Oligonucleotide Tagging for Copper-Free Click Conjugation

Copper-free click chemistry between cyclooctynes and azide is a mild, fast and selective technology for conjugation of oligonucleotides. However, technology for site-specific introduction of the requisite probes by automated protocols is scarce, while the reported cyclooctynes are large and hydrophobic. In this work, it is demonstrated that the introduction of bicyclo[6.1.0]nonyne (BCN) into synthetic oligonucleotides is feasible by standard solid-phase phosphoramidite chemistry. A range of phosphoramidite building blocks is presented for incoporation of BCN or azide, either on-support or in solution. The usefulness of the approach is demonstrated by the straightforward and high-yielding conjugation of the resulting oligonucleotides, including biotinylation, fluorescent labeling, dimerization and attachment to polymer.


Introduction
Synthetic DNA and RNA oligonucleotides (ONs) are key tools in a broad variety of diagnostic and therapeutic applications, including microarray technology [1], antisense and gene-silencing therapies [2], nanotechnology [3] and materials sciences [4,5].Generally, such applications require the introduction of a suitable handle in an oligonucleotide to enable selective conjugation to a functionality of interest [6][7][8].For example, attachment of a cell-penetrating ligand is the most commonly applied strategy to tackle the low internalization rate of ONs into target cells [2], currently the main bottleneck in oligonucleotide-based therapeutics (antisense, siRNA).Similarly, the preparation of oligonucleotide-based microarrays requires the selective immobilization of ONs on a suitable solid surface, e.g., glass [1].Conventional post-synthetic labeling protocols, based on amide bond formation or sulfide-based chemistry [8][9][10] typically suffer from low yield and long reaction times and often require a high concentration of the biomolecule in combination with a large excess of coupling partner.One promising alternative to the traditional conjugation technologies involves the copper-catalyzed cycloaddition of alkynes and azides, a procedure commonly referred to as "click reaction" [11,12].However, the use of copper for oligonucleotide conjugation may be compromised due to potential metal-catalyzed strand degradation and/or difficulties in final purification [13][14][15].Although new ligands reduce the chance of undesired chain cleavage during copper-catalyzed click reaction [16][17][18], strain-promoted azide-alkyne cycloaddition (SPAAC) offers the possibility of oligonucleotide conjugation in the absence of copper [19][20][21][22][23] as demonstrated for oligonucleotides labeled with plain cyclooctyne (OCT) [15] or the more reactive dibenzofused cyclooctyne DIBO [24].Most recently, Brown et al. [25,26] further extended the latter approach by ON incorporation of aminoalkyl thymidine derivatives, followed by selective N-acylation with azide or cyclooctyne after cleavage from support.Alternative approaches for the preparation of azide-containing nucleotides-compromised by the incompatibility of azide with phosphoramidite chemistry-involve post-synthetic nucleophilic substitution [27][28][29] or selective diazotransfer reaction [30] or phosphonate-based coupling chemistry [31][32][33][34] However, a simple and general strategy for the on-support, automated synthesis of oligonucleotides with readily accessible building blocks, and suitable for introduction of any functional group (including cyclooctyne and azide), is still desirable.
We here report two versatile approaches for conjugation of oligonucleotides by strain-promoted azide-alkyne cycloaddition.First, a range of novel phosphoramidite building blocks was developed for incorporation of bicyclo[6.1.0]nonyne(BCN) [35] and an adenosine-based building block is presented suitable for BCN or azide introduction following standard oligonucleotide synthesis protocols, and allowing multiple nucleotide 2′-functionalization (Figure 1).The ease of operation of copper-free click conjugation is demonstrated for a range of functional groups, by oligonucleotide dimerization, and by the preparation and characterization of an amphiphilic polythiophene-oligonucleotide hybrid polymer.

Preparation of BCN-Phosphoramidites
Earlier reported approaches for copper-free conjugation of oligonucleotides were based solely on DIBO, a dibenzofused cyclooctyne that inevitably leads to a mixture of regioisomeric and diastereomeric adducts upon reaction with azide.We reasoned that bicyclo[6.1.0]nonyne(BCN) has particular potential for oligonucleotide conjugation, due to its higher reactivity in comparison to DIBO, its relatively low lipophilicity in comparison all dibenzofused cyclooctynes [36], and its plane-symmetry, which precludes the formation of regioisomers upon cycloaddition.Thus, phosphoramidite derivative 1 was, prepared in a single step from commercially available BCN alcohol (R=OH) with 81% yield (Scheme 1), as well as a diethyleneglycol chain-extended phosphoramidite 2. Scheme 1. Synthesis of BCN-phosphoramidites 1 and 2.

