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Automated Solid-Phase Click Synthesis of Oligonucleotide Conjugates: From Small Molecules to Diverse N‑Acetylgalactosamine Clusters Valentina M. Farzan,†,# Egor A. Ulashchik,‡,# Yury V. Martynenko-Makaev,‡,# Maksim V. Kvach,‡ Ilya O. Aparin,§ Vladimir A. Brylev,§ Tatiana A. Prikazchikova,† Svetlana Yu. Maklakova,⊥ Alexander G. Majouga,⊥,∥ Alexey V. Ustinov,§ German A. Shipulin,▽ Vadim V. Shmanai,‡ Vladimir A. Korshun,*,§,○ and Timofei S. Zatsepin*,†,⊥,▽ †

Center of Translational Biomedicine, Skolkovo Institute of Science and Technology, Skolkovo, Moscow 143026, Russia Institute of Physical Organic Chemistry, National Academy of Sciences of Belarus, Surganova 13, Minsk 220072, Belarus § Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Miklukho-Maklaya 16/10, Moscow 117997, Russia ⊥ Department of Chemistry, Lomonosov Moscow State University, Leninskie gory 3, Moscow 119992, Russia ∥ National University of Science and Technology “MISiS”, Leninskiy Prospect 4, Moscow 119991, Russia ▽ Central Research Institute of Epidemiology, Novogireevskaya 3a, Moscow 111123, Russia ○ Gause Institute of New Antibiotics, Bolshaya Pirogovskaya 11, Moscow 119021, Russia ‡

S Supporting Information *

ABSTRACT: We developed a novel technique for the efficient conjugation of oligonucleotides with various alkyl azides such as fluorescent dyes, biotin, cholesterol, N-acetylgalactosamine (GalNAc), etc. using copper-catalysed alkyne−azide cycloaddition on the solid phase and CuI·P(OEt)3 as a catalyst. Conjugation is carried out in an oligonucleotide synthesizer in fully automated mode and is coupled to oligonucleotide synthesis and on-column deprotection. We also suggest a set of reagents for the construction of diverse conjugates. The sequential double-click procedure using a pentaerythritol-derived tetraazide followed by the addition of a GalNAc or Tris−GalNAc alkyne gives oligonucleotide−GalNAc dendrimer conjugates in good yields with minimal excess of sophisticated alkyne reagents. The approach is suitable for highthroughput synthesis of oligonucleotide conjugates ranging from fluorescent DNA probes to various multi-GalNAc derivatives of 2′-modified siRNA.

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INTRODUCTION Modified oligonucleotides are widely used in life science, for in vitro diagnostics, and as therapeutics. The attachment of dyes, ligands, peptides, or proteins results in superior oligonucleotide suitability for assays1 or therapeutic2−5 purposes. Various chemistries are being used to synthesize oligonucleotide conjugates,6−9 but “click chemistry” has become an invaluable tool for biorthogonal conjugation during the past decade.10−14 Copper-catalyzed alkyne−azide cycloaddition (CuAAC) is very popular in nucleic acid chemistry due to regioselectivity and high yields.15−20 Additionally, reactive groups (terminal alkynes and azides) are fully compatible with phosphoramidite oligonucleotide chemistry and postsynthetic DNA and RNA deprotection. In the case of azide derivatives, some precautions are needed during oligonucleotide synthesis as azide phosphoramidites undergo Staudinger transformation in acetonitrile, which results in degradation.17,19,21 However, degradation can be avoided by immobilizing azide derivatives on solid supports © XXXX American Chemical Society

or using a solution of azide phosphoramidite in dichloromethane.19,22 The reaction rate for CuAAC can be further increased by the use of copper-chelating azides,23,24 but this approach suffers from constrained copper removal. Even after the common solution-phase CuAAC, thorough copper removal from oligonucleotide conjugates is necessary, especially for in vivo applications.25 Copper-free strain-promoted alkyne−azide cycloadditions (SPAAC), inverse electron-demand Diels−Alder (IEDDA) and nitrile imine−alkene cycloadditions with increased specificity and reaction rates were developed as a CuAAC alternative for oligonucleotide conjugation.10−14 However, more-reactive IEDDA reagents such as transcyclooctene moieties are less-tolerated in oligonucleotide synthesis;26 thus, they are reliably introduced by post-synthetic Received: August 3, 2017 Revised: September 12, 2017 Published: September 18, 2017 A

