I, their corresponding enantiomers and/or optical isomers,

wherein R is¹Is a hydroxyl protecting group, n is an integer of 0 to 10, and m is an integer of 0 to 20.

The GalNAc phosphoramidite of formula I carries a GalNAc moiety, which is the targeting moiety for conjugates comprising a GalNAc moiety. The GalNAc moiety, due to its affinity to asialoglycoprotein receptors located on hepatocytes, is capable of functionally delivering the oligonucleotide conjugate to hepatocytes. Such GalNAc cluster conjugates have the potential to act as pharmacokinetic modulators and are therefore therapeutically valuable compounds, as described for example in PCT publication WO 2017/084987.

Due to the unique combination of GalNAc moieties and phosphoramidites, GalNAc phosphoramidites of formula I can be introduced directly as building blocks along with nucleoside building blocks in solid phase oligonucleotide synthesis. Thus, a separate conjugation step to introduce GalNAc moieties can be avoided.

According to the current process described in PCT publication WO 2017/084987, GalNAc phosphoramidites of formula I can be prepared by the following steps:

a) GalNAc acid derivatives of formula A

Wherein R is¹Is a hydroxyl protecting group, and n is an integer of 0 to 10,

reaction with an amine of the formula IV

Wherein R is³Is a hydroxy protecting group, and m is an integer from 0 to 20, to form an amide of formula C

Wherein R is¹、R³N and m are as above;

b) removal of the hydroxy-protecting group R³To form a GalNAc amide of formula D

Wherein R is¹N and m are as above and

c) reacting the GalNAc amide of formula D with a phosphoramiditing agent to form a GalNAc phosphoramidite derivative of formula I.

This method was found to result in racemization at the designated chiral center (arrow in formula I below) in coupling step a).

It is therefore an object of the present invention to provide a novel process for producing structural units in epimerically pure form.

This object is achieved by a novel process for the preparation of epimerically pure GalNAc phosphoramidite epimers of the formula I, which comprises

a) The compound of the coupling formula II is coupled,

wherein R is³Is a hydroxy protecting group, and m is as above, or a salt thereof

With a GalNAc moiety of formula III

Wherein R is¹And n is as above, to form a GalNAc amide of formula IV

Wherein R is¹、R³N and m are as above; and

b) removal of the hydroxy-protecting group R³To form the free alcohol GalNAc amide of the formula IV and

c) reacting the free alcohol of the GalNAc amide of formula IV with a phosphoramidite reagent to form the GalNAc phosphoramidite epimer of formula I.

The following definitions are set forth to illustrate and define the meaning and scope of the various terms used to describe the invention.

When a chiral carbon is present in a chemical structure, it is meant that all stereoisomers associated with the chiral carbon are encompassed in the structure as pure stereoisomers as well as mixtures thereof.

The term epimer refers to one of a pair of stereoisomers, wherein the configuration of the isomer at only one stereocenter is different, and wherein all other stereocenters in the molecule are the same.

The term "C_1-12-alkyl "denotes a monovalent linear or branched saturated hydrocarbon radical of 1 to 12 carbon atoms, and the term" C_1-6-alkyl "denotes a monovalent linear or branched saturated hydrocarbon radical of 1 to 6 carbon atoms.

“C_1-12-alkyl "or" C_1-6Examples of-alkyl "include methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl and pentyl and hexyl and isomers thereof.

The term "acyl" denotes a carbonyl group attached to an alkyl group. The term especially stands for C_1-12-alkylcarbonyl, more particularly representing C_1-6-alkylcarbonyl optionally substituted with C_1-6-alkyl substituted or optionally substituted by phenyl. Examples of acyl are acetyl, pivaloyl or benzoyl. Optional substituents of phenyl are halogen such as chlorine, bromine or iodine orC_1-6-an alkyl group. Acyl preferably represents acetyl.

The term "hydroxy protecting group" denotes a group intended to protect a hydroxy group and includes both ester forming groups and ether forming groups, in particular tetrahydropyranyl, acyl (e.g. benzoyl, acetyl, carbamoyl), benzyl and silyl ether (e.g. TBS, TBDPS) groups. Other examples of such Groups can be found in t.w.greene and p.g.m.wuts, "Protective Groups in Organic Synthesis", 2 nd edition, John Wiley & Sons, inc., New York, NY, 1991, chapters 2-3; haslam, "Protective Groups in Organic Chemistry", J.G.W.McOmie, eds., Plenum Press, New York, NY, 1973, Chapter 5, and T.W.Greene, "Protective Groups in Organic Synthesis", John Wiley and Sons, New York, NY, 1981.

The term "amino-protecting group" means a group intended to protect an amino group, and includes benzoyl, benzyloxycarbonyl, carbonylbenzyloxy (CBZ or Z), 9-Fluorenylmethoxycarbonyl (FMOC), p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, tert-Butoxycarbonyl (BOC) and trifluoroacetyl. Other examples of such Groups can be found in t.w.greene and p.g.m.wuts, "Protective Groups in Organic Synthesis", 2 nd edition, John Wiley & Sons, inc., New York, NY, 1991, chapter 7; haslam, "Protective Groups in Organic Chemistry", J.G.W.McOmie, eds., Plenum Press, New York, NY, 1973, Chapter 5, and T.W.Greene, "Protective Groups in Organic Synthesis", John Wiley and Sons, New York, NY, 1981.

The GalNAc phosphoramidite epimers of formula I are preferably those of the following: wherein R is¹Is optionally substituted by C_1-6-alkyl or phenyl substituted C_1-6-alkylcarbonyl, n is an integer from 0 to 5 and m is an integer from 0 to 10; more preferably those of the following: wherein R is¹Is acetyl, n is 2, and m is 5.

