Highly pure n,n-diisopropylphosphoramidite compounds

Abstract

The disclosure relates to the production of highly pure N,N-diisopropylphosphoramidite compounds, such as 6-bromohexyl 2-cyanoethyl N,N-diisopropylphosphoramidite, and highly pure starting material used therein, such as 6-bromohexan-1-ol. The resulting N,N- diisopropylphosphoramidite compounds are useful, for example, for the improved preparation of expandable nucleotide triphosphates (XNTPs) for use in sequencing by expansion.

Description

P39849-WO-1 (SMH)HIGHLY PURE N,N-DHSOPROPYLPHOSPHORAMIDITE COMPOUNDSFIELD OF THE INVENTION

[0001] The disclosure relates to the production of highly pure / VN-di isopropyl phosphorami di te compounds, such as 6-bromohexyl 2-cyanoethyl A,7V-diisopropylphosphoramidite, and highly pure starting material used therein, such as 6-bromohexan-l-ol. The resulting N,N-diisopropylphosphoramidite compounds are useful, for example, for the improved preparation of expandable nucleotide triphosphates (XNTPs) for use in sequencing by expansion.BACKGROUND

[0002] Over the last two decades, biological membranes have emerged as an important tool in a variety of biomedical applications. This includes the use of lipid bilayer membranes in nanopore based sequencing applications, where nanopores provide a constant and reproducible physical aperture, through which a target molecule can be directed and sequenced.One approach for nanopore-based sequencing of, for example, nucleic acids involves a sequencing-by-expansion approach by transcribing the sequence of nucleic acids into a simple to measure polymer molecule called an Xpandomer. Much like with polymerase chain reaction (PCR), Xpandomer synthesis is based on the natural function of DNA replication where expandable nucleoside triphosphates (XNTPs) act as substrates for replication.Xpandomer synthesis is based on four easily differentiated XNTPs that include High Signal-to-Noise Reporters, one for each DNA base. Engineered polymerases incorporate these modified nucleotides into Xpandomers, producing a copy of the target nucleic acid template from the library. As the Xpandomer molecule transits through the nanopore, the distinct electrical signal of each base reporter is easily identifiable to enable highly accurate and high throughput nanopore-based nucleic acid sequencing. See, e.g., U.S. Pat. No. 7,939,259, titled “High Throughput Nucleic Acid Sequencing by Expansion;” and PCT publication WO 2020 / 236526 Al, titled “Translocation control elements, reporter codes, and further means for translocation control for use in nanopore sequencing”, both of which are hereby incorporated herein in their entirety.

[0003] XNTPs are typically designed to include a cleavable bond between the a-phosphate and the nucleotide sugar, as well as an additional linkage between the a-phosphate and another part ofthe nucleotide, such as the sugar or the nucleobase, typically the nucleobase. While cleavage of the cleavable bond in the context of oligo- or polymers results in the disruption of the standard phosphate backbone, the additional linkage is maintained as a connector between the - now expanded - XNTP units. The additional linkage can be a tether molecule, for example, such as a symmetrically synthesized reporter tether (SSRT) as disclosed in WO 2020 / 236526 Al. To simplify the synthesis of XNTPs, the tether molecule may be attached to the a-phosphate and the other part of the nucleotide via click chemistry. In other words, two clickable groups, such as alkyne groups, comprised in a XNTP precursor nucleotide, one at the a-phosphate and one at the other part of the nucleotide, may be clicked to two compatible clickable groups, such as azido groups, comprised in the tether molecule, e.g. at both ends of the tether.

[0004] 6-bromohexyl 2-cyanoethyl / V, / V-diisopropylphosphoramidite is being used as a powerful reagent for preparation of oligonucleotides with 5' -azido modifications (see Tetrahedron Letters, 2007, 48, 8795-8798). After its coupling to an oligomer using standard automated phosphoramidite-based solid-phase-supported DNA synthesis methods, the bromine can be displaced by an azide, e.g. using sodium azide, to yield the 5' -azido hexyl oligomer on solid support. 5 ’-azido hexyl oligomers maybe used, for example, as part of tether molecules for clicking to XNTP precursor nucleotides as mentioned above.

[0005] 6-bromohexyl 2-cyanoethyl / V, / V-diisopropylphosphoramidite can be synthesized by reacting 6-bromohexan-l-ol with a phosphoramidite precursor. Impurities present in a 6-bromohexan-l-ol starting material may also react with the phosphoramidite precursor, resulting in undesired N, A-diisopropylphosphoramidite byproducts. Other types of undesired byproducts may also be formed during synthesis, work-up and purification of the 7V,7V-diisopropylphosphoramidites. When used for XNTP synthesis, such byproducts may negatively influence the sequencing performance, e.g. by causing strand breaks after expansion of the XNTPs or negatively influencing the polymerase-mediated XNTP incorporation reaction.

[0006] There are several methods described in the literature allowing the synthesis of 6-bromohexan-l-ol (see Angew. Chem. Int. Ed. 2022, 61, e202207647; Russian Journal of Bioorganic Chemistry Vol. 34 No. 1 2008; J. Org. Chem. 2000, 65, 5837-5838; Synthesis, 1985, 12 1161-1162). The predominant, commercially most attractive path is the treatment of 1,6-hexandiol with 48% HBr, with known byproducts being 1,6-dibromohexane and remaining 1,6-hexandiol (J. Org. Chem. 2000, 65, 5837-5838; Synthesis, 1985, 121161-1162). See the following scheme:HBr

[0007] The present disclosure unveils undesired / V, / V-di isopropylphosphoramidite byproducts that were not expected in light of the prior art. Improved methods are thus desirable for producing highly pure VN-diisopropylphosphoramidite compounds, such as 6-bromohexyl 2-cyanoethyl / V, / V-diisopropylphosphoramidite, including highly pure reactants used therein, such as 6-bromohexan- 1 -ol.SUMMARY OF THE INVENTION

[0008] .V, V-diisopropylphosphoramidites, such as 6-bromohexyl 2-cyanoethyl N,N-diisopropylphosphoramidite, can be incorporated in oligomers called SSRTs, which can be used as building blocks for XNTPs that are used in nanopore sequencing, especially within the technology of sequencing by expansion (see e.g. WO 2024 / 074412 Al). The linkers used for linking the SSRT to the nucleotide may influence the incorporation kinetics, accuracy and processivity of the polymerase. Furthermore, since sequencing by expansion is a single molecule detection technology, structural variants of the SSRT oligomers may behave differently when they translocate through the nanopore or may even cause strand break after expansion of the XNTPs. Therefore, SSRT oligomers and their building blocks - including the phosphorami dites - of the highest purity are desirable.

[0009] It was discovered that commercially available 6-bromohexyl 2-cyanoethyl N,N-diisopropylphosphoramidite ordered from several manufacturers contained the following critical impurities of formulas (Vc), (Vic) and (Vila) that were characterized byJH NMR spectroscopy and verified by the synthesis of reference compounds:

[0010] Phosphoramidite (Vc) was identified as critical as it is part of the XNTP linker that binds to the polymerase pockets close to the active site. Phosphoramidite (Vc) leads to SSRT impurities with a longer linker (+100 Da mass difference), which are difficult to separate from crude SSRT products. Phosphoramidite (Vc), found in the commercial materials, is being incorporated in the oligomers and polymers in the same way as 6-bromohexyl 2-cyanoethyl V,V-diisopropylphosphoramidite. Purification attempts result in a lower recovery of the desired full length products. Fig. 1 shows analytical chromatograms of crude SSRT products synthesized with 6-bromohexyl 2-cyanoethyl V,V-diisopropylphosphoramidite from different commercial suppliers. The +100 Da impurity caused by impurity (Vc) is marked at a retention time of 32.2 to 32.3 min. Fig. 2 showsJH NMR (CD3CN) of the commercially available phosphoramidite from supplier 1. The impurity is carried over to the XNTP synthesis and can be hardly separated from the final desired XNTP reagent. The occurrence of this impurity may be due to the presence of the impurity 6-((6-bromohexyl)oxy)hexan-l-ol (formula (IVc)) in the 6-bromohexan-l-ol preparation used for synthesis. Impurity (Vc) was unexpected since 6-((6-bromohexyl)oxy)hexan-l-ol was not described as an impurity occurring in the course of the known synthetic procedure of 6-bromohexan-l-ol (J. Org. Chem. 2000, 65, 5837-5838; Synthesis, 1985, 12 1161-1162). Phosphoramidite (Vila) is another unexpected impurity that may be due to the presence of an impurity, i.e. 6-methoxyhexan-l-ol (formula (Villa)), in the 6-bromohexan-l-ol preparation used for synthesis.

[0011] Phosphoramidite impurity (Vic) is an impurity that can be formed during synthesis, workup and purification of 6-bromohexyl 2-cyanoethyl V,V-diisopropylphosphoramidite. It can further undergo Hofmann elimination to give a terminal olefin (e.g. during basic cleavage and deprotection after SSRT synthesis). Both impurities (as well as phosphoramidite impurity (Vila)) can lead to uncyclized XNTPs. When incorporated in an Xpandomer, such uncyclized XNTPs lead to strand breaks and therefore shorter average read length.

