Synthesis process of oligonucleotide with sulfo-PMO structure

Abstract

The invention discloses an oligonucleotide synthesis process of a sulfo-PMO structure. In the oligonucleotide synthesis process of the sulfo-PMO structure, the molecular structural formula of sulfo-PMO is shown in the specification. According to the invention, the molecular structure of thio-PMO is specifically limited, and the synthesis of nucleotide phosphate amide and (thio) phosphoric acid amide in the solid-phase synthesis oligonucleotide is carried out along the direction from 5'to 3 ', that is, in each cycle period, the O5'or O6' position of the nucleotide phosphate amide and (thio) phosphoric acid amide monomer is firstly subjected to coupling reaction, and then the protecting group is removed at the O3 'or N3' position, so that the oligonucleotide phosphate amide and (thio) phosphoric acid amide are obtained. And releasing active hydroxyl or amino / amido to enter the next cycle. The protecting group adopted by the sulfo-PMO molecule is consistent with the protecting groups of other nucleotides, so that the conversion rate of each step of coupling reaction can be conveniently detected on line.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of solid-phase synthesis of oligonucleic acids, and particularly relates to a synthesis process of oligonucleic acids with a thio-PMO structure. BACKGROUND

[0002] Phosphorodiamidate morpholino oligonucleotide (PMO) adopts a morpholine ring to replace a deoxyribose or ribose structure, and a phosphorodiamide group as a connecting unit. PMO replaces a negatively charged phosphate group with an electrically neutral morpholine phosphorodiamide amine, and shows characteristics such as high affinity and strong enzymatic stability. PMO has great potential for drug development as a structural unit of chemical modification of nucleic acid drugs.

[0003] At present, most PMO protecting groups are triphenylmethyl Tr. In the process of monomer coupling, the maximum absorption wavelength of the protecting group Tr cation removed under acidic conditions is between 400-435 nm. In the process of preparing and synthesizing nucleic acid molecules of a target sequence on a solid-phase synthesizer, other nucleotides generally use a protecting group dimethoxytrityl DMTr, and the maximum absorption wavelength of the cation is between 490-515 nm. The absorption coefficients of the two protecting groups triphenylmethyl Tr and dimethoxytrityl DMTr at a specific wavelength are different, and the use of light of a certain wavelength for colorimetric detection of the coupling efficiency of each step will produce inconsistencies.

[0004] In addition, in the traditional solid-phase synthesis of nucleotide phosphoramidite, the synthesis of oligonucleic acids is carried out in the direction of 3' to 5', and only the morpholine ring can be used to replace the deoxyribose structure in the synthesis sequence, and the coexistence of the morpholine ring and the deoxyribose structure cannot be realized.

[0005] Therefore, the present application is provided. SUMMARY

[0006] The present application aims to provide a synthesis process of oligonucleic acids with a thio-PMO structure. In the synthesis process, the synthesis of nucleotide phosphoramidite and (thio)phosphoramidite is carried out in the direction of 5' to 3' in the solid-phase synthesis of oligonucleic acids, that is, in each cycle, the O5' or O6' position of the nucleotide phosphoramidite and (thio)phosphoramidite monomer is first subjected to a coupling reaction, and then the protecting group is removed from the O3' or N3' position to release an active hydroxyl group or an amino / amine group for the next cycle. Moreover, the protecting group of the (thio)PMO monomer is consistent with the protecting group of other nucleotide monomers, which facilitates the use of colorimetric online detection of the conversion rate of each coupling reaction step.

[0007] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted: The first aspect of this invention provides a process for synthesizing oligonucleotides with a thiolated PMO structure, wherein the thiolated PMO molecular structure used in the synthesis process is shown in Formula I: …….Formula I; In Formula I, Base is a nucleotide base or its derivative, wherein the nucleotide base is adenine (A), guanine (G), cytosine (C), 5-methylcytosine (m5C), thymine (T), uracil (U), xanthine (Xan), hypoxanthine (I), pseudouracil (Ψ) or N1-methylpseudouracil (m1Ψ) or other nucleotide bases and their derivatives; R1 and R2 are respectively halogen, cyanoalkyloxy, hydroxyl, alkoxy, alkenyloxy, amino / amine, alkylamino / amine, alkenylamino / amine, cycloalkoxy, cycloalkylamino, or alkoxyalkoxy. R3 can be hydrogen, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aromatic, alkoxy, alkoxyalkyl, alkenyloxy, silyloxy, silyloxymethyleneoxy, alkoxyalkoxy, azide, cyano, amino, mercapto, alkylmercapto, selenyl, alkylselenoyl, acyloxy, ester, amide, aminoacyl, etc. R4 can be triphenylmethyl Tr, methoxytriphenylmethyl MMTr, dimethoxytriphenylmethyl DMTr, trimethoxytriphenylmethyl TMTr, allyl, allyloxycarbonyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, acyl, ester, alkoxyacyl, etc.

[0008] Preferably, the protecting group is selected from one of 4,4'-dimethoxytriphenylmethylDMTr, triphenylmethylTr, allyl, allyloxycarbonyl, tert-butyldimethylsilyl, and tert-butyldiphenylsilyl.

[0009] Preferably, the thio-PMO molecular structure includes: , , , , , , , , , , , , , .

[0010] Preferably, the thio-PMO molecular structure includes: , 、 、 、 、 、 、 、 、 、 、 、 、 .

