Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX
pubs.acs.org/OrgLett
Synthetic Method for 2′-Amino-LNA Bearing Any of the Four Nucleobases via a Transglycosylation Reaction Hiroaki Sawamoto,*,† Yuuki Arai,‡ Shuhei Yamakoshi,‡ Satoshi Obika,§ and Eiji Kawanishi‡ †
Research Unit/Innovative Medical Science, Sohyaku. Innovative Research Division, Mitsubishi Tanabe Pharma Corporation, 1000, Kamoshida-cho, Aoba-ku, Yokohama 227-0033, Japan ‡ Research Unit/Immunology & Inflammation, Sohyaku. Innovative Research Division, Mitsubishi Tanabe Pharma Corporation, 1000, Kamoshida-cho, Aoba-ku, Yokohama 227-0033, Japan § Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-0871, Japan S Supporting Information *
ABSTRACT: A transglycosylation reaction of 2′-amino-locked nucleic acid (LNA) from thymine (T) to other nucleobases adenine (A), guanine (G), and 5-methylcytosine (mC) has been developed. This reaction proceeds in high yield and with high βselectivity. The mild reaction conditions enable the coexistence of acid-labile protecting groups, including a 4,4′-dimethoxytrytyl (DMTr) group. 2′-Amino-LNAs bearing any nucleobase can now be easily synthesized.
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Although many efforts have been made in the development of 2′-amino-LNA derivatives, their practical use remains questionable. One reason for this may be the difficulty in preparing purine nucleosides that bear proper protecting groups tolerable to the oligonucleotide synthesis.7 We have developed an efficient synthetic method for the preparation of 2′-amino-LNA that is not limited to pyrimidine, but applicable to purine nucleobases as well. To synthesize bridged nucleic acids bearing any four bases, it takes many steps generally, because the introduction of nucleobases is carried out at an early stage.8 A common intermediate that could be obtained at the later steps could allow the number of total steps to be reduced. The most effective way to synthesize 2′-amino LNA bearing all types of nucleobases is to implement a transglycosylation reaction of 2′amino-LNA-T as a glycosyl donor after construction of the 2′4′ bridge. However, this is anticipated to be difficult. First, the rigid conformation inhibits the generation of an oxonium cation on the 1′-position of the 2′-amino-LNA, and the desired reaction hardly occurs,8e,9 leading to limited glycosylation with the 2′,4′-bridged [2.1.2]-bicyclic system. The glycosylation
atural DNA and RNA are composed of four nucleosides bearing four nucleobases: adenine (A), guanine (G), cytosine (C), and thymine (T) for DNA or uracil (U) for RNA. Oligonucleotide medicines, based on nucleotide sequences complementary to the target DNA or RNA, represent a new therapy modality with better efficacy outcomes than other drugs. Several oligonucleotide medicines have been launched to date.1 Recently, Nusinersen received FDA approval based on very positive clinical outcomes for the treatment of spinal muscular atrophy.2 2′-Amino-locked nucleic acid (2′-amino-LNA) was synthesized by Wengel in 19983 and has many interesting properties: (i) 2′,4′-bridged structure contributes to high binding affinity to natural RNA, (ii) 2′-amino groups are weakly basic, and (iii) various groups can substitute on the 2′-position. Furthermore, oligonucleotides containing 2′-amino-LNA have unique biodistribution.4 Therefore, many 2′-amino-LNA derivatives have been reported to date.5 In addition, GuNA variants, comprising a guanidine moiety at the 2′-position of a 2′-amino-LNA, not only improved nuclease resistance and organization potency of the complementary strand, but also improved the membrane permeability not achieved by other artificial nucleic acids until now.6 © XXXX American Chemical Society
Received: February 8, 2018
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DOI: 10.1021/acs.orglett.8b00476 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters Table 1. Transglycosylation Reaction from T to Aa