In a more preferred embodiment, the GalNAc phosphoramidite epimer of formula I comprises a compound of formula Ib to formula Ie.

Even more preferred are the epimers of formula Ib and formula Ic.

Step a)

Step a) is characterized by coupling a compound of formula II or a salt thereof with a GalNAc moiety of formula III to form a GalNAc amide of formula IV.

The compound of formula II or a salt thereof may be prepared by the following steps:

a1) lysine compounds coupled to V

Wherein R is²And R⁴Is an amino protecting group with an amine of formula VI

Wherein R is³Is a hydroxy protecting group, and m is as above

To form carboxamides of formula VII;

wherein R is²、R³、R⁴And m is as above; and is prepared by the following steps:

b1) removal of the amino-protecting group R⁴To form an amine of the formula VIII

Wherein R is²And R³And m is as above;

c1) coupling an amine of formula VIII with an amino-protected lysine to form a dipeptide of formula IX

Wherein R is²And R³And m is as above; and

d1) removal of the amino-protecting group R²To form a compound of formula II.

In a preferred embodiment, the coupling steps a1) and c1) are carried out in the presence of a peptide coupling agent, an amine base and an organic solvent.

The coupling may be carried out following classical methods known to the person skilled in the art, using carbodiimide coupling reagents, such as DCC (N, N '-dicyclohexylcarbodiimide) or EDC (N- (N', N "-dimethylaminopropyl-N '-ethylcarbodiimide), with or without additives such as HOBt (1-hydroxybenzotriazole) or HOSu (N-hydroxysuccinimide), TBTU (N, N' -tetramethyl-O- (benzotriazol-1-yl) uronium tetrafluoroborate), HBTU (2- (1H-benzotriazol-1-yl) -1,1,3, 3-tetramethyluronium hexafluorophosphate) or HOAt (1-hydroxy-7-azabenzotriazole), and common combinations thereof (such as TBTU/HOBt or HBTU/HOAt).

In a preferred embodiment, N-propylphosphonic anhydride (T3P) is selected as the coupling agent together with a tertiary amine as the amine base, such as triethylamine, N-methylmorpholine or N-diisopropylethylamine, but preferably together with N-diisopropylethylamine.

The coupling reaction generally takes place at a reaction temperature in the range of 20 ℃ and 70 ℃, preferably in the range of 20 ℃ and 40 ℃ in a polar aprotic solvent such as acetonitrile, ethyl acetate or tetrahydrofuran or mixtures thereof.

The products from coupling steps a1) and c1) can be obtained from the organic layer of the reaction mixture using methods known to the person skilled in the art, for example by washing the organic phase and subsequently removing the solvent by evaporation.

Amino protecting group R⁴Usually ammonia which is cleavable under basic conditionsA radical protecting group. FMOC is the most preferred amino protecting group. Basic conditions generally involve treatment with a secondary aliphatic amine in the presence of an organic solvent, such as with piperidine, 4-methylpiperidine, pyrrolidine or diethylamine, but diethylamine is preferred. Suitable solvents are polar aprotic solvents such as acetonitrile or tetrahydrofuran or mixtures thereof.

Amino protecting group R²Typically an amino protecting group cleavable under acidic conditions, preferably tert-butyloxycarbonyl (Boc).

Thus, removal of the amino protecting group R²It can be carried out in a polar aprotic solvent (such as acetonitrile) with a suitable acid, for example selected from hydrochloric acid, trifluoroacetic acid, sulfonic acids such as p-toluenesulfonic acid or methanesulfonic acid.

In a preferred embodiment, methanesulfonic acid is used.

The acidic treatment forms the triammonium salt of the dipeptide of formula IX with the corresponding acid, and for a preferred embodiment the triammonium salt of the dipeptide of formula IX with methanesulfonic acid.

The triammonium salt of the dipeptide of formula IX can be isolated by applying methods known to the person skilled in the art, such as crystallization, or applied directly to the coupling in step b) with the GalNAc moiety of formula III.

The GalNAc moiety of formula III can be prepared according to the disclosure in PCT international publication WO 2017/084987, in particular according to example 7 thereof.

The final coupling of the compound of formula II or a salt thereof, such as the triammonium salt of the dipeptide of formula IX with the GalNAc moiety of formula III, can be carried out under the coupling conditions described above. In addition, the above-mentioned preferable reaction conditions can be applied to the coupling reaction.

The GalNAc amide of formula IV can be further purified by reverse phase chromatography, and the product-containing fraction can be, for example, lyophilized to obtain the purified GalNAc amide of formula IV.

Step b)

Step b) requiring removal of the hydroxy-protecting group R³To form the free alcohol of the GalNAc amide of formula IV.

Importantly, the hydroxyl protecting group R³With a hydroxy-protecting group R¹Chemically different, and therefore the protection of the group R with a hydroxyl group can be chosen³Is cleaved to form a hydroxy protecting group R¹The removal conditions remain unaffected.

Suitable hydroxy-protecting groups R³Is benzyl, optionally substituted by halogen or C_1-6Alkyl substitution, or is benzhydryl or trityl, i.e. a group that can be cleaved by hydrogenolysis.

In a preferred embodiment, R³Is benzyl and the hydrogenolysis is a catalytic hydrogenation with hydrogen in the presence of a suitable hydrogenation catalyst.

A suitable hydrogenation catalyst for removing benzyl groups is palladium on carbon (Pd/C).

The reaction is generally carried out at a reaction temperature of between 0 ℃ and 40 ℃ (preferably between 10 ℃ and 30 ℃) and at a hydrogen pressure of between 10bar and 100bar (preferably between 30bar and 80bar) in the presence of a polar protic solvent such as an aliphatic alcohol such as 2-propanol or a polar aprotic solvent such as THF or ethyl acetate.

The free alcohol of the GalNAc amide of formula IV can be obtained by filtering off the catalyst and concentration of the filtrate by evaporation in vacuo.

