FIELD OF THE INVENTION

The present disclosure relates to compounds and methods for purification of biopolymers, such as RNA and DNA.

BACKGROUND

Solid-phase synthesis is an invaluable tool which can be used to prepare custom oligonucleotide sequences (e.g., custom RNA and DNA), custom peptides, custom oligosaccharides, and various metabolites. The chemical synthesis of oligonucleotides generally occurs in a sequential fashion in which one end of the growing chain is attached to a solid surface, and reactive nucleotide monomers are condensed sequentially using a repetitive synthesis cycle. These nucleotide monomers contain a reactive phosphorus group such as phosphoramidite, H-phosphonate, or other reactive phosphorus or modified groups well known in the art, and the building blocks are derived from protected, 2′-deoxynucleosides (dA, dC, dG, and T), protected ribonucleosides (A, C, G, and U), or protected chemically modified nucleosides. The typical desired oligonucleotide product contains an internucleotide bond between the 5′-hydroxyl and 3′-hydroxyl of adjacent nucleotides. However, it may also be desirable to form unnatural internucleotide linkages between any of the hydroxyl groups on adjacent nucleotides. To accomplish the synthesis, the first step of the synthesis cycle is generally the reaction of a protected monomer with a nucleoside attached to the surface or reaction of a protected monomer with a hydroxyl group on the surface. The hydroxyl group on the surface can either be part of a cleavable universal linker or non-cleavable surface attachment. After initial coupling of the reactive phosphorus group with a hydroxyl on the surface, the steps that follow typically include capping of unreacted hydroxyl groups and then oxidation of the reactive phosphorus intermediate. Under certain conditions where certain modified phosphorus groups are being used, it may be necessary to oxidize prior to capping, especially in the case where the oxidation reagent produces a modified phosphorus group such as a phosphorothioate, boranophosphonate, or phosphoramidate. The final step is usually the deblocking of the hydroxyl group that will couple to the next protected nucleotide monomer. The protecting group removed in this final step is generally a dimethoxytrityl group (DMT). However, many other protective groups are well known in the art such as, for example, the 9-phenylxanthyl group (Pixyl), benzhydryloxy-bis(trimethylsilyloxy)silyl (BZH). Upon the completion of the chain assembly, the desired product is released from the solid phase, deprotected, and utilized in further biological applications.

The oligonucleotide product should be free of shorter, truncated sequences and of sequences having a different sequence than the desired product since the presence of side-products may cause undesired effects when used in in vitro or in vivo experiments. A desirable oligonucleotide synthesis would provide the chosen oligonucleotide in quantitative yield, such that the completed synthetic composition contains only the target oligonucleotide product. However, chemical methods generally provide less than quantitative yield and are usually in the range of 95% to 99% yield. The crude reaction mixture often comprises a mixture of desired oligonucleotide and truncated or failure sequences and requires purification to obtain a clean oligonucleotide target product.

The most common method of purifying the desired oligonucleotide product out of the crude synthesis products is high performance liquid chromatography (HPLC). However, isolation of the target full-length oligonucleotide from shorter oligonucleotide sequences often requires chromatographic conditions that are laborious and time consuming. For longer oligonucleotides such as oligonucleotides having 100 or more nucleotides, HPLC elution gradients can take two to three hours for each run, and the process requires precise collection and fraction analysis to obtain acceptable levels of purity. Moreover, such chromatographic methods often require stationary phases having very small particle sizes and abundant theoretical plates.

In standard oligonucleotide synthesis methods, the failure sequences are capped with an acetyl group or other acid-stable reactive functional groups and the full-length oligonucleotide comprises a dimethoxytrityl group, 9-phenylxanthyl group (Pixyl), benzhydryloxy-bis(trimethylsilyloxy)silyl (BZH) or other well-known groups at the 5′-hydroxyl position. Researchers have proposed that the full-length oligonucleotide product may be distinguished from the failure sequences by the chemical group on the 5′-hydroxyl of the oligonucleotide molecule at the completion of the synthesis cycle. Since groups like the dimethoxytrityl group are significantly more hydrophobic than the acetyl group, this difference can theoretically be used to separate the full-length oligonucleotide products from the capped failure products using a chromatographic method such as reverse-phase HPLC (J. Chrom. 326, 293-299; 1985). However, the use of DMT for HPLC purification has several drawbacks. While the DMT group is easily removed while the nucleotide is on a solid support due to favorable equilibrium conditions, the DMT group is significantly harder to remove after removal of the oligonucleotide from the solid support and requires strong acid conditions and long reaction times. Unfortunately, the conditions required to remove the DMT group in solution can degrade the purified oligonucleotide ultimately resulting in a significant loss of purity. For DNA, such conditions are especially harsh on purine bases, resulting in cleavage of the glycosidic bond. For RNA, exposure to acid is even more problematic since such exposure can directly cleave the internucleotide bond and catalyze isomerization of the internucleotide bond resulting in a mixture of 5′-3′ and 5′-2′ linked RNA products.