DOI: 10.1021/acs.bioconjchem.7b00462 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry procedures26 or enzymatically.27 Also, solution-phase conjugation can be hardly applied to hydrophobic molecules due to their poor solubility in water or in water and organic mixtures. Solid-phase functionalization is widely used in oligonucleotide chemistry28 and can become a method of choice to overcome challenges in CuAAC. These techniques (for example, common acylation29 or aldehyde couplings)30,31 were developed along with metal-catalyzed reactions.32 To date, a number of procedures for solid-phase oligonucleotide functionalization by Sonogashira,33−38 Stille,39,40 GlaserHey,41,42 CuAAC,43−52 and SPAAC and nitrile oxide and alkyne couplings53−55 have been reported. Among them, only Sonogashira coupling was adopted for the automated DNA synthesizer in pioneer studies by Grinstaff and co-workers.33,34 Most of the approaches mentioned above were based on offsynthesizer techniques (including synthesizer-incompatible microwave irradiation or heating) or required a prolonged reaction time (up to 24 h). Moreover, in most cases, the final product is a mixture of regioisomers, which complicates routine high-performance liquid chromatography (HPLC) purification due to the broadening or doubling of the peaks. Nowadays, many routine procedures are being replaced by automated ones. Automated platforms can be used not only for the synthesis of biopolymers but also for small molecules and defined polymers by various chemistries.56,57 The aim of this study was to develop an in-synthesizer solid-phase CuAAC oligonucleotide conjugation procedure that provides a single regioisomer and can complement the phosphoramidite method in simplicity, wide applicability and cost of labeling and conjugation (Figure 1). First, there are molecules that are not

date for solid-phase CuAAC on oligonucleotides do not meet the above criteria.

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RESULTS AND DISCUSSION Optimization of Solid-Phase Oligonucleotide Labeling by CuAAC. First, we tried to develop the synthesis of phosphoramidite based on propiolic acid in which terminal alkyne is more reactive in CuAAC58 due to the presence of an electron-withdrawing group. However, acylation of the 6aminohexanol or trans-4-aminocyclohexanol with propiolic anhydride led to a complex reaction mixture, a result of the alkyne side reaction with the hydroxyl group.59 Thus, we abandoned the idea of a more-reactive alkyne. Initially tested mixtures of t-BuOH and water and of DMSO and water as media for solid-phase CuAAC showed high viscosity and surface tension resulting in droplet formation and incorrect reagent delivery in a DNA synthesizer. Therefore, we studied solid-phase CuAAC in several aprotic polar solvents: formamide, MeCN, dimethyl sulfoxide (DMSO), dimethylformamide (DMF), and dimethylamylamine (DMAA). Because most of the reaction components including dye azides were poorly soluble in formamide and MeCN, these solvents were excluded from the study. Then we screened a number of copper catalysts (including CuCl, Cu(MeCN)4PF6, CuI/N,N-diisopropylethylamine (DIPEA), CuCl·tris-[(1-benzyl-1H-1,2,3-triazol4-yl) methyl]amine (TBTA), CuBr·Me2S,60 CuBr·PhSMe, CuBr·TBTA, and CuI·P(OEt)361) to find preferable ones. In the case of CuCl, Cu(MeCN)4PF6, CuI/DIPEA, and CuCl· TBTA, the yields of the product were less than 5−30%, while for CuBr·PhSMe, CuBr·TBTA, and CuI·P(OEt)3, conversion exceeded 95%. To optimize solid-phase conjugation, we started with relatively high concentrations of the catalysts (100 mM) and 6-R6G azide62 (50 mM, 4 × 100 μL; azide/alkyne ratio of 20:1) (Table 1) and gradually lowered the concentrations. The thioanisole complex was omitted as yields significantly dropped down for low concentrations of the reagents; the formation of some by-products was observed, and a long-lasting offensive odor made the work uncomfortable. DMSO was found to be a poor choice for both CuBr·TBTA and CuI·P(OEt)3 because some precipitates were formed in solutions overnight, probably as a result of catalyst oxidation. These two catalysts gave similar yields, and CuBr·TBTA appeared to be slightly better in terms of conversion. However, application of CuBr·TBTA to solidphase CuAAC with rhodamine dye azides led to their partial oxidation (Figure 2). Up to 10% of the by-product with molecular weight M+16 (Figure 2, trace 4) was observed for dye−oligonucleotide conjugates if the solutions were not thoroughly degassed or if they were left in the synthesizer for a prolonged period. In the case of dual-labeled probes, this byproduct cannot be separated by reverse-phase HPLC (RPHPLC) and decreases the performance of the probes in qPCR assay. This problem can be overcome by thorough oxygen removal just before the conjugation. Therefore, we moved on with CuI·P(OEt)3/DMAA because this always yielded quality conjugates without special precautions. We performed an analysis of reaction mixtures by RP-HPLC, but the presence of admixtures with close retention time (truncated oligonucleotides, traces of N3-cyanoethyl adducts, etc.) complicated the calculation of conversion into solid-phase CuAAC. Therefore, we used liquid chromatography−mass spectrometry (LC−MS) for the estimation of conversion in conjugation. We calculated conversion as a ratio of the conjugate to initial alkyne oligonucleotide, excluding any other oligonucleotide species.