Step c)

Step c) entails reacting the free alcohol of the GalNAc acid amide of formula IV with a phosphoramidite reagent to form the GalNAc phosphoramidite epimer of formula I.

The phosphoramidite may be selected from 2-cyanoethyl-N, N-bis- (2-propyl) chlorophosphite or from 2-cyanoethyl-N, N, N ', N' -tetrakis- (2-propyl) phosphoramidite.

In a preferred embodiment, the phosphoramidite is 2-cyanoethyl-N, N, N ', N' -tetrakis (2-propyl) phosphoramidite in combination with an activator.

The activator may be selected from acidic ammonium salts of secondary amines, preferably acidic ammonium salts of secondary aliphatic amines such as diisopropylamine, preferably diisopropylammonium tetrazolium. Alternatively, other tetrazole type activators may be used, such as tetrazole, 5- (ethylthio) -1H-tetrazole, 5- (benzylthio) -1H-tetrazole, or 4, 5-dicyanoimidazole.

The reaction can be carried out in a polar aprotic solvent such as dichloromethane, tetrahydrofuran or acetonitrile at a reaction temperature between-20 ℃ and 50 ℃, preferably between 10 ℃ and 30 ℃.

The separation of the product from the reaction mixture can be carried out by evaporation. However, the product is usually left in solution and further purified by preparative chromatography.

Alternatively, the reaction mixture of the above phosphoramidite reaction can be used directly for solid phase oligonucleotide synthesis without chromatographic purification.

In a preferred embodiment, the chromatographically purified product GalNAc phosphoramidite epimer of formula I is dissolved in a polar aprotic solvent such as dichloromethane or acetonitrile or a mixture thereof and applied directly for the preparation of a GalNAc-cluster oligonucleotide conjugate. Alternatively, desiccants such as molecular sieves (A), (B), (C), (D) and (D) can be usedOr) Anhydrous K₂CO₃Alkaline activated alumina, CaCl₂Or CaH₂Preferably CaH₂OrAnd (4) sieving the molecular sieve, and drying the product solution.

Preparation of GalNAc-cluster oligonucleotide conjugates comprises

a) Preparation of GalNAc phosphoramidite epimer of formula I;

b) the use of GalNAc phosphoramidite epimers of formula I together with a desired nucleoside building block of a desired sequence in solid phase oligonucleotide synthesis to form a desired GalNAc-cluster oligonucleotide conjugate bound to a solid support, and finally

c) The GalNAc-cluster oligonucleotide conjugate was cleaved from the solid support and deprotected completely and purified.

As used herein, the term oligonucleotide is defined as a molecule comprising two or more covalently linked nucleosides as is commonly understood by a skilled artisan. For use as therapeutically valuable oligonucleotides, the synthetic oligonucleotides are typically 7-30 nucleotides in length. Oligonucleotides are usually prepared in the laboratory by solid phase chemical synthesis followed by purification. When referring to the sequence of an oligonucleotide, reference is made to the nucleobase portion of a covalently linked nucleotide or nucleoside or a modified sequence or order thereof. The oligonucleotides of the invention are artificial, chemically synthesized, and usually purified or isolated. The oligonucleotides of the invention may comprise one or more modified nucleosides or nucleotides. In some embodiments, the oligonucleotide is an antisense oligonucleotide.

Oligonucleotides may be composed of DNA, RNA, modified RNA, or LNA nucleoside monomers, or combinations thereof. LNA nucleoside monomers are modified nucleosides that include a linker group (called a diradical or bridge) between C2 'and C4' of the ribose ring of the nucleotide. These nucleosides are also referred to in the literature as bridged nucleic acids or Bicyclic Nucleic Acids (BNA).

In non-limiting examples, the GalNAc-cluster oligonucleotide conjugate can be selected from the group consisting of:

5’-(S,S)-GalNAc-C6-caG_s ^MeC_sG_st_sa_sa_sa_sg_sa_sg_sa_sG_sG-3’

5’-(S,S)-GalNAc-C6-caG_s ^MeC_sG_st_sa_sa_sa_sg_sa_sg_sa_sG_sG-3’

5’-(R,S)-GalNAc-C6-caG_s ^MeC_sG_st_sa_sa_sa_sg_sa_sg_sa_sG_sG-3’

5’-(S,S)-GalNAc-C6-ca^MeC_s ^MeC_st_sa_st_st_st_sa_sa_sc_sa_st_sc_sA_s G_sA_s ^MeC-3’

5’-(R,S)-GalNAc-C6-ca^MeC_s ^MeC_st_sa_st_st_st_sa_sa_sc_sa_st_sc_sA_s G_sA_s ^MeC-3’

5’-(S,S)-GalNAc-C6-caT_s ^MeC_sA_sa_sc_st_st_st_sc_sa_sc_st_st_s ^MeC_sA_sG_s-3’

5’-(R,S)-GalNAc-C6-caT_s ^MeC_sA_sa_sc_st_st_st_sc_sa_sc_st_st_s ^MeC_sA_sG_s-3’

5’-(S,S)-GalNAc-C6_s-A_sA_sT_sg_sc_st_sa_sc_sa_sa_sa_sa_sc_s ^MeC_s ^MeC_sA-3’

5’-(R,S)-GalNAc-C6_s-A_sA_sT_sg_sc_st_sa_sc_sa_sa_sa_sa_sc_s ^MeC_s ^MeC_sA-3’

wherein capital letters denote β -D-oxy-LNA units; lower case letters represent DNA units; the subscript "s" represents a phosphorothioate linkage; the superscript Me denotes a DNA containing a 5-methylcytosine base or a β -D-oxy-LNA unit, and C6 denotes a 6-aminohexyl-1-phosphate linkage.

