Stereocontrolled Synthesis of Boranophosphate DNA by an

Article Cite This: J. Org. Chem. 2019, 84, 7971−7983

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Stereocontrolled Synthesis of Boranophosphate DNA by an Oxazaphospholidine Approach and Evaluation of Its Properties Rintaro Iwata Hara,†,‡ Tatsuya Saito,† Tomoki Kogure,† Yuka Hamamura,§ Naoki Uchiyama,§ Yohei Nukaga,† Naoki Iwamoto,§ and Takeshi Wada*,† †

Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda, Chiba 278-8510, Japan Department of Neurology and Neurological Science, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan § Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan Downloaded via UNIV OF SOUTHERN INDIANA on July 17, 2019 at 09:30:44 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

S Supporting Information *

ABSTRACT: In this paper, we describe the first stereocontrolled synthesis and properties of boranophosphate DNA (PB-DNA), which contains all of the four nucleobases longer than 10mer. Synthesis was accomplished via an oxazaphospholidine approach combined with acid-labile protecting groups on nucleobases. It was demonstrated that there were significant differences between all(Rp)- and all-(Sp)-PB-DNA in terms of the duplex-formation ability, nuclease resistance, and ribonuclease H (RNase H) activity. In particular, all-(Sp)-PB-DNA was demonstrated to show a duplexformation ability with RNA and RNase H activity, both of which are necessary for antisense-type nucleic acid therapeutics.

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expression.9,10 In the RNase H-dependent mechanism, an antisense oligonucleotide (ASO) binds to its target RNA, and then the ASO/RNA hybrid duplex is recognized by the RNase H to cleave the RNA strand. In the RNase H-independent mechanism, ASOs are also called steric blocking oligonucleotides, which inhibit the gene function by simply masking the target mRNA. In both mechanisms, the duplex-formation ability of ASO is quite important. Of course, in the RNase Hdependent mechanism, the RNase H activity of ASO/RNA hybrids is directly linked to the efficacy of antisense therapeutics. Due to the fact that nucleic acid molecules are susceptible to a variety of endogenous nucleases, the use of chemically modified nucleic acids is necessary to protect nucleic acid drugs from nuclease digestion. Among the FDA-approved nucleic acid drugs, fomivirsen, mipomersen, nusinersen, and inotersen have phosphorothioate (PS) linkages instead of phosphodiesters (Figure 1). Pegaptanib, mipomersen, nusinersen, and inotersen have 2′-O-modified ribonucleosides. Eteplirsen is based on the oligomer of morpholino nucleic acids via phosphorodiamidate linkages. Generally, these chemical modifications also alter a variety of properties, including the duplex-formation ability, cytotoxicity, and pharmacokinetics.11−13 Modification of phosphorus atoms of nucleic acids is particularly necessary to increase their nuclease resistance, and therefore all of the approved antisense drugs

INTRODUCTION A few decades have already passed since the development of nucleic acid therapeutics started, and there have been remarkable advances in this field in the last few years. Nucleic acid therapeutics are literally drugs composed of nucleic acid oligomers, which, according to their action mechanisms, are classified into antisense drugs, RNA interference (RNAi) drugs, aptamer drugs, and so on.1 By 2018, six nucleic acid drugs have been approved for clinical use by the Food and Drug Administration (FDA). The first FDA-approved nucleic acid was fomivirsen (Vitravene), which is an antisense drug based on a 21mer DNA oligomer, which was approved in 1998.2 In 2004, the aptamer drug pegaptanib (Macugen) was approved by the FDA,3 but it is the only example of an FDAapproved aptamer drug at present. Mipomersen (Kynamro),4 eteplirsen (Exondys 51), 5 nusinersen (Spinraza),6 and inotersen (TEGSEDI)7 were approved by the FDA in 2013, 2016, 2016, and 2018, respectively, as new antisense drugs long since fomivirsen. In addition, the first RNAi therapeutic patisiran (Onpattro) has been recently approved by the FDA in 2018.8 Among nucleic acid therapeutics, antisense drugs are the most successful ones currently, and the number of antisense drugs in clinical trials have been increasing over the past several years. Typical antisense drugs work by binding to their complementary region of messenger RNA (mRNA) in cell nuclei to decrease disease-related protein production. Mechanisms dependent and independent of ribonuclease H (RNase H) are known for their action mechanisms to inhibit gene © 2019 American Chemical Society