[0012] The use of the commercially available 6-bromohexyl 2-cyanoethyl N,N-diisopropylphosphoramidite within the sequencing by expansion technology thus lead to oligomers with lower purity, negatively affecting sequencing performance.

[0013] Attempted purification of the commercial 6-bromohexyl 2-cyanoethyl N,N-diisopropylphosphoramidite did not deliver the highly pure material that was desired for the synthesis of the polymers for sequencing by expansion technology. Additionally, the direct phosphitylation of commercially available 6-bromohexan- 1 -ol, using the standard synthetic procedure according to the following scheme, did not yield the 6-bromohexyl 2-cyanoethyl N,N-diisopropylphosphoramidite in the desired quality either:

[0014] The present disclosure therefore aims at providing highly pure N,N-diisopropylphosphoramidites, such as 6-bromohexyl 2-cyanoethyl A,A-diisopropylphosphoramidite, useful for terminal modification of oligomers or polymers, such as SSRTs.

[0015] To this end, commercially available linkers, such as 6-bromohexan- l-ol, that are used as starting material for the synthesis of the highly pure / V, / V-diisopropylphosphoramidite compound, are purified to remove undesired impurities already present in the starting material, such as 6-((6-bromohexyl)oxy)hexan-l-ol (formula (IVc)), or 6-methoxyhexan-l-ol (formula (Villa)). Prolonged storage of pure 6-bromohexan- l-ol should be avoided, as impurity 6-((6-bromohexyl)oxy)hexan-l-ol can also form during storage, especially at ambient temperatures.

[0016] Moreover, the phosphitylation reaction of the purified 6-bromohexan- l-ol can be performed at low temperatures, such as 0 °C, to avoid the formation of the critical impurity 6-((6-bromohexyl)oxy)hexan-l-ol during the reaction. More bulky, less nucleophilic or non-nucleophilic tertiary amines such as DIPEA are recommended as additives during the reaction instead of, e.g. tri ethylamine, to reduce nucleophilic substitution of the bromine.

[0017] Triethylamine that is commonly used also as an additive in the mobile phase during purification of the / V, / V-diisopropylphosphoramidite compound by chromatography causes the formation of a further impurity, impurity (Vic) in the case of 6-bromohexyl 2-cyanoethyl N,N-diisopropylphosphoramidite, when present in high concentration during the evaporation of the solvent on the rotary evaporator at elevated temperatures. To avoid the formation of this impurity, the product containing fractions after the chromatography can be extensively washed with water or mixtures of water and organic solvents, such as DMF. To avoid hydrolysis of the 7V,7V-diisopropylphosphoramidite compound, concentration and drying (e.g. by azeotropic coevaporation) can be done at mild temperatures, such as 30 °C. Optionally, the chromatography can also be done with more bulky, less nucleophilic or non-nucleophilic tertiary amines such as DIPEA to reduce nucleophilic substitution of bromine, if the described extraction procedure after chromatography is followed.

[0018] Exemplary results ofJH NMR (CD3CN) of 6-bromohexyl 2-cyanoethyl N,N-diisopropylphosphoramidite produced by the method disclosed herein and isolated after the column chromatography are shown in Fig. 3. Fig. 4 shows an exemplary analytical chromatogram of crude SSRT, which has been synthesized using the highly pure 6-bromohexyl 2-cyanoethyl MV-diisopropylphosphoramidite, showing the absence of the +100 Da impurity.

[0019] The use of highly pure N, 7V-di isopropyl phosphorami di te compounds described herein in the synthesis of oligomers results in an easier purification and higher yields of the desired polymeric products and, additionally, the isolated products show higher purity (detected by LC-MS). The expected advantage when used in the synthesis of XNTPs is improved nanopore sequencing, for example generating sequences of higher read length. The results described herein may plausibly extend to comparable compounds with leaving groups other than Br and / or having a different length than the 6-bromohexan-l-ol exemplified herein, for which comparable impurities can be expected.

[0020] Exemplary embodiments of the disclosure are described by the following items:[1] A method for producing a compound of formula (I), wherein LG1is a leaving group and n is an integerthe method comprising the following steps in order:(a) Purifying a compound of formula (II):(II), and(b) Reacting the purified compound of formula (II) with a compound of formula (III), wherein LG2is a leaving group:(III).[2] The method of item 1, wherein the compound of formula (II) is purified in step (a) by distillation, chromatography or precipitation, or a combination thereof.[3] The method of any one of the preceding items, wherein step (a) removes a compound of structure (IV) and / or (VIII):[4] The method of any one of the preceding items, wherein step (b) is conducted at a temperature of 0 °C to 25 °C, such as 0 °C to 4 °C.[5] The method of any one of the preceding items, wherein step (b) is conducted in the presence of an amine compound, such as A / A-diisopropylethylamine (DIPEA), l,8-diazabicycloundec-7-ene (DBU), l,5-diazabicyclo(4.3.0)non-5-ene (DBN), pyridine or 2,6-lutidine.[6] The method of any one of the preceding items, wherein step (b) is conducted without tri ethylamine.[7] The method of any one of the preceding items, wherein the compound of structural formula (III) is 2-cyanoethyl N, A-di isopropyl ch lorophosphoramidite or activated 2-cyanoethyl N,N,N’,N’-tetraisopropylphosphordiamidite.[8] The method of any one of the preceding items, wherein the relative amount of substance of (each of) the compound(s) of formula (V) and / or (VII) is 0.1 % or less, in relation to the amount of substance of the compound of formula (I), in the product of step (b):[9] The method of any one of the preceding items, wherein n is an integer of 3-5, such as 4.

[0010] The method of any one of the preceding items, wherein LG1is a halide, tosylate or mesylate.

[0011] The method of any one of the preceding items, wherein LG1is selected from I, Br, tosylate and mesylate.

[0012] The method of any one of the preceding items, wherein LG1is Br.

[0013] The method of any one of the preceding items, wherein n = 4 and LG1is Br.

[0014] The method of any one of the preceding items, wherein the compound of formula (I) has the structure of formula (Ic), and the compound of formula (II) has the structure of formula (lie):).

[0015] The method of any one of the preceding items, further comprising the step:(c) Purifying the product of step (b).

[0016] The method of item 15, wherein the product of step (b) is purified by chromatography.

[0017] The method of item 16, wherein the chromatography is column chromatography.

[0018] The method of any one of items 15-17, wherein the product-containing fractions are washed with water and / or a mixture of water and an organic solvent, such as a water / dimethylformamide (DMF) mixture.

[0019] The method of any one of items 16-18, wherein an amine compound is used as an additive in the mobile phase of the chromatography, wherein the amine compound is optionally DIPEA, l,8-diazabicycloundec-7-ene (DBU), l,5-diazabicyclo(4.3.0)non-5-ene (DBN), pyridine or 2,6-lutidine.

[0020] The method of any one of items 16-19, wherein the mobile phase of the chromatography does not comprise triethylamine.

[0021] The method of any one of items 15-20, wherein the relative of amount of substance of the compound of formula (VI) is 0.1 % or less, in relation to the amount of substance of the compound of formula (I), in the product of step (c):wherein (i) each R independently represents a substituted or unsubstituted hydrocarbon, or wherein (ii) all three R together represent a substituted or unsubstituted ring structure.

[0022] A preparation of the compound of formula (I) obtainable by the method of any one of items 1-21.

[0023] The preparation of item 22, wherein the relative amount of substance of (each of) the compound(s) of formula (V), (VI) and / or (VII) is 0.1 % or less, in relation to the amount of substance of the compound of formula (I):I),wherein LG1is a leaving group and n is an integer of 1-18, andwherein (i) each R independently represents a substituted or unsubstituted hydrocarbon, or wherein (ii) all three R together represent a substituted or unsubstituted ring structure.

[0024] A preparation of the compound of formula (I), wherein the relative amount of substance of (each of ) the compound(s) of formula (V), (VI) and / or (VII) is 0.1 % or less, in relation to the amount of substance of the compound of formula (I):)I),wherein LG1is a leaving group and n is an integer of 1-18, andwherein (i) each R independently represents a substituted or unsubstituted hydrocarbon, or wherein (ii) all three R together represent a substituted or unsubstituted ring structure.

[0025] A preparation of the compound of formula (I), wherein the compound is at least 98.0 %, such as at least 99.0 % pure, optionally as determined by nuclear magnetic resonance (NMR) spectroscopy.BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Fig. 1: analytical chromatograms of crude SSRT products synthesized with 6-bromohexyl 2-cyanoethyl A,A-diisopropylphosphoramidite from different commercial suppliers. 1 A: Supplier 1; IB: Supplier 2.

[0022] Fig. 2:JH NMR (CD3CN) of the commercially available phosphoramidite from supplier 1. 2A: Entire graph; 2B: zoom onto relevant region of graph of 2A.