[0011] The oligonucleic acid of the thio-PMO structure can be coupled with at least one of Formula II to Formula V to form a chimeric oligonucleic acid molecule: Formula II; In Formula II, Base is a nucleotide base or a derivative thereof, the nucleotide base being adenine (A), guanine (G), cytosine (C), 5-methylcytosine (m5C), thymine (T), uracil (U), xanthine (Xan), hypoxanthine (I), pseudouracil (Ψ), or N1-methylpseudouracil (m1Ψ), and the like, and derivatives thereof; PG is trityl Tr, methoxytrityl MMTr, dimethoxytrityl DMTr, trimethoxytrityl TMTr, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, t-butyldimethylsilyl, or t-butyldiphenylsilyl; R1 and R2 are each hydrogen, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, alkoxy, alkoxyalkyl, alkenyloxy, silyloxy methyleneoxy, alkoxyalkoxy, azido, cyano, amine, thiol, alkylthiol, seleno, alkylseleno, acyloxy, ester, amido, amidoacyl, and the like; LG1 is secondary amine, halogen; LG2 is 2-cyanoethyl, 2-cyanopropyl, cyanoisobutyl, (CH3)2CCH2CN, arylethyl, or allyl; Formula III; In Formula III, Base is a nucleotide base or a derivative thereof, the nucleotide base being adenine (A), guanine (G), cytosine (C), 5-methylcytosine (m5C), thymine (T), uracil (U), xanthine (Xan), hypoxanthine (I), pseudouracil (Ψ), or N1-methylpseudouracil (m1Ψ), and the like, and derivatives thereof; PG is triphenylmethyl Tr, methoxytriphenylmethyl MMTr, dimethoxytriphenylmethyl DMTr, trimethoxytriphenylmethyl TMTr, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, or tert-butyldiphenylsilyl; R1, R2, and R3 are each an alkyl group, an alkenyl group, a cycloalkyl group, an alkoxyalkyl group, or an aromatic group; LG1 is a secondary amine, a halogen; LG2 is a 2-cyanoethyl group, a 2-cyanopropyl group, a cyanoisobutyl group, a (CH3)2CCH2CN group, an aromatic ethyl group, or an allyl group; … Formula IV; In Formula IV, Base is a nucleotide base or a derivative thereof, the nucleotide base being adenine (A), guanine (G), cytosine (C), 5-methylcytosine (m5C), thymine (T), uracil (U), xanthine (Xan), hypoxanthine (I), pseudouracil (Ψ), or N1-methylpseudouracil (m1Ψ), and the like, and derivatives thereof; PG is triphenylmethyl Tr, methoxytriphenylmethyl MMTr, dimethoxytriphenylmethyl DMTr, trimethoxytriphenylmethyl TMTr, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, or tert-butyldiphenylsilyl; R1 and R2 are each an alkyl group, an alkenyl group, a cycloalkyl group, an alkoxyalkyl group, or an aromatic group; R3 and R4 are each hydrogen, a halogen, an alkyl group, an alkenyl group, an alkynyl group, a cycloalkyl group, a cycloalkenyl group, an aromatic group, an alkoxy group, an alkoxyalkyl group, an alkenyloxy group, a silyloxy methyleneoxy group, an alkoxyalkoxy group, an azido group, a cyano group, an amine group, a mercapto group, an alkylmercapto group, a seleno group, an alkylseleno group, an acyloxy group, an ester group, an amido group, an amidoacyl group, and the like; X is a halogen, a hydroxyl group, an alkoxy group, an alkenyloxy group, an amine group, an alkylamine group, a cycloalkoxy group, or a cycloalkylamine group; Y is oxygen O or sulfur S; … Formula V; In Formula V, Base is a nucleotide base or a derivative thereof, the nucleotide base being adenine (A), guanine (G), cytosine (C), 5-methylcytosine (m5C), thymine (T), uracil (U), xanthine (Xan), hypoxanthine (I), pseudouracil (Ψ), or N1-methylpseudouracil (m1Ψ), and the like, and derivatives thereof; PG is triphenylmethyl Tr, methoxytriphenylmethyl MMTr, dimethoxytriphenylmethyl DMTr, trimethoxytriphenylmethyl TMTr, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, or tert-butyldiphenylsilyl; R1, R2, R3, R4, and R5 are each alkyl, alkenyl, cycloalkyl, alkoxyalkyl, or aryl; X is halogen, hydroxyl, alkoxy, alkenyloxy, amino, alkylamino, cycloalkoxy, or cycloalkylamino; Y is oxygen O or sulfur S.

[0012] Preferably, the protecting group is selected from one of 4,4'-dimethoxytriphenylmethyl DMTr, triphenylmethyl Tr, allyl, allyloxycarbonyl, tert-butyldimethylsilyl, and tert-butyldiphenylsilyl.

[0013] The nucleotide phosphoramidite monomers and thiophosphoramidite monomers include: , , , , , , , , , , , , , .

[0014] The method for preparing the nucleotide phosphoramidite monomers and thiophosphoramidite monomers includes linking a pentavalent phosphorus compound at the O5' position; and linking a protecting group PG at the O3' position, the protecting group PG being triphenylmethyl Tr, methoxytriphenylmethyl MMTr, dimethoxytriphenylmethyl DMTr, trimethoxytriphenylmethyl TMTr, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, or tert-butyldiphenylsilyl; Y in the nucleotide monomer is oxygen O or sulfur S; and R is hydrogen, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, alkoxy, alkoxyalkyl, alkenyloxy, silyloxy, silyloxy methyleneoxy, alkoxyalkoxy, azido, cyano, amine, thiol, alkylthiol, seleno, alkylseleno, acyloxy, ester, amido, amidoacyl, etc.

[0015] Preferably, the nucleotide phosphoramidite and thiophosphoramidite monomers include: , , , , , 、 、 、 、 、 、 、 、 ; Y is oxygen O or sulfur S; and R is hydrogen, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, alkoxy, alkoxyalkyl, alkenyloxy, siloxy, siloxymethylenoxymethylenoxymethylenoxy, alkoxyalkoxy, azido, cyano, amine, thiol, alkylthiol, seleno, alkylseleno, acyloxy, ester, amido, amidoacyl, and the like.