Accordingly, there is a need for methods and compounds for mild and effective purification of oligonucleotides to produce oligonucleotides of high purity. The present disclosure provides methods and compounds for purifying oligonucleotides using an affinity tag which is cleavable under mild conditions and has little or no overall effect on the purity or integrity of the oligonucleotide product. These compounds can also be reacted with nascently synthesized biopolymers and biologically active metabolites which contain an accessible hydroxyl group. The affinity tag can be used to purify the biopolymer or metabolite.

SUMMARY

These and other features and advantages of the present methods and compounds will be apparent from the following detailed description, in conjunction with the appended claims.

In one aspect, this disclosure provides a method of purifying a target oligonucleotide. The method comprises the following steps:1. synthesizing the target oligonucleotide on a solid support and obtaining a mixture comprising the target oligonucleotide and truncated oligonucleotides;2. reacting the target oligonucleotide with an orthoester linker, and thereby forming an oligonucleotide-orthoester linker conjugate, wherein the orthoester linker either comprises an affinity tag at the time of conjugation or the affinity tag is reacted with the oligonucleotide-orthoester linker conjugate in a second reaction after the conjugation reaction;3. cleaving the oligonucleotide-orthoester linker conjugate and the truncated oligonucleotides from the solid support;4. loading the oligonucleotide-orthoester linker conjugate and the truncated oligonucleotides onto a chromatographic column or an affinity capture support and binding the oligonucleotide-orthoester linker conjugate by the affinity tag to the affinity capture support;5. washing off the truncated oligonucleotides form the column or the affinity capture support; and6. cleaving the orthoester linker from the target oligonucleotide and eluting the target oligonucleotide from the chromatographic column or the affinity capture support, and thereby releasing the purified target oligonucleotide.

In the method, the affinity tag may be one or more groups in the orthoester linker or the affinity tag is linked with a linker to the orthoester linker.

In any of these methods, the second reaction is conducted before or after the oligonucleotide-orthoester linker conjugate is cleaved from the solid support.

In any of these methods, the orthoester linker may be attached at the 5′-hydroxyl of the target oligonucleotide or the orthoester linker is attached at the 3′-hydroxyl of the target oligonucleotide.

In any of the methods provided in this disclosure, the affinity tag may comprise one or more of the following: a fluorous tag, a hydrophobic tag, a biotin tag, a glutathione tag, a maltose tag, an arylboronic acid tag, a poly-histidine peptide tag, a poly-sulfhydryl tag, a cyclodextrin tag, an adamantane tag, a polyamine tag, a maleimide tag, an alkyne tag, an azido tag, a hydrazide tag, an amino tag, a diol tag, a thiol tag, or any combination thereof.

The present methods can be performed with the target oligonucleotide comprising an oligoribonucleotide (RNA). This RNA may comprise a 2′-hydroxyl protecting group selected from a thionocarbamate (TC) protecting group, bis(2-acetoxyethoxy)methyl (ACE) protecting group, t-butyldimethylsilyl (TBDMS) protecting group, triisopropylsilyloxymethyl (TOM) protecting group, pivaloyloxymethyl (PivOM) protecting group and 2-cyanoethoxymethyl (CEM) protecting group. The RNA may further comprise a phosphorous protecting group, a nucleobase protecting group, or a combination thereof. In the RNA, the phosphorous protecting group may be deprotected before reacting the oligonucleotide with the orthoester linker. In the RNA, the nucleobase protecting group and optionally the phosphorous protecting group may be deprotected after reacting the target oligonucleotide with the orthoester linker. In these methods, the cleavage of the oligonucleotide-orthoester linker conjugate from the solid support and the deprotection of the nucleobase protecting group and optionally of the phosphorus protecting group may be performed in a single reaction. The RNA may comprise at least 70 nucleotides.