[0023] Fig. 3: Exemplary results ofJH NMR (CD3CN) of 6-bromohexyl 2-cyanoethyl N,N-diisopropylphosphoramidite produced by the method disclosed herein and isolated after the column chromatography. 3A: Product not washed with water after chromatography, entire graph; 3B: zoom onto relevant region of graph of 3A; 3C: Product washed with water after chromatography, entire graph; 3D: zoom onto relevant region of graph of 3C.

[0024] Fig. 4: Exemplary analytical chromatogram of crude SSRT, which has been synthesized using the highly pure 6-bromohexyl 2-cyanoethyl A,7V-diisopropylphosphoramidite, showing the absence of the +100 Da impurity.

[0025] Fig. 5: NMR of commercial 6-bromohexan-l-ol (formula (lie)), Apollo Scientific:JH NMR (CD3CN, 400 MHz, 32 scans, 0.1 mol / 0.65 mL). 5A: Entire graph; 5B: zoom onto relevant region of graph of 5A.

[0026] Fig. 6: 6A and 6B: NMR of 6-bromohexan-l-ol (formula (lie)) after distillation: 'H NMR (CD3CN, 400 MHz, 32 scans, 0.1 mol / 0.65 mL). 6A: Entire graph; 6B: zoom onto relevant region of graph of 6A; 6C and 6D: NMR of 6-bromohexan-l-ol (formula (lie)) after chromatography and distillation:1HNMR(CD3CN, 400 MHz, 64 scans, 0.1 mol / 0.65 mL). 6C: Entire graph; 6D: zoom onto relevant region of graph of 6C.

[0027] Fig. 7: NMR of produced phosphoramidite (formula (Ic)); 7A: 'H NMR (CD3CN, 500 MHz, 32 scans, 0.1 mol / 0.65 mL) from Example 2; 7B: zoom on relevant region of graph of 7A; 7C:31P NMR (CD3CN, 500 MHz, 1024 scans, 0.1 mol / 0.65 mL) from Example 2; 7D: 'H NMR (CD3CN, 400 MHz, 32 scans, 0.1 mol / 0.65 mL) from Example 4; 7E: zoom on relevant region of graph of 7D; 7F:31P NMR (CD3CN, 400 MHz, 32 scans, 0.1 mol / 0.65 mL) from Example 4.

[0028] Fig. 8: NMR of produced phosphoramidite (formula (Ic)); 8A: 'H NMR (CD3CN) from Example 5; 8B: zoom on relevant region of graph of 8A; 8C:31P NMR (CD3CN) from Example 5.

[0029] Fig. 9: 'H NMR (CD3CN) of 6-bromohexyl 2-cyanoethyl N, A-diisopropylphosphoramidite (analyte); 9A: Entire graph; 9B: zoom on region of analyte and impurity (Vc); 9C: zoom on region of analyte and impurity (Vic).

[0030] Fig. 10: 'H NMR (CD3CN, 400 MHz, 32 scans, 0.1 mmol / 0.65 ml) of the commercial 6-bromohexan-l-ol. 10A: Entire graph; 10B: zoom onto relevant region of graph of 10A.

[0031] Fig. 11:1HNMR(CD3CN, 400 MHz, 32 scans, 0.1 mmol / 0.65 ml) of 6-bromohexan-l-ol after the column chromatography.

[0032] Fig. 12: 'H NMR (CD3CN, 400 MHz, 32 scans, 0.1 mmol / 0.65 ml) of 6-bromohexan-l-ol after column-chromatography and distillation according to example 7. 12A: Entire graph; 12B: zoom onto relevant region of graph of 12 A.

[0033] Fig. 13: 'H NMR (CD3CN, 500 MHz, 32 scans, 0.1 mmol / 0.65 ml) of the purified 6-BrHexPPA according to example 7 and 8. 13 A: Entire graph; 13B: zoom onto relevant region of graph of 13 A.

[0034] Fig. 14:31P NMR (CD3CN, 500 MHz, 1024 scans, 0.1 mol / 0.65 ml) of the purified 6-BrHexPPA according to example 7 and 8.DETAILED DESCRIPTION OF THE INVENTION

[0035] The disclosure will now be described in detail by way of reference only using the following definitions and examples. All patents and publications, including all sequences disclosed within such patents and publications, referred to herein are expressly incorporated by reference.

[0036] Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton (Singleton et al., Dictionary of microbiology and molecular biology, 2nd ed., 1994, John Wiley and Sons, New York), Hale (Hale and Marham, The Harper Collins dictionary of biology, 1991, Harper Perennial, NY) and Walker (Walker and Cox, The Language of Biotechnology: A Dictionary of Terms. 1988, American Chemical Society, Washington, D.C. ISBN-0-8412-1499-1) provide one of skill with a general dictionary of many of the terms used in this invention. Practitioners are particularly directed to Sambrook (Sambrook et al., Molecular cloning: A laboratory manual, 1989, Cold Spring Harbor Laboratory Press), and Ausubel (Ausubel et al., Current protocols in molecular biology, 1993, John Wiley & Sons, Inc.), for definitions and terms of the art. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary.

[0037] As used herein, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

[0038] In chemical structures shown herein, when not all natural valencies of an atom are filled by named groups, it should be understood that the unfilled valencies are filled by hydrogen. When a wavy line in a structure intersects a bond, then the intersected bond is the location where the structure joins to the remainder of a molecule.

[0039] Reference throughout this specification to "one embodiment" or "an embodiment" and variations thereof means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances ofthe phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0040] The headings provided herein are not limitations of the various aspects or embodiments of the invention, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.I. Terms

[0041] As used herein, the term "aliphatic" means a straight or branched hydrocarbon chain, which may be saturated or mono- or polyunsaturated. An unsaturated, aliphatic group contains one or more double and / or triple bonds. The branches of the hydrocarbon chain may include linear chains as well as non-aromatic cyclic elements. The hydrocarbon chain may, unless otherwise stated, be of any length, and contain any number of branches. Both the main chain as well as the branches may furthermore contain heteroatoms as for instance B, N, O, P, S, Se or Si.

[0042] As used herein, the term "alkyl" includes saturated aliphatic groups, including straightchain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.), branched- chain alkyl groups (isopropyl, tert-butyl, isobutyl, etc.), cycloalkyl (alicyclic) groups (cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl), alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. The term alkyl further includes alkyl groups, which can further include oxygen, nitrogen, sulfur or phosphorous atoms replacing one or more carbons of the hydrocarbon backbone. In certain embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1-C30 for straight chain, C1-C30 for branched chain). Moreover, the term alkyl includes both "unsubstituted alkyls" and "substituted alkyls", the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, alkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinate, cyano, amino (including alkyl amino,dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamide, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. An "alkylaryl" or an "arylalkyl" moiety is an alkyl substituted with an aryl (e.g., phenylmethyl (benzyl)). The term "alkyl" also includes the side chains of natural and unnatural amino acids.

[0043] As used herein, the term "alkenyl" includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that contain at least one double bond. For example, the term "alkenyl" includes straight- chain alkenyl groups (e.g., ethylenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, etc.), branched-chain alkenyl groups, cycloalkenyl (alicyclic) groups (cyclopropenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl), alkyl or alkenyl substituted cycloalkenyl groups, and cycloalkyl or cycloalkenyl substituted alkenyl groups. The term alkenyl further includes alkenyl groups which include oxygen, nitrogen, sulfur or phosphorous atoms replacing one or more carbons of the hydrocarbon backbone. In certain embodiments, a straight chain or branched chain alkenyl group has 30 or fewer carbon atoms in its backbone (e.g., C2-C30 for straight chain, C3-C30 for branched chain). Moreover, the term alkenyl includes both "unsubstituted alkenyls" and "substituted alkenyls," the latter of which refers to alkenyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, alkyl groups, alkenyl groups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinate, cyano, amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamide, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Other examples of alkenyl groups include, but are not limited to, ethenyl, 1 -propenyl, 2-propenyl, 1 -methyl-ethenyl, 1 -butenyl, 2-butenyl, 3 -butenyl, 1 -methyl- 1 -propenyl, 2-methyl-l-propenyl, 1 -methyl-2-propenyl, 2-methyl-2-propenyl; 1 -pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1 -methyl-l-butenyl, 2-methyl-l-butenyl, 3-methyl-l-butenyl, 1 -methyl-2-butenyl, 2-methyl-2-butenyl, 3-methyl-2-butenyl, l-methyl-3-butenyl, 2-methyl-3-butenyl, 3-methyl-3-butenyl, l,l-dimethyl-2-propenyl, 1,2-dimethyl-l -propenyl, 1 ,2-dimethyl-2-propenyl, 1 -ethyl- 1-propenyl, 1 -ethyl-2-propenyl, 1 -hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5 -hexenyl, 1-methyl- 1-pentenyl, 2-methyl-l-pentenyl, 3-methyl-l-pentenyl, 4-methyl-l -pentenyl, 1 -methyl-2-pentenyl, 2-methyl-2-pentenyl, 3-methyl-2-pentenyl, 4-methyl-2-pentenyl, 1-methy 1-3 -pentenyl, 2-methyl- 3 -pentenyl, 3 -methyl-3 -pentenyl, 4-methy 1-3 -pentenyl, 1 -methyl-4-pentenyl, 2-methyl-4-pentenyl, 3-methyl-4-pentenyl, 4-methyl-4-pentenyl, 1 , 1 -dimethyl-2-butenyl, l,l-dimethyl-3-butenyl, 1 ,2-dimethyl- 1 -butenyl, 1 ,2-dimethyl-2-butenyl, l,2-dimethyl-3-butenyl, 1,3-dimethyl-l-butenyl, l,3-dimethyl-2-butenyl, l,3-dimethyl-3-butenyl, 2, 2-dimethyl-3 -butenyl, 2,3-dimethyl-l-butenyl, 2,3-dimethyl-2-butenyl, 2,3-dimethyl-3-butenyl, 3,3-dimethyl-l-butenyl, 3,3-dimethyl-2-butenyl, 1-ethyl-l-butenyl, 1 -ethyl-2-butenyl, l-ethyl-3 -butenyl, 2-ethyl-l -butenyl, 2-ethyl-2-butenyl, 2-ethyl-3 -butenyl, 1 , 1 ,2-trimethyl-2-propenyl, 1 -ethyl- 1 -methyl-2-propenyl, l-ethyl-2-methyl-1 -propenyl and 1 -ethyl-2-methyl-2-propenyl groups. Groups containing multiple double bonds may include but are not limited to buta-1, 3-dienyl, penta- 1,3 -di enyl or penta- 1,4-dienyl groups.

