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Efficient conjugation to phosphorothioate oligonucleotides by Cu-catalyzed Huisgen 1,3-dipolar cycloaddition. Malgorzata Honcharenko, Dmytro Honcharenko, and Roger Stromberg Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/ acs.bioconjchem.9b00217 • Publication Date (Web): 08 May 2019 Downloaded from http://pubs.acs.org on May 12, 2019
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Bioconjugate Chemistry
Efficient conjugation to phosphorothioate oligonucleotides by Cu-catalyzed Huisgen 1,3-dipolar cycloaddition. Malgorzata Honcharenko*1, Dmytro Honcharenko1 and Roger Strömberg*1, 1 Department of Biosciences and Nutrition, Karolinska Institute, SE-14183 Huddinge, Sweden
* Corresponding authors: Malgorzata Honcharenko and Roger Strömberg, Tel: +46-8-52481025; +468-52481024. E-mail:
[email protected] ;
[email protected].
Abstract Improving oligonucleotide delivery is critical for the further development of oligonucleotidebased therapeutics. Covalent attachment of reporter molecules is one of the most promising approaches towards efficient oligonucleotide-based therapies. An efficient methods for the attachment of a variety of reporter groups is Cu(I)-catalyzed Huisgen azide‒alkyne 1,3dipolar cycloaddition. However, the majority of potential oligonucleotide (ON) therapeutics in clinical trials are carrying phosphorothioate (PS) linkages, and this robust conjugation method is not yet established for these ONs due to a general concern of Cu-S interaction. Here, we developed a method allowing for efficient conjugation of peptides to PS oligonucleotides. The method utilizes solid supported oligonucleotides that can be readily transformed into “clickable ONs” by simple linker conjugation and further reacted with an azido containing moiety (e.g, a peptide) using the CuBr×Me2S complex as a superior catalyst in that reaction. This study opens the way for further development of PS oligonucleotideconjugates by means of efficient Cu(I)-catalyzed Huisgen azide‒alkyne 1,3-dipolar cycloaddition. Introduction Nucleic acid therapeutics is currently experiencing a tremendous expansion not least encouraged by several recent FDA approvals of oligonucleotide (ON) drugs1, 2 and there are numerous ongoing clinical trials3, 4. Despite recent achievement, delivery to the site of action is in general insufficient and limits the ON efficacy. Being polyanionic macromolecules with poor drug-like properties, the ONs fundamentally differ5 from the typical small molecule drugs that have been the backbone of the pharmaceutical industry. The conjugation of different uptake promoting molecules to ONs is a promising approach to enhance their delivery, as exemplified by targeting the liver by use of N-acetylgalactosamine (GalNAc) conjugated ONs6 and to the pancreatic β-cells by conjugation of ONs to a glucagon-like peptide-1 (GLP-1)7. A number of various methods for the conjugation of specific molecules, especially peptides, to ONs have been developed, both, by in-line conjugation8, and by postconjugation in solution9 and on solid phase10, 11. The use of Cu(I)-catalyzed Huisgen azidealkyne 1,3-dipolar cycloaddition12, known as “click” reaction, has been proven to be a highly
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efficient approach for conjugation of ONs to peptides11, 13, other ONs14, the m3G-Cap15, small molecules like biotin16, 17 or fluorophores18, carbohydrates19-22, and even for the preparation of multiple conjugates10. Phosphorothioate (PS) linkage, where a non-bridging phosphodiester oxygen is replaced by sulfur, is one of the most widely used nucleic acid modification23. Combined with 2′-O-alkyl (e.g., 2′-O-methyl, 2′-O-methoxyethyl) modification, the phosphorothioate oligonucleotides (PS-ONs) are also the most exploited ONs in clinical trials3, 4. This is not least due to PS-ONs increased stability to nucleases, RNase H-mediated degradation of RNA in a DNA/RNA hybrid duplex, and favorable pharmacokinetic properties5. However, the Cu-catalyzed azidealkyne cycloaddition reaction is virtually not represented in conjugation chemistry involving PS-ONs. One report, where oligodeoxyribonucleotides containing only few phosphorothioate internucleotide linkages were subjected to Cu-catalyzed cycloaddition, describes a new phosphoramidite synthon, the alkynyl phosphinoamidite, for