Synthesis and Excellent Duplex Stability of Oligonucleotides Containing 2′-Amino-LNA Functionalized with Galactose Units

A convenient method for the preparation of oligonucleotides containing internally-attached galactose and triantennary galactose units has been developed based on click chemistry between 2′-N-alkyne 2′-amino-LNA nucleosides and azido-functionalized galactosyl building blocks. The synthesized oligonucleotides show excellent binding affinity and selectivity towards complementary DNA/RNA strands with an increase in the melting temperature of up to +23.5 °C for triply-modified variants.

Herein, we report synthesis and characterization of ONs modified via copper(I)-catalyzed click chemistry with galactose derivatives M 2 and M 3 .

Scheme 1. Synthesis of galactopyranoside intermediates.
The amino group of compound 4 [62] was subsequently reacted with 2-azidoacetic acid in (HATU) and N,N-diisopropylethylamine (DIPEA) to afford azido derivative 5 in 76% yield. The ester groups were next converted into the corresponding acids to give 6, followed by reaction with pentafluorophenol (PFP) in dichloromethane (DCM) in the presence of N,N-diisopropylcarbodiimide (DIC) to afford tris-pentafluorophenol ester 7 in 77% yield. The Boc-protecting group of galactopyranoside 3 was removed by standard conditions (1:3 trifluoroacetic acid (TFA)/DCM, 1 h) yielding the corresponding amino galactopyranoside intermediate 3a. Further reaction with tris-pentafluorophenol ester 7 furnished the triantennary compound 8 in 69% yield, which upon ester cleavage was converted into the desired triantennary azido galactopyranoside 9 in 75% yield (Scheme 2).

Synthesis of Azido-Functionalized Galactose Derivatives
(1) and 2-[2-(2-azidoethoxy)ethoxy]ethoxy-β-D-galactopyranoside (2) were synthesized according to procedures in the literature [60,61]. For synthesis of compound 3 we first tried the reduction of azido group 1 into an amino group with 10% Pd/C in different organic solvents (MeOH/EtOH/EtOAc) in an H 2 atmosphere. We observed in every case intramolecular acetyl migration from the sugar to the amine. To avoid this acetyl migration the amine was directly equipped with a Boc-protecting group after azide reduction with Pd/C in EtOAc furnishing galactopyranoside 3 in 79% yield (Scheme 1). Among multiple DNA linking strategies developed to date, the copper(I)-catalyzed azide alkyne cycloaddition (CuAAC) reaction is a robust and highly adaptable bioconjucation process ("click reactions") for ONs allowing preparation of various conjugates in high yields [50][51][52][53], and single to triple modification of oligonucleotides by this reaction has been reported [54][55][56][57][58][59].
Herein, we report synthesis and characterization of ONs modified via copper(I)-catalyzed click chemistry with galactose derivatives M 2 and M 3 .   [60,61]. For synthesis of compound 3 we first tried the reduction of azido group 1 into an amino group with 10% Pd/C in different organic solvents (MeOH/EtOH/EtOAc) in an H2 atmosphere. We observed in every case intramolecular acetyl migration from the sugar to the amine. To avoid this acetyl migration the amine was directly equipped with a Boc-protecting group after azide reduction with Pd/C in EtOAc furnishing galactopyranoside 3 in 79% yield (Scheme 1).  The amino group of compound 4 [62] was subsequently reacted with 2-azidoacetic acid in N,N-dimethylformamide/tetrahydrofuran (DMF/THF) in the presence of 1-[bis(dimethylamino) methylene]-1H-1,2,3-triazolo-[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) and N,N-diisopropylethylamine (DIPEA) to afford azido derivative 5 in 76% yield. The ester groups were next converted into the corresponding acids to give 6, followed by reaction with pentafluorophenol (PFP) in dichloromethane (DCM) in the presence of N,N-diisopropylcarbodiimide (DIC) to afford tris-pentafluorophenol ester 7 in 77% yield. The Boc-protecting group of galactopyranoside 3 was removed by standard conditions (1:3 trifluoroacetic acid (TFA)/DCM, 1 h) yielding the corresponding amino galactopyranoside intermediate 3a.

