Synthesis of Oligonucleotides Containing 2′-N-alkylaminocarbonyl-2′-amino-LNA (2′-urea-LNA) Moieties Using Post-Synthetic Modification Strategy

The post-synthetic modification of an oligonucleotide is a powerful strategy for the synthesis of various analogs of the oligonucleotide, aiming to achieve the desired functions. In this study, we synthesized the thymidine phosphoramidite of 2′-N-pentafluorophenoxycarbonyl-2′-amino-LNA, which was introduced into oligonucleotides. Oligonucleotides containing a 2′-N-pentafluorophenoxycarbonyl-2′-amino-LNA unit could be isolated under ultra-mild deprotection conditions (50 mM K2CO3 in MeOH at room temperature for 4 h). Moreover, by treatment with various amines as a post-synthetic modification, the oligonucleotides were successfully converted into the corresponding 2′-N-alkylaminocarbonyl-2′-amino-LNA (2′-urea-LNA) derivatives. The duplex- and triplex-forming abilities of the synthesized oligonucleotides were evaluated by UV-melting experiments, which showed that 2′-urea-LNAs could stabilize the nucleic acid complexes, similar to the proto-type, 2′-amino-LNA. Thus, 2′-urea-LNAs could be promising units for the modification of oligonucleotides; the design of a substituent on urea may aid the formation of useful oligonucleotides. In addition, pentafluorophenoxycarbonyl, an amino moiety, acted as a precursor of the substituted urea, which may be applicable to the synthesis of oligonucleotide conjugates.


Introduction
Chemically modified oligonucleotides have been widely used in areas such as nanotechnology and drug development. The purpose of such chemical modification is to realize the desired functions depending on the specific applications. The functions of small molecules can be explored using a large number of derivatives; however, this is not easy for many modified oligonucleotides because of their synthetic difficulty. The preparation of a modified oligonucleotide is time-consuming as it involves these processes-(i) synthesis of the modified building block, and (ii) synthesis of the oligonucleotide including the building block employing an oligonucleotide synthesizer. Under these circumstances, chemical modification following the synthesis of the oligonucleotide-called "post-synthetic modification"-is a powerful strategy enabling us to prepare various derivatives from a single oligonucleotide encompassing a reactive site [1][2][3][4][5][6][7][8].
sugar and the bulkiness of the bridge moiety are expected to improve the hybridizing ability of the oligonucleotides to target nucleic acids and reduce nuclease degradation [9][10][11][12][13]. In particular, 2′amino-LNA, a 2′,4′-bridged nucleic acid, can have various substituents of the 2′-amino group [14]; therefore, 2′-amino-LNA would be a useful scaffold to explore oligonucleotides possessing the desired properties. Previous studies have reported oligonucleotides containing 2′-N-substituted 2′amino-LNA derivatives, such as 2′-N-alkyl, 2′-N-acyl, and 2′-N-alkoxycarbonyl derivatives [15][16][17][18][19][20][21][22]. In general, the synthesis was based on a common method using each modified phosphoramidite; however, post-synthetic approaches using click chemistry [20,21,23] and amidation [24] were also applied to the synthesis of the 2′-N-substituted 2′-amino-LNA derivatives in the oligonucleotides ( Figure 1). The substrates containing the reactive sites are somewhat specific, and the 1,2,3-triazole and glycyl units remain after post-synthetic modification. Thus, the development of a new postsynthetic modification method for the 2′-N-substituted 2′-amino-LNA is essential. We considered that 2′-amino-LNA bearing an active carbamate, like a pentafluorophenyl carbamate, could be converted into 2′-N-alkylaminocarbonyl-2′-amino-LNA (2′-urea-LNA) via the post-synthetic treatment with amines. With this method, various amines that are commercially available or easily synthesized can be used and the procedure is simple to perform (amine treatment). Moreover, urea is the only unit that remains on the oligonucleotide. We synthesized oligonucleotides containing various 2′-urea-LNA derivatives using post-synthetic modification and evaluated their duplex-and triplex-forming ability. The details are described herein.