After solid phase synthesis, the GalNAc-cluster oligonucleotide conjugate remains bound to the solid support and still carries, for example, a hydroxyl protecting group R¹A protecting group of (1).

Cleavage and deprotection from the support can occur using methods known to those skilled in the art and described in the literature, such as in Wincott et al; acids Res. (1995)23(14): 2677-. Typically, the GalNAc-cluster oligonucleotide conjugate is obtained in the form of a suitable salt such as an ammonium salt or an alkali metal salt such as a sodium or potassium salt.

The compounds disclosed herein have a nucleobase sequence selected from the group consisting of SEQ ID NOs 1,2,3 and 4.

SEQ ID NO 1:cagcgtaaagagagg

SEQ ID NO 2:cacctatttaacatcagac

SEQ ID NO 3:catcaactttcacttcag

SEQ ID NO 4:aatgctacaaaaccca

Examples of the invention

Abbreviations:

DIPEA diisopropylethylamine

DMAP 4- (dimethylamino) -pyridine

ESI electrospray ionization

EtOAc ethyl acetate

EtOH ethanol

HRMS high resolution mass spectrometry

HSQC-NMR heteronuclear single quantum coherent-nuclear magnetic resonance

MeOH methanol

MS molecular sieve

MsOH methanesulfonic acid

rt Room temperature (20-25 deg.C)

SPOS solid phase oligonucleotide Synthesis

T3P n-propylphosphonic anhydride

THF tetrahydrofuran

TBME methyl tert-butyl ether

The method comprises the following steps:

example 1a

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

To a solution of Fmoc-L-Lys (Boc) -OH (54g, 115mmol), 6-benzyloxyhexyl-1-amino hydrochloride (prepared according to WO2017084987A 1) (29.5g, 121mmol) and N-ethyldiisopropylamine (78.4mL, 461mmol) in THF (540mL) was added N-propylphosphonic anhydride (cyclic trimer in EtOAc 50%, 122mL, 207mmol) at 20-25 ℃ over 30 s. The resulting pale yellow solution, pH 7-8, was stirred at 20-25 ℃ for 1 hour. To the reaction mixture were added water (540mL), TBME (135mL) and n-heptane (540mL) in that order and the biphasic mixture was extracted. The organic layer was concentrated and concentrated in vacuo to give the target (S) -amide (79g), which was used without further purification. Hrms (esi): c₃₉H₅₁N₃O₆(MH⁺) Calculated values: 657.3778, respectively; the found value is: 657.3781.

example 1b

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

In the same manner as in example 1a, the crude (R) -amide was obtained as a white solid and used without further purification. Hrms (esi): c₃₉H₅₁N₃O₆(MH⁺) Calculated values: 657.3778, respectively; the found value is: 657.3789.

example 2a

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

To a solution of the above crude amide (79g, 120mmol) in THF (237mL) was added diethylamine (251mL, 2.4mol) and the colorless solution was stirred at 20-25 deg.C for 1.5 h. The reaction mixture was then concentrated and dried in vacuo to give a pale yellow oil, which was redissolved in TBME (521mL) and water (521 mL). Methanesulfonic acid (7.02mL, 108mmol) was added to pH 4 and the layers were separated. The aqueous layer was re-extracted with TBME (521mL) and then basified with sodium hydroxide (32% aq, 12.9mL, 139mmol) to pH 14. The aqueous phase was extracted with TBME (521mL), the organic layer was separated, dried over sodium sulfate, filtered, concentrated and dried in vacuo to give (S) -B as a colorless oil (48.5g, 97% yield over 2 steps). Hrms (esi): c₂₄H₄₁N₃O₆(MH⁺) Calculated values: 435.3097, respectively; the found value is: 435.3121.

example 2b

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

In the same manner as in example 2a, (R) -B was obtained as a pale yellow oil (923mg, 85% over two steps). Hrms (esi): c₂₄H₄₁N₃O₆(MH⁺) Calculated values: 435.3097, respectively; the found value is: 435.3113.

example 3a

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

(S) -B (48g, 110mmol), DIPEA (75mL, 441mmol) and Boc-To a solution of L-Lys (Boc) -OH (45.8g, 132mmol) in THF (480mL) was added T3P (50% in ethyl acetate, 97.4mL, 165mmol), and the colorless solution was stirred for 45 min. Water (480mL) was then added and the biphasic mixture was stirred for 5 min. N-heptane (480mL) was added and the layers were separated. The organic layer was washed with 0.5M HCl water (230mL), 0.5M NaOH (230mL), dried over sodium sulfate, filtered and concentrated. The crude product was dissolved in EtOH (45mL) and n-heptane (428mL) was added. The resulting white suspension was stirred at 20-25 ℃ for 16 hours. The suspension was filtered, the filter cake was washed with EtOH/n-heptane (0.5/9.5, 50ml), and the white solid was dried in vacuo to give (S, S) -C (63.3g, 75% yield) as a white crystalline solid. Hrms (esi): c₄₀H₆₉N₅O₉(MH⁺) Calculated values: 763.5095, respectively; the found value is: 763.5098.

example 3b

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

In the same manner as in example 3a, (R, S) -C was obtained as a white solid (16.4g, 71%). Hrms (esi): c₄₀H₆₉N₅O₉(MH⁺) Calculated values: 763.5095, respectively; the found value is: 763.5076.

example 4a

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

(S, S) -C (36.1g, 47.3mmol) was suspended in acetonitrile (366mL) and methanesulfonic acid (15.4mL, 237mmol) was added. The resulting pale yellow, turbid solution was heated to 55-60 ℃. After 20 minutes, additional acetonitrile (366mL) was added to enable stirring. After 2 hours, the oil bath was removed and a white slurry was used for coupling. DIPEA (137mL, 804mmol) and F solution (which has been prepared according to WO2017084987A (7.6% w/w, 1.35kg, 191mmol) are added to the above reaction mixture and the light yellow solution is warmed to 40-45 ℃ then T3P (50% in ethyl acetate, 139mL, 237mmol) is added over 5min and the colorless solution is stirred at 40-45 ℃ after 30 min the reaction mixture is cooled to 20-25 ℃ and concentrated to about 500g in vacuo and the crude solution is dissolved in 1M sodium bicarbonate (236mL, 236mmol) and purified in 4 portions by reverse phase chromatography (Redispe R_f C18，360g，H₂O/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 azeotroped with acetonitrile (2 ×) to give partially deacetylated (S, S) -G (77.1G) as a white foam.