Received: March 25, 2019 Published: May 29, 2019 7971

DOI: 10.1021/acs.joc.9b00658 J. Org. Chem. 2019, 84, 7971−7983

Article

The Journal of Organic Chemistry

clearly revealed that each stereoregular PS oligonucleotide is significantly different in its properties, including the affinity to its complementary DNA and RNA, nuclease resistance, and RNase H activity.16,17,20−23 Quite recently, another synthetic strategy has also been reported to construct stereoregular PSDNA backbone using P(V)-based reagents, which is different from conventional phosphoramidite chemistry.24 Some stereoregular PS nucleic acid therapeutics are still undergoing clinical trials, and therefore, stereoregular PS-oligonucleotides have the potential to be the next gold standard as nucleic acid therapeutics. Another promising phosphorus atom modification other than PS is boranophosphate (PB) modification, in which one of the nonbridging oxygen atoms is replaced with a borane group. There have been a few reports concerning the preparation of PB oligonucleotides and their properties.25−37 In particular, PB-RNA was reported to be synthesizable using an enzymatic transcription system, and even highly modified PB-siRNAs were shown to have a silencing activity and to be less cytotoxic.27 However, enzymatic synthesis of PB-RNA is limited in the regard that only one isomer (Sp isomer) can be synthesized and that other oligonucleotides containing DNA and a variety of artificial nucleic acids cannot be synthesized. Chemical synthesis of PB oligonucleotides can be an approach to obtain a wide variety of PB-nucleic acid therapeutics. Shaw et al. and Caruthers et al. reported the synthesis of PB-modified homo-T oligomers via an Hphosphonate approach.30,33 Caruthers et al. reported the synthesis of PB-DNA containing four nucleobases using a phosphoramidite method.25,32 We also reported the synthesis of PB-DNA containing four nucleobases via an H-boranophosphonate method31 and a boranophosphotriester method.34−37 All of these synthetic methods are of nonstereocontrolled synthesis of PB-DNAs and afford 2n stereoisomers same as the old-time synthesis of PS oligonucleotides.

Figure 1. Chemical structure of phosphate (PO)-DNA (unmodified DNA), PS-DNA, and boranophosphate (PB)-DNA.

have this kind of modification. Among them, PS modification, in which one of the nonbridging oxygen atoms in a phosphodiester bond is replaced with a sulfur atom, is kind of a gold standard, including the approved antisense drugs. These P-atom-modified nucleic acids have stereogenic Pcenters and correspondingly two diastereomers (Sp and Rp) per modification, and they are dramatically different in many properties. All PS nucleic acids in early days, including the approved antisense drugs, were used as diastereomixtures. This means that, for example, mipomersen, a 20mer PSoligonucleotide, is a mixture of 524 288 (219) kinds of different compounds with individually different properties. Therefore, stereoregular PS nucleic acids must be more druggable than diastereomixture ones, although the stereoselective synthesis of PS nucleic acids is difficult. We previously reported an oxazaphospholidine approach for the stereocontrolled synthesis of PS-oligonucleotides.14−17 This approach is a type of phosphoramidite method whose features include the use of oxazaphospholidine monomer units,18,19 whose phosphorus atom is in a stereocontrolled manner, derived from optically pure aminoalcohols, and N-cyanomethylpyrrolidinium triflate (CMPT), an acidic activator with low nucleophilicity. Mainly on the basis of the oxazaphospholidine approach, a few groups

Figure 2. Oxazaphospholidine approach for the synthesis of PB-DNA. 7972


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The Journal of Organic Chemistry Scheme 1. Synthesis of Aminoalcoholsa

Although there has been an attempt for the stereocontrolled synthesis of PB oligonucleotides via an oxazaphospholidine approach, it is more difficult than that of PS oligonucleotides.38−40 The main difficulty in the synthesis other than that of PS nucleic acids derives from the fact that the boronating reagent also works as a reducing reagent. It was previously reported that acyl-type protecting groups on nucleobases were susceptible to borane reagents and that acyl groups were easily converted to stable alkyl groups.41 To eliminate the reduction problems, we previously reported the use of acid-labile trityltype protecting groups on nucleobases and two substituents on the oxazaphospholidine ring to generate H-phosphonate intermediates with unprotected nucleobases after acid treatment in each detritylation step (Figure 2).39,40 In this approach, the boronating reaction can be conducted after all of the base-protecting groups are removed, different from the conventional phosphoramidite method. Unprotected nucleobases were previously demonstrated not to cause any side reactions in the coupling reaction.42−44 The strategy worked to some extent and homo-T and 2′-OMe-modified homo-rU 12mers, and PB-dCAGT (4mer) could be synthesized in a stereocontrolled manner.39,40,45 However, the coupling efficiency was not enough to synthesize nucleic acid oligomers with all four nucleobases longer than 4mer by this strategy. One probable reason for the low coupling efficiency is that the steric hindrance of both protecting groups on nucleobases and substituents on the oxazaphospholidine ring is larger than that of the conventional oxazaphospholidine approach. In this study, we aimed to develop oxazaphospholidine monomers from which stereoregular PB-DNA oligomers can be efficiently synthesized. We designed novel oxazaphospholidine monomers that had a 4-methoxyphenyl substituent on its oxazaphospholidine ring to be less sterically hindered than the two-substituent-type monomers while keeping the acid lability. In addition, we studied the introduction of 4methoxybenzyloxycarbonyl (MCbz) group on nucleobases instead of trityl-type groups. The MCbz group was also expected to be less sterically hindered than the trityl-type groups and to be cleaved under acidic conditions.