[0044] As used herein, the term "alkynyl" includes unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but which contain at least one triple bond. For example, the term "alkynyl" includes straight- chain alkynyl groups (e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl, nonynyl, decynyl, etc.), branched-chain alkynyl groups, and cycloalkyl or cycloalkenyl substituted alkynyl groups. The term alkynyl further includes alkynyl groups which include oxygen, nitrogen, sulfur or phosphorous atoms replacing one or more carbons of the hydrocarbon backbone. In certain embodiments, a straight chain or branched chain alkynyl group has 30 or fewer carbon atoms in its backbone (e.g., C2-C30 for straight chain, C3-C30 for branched chain). Moreover, the term alkynyl includes both "unsubstituted alkynyls" and "substituted alkynyls", the latter of which refers to alkynyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, alkyl groups, alkenyl groups, alkynyl groups, halogens, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxy carbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinate, cyano,amino (including alkyl amino, dialkylamino, arylamino, diarylamino, and alkylarylamino), acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamide, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Groups containing multiple triple bonds may include but are not limited to buta- 1,3 -diynyl, penta- 1,3 -diynyl or penta- 1,4-diynyl groups.

[0045] As used herein, the term "aromatic" means, unless otherwise stated, a planar cyclic hydrocarbon moiety of conjugated double bonds, which may be a single ring or include multiple fused or covalently linked rings. The main chain of the cyclic hydrocarbon moiety may, unless otherwise stated, be of any length and contain any number of heteroatoms, as for instance N, O and S. The aromatic group may be substituted by alkyl groups or heteroatoms like O, S, N, P or Si.

[0046] Each of the terms (e.g., "alkyl," "aromatic," etc.) includes both substituted and unsubstituted forms of the indicated radical. In that regard, whenever a group or moiety is described as being "substituted" or "optionally substituted" (or "optionally having" or "optionally comprising") that group may be unsubstituted or substituted with one or more of the indicated substituents. Likewise, when a group is described as being "substituted or unsubstituted" if substituted, the substituent(s) may be selected from one or more of the indicated substituents. If no substituents are indicated, it is meant that the indicated "optionally substituted" or "substituted" group may be substituted with one or more group(s) individually and independently selected from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, hydroxy, protected hydroxyl, alkoxy, aryloxy, acyl, mercapto, alkylthio, arylthio, cyano, cyanate, halogen, thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, protected C-carboxy, O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, silyl, sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy, trihalomethanesulfonyl, trihalomethanesulfonamido, an ether, amino (e.g. a mono-substituted amino group or a di-substituted amino group), and protected derivatives thereof. Any of the above groups may include one or more heteroatoms, including O, N, or S. For example, where a moiety is substituted with an alkyl group, that alkyl group may comprise a heteroatom selected from O, N, or S (e.g. -(CH2-CH2-O-CH2-CH3)).

[0047] As used herein, the term "heteroatom" is meant to include atoms other than carbon, for example, boron (B), oxygen (O), nitrogen (N), sulfur (S), phosphorus (P), and silicon (Si). In some embodiments, a “heterocyclic ring” may comprise one or more heteroatoms. In other embodiments, an aliphatic group may comprise or be substituted by one or more heteroatoms.

[0048] As used herein, the term "nucleobase" refers to a heterocyclic moiety capable of non-covalently pairing with another nucleobase. The term "nucleobase" encompasses both "unmodified nucleobases" and "modified nucleobases." A "naturally occurring nucleobase" or an "unmodified nucleobase" (used interchangeably) refer to a nucleobase that is unmodified relative to its naturally occurring form. Likewise, a "modified nucleobase" means any substitution and / or change from a natural nucleobase. Nucleobase (or base) modifications or substitutions are structurally distinguishable from, yet functionally interchangeable with, naturally occurring or synthetic unmodified nucleobases. Both natural and modified nucleobases are capable of participating in hydrogen bonding. Such nucleobase modifications may impart nuclease stability, binding affinity or some other beneficial biological property to oligonucleotides.

[0049] As used herein, the term "sequencing" refers to the determination of the identity and position of nucleobases in a nucleic acid.

[0050] Phosphate: A “phosphate” includes an “organophosphate” as well as variants thereof, such as an “amidophosphate” (which is a synonym for “phosphoramidate”). A phosphate can include a side chain, such as a side chain linking the a-phosphate to the nucleotide sugar or nucleobase. The first, second and third phosphate counted from the 5’ end of a nucleoside are also referred to as a-phosphate, [3-phosphate and y-phosphate, respectively (or, in case the a-phosphate is a phosphoramidate, it can also be referred to as “a-phosphoramidate”).

[0051] Expandable nucleoside triphosphate: An “expandable nucleoside triphosphate”, “expandable NTP” or “XNTP” refers to a 5' phosphate modified non-natural nucleoside triphosphate (NTP) molecule (typically a non-natural 2’ -deoxynucleoside triphosphate (dNTP) molecule) compatible with template-dependent enzymatic polymerization. Each XNTP has two distinct functional regions, i.e., a selectively cleavable bond (e.g. a phosphoramidate bond) linking the 5’ a-phosphate to a sugar comprised in a nucleoside and a tether that is attached within the XNTP at positions that allow for controlled expansion by cleavage of the cleavable bond (e.g. a tether linking the 5’ a-phosphate and the nucleobase). A tether typically comprises a polymer. An XNTP can thus be present in a constrained configuration (when the cleavable bond is still intact)or in an expanded configuration (when the cleavable bond has been cleaved, e.g. via acid treatment).

[0052] Xpandomer: An “Xpandomer” or “Xp” refers to a molecule consisting of at least two monomers derived from XNTPs. An Xpandomer is obtainable, for example, by polymerase-mediated replication of a template nucleic acid using XNTPs as polymerase substrates to yield a Xp complementary strand. An expanded configuration of the Xpandomer can be obtained by cleavage of the cleavable bond in the XNTPs, e.g. via acid treatment.II. Methods

[0053] In some embodiments, the disclosure relates to a method for producing a compound of formula (I), wherein LG1is a leaving group and n is an integer of 1-18:the method comprising the following steps in order:(a) Purifying a compound of formula (II):(II), and(b) Reacting the purified compound of formula (II) with a compound of formula (III), wherein LG2is a leaving group:(III).

[0054] In some embodiments, the compound of formula (II) is purified in step (a) by distillation, chromatography or precipitation, or a combination thereof. In some embodiments, the compound of formula (II) is purified in step (a) by distillation, such as vacuum distillation. In someembodiments, the compound of formula (II) is purified in step (a) by a combination of chromatography and distillation, such as vacuum distillation. As shown in the Examples, a combination of chromatography and distillation could remove different types of impurities that could not be removed entirely by one type of purification alone. In some embodiments, the chromatography is column chromatography, such as flash column chromatography. In some embodiments, the distillation is a vacuum distillation.

[0055] The present disclosure surprisingly found previously undescribed impurities in preparations of 6-bromohexan-l-ol. Impurities that have been detected or may be expected include 6-((6-bromohexyl)oxy)hexan-l-ol (detected), 6-methoxyhexan- 1 -ol (detected), 5-methoxy-3-methylpentan- 1 -ol (isomer of 6-methoxyhexan- 1 -ol), hexan-l-ol, 5-hexen-l-ol, hexan-l,6-diol (detected), 1,6- dibromohexane (detected), 6-bromohex-l-ene (detected), 6-(hex-5-en-lyloxy)hexan-l-ol, 6-chlorohexan- 1 -ol, 5-bromopentan-l-ol, heptan-l-ol, 7-bromoheptan-l-ol, and 4-methyldecan-5-ol (detected).