Activation and Incorporation of BCN-Phosphoramidites
Next, compound 1 was activated with 5-ethylthiotetrazole (ETT) and attached to hexathymidine nucleotide (3, n = 6), supported on controlled pore glass (Scheme 2), leading to a single ON after oxidation and cleavage from support, as indicated by HPLC.However, mass spectrometry indicated that not the expected BCN-containing diester 5, but 5′-monophosphate 4 had been isolated instead.We attribute the formation of 4 to rapid hydrolysis of the transiently formed diester phosphate 5, presumably involving a heterolytic cleavage mechanism with formation of a surprisingly stable BCN-derived cyclopropylmethyl cation [37].Based on the latter assumption, phosphoramidite 2 was next subjected to the same oligonucleotide synthesis protocol, now leading to the isolation of the desired 5′-BCN-containing hexanucleotide 6 in high yield.Another successful strategy to avoid the formation of a cyclopropylmethyl cation involved the preparation of hexathymidine conjugate ON 7, containing a homologated BCN ethyl derivative (Scheme 3).Scheme 2. Solid-phase synthesis of BCN-charged oligonucleotides 5-8.Scheme 3. Synthesis of exo-BCN-ethanol phosphoramidite for the preparation of 7.

Comparison of BCN-Containing Oligonucleotides to Dibenzofused Cyclooctynes
Now the stage was set to compare the lipophilicity of the oligonucleotides 6 and 7 containing a BCN-type cyclooctyne to a dibenzoannulated cyclooctyne.To this end, we prepared DBCO-containing hexa-T (compound 8 in Scheme 2) from commercially available DBCO-phosphoramidite. To our satisfaction, C18-reversed phase HPLC analysis confirmed the higher polarity of BCN-containing ONs 6 and 7 (elution after 13.2 and 12.0 min, respectively) with respect to DBCO-containing ON 8 (19.6 min).The usefulness of BCN-containing ONs for metal-free cycloaddition with azide was also evaluated, by addition of desthiobiotin azide 9 to BCN-charged ON 7 (Figure 2B).HPLC analysis indicated a rapid and quantitative cycloaddition of 6 and 9 (Figures 2C and S1), to give the expected triazole adduct 10 in only 75 min, thereby corroborating the usefulness of 5′-BCN incorporation for copper-free conjugation of oligonucleotides.

3′-Fmoc-Aminopropyl Adenosine for Internal Labeling and Conjugation of Oligonucleotides
Despite the promising results with BCN-derived phosphoramidites for the preparation of 5′-functionalized ONs, the introduction of BCN at other positions in an oligonucleotide (3′-end or internally) is not readily accessible with simple building blocks.Moreover, it is clear that a phosphoramidite-based strategy for the introduction of an azide group, the complementary partner for SPAAC, is hampered by competitive Staudinger reduction of azide with P III -type reagents [27,31].Therefore, our next aim was to develop a generic building block for internal incorporation in an ON chain, to facilitate subsequent on-support derivatization with any functional group of choice, including BCN or azide.

Preparation, Incorporation and Model Studies
Based on the above reasoning, a straightforward synthetic route towards Fmoc-protected 2′-O-aminopropyl adenosine-based building block 13 was designed (Scheme 4).Importantly, the 2′-aminopropyl group would ensure subsequent selective functionalization after Fmoc-deprotection.A similar strategy was recently reported based on a 2′-aminoethyl thymidine building block [25], but to the best of our knowledge on-support oligonucleotide functionalization has not been reported to date.An advantage of modification via 2′-OH of ribose, instead of conjugation via the nucleobase, is that negligible interference with hybridization is expected, due to the direction of a 2′-O-functional group towards the minor groove of a DNA duplex.
Thus, starting phosphoramidite 13 was conveniently prepared in only three high-yielding steps from readily available 2′-(3-azidopropyl) adenosine building block 11 [38] by Staudinger reduction, Fmoc-protection and phosphitylation (Scheme 4).Next, CPG-immobilized thymidine (3) was 5′-chain extended with building block 13 (Scheme 5) under standard conditions.The successful formation of the projected phosphate diester 14 was corroborated by Fmoc removal (20% piperidine in DMF) to give intermediate 15, followed by cleavage from CPG, leading to the free amino-derivative 16 in high purity, as confirmed by HPLC and HRMS (Figure S2 and Table S1).Alternatively, the Fmoc-deprotected dinucleotide 15 was subjected to on-support acylation before cleavage with NH 4 OH, thereby generating a range of DMT-on dinucleotides functionalized with phenylalanine (17), biotin (18) or fluorescein (19) 22) afforded the anticipated triazole adduct in quantitative yield (Figure S2 and Table S1).A similar smooth reaction was observed for dimerization of BCN-charged dinucleotide 20 and azide-charged dinucleotide 21, affording exclusively the (3+2) cycloaddition product, as confirmed by HPLC and LC-MS analysis (Figure S2 and Table S1).

Oligonucleotide Dimerization
Now the stage was set to evaluate the scope of building block 13 for the synthesis and functionalization of longer oligonucleotides, and to explore the usefullness of SPAAC to obtain site-specifically conjugated oligonucleotides (Scheme 6).As anticipated, attachment of Fmoc building block 13 to a CPG-tetranucleotide, and subsequent repetitive coupling with standard ON building blocks, proceeded smoothly to give the 12-mer ON 23 with sequence d(AGTATTGX*CCTA) (X* = 2′-Fmoc-N-propyladenosine), as corroborated by cleavage from CPG of an analytical sample.Next, undecanucleotide 23 was on-support Fmoc-deprotected with piperidine (to 24) and coupled with azidohexanoic acid or a BCN-derived active carbonate and cleaved from support, to give the respective azide derivative 25 and BCN-derivative 26, respectively.As anticipated, overnight stirring of a 1:1 mixture of the BCN-and the azide-functionalized ON conjugates 26 and 25, respectively, was found to give the desired oligonucleotide dimer 27, as confirmed by HPLC and MALDI-TOF analysis (Table S2), thereby demonstrating for the versatility of our approach for conjugation of oligonucleotides at any adenosine in the ribose backbone.Scheme 6. On-support preparation of azide or BCN-charged oligonucleotides and dimerization in solution.