a Reagents and conditions: (a) SOCl2, MeOH, 0 °C to room temperature (rt); (b) MMTrCl, Et3N, CH2Cl2, rt; (c) LiAlH4, tetrahydrofuran (THF), 0 °C to rt; (d) SO3−pyridine, Et3N, dimethyl sulfoxide (DMSO)−CH2Cl2, 0 °C to rt; (e) 4-methoxyphenylmagnesium bromide, THF−Et2O, −78 °C to rt; (f) (i) dichloroacetic acid, CH2Cl2, rt, (ii) NaOH(aq); (g) (i) trans-cinnamic acid, EtOH, reflux, (ii) KOH(aq), 41% ((αR,2S)-7 from L-proline), 33% ((αS,2R)-7 from D-proline).

Scheme 2 shows the synthesis of dC and dA derivatives. Both nucleobases were protected with an acid-labile MCbz group. 3′,5′-tert-Butyldimethylsilyl (TBDMS)-protected dC (8)47 was treated with carbonyldiimidazole (CDI) followed by 4-methoxybenzyl alcohol to afford MCbz-protected dC derivative 9. The TBDMS groups of 9 were removed in the presence of tetrabutylammonium fluoride (TBAF), and then the 5′-hydroxyl group was protected with a 4,4′-dimethoxytrityl (DMTr) group to obtain dC derivative 10. Synthesis of the protected dA was conducted in a manner similar to the case of dC. The amino group of 3′,5′-TBDMS-dA (11)47 was protected by an MCbz group using CDI and 4-methoxybenzyl alcohol to afford compound 12. Its TBDMS groups were removed by TBAF followed by tritylation to obtain 3′-OH dA derivative 13. O6-Trimethylsilylethyl (Tse)-protected dG was prepared according to our previous report.39 Oxazaphospholidine monomer units were synthesized using (αR,2S)-7 or (αS,2R)-7 and protected deoxyribonucleotide derivatives (Table 1). Except for the dG derivatives, the reactions were conducted initially at −78 °C and finally at room temperature through gradual warming. In the cases of the dG derivatives, the reactions were completed at −78 °C to prevent phosphitylation at the unprotected 2-NH2 group. As shown in Table 1, all of the oxazaphospholidine monomers were synthesized in high stereopurity and moderate isolate yields. Next, dinucleoside boranophosphates were synthesized via solid-phase synthesis using the oxazaphospholidine monomers. As shown in Scheme 3, each monomer (15−18) was reacted with thymidine anchored on a controlled pore glass in the presence of CMPT as an acidic activator.11 After condensation,

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RESULTS AND DISCUSSION First, we synthesized aminoalcohols from L- or D-proline, consulting the literature (Scheme 1).46 L- or D-proline (1) was converted to the corresponding methyl ester using thionyl chloride, and then the amino group was tritylated using 4methoxytrityl chloride (MMTrCl). The methyl ester (2S)-3 and (2R)-3 was reduced to alcohol (2S)-4 and (2R)-4, followed by the Parikh−Doering oxidation to afford aldehyde (2S)-5 and (2R)-5, respectively. The aldehyde was then reacted with 4-methoxyphenyl Grignard reagent to afford (2S)6 and (2R)-6. Finally, the MMTr group was removed by treatment with dichloroacetic acid (DCA) and then the resulting crude product was purified by recrystallization with trans-cinnamic acid to afford pure aminoalcohol (αR,2S)-7 in a 41% yield from L-proline, and (αS,2R-7) in a 36% yield from Dproline, respectively. Except for the final recrystallization step, all steps were conducted without any purification manipulation such as column chromatography, recrystallization, or reprecipitation. It should be noted that some epimerization reactions derived from stability of 4-methoxybenzyl cation occurred during detritylation and the epimer was removed in the recrystallization step. 7973


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The Journal of Organic Chemistry Scheme 2. Synthesis of dC and dA Derivative 10 and 13a

a

Reagents and conditions: (a) (i) carbonyldiimidazole, 1,2-dichloroethane, reflux, 20 h, (ii) 4-methoxybenzyl alcohol, 1,2-dichloroethane, reflux, 18 h, 75%, two steps; (b) (i) TBAF, THF, rt, 1 h, (ii) DMTrCl, pyridine, rt, 14 h, 70%, two steps; (c) (i) carbonyldiimidazole, 1,2-dichloroethane, reflux, 17 h, (ii) 4-methoxybenzyl alcohol, 1,2-dichloroethane, reflux, 20 h, 75%, two steps; (d) (i) TBAF, THF, rt, 1 h, (ii) DMTrCl, pyridine, rt, 18 h, 64%, two steps.