Figure 1. Solid-phase CuAAC modification of oligonucleotides. (i) Automated oligonucleotide synthesis, (ii) automated click reaction, and (iii) deprotection and purification.

compatible with the chemistry of the phosphoramidite synthetic cycle (mainly with the oxidation and detritylation steps). Second, the synthesis of a series of phosphoramidites is trickier for screening ligands or dyes, and more starting material is needed in comparison to post-synthetic labeling. However in case of screening solution-phase conjugation is a laborious procedure, so solid phase approaches are preferable. Also, the high stability of azide solutions at room temperature in comparison to phosphoramidites should decrease the cost for rarely used modifiers due to prolonged installation at the synthesizer. The only drawback of solid-phase conjugation is the requirement of conjugate stability during post-synthetic ammonia deprotection. To ensure that the solid-phase conjugation step would not slow down common oligonucleotide synthesis, we defined the optimal reaction time for CuAAC as less than 3 h. In this case, one can run oligonucleotide synthesis overnight, including the labeling stage plus on-synthesizer deprotection during postsynthesis procedures. Also, solutions should be stable, and the concentration of azide derivatives should not exceed 0.05 M to make the method cost-competitive with phosphoramidite solid phase synthesis (SPS). Unfortunately, all methods published to B


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Bioconjugate Chemistry Table 1. Optimization of Solid-Phase Oligonucleotide Labeling by CuAACa catalyst concentration 100 mM