[0016] Preferably, the nucleotide phosphoramidate and thiophosphoramidate monomers include: 、 、 、 、 、 、 、 、 、 、 、 、 、 ; Y is oxygen O or sulfur S; and R is hydrogen, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, alkoxy, alkoxyalkyl, alkenyloxy, siloxy, siloxymethylenoxymethylenoxymethylenoxy, alkoxyalkoxy, azido, cyano, amine, thiol, alkylthiol, seleno, alkylseleno, acyloxy, ester, amido, amidoacyl, and the like.

[0017] The second aspect of the present application provides a thio-PMO structure oligonucleotide, which comprises a structure shown in Formula VI to Formula VIII: Formula VI; Formula VII; Formula VIII; In Formula VI to Formula VIII, Base is a nucleotide base or a derivative thereof, which is adenine (A), guanine (G), cytosine (C), 5-methylcytosine (m5C), thymine (T), uracil (U), xanthine (Xan), hypoxanthine (I), pseudouracil (Ψ), or N1-methylpseudouracil (m1Ψ), and the like, and derivatives thereof. R and R' are independently hydrogen, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, alkoxy, alkoxyalkyl, alkenyloxy, silyloxy, siloxymethyloxy, alkoxyalkoxy, azido, cyano, amido, mercapto, alkylmercapto, seleno, alkylseleno, acyloxy, ester, amido, amidoacyl, etc. R1and R2are independently alkyl, alkenyl or cycloalkyl; R3and R4are independently hydrogen, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, alkoxy, alkoxyalkyl, alkenyloxy, silyloxy, siloxymethyloxy, alkoxyalkoxy, azido, cyano, amido, mercapto, alkylmercapto, seleno, alkylseleno, acyloxy, ester, amido, amidoacyl, etc.

[0018] Compared with the prior art, the present application has at least the following beneficial effects: In the present application, the synthesis of nucleotide phosphoramidite, (thio)phosphoramidite in the solid-phase synthesis of oligonucleic acid is carried out in the direction of 5' to 3', i.e. in each cycle, the O5' or O6' position of the nucleotide phosphoramidite, (thio)phosphoramidite monomer is first coupled, and then the O3' or N3' position is deprotected to release the active hydroxyl or amino / amine group for the next cycle. Moreover, the protecting group of the PMO monomer of the present application is consistent with the protecting group of other nucleotides, which facilitates the online detection of the conversion rate of each coupling reaction in the synthesis process. BRIEF DESCRIPTION OF DRAWINGS

[0020] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings required to be used in the specific embodiments or prior art description will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual proportion.

[0021] Figure 1 The flow chart of the solid-phase synthesis of oligonucleic acid in the present application. DETAILED DESCRIPTION

[0022] The technical solutions of the present application will be described in detail below with reference to the embodiments. The following embodiments are only used to more clearly illustrate the technical solutions of the present application, and therefore only serve as examples, but cannot limit the protection scope of the present application.

[0023] It should be noted that, unless otherwise specified, the technical terms or scientific terms used in the present application should be understood as the usual meanings understood by the skilled in the art to which the present application belongs.

[0024] The substances corresponding to the letter abbreviations in the present application are as follows: PMO: phosphorodiamidate morpholinooligonucleotide; DBU: 1,8-diazobispiro[5.4.0]undecyl-7-ene; CPG: controlled pore glass. (glass); PS: polystyrene; DIEA: N,N-diisopropylethylamine; DMI: 1,3-dimethyl-2-imidazolone; NMP: N-methylpyrrolidone; Ac: acetyl; Alkyl: alkyl; Allyl: allyl; Alloc: allyloxycarbonyl; Bz: benzoyl; Tr: triphenylmethyl; MMTr: methoxytriphenylmethyl; DMTr: dimethoxytriphenylmethyl; TMTr: trimethoxytriphenylmethyl; Ph: aromatic; TBDMS: tert-butyldimethylsilyl; TBDPS: tert-butyldiphenylsilyl; DCM: dichloromethane; DMF: N,N-dimethylformamide; DMAP: 4-dimethylaminopyridine; THF: tetrahydrofuran; TFA: trifluoroacetic acid; TFE: 2,2,2-trifluoroethanol; iPrOH: isopropanol; TLC: thin-layer chromatography; HPLC: high-performance liquid chromatography; A: Adenine Adenine; G: Guanine; C: Cytosine; T: Thymine; U: Uracil; I: Hypoxanthine; Ap: A PMO; Gp: G PMO; Cp: C PMO; Up: U PMO; Tp: T PMO; Ip: I PMO; O: Oxygen; S: Sulfur; Aps: Thio-A PMO; Gps: Thio-G PMO; Cps: Thio-C PMO; Ups: Thio-U PMO; Tps: Thio-T PMO; Ips: Thio-I PMO; SP NMe2 Thiophosphoryl dimethylamine.

[0025] Example 1: Synthesis of N3'-DMTr-N6-Bz-A-PMO phosphoramidochloridothioate This embodiment describes a PMO molecule for solid-phase synthesis of oligonucleic acids. The PMO molecule has the following structural formula: ; The above PMO molecule synthesis route is as follows: ; Specific synthesis methods include: Under nitrogen protection, 263 mg of N3'-DMTr-N6-Bz-A-PMO (0.4 mmol, 1.0 eq.) was added into dry mixed solvent (3 mL of dichloromethane and 3 mL of acetonitrile), and 70 mg of lithium bromide (0.8 mmol, 2.0 eq.) was added at 0°C. The mixture was stirred for 5 min. 0.12 mL of DBU (0.8 mmol, 2.0 eq.) and 0.08 mL of dimethylamino thiophosphoryl dichloride (0.6 mmol, 1.5 eq.) dissolved in 0.5 mL of dry dichloromethane were added in sequence. The mixed solution was stirred at 0°C for 40 min under nitrogen protection. The reaction solvent was removed under reduced pressure, 30 mL of ethyl acetate was added to the residue, and the organic phase was dried over sodium sulfate and concentrated under reduced pressure. The target product was obtained by silica gel chromatography separation using dichloromethane / acetone / methanol mixed solvent as the mobile phase (purity >99%, yield 63%).