[0056] Therefore, in some embodiments, step (a) is suitable for removing a compound of formula (IV) and / or of formula (VIII):

[0057] In some embodiments, step (a) is suitable for removing a compound selected from 6-((6-bromohexyl)oxy)hexan-l-ol, 6-methoxyhexan- l-ol, 5-methoxy-3-methylpentan-l-ol, hexan-l-ol, 5-hexen-l-ol, hexan-l,6-diol, 1,6-dibromohexan, 6-bromohex-l-ene, 6-(hex-5-en-lyloxy)hexan-l-ol, 6-chlorohexan- l-ol, 5-bromopentan-l-ol, heptan-l-ol, and 7-bromoheptan-l-ol.Therefore, in some embodiments, step (a) removes a compound of formula (IV) and / or of formula (VIII):

[0058] In some embodiments, step (a) removes a compound selected from 6-((6-bromohexyl)oxy)hexan-l-ol, 6-methoxyhexan-l-ol, 5-methoxy-3-methylpentan-l-ol, hexan-l-ol, 5-hexen-l-ol, hexan-l,6-diol, 1,6-dibromohexan, 6-bromohex-l-ene, 6-(hex-5-en-lyloxy)hexan-l-ol, 6-chlorohexan-l-ol, 5-bromopentan-l-ol, heptan-l-ol, and 7-bromoheptan-l-ol.

[0059] In some embodiments, the compound of formula (IV) is removed in step (a) by distillation. In some embodiments, the compound of formula (VIII) is removed in step (a) by chromatography.

[0060] In addition, or instead of impurity (VIII), isomers of impurity (VIII) may be present in the starting material. In some embodiments, such isomers may be removed in step (a) as well (e.g. by chromatography). In some embodiments, step (a) is suitable for removing 6-methoxyhexan-l-ol. In some embodiments, step (a) removes 6-methoxyhexan-l-ol. In some embodiments, step (a) is suitable for removing 5-methoxy-3-methylpentan-l-ol. In some embodiments, step (a) removes 5-methoxy-3-methylpentan-l-ol. In some embodiments, step (a) is suitable for removing 6-methoxyhexan-l-ol and 5-methoxy-3-methylpentan-l-ol. In some embodiments, step (a) removes 6-methoxyhexan-l-ol and 5-methoxy-3-methylpentan-l-ol.

[0061] It has been found that step (b) works in a range of temperatures, but is preferably conducted at low temperatures in order to reduce the formation of new impurities. In some embodiments, step (b) is conducted at a temperature of 0 °C to 25 °C, such as 0 °C to 4 °C.

[0062] Step (b) may be conducted for at least 30 min, for example, such as 30 to 90 min, or 45 min to 60 min.

[0063] In some embodiments, step (b) is conducted in the presence of an additive, for example an amine compound. In some embodiments, the amine compound has the formula NR3, wherein (i) each R independently represents a substituted or unsubstituted hydrocarbon, or (ii) all three R together with the nitrogen represent a substituted or unsubstituted ring structure.

[0064] In some embodiments, each R is independently a C1-C3 alkyl group. In some embodiments, each R is independently a C2-C3 alkyl group. In some embodiments, each R is independently an ethyl or an isopropyl group. When all three R together with the nitrogen form a substituted or unsubstituted ring structure, the ring structure may be a single ring structure, for example. In some embodiments, the ring structure is a substituted or unsubstituted aromatic ring structure, such as pyridine or 2,6-lutidine. In some embodiments, the ring structure is a substituted or unsubstituted aromatic single ring structure. In some embodiments, the ring structure is a substituted orunsubstituted aliphatic ring structure, such as / V-methyl-morpholin. In some embodiments, the substitutions in a substituted ring structure are C1-C3 alkyl groups, such as methyl groups.

[0065] The amine compound may be, for example, a tertiary amine including triethylamine (TEA), A,A-diisopropylethylamine (DIPEA), l,8-diazabicycloundec-7-ene (DBU), 1,5-diazabicyclo(4.3.0)non-5-ene (DBN), pyridine or 2,6-lutidine. In some embodiments, the amine compound is less nucleophilic than TEA, such as an amine compound that is non-nucleophilic. Nucleophilicity is a well-known parameter in the art, and may be determined e.g. by the general Equation (6) as described by Phan et al., 2006 (Angew Chem Int Ed Engl., 2006; 45(23):3869-74). In some embodiments, the amine compound is VA-diisopropylethylamine (DIPEA), 1,8-diazabicycloundec-7-ene (DBU), l,5-diazabicyclo(4.3.0)non-5-ene (DBN) or 2,6-lutidine. In some embodiments, the amine compound is DIPEA. In some embodiments, step (b) is conducted in the absence of TEA.

[0066] In any of the structures given herein, n can be an integer of 1-18. In some embodiments, n is an integer of 1-15 or 2-10. In some embodiments, n is an integer of 3-5. In some embodiments, n is 4. Generally, n will be the same integer for all compounds involved in a given method or preparation disclosed herein. Thus, for example, when n is 4, this applies to the compounds of formula (I) and (II), as well as to the impurities disclosed herein, when present. Accordingly, when n is 4, for example, the compound of formula (II) is a hexan-l-ol with a leaving group LG1at position 6, and the compound of formula (I) is a phosphoramidite compound derived from reacting said hexan-l-ol with a leaving group LG1at position 6 with a phosphoramidite precursor as disclosed herein. Accordingly, in some embodiments, the compounds of formulas (I) and (II) have the following structures (la) and (Ila), respectively:

[0067] In some embodiments, the compound of formula (IV) has the structure of formula (IVa):

[0068] In some embodiments, the compound of formula (VIII) has the structure of formula (Villa):

[0069] In any of the structures given herein, LG1is a leaving group. The type of leaving group is not particularly limited, and includes halides, tosylate, mesylate, and triflate, for example. The halide may be, for example, I, Br or Cl, and in particular I or Br. In some embodiments, LG1is I, Br, Cl, tosylate or mesylate. In some embodiments, LG1is I, Br, tosylate or mesylate. In some embodiments, LG1is a halide. In some embodiments, LG1is Br. Generally, LG1will be the same for all compounds involved in a given method or preparation disclosed herein. Thus, for example, when LG1is Br, this applies to the compounds of formula (I) and (II), as well as to the impurities disclosed herein, when present. Accordingly, when LG1is Br, for example, the compounds of formulas (I) and (II) have the following structures (lb) and (lib), respectively:(lb)(lib)

[0070] In some embodiments, the compound of formula (IV) has the structure of formula (IVb):

[0071] In some embodiments, n is 4 and LG1is Br. Accordingly, in some embodiments, the compound of formula (I) has the structure of formula (Ic), and the compound of formula (II) has the structure of formula (lie):).

[0072] In some embodiments, the compound of formula (IV) has the structure of formula (IVc):

[0073] In any of the structures given herein, LG2is a leaving group. The type of leaving group is not particularly limited, and includes halides and amides, for example. In some embodiments, LG2is Cl or diisopropylamide. In some embodiments, LG2is Cl. In some embodiments, the compound of structural formula (III) is 2-cyanoethyl N, V-diisopropylchlorophosphoramidite (formula (Illa)) or activated 2-cyanoethyl N,N,N’,N ’-tetraisopropylphosphor diamidite (formula Illb).

[0074] 2-cyanoethyl V,V,V’,V’-tetraisopropylphosphordiamidite (formula Illb) can be activated, for example, by diisopropyl ammonium tetrazolide, e.g. diisopropyl ammonium tetrazolide in dichloromethane (DCM).

[0075] The purification of the starting material in step (a) generally results in a starting material comprising less impurities, including less compound of structure (IV) and / or (VIII). Thereby, certain impurities in the product of step (b), such as a compound of structure (V) or (VII), arising from impurities in the starting material may be reduced. Thus, in some embodiments, the relative amount of substance of (each of) the compound(s) of formula (V) and / or (VII) is 0.1 % or less, in relation to the amount of substance of the compound of formula (I), in the product of step (b):I)

[0076] When n is 4, for example, the compound of formula (V) has the structure of formula (Va):

[0077] When n is 4, for example, the compound of formula (VII) has the structure of formula (Vila):

[0078] When LG1is Br, for example, the compound of formula (V) has the structure of formula (Vb):

[0079] When n is 4 and LG1is Br, for example, the compound of formula (V) has the structure of formula (Vc):

[0080] The product of step (b) can be further purified, for example, to remove undesired byproducts, unreacted starting material and / or reaction additives. Accordingly, in some embodiments, the method further comprises the step:(c) Purifying the product of step (b).