reaction with phenyl 3,5-di-O-benzyl-2-O,4-C-methylene-β-Dribofuranoside (which is the intermediate of 2′,4′-BNA/LNA) as a glycosyl donor resulted in moderate yield and poor βselectivity (α:β ≈ 2:1).10 Second, the control of the stereochemistry at the 1′-position of the nucleoside is usually achieved by the neighboring group participation from a 2′-Oacyl moiety.11 In the case of the bridge substrate, it is unclear if the effect works. Furthermore, acylation of the 2′-position causes a reduction of the reactivity,12 suggesting that reactivity and stereoselectivity are opposite and not compatible. Incidentally, α-aminoacetals are activated by Lewis acids and attacked by nucleophiles, giving acetal substitution products.13 In this reaction, the amino group contributes to the stabilization of α-oxocarbenium intermediates, and the reaction proceeds with high reactivity and high stereoselectivity. Therefore, we investigated the transglycosylation reaction with N-unsubstituted 2′-amino-LNA as a glycosyl donor. 2′-Amino-LNA-T was synthesized from the known substrate 1 (see Scheme 1).14 First, compound 2 was obtained by
Isolated Yield (%) entry 1 2 3 4
solvent DCE DCE DCE toluene
reaction temperature b
rt rtb 60 °C 60 °C
reaction time
6a
6b
2h 24 h 20 min 20 min
21 83 78 89
29 trace trace trace
a
Amount of reagent: N-(9H-Purin-6-yl)benzamide (1.5 equiv), N,Obis(trimethylsilyl)acetamide (BSA) (6 equiv), TMSOTf (0.1 equiv). b Room temperature.
material 4 was reduced, and 6b was gradually converted to the desired product 6a with an anomeric β-configuration (entry 1). After 24 h, almost all of the starting material 4 and isomers 6b disappeared, and the target material 6a was mainly obtained (entry 2). In the crude 400 MHz 1H NMR spectrum in CDCl3, the main singlet peak corresponding to an anomeric proton was detected at ∼6.0 ppm; however, the doublet peak indicating an α-anomer19 was not detected. The stereochemistry was determined by the NOE relationship between the ribose 1′and 3′-positions and between the ribose 6′-position and the adenine 8-position (see Figure 1). The reaction implemented at
Scheme 1. Synthesis of 2′-Amino LNA-T
Figure 1. Stereochemistry of each product was confirmed by 1H NMR NOESY spectrum.
60 °C gave the desired N9-product 6a as the main product after only 20 min; only trace amounts of the starting material 4 and regioisomers 6b were detected by LC-MS (entry 3 in Table 1). The yield of the targeted material was slightly improved by changing the solvent to toluene (entry 4 in Table 1). Guanosine derivative 6c was also obtained using O6diphenylcarbamoyl (DPC)-N2-isobutyrylguanine as a nucleophile in a similar manner. This high reactivity realized the conversion, even to the pyrimidine base. Using large amounts of 5-methylcytosine (mC), the desired product 6d was obtained in moderate yield via the transglycosylation reaction. In this case, the reaction proceeded with high stereoselectivity, and no byproducts other than starting materials 4 were obtained (see Scheme 2). Deprotection of the 3′,5′-dibenzyl group sometimes does not occur in the case of substrates bearing nucleobases other than T, because of the severe reaction conditions. When we investigated the potential deprotection of 2′-amino-LNA-A (6a), the substrate decomposed, and no target product was obtained (data not shown). Therefore, transglycosylation is preferably performed with substrates protected with a 5′-DMTr protective group, which is essential for the oligonucleotide synthesis.20 The DMTr group, which is unstable under acidic
removal of the tert-butyldiphenylsilyl (TBDPS) group of compound 1. An azido group was chosen as a nitrogen source to introduce the 2′-amino moiety, and compound 3 was synthesized via a Mitsunobu reaction with diphenyl phosphoryl azide (DPPA).15 The conversion of the azido group to an amino intermediate was performed by reduction,16 followed by intramolecular nucleophilic addition of the amino group into the 2′-position to give compound 4. The removal of the two benzyl groups via hydrogenation in acetic acid with Pearlman’s catalyst17 was followed by 5′-DMTr protection to give compound 5. These two reactions required long reaction times. First, we investigated the transglycosylation reaction from 2′amino-LNA-T to 2′-amino-LNA-A (see Table 1). At room temperature, the reaction proceeded slowly in the presence of a catalytic amount of trimethysilyl trifluoromethanesulfonate (TMSOTf)18 in 1,2-dichloroethane (DCE). By analyzing the reaction by LC-MS (see Figure S1 in the Supporting Information), a mixture of N1 and N7-regioisomers 6b was mainly detected at the initial stage; subsequently, the starting B