Synthesis
The synthesis of thymidine phosphoramidites with various 2′-N-alkoxycarbonyl-2′-amino-LNA modifications was previously reported by us [22]. Thus, according to the procedure, a thymidine phosphoramidite with 2′-N-pentafluorophenoxycarbonyl-2′-amino-LNA modification was synthesized as shown in Scheme 1. Compound 1 was treated with bis(pentafluorophenyl) carbonate in the presence of Et3N to produce the 2′-N-pentafluorophenoxycarbonyl derivative (2) in 89% yield. In this reaction, no 3′-O-pentafluorophenoxycarbonyl derivative was obtained, unlike the case of other alkoxycarbonyl derivatives [22]. This was probably because of the poor stability of the 3′-O- We considered that 2 -amino-LNA bearing an active carbamate, like a pentafluorophenyl carbamate, could be converted into 2 -N-alkylaminocarbonyl-2 -amino-LNA (2 -urea-LNA) via the post-synthetic treatment with amines. With this method, various amines that are commercially available or easily synthesized can be used and the procedure is simple to perform (amine treatment). Moreover, urea is the only unit that remains on the oligonucleotide. We synthesized oligonucleotides containing various 2 -urea-LNA derivatives using post-synthetic modification and evaluated their duplex-and triplex-forming ability. The details are described herein.

Synthesis
The synthesis of thymidine phosphoramidites with various 2 -N-alkoxycarbonyl-2 -amino-LNA modifications was previously reported by us [22]. Thus, according to the procedure, a thymidine phosphoramidite with 2 -N-pentafluorophenoxycarbonyl-2 -amino-LNA modification was synthesized as shown in Scheme 1. Compound 1 was treated with bis(pentafluorophenyl) carbonate in the presence Molecules 2020, 25, 346 3 of 10 of Et 3 N to produce the 2 -N-pentafluorophenoxycarbonyl derivative (2) in 89% yield. In this reaction, no 3 -O-pentafluorophenoxycarbonyl derivative was obtained, unlike the case of other alkoxycarbonyl derivatives [22]. This was probably because of the poor stability of the 3 -O-pentafluorophenoxycarbonyl derivative, resulting from the good leaving ability of the pentafluorophenoxy group. Phosphitylation of 2 using (i-Pr 2 N) 2 POCH 2 CH 2 CN and 5-(ethylthio)tetrazole afforded the desired phosphoramidite (3), which is a suitable building block for the synthesis of oligonucleotides, in 75% yield. Next, we synthesized the oligonucleotides using common phosphoramidite chemistry on an oligonucleotide synthesizer; the sequences of the oligonucleotides are shown in Scheme 1. Phenoxyacetyl (Pac) and isopropylphenoxyacetyl (i-PrPac) protections were used for the dA and dG phosphoramidites, respectively. Furthermore, the nucleobases in dC and 2′-deoxy-5-methylcytidine (d m C) phosphoramidites were acetyl-protected. The coupling time was increased from 25 s to 10 min when phosphoramidite (3) was introduced into the oligonucleotides, and the coupling efficiency was estimated to be over 95%, based on the trityl monitoring observed in the removal of the 5′-DMTr group. After the synthesis of the oligonucleotides on a DNA synthesizer, the fully protected oligonucleotides attached to control pore glass (CPG) resin were subjected to ultra-mild conditions (50 mM K2CO3 in MeOH at room temperature for 4 h) to produce the corresponding 5′-O-DMTroligonucleotides via the removal of the cyanoethyl groups in the phosphotriester moieties and the protecting groups in nucleobases, followed by cleavage from the resin. The 2′-Npentafluorophenoxycarbonyl unit was tolerant of the base treatment. The DMTr-removal and purification step yielded the desired oligonucleotides modified with 2′-Npentafluorophenoxycarbonyl-2′-amino-LNA.