In any of the purification methods provided in this disclosure, the orthoester linker may be a compound of formula (Ia):



wherein each of R, R′, R″, and R″′ is independently a C1-C24alkyl, a C2-C24alkenyl, a C2-C24alkynyl, a halosubstituted C1-C24alkyl, a halosubstituted C2-C24alkenyl, a halosubstituted C2-C24alkynyl, a carbocyclyl, a heteroalkyl, an aryl, a heteroaryl, a heterocycle or any substituted equivalents, with a proviso that R may be a hydrogen.

In the compound of formula (Ia), R may be H or C1-C3alkyl and R′, R″, R″′ are each independently may be an aryl or a substituted aryl. In the compound of formula (Ia), R may be wherein R is H or CH3and R′, R″, R″′ may be phenyl or a substituted phenyl. In the compound of formula (Ia), at least one of R, R′, R″, and R″′ may be a hydrophobic tag, partial hydrophobic tag, fluorous tag, or partial fluorous tag.

In any of the purification methods provided in this disclosure, the orthoester linker may be a compound of formula (Ib):



wherein AfTg is an affinity tag and R, R′, R″ are each independently AfTg-L or a C1-C24alkyl, a C2-C24alkenyl, a C2-C24alkynyl, a halosubstituted C1-C24alkyl, a halosubstituted C2-C24alkenyl, a halosubstituted C2-C24alkynyl, a carbocyclyl, a heteroalkyl, an aryl, a heteroaryl, a heterocycle, any substituted equivalents or a combination thereof provided that the total number of carbons doesn't exceed 24; and

wherein L is a covalent bond or a straight, branched, mono- or poly-cyclic, saturated, partially unsaturated or unsaturated C1-C12hydrocarbon chain that is optionally substituted with F, Cl, Br, I or C1-C3alkyls and optionally interspersed with heteroatoms independently selected from: O, S, N, or with groups independently selected from S—S, NRa, NRa—CO, —CO—NRa—, NRa—CO—NRb, CO wherein Ra, Rbare each independently H or C1-C6alkyl.

In any of the purification methods provided in this disclosure, the orthoester linker may be a compound of formula (I)



wherein each of R1, R2, R3, R4, R5, R6and R7is independently H, C1-C24alkyl, C1-C24heteroalkyl, C2-C24alkenyl, C2-C24heteroalkenyl, C2-C24alkynyl, C2-C24heteroalkynyl, halogen, aryl, heteroaryl, heterocyclyl, carbocyclyl, or any substituted equivalents or combination thereof provided that the total number of carbons doesn't exceed 24; R8and R9are H; X is H, methyl or an electron withdrawing group; and n is 0, 1, or 2, wherein at least one of R1, R2, R3, R4, R5, R6or R7comprises an affinity tag. In some embodiments, at least one of R1, R2, R3, R4, R5, R6or R7in the compound of the formula (I) is a fluorosubstituted alkyl, a fluorosubstituted alkenyl, a fluorosubstituted alkynyl, a fluorosubstituted carbocyclyl, a fluorosubstituted heteroalkyl, a fluorosubstituted heteroalkenyl, a fluorosubstituted heteroalkynyl, or a fluorosubstituted heterocyclyl.

In the purification methods provided in this disclosure, the the affinity tag may be a fluorous or a hydrophobic tag with a c Log P value of at least 3. In the purification methods according to this disclosure, the affinity tag may be a fluorosubstituted alkyl, a fluorosubstituted alkenyl, a fluorosubstituted alkynyl, or a fluorosubstituted carbocyclyl.