Oligonucleotide-Polythiophene Hybrid
Finally, we were intrigued by the idea of applying SPAAC for the synthesis of functional oligonucleotide-containing materials, in particular toward the construction of bioresponsive films.Poly(3-hexylthiophene) (P3HT) is a well known electroconductive material which can be found in solar cells and other nanoelectronic devices but is known to be notoriously insoluble in aqueous systems.One potential strategy for solubilization would involve attachment of oligonucleotides to these synthetic lipophilic polymers.To this end, azido-functionalized P3HT (28) was treated with BCN-conjugated ON 5′-d(AGTATTGXCCTA)-3′ (26) and the reaction was monitored by color change as well as UV-VIS spectroscopy (Figure 3).To our satisfaction, mixing 28 and 26 led to the formation of a yellowish solution, thereby indicating the formation of composites through (3+2) cycloaddition between P3HT and ON as the result of the slow solubilization of the otherwise completely water-insoluble hydrophobic polymer 28.More conclusive support for succesfull conjugation was obtained by UV-VIS spectroscopy, which clearly revealed the presence of P3HT in the aqueous solution as indicated by the appearance of absorption peak at 455 nm (Figure 3C), as well as by MALDI-TOF analysis (Figure S4).Automated ON synthesis was completed using a Millipore Expedite (8900 series) nucleic acid synthesis system using the recommended thymidine conditions for each synthesis cycle in a DMT-on protocol for 0.2 Mol columns.Standard synthesizer reagents and thymidine-CPG were obtained from Glen Research Corporation.The ONs were deprotected and cleaved from the CPG support by manually passing conc.NH 4 OH back and forth through the column with a pair of syringes for 15 min.The resulting ON solutions were sparged with N 2 for 3 hours to remove excess NH 3 .The concentrated solutions were frozen and lyophilized.

General procedure for the synthesis of 17-21
A solution of corresponding carboxylic acid (0.019 mmol), HATU (0.019 mmol) and NMM (5 L) in DMF (200 L), was added to the dried Fmoc-off CPG 15.The resulting mixture was stirred for 2 h at rt and filtered off.It was further washed with DMF (2 × 1 mL), MeCN (2 × 1 mL), CH 2 Cl 2 (2 × 1 mL) and dried.The corresponding coupled dinucleotides 17-21 were cleaved from CPG with aq.NH 4 OH solution (0.5 mL) for 12 h, 55 °C and confirmed by HPLC and HR-MS analysis.

SPAAC reactions and analysis of 20 with 21 and 22
The solutions of 20 (50 mL) and 21 (100 mL) or 22 (100 mL, 1 mg/100 mL) in water were mixed for 2 min to afford the (3+2) cycloaddition product, as confirmed by HPLC and HR-MS analysis.oligonucleotides were found to undergo fast SPAAC functionalization or dimerization, and were even suitable for conjugation to lipophilic polymers.The coupling of oligonucleotide to azide-substituted polythiophene opens the possiblity for the construction of a variety of simple field effect transistor biodevices in which sequence specific ON functionalized conjugated polymers are the key component.In a broader context, we have demonstrated the unique combination of reaction efficiency and selectivity of cyclooctyne-based chemistry for the conjugation of sensitive (bio)molecules in aqueous systems, which may be readily extended toward the conjugation of BCN-oligonucleotides to azidecontaining solid surfaces, polymers and large proteins.Finally, we [39] and others [40,41] recently demonstrated that cycloadditions of BCN is not limited to azides, but BCN also undergoes extremely fast strain-promoted inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC) with tetrazines.In contrast, benzofused cyclooctynes DBCO and DIBO are inreactive towards tetrazine [42], which further lifts the potential of BCN-modified oligonucleotides for fast and selective bioconjugations, potentially also in vivo [43].Research along this line, as well as extension of the strategy towards other 2′-O-alkylated nucleobases, is currently ongoing in our laboratory.

Figure 1 .
Figure 1.Structures of BCN-and adenosine-based phosphoramidites for incorporation into and copper-free conjugation of oligonucleotides.

Scheme 5 .
Scheme 5. On-support incorporation and conjugation of 13 into a dinucleotide.

Figure 3 .
Figure 3. (A) Conjugation of azido-terminated P3HT 28 and BCN-containing oligonucleotide 26 leading to 29. (B) Aqueous solution of 28 turns yellow as a consequence of spontaneous (3+2) cycloaddition leading to solubilization.(C) UV-VIS spectrum of aqueous solution shows the appearance of the typical absorption band of P3HT at 455 nm.