Table 1. Synthesis of Oxazaphospholidine Monomers

entry

14

1 2 3 4a 5 6 7 8a

(4S,5R) (4S,5R) (4S,5R) (4S,5R) (4R,5S) (4R,5S) (4R,5S) (4R,5S)

nucleobase

product

yield (%)

Rp/Spb

Th CyMcbz AdMcbz GuTse Th CyMcbz AdMcbz GuTse

(Rp)-15 (Rp)-16 (Rp)-17 (Rp)-18 (Sp)-15 (Sp)-16 (Sp)-17 (Sp)-18

43 47 44 37 67 40 48 38

>99:1 >99:1 >99:1 >99:1 >1:99 >1:99 >1:99 >1:99

(10) (13)

(10) (13)

Phosphitylation reaction was conducted at −78 °C. bDetermined by 31P NMR spectroscopy.

a

trifluoroacetic acid treatment was conducted for deprotection of the nucleobase and the 5′-hydroxyl group. The resulting Hphosphonate intermediates on the support were converted into the corresponding boranophosphates by treatment with BH3· SMe2 in the presence of N,O-bis(trimethylsilyl)acetamide, a silylating reagent to prevent side reactions on nucleobases.39 Finally, ammonia treatment was conducted to afford the dimers cleaved from the support. It should be noted that borane adducts on a nucleobase were observed after conventional cleavage conditions in the case of dAPBT (Figure S8). Therefore, longer ammonia treatment time with higher temperature was used in the synthesis of dAPBT and dGPBT to remove the borane group on the nucleobases. As summarized in Table 2, all (Rp)- and (Sp)-PB dimers were synthesized with good yields and high stereopurity, as confirmed by reversedphase high-performance liquid chromatography (RP-HPLC),

and all dimers were successfully identified by mass spectrometry. Based on the results, we tried to synthesize longer PBmodified oligomers, as shown in Figure 3. Figures S9−S12 show the HPLC profiles of all-(Rp)- and all-(Sp)-PB 4mers, CPBAPBGPBT containing all of the four nucleobases, using the same oxazaphospholidine approach. In the HPLC analysis, each 4mer was observed as the main product with a small amount of failure sequences. The 4mers were isolated in 14% (all-(Rp)-PB 4mer) and 23% (all-(Sp)-PB 4mer) yields, respectively. In a similar manner, we tried synthesizing 12mer PB-DNA oligomers containing four nucleobases, GPBTPBAPBCPBTPBAPBCPBTPBAPBCPBTPBT. After RP-HPLC purification, we successfully obtained the stereoregular all(Rp)-PB- and all-(Sp)-PB-DNA 12mers in 2 and 3% yields, respectively. These two oligomers were found in quite different 7974


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The Journal of Organic Chemistry Scheme 3. Synthesis of PB-DNA Dimers by the Oxazaphosphlidine Approach

Table 2. Synthesis of PB-DNA Dimers Using the Oxazaphospholidine Monomers entry

monomer

product

1 2 3 4 5 6 7 8

(Rp)-19 (Rp)-20 (Rp)-21 (Rp)-22 (Sp)-19 (Sp)-20 (Sp)-21 (Sp)-22

(Rp)-TPBT (Rp)-dCPBT (Rp)-dAPBT (Rp)-dGPBT (Sp)-TPBT (Sp)-dCPBT (Sp)-dAPBT (Sp)-dGPBT

concd NH3−EtOH (3:1, v/v) 25 25 55 55 25 25 55 55

°C, °C, °C, °C, °C, °C, °C, °C,

3h 3h 12 h 12 h 3h 3h 12 h 12 h

HPLC yield (%)

Rp/Spa

m/z calcd [M − H]−

m/z found

91 91 88 77 77 93 87 77

98:2 >99:1 >99:1 >99:1 >1:99 2:98 >1:99 3:97

543.1669 528.1671 552.1785 568.1734 543.1669 528.1671 552.1785 568.1734

543.1677 528.1673 552.1793 568.1744 543.1681 526.1681 552.1780 568.1748

a

Determined by RP-HPLC.

of PO-DNA/RNA (−18.2 °C in Rp and −7.4 °C in Sp); comparing the two oligomers, the all-(Sp)-PB-DNA/RNA hybrid was more thermodynamically stable than all-(Rp)-PBDNA/RNA hybrid. It was previously shown that the Tm values of the stereorandom-PB-DNA/RNA hybrid was significantly lower than those of the PO-DNA/RNA hybrid31 and that stereoregular all-(Sp)-PB-T12 could form a duplex with rU12 while all-(Rp)-PB-T12 could not.39 The results from this study are not in conflict with those of the previous studies. In this study, the Tm values of the stereoregular PB-DNA/RNA hybrids were compared to those of the PS-DNA/RNA hybrids. As shown in Table 4, the Tm value of the all-(Rp)-PB-DNA/ RNA hybrid is close to that of the all-(Sp)-PS-DNA/RNA hybrid and the Tm value of the all-(Sp)-PB-DNA/RNA hybrid is close to that of the all-(Rp)-PS-DNA/RNA hybrid. These results were interesting from the viewpoint that the position of the BH3 group in (Rp)-PB-DNA corresponds to that of the sulfur atom in (Sp)-PS-DNA. Therefore, stereoregular PBDNA was revealed to have similar properties as stereoregular PS-DNA in duplex-formation ability for complementary RNA, although PB-DNA/RNAs were slightly less stable than PSDNA/RNAs. Nuclease digestion experiments were conducted using nuclease P1 (nP1) and snake venom phosphodiesterase (SVPDE). nP1 is an (Sp)-PS-specific nuclease and SVPDE is