[0081] Purification can be achieved by any suitable means known in the art. For example, the product of step (b) may be purified by chromatography, such as column chromatography. In some embodiments, the column chromatography is flash column chromatography.Chromatography is a technique for separating the components of a mixture by a stationary phase and mobile phase. The mobile phase moves through the stationary phase (e.g. in a column) and the components present in the mobile phase are differentially retained, thereby enabling separation from one another. Flash chromatography is commonly known in the art (see e.g. Roge et al., IJPSR, 2011; Vol. 2(8): 1930-1937), and is a type of chromatography in which mobile phase is pushed through the stationary phase by positive (air) pressure to allow faster chromatography runs. Examples of suitable stationary phases include a silica gel. Examples of suitable solvents used as mobile phases include n-hexane (nHex) or n-heptane (nHep). The solvent may be mixed with additives, such as isopropanol (iPrOH) (e.g. in step (a)), e.g. at a ratio of nHex:iPrOH of 90:10 to97:3, for example 95:5, or an amine compound, such as TEA, (e.g. in step (c)), e.g. at a ratio of nHex:amine or n-Hep:amine of 95:5 to 99:1, for example 97:3 or 98.5:1.5. As mentioned elsewhere in the specification, the use of amine compounds other than TEA in step (b) or (c) may be beneficial in terms of byproduct formation, although it could be shown that the use of n-Hep, and not n-Hex, in the mobile phase of the (column) chromatography of step (c) mitigates the byproduct formation associated with TEA.Undesired byproducts may form, for example, by reaction of LG1with reaction additives and / or purification additives, such as additives in the mobile phase in chromatography. In step (b), such additive may in particular be an amine compound of the formula NR3, wherein (i) each R independently represents a substituted or unsubstituted hydrocarbon, or (ii) all three R together with the nitrogen represent a substituted or unsubstituted ring structure. Accordingly, undesired side products may include a compound of formula (VI):wherein LG1is a leaving group, n is an integer of 1-18, and wherein (i) each R independently represents a substituted or unsubstituted hydrocarbon, or (ii) all three R together with the nitrogen represent a substituted or unsubstituted ring structure.

[0082] In some embodiments, each R is independently a C1-C3 alkyl group. In some embodiments, each R is independently a C2-C3 alkyl group. In some embodiments, each R is independently an ethyl or an isopropyl group. When all three R together with the nitrogen form a substituted or unsubstituted ring structure, the ring structure may be a single ring structure, for example. In some embodiments, the ring structure is a substituted or unsubstituted aromatic ring structure, such as pyridine or 2,6-lutidine. In some embodiments, the ring structure is a substituted or unsubstituted aromatic single ring structure. In some embodiments, the ring structure is a substituted or unsubstituted aliphatic ring structure, such as V-methyl-morpholin. In some embodiments, the substitutions in a substituted ring structure are C1-C3 alkyl groups, such as methyl groups.

[0083] Concrete amine compounds include triethylamine (TEA) (commonly used as additive in the mobile phase), but also other amines, such as DIPEA, DBU, DBN, pyridine and 2,6-lutidine.Side products that may arise from the presence of TEA or pyridine are shown by the structures (VI- 1) and (VI-2), respectively:

[0084] When n is 4, for example, the compound of formula (VI) has the structure of formula (Via):

[0085] When n is 4, for example, the compound of formula (VI-1) has the structure of formula (VIa-1):(VIa-1)

[0086] When LG1is Br, for example, the compound of formula (VI) has the structure of formula (VIb):

[0087] When LG1is Br, for example, the compound of formula (VI- 1) has the structure of formula (VIb-1):(VIb-1)

[0088] When n is 4 and LG1is Br, for example, the compound of formula (VI) has the structure of formula (Vic):

[0089] When n is 4 and LG1is Br, for example, the compound of formula (VI- 1) has the structure of formula (VIc-1):(VIc-1)

[0090] The above applies mutatis mutandis also to compounds of structure (VI-2).

[0091] Depending on the presence of amine compounds, corresponding byproducts may arise during step (b). Byproducts formed in step (b) may be removed in step (c), for example. However, when amine compounds, such as TEA, are present in step (c), e.g. in the mobile phase of chromatography and subsequent work-up, the corresponding byproduct may form during step (c), for example during evaporation of the solvent after chromatography. It has been found that suchbyproducts may be removed by additional washing of the product- containing fractions with water and / or a mixture of water and organic solvents, such as dimethylformamide (DMF). Thus, in some embodiments, the product-containing fractions from the chromatography are washed with water and / or a water: dimethylformamide (DMF) mixture. In some embodiments, the product-containing fractions from the chromatography are washed with water. The water: DMF mixture may be 10:1 to 8:1 mixture, for example, such as a 9:1 mixture. In some embodiments, the product-containing fractions from the chromatography are washed with water, then with a water: DMF mixture, and then again with water.

[0092] In order to reduce or even entirely avoid TEA-derived side products, the use of TEA may be reduced or avoided entirely. Thus, in some embodiments, step (b) is conducted without TEA. In some embodiments, step (c) is conducted without TEA. In some embodiments, the mobile phase of the (column) chromatography comprises 1.5% of TEA or less. In some embodiments, the product is eluted via a mobile phase of the (column) chromatography comprising 1.5% of TEA or less. In some embodiments, the mobile phase of the (column) chromatography does not comprise TEA. In some embodiments, the entire method is conducted without TEA.

[0093] Instead of TEA, other types of amine compounds (that are less nucleophilic than TEA, such as non-nucleophilic) may be used as additives in step (a), (b) and / or (c). In some embodiments, a (less nucleophilic) tertiary amine, such as DIPEA, DBU, DBN, pyridine or 2,6-lutidine is used as an additive in step (c). In some embodiments, a (less nucleophilic) tertiary amine, such as DIPEA, DBU, DBN, pyridine or 2,6-lutidine is used as an additive in the mobile phase of the (column) chromatography.

[0094] In embodiments wherein the use of TEA is reduced or entirely avoided, the additional washing with water and / or a mixture of water and an organic solvent after chromatography may be omitted. Especially in such embodiments it may be beneficial, though, to wash the crude product with water and / or a mixture of water and organic solvents, such as dimethylformamide (DMF), i.e. before further purification, e.g. by chromatography. Thus, in some embodiments, the crude product is washed with water and / or a water: dimethylformamide (DMF) mixture. The water: DMF mixture may be 10:1 to 8:1 mixture, for example, such as a 9:1 mixture.

[0095] It has further been surprisingly found that by using n-Hep instead of n-Hex as the solvent in the mobile phase of the (column) chromatography in step (c) can reduce the amount of byproducts (despite the presence of TEA). This is shown in Example 8 wherein nHeptane:TEA isexemplarily used at a 97:3 ratio. Thus, in some embodiments, the (column) chromatography in step (c) is conducted using n-Hep as the solvent in the mobile phase. In some embodiments, the (column) chromatography in step (c) is conducted without using n-Hex in the mobile phase. In some embodiments, the ratio of n-Hep:amine (such as TEA) in the mobile phase is 95:5 to 99:1, such as 97:3.

[0096] In some embodiments, the relative amount of substance of the compound of formula (VI) (such as a compound of any one of formulas (VI- 1) to (VI-5)) is 0.1 % or less, in relation to the amount of substance of the compound of formula (I), in the product of step (c). When more than one type of impurity is considered, in some embodiments, the relative amount of substance of each compound falling under formula (VI) is 0.1 % or less, in relation to the amount of substance of the compound of formula (I), in the product of step (c). In particular, in some embodiments, the relative amount of substance of the compound of formula (VI- 1) (such as (VIa-1), (VIb-1) or (VIc-1)) is 0.1 % or less, in relation to the amount of substance of the compound of formula (I), in the product of step (c).

[0097] In some embodiments, the relative amount of substance of the compound of formula (V) and / or (VI) is 0.1 % or less, in relation to the amount of substance of the compound of formula (I), in the product of step (c).

[0098] In general, when more than one type of impurity is considered, the relative amount of substance of each impurity may be 0.1 % or less, in relation to the amount of substance of the compound of formula (I), e.g. in the product of step (b) or (c).

[0099] The relative amount (%) of substance is independent from the actual method used. For example, the relative amount of substance of impurities, e.g. formula (V), (VI) and / or (VII), may be determined by nuclear magnetic resonance (NMR) spectroscopy, such asJH NMR spectroscopy. In some embodiments, the relative amount of substance of impurities of 0.1 % or less, in relation to the amount of substance of the compound of formula (I), is determined according to Example 5.III. Preparations

[0100] The disclosure also provides a preparation of the compound of formula (I) obtainable by the method disclosed herein. The preparation is characterized by comprising low proportions of undesired byproducts. In some embodiments, the relative amount of substance of (each of) thecompound(s) of formula (V), (VI) and / or (VII) is 0.1 % or less, in relation to the amount of substance of the compound of formula (I), in the preparation obtainable by the method disclosed herein.