ON Synthesis
Synthesis of 9-mer ONs was performed at a 1.0 μmol scale using standard solid-phase phosphoramidite chemistry on an automated DNA synthesizer. Phosphoramidite building block 10 [54] showed a satisfactory step-wise coupling yield of >94% using 1H-tetrazole as the activator and a coupling time of 20 min used for incorporation of monomer M 1 into ONs, one or three times according to Lou et al. [63] to afford ON1 (5′-GTGAM 1 ATGC) and ON2 (5′-GM 1 GAM 1 AM 1 GC) (Scheme 3). The synthesized ONs were passed through NAP-10 column, and their purity (>90%) and composition was confirmed by ion-exchange (IE) HPLC and MALDI-MS (see Section 3.8), respectively. Scheme 2. Synthesis of triantennary azido galactopyranoside.

ON Synthesis
Synthesis of 9-mer ONs was performed at a 1.0 µmol scale using standard solid-phase phosphoramidite chemistry on an automated DNA synthesizer. Phosphoramidite building block 10 [54] showed a satisfactory step-wise coupling yield of >94% using 1H-tetrazole as the activator and a coupling time of 20 min used for incorporation of monomer M 1 into ONs, one or three times according to Lou et al. [63] to afford ON1 (5 -GTGAM 1 ATGC) and ON2 (5 -GM 1 GAM 1 AM 1 GC) (Scheme 3). The synthesized ONs were passed through NAP-10 column, and their purity (>90%) and composition was confirmed by ion-exchange (IE) HPLC and MALDI-MS (see Section 3.8), respectively.

Binding Affinity
The effect upon incorporation of one (ON3 and ON5) or three (ON4 and ON6) galactopyranosyl modifications into mixed sequence 9-mer ONs on the thermal stability and base-pairing specificity of duplexes with DNA and RNA complements was evaluated by UV-VIS thermal denaturation experiments (Tables 1 and 2). All changes in thermal denaturation temperatures (Tm) of modified nucleic acid duplexes are discussed relative to the Tm values measured for the unmodified reference duplexes.

Binding Affinity
The effect upon incorporation of one (ON3 and ON5) or three (ON4 and ON6) galactopyranosyl modifications into mixed sequence 9-mer ONs on the thermal stability and base-pairing specificity of duplexes with DNA and RNA complements was evaluated by UV-VIS thermal denaturation experiments (Tables 1 and 2). All changes in thermal denaturation temperatures (T m ) of modified nucleic acid duplexes are discussed relative to the T m values measured for the unmodified reference duplexes.
ON7 and ON8 containing one or three non-conjugated 2 -amino-LNA-T monomers showed a substantially increased thermal affinity towards DNA (∆T m value +9.5 • C with three monomers M 4 ) and RNA complementary strands (∆T m value +20.0 • C with three monomers M 4 ) ( Table 1). Remarkably, the corresponding ONs containing one or three monomer(s) M 2 or M 3 (ON3-ON6) display similar increases in thermal affinity, with ON5:DNA (single incorporation of M 3 ) as the only exception (∆T m value of only +2.0 • C (Table 1)). These results demonstrate that both the mono-and triantennary galactopyranosyl unit are well tolerated in a short ON aimed at DNA or RNA targeting.