DOI: 10.1021/acs.orglett.8b00476 Org. Lett. XXXX, XXX, XXX−XXX
Letter
Organic Letters Scheme 2. Transglycosylation Reaction from T to G and m a C
In the transglycosylation reaction with 2′-amino-LNA-T as a glycosyl donor, the nucleophilicity of the 2′-amino group is very important. In the case of substrate 8, which is acetylated at the 2′-position, the transglycosylation reaction does not proceed, and the starting materials are recovered. In addition, in all of the cases described here, the reaction proceeds in a βselective manner. We speculate that neighboring group participation of the 2′-amino group may be contributing to both the reactivity and the promoting high stereoselectivity. The precise reaction mechanism is currently under investigation. In conclusion, we have developed an efficient method for synthesizing 2′-amino-LNAs bearing not only pyrimidine, but also purine nucleobases. Introduction of a nucleobase is the key step in the preparation of any artificial nucleic acids. The synthesis of 2′-amino-LNAs with a nucleobase (A, T, G, and m C) incorporated at an early stage of the reaction scheme increases the number of reaction steps, the reaction cost, and the occurrence of undesired situations. In the method described here, the introduction of the nucleobase is one of the final steps in the reaction process. Therefore, 2′-amino-LNAs bearing any of the four nucleobases can be synthesized very efficiently. One of the problems in utilizing 2′-amino-LNA has been resolved, and further applications of 2′-amino-LNA and its derivatives will be promoted by this discovery.
a
Reaction conditions for Method A, the synthesis of 6c (denoted by an asterisk symbol (*)): O6-DPC-N2-isobutyrylguanine (1.5 equiv), BSA (6 equiv), TMSOTf (0.1 equiv), 60 °C, 15 min. Reaction conditions for Method B, the synthesis of 6d (denoted by a double asterisk symbol (**)): 5-methylcytosine (3 equiv), BSA (12 equiv), TMSOTf (0.2 equiv), 60 °C, 150 min.
conditions, may be tolerable in the transglycosylation reaction that we have developed, because the reaction occurs with catalytic amounts of Lewis acid, despite the presence of basic 2′-amino groups. Therefore, we investigated the reaction with the 5′-DMTr protected substrate 5 (see Scheme 3). Using Scheme 3. Transglycosylation Reaction from 2′-AminoLNA-T to A, G and mC Using Substrate 5 as a Glycosyl Donora
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.8b00476. Typical experimental procedures, preparations, and characterization of compounds and spectroscopic data (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
a
Reaction conditions for Method A (denoted by an asterisk symbol (*)): purine base (1.5 equiv), BSA (7 equiv), TMSOTf (0.1 equiv), toluene, 60 °C, 10−15 min. Reaction conditions for Method B (denoted by a double asterisk symbol (**)): mC (3 equiv), BSA (13 equiv), TMSOTf (0.2 equiv), toluene, 60 °C, 50 min.
Hiroaki Sawamoto: 0000-0003-2168-1207 Satoshi Obika: 0000-0002-6842-6812 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We thank Ms. Megumi Furui (Mitsubishi Tanabe Pharma Corporation) for the determination of the stereochemistry via NMR analysis.
benzoyl A as a glycosyl acceptor, the transglycosylation reaction occurred promptly, and the desired β-anomer 7a was selectively obtained in high yield without decomposition of substrates. Guanosine derivative 7b was also obtained in a similar manner. Using mC as a glycosyl acceptor, 7c was also obtained. As far as we know, this glycosylation reaction with substrates bearing a 5′-DMTr protecting group is the first example described in nucleic acid chemistry.
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REFERENCES
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DOI: 10.1021/acs.orglett.8b00476 Org. Lett. XXXX, XXX, XXX−XXX
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DOI: 10.1021/acs.orglett.8b00476 Org. Lett. XXXX, XXX, XXX−XXX