[0026] Example 2: Synthesis of N3'-DMTr-U-PMO phosphoramidochloridothioate The PMO molecule of the present example is used for solid-phase synthesis of an oligonucleic acid, and the structure of the PMO molecule is The synthesis route of the PMO molecule is as follows: The specific synthesis method includes: Under nitrogen protection, 263 mg of N3'-DMTr-N6-Bz-A-PMO (0.4 mmol, 1.0 eq.) was added into dry mixed solvent (3 mL of dichloromethane and 3 mL of acetonitrile), and 70 mg of lithium bromide (0.8 mmol, 2.0 eq.) was added at 0°C. The mixture was stirred for 5 min. 0.12 mL of DBU (0.8 mmol, 2.0 eq.) and 0.08 mL of dimethylamino thiophosphoryl dichloride (0.6 mmol, 1.5 eq.) dissolved in 0.5 mL of dry dichloromethane were added in sequence. The mixed solution was stirred at 0°C for 40 min under nitrogen protection. The reaction solvent was removed under reduced pressure, 30 mL of ethyl acetate was added to the residue, and the organic phase was dried over sodium sulfate and concentrated under reduced pressure. The target product was obtained by silica gel chromatography separation using dichloromethane / acetone / methanol mixed solvent as the mobile phase (purity >99%, yield 63%).

[0027] Example 3: Synthesis of N3'-DMTr-T-PMO phosphoramidochloridothioate The PMO molecule of the present example is used for solid-phase synthesis of an oligonucleic acid, and the structure of the PMO molecule is​​ The synthesis route of the above PMO molecule is as follows: The specific synthesis method includes: Under nitrogen protection, 272 mg of N3'-DMTr-T-PMO (0.5 mmol, 1.0 eq.) was added into a dry mixed solvent (3 mL of dichloromethane and 3 mL of acetonitrile), and the temperature was controlled at 0°C. Then 88 mg of lithium bromide (1.0 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 minutes. Then 0.15 mL of DBU (1.0 mmol, 2.0 eq.) and 0.10 mL of dimethylamino thiophosphoryl dichloride (0.75 mmol, 1.5 eq.) dissolved in 0.5 mL of dry dichloromethane were added in sequence. The mixed solution was stirred at 0°C for 40 minutes under nitrogen protection. The reaction solvent was removed under reduced pressure, 30 mL of ethyl acetate was added to the residue, and after washing with 15 mL of cold water, the organic phase was dried over sodium sulfate and concentrated under reduced pressure. Silica gel chromatography was used for separation, and the mobile phase was selected as a mixture of dichloromethane / acetone / methanol to obtain 218 mg of the target product (purity >99%, yield 64%).

[0028] Further, the above-mentioned synthesis method was used to prepare the following PMO monomers:

[0029] Example 4: Synthesis of N3'-Tr-U-PMO phosphoramidochloridothioate This example is a PMO molecule for solid-phase synthesis of oligonucleic acids, and the structural formula of the PMO molecule is as follows: The synthesis route of the above PMO molecule is as follows: The specific synthesis method includes: ​​​​​​​​​​​​​​​Under nitrogen protection, 235 mg of N3'-Tr-U-PMO (0.5 mmol, 1.0 eq.) was added into dry mixed solvent (3 mL of dichloromethane and 3 mL of acetonitrile), temperature was controlled at 0°C, 88 mg of lithium bromide (1.0 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 minutes. 0.15 mL of DBU (1.0 mmol, 2.0 eq.) and 0.10 mL of dimethylamino thiophosphoryl dichloride (0.75 mmol, 1.5 eq.) dissolved in 0.5 mL of dry dichloromethane were added in sequence. The mixed solution was stirred at 0°C for 40 minutes under nitrogen protection. The reaction solvent was removed under reduced pressure, 30 mL of ethyl acetate was added into the residue, and after washing with 15 mL of cold water, the organic phase was dried over sodium sulfate and concentrated under reduced pressure. The target product was obtained by silica gel chromatography separation using dichloromethane / acetone / methanol mixed solvent as the mobile phase (purity >99%, yield 66%).

[0030] Example 5: Synthesis of N3'-Tr-T-PMO phosphoramidochloridothioate This example is a PMO molecule for solid-phase synthesis of an oligonucleic acid, and the structure of the PMO molecule is ; The synthesis route of the above PMO molecule is as follows: ; The specific synthesis method includes: Under nitrogen protection, 242 mg of N3'-Tr-T-PMO (0.5 mmol, 1.0 eq.) was added into dry mixed solvent (3 mL of dichloromethane and 3 mL of acetonitrile), temperature was controlled at 0°C, 88 mg of lithium bromide (1.0 mmol, 2.0 eq.) was added, and the mixture was stirred for 5 minutes. 0.15 mL of DBU (1.0 mmol, 2.0 eq.) and 0.10 mL of dimethylamino thiophosphoryl dichloride (0.75 mmol, 1.5 eq.) dissolved in 0.5 mL of dry dichloromethane were added in sequence. The mixed solution was stirred at 0°C for 40 minutes under nitrogen protection. The reaction solvent was removed under reduced pressure, 30 mL of ethyl acetate was added into the residue, and after washing with 15 mL of cold water, the organic phase was dried over sodium sulfate and concentrated under reduced pressure. The target product was obtained by silica gel chromatography separation using dichloromethane / acetone / methanol mixed solvent as the mobile phase (purity >99%, yield 66%).

[0031] Further, the above-described synthesis method was used to prepare the following PMO monomers: 、 、 、 、 , 、 、 、 、 、 、 .