[0101] The disclosure also provides a preparation of the compound of formula (I), wherein the relative amount of (each of) the compound(s) of formula (V), (VI) and / or (VII) is 0.1 % or less, in relation to the amount of substance of the compound of formula (I).

[0102] In some embodiments, the relative amount of substance of the compound of formula (V) is 0.1 % or less, in relation to the amount of substance of the compound of formula (I). In some embodiments, the relative amount of substance of the compound of formula (VI) (such as a compound of any one of formulas (VI-1) to (VI-5)) is 0.1 % or less, in relation to the amount of substance of the compound of formula. In particular, in some embodiments, the relative amount of substance of the compound of formula (VI- 1) (such as (VIa-1), (VIb-1) or (VIc-1)) is 0.1 % or, in relation to the amount of substance of the compound of formula, in the preparation. In some embodiments, the relative amount of substance of the compound of formula (VII) is 0.1 % or less, in relation to the amount of substance of the compound of formula (I). In some embodiments, the relative amount of substance of each of compounds of formula (V), (VI) (such as a compound of any one of formulas (VI-1) to (VI-5)) and (VII) is 0.1 % or less, in relation to the amount of substance of the compound of formula (I).

[0103] Generally, when more than one type of impurity is considered, the relative amount of substance of each compound may be 0.1 % or less, in relation to the amount of substance of the compound of formula (I), in the preparation.

[0104] The disclosure also provides a preparation of the compound of formula (I), wherein the compound is at least 98.0 %, such as at least 99.0 % pure. Purity may be determined, for example, by nuclear magnetic resonance (NMR) spectroscopy, such as31P NMR spectroscopy. For example, the % purity may represent the area% determined by31P NMR spectroscopy.IV. Examples

[0105] Example 1 : Purification of 6-bromohexan- 1 -ol (formula (lie))

[0106] 6-Bromohexan-l-ol (Apollo Scientific) was distilled in vacuo in order to remove the critical impurity 6-((6-bromohexyl)oxy)hexan- 1 -ol (formula (IVc)), as well as other impurities, that are usually present in the commercially available material.

[0107] Vigreux distillation column 30 cm long was used.Pressure: 0.2 mbarTemperature (heating block): 150 °CTemperature (vapour): 64 °C

[0108] Fractions, that did not contain the critical impurity of formula (IVc) (see the 1H NMR of the purified material), were used in the phosphitylation step (see Example 2). (store at -20 °C) NMR of commercial 6-bromohexan-l-ol (formula (lie)), Apollo Scientific: 1H NMR (CD3CN, 400 MHz, 32 scans, 0.1 mol / 0.65 mL), see Fig. 5.NMR of 6-bromohexan-l-ol (formula (lie)) after distillation: 1H NMR (CD3CN, 400 MHz, 32 scans, 0.1 mol / 0.65 mL); see Fig. 6A and 6B.

[0109] Example 2: Synthetic procedure: Phosphitylation using DIPEA and PPAC1DIPEA

[0110] 6-bromohexan-l-ol (8.54 g, 47.2 mmol) obtained in Example 1 was dissolved in DCM (236 mL), cooled in an ice bath, DIPEA (10.7 mL, 61.3 mmol, 1.3 equiv) was added. 2-Cyanoethyl / V, / V-di isopropyl chlorophosphorami di te (1 1.6 mL, 51.9 mmol, 1.1 equiv) was added drop wise over 5 min. Reaction mixture was stirred at 0-4 °C for 60 min, sat. NaHCCh solution was added, the organic phase was separated and the aqueous phase was extracted 2 times with DCM. The organic phase was washed with brine, dried over Na2SC>4, filtered and concentrated in vacuo (water bath temperature: 30 °C). The crude product was purified through flash column chromatography (SiCh, nHex: TEA 97:3)

[0111] Fractions containing the product were united (1500 mL) and washed 4 times with 500 ml water, 1 time with 300 mL water: DMF 9: 1 and 2 times with 500 mL water. The organic phase was dried over NaSCL filtered and concentrated in vacuo (water bath temperature: 30 °C). As soon as the volume was reduced to app. 100 mL, 50 mL of toluene were added and the rest of the solvent was distilled off. Yield: 13.1 g (72%, colourless liquid)

[0112] NMR of produced phosphoramidite (formula (Ic)):JH NMR (CD3CN, 500 MHz, 32 scans, 0.1 mol / 0.65 mL); see Fig. 7A and 7B.31P NMR (CD3CN, 500 MHz, 1024 scans, 0.1 mol / 0.65 mL); see Fig. 7C.

[0113] Example 3: Dual purification of 6-bromohexan-l-ol

[0114] 6-Bromohexan-l-ol (Biosynth) was purified through flash column chromatography (SiCh, nHex:iPrOH 95:5) to remove the critical impurity Villa.

[0115] The product from the column chromatography was subsequently distilled in vacuo in order to remove the critical impurity 6-((6-bromohexyl)oxy)hexan-l-ol (formula (IVc)), as well as other impurities, that were still present in the material after the first purification.

[0116] Vigreux distillation column 30 cm long was used.Pressure: 0.1 mbarTemperature (oil bath): 105 °CTemperature (vapour): 61-63 °C

[0117] Fractions, that did not contain the critical impurity of formula (IVc) (see the 'H NMR of the purified material), were used in the phosphitylation step, (store at -20 °C)NMR of 6-bromohexan-l-ol (formula (lie)) after distillation: 'H NMR (CD3CN, 400 MHz, 64 scans, 0.1 mol / 0.65 mL); see Fig. 6C and 6D.

[0118] Example 4: Improved 6-bromohexyl 2-cyanoethyl N.N-diisopropylphosphoramidite DIPEA

[0119] 6-bromohexan-l-ol (10.00 g, 55.2mmol) obtained in Example 3 was dissolved in DCM (250 mL), cooled in an ice bath, DIPEA (12.5 mL, 71.8 mmol, 1.3 equiv) was added. 2-Cyanoethyl A,A-diisopropylchlorophosphoramidite (13.6 mL, 60.8 mmol, 1.1 equiv) was added dropwise over 5 min. Reaction mixture was stirred at 0-4 °C for 45 min, sat. NaHCCh solution was added, the organic phase was separated and the aqueous phase was extracted 2 times with DCM. The organic phase was washed with brine, dried over Na2SC>4, filtered and concentrated in vacuo (water bath temperature: 30 °C). The crude product was purified through flash column chromatography (SiCh, nHex:TEA 97:3)

[0120] Fractions containing the product were united (1200 mL) and washed 4 times with 500 mL water, 1 time with 300 mL water: DMF 9: 1 and 2 times with 500 mL water. The organic phase was dried over Na2SC>4 filtered and concentrated in vacuo (water bath temperature: 30 °C). As soon as the volume was reduced to app. 100 mL, 50 mL of toluene were added and the rest of the solvent was distilled off. Yield: 11.10 g (53%, colourless liquid)

[0121] NMR of produced phosphoramidite (formula (Ic)):JH NMR (CD3CN, 400 MHz, 32 scans, 0.1 mol / 0.65 mL); see Fig. 7D and 7E.31P NMR (CD3CN, 400 MHz, 32 scans, 0.1 mol / 0.65 mL); see Fig. 7F.

[0122] Example 5: 6-bromohexyl 2-cyanoethyl N.N-diisopropylphosphoramidite synthesis without washing step

[0123] 6-bromohexan-l-ol was dissolved in DCM, cooled in an ice bath, DIPEA (1.5 equiv) was added. 2-Cyanoethyl N, A-diisopropylchlorophosphoramidite (0.95 equiv) was added dropwise. Reaction mixture was stirred at 0 °C for 60 min. The reaction mixture was concentrated, taken upin 500mL hexane, and washed with DMF / water (lOx 100 mL) to remove P(V) impurities (at ~14 ppm in31P NMR). The crude product was then dried over MgSC>4, filtered and concentrated.

[0124] The crude product was purified through a 220 g silica gel column dried at 120 °C over the weekend. The column was pre-equilibrated with 3% TEA in hexanes (trap pak 24hrs), reequilibrated with 1.5 % TEA in hexanes (trap pak 24hrs), and run with 0-10 % gradient (trap pak 24hrs). Conservative peaks were collected, leaving out the front and lag fractions. Fractions were concentrated fractions, followed by covap from ACN (3x 50 mL), and dried by HIV AC overnight.

[0125] NMR of produced phosphoramidite (formula (Ic)):XH NMR see Fig. 8Aand 8B.31PNMR see Fig. 8C.

[0126] Example 6: Threshold level determination of Br-Hex-PPA impurities with 'H-NMR

[0127] The measurement is performed on a 500 MHz NMR Spectrometer or higher.

[0128] Impurity 1 (formula (Vc)): Br-Hex-O-Hex-PPA

[0130] Analyte (formula (Ic)): Br-Hex-PPA

[0131] The threshold levels measured with this method is 0.1 % with regard to the analyte.