50 mM

10 mM

addition (S, single; D, double) catalyst

solvent

CuBr/PhSMe

CuBr/TBTA

CuI/P(OEt)3

6-R6G azide concentration, mM

S

D

DMSO DMF DMAA

10

6.3 1.7 2.0

13.5 3.5 2.1

ns ns ns

ns ns ns

ns ns ns

ns ns ns

DMSO DMF DMAA

50

35.3 11.9 9.2

62.5 17.5 17.3

ns ns ns

ns ns ns

ns ns ns

ns ns ns

80.7 88.5

98.6 98.2

49.9 59.7

88.1 90.6

9.6 27.7

28.6 54.2

DMF DMAA

5

S

D

S

D

DMSO DMF DMAA

10

47.9 95.6 98.7

81.3 98.4 99.4

ns 68.5 73.6

ns 94.4 94.7

ns 19.4 34.1

ns 48.1 63.5

DMSO DMF DMAA

50

77.0 97.5 97.6

98.4 98.5 98.3

ns ns ns

ns ns ns

ns ns ns

ns ns ns

22.5 44.8

42.0 72.7

17.1 40.9

32.6 61.5

7.5 21.3

11.6 30.1

DMF DMAA

5

DMSO DMF DMAA

10

52.9 89.0 95.7

89.7 98.0 99.1

ns 73.8 90.8

ns 91.7 98.2

ns 20.5 28.9

ns 29.8 48.5

DMSO DMF DMAA

50

78.9 96.1 98.0

96.7 96.8 97.9

ns ns ns

ns ns ns

ns ns ns

ns ns ns

a

Reaction time of 2 h; conversion rates are based on electrospray ionization mass spectrometry (ESI-MS) data, calculated as an average for three repeats. ns: not studied. Values exceeding 95% are marked in bold.

optimization, the conversion exceeded 95% for most conjugates. Thus, such probable errors do not significantly influence the evaluation of the developed procedure. At optimized conditions, conversion exceeds 95% within 2 h at a 1 μmol oligonucleotide scale with 10 mM solution of 6R6G azide (2 equiv) in the presence of 100 mM CuI·P(OEt)3 (Table 1). Most azides were found to be stable in solutions at least for 1 month; the copper catalyst should be replaced twice a week for CuI·P(OEt)3 or daily for CuBr·TBTA. Because solutions of azides are stable in amber bottles in comparison to catalysts, we dissected azide derivatives and copper catalysts into different vessels. Solutions were delivered to the column with alkyne-modified oligonucleotides sequentially in several pulses, similar to phosphoramidite and activator in the coupling step of oligonucleotide assembly. Excellent yields of solid-phase CuAAC oligonucleotide functionalization with 6-R6G azide encouraged us to apply the developed procedure to synthesize other labeled oligonucleotides. A toolkit of alkyne and azide reagents (Figure 3) was used to prepare a number of 5-FAM, 6-R6G, 6-TAMRA, 6-ROX, Cy5, biotin, pyrene, 1-phenylethynylpyrene, and cholesterol oligonucleotide derivatives with excellent conversion (Table S1). For most of the conjugates (except rhodamine and cyanine dyes), we performed on-column deprotection with

Figure 2. HPLC profiles of the crude reaction mixtures after deprotection of the oligonucleotides: (1) 5′-alkyne-T10; (2, 3) 5′alkyne-T10 after CuAAC, 58% and 98% conversion in the presence of CuI·P(OEt)3; and (4) 5′-alkyne-T10 after CuAAC, 98% conversion in the presence of CuBr·TBTA. Peak I: 5′-alkyne-T10. Peak II: oxidized 5′-R6G-T10. Peak III: 5′-R6G-T10.

We performed LC−MS analysis for several model 6-R6G conjugate and alkyne T20 oligonucleotide mixtures and found only a negligible influence of 6-R6G on the total ion count for oligonucleotides. Naturally, various conjugated residues can influence the observed ion counts in different manner, but after C


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Bioconjugate Chemistry

such conjugates, but optimization of the carbohydrate attachment to the oligonucleotides is still under development.8 The final goal of our study was to develop novel GalNAc− siRNA conjugates that will demonstrate improved delivery in vivo. The number of ASGPR receptors in the hepatocytes in vivo or in freshly prepared primary hepatocytes is about 0.5−1 milliliter per cell, while immortalized hepatocyte cell lines (either human Hep3B, HepG2, or mouse Hepa1−6) have at least 1 to 2 orders of magnitude fewer receptors (10 000− 30 000 per cell).5 In addition, primary hepatocytes lose receptors at a high rate that results in 30 000−50 000 receptors per cell in less than 1 week. As a result, data on conjugate delivery in vitro are poorly translated to that in vivo, so we wanted to develop a procedure that can be easily scaled up to the milligram scale. To perform such conjugation, we synthesized model T20 oligonucleotides bearing one, two, or three alkyne moieties to optimize conditions of conjugation and two sense strands of siRNAs for further studies on in vitro and in vivo targeted delivery (Table 2). Table 2. Alkyne-Modified DNA and RNA Oligonucleotides Used in the Studya T20 T20bis Tris−T20 alkyne− AHSA AHSA− (Pro)3 Tris−AHSA