[0032] Further, the following PMO monomers are prepared by the above described synthetic method: ; wherein Base is a nucleotide base or derivative thereof, the nucleotide base is adenine (A), guanine (G), cytosine (C), 5-methylcytosine (m5C), thymine (T), uracil (U), xanthine (Xan), hypoxanthine (I), pseudouracil (Ψ), or N1-methylpseudouracil (m1Ψ), and derivatives thereof; R1and R2are independently halogen, cyanoalkyloxy, hydroxy, alkoxy, alkenyloxy, amino / amine, alkylamino, alkenylamino, cycloalkyloxy, cycloalkylamino, or alkoxyalkyloxy; R3is hydrogen, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, alkoxy, alkoxyalkyl, alkenyloxy, silyloxy, silyloxymethylenoxyl, alkoxyalkyloxy, azido, cyano, amine, thiol, alkylthiol, seleno, alkylseleno, acyloxy, ester, amido, amidoacyl, and the like; R4is trityl Tr, methoxytrityl MMTr, dimethoxytrityl DMTr, trimethoxytrityl TMTr.

[0033] Example 6: Synthesis of O3'-DMTr-T phosphoramidochloridothioate This example is a nucleotide monomer for solid phase synthesis of oligonucleic acid, the structural formula of the nucleotide monomer is ; The synthetic route of the above nucleotide monomer is: ; The specific synthesis method includes: Under nitrogen protection, 280 mg of O2'-Me-O3'-DMTr-U (0.5 mmol, 1.0 eq.) was added into dry mixed solvent (3 mL of dichloromethane and 3 mL of acetonitrile), and 88 mg of lithium bromide (1.0 mmol, 2.0 eq.) was added at 0°C. After stirring for 5 min, 0.15 mL of DBU (1.0 mmol, 2.0 eq.) and 0.10 mL of dimethylamino thiophosphoryl dichloride (0.75 mmol, 1.5 eq.) dissolved in 0.5 mL of dry dichloromethane were added in sequence. The mixed solution was stirred at 0°C for 40 min under nitrogen protection. The solvent was removed under reduced pressure, 30 mL of ethyl acetate was added to the residue, and after washing with 15 mL of cold water, the organic phase was dried over sodium sulfate and concentrated under reduced pressure. The target product was separated by silica gel chromatography using a dichloromethane / acetone / methanol mixed solvent as the mobile phase, and 223 mg of the target product was obtained (purity >99%, yield 67%).

[0034] Example 7: Synthesis of O2'-Me-O3'-DMTr-U phosphoramidochloridothioate This example is a nucleotide monomer for solid-phase synthesis of oligonucleic acids, and the structure of the nucleotide monomer is ; The synthesis route of the above nucleotide monomer is as follows: ; The specific synthesis method includes: Under nitrogen protection, 280 mg of O2'-Me-O3'-DMTr-U (0.5 mmol, 1.0 eq.) was added into dry mixed solvent (3 mL of dichloromethane and 3 mL of acetonitrile), and 88 mg of lithium bromide (1.0 mmol, 2.0 eq.) was added at 0°C. After stirring for 5 min, 0.15 mL of DBU (1.0 mmol, 2.0 eq.) and 0.10 mL of dimethylamino thiophosphoryl dichloride (0.75 mmol, 1.5 eq.) dissolved in 0.5 mL of dry dichloromethane were added in sequence. The mixed solution was stirred at 0°C for 40 min under nitrogen protection. The solvent was removed under reduced pressure, 30 mL of ethyl acetate was added to the residue, and after washing with 15 mL of cold water, the organic phase was dried over sodium sulfate and concentrated under reduced pressure. The target product was separated by silica gel chromatography using a dichloromethane / acetone / methanol mixed solvent as the mobile phase, and 223 mg of the target product was obtained (purity >99%, yield 67%).

[0035] Further, the following nucleotide monomers were prepared by using the above-mentioned similar preparation method: 、 、 、 、 、 、 、 、 、 、 、 、 、 ; R is hydrogen, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, alkoxy, alkoxyalkyl, alkenyloxy, silyloxy, siloxymethylenoxymethylenoxymethylenoxy, alkoxyalkoxy, azido, cyano, amine, thiol, alkylthiol, seleno, alkylseleno, acyloxy, ester, amido, amidoacyl, etc.

[0036] Further, the above-mentioned similar preparation method is used to prepare the following nucleotide monomers: 、 、 、 、 、 、 、 、 、 、 、 、 、 ; R is hydrogen, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, alkoxy, alkoxyalkyl, alkenyloxy, silyloxy, siloxymethylenoxymethylenoxymethylenoxy, alkoxyalkoxy, azido, cyano, amine, thiol, alkylthiol, seleno, alkylseleno, acyloxy, ester, amido, amidoacyl, etc.

[0037] The synthesis process of the oligonucleic acid with a thio PMO structure provided in the application, the solution system used by the PMO in the process refers to the literature "Journal of Pharmacy of the Chinese People's Liberation Army", Vol. 35, No. 6, 493-496, 2022.

[0038] The carrier for solid-phase synthesis is cross-linked polystyrene PS; 0.050 grams of modified solid-phase synthesis resin is weighed and added to a solid-phase synthesis tube; after 2.0 milliliters of NMP is added, swelling is carried out at room temperature for 2 hours, and then the solution is pumped and filtered to empty the solution. 3.0 milliliters of dichloromethane is used to wash the resin, and the washing process is repeated 3 times to start solid-phase synthesis.

[0039] The process route for solid-phase synthesis, please refer to the attached Figure 1 .

[0040] The monomers introduced in the solid-phase synthesis are phosphoramidites P(III) and phosphoramidites P(V).

[0041] The process conditions for phosphorus amide monomers are as follows: Reagents and monomers: Prepare a 0.15 mol / L monomer / acetonitrile solution; the reagent for removing the DMTr (or Tr) protecting group is a 3% dichloroacetic acid / toluene solution (v / v); the activator is a 0.25 mol / L 5-ethylthiotetrazole / acetonitrile solution; the oxidizing agent is a 0.05 mol / L iodine solution of water / pyridine (10 / 90, v / v); the sulfiding agent is a 3% hydroflavin / pyridine solution; capping agent A is a 10% acetic anhydride / acetonitrile solution (v / v), and capping agent B is a 1-methylimidazolium / pyridine / tetrahydrofuran solution (16 / 10 / 74, v / v / v).