retention times in HPLC analysis as expected (Figure 4), and both oligomers were identified by mass spectrometry (Table 3). We also synthesized stereoregular PS-DNA oligomers with the same sequence via the oxazaphospholidine method, applying S8 treatment of H-phosphonate intermediates place of BH3 treatment. The all-(Rp)-PS- and all-(Sp)-PS-DNA 12mers were obtained in similar yields to PB-DNAs (3 and 2%, respectively). Comparing these similar yields in the PS-DNA synthesis and the PB-DNA synthesis, we consider that the low yield of PB-DNA in this strategy would be derived from failure in coupling rather than side reactions during the boronation or other manipulations. Next, we studied the properties of the stereopure PB-DNAs. UV melting analyses were conducted to study their doublestrand formation ability for their complementary DNA and RNA. The melting curves of DNA/DNA duplexes are shown in Figure S13. Any appreciable melting point was not observed, and therefore, it was suggested that both PB-DNAs did not form stable duplexes under these conditions. The melting curves of DNA/RNA hybrid duplexes, whose properties are quite important for antisense therapeutics, are shown in Figure 5. In this case, both curves of PB-DNAs suggested duplex formation, and the Tm values of the hybrid duplexes were 22.7 and 33.5 °C in all-(Rp)-PB-DNA/RNA and all-(Sp)-PB-DNA/ RNA hybrids, respectively. Both Tm values are lower than that 7975


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Figure 3. Synthesis of PB-DNA oligomers by the oxazaphospholidine approach.

an (Rp)-PS-specific nuclease,48−50 and therefore the stereochemistry of PS-DNAs can be determined by these nucleases. These nucleases are also reported to distinguish the difference between (Sp)- and (Rp)-PB DNAs.51,52 After the stereoregular PB-DNAs were treated with these nucleases, the resulting products were analyzed using HPLC. As shown in Figures 6 and 7, the all-(Rp)-PB-DNA 12mer was resistant to SVPDE and susceptible to nP1; on the contrary, the all-(Sp)-PB-DNA 12mer was resistant to nP1 and susceptible to SVPDE. In addition, in these experiments, the results of (Rp)-PB-DNA and (Sp)-PB-DNA were quite similar to those of (Sp)-PSDNA and (Rp)-PS-DNA, respectively (Figures S14 and S15). These results corroborate the fact that PB-DNAs were successfully synthesized in a stereocontrolled manner. Finally, we studied the RNase H activity, which is undoubtedly important for the efficacy of RNase H-dependent antisense therapeutics. In this study, we used Escherichia coli RNase H and all DNA 12mer and 10 equiv of the complementary RNA were treated with RNase H at 20 °C. After 30 min, in the case of PO-DNA and all-(Rp)-PS-DNA, the peak of the intact RNA completely disappeared, as shown in Figures 8a and S16a. In addition, in all-(Sp)-PB-DNA, a large amount of the intact RNA was cleaved (Figure 8c). On the other hand, all-(Rp)-PB-DNA and all-(Sp)-PS-DNA showed a weak cleavage activity (Figures 8b and S16b). The differences in the E. coli RNase H activity between two PS stereoisomers are consistent with the previous report of stereochemically enriched PS-DNAs. In addition to the abovementioned properties, the properties of all-(Sp)-PB-DNA are similar to those of all-(Rp)-PS-DNA, and all-(Rp)-PB-DNA are similar to those of all-(Sp)-PS-DNA. These results are

reasonable from the viewpoint that these nucleases unequally recognize two nonbridging oxygen atoms of a phosphate group in the DNA. The reason why all-(Sp)-PB-DNA was lower in RNase H activity than all-(Rp)-PS-DNA might be derived from the difference in the RNase H recognition,22,53,54 and/or from the thermodynamic properties of the hybrid strands, which were suggested from the different shapes of their UV melting curves. These differences were comparable to PSDNA, and therefore, it was shown that PB-DNA has potential to cleave its complementary RNA by RNase H in this study.