Figure 3. Toolkit of azide and alkyne reagents. alkyne−Luc Luc−(Pro)3

1,2-ethylenediamine in toluene63 in the synthesizer to make a straightforward automated procedure for conjugate preparation. The solid-phase click chemistry of coumarin 343 to oligonucleotides was recently reported;52 however, we failed to find deprotection conditions for hetero oligonucleotides because dye degraded up to 80% faster than during 2 h of exposure to concentrated aqueous ammonia at room temperature (conditions for ultramild protected phosphoramidites). In the case of coumarin 343 azide, we also performed common solution CuAAC reaction with 5′-alkyne T20 oligonucleotide to obtain the desired reference conjugate. Oligonucleotide Multi-Labeling by Solid-Phase CuAAC. Targeted delivery is a key point for oligonucleotide therapeutics. Lipid nanoparticles and N-acetylgalactosamine (GalNAc) conjugates have been proven efficient for antisense oligonucleotide- and siRNA-targeted delivery to the liver in various clinical trials.2−5 Today, several siRNA−GalNAc conjugates are at the II and III phases of clinical trials.5 The main feature of heavily modified siRNA-GalNAc conjugates is an extremely prolonged action in vivo after subcutaneous injection (up to 9 months) at low ED50 0.5−2 mg/kg.64 Such a remarkable effect results from their enhanced stability and efficient receptor-mediated endocytosis by the ASGPR receptor in hepatocytes. Multi-GalNAc conjugates with defined geometry have a significantly increased affinity to ASGPR due to the receptor’s oligomerization in the cell membrane. Recent studies64−67 confirmed that specific linkages with different rigidity and geometry determine the efficacy of binding to the receptor and thus influence the applicability of the conjugates in vivo. Various methods were developed for the synthesis of

Tris−Luc

(alkyne)−T20 (alkyne)−T20−(Pro−alkyne) (Tris−alkyne)−T20 (alkyne)−gg(Af)u(Gf)a(Af)g(Uf)g(Gf)a(Gf)a(Uf)u(Af)g(Uf) (invT) gg(Af)u(Gf)a(Af)g(Uf)g(Gf)a(Gf)a(Uf)u(Af)g(Uf)(invT)− (Pro−alkyne)3 (Tris−alkyne)−gg(Af)u(Gf)a(Af)g(Uf)g(Gf)a(Gf)a(Uf) u(Af)g(Uf)(invT) (alkyne)−cu(Uf)a(Cf)g(Cf)u(Gf)a(Gf)u(Af)c(Uf)u(Cf) g(Af)(invT) cu(Uf)a(Cf)g(Cf)u(Gf)a(Gf)u(Af)c(Uf)u(Cf)g(Af)(invT)− (Pro−alkyne)3 (Tris−alkyne)−cu(Uf)a(Cf)g(Cf)u(Gf)a(Gf)u(Af)c(Uf) u(Cf)g(Af)(invT)

a

5′→3′ sequences; n, 2′-OMe−nucleotide, Nf, 2′-deoxy-2′-fluoronucleotide; invT, inverted dT (3′−3′ linkage).

After penetration to the cytosol, the antisense strand of siRNA is recruited by the RNAi protein machinery, while the sense strand is omitted and degraded. Thus, a delivery moiety should be attached to the sense strand to minimize the influence on the protein−RNA interactions. The AHSA oligonucleotide is a sense strand of the widely used siRNA for the targeting activator of the 90 kDa heat shock protein ATPase homologue 1 (AHA1 or AHSA1) in mice.68−70 This protein is evenly expressed in most tissues and is frequently used to study the specificity of siRNA targeted delivery in vivo. Luc is a sense strand of luciferase siRNA that is used as a negative control for in vitro and in vivo studies because luciferase is absent in wild-type mice. In this study, 5′- or 3′alkyne AHSA and Luc oligonucleotides were fully 2′-modified to maximize nuclease stability in vitro and in vivo (Table 2). For the synthesis of diverse branched oligonucleotide− GalNAc conjugates, we developed two solid-phase CuAAC conjugation procedures (Figure 4). The first one is a direct conjugation of GalNAc azides to oligonucleotides bearing several alkyne residues originating from either multiple couplings of Pro−alkyne reagent (Figure 4A) or the single coupling of Tris−alkyne reagent (Figure 4B). The second one is based on two sequential CuAAC additions to a mono alkyne oligonucleotide: diazide or tetraazide71 derivatives followed by D