In some embodiments, the purification methods are conducted with the following orthoester linker:



In further aspects, the present disclosure provides an oligonucleotide orthoester linker conjugate comprising an oligonucleotide which comprises at its 5′ or 3′end a moiety of formula (IV).



wherein the oxygen linked to the squiggly line is the oxygen of the 5′end or the 3′end of the oligonucleotide;

wherein at least one of R1, R2, R3, R4, R5, R6and R7is C1-C24alkyl, C1-C24heteroalkyl, C2-C24alkenyl, C2-C24heteroalkenyl; C2-C24alkynyl, C2-C24heteroalkynyl, halogen, aryl, heteroaryl, heterocyclyl, carbocyclyl, any substituted equivalent or a combination thereof and the remaining R1, R2, R3, R4, R5, R6and R7are H, C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, C6-C12arylalkyl, carbocyclyl, heterocyclyl, any substituted equivalent or combination thereof; and n is 0, 1, or 2, wherein at least one of R1, R2, R3, R4, R5, R6or R7comprises an affinity tag; and with the proviso that said moiety is not



The oligonucleotide orthoester linker conjugates of with the moiety of the formula (IV) include compounds, wherein at least one of R1, R2, R3, R4, R5or R6is a fluorosubstituted alkyl, a fluorosubstituted alkenyl, a fluorosubstituted alkynyl, or a fluorosubstituted aryl, a fluorosubstituted heteroalkyl, a fluorosubstituted heteroalkenyl, a fluorosubstituted heteroalkynyl, or a fluorosubstituted heterocyclyl. In further embodiments, the oligonucleotide orthoester linker conjugates with the moiety of the formula (IV) include compounds, wherein at least one of R1, R2, R3, R4, R5or R6is a fluorous tag and the remaining R groups including R7are H. In further embodiments, the oligonucleotide orthoester linker conjugates of the formula (IV) include compounds, wherein at least two of R1, R2, R3, R4, R5or R6are a fluorous tag. The oligonucleotide orthoester linker conjugates with the moiety of the formula (IV) include compounds, wherein n=0 and the affinity tag is a fluorous or a hydrophobic tag with a c Log P value of at least 3.

In some embodiments, the oligonucleotide in the oligonucleotide orthoester linker conjugate with the moiety of the formula (IV) comprises an oligoribonucleic acid (RNA).

In some embodiments of the oligonucleotide orthoester linker conjugates, the moiety of the formula (IV) is selected from the group consisting of:


wherein R11and R12are each independently H, C1-C23alkyl, C1-C23heteroalkyl, C1-C23substituted alkyl, C2-C23alkenyl, C2-C23heteroalkenyl, C2-C23substituted alkenyl, C2-C23alkynyl, C2-C23heteroalkynyl, C2-C23substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl or substituted heterocyclyl provided that at least one of R11and R12comprises an affinity tag; R7is H, methyl, ethyl, n-propyl, phenyl or benzyl; Z are each independently CRaRb, O, S or NRawherein Raand Rbare each independently H, C1-C6alkyl, or Raand R11or Raand R12together form a heterocycle with N; n is 0, 1, or 2.

In some embodiments of the oligonucleotide orthoester linker conjugates with the moiety of the formula (IV), the moiety is:


wherein X1is H, F, an azido, a protected sulfhydryl, a protected poly-sulfhydryl, a poly-histidine, a protected amino group, a protected hydrazide group, a protected oxyamine group, a maleimide, a cyclooctyne, a conjugated diene, a C2alkenyl group, a C2substituted alkenyl group, a C2alkynyl group or a C2substituted alkynyl group; R18and R19are each independently H, F, C1-3heteroalkyl or C1-3substituted alkyl; n is 1 or 2; and m is an integer ranging from 0 to 12.

In some embodiments of the oligonucleotide orthoester linker conjugates with the moiety the formula (IV), said moiety is selected from the group consisting of:



Further aspects of this disclosure relate to an orthoester linker of formula (Id)



wherein at least one of R1, R2, R3, R4, R5, R6and R7is C1-C24alkyl, C1-C24heteroalkyl, C2-C24alkenyl, C2-C24heteroalkenyl, C2-C24alkynyl, C2-C24heteroalkynyl, halogen, aryl, heteroaryl, heterocyclyl, carbocyclyl, any substituted equivalent or any combination thereof provided that the total number of carbons doesn't exceed 24 and the remaining R1, R2, R3, R4, R5, R6and R7are H, C1-C6alkyl, C2-C6alkenyl, C2-C6alkynyl, halo- or hetero-substituted equivalents, C3-C12carbocyclyl or heterocyclyl and substituted equivalents with the proviso that two R groups of R1to R7groups do not form a fused ring with the cyclic orthoester as shown below:



X is F, Cl, Br, or a mono-, bis or tris-halosubsituted methyl; and n is 0 or 1, wherein at least one of R1, R2, R3, R4, R5, R6or R7comprises an affinity tag. The orthoester linker of the formula (Id) may have the following structure:



wherein R11is H, C1-C23alkyl, C1-C23heteroalkyl, C1-C23substituted alkyl, C2-C23alkenyl, C2-C23heteroalkenyl, C2-C23substituted alkenyl, C2-C23alkynyl, C2-C23heteroalkynyl, C2-C23substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl or substituted heterocyclyl; R7is H or CH3. Z is CRaRb, O, S, NRa, NRaCO, CONRa, where in Raand Rbare each independently H, C1-C6alkyl, or Raand R11together form a heterocycle with N; X is F, Cl, Br, or a mono-, bis or tris-halosubsituted methyl; and n is 0 or 1, R11comprises an affinity tag.

The orthoester linker of the formula (Id) may have the structure:



wherein R11and R12are each independently H, C1-C23alkyl, C1-C23heteroalkyl, C1-C23substituted alkyl, C2-C23alkenyl, C2-C23heteroalkenyl, C2-C23substituted alkenyl, C2-C23alkynyl, C2-C23heteroalkynyl, C2-C23substituted alkynyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclyl or substituted heterocyclyl provided that at least one of R11and R12comprises an affinity tag; R7is H or CH3. Z are each independently CRaRb, O; S, NRaCO, CONRaor NRawherein Raand Rbare each independently H, C1-C6alkyl, or Raand R11or Raand R12together form a heterocycle with N; X is F, Cl, Br, or a mono-, bis or tris-halosubstituted methyl; and n is 0 or 1.

Any of the orthoester linkers of the formula (Id), may have the affinity tag wherein the affinity tag is a fluorous tag or a hydrophobic tag with a c Log P value of at least 3.

Some of the orthoester linkers of the formula (Id) include compounds, wherein n is 0 and at least two of R1, R2, R3, R4, R5, and R6are each independently C1-C24alkyl, C1-C24heteroalkyl, C2-C24alkenyl, C2-C24heteroalkenyl, C2-C24alkynyl, C2-C24heteroalkynyl, halogen, aryl, heteroaryl, heterocyclyl, carbocyclyl or any substituted equivalent, and all the remaining R groups of R1, R2, R3, R4, R5, and R6are H, provided that the two R groups that are not H are not linked together to form a fused ring with the cyclic orthoester. Some of the orthoester linkers of the formula (Id) include compounds, wherein one of R1, R2, R3, R4, R5or R6is a fluorosubstituted alkyl, a fluorosubstituted alkenyl, a fluorosubstituted alkynyl, or a fluorosubstituted aryl.

Further aspects of this disclosure include a fluorous orthoester linker having the structure:



wherein Z is CH2, O, S, NRa, or NRaCO wherein Rais H or C1-C6alkyl; R′ is H, methyl, ethyl. n-propyl, phenyl or benzyl; X is H, F, Cl, Br, or a mono-, bis or tris-halosubstituted methyl or cyano; m is an integer from 0 to 12; n is 1 or 2.

Further aspects of this disclosure include an orthoester linker having the structure selected from:



Further aspects of this disclosure include an orthoester linker of formula (IIIa):



wherein X1is H, F, an azido, a protected sulfhydryl, a protected poly-sulfhydryl, a poly-histidine, a protected amino group, a protected hydrazide group, a protected oxyamine group, a cyclooctyne, a conjugated diene, a C2alkenyl group, a C2substituted alkenyl group, a C2alkynyl group or a C2substituted alkynyl group; R18and R19are each independently H, F, C1-3heteroalkyl or C1-3substituted alkyl; X2is H, CH3, F, Cl, Br, or a mono-, bis or tris-halosubstituted methyl or cyano; n is 1 or 2; and m is an integer ranging from 0 to 12.

The orthoester linker of the formula (IIIa) may be the following compound:



These and other features and advantages of the present methods and compounds will be apparent from the following detailed description, in conjunction with the appended claims.