[0132] Sample preparation

[0133] Sample solution:

[0134] A defined amount of analyte is weighed in an appropriate vial (e.g. 1.5 mL HPLC-Vial). Acetonitrile-d3 is added to achieve a concentration of 100 mM. Volume minimum is 600 pL.

[0135] The sample is shaken to fully dissolve the product and measurement has to occur directly after sample preparation.

[0136] Acquisition and Processing Parameter:Acquis WonProcessing!

[0137] Correct phase- and baseline correction for the processing should be taken care of.

[0138] AnalysisIdentity

[0139] Referencing of the chemical shift to the solvent signal of ACN (1.94 ppm).

[0140] The identity of the 1H-NMR is checked by the operator to be in accordance to the reference spectrum (Fig. 9A).

[0141] -Determination of the impurity Br-Hex-O-Hex-PPA in Br-Hex-PPA

[0142] The impurity Br-Hex-O-Hex-PPA shows a signal (triplet) at approx. 3.35 ppm, 3.36 ppm und 3.37 ppm (Fig. 9B).

[0143] For the determination of the threshold level, the S / N ratio of the signals at 3.315 ppm (satellite signal of the analyte) and 3.360 ppm is determined:3.360 ppm)3.315 ppm)

[0144] For the threshold level of 0.1 % of impurities: R < 0.2.Determination of impurity Et3N-Hex-PPA in Br-Hex-PPA

[0145] The impurity EtsN-Hex-PPA shows a signal (quartet) at approx. 3.15 ppm, 3.16 ppm, 3.18 ppm and 3.19 ppm (see Fig. 9C).

[0146] For the determination of the threshold level, the S / N ratio of the signals at 3.315 ppm (satellite signal of the analyte) and at 3.175 ppm is determined:S / N {Signal 3.175 ppm)R(Et3NHexPPA)S / N (Signal 3.315 ppm)

[0147] For the threshold level of 0.1 % of impurities: R < 0.2.

[0148] Example 7: Dual purification of 6-Bromohexan-l-ol

[0149] 1.1 Column chromatography

[0150] Column chromatography using »Hex: / PrOH 95:5 was performed to purify away 6-methoxyhexan-l-ol (can be detected inJH NMR, singulet at 3.27 ppm), an impurity that might be present in the commercially obtained 6-bromohexan-l-ol.

[0151] 1.2 Distillation in vacuo

[0152] After the column chromatography, 6-Bromohexan- 1 -ol was distilled in vacuo in order to get rid of the critical impurity 6-bromohexoxyhexanol as well as other impurities that might still be present.

[0153] Vigreux distillation column 30 cm long was used.

[0154] Pressure: 0.06-0.08 mbar

[0155] Temperature oil bath): 90-105 °C

[0156] Temperature (vapour): 53-58 °C

[0157] Fractions that did not contain the critical impurity 6-bromohexoxyhexanol (see theJH NMR of the purified material), were used in the phosphitylation step (store at -20 °C).

[0158] Fig. 10 shows theJH NMR of the commercial 6-bromohexan-l-ol. Fig. 11 shows theJH NMR of 6-bromohexan-l-ol after the column chromatography. Fig. 12 shows theJH NMR of 6-bromohexan-l-ol after column-chromatography and distillation.JH NMR (CD3CN, 400 MHz, 32 scans, 0.1 mmol / 0.65 mL)

[0159] Example 8: Synthetic procedure: Phosphitylation using DIPEA and PPAC1

[0160] 6-Bromohexan- 1 -ol (14.00 g g, 77.32 mmol) obtained in Example 7 was dissolved in DCM (380 ml), cooled in ice bath, DIPEA (17.5 ml, 100.5 mmol, 1.3 equiv) was added. 2-Cyanoethyl N,N-diisopropylchlorophosphoramidite (18.97 ml, 85.05 mmol, 1.1 equiv) was added dropwise over 5 min. Reaction mixture was stirred at 0 °C for 60 min, sat. NaHCCh solution was added, the organic phase was separated and aqueous phase was extracted two times with DCM. Organic phase was washed with brine, dried over Na2SO4, filtered and concentrated in vacuo (water bath temperature: 30 °C). The crude product was purified through flash column chromatography (SiO2, «Heptane:TEA 97:3). Yield: 20.0 g (68%, colourless liquid).

[0161] Fig. 13 shows the H NMR and Fig. 14 the31P NMR of the purified 6-BrHexPPA.

Claims

PATENT CLAIMS1. A method for producing a compound of formula (I), wherein LG1is a leaving group and n is an integer of 1-18:the method comprising the following steps in order:(a) Purifying a compound of formula (II):(II), and(b) Reacting the purified compound of formula (II) with a compound of formula (III), wherein LG2is a leaving group:(III).

2. The method of claim 1, wherein the compound of formula (II) is purified in step (a) by distillation, chromatography or precipitation, or a combination thereof.

3. The method of claim 1 or 2, wherein the compound of formula (II) is purified in step (a) by a combination of distillation and chromatography.

4. The method of any one of the preceding claims, wherein step (a) removes a compound of structure (IV) and / or (VIII):.

5. The method of any one of the preceding claims, wherein step (b) is conducted in the presence of an amine compound, such as / V,V-diisopropylethylamine (DIPEA), l,8-diazabicycloundec-7-ene (DBU), l,5-diazabicyclo(4.3.0)non-5-ene (DBN), pyridine or 2,6-lutidine.

6. The method of any one of the preceding claims, wherein step (b) is conducted without triethylamine.

7. The method of any one of the preceding claims, wherein the compound of structural formula (III) is 2-cyanoethyl A,A-diisopropylchlorophosphoramidite or activated 2-cyanoethyl N,N,N’,N’-tetraisopropylphosphordiamidite.

8. The method of any one of the preceding claims, wherein the relative amount of substance of (each of) the compound(s) of formula (V) and / or (VII) is 0.1 % or less, in relation to the amount of substance of the compound of formula (I), in the product of step (b):""9. The method of any one of the preceding claims, wherein n is an integer of 3-5, such as 4.

10. The method of any one of the preceding claims, wherein LG1is a halide, tosylate, mesylate or triflate.

11. The method of any one of the preceding claims, wherein LG1is selected from I, Br, tosylate and mesylate.

12. The method of any one of the preceding claims, wherein LG1is Br.

13. The method of any one of the preceding claims, wherein n = 4 and LG1is Br.

14. The method of any one of the preceding claims, wherein the compound of formula (I) has the structure of formula (Ic), and the compound of formula (II) has the structure of formula (lie):

15. The method of any one of the preceding claims, further comprising the step:(c) Purifying the product of step (b).

16. The method of claim 15, wherein the product of step (b) is purified by chromatography.

17. The method of claim 16, wherein the chromatography is column chromatography.

18. The method of any one of claims 15-17, wherein the product-containing fractions are washed with water and / or a mixture of water and an organic solvent, such as a water / dimethylformamide (DMF) mixture.

19. The method of any one of claims 16-18, wherein an amine compound is used as an additive in the mobile phase of the chromatography, wherein the amine compound is optionally DIPEA, 1,8-diazabicycloundec-7-ene (DBU), l,5-diazabicyclo(4.3.0)non-5-ene (DBN), pyridine or 2,6-lutidine.

20. The method of any one of claims 16-19, wherein the mobile phase of the chromatography comprises 1.5 % tri ethylamine or less.

21. The method of any one of claims 16-20, wherein the mobile phase of the chromatography does not comprise triethylamine.

22. The method of any one of claims 16-21, wherein the chromatography in step (c) is conducted using n-Heptane as the solvent in the mobile phase.

23. The method of any one of claims 15-22, wherein the relative of amount of substance of the compound of formula (VI) is 0.1 % or less, in relation to the amount of substance of the compound of formula (I), in the product of step (c):wherein (i) each R independently represents a substituted or unsubstituted hydrocarbon, or wherein (ii) all three R together represent a substituted or unsubstituted ring structure.

24. A preparation of the compound of formula (I) obtainable by the method of any one of claims 1-23.

25. The preparation of claim 24, wherein the relative amount of substance of (each of) the compound(s) of formula (V), (VI) and / or (VII) is 0.1 % or less, in relation to the amount of substance of the compound of formula (I):I),wherein LG1is a leaving group and n is an integer of 1-18, andwherein (i) each R independently represents a substituted or unsubstituted hydrocarbon, or wherein (ii) all three R together represent a substituted or unsubstituted ring structure.

26. A preparation of the compound of formula (I), wherein the relative amount of substance of (each of) the compound(s) of formula (V), (VI) and / or (VII) is 0.1 % or less, in relation to the amount of substance of the compound of formula (I):I),wherein LG1is a leaving group and n is an integer of 1-18, andwherein (i) each R independently represents a substituted or unsubstituted hydrocarbon, or wherein (ii) all three R together represent a substituted or unsubstituted ring structure.

27. A preparation of the compound of formula (I), wherein the compound is at least 98.0 %, such as at least 99.0 % pure, optionally as determined by nuclear magnetic resonance (NMR) spectroscopy.

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