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Figure 5. Sophisticated multi-GalNAc−oligonucleotide conjugates prepared in the study.

the reagent toolkit, can be illustrated on 3 9-GalNAc AHSA conjugates (Figure 6).

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CONCLUSIONS We have developed a simple solid-phase CuAAC procedure for the preparation of highly branched oligonucleotide conjugates in high yields. The scope of the technique has been demonstrated by the synthesis of single or multilabeled modified oligonucleotides as well as oligonucleotide−GalNAc dendrimer conjugates with various topologies. This fast and convenient double-click procedure (Figure 4C) provided excellent yields with a minimal consumption of reagents. The double-click technique can be scaled up only in the solid-phase approach because solution-phase click chemistry with a tetraazide leads to a complex mixture of products.59 We emphasize that the conjugation is carried out in an oligonucleotide synthesizer in fully automated mode and is coupled to oligonucleotide synthesis and on-column deprotection. Remarkably, the conjugates are fully assembled and deprotected overnight without human intervention and then undergo only elution and final HPLC purification. Conjugation is carried out for totally protected oligonucleotides, which decreases copper retaining in the sample. Several additional washings with any solvents or chelators can further improve copper removal. The toolkit of azide and alkyne reagents (Figure 3) could be also used to build more-diverse

Figure 4. Pathways for the synthesis of oligonucleotide−GalNAc conjugates with various topologies by solid-phase CuAAC.

GalNAc alkynes (Figure 4C). Diazide and tetraazide derivatives are used in large excess (10 or 20 equiv per alkyne) to exclude interoligonucleotide cross-linking, while more hard-to-access GalNAc alkynes are added in minimal excess (2 equiv and 3.3 equiv per azide group for diazide and tetraazide, respectively). Good yields of GalNAc modification prompted us to synthesize a number of multi-GalNAc−oligonucleotide conjugates (Table 3) with different topologies (Figure 5, framed in red). Conversely, the tetraazide-based solid-phase CuAAC approach (Figure 4) allowed us to synthesize even an oligonucleotide conjugate with six Tris−GalNAc derivatives attached to both the 5′- and the 3′- ends (three at each end). This results in 18 GalNAc residues per oligonucleotide after one addition (Figure 5). Topology variations account for the tunable hydrophobicity of the conjugate, as evaluated by RPHPLC (Figure S1), and may influence the behavior of the conjugate in vivo. The complexity of oligonucleotide conjugates, easily accessible with solid-phase CuAAC using

Table 3. Conversion in Multiple and Multi-Stage Conjugation of GalNAc Derivatives to Modified Oligonucleotidesa azide (or azide + alkyne) reagents

concentration, mM (equiv)

T20

T20bisb

Tris− T20

alkyne AHSA

AHSA− (Pro)3

Tris− AHSA

alkyne Luc

Luc− (Pro)3

Tris− Luc

GalNAc−N3 tGalNAc−N3 diazide diazide + GalNAc−alkyne diazide + bt1GalNAc−alkyne diazide + bt2GalNAc−alkyne tetraazide tetraazide + GalNAc−alkyne tetraazide + bt1GalNAc− alkyne tetraazide + bt2GalNAc− alkyne

10 (2) 10 (2) 50 (10) 10 (2) 10 (2) 10 (2) 100 (20) 50 (10) 50 (10)

95.5 97.2 96.8 92.5 93.7 91.4 96.1 89.2 88.2

96.1 94.8 97.1 92.1 90.0 89.4 93.1 86.1 86.5

91.5 92.7 nsc nsc nsc nsc nsc nsc nsc

95.9 96.2 95.4 92.0 93.1 89.5 93.2 87.9 88.1



96.2 97.7 96.2 90.3 93.2 92.8 96.8 91.5 89.1



50 (10)