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CONCLUSIONS In this paper, we presented the first example of the stereocontrolled synthesis of PB-DNAs containing all of the four nucleobases, which were long enough to show duplexformation ability with their complementary RNA. Synthesis was accomplished according to an oxazaphospholidine method combined with acid-labile base-protecting and chiral auxiliary groups. We revealed the difference in the properties between two stereoisomers as follows: Tm values of DNA/RNA hybrids: PO-DNA > all-(Sp)-PB-DNA > all-(Rp)-PB-DNA; E. coli RNase H activity: PO-DNA > all-(Sp)-PB-DNA > all-(Rp)PB-DNA. This is also the first example where it was clearly indicated that RNase H activity of PB-DNA is different between two stereoisomers. These results showed the potential of PB-DNA as nucleic acid therapeutics including ASOs.

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EXPERIMENTAL SECTION

General Information. All solution-phase reactions were conducted under an argon atmosphere. 1H NMR spectra were obtained using JEOL 300 MHz, 400 MHz, or Bruker 500 MHz instrument with tetramethylsilane as the internal standard in CDCl3. 31P NMR spectra 7976


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Figure 4. RP-HPLC profiles at 260 nm of PB-DNA 12mers: (a) crude products for synthesis of all-(Rp)-PB-DNA 12mer, (b) purified all-(Rp)-PBDNA 12mer, (c) crude products for synthesis of all-(Sp)-PB-DNA 12mer, and (d) purified all-(Sp)-PB-DNA 12mer. RP-HPLC was performed with a linear gradient of 0−40% acetonitrile for 60 min in 0.1 M triethylammonium acetate buffer (pH 7.0) at 30 °C at a flow rate of 0.5 mL/min.

Table 3. Synthesis of PB-DNA Oligomers by the Oxazaphospholidine Approach entry

PB-DNA

isolated yield (%)

1 2 3 4

all-(Rp)-d(CPBAPBGPBT) all-(Sp)-d(CPBAPBGPBT) all-(Rp)-d(GPBTPBAPBCPBTPBAPBCPBTPBAPBCPBTPBT) all-(Sp)-d(GPBTPBAPBCPBTPBAPBCPBTPBAPBCPBTPBT)

14 23 3 2

were obtained with 85% H3PO4 as an external standard (δ 0.0). 13C NMR spectra were obtained with CDCl3 solvent as an internal standard (δ 77.0). Reagents and solid supports were purchased from Wako Pure Chemical Industries, Tokyo Chemical Industry, SigmaAldrich, and Glen Research. Oligonucleotides were purchased from Hokkaido System Science and Japan BioServices. Silica gel column chromatography was carried out using silica gel 60N (63−210 or 40− 50 μm) as a neutral silica gel, and Chromatorex NH-DM1020 as an NH-silica gel. RP-HPLC for analysis and purification was performed using a μBondasphere 5 μm C18, 100 Å, 19 × 150 mm2 (Waters) or Source 5RPC ST 4.6/150 (GE Healthcare). Organic solvents were purified and dried using the appropriate procedures. Mass spectra were recorded on a 910-MS FTMS system (Varian, electrospray ionization mass spectrometer, Fourier transform ion cyclotron resonance mass spectrometer) or JMS-700 (JEOL, Fast atom

m/z calcd 1166.3531 1166.3531 1189.3481 1189.3481

([M ([M ([M ([M

− − − −

m/z found H]−) H]−) 3H]3−) 3H]3−)

1166.3647 1166.3542 1189.3484 1189.3520

bombardment-mass spectrometry (FAB-MS), double-focusing mass spectrometer). Synthesis of Compounds. (R)-(4-Methoxyphenyl)((S)-pyrrolidin-2-yl)methanol and (S)-(4-Methoxyphenyl)((R)-pyrrolidin-2-yl)methanol ((αR,2S)-7 and (αS,2R)-7). Both aminoalcohols, (αR,2S)-7 and (αS,2R)-7 were synthesized in the same manner, except for the stereochemistry of the starting materials (L-proline or D-proline). We will describe the detailed synthetic procedure of (αS,2R)-7 as an example. D-Proline (D-1, 23.0 g, 200 mmol) was dissolved in MeOH (200 mL). The solution was cooled down to 0 °C and thionyl chloride (29 mL, 400 mmol) was added dropwise. The mixture was then warmed to room temperature and stirred for 15 h. The solvent was removed under reduced pressure, and the mixture was dried by repeated coevaporation with CHCl3. Crude products containing compound (2R)-2 were dissolved in CH2Cl2 (200 mL), followed by the addition 7977


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Figure 5. UV melting curves of the duplexes of PO-, PB-, or PS-DNA 12mer and their complementary RNA 12mer.