Binding Specificity
Next, the binding specificities of single-and triple-modified oligonucleotides were evaluated against centrally-positioned mismatched bases (Table 2). We found, in general, very similar pairing selectivity for all sequences (ON ref , ON3-ON8) with the notable exceptions that the modified strands all showed better discrimination against a DNA or RNA G mismatch than the control, and that the control showed better discrimination against an RNA C mismatch than the modified sequences.
All DNA ON ref displayed a thermal denaturation temperature of 29.0 • C with a DNA complementary strand, and an RNA counterpart at 27.0 • C. The results of ON3 showed that the monoantennary galactopyranosyl functionalized triazole-linked 2 -amino-LNA was well tolerated in a DNA/DNA duplex as the thermal denaturation temperature was comparable with that of ON ref (Table 1). A remarkable stabilization at 8.5 • C was observed with the RNA complement. The corresponding three monoantennary galactopyranosyl units in ON4 was better tolerated as the thermal denaturation temperature was induced by 11.0 • C against a DNA complement and 23.5 • C against an RNA complement. In general, similar trends were observed in ON5-ON8 showed increased thermal duplex stability against both DNA and RNA relative to ON ref , but the sequence containing a singly-incorporated triantennary galactopyranosyl unit in ON5 stabilized less than the non-conjugated 2 -amino-LNA ON7. Additionally, the corresponding non-conjugated 2 -amino-LNA (ON8) was better tolerated than the triply-incorporated triantennary galactopyranosyl unit in ON6 against a DNA complement, and a reverse trend was observed in an RNA complement, respectively (Table 1). For evaluation of the mismatch discriminative properties of these mono-/triantennary galactopyranosyl-conjugated and non-conjugated 2 -amino-LNA ONs were chosen with one mismatched nucleotide opposite the sugar modification. All modifications showed similar, or even enhanced, mismatch discrimination relative to the unmodified strand ON ref , with the discriminative power generally superior against DNA strands (Table 2).
In summary, singly-or triply-incorporated galactopyranose moieties induced significantly increased duplex stability relative to the reference duplexes, thus expanding the results reported for galactopyranose-modified carbohydrate moieties attached to the 2 -position of nucleotides via triazole [64] and amide linkages [65,66]. The data reported herein, in combination with the remarkable exonuclease resistance induced by other N-functionalized 2 -amino-LNA nucleotides [63,65], establish monomers M 2 and M 3 as promising constituents of future RNA-targeting oligonucleotide drugs.

General Information
All reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification. Dichloromethane, N,N-diisopropylethylamine (DIPEA), THF, methanol, N,N-dimethylformamide (DMF) and pyridine were dried over activated 4 Å molecular sieves, and petroleum ether (PE) and ethylacetate (EtOAc) were used as received. All reactions conducted in anhydrous solvents were carried out under an argon atmosphere. Silica gel column chromatography was performed using Merck Millipore silica gel 60 (0.040-0.063 mm) (Darmstadt, Germany). Thin layer chromatography (TLC) was performed using Merck silica gel 60 F254 (0.22 mm thickness, aluminum-backed) (Darmstadt, Germany). Compounds were visualized at 254 nm or stained with 5% sulfuric acid in EtOH. 1 H-NMR spectra were measured at 400 MHz, 13 C-NMR spectra were measured at 101 MHz, and 19 F-NMR spectra were measured at 376 MHz, all on a Bruker AVANCE III 400 spectrometer (Billerica, MA, USA). Chemical shifts are given in ppm and J values are given in Hz. All assignments for 1 H-NMR and 13 C-NMR have been confirmed by COSY, HSQC, and HMBC. HRMS-ESI spectra were recorded on a Bruker APEX III FT-ICR mass spectrometer (Billerica, MA, USA). (3) 2-[2-(2-Azidoethoxy)ethoxy]ethoxy-2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside (1, 500 mg, 0.99 mmol) was dissolved in EtOAc (5.0 mL), and 10% Pd/C (6.0 mg) and di-tert-butyl dicarbonate (324 mg, 1.48 mmol) were added. The reaction mixture was stirred at r.t. in an H 2 atmosphere for 6 h according to a literature procedure [67]. The reaction mixture was passed through a celite pad and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography using 20-40% EtOAc in petroleum ether (v/v) as eluent to afford compound 3 as a viscous oil (451 mg) in 79% yield.