87.9

85.2

nsc

89.7

nsc

nsc

88.1

nsc

nsc

a

Conversion in solid-phase CuAAC, percent values (calculated as an average for three repetitions based on ESI-MS data). bDouble-addition. cns: not studied. E


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Figure 6. Ball-and-stick representation of three 9-GalNAc AHSA conjugates, top to bottom: alkyne−AHSA + tetraazide + bt1GalNAc−alkyne, AHSA−(Pro−alkyne3 + tGalNAc−azide), and (Tris−alkyne)−AHSA + tGalNAc−azide; atoms: carbon (gray), hydrogen (not shown), oxygen (red), nitrogen (blue), phosphorus (yellow), and fluorine (green).

in-synthesizer) 5 times every 30 min. After 3 h, the solid support was thoroughly washed out (10 × 150 μL of DMSO, then 3 × 150 μL of MeCN) followed by the deprotection of cyanoethyl groups (2 × 10% diethylamine in MeCN (v/v), 10 min), and full deprotection using the TAMRA cocktail (tbutylamine/water, 1:3 v/v) at 55 °C overnight.62 The solution was evaporated, and an aliquot was analyzed by HPLC and ESIMS. Solid-Phase CuAAC Modification of Model 5′-AlkyneModified T20 Oligonucleotides with Small Molecules. The model DNA oligonucleotide T20 was prepared in the solid phase and coupled with 5′-alkyne phosphoramidite. Click modification was performed on the solid phase by adding 10 mM solution of azide, 100 mM CuI·P(OEt)3 4 × 100 μL (azide = 2 equiv) for 4 h followed by washings as described above. In most cases, the oligonucleotides were cleaved from the support and deprotected in the synthesizer using 1,2-ethylene diamine/ toluene, 1:1 v/v, for 2 h at room temperature followed by washings (4 × 150 μL of MeCN) and elution with 50% acetonitrile in water or alternatively deprotected using common AMA solution (concentrated aqueous ammonia and 40% aqueous methylamine, 1:1 v/v) for 30 min at 65 °C. In the case of rhodamine dyes (R6G, TAMRA, and ROX), deprotection was performed using the TAMRA cocktail at 55 °C overnight. In the case of coumarin 343 and Cy5 azide, we used two deprotection schemes (concentrated aqueous ammonia at room temperature for 2 and 18 h), which are typically used in cases of ultramild (CAc, APAC, and GiPr‑PAC) and mild (CAc, ABz, and GDMF) DNA phosphoroamidites. For the solution-phase click for coumarin 343 azide, 5′alkyne-modified oligonucleotide (2 nmol) was dissolved in 1 M triethylammonium acetate (30 μL). Then, 20 μL of copper catalyst solution in 60% DMSO (15 mM TBTA and 13 mM copper(II) sulfate) was added followed by 4 μL of 10 mM coumarin 343 azide in DMSO and 140 μL of 80% DMSO. The solution was degassed and filled with argon twice in a vacuum centrifuge concentrator, and then degassed ascorbic acid solution (0.25 M, 6 μL) was added. The reaction was left overnight at ambient temperature, and the oligonucleotide material was precipitated with 4% LiClO4 in acetone (1 ml). HPLC purification and analysis was performed as described above.

oligonucleotide conjugates versus these listed in Table 3, e.g., those containing internal modifications. Moreover, the set of the reagents can be easily extended by preparing new azide and alkyne derivatives suitable for solid-phase CuAAC. The only drawback for this fully automated approach is that the conjugate must be stable during the final ammonia treatment for all oligonucleotides and additional fluoride deprotection for RNA. This approach can be useful for the CuAAC or SPAAC synthesis of DNA probes, self-assembling nanostructures, fast screening of oligonucleotide conjugates with novel ligands72 to improve in vivo delivery, etc.