[0042] The operational sequence for the solid-phase synthesis of phosphorous amide monomers is as follows: 1) Deprotection: 3% dichloroacetic acid toluene solution was used to remove the DMTr protecting group, followed by washing with acetonitrile.

[0043] 2) Coupling: Using 0.25 mol / L 5-ethylthiotetrazole as the activator, according to the sequence design, nucleotide monomers / acetonitrile solutions were fed into the cycle for coupling, followed by rinsing with acetonitrile.

[0044] 3) Oxidation / Sulfidation: P(III) was oxidized using a 0.05 mol / L iodine solution in water / pyridine (v / v, 90 / 10) as the oxidant, followed by rinsing with acetonitrile. P(III) was sulfided using a 3% hydroflavin pyridine solution as the sulfiding agent, followed by rinsing with acetonitrile.

[0045] 4) Capping: Capping agent A and capping agent B are used as capping reagents to protect the unreacted active groups, followed by rinsing with acetonitrile.

[0046] The process conditions for using phosphoramide monomers in the solid-phase synthesis of oligonucleotides are as follows: DMTr removal solution: 5.355 g of 4-cyanopyridine was added to 100.0 mL of dichloromethane, and then 4.900 g of trifluoroacetic acid was added while stirring at room temperature. After stirring for 1 hour, 4.5 mL of ethanol and 100 mL of trifluoroethanol were added, and the volume was adjusted to 500 mL with dichloromethane to prepare a trifluoroethanol / dichloromethane solution of 4-cyanopyridine trifluoroacetate (2%) and ethanol (0.9%), which was used as the DMTr removal solution.

[0047] Neutralization solution: Prepare a diisopropylethylamine (5%) isopropanol / dichloromethane solution by diluting 25.0 mL of diisopropylethylamine and 125.0 mL of isopropanol to 500 mL with dichloromethane.

[0048] Coupling solvent: 2.303 g of N-ethylmorpholine was dissolved in 1,3-dimethyl-2-imidazolidinone (DMI) to make 50 mL of N-ethylmorpholine DMI solution (0.40 mol / L) as the coupling solvent.

[0049] Lysis solution: 0.154 g of dithiothreitol was dissolved in 5 mL of N-methyl pyrrolidone (NMP), 0.739 g of triethylamine was added, and the solution was diluted with NMP to 10 mL to obtain an NMP solution containing 0.10 mol / L of dithiothreitol and 0.73 mol / L of triethylamine, which was prepared immediately before use.

[0050] The process operation of solid-phase synthesis of (thio)phosphoramidite monomers includes: According to the base sequence of the target sequence, the nucleotide monomers were coupled one by one through the cycle operation process of DMTr removal, neutralization, washing, coupling, neutralization, and washing. After the coupling of the last monomer molecule was completed, the terminal DMTr was removed, and 3.0 mL of p-methoxytrityl chloride capping solution (0.32 mol / L) was added to the reaction tube. After reaction at 45°C for 90 minutes, the reaction solution was emptied, and NMP was used for washing 8 times, each time 4.0 mL.

[0051] The above operation was repeated according to the set sequence to obtain the oligonucleotide product of the target sequence.

[0052] Deprotection is to transfer the solid-phase carrier loaded with the oligonucleotide product to a reactor, add concentrated ammonia water (28%), control the temperature at 60°C for 12 hours of ammonolysis, then cool to room temperature, filter, and rinse the solid with a mixture of purified water and ethanol. The combined filtrate was concentrated under reduced pressure at low temperature to obtain the crude oligonucleotide of the designed sequence.

[0053] TBDMS or other silyl ether protecting group removal: TBAF / THF solution was added to the oligonucleotide product containing silyl ether protecting group, and the solution was treated at room temperature for 2-6 hours.

[0054] Purification: The crude product after deprotection was dissolved in purified water, and HPLC purification was performed. The product peak solution was collected, and the product peak solution was concentrated under reduced pressure and freeze-dried to obtain the product.

[0055] The above solid-phase synthesis is also applicable to Tr, MMTr, and TMTr-protected monomers.

[0056] In the example of solid-phase synthesis, the PMO monomers and other nucleotide monomers used are represented by the following structures: , , 、 、 、 、 .

[0057] Example 8

[0058] Monomer Up-0 was loaded on cross-linked polystyrene PS, and N3'-DMTr was removed by trifluoroacetic acid solution. After washing, monomer A-3 was pumped in for coupling. The loaded nucleic acid molecule was treated by ammonolysis and deprotection. The crude product was concentrated under reduced pressure and purified by HPLC. The main fraction was collected, concentrated and lyophilized to obtain the product. The product was confirmed as 6'-Up-SP of the target sequence by detection and analysis. NMe2 A-3' nucleic acid molecule.

[0059] Example 9

[0060] Monomer U-3 was loaded on cross-linked polystyrene PS, and the protecting group DMTr was removed by dichloroacetic acid solution. After washing, monomer Aps-1 was pumped in for coupling. The loaded oligonucleic acid molecule was treated by ammonolysis and deprotection. The crude product was concentrated under reduced pressure and purified by HPLC. The main fraction was collected, concentrated and lyophilized to obtain the product. The product was confirmed as 5'-U-SP of the target sequence by detection and analysis. NMe2 Ap-3' nucleic acid molecule.

[0061] Example 10

[0062] Monomer Up-0 was loaded on cross-linked polystyrene PS, and N3'-DMTr was removed by trifluoroacetic acid solution. After washing, monomer Ups-1 was pumped in for coupling. The loaded nucleic acid molecule was treated by ammonolysis and deprotection. The crude product was concentrated under reduced pressure and purified by HPLC. The main fraction was collected, concentrated and lyophilized to obtain the product. The product was confirmed as 6'-Up-SP of the target sequence by detection and analysis. NMe2 Up-3' nucleic acid molecule.