Synthesis and Purification of ON1-ON2
ONs were synthesized on a DNA synthesizer (PerSpective Biosystems Expedite 8909, (Framingham, MA, USA)) in 1.0 µmol scale using manufacturer's standard protocols. For incorporation of monomer M 1 [56] a hand-coupling procedure [68] was used (20 min coupling time and 5-[3,5-bis(trifluoromethyl)phenyl]-1H-tetrazole (0.25 M, in anhydrous CH 3 CN) as activator). The coupling efficiencies of standard DNA phosphoramidites and phosphoramidite 10 based on the absorbance of the dimethoxytrityl cation released after each coupling varied between 95% and 98%. Cleavage from solid support and removal of nucleobase protecting groups was performed using 32% aq. ammonia for 16 h at 55 • C. The resulting oligonucleotides were purified by DMT-ON RP-HPLC using Waters System 600 (Milford, MA, USA) equipped with Xterra MS C18-column (5 µm, 150 mm × 7.8 mm, (Milford, MA, USA)). Elution was performed starting with an isocratic hold of A-buffer for 5 min followed by a linear gradient to 70% B-buffer over 40 min at a flow rate of 1.0 mL/min (A-buffer: 0.05 M triethyl ammonium acetate, pH 7.4; B-buffer: 25% buffer A, 75% CH 3 CN). RP-purification was followed by detritylation (80% aq. AcOH, 30 min) and precipitation (acetone, −18 • C, 12 h). The identity and purity of ON1 and ON2 were verified by MALDI-TOF mass spectrometry (Billerica, MA, USA) ( Table 3) and IE HPLC, respectively. IE HPLC was performed using a Merck Hitachi LaChrom instrument (Tokyo, Japan) equipped with a Dionex DNAPac Pa-100 column (250 mm × 4 mm, Sunnyvale, CA, USA). Elution was performed starting with an isocratic hold of A-and C-buffers for 2 min followed by a linear gradient to 60% B-buffer over 28 min at a flow rate of 1.0 mL/min (A-buffer: Milli-Q water; B-buffer: 1 M NaClO 4 , C-buffer: 25 mM Tris-Cl, pH 8.0). MALDI-TOF mass-spectrometry was performed using a MALDI-LIFT system on the Ultraflex II TOF/TOF instrument from Bruker (Billerica, MA, USA) using an HPA-matrix (10 mg 3-hydroxypicolinic acid, 50 mM ammonium citrate in 70% aq. CH 3 CN).

Thermal Denaturation Studies
Thermal denaturation studies were carried out on a Perkin Elmer Lambda 35 UV-VIS spectrometer (Shelton, CT, USA) using a Hellma SUPRASIL synthetic quartz 10 mm path length cuvettes. The oligonucleotides (1.0 µM per strand) were dissolved in medium salt buffer [NaCl (100 mM), EDTA (0.1 mM), NaH 2 PO 4 (10 mM), Na 2 HPO 4 (5 mM), pH 7.0] and the resulting solution heated to 90 • C for 10 min and then slowly cooled down to 5 • C. Concentrations of oligonucleotides were calculated using the extinction coefficients. UV absorbance at 260 nm as a function of time was recorded and the thermal denaturation temperatures (T m ) were determined as the maxima of the first derivative of the UV 260 vs. temperature curves. The T m values are given as averages of two measurements within ±0.5 • C.

Postsynthetic Click Procedure in Solution
ON ON1 or ON2 (40 nmol) was dissolved in fresh MQ water (190 µL for one incorporation and 151 µL for three incorporations) in a 1.0 mL microwave vial, and 2 M tetra triethylammonium acetate buffer (pH 7.4; 20 µL), DMSO (84 µL for one incorporation and 52 µL for three incorporations), the corresponding azide 2 or 9 (16 µL (ON1) for one incorporation and 48 µL (ON2) for three incorporations of 10 mM solution in DMSO), CuSO 4 -TBTA equimolar complex (10 µL of 10 mM stock solution) and ascorbic acid (10 µL of 25 mM freshly prepared stock solution) were subsequently added under an argon atmosphere. The resulting mixture was deaerated, tightly closed, and subjected to microwave conditions (microwave reactor, 60 • C, 2 h, Biotage initiator microwave synthesizer). Thereafter, the reaction was cooled to r.t. and then filtrated through an IIIustra NAP-10 column following manufacture's protocol. The resulting solution was evaporated to dryness at r.t. under a nitrogen atmosphere. The resulting conjugates ON3-ON6 were analyzed by MALDI TOF mass spectrometry (Table 4) and IE HPLC. The ONs (ON4 and ON6) were further purified by RP HPLC. The yields of the click reactions were determined based on the absorbance of the ONs at 260 nm: 92% (ON3), 20% (ON4), 77% (ON5), and 17% (ON6).

Conclusions
In summary, we have successfully developed the copper(I)-catalyzed azide alkyne cycloaddition click chemistry conjugation of mono-and triantennary galactospyranosyl units to internally-positioned amino-LNA nucleotides. This method is efficient and provides the desired products in a remarkable purity. High-affinity recognition of complementary DNA and RNA strands has been demonstrated, thus confirming the suitability of the N2 -position 2 -amino-LNA nucleotides for attachment of targeting ligands for therapeutic oligonucleotide constructs.

Conflicts of Interest:
The authors declare no conflict of interest.