The 2′-N-Pentafluorophenoxycarbonyl-2′-amino-LNA was converted within an oligonucleotide (ON1) by the treatment with various amines (Scheme 2 and Table 1). The treatment with 10 M NH3 aq. at 30 °C for 4 h left the unreacted oligonucleotide (ON1), although the production of the corresponding unsubstituted 2′-urea-LNA was also observed (ON3a) (Figure 2a). The prolonged reaction time to 24 h yielded the desired ON3a with high efficiency, and it was isolated in 76% yield ( Figure 2b). More than half of the ON1 remained in the treatment with 0.1 M MeNH2 aq. at 30 °C for 2 h (Figure 2c). ON1 almost disappeared at an increased concentration of MeNH2 aq. to 0.5 M. Finally, ON1 was treated with 0.5 M MeNH2 aq. at 30 °C for 4 h to produce the desired methylurea ON3b (Figure 2d) in 86% yield, without any unreacted ON1 left. The use of 0.5 M Me2NH aq. as a secondary amine successfully gave the dimethylurea (ON3c) in 68% yield. When pyrrolidine, piperazine, ethylenediamine, 1,3-propanediamine, 3,3′-diamino-N-methyldipropylamine, and tris(3aminopropyl)amine were used, oligonucleotides containing the corresponding 2′-urea-LNA with the respective substituents on the urea moieties were obtained. These results suggested that 2′-Npentafluorophenoxycarbonyl-2′-amino-LNA was a good precursor for the construction of 2′-amino-LNA analogs with a substituted urea unit by post-synthetic modification. In contrast, when an Scheme 1. Synthesis of thymidine phosphoramidite 3 and modified oligonucleotides.
Next, we synthesized the oligonucleotides using common phosphoramidite chemistry on an oligonucleotide synthesizer; the sequences of the oligonucleotides are shown in Scheme 1. Phenoxyacetyl (Pac) and isopropylphenoxyacetyl (i-PrPac) protections were used for the dA and dG phosphoramidites, respectively. Furthermore, the nucleobases in dC and 2 -deoxy-5-methylcytidine (d m C) phosphoramidites were acetyl-protected. The coupling time was increased from 25 s to 10 min when phosphoramidite (3) was introduced into the oligonucleotides, and the coupling efficiency was estimated to be over 95%, based on the trityl monitoring observed in the removal of the 5 -DMTr group. After the synthesis of the oligonucleotides on a DNA synthesizer, the fully protected oligonucleotides attached to control pore glass (CPG) resin were subjected to ultra-mild conditions (50 mM K 2 CO 3 in MeOH at room temperature for 4 h) to produce the corresponding 5 -O-DMTr-oligonucleotides via the removal of the cyanoethyl groups in the phosphotriester moieties and the protecting groups in nucleobases, followed by cleavage from the resin. The 2 -N-pentafluorophenoxycarbonyl unit was tolerant of the base treatment. The DMTr-removal and purification step yielded the desired oligonucleotides modified with 2 -N-pentafluorophenoxycarbonyl-2 -amino-LNA.
The 2 -N-Pentafluorophenoxycarbonyl-2 -amino-LNA was converted within an oligonucleotide (ON1) by the treatment with various amines (Scheme 2 and Table 1). The treatment with 10 M NH 3 aq. at 30 • C for 4 h left the unreacted oligonucleotide (ON1), although the production of the corresponding unsubstituted 2 -urea-LNA was also observed (ON3a) (Figure 2a). The prolonged reaction time to 24 h yielded the desired ON3a with high efficiency, and it was isolated in 76% yield (Figure 2b). More than half of the ON1 remained in the treatment with 0.1 M MeNH 2 aq. at 30 • C for 2 h (Figure 2c). ON1 almost disappeared at an increased concentration of MeNH 2 aq. to 0.5 M. Finally, ON1 was treated with 0.5 M MeNH 2 aq. at 30 • C for 4 h to produce the desired methylurea ON3b (Figure 2d) in 86% yield, without any unreacted ON1 left. The use of 0.5 M Me 2 NH aq. as a secondary amine successfully gave the dimethylurea (ON3c) in 68% yield. When pyrrolidine, piperazine, ethylenediamine, 1,3-propanediamine, 3,3 -diamino-N-methyldipropylamine, and tris(3-aminopropyl)amine were used, oligonucleotides containing the corresponding 2 -urea-LNA with the respective substituents on the urea moieties were obtained. These results suggested that 2 -N-pentafluorophenoxycarbonyl-2 -amino-LNA was a good precursor for the construction of 2 -amino-LNA analogs with a substituted urea unit by post-synthetic modification. In contrast, when an oligonucleotide containing 2 -N-phenoxycarbonyl-2 -amino-LNA (ON1 OPh ) was treated with 10 M NH 3 aq. at 30 • C for 24 h and 0.5 M MeNH 2 aq. at 30 • C for 24 h, almost no reaction was observed in all cases (Figure 2e,f); this could be due to the low reactivity of the phenyl carbamate.  (Figure 2e,f); this could be due to the low reactivity of the phenyl carbamate. A 14-mer homopyrimidine oligonucleotide (ON2) was converted into oligonucleotides (ON4ai) containing the substituted analogs of 2′-urea-LNA under the same conditions (Scheme 2 and Figure  S1 (Supplementary Materials)). The isolated yields are shown in Table 1.    Figure  S1 (Supplementary Materials)). The isolated yields are shown in Table 1.     A 14-mer homopyrimidine oligonucleotide (ON2) was converted into oligonucleotides (ON4a-i) containing the substituted analogs of 2 -urea-LNA under the same conditions (Scheme 2 and Figure S1 (Supplementary Materials)). The isolated yields are shown in Table 1.