And (3) re-acetylation:

(S, S) -G (77.1G) obtained above was dissolved in acetonitrile (231mL) and treated with DMAP (465mg, 3.81mmol), DIPEA (4.86mL, 28.6mmol) and acetic anhydride (2.51mL, 26.7mmol) at 20-25 ℃ for 1 h. After dilution with water (1.0L), the solution was repurified in 5 portions by reverse phase chromatography (Redispe R)_fC18，360g，H₂O/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 azeotroped with acetonitrile to give (S, S) -G (67.0G, 87%) as a white foam. Hrms (esi): c₉₁H₁₄₄N₈O₄₂((M+2H)/2²⁺) Calculated values: 1011.4762, respectively; the found value is: 1011.4761.

example 4b

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

In the same manner as example 4a but without a reacetylation procedure, (R, S) -G was obtained as a white foam (29.1G, 66%). Hrms (ei): c₉₁H₁₄₄N₈O₄₂(M⁺) Calculated values: 2020.9378, respectively; the found value is: 2020.9365.

example 5a

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

(S, S) -G (67.0G, 33.1mmol) was dissolved in 2-propanol (670ml) and palladium on carbon 10% (3.8G, 3.57mmol) was added. The mixture was dried at 20 ℃ under 60bar of H₂Hydrogenation was carried out in a pressurized reactor 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 was azeotroped with acetonitrile (3 × 500 ml) to give crude (S, S) -H (61.1g, 95%) as a white foam which was stored without further purification at-20 ℃. Hrms (esi): c₈₄H₁₃₉N₈O₄₂(MH⁺) Calculated values: 1930.8908, respectively; the found value is: 1931.9004.

example 5b

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

In the same manner as in example 5a, crude (R, S) -H was obtained as a white foam (29.1g, quantitative). LC-MS (ESI): c₈₄H₁₃₉N₈O₄₂(MH⁺) Calculated values: 1931.9, respectively; the found value is: 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 ] acetyl ] amino ] -N- [ (1S) -1- [6- [ 2-cyanoethoxy- (diisopropylamino) phosphino ] oxyhexylcarbamoyl ] -5- [ [2- [2- [2- [2- [ (2R,3R,4S,5R,6R) -3-acetamido-6-ethyl-4, 5-dimethyl-tetrahydropyran-2-yl ] oxyethoxy ] ethoxy ] N-a-4, 5-dimethyl-tetrahydropyran-2-yl ] oxyethoxy ] ethoxy Acetyl-amino-pentyl caproamide

To a solution of (S, S) -H (3.4g, 1.76mmol) in dry dichloromethane (20ml) was added 3- ((bis (diisopropylamino) phosphino) oxy) propionitrile (902mg, 2.99mmol) and diisopropyl-ammonium tetrazolium (151mg, 0.88 mmol). The pale yellow solution was stirred at 20-25 ℃ for 1 hour. The reaction mixture was diluted with TBME and directly purified by preparative chromatography (Redispe R)_fGold Cyano, 275g, TBME (containing 1% v/v NEt)₃) Acetonitrile 90:10 to 70: 30). The product-containing fractions were concentrated in vacuo to give (S, S) -I (3.0g, 80%) as a white foam.³¹P NMR(162MHz,DMSO-d6)：ppm 146.32, a first step of removing the first layer; HRMS (from anhydrous CHCl)₃Nano-spraying of (a): c₉₁H₁₄₄N₈O₄₂((M+2H)/2²⁺) Calculated values: 1066.5066, respectively; the found value is: 1066.5078.

example 6b

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

To a solution of (R, S) -H (2.5g, 1.29mmol) in acetonitrile (20ml, using CaH)₂Dry) to a solution was added 3- ((bis (diisopropylamino) phosphino) oxy) propionitrile (624mg, 2.07mmol) and diisopropyl-ammonium tetrazolium (44.3mg, 0.26 mmol). The colorless solution was stirred at 20-25 ℃ for 1.5 h. The reaction mixture was concentrated in vacuo to a volume of 12mL and preparative chromatography (Redispe R) was applied_fGold Cyano, 275g, TBME/acetonitrile 95:5 to 75: 25). The product-containing fractions were concentrated in vacuo to give (R, S) -I (2.1g, 76%) as a colorless wax.³¹P NMR(162MHz,DMSO-d6)：ppm 146.83。

For Solid Phase Oligonucleotide Synthesis (SPOS), (S, S) -I and (R, S) -I were dissolved in anhydrous MeCN or CH₂Cl₂To obtain a 0.1-0.2M solution. Applying the solution toMS、MS, anhydrous K₂CO₃Alkaline activated alumina, CaCl₂Or CaH₂Dried for one hour and then directly on the oligonucleotideFor use on a synthesizer.