[0063] Example 11

[0064] The monomer Up-0 is loaded on cross-linked polystyrene PS, and N3'-DMTr is removed by trifluoroacetic acid solution. After washing, the monomer Up-2 is pumped in for coupling. N3'-DMTr is removed by trifluoroacetic acid solution. After washing, the monomer Ups-1 is pumped in for coupling. N3'-DMTr is removed by trifluoroacetic acid solution. After washing, the monomer A-4 is pumped in for coupling. After the loaded nucleic acid molecule is subjected to ammonolysis and deprotection, the crude product concentrated under reduced pressure is separated and purified by HPLC. The main fraction is collected, concentrated and lyophilized to obtain the product. After detection and analysis, it is confirmed that the product is the 6'-Up-P of the target sequence. NMe2 -Up-SP NMe2 -Up-P NMe2 -A-3' nucleic acid molecule.

[0065] Finally, it should be noted that: the above examples are only used to illustrate the technical solutions of the present application, and not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that: it can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacement for part or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present application, and they should be covered in the scope of the claims and the specification of the present application.

Claims

1. A process for synthesizing oligonucleotides with a thioprenocorticoid PMO structure, characterized in that, The PMO molecular structure used in the oligonucleotide synthesis process of the thio-PMO structure is shown in Formula I: ...Equation I; In Formula I, Base is a nucleotide base or its derivative, wherein the nucleotide base is adenine (A), guanine (G), cytosine (C), 5-methylcytosine (m5C), thymine (T), uracil (U), xanthine (Xan), hypoxanthine (I), pseudouracil (Ψ) or N1-methylpseudouracil (m1Ψ) or other nucleotide bases and their derivatives; R1 and R2 are respectively halogen, cyanoalkyloxy, hydroxyl, alkoxy, alkenyloxy, amino / amine, alkylamino / amine, alkenylamino / amine, cycloalkoxy, cycloalkylamino, or alkoxyalkoxy. R3 can be hydrogen, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aromatic, alkoxy, alkoxyalkyl, alkenyloxy, silyloxy, silyloxymethyleneoxy, alkoxyalkoxy, azide, cyano, amino, mercapto, alkylmercapto, selenyl, alkylselenoyl, acyloxy, ester, amide, aminoacyl, etc. R4 can be triphenylmethyl Tr, (4-methoxyphenyl)diphenylmethyl MMTr, 4,4'-dimethoxytriphenylmethyl DMTr, 4,4',4''-trimethoxytriphenylmethyl TMTr, allyl, allyloxycarbonyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, acyl, ester, alkoxyacyl, etc.

2. The oligonucleotide synthesis process with a thio-PMO structure according to claim 1, characterized in that, The protecting group is selected from one of 4,4'-dimethoxytriphenylmethylDMTr, triphenylmethylTr, allyl, allyloxycarbonyl, tert-butyldimethylsilyl, and tert-butyldiphenylsilyl.

3. The oligonucleotide synthesis process with a thio-PMO structure according to claim 1, characterized in that, The thiolated PMO molecular structure includes, but is not limited to, , , , , , , , , , , , , , .

4. The oligonucleotide synthesis process of the thio-PMO structure according to claim 1, characterized in that, The thiolated PMO molecular structure includes, but is not limited to, , , , , , , , , , , , , , .

5. The oligonucleotide synthesis process with a thioprenocorticoid PMO structure according to claim 1, characterized in that, The oligonucleotide synthesis process of the thio-PMO structure can incorporate other types of nucleotide monomers, including at least one of Formulas II to V: ...Formula II; In Formula II, Base is a nucleotide base or its derivative, wherein the nucleotide base is adenine (A), guanine (G), cytosine (C), 5-methylcytosine (m5C), thymine (T), uracil (U), xanthine (Xan), hypoxanthine (I), pseudouracil (Ψ) or N1-methylpseudouracil (m1Ψ) or other nucleotide bases and their derivatives; PG is triphenylmethyl Tr, methoxytriphenylmethyl MMTr, dimethoxytriphenylmethyl DMTr, trimethoxytriphenylmethyl TMTr, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl; R1 and R2 are hydrogen, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aromatic, alkoxy, alkoxyalkyl, alkenyloxy, silyloxymethyleneoxy, alkoxyalkoxy, azide, cyano, amino, mercapto, alkylmercapto, selenyl, alkylselenoyl, acyloxy, ester, amide, aminoacyl, etc. LG1 is a secondary amine and halogen; LG2 is 2-cyanoethyl, 2-cyanopropyl, cyanoisobutyl, (CH3)2CCH2CN, aromatic ethyl or allyl; ...Formula III; In Formula III, Base is a nucleotide base or its derivative, wherein the nucleotide base is adenine (A), guanine (G), cytosine (C), 5-methylcytosine (m5C), thymine (T), uracil (U), xanthine (Xan), hypoxanthine (I), pseudouracil (Ψ) or N1-methylpseudouracil (m1Ψ) and its derivatives. PG is triphenylmethyl Tr, methoxytriphenylmethyl MMTr, dimethoxytriphenylmethyl DMTr, trimethoxytriphenylmethyl TMTr, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl; R1, R2 and R3 are alkyl, alkenyl, cycloalkyl, alkoxyalkyl or aromatic groups, respectively; LG1 is a secondary amine and halogen; LG2 is 2-cyanoethyl, 2-cyanopropyl, cyanoisobutyl, (CH3)2CCH2CN, aromatic ethyl or allyl; ...Form IV; In Formula IV, Base is a nucleotide base or its derivative, wherein the nucleotide base is adenine (A), guanine (G), cytosine (C), 5-methylcytosine (m5C), thymine (T), uracil (U), xanthine (Xan), hypoxanthine (I), pseudouracil (Ψ) or N1-methylpseudouracil (m1Ψ) or other nucleotide bases and their derivatives; PG is triphenylmethyl Tr, methoxytriphenylmethyl MMTr, dimethoxytriphenylmethyl DMTr, trimethoxytriphenylmethyl TMTr, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl; R1 and R2 are alkyl, alkenyl, cycloalkyl, alkoxyalkyl, or aromatic groups, respectively. R3 and R4 are respectively hydrogen, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aromatic, alkoxy, alkoxyalkyl, alkenyloxy, silyloxymethyleneoxy, alkoxyalkoxy, azide, cyano, amino, mercapto, alkylmercapto, selenyl, alkylselenoyl, acyloxy, ester, amide, aminoacyl, etc. X is a halogen, hydroxyl, alkoxy, alkenyloxy, amino, alkylamino, cycloalkoxy, or cycloalkylamino. Y represents oxygen (O) and sulfur (S); ...Form V; In Formula V, Base is a nucleotide base or its derivative, wherein the nucleotide base is adenine (A), guanine (G), cytosine (C), 5-methylcytosine (m5C), thymine (T), uracil (U), xanthine (Xan), hypoxanthine (I), pseudouracil (Ψ) or N1-methylpseudouracil (m1Ψ) and its derivatives. PG is triphenylmethyl Tr, methoxytriphenylmethyl MMTr, dimethoxytriphenylmethyl DMTr, trimethoxytriphenylmethyl TMTr, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl or tert-butyldiphenylsilyl; R1, R2, R3, R4 and R5 are alkyl, alkenyl, cycloalkyl, alkoxyalkyl or aromatic groups, respectively; X is a halogen, hydroxyl, alkoxy, alkenyloxy, amino, alkylamino, cycloalkoxy, or cycloalkylamino. Y represents oxygen (O) and sulfur (S).