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MATERIALS AND METHODS The synthesis of azide, alkyne, and phosphoramidite derivatives (Figure 3) is presented in the Supporting Information. HPLC analysis and the purification of oligonucleotides was carried out as described previously.62 Electrospray ionization mass spectrometry (ESI-MS) analysis for the oligonucleotides was performed using an Agilent 1260-Bruker Maxis Impact system as described earlier62 with minor modifications. The HPLC instrument was equipped with the 2.1 × 50 mm Jupiter C18 column (5 μm, Phenomenex); buffer A: 10 mM diisopropylamine and 15 mM 1,1,1,3,3,3-hexafluoroisopropanol; buffer B: 10 mM diisopropylamine, 15 mM 1,1,1,3,3,3-hexafluoroisopropanol, and 80% MeCN. Salts were washed out with buffer A (4 CV) followed by a step of 100% buffer B (2 CV) with a flow rate of 0.3 mL/min and a temperature of 45 °C. The MS analysis of the oligonucleotides was carried out in negative mode (capillary voltage of 3500 V and dry temp of 160 °C), and raw spectra were deconvoluted by the maximum entropy method. Optimization of Solid-Phase CuAAC for 5′-AlkyneModified T10 Oligonucleotide. The 5′-alkyne-modified T10 oligonucleotide was assembled in ASM-2000 (Biosset) or MM12 (Bioautomation) oligonucleotide synthesizers using the phosphoramidite method. Protected 2′-deoxyribonucleoside 3′phosphoramidites, Unylinker-CPG (500 Å) and S-ethylthio1H-tetrazole were purchased from ChemGenes. A solution of 6-R6G azide (100 mM) and a copper catalyst (100 mM) in an aprotic bipolar solvent (DMSO, DMF, or DMAA, 100 μL) was added to the 5′-alkyne-modified T10 DNA oligonucleotides bound to the solid phase in the column (1 μmol synthesis scale, F


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Solid-Phase CuAAC Synthesis of GalNAc Oligonucleotide Conjugates. Alkyne-modified oligonucleotides were prepared on the solid phase (Table S2) followed by CuAAC conjugation on the solid phase in the presence of 100 mM CuI· P(OEt)3 in DMAA (4 × 100 μL, 2 h). We always used at least 2 equiv of the dissolved component (either azide or alkyne), so the concentration of azide varied for each reagent (Table S3). In the case of diazide and tetrazide conjugation, concentration was increased up to 50 mM to exclude simultaneous coupling to neighbor oligonucleotides or to several internal alkyne moieties. Homo-T oligonucleotides were cleaved from the support and deprotected using common AMA solution (concentrated aqueous ammonia and 40% aqueous methylamine, 1:1 v/v) for 30 min at 65 °C. In the case of 2′-fluoromodified oligonucleotides, deprotection was performed using concentrated aqueous ammonia at 25−27 °C for 24 h. HPLC purification and analysis was performed as described above.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.7b00462. Additional details on experimental procedures, NMR spectra of all new compounds, and HPLC profiles. Tables showing the conjugation of small molecules, alkyne-modified oligonucleotides, the conjugation of GalNAc. Figures showing HPLC traces. (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Ilya O. Aparin: 0000-0001-8818-485X Vladimir A. Korshun: 0000-0001-9436-6561 Timofei S. Zatsepin: 0000-0003-0030-9174 Author Contributions #

V.M.F., E.A.U, and Y.V.M. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The research was supported by a Skoltech internal grant (to T.S.Z.) and in part by Russian Science Foundation (to V.A.K., project no. 15-15-00053), RFBR (to V.A.B., project no. 17-5404111), and BRFFR (to Y.V.M., project no. M17PM-047). We are grateful to Ms. D. Leboeuf (Skoltech) for critical reading of the manuscript.

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ABBREVIATIONS CuAAC, copper-catalyzed alkyne−azide cycloaddition; GalNAc, N-acetylgalactosamine; SPAAC, strain-promoted alkyne− azide cycloaddition; TBTA, Tris[(1-benzyl-1H-1,2,3-triazol-4yl)methyl]amine

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REFERENCES

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