Solid phase oligonucleotide synthesis

GalNAc-cluster modified LNA/DNA was produced on solid phase on 1 or 20. mu. mol scale or on 0.2, 0.95 or 1.9mmol scale on BioAutomation Mermade 12 by standard phosphoramidite chemistry (see WO2017084987A1 and WO2018215391A1) using 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 Nitto phase HL Unylinker 400. Using the corresponding phosphoramidite, oligonucleotides (Sigma-Aldrich, SAFC, Hamburg, Germany) and DNA (Sigma-Aldrich, SAFC, Hamburg, Germany) containing a 2-OCH2-4 bridging nucleotide were generated. The above prepared GalNAc-cluster phosphoramidites (S, S) -I and MeCN or CH of (R, S) -I are reacted using 1.5-4.0 equivalents of phosphoramidite, a ratio of phosphoramidite to activator of 30/70-40/60, and a coupling time of 10-30 minutes₂Cl₂The solution is used with a standard SPOS activator such as 4, 5-dicyanoimidazole (with and without N-methylimidazole) or a tetrazole activator such as 5- (benzylthio) -1H-tetrazole or activator 42. By organic oxidants such as camphorsulfonyl chloride oxaziridine, cumene hydroperoxide, tert-butyl hydroperoxide or iodine in pyridine/H₂Oxidation in O (9: 1). Thiolation can be achieved by standard thiolating reagents for SPOS such as 3-amino-1, 2, 3-dithiazole-5-thione (xhathane hydride), 3-dimethylamino-1, 2, 3-dithiazole-5-thione, 3-ethoxy-1, 2, 4-dithiazolin-5-one, Beaucage reagent, or phenylacetic acid disulfide in their respective solvents. No capping step was used for coupling of GalNAc-cluster phosphoramidites. By methods known in the art (Wincott F. et al Nucleic Acid Research, 1995, 23,14,2677-84), such as concentrated NH₄OH (28-33%) achieves cleavage and deprotection at temperatures between 25-55 ℃. The deprotected and dried crude GalNAc-cluster modified LNA, which is an ammonium salt, was characterized by ion pair HPLC-MS and its properties confirmed. They can be purified by standard purification methods for oligonucleotides (see, e.g., WO2018215391A 1).

(capital letters indicate beta-D-oxy-LNA units; lowercase letters indicate DNA units; subscript "s" indicates a phosphorothioate linkage; superscript Me indicates a DNA containing a 5-methylcytosine base or a beta-D-oxy-LNA unit, and AM-C6 indicates a 6-aminohexyl-l-phosphate linkage).

Example 7a：

5’-(S,S)-GalNAc-C6-caG_s ^MeC_sG_st_sa_sa_sa_sg_sa_sg_sa_sG_sSynthesis of G-3

The title product was synthesized on a 1.9mmol scale on AKTA Oligopilot 100(GE Healthcare, Freiburg, Germany) according to the standard SPOS conditions described above using (S, S) -I. The deprotected and concentrated crude oligonucleotide as ammonium salt was purified by reverse phase HPLC and, after ultrafiltration/diafiltration and lyophilization, gave 5' - (S, S) -GalNAc-C6-caG_s ^MeC_sG_st_sa_sa_sa_sg_sa_sg_sa_sG_sThe sodium salt of G-3' (1.7G, 87.0 a% HPLC purity) was a white lyophilizate. IP-RP-HPLC-HRMS (ESI): c₂₂₀H₃₀₃N₇₆O₁₁₁P₁₅S₁₂(M^-) Calculated values: 6633.3115, respectively; the found value is: 6633.3089. by passing¹H-¹³C-HSQC-NMR determination of the epimeric purity of>95% (LOD). In addition, hydrolysis in 6M HCl, derivatization of the free amino acids, and gas chromatographic separation of the lysine enantiomer at CHIRASIL VAL showed an epimeric purity of 99.8%.

Example 7b5’-(R,S)-GalNAc-C6-caG_s ^MeC_sG_st_sa_sa_sa_sg_sa_sg_sa_sG_sSynthesis of G-3

The title product was synthesized on a 1.9mmol scale on AKTA Oligopilot 100(GE Healthcare, Freiburg, Germany) according to the standard SPOS conditions described above using (R, S) -I. Purification by reverse phase HPLC as deprotection and concentration of the ammonium saltCrude oligonucleotide and, after ultrafiltration/diafiltration and lyophilization, 5' - (R, S) -GalNAc-C6-caG was obtained_s ^MeC_sG_st_sa_sa_sa_sg_sa_sg_sa_sG_sThe sodium salt of G-3' (1.6G, 91.6 a% HPLC purity) was a white lyophilizate. IP-RP-HPLC-HRMS (ESI): c₂₂₀H₃₀₃N₇₆O₁₁₁P₁₅S₁₂(M^-) Calculated values: 6633.3115, respectively; the found value is: 6633.3134. by passing¹H-¹³C-HSQC-NMR determination of the epimeric purity of>95% (LOD). In addition, hydrolysis in 6M HCl, derivatization of the free amino acids, and gas chromatographic separation of the lysine enantiomer at CHIRASIL VAL are shown<Epimeric purity of 95.6%.

Example 8a5’-(S,S)-GalNAc-C6-ca^MeC_s ^MeC_st_sa_st_st_st_sa_sa_sc_sa_st_sc_sA_s G_sA_s ^MeSynthesis of C-3

The title product was synthesized on a 0.2mmol scale on AKTA Oligopilot 100(GE Healthcare, Freiburg, Germany) according to the standard SPOS conditions described above using (S, S) -I. The deprotected and concentrated crude oligonucleotide as ammonium salt was purified by IEX-MPLC and, after ultrafiltration/diafiltration and lyophilization, 5' - (S, S) -GalNAc-C6-ca was obtained^MeC_s ^MeC_st_sa_st_st_st_sa_sa_sc_sa_st_sc_sA_sG_sA_s ^MeThe sodium salt of C-3' (350mg, 79.1 a% HPLC purity) was a white lyophilizate. IP-RP-HPLC-HRMS (ESI): c₂₅₉H₃₅₉N₇₆O₁₃₅P₁₉S₁₆(M^-) Calculated values: 7793.4109, respectively; the found value is: 7793.4127. by passing¹H-¹³C-HSQC-NMR determination of the epimeric purity of>96% (LOD). Hydrolysis in 6M HCl, free amino acidsDerivatization and gas chromatographic separation of the lysine enantiomer at CHIRASIL VAL showed an epimeric purity of 99.8%.