Table 4. Tm Values of the Duplexes of PO-, PB-, or PS-DNA 12mer and Their Complementary RNA 12mer entry 1 2 3 4 5

PO-DNA (Rp)-PB-DNA (Sp)-PB-DNA (Rp)-PS-DNA (Sp)-PS-DNA

Tm (°C)

ΔTm (°C)

ΔTm per mod. (°C)

40.9 22.7 33.5 31.0 23.4

−18.2 −7.4 −9.9 −17.5

−1.7 −0.7 −0.9 −1.6

Figure 7. RP-HPLC profiles at 260 nm of DNA 12mers after treatment with SVPDE: (a) PO-DNA 12mer, (b) all-(Rp)-PB-DNA 12mer, and (c) all-(Sp)-PB-DNA 12mer. RP-HPLC was performed with a linear gradient of 0−30% acetonitrile for 60 min in 0.1 M triethylammonium acetate buffer (pH 7.0) at 50 °C at a flow rate of 0.5 mL/min.

Figure 6. RP-HPLC profiles of DNA 12mers after treatment with nP1: (a) PO-DNA 12mer, (b) all-(Rp)-PB-DNA 12mer, and (c) all(Sp)-PB-DNA 12mer. RP-HPLC was performed with a linear gradient of 0−40% acetonitrile for 60 min in 0.1 M triethylammonium acetate buffer (pH 7.0) at 50 °C at a flow rate of 0.5 mL/min.

Figure 8. RP-HPLC profiles of RNA 12mers after treatment with E coli. RNase H in the presence of (a) PO-DNA 12mer, (b) all-(Rp)PB-DNA 12mer, and (c) all-(Sp)-PB-DNA 12mer. RP-HPLC was performed with a linear gradient of 0−30% acetonitrile for 60 min in 0.1 M triethylammonium acetate buffer (pH 7.0) at 30 °C at a flow rate of 0.5 mL/min.

of Et3N (112 mL, 800 mmol). MMTrCl (67.9 g, 220 mmol) was added to the solution; then, after 19 h, the reaction was quenched by the addition of sat. NH4Cl(aq)−conc NH3(aq) (2:1, v/v, 200 mL). The mixture was then extracted with CH2Cl2, and the collected organic layer was washed with a saturated aqueous (aq) solution of NaHCO3. The resulting organic solution was then dried over Na2SO4, filtered, and concentrated. The crude product containing ester (2R)-3 was dried by repeated coevaporation with toluene, CHCl3, and tetrahydrofuran (THF), and then dissolved in THF (200 mL).

Lithium aluminum hydride (8.35 g, 220 mmol) was suspended in THF (200 mL) in another flask. A THF solution of (2R)-3 was added dropwise to the solution at 0 °C, and then the mixture was warmed to room temperature. After 18 h, water was added dropwise (8.4 mL), followed by the addition of a 15% aqueous solution of NaOH (8.4 mL) and water (25.2 mL). MgSO4 was added to the mixture, and 7978