Oligonucleotides 2 T m (∆T m 3 ) with ssDNA T m (∆T m 3 ) with ssRNA
ON3a natural 51 • C 52 • C hybridizing ability to ssDNA as 2′-(methoxycarbonyl)amino-LNA (ON5) and the parent 2′-amino-LNA (ON6), which suggested that the 2′-urea unit favored the formation of the duplex with ssDNA. The introduction of an amino group into the N-substituents of urea stabilized the duplexes. For example, the Tm of aminoethyl urea (ON3f) and aminopropyl urea (ON3g) (55 °C) was slightly higher than that of the same monosubstituted methylurea (ON3b) (53 °C). No further stabilization occurred in ON3h and ON3i which contained more amino groups.     In the case of duplex formation with ssRNA, although ON3i (bearing a branched bis(aminopropyl)amino group) showed a decreased Tm (55 °C), the stabilization abilities by other 2′urea-LNA derivatives were comparable to that by carbamate (ON5) or unsubstituted 2′-amino-LNA (ON6). For all 2′-urea-LNA derivatives, the duplexes were significantly stabilized when compared with the natural duplex by ON7. The results implied that a linear long chain on the urea unit might not influence the stability of the duplex formed with ssRNA.
In the case of duplex formation with ssRNA, although ON3i (bearing a branched bis(aminopropyl)amino group) showed a decreased T m (55 • C), the stabilization abilities by other 2 -urea-LNA derivatives were comparable to that by carbamate (ON5) or unsubstituted 2 -amino-LNA (ON6). For all 2 -urea-LNA derivatives, the duplexes were significantly stabilized when compared with the natural duplex by ON7. The results implied that a linear long chain on the urea unit might not influence the stability of the duplex formed with ssRNA.

General
All moisture-sensitive reactions were conducted in well-dried glassware under an Ar atmosphere. Anhydrous CH 2 Cl 2 and MeCN were used as purchased. 1 H-NMR, 13 C-NMR and 31 P-NMR spectra were recorded on a Bruker AVANCE III HD 500 MHz spectrometer equipped with a BBO cryoprobe, and an Agilent/Varian 400 MHz spectrometer. The chemical shift values were reported in ppm, relative to the internal tetramethylsilane (δ = 0.00 ppm) or solvent residual signals (δ = 3.31 ppm for CD 3 OD) for 1 H-NMR, solvent residual signals (δ = 77.0 ppm for CDCl 3 and δ = 49.0 ppm for CD 3 OD) for 13 C-NMR, and external 5% H 3 PO 4 (δ = 0.00 ppm) for 31 P-NMR. High-resolution mass spectrometry was performed on a Waters SYNAPT G2-Si (Quadrupole/TOF). For column chromatography, silica gel PSQ-60B (Fuji Silysia) was used. The progress of the reaction was monitored by analytical thin-layer chromatography (TLC) on pre-coated aluminum sheets (Silica gel 60 F254 by Merck). For HPLC, a JASCO EXTREMA (PU-4180, CO-4060 or CO-4061, UV-4075, and AS-4050) instrument with a CHF122SC (ADVANTEC) fraction collector was used. UV-melting experiments were carried out using a JASCO V-730 UV/VIS spectrophotometer equipped with a T m analysis accessory. The synthesis of oligonucleotides was performed on an automated DNA synthesizer (Gene Design nS-8II).