Example 8b5’-(R,S)-GalNAc-C6-ca^MeC_s ^MeC_st_sa_st_st_st_sa_sa_sc_sa_st_sc_sA_sG_s A_s ^MeSynthesis of C-3

The title product was prepared synthetically on a 0.2mmol scale on AKTA Oligopilot 100(GE Healthcare, Freiburg, Germany) according to the above standard SPOS conditions using (R, S) -I. The deprotected and concentrated crude oligonucleotide as ammonium salt was purified by IEX-MPLC and, after ultrafiltration/diafiltration and lyophilization, 5' - (R, S) -GalNAc-C6-ca was obtained^MeC_s ^MeC_st_sa_st_st_st_sa_sa_sc_sa_st_sc_sA_sG_sA_s ^MeThe sodium salt of C-3' (630mg, 82.9 a% HPLC) was white lyophilizate. IP-RP-HPLC-HRMS (ESI): c₂₅₉H₃₅₉N₇₆O₁₃₅P₁₉S₁₆(M^-) Calculated values: 7793.4109, respectively; the found value is: 7793.4127. by passing¹H-¹³C-HSQC-NMR determination of the epimeric purity of>96% (LOD). In addition, hydrolysis in 6M HCl, derivatization of the free amino acids, and gas chromatographic separation of the lysine enantiomer at CHIRASIL VAL are shown>Epimeric purity of 95.4%.

Example 9a5’-(S,S)-GalNAc-C6-caT_s ^MeC_sA_sa_sc_st_st_st_sc_sa_sc_st_st_s ^MeC_sA_sG_sSynthesis of (E) -3

The title product was synthesized on a 2X 1.9mmol scale on AKTA Oligopilot 100(GE Healthcare, Freiburg, Germany) according to the standard SPOS conditions described above using (S, S) -I. Purification of the deprotected and concentrated crude oligonucleotide as ammonium salt by IEX-MPLC followed by reverse phase HPLC and after ultrafiltration/diafiltration and lyophilization gave 5' - (S, S) -GalNAc-C6-caT_s ^MeC_sA_sa_sc_st_st_st_sc_sa_sc_st_st_s ^MeC_sA_sG_sThe sodium salt of-3' (12.5g, 92.7 a% HPLC purity) was a white lyophilizate. IP-RP-HPLC-HRMS (ESI): c₂₄₈H₃₄₆N₆₈O₁₃₃P₁₈S₁₅(M^-) Calculated values: 7441.3490, respectively; the found value is: 7441.3730. by passing¹H-¹³C-HSQC-NMR determination of the epimeric purity of>98% (LOD). In addition, hydrolysis in 6M HCl, derivatization of the free amino acids, and gas chromatographic separation of the lysine enantiomer at CHIRASIL VAL showed an epimeric purity of 99.6%.

Example 9b5’-(R,S)-GalNAc-C6-caT_s ^MeC_sA_sa_sc_st_st_st_sc_sa_sc_st_st_s ^MeC_sA_sG_sSynthesis of (E) -3

The title product was prepared synthetically on the 1.9mmol scale on AKTA Oligopilot 100(GE Healthcare, Freiburg, Germany) according to the above standard SPOS conditions using (R, S) -I. The deprotected and concentrated crude oligonucleotide as ammonium salt was purified by reverse phase HPLC and, after ultrafiltration/diafiltration and lyophilization, gave 5' - (R, S) -GalNAc-C6-caT_s ^MeC_sA_sa_sc_st_st_st_sc_sa_sc_st_st_s ^MeC_sA_sG_sThe sodium salt of-3' (6.0g, 82.0 a% HPLC) was white lyophilizate. IP-RP-HPLC-HRMS (ESI): c₂₄₈H₃₄₆N₆₈O₁₃₃P₁₈S₁₅(M^-) Calculated values: 7441.3490, respectively; the found value is: 7441.3508. by passing¹H-¹³C-HSQC-NMR determination of the epimeric purity of>98% (LOD). In addition, hydrolysis in 6M HCl, derivatization of the free amino acids, and gas chromatographic separation of the lysine enantiomer at CHIRASIL VAL are shown>Epimeric purity of 97.0%.

Example 10a5’-(S,S)-GalNAc-C6_s-A_sA_sT_sg_sc_st_sa_sc_sa_sa_sa_sa_sc_s ^MeC_s ^MeC_sSynthesis of A-3

The title product was synthesized on a 0.95mmol scale on AKTA Oligopilot 100(GE Healthcare, Freiburg, Germany) according to the standard SPOS conditions described above using (S, S) -I. The deprotected and concentrated crude oligonucleotide as ammonium salt was purified by reverse phase HPLC and, after ultrafiltration/diafiltration and lyophilization, gave 5' - (S, S) -GalNAc-C6_s-A_sA_sT_sg_sc_st_sa_sc_sa_sa_sa_sa_sc_s ^MeC_s ^MeC_sThe sodium salt of A-3' (1.6g, 87.7 a% HPLC purity) was a white lyophilizate. IP-RP-HPLC-HRMS (ESI): c₂₂₉H₃₁₈N₇₂O₁₁₃P₁₆S₁₆(M^-) Calculated values: 6891.2684, respectively; the found value is: 6891.2714. by passing¹H-¹³C-HSQC-NMR determination of the epimeric purity of>94% (LOD). In addition, hydrolysis in 6M HCl, derivatization of the free amino acids, and gas chromatographic separation of the lysine enantiomer at CHIRASIL VAL showed an epimeric purity of 99.6%.