6. The oligonucleotide synthesis process with a thioprenocorticoid PMO structure according to claim 5, characterized in that, The protecting group is selected from one of 4,4'-dimethoxytriphenylmethylDMTr, triphenylmethylTr, allyl, allyloxycarbonyl, tert-butyldimethylsilyl, and tert-butyldiphenylsilyl.

7. The oligonucleotide synthesis process for the thio-PMO structure according to claim 5, wherein the oligonucleotide synthesis process for the thio-PMO structure can incorporate other types of nucleotide monomers, characterized in that, The nucleotide monomers include, but are not limited to, 、 、 、 、 、 、 、 、 、 、 、 、 、 ； In the nucleotide monomers, PG is triphenylmethyl Tr, methoxytriphenylmethyl MMTr, dimethoxytriphenylmethyl DMTr, trimethoxytriphenylmethyl TMTr, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, or tert-butyldiphenylsilyl; R is hydrogen, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aromatic, alkoxy, alkoxyalkyl, alkenyloxy, silyloxy, silyloxymethyleneoxy, alkoxyalkoxy, azide, cyano, amino, mercapto, alkylmercapto, selenyl, alkylselenoyl, acyloxy, ester, amide, aminoacyl, etc.

8. The oligonucleotide synthesis process for the thio-PMO structure according to claim 5 is capable of incorporating other types of nucleotide monomers, characterized in that... The nucleotide monomers include, but are not limited to, 、 、 、 、 、 、 、 、 、 、 、 、 、 ； The preparation method includes linking a pentavalent (thio)phosphorus compound at the O5' position; and linking a protecting group PG at the O3' position, wherein the protecting group PG is triphenylmethyl Tr, methoxytriphenylmethyl MMTr, dimethoxytriphenylmethyl DMTr, trimethoxytriphenylmethyl TMTr, allyl, allyloxycarbonyl, trimethylsilyl, triethylsilyl, triisopropylsilyl, tert-butyldimethylsilyl, or tert-butyldiphenylsilyl; wherein R in the nucleotide monomer is hydrogen, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aromatic, alkoxy, alkoxyalkyl, alkenyloxy, silyloxymethyleneoxy, alkoxyalkoxy, azide, cyano, amino, mercapto, alkylmercapto, selenyl, alkylselenoyl, acyloxy, ester, amide, aminoacyl, etc.; and Y is oxygen (O) or sulfur (S).

9. The oligonucleotide synthesis process of the thio-PMO structure according to claim 1, characterized in that, The (thio)PMO monomer can be intercalated with other nucleotide phosphorylated amides and (thio)phosphoramides using the same protecting group, which facilitates online detection of the coupling efficiency of each cycle of solid-phase synthesis. The protecting group is selected from one of 4,4'-dimethoxytriphenylmethylDMTr, triphenylmethylTr, allyl, allyloxycarbonyl, tert-butyldimethylsilyl, and tert-butyldiphenylsilyl.

10. A process for synthesizing oligonucleotides with a thioprenocorticoid PMO structure, characterized in that, The oligonucleic acid with the thio-PMO structure includes the structures shown in Formulas VI to VIII. ……Formula VI; ...Equation VII; ...Form VIII; In Formulas VI to VIII, Base is a nucleotide base or its derivative, wherein the nucleotide base is adenine (A), guanine (G), cytosine (C), 5-methylcytosine (m5C), thymine (T), uracil (U), xanthine (Xan), hypoxanthine (I), pseudouracil (Ψ), or N1-methylpseudouracil (m1Ψ) and its derivatives. R and R´ represent hydrogen, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aromatic, alkoxy, alkoxyalkyl, alkenyloxy, silyloxy, silyloxymethyleneoxy, alkoxyalkoxy, azide, cyano, amino, mercapto, alkylmercapto, selenyl, alkylselenoyl, acyloxy, ester, amide, aminoacyl, etc. R1 and R2 are alkyl, alkenyl, or cycloalkyl, respectively; R3 and R4 are hydrogen, halogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aromatic, alkoxy, alkoxyalkyl, alkenyloxy, silyloxy, silyloxymethyleneoxy, alkoxyalkoxy, azide, cyano, amino, mercapto, alkyl mercapto, selenyl, alkylselenoyl, acyloxy, ester, amide, aminoacyl, etc.

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