solid-phase ON synthesis. The alkynylphosphonate was functionalized on solid support to generate the 1,2,3-triazolyl or substituted 1,2,3-triazolyl phosphonate-2′-deoxyribonucleotide internucleotide linkage24. Phosphorothioate linkages are of a similar chemical reactivity as phosphodiesters under hydrolytic conditions25, 26. However, it can in principle be a concern that the presence of metal ions can cause cleavage and desulfurization of PS linkages27. This could be a reason why little work has been reported on Cu(I) “click chemistry” conjugation with PS-ONs, and the lack of literature examples may give the impression that PS-ONs are not compatible with Cu-catalyzed reactions. Results and discussion. In this work, we evaluate and fine-tune the reaction conditions for the synthesis of peptide conjugates of PS-ONs by means of Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition, and established a protocol that can be generally used for efficient conjugation. The reaction is performed on solid support using commercial or in-house made PS-ONs, commercial reagents, and readily made linkers. The initial plan was to directly adopt our strategy for synthesis of phosphodiester ON conjugates for the synthesis of peptide PS-ON conjugates (Scheme S1, Supporting Information). The acetylated form of the MIF-1 peptide (MIF-1 = Pro-Leu-Gly-NH2, that is a blood-brain barrier (BBB) penetrating peptide28) called MIF-aza (AcPro-Leu-Gly-CH2CH2N3, 3, Figure 1) and that contains an azido group is used in this study. This peptide was used for optimizing Cu-catalyzed “click” reactions between peptides and ONs on solid support11. The starting point for the evaluation of “click” conditions was a solid-supported PS-ON which was first equipped with an amino linker by conjugation to the H-phosphonate linker 111 exploiting pivaloyl chloride as an activator. After H-phosphonate oxidation with iodine/water/pyridine and removal of 4-monometoxytrityl the solid-supported ON was further reacted with pre-activated triple bond donor29, 4-((2-(prop-2-yn-1yloxy)acetamido)methyl) benzoic acid (PAMBA, 2)10 to give ON-PAMBA conjugate (Scheme S1, Supporting Information). The ON equipped with the triple bond linker (ON-
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Bioconjugate Chemistry
PAMBA) can then be either examined in a “click” reaction on solid support or deprotected, cleaved from the solid support and freeze-dried to be studied in “click” reactions in solution.
Figure1: A: Synthetic steps leading to “click” reaction on solid support for which different Cu(I) sources and conditions were investigated. B: Example of crude chromatogram depicting CuI catalyzed reaction between all-PS-ON1 and MIF-N3 on solid support and corresponding mass spectra of the main product. Except of the mass corresponding to all-PS-ON1-MIF conjugate, masses of conjugates corresponding to 1 and 2 desulfurization products are as well detected. ON1= 12-mer, 5 ′ dGGdCMeCdGCUTdCCTC, deoxynucleosides (dX), LNA nucleotides in bold and 2 ′ -O-methyl nucleotide units in italic. ON2 = 18-mer, 2'-OMe-RNA, DNA dX gapmer, 5′CCUCUUdAdCdCTdCdAGUUACA.
At first, the conjugation of PS-ON with azido-containing peptide MIF-aza (3) was performed under previously described “click” reaction conditions for the solid-supported conjugation of phosphodiester ONs10, 11, 14, 15, 17 using tert-butanol/water (1:1) mixture as solvent, 24 h reaction time, and 2 equiv. of copper(I) iodide (CuI) as a copper source (Figure 1). The chromatogram of the crude deprotected phosphorothioate oligonucleotide conjugate (PSON1-MIF) showed good conversion of PS-ON1 to the peptide-conjugate product. However, further analysis by mass spectrometry of the main product revealed that more than half of the material displayed a mass smaller by 16 Da or a multiple thereof (Figure S1, line A and B,
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Supporting Information). This indicates that, apart from peptide conjugation, substantial desulfurization of the product was the outcome of the reaction. Partially desulfurized oligonucleotides are commonly observed impurities of synthetic PSONs30. It is known that under certain conditions PS-ONs can undergo desulfurization, e.g., during repeated treatment with an aged solution of trichloroacetic acid (TCA)31. In addition, caution needs to be employed in assessing minor presence (