Example 10b5’-(R,S)-GalNAc-C6_s-A_sA_sT_sg_sc_st_sa_sc_sa_sa_sa_sa_sc_s ^MeC_s ^MeC_sSynthesis of A-3

According to the standard SPOS conditions described above using (R, S) -I, in AKTA OligoThe title product was prepared synthetically on a 0.95mmol scale on a pilot 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, gave 5' - (R, S) -GalNAc-C6_s-A_sA_sT_sg_sc_st_sa_sc_sa_sa_sa_sa_sc_s ^MeC_s ^MeC_sThe sodium salt of A-3' (2.0g, 88.0 a% HPLC) was a white lyophilizate. IP-RP-HPLC-HRMS (ESI): c₂₂₉H₃₁₈N₇₂O₁₁₃P₁₆S₁₆(M^-) Calculated values: 6891.2684, respectively; the found value is: 6891.2715. by passing¹H-¹³C-HSQC-NMR determination of the epimeric purity of>95% (LOD). In addition, hydrolysis in 6M HCl, derivatization of the free amino acids, and gas chromatographic separation of the lysine enantiomer at CHIRASIL VAL showed an epimeric purity of 99.4%.

Claims (22)

1. A process for the preparation of epimerically pure GalNAc phosphoramidite epimers of the formula I, their corresponding enantiomers and/or optical isomers,

wherein R is¹Is a hydroxyl protecting group, n is an integer from 0 to 10, and m is an integer from 0 to 20, the process comprising

a) The compound of the coupling formula II is coupled,

wherein R is³Is hydroxyA radical protecting group, and m is as above, or a salt thereof

With a GalNAc moiety of formula III

Wherein R is¹And n is as above, to form a GalNAc amide of formula IV

Wherein R is¹、R³N and m are as above; and

b) removing the hydroxyl protecting group R³To form the free alcohol of said GalNAc amide of formula IV, and

c) reacting the free alcohol of the GalNAc amide of formula IV with a phosphoramidite reagent to form the GalNAc phosphoramidite epimer of formula I.

2. The method of claim 1, wherein R¹Is an acyl group.

3. The method of claim 1 or 2, wherein R¹Is C_1-6-alkylcarbonyl optionally substituted with C_1-6-alkyl or phenyl substitution.

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

5. The method of any one of claims 1 to 4, wherein R¹Is acetyl, n is 2 and m is 5.

6. The method of any one of claims 1 to 5, wherein R³Is benzyl.

7. The process of any one of claims 1 to 6, wherein the formula I comprises GalNAc phosphoramidite epimers of formula Ib to formula Ie.

8. The process according to any one of claims 1 to 7, wherein the compound of formula II is prepared by:

a1) lysine compounds coupled to V

Wherein R is²And R⁴Is a protecting group for amino

With amines of said formula VI

Wherein R is³And m is as above

To form carboxamides of formula VII;

wherein R is²、R³、R⁴And m is as above, an

b1) Removal of amino groupsProtect R⁴To form an amine of the formula VIII

Wherein R is²、R³And m is as above;

c1) coupling the amine of formula VIII with an amino-protected lysine to form a dipeptide of formula IX

Wherein R is²And R³And m is as above; and

d1) removal of the amino-protecting group R²To form a compound of formula II.

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

10. The process of claim 8 or 9, wherein the peptide coupling agent is n-propyl phosphoric anhydride, the amine base is a tertiary amine, the organic solvent is a polar aprotic solvent, and the reaction temperature is selected from 20 ℃ to 70 ℃.

11. The method of claims 8-10, wherein R⁴For amino protecting groups cleavable under basic conditions, FMOC is preferred.

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

13. The process according to any one of claims 8 to 12, wherein the amino protecting group R²Is tert-butoxycarbonyl.

14. The process according to claim 8, wherein in step d1) the amino protecting group R is removed under acidic conditions²And a triammonium salt of the dipeptide of formula IX with the corresponding acid is formed.

15. The process according to claim 14, wherein the acid is a sulfonic acid, preferably methanesulfonic acid.

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

17. The process of claim 16, wherein the peptide coupling agent is n-propyl phosphoric anhydride, the amine base is a tertiary amine, the organic solvent is a polar aprotic solvent, and the reaction temperature is selected from 20 ℃ to 70 ℃.

18. The process according to claims 1 to 7, wherein the removal of the hydroxyl protecting group R in step b)³To form the free alcohol is carried out by means of catalytic hydrogenation with hydrogen in the presence of a hydrogenation catalyst.

19. The process according to claims 1 to 7, wherein the phosphoramidite in step c) is selected from 2-cyanoethyl-N, N-bis- (2-propyl) chlorophosphite or from 2-cyanoethyl-N, N, N ', N' -tetrakis (2-propyl) phosphoramidite.

20. The process according to claim 19, wherein the reaction in step c) is carried out with 2-cyanoethyl-N, N' -tetrakis (2-propyl) phosphoramidite in the presence of an acidic ammonium salt of a secondary amine and a polar aprotic solvent at a reaction temperature between-20 ℃ and 50 ℃.

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

22. The use of claim 21, wherein said preparation of a GalNAc-cluster oligonucleotide conjugate comprises

a) Preparation of the GalNAc phosphoramidite epimer of formula I according to claims 1 to 20;

b) using said GalNAc phosphoramidite epimer of formula I together with a desired nucleoside building block of a desired sequence in solid phase oligonucleotide synthesis to form a desired GalNAc-cluster oligonucleotide conjugate bound to a solid support, and finally

c) Cleaving the GalNAc-cluster oligonucleotide conjugate from the solid support and deprotecting and purifying completely.