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The Journal of Organic Chemistry then the suspension was filtered over celite and washed with AcOEt. The filtrate was concentrated and diluted with chloroform (300 mL), and then the solution was washed with a saturated aqueous solution of NaHCO3. The resulting organic solution was dried over Na2SO4, filtered, and concentrated to afford a crude product containing alcohol (2R)-4. The crude was dissolved in CH2Cl2 (200 mL) and then Et3N (167 mL) was added. A dimethyl sulfoxide (DMSO) solution of sulfur trioxide complex (95.5 g in 150 mL DMSO, 600 mmol) was added to the solution at 0 °C and stirred for 2.5 h after warming to room temperature. The solution was then cooled to 0 °C and the reaction was quenched by the addition of water. The mixture was washed with a saturated aqueous solution of NaCl, and the water layers were backextracted with CH2Cl2. The combined organic layer was dried over Na2SO4, filtered, and concentrated. The mixture was diluted with Et2O and washed with a saturated aqueous solution of NaCl, and then the water layers were backextracted with Et2O. The collected organic layer was dried over Na2SO4, filtered, and concentrated to afford the crude product containing aldehyde (2R)5. The crude was then dried by repeated coevaporation with toluene and dissolved in Et2O (200 mL). The solution was cooled down to −78 °C and then a 0.5 M solution of 4-methoxyphenylmagnesium bromide in THF (700 mL) was added dropwise over 3 h. The mixture was gradually warmed to room temperature. After 16 h, sat. NH4Cl(aq)−conc. NH3(aq) (2:1, v/v, 180 mL) was added at 0 °C, and the suspension was filtered over celite. The filtrate was washed with a saturated aqueous solution of NaCl and the water layers were backextracted with CH2Cl2. The collected organic layer was dried over Na2SO4, filtered, and concentrated. 3% DCA solution in CH2Cl2 (700 mL) was added to the crude product containing compound (αS,2R)-6 at 0 °C. After 30 min, the mixture was extracted with water. A 5 M aqueous solution of NaOH was added to the aqueous solution until the pH exceeded 11. The solution was then extracted with CH2Cl2 and then the collected organic layer was washed with water. The resulting organic solution was dried over Na2SO4, filtered, and concentrated to afford crude products containing aminoalcohol (αS,2R)-7 (24.6 g, not diastereopure). The crude and trans-cinnamic acid (17.6 g) were dissolved in EtOH (30 mL) at reflux, and then the solution was gradually cooled down to 0 °C. The mixture was then filtered and the solid residue was washed with cold EtOH. The residue was redissolved in CH2Cl2, followed by the addition of a 2 M KOH solution. The product was extracted with CH2Cl2, and the collected organic solution was washed with a saturated aqueous solution of NaCl. The resulting organic solution was dried over Na2SO4, filtered, and concentrated to afford pure (αS,2R)-7 (13.6 g, 66 mmol, 33%). (αR,2S)-7. 1H NMR (300 MHz, CDCl3) δ 7.31−7.26 (m, 2H), 6.90−6.84 (m, 2H), 4.64 (d, J = 4.8 Hz, 1H), 3.80 (s, 3H), 3.39−3.31 (m, 1H), 3.04−2.85 (m, 2H), 2.80−2.60 (br, 2H), 1.79−1.45 (m, 4H). 13C{1H} NMR (125 MHz, CDCl3) δ 158.5, 135.1, 127.0, 113.4, 73.6, 64.3, 55.1, 46.5, 25.3, 25.2. FAB-high-resolution mass spectrometry (HRMS): calcd for C12H18NO2+ [M + H]+, 208.1332; found, 208.1337. (αS,2R)-7. 1H NMR (400 MHz, CDCl3) δ7.30−7.27 (m, 2H), 6.89−6.86 (m, 2H), 4.65 (d, J = 4.8 Hz, 1H), 3.80 (s, 3H), 3.40−3.35 (m, 1H), 3.03−2.98 (m, 1H), 2.94−2.88 (m, 1H), 2.60−2.46 (br, 2H), 1.79−1.61 (m, 3H), 1.55−1.46 (m, 1H). 13C{1H} NMR (125 MHz, CDCl3) δ 158.6, 134.9, 127.0, 113.5, 73.8, 64.2, 55.1, 46.6, 25.3. FAB-HRMS: calcd for C12H18NO2+ [M + H]+, 208.1332; found, 208.1338. 3′,5′-Bis-O-(tert-butyldimethylsilyl)-N 4 -((4-methoxybenzyl)oxycarbonyl)-2′-deoxycytidine: 9. Compound 8 (4.55 g, 10 mmol) was dried by repeated coevaporation with pyridine, toluene, and CHCl3, and then dissolved in 1,2-dichloroethane (100 mL). CDI (2.60 g, 16 mmol) was added to the solution and the mixture was stirred for 20 h at reflux. 4-Methoxybenzyl alcohol (2.0 mL, 16 mmol) was added and then the solution was stirred for 18 h at reflux. The solution was cooled to room temperature and washed with a saturated aqueous solution of NaHCO3. The aqueous solution was then backextracted with CH2Cl2, and all of the collected organic solution was dried over Na2SO4, filtered, and concentrated. The crude was

purified by silica gel column chromatography (neutral silica gel, hexane−AcOEt (1:1, v/v)) to afford 9 (4.64 g, 7.5 mmol, 75%). 1 H NMR (300 MHz, CDCl3) δ 8.32 (d, J = 5.4 Hz, 1H), 8.00 (br. 1H), 7.34 (d, J = 6.6 Hz, 2H), 7.15 (br, 1H), 6.85 (d, J = 6.6 Hz, 2H), 6.21−6.18 (m, 1H), 5.11 (s, 2H), 4.38−4.34 (m, 1H), 3.94−3.90 (m, 2H), 3.77−3.72 (m, 4H), 2.51−2.40 (m, 1H), 2.13−2.05 (m, 1H), 0.90−0.85 (m, 18H), 0.10−0.02 (m, 12H). 13C{1H} NMR (75 MHz, CDCl3) δ162.1, 159.7, 154.7, 152.3, 144.2, 130.0, 128.4, 127.0, 113.9, 113.6, 112.6, 94.3, 87.5, 86.5, 69.8, 67.5, 61.6, 55.1, 42.1, 25.7, 25.6, 18.2, 17.8, −4.7, −5.1, −5.6, −5.7. FAB-HRMS: calcd for C30H49N3O7Si2 [M + H]+, 620.3182; found, 620.3187. 5′-O-(4,4′-Dimethoxytrityl)-N4-((4-methoxybenzyl)oxycarbonyl)2′-deoxycytidine: 10. Compound 9 (4.34 g, 7.0 mmol) was dissolved in THF (35 mL), followed by the addition of a 1 M TBAF solution in THF (35 mL). After 1 h, the solution was diluted with AcOEt (300 mL) and then washed with a saturated aqueous solution of NaHCO3. The aqueous solution was then backextracted with AcOEt, and all of the collected organic solution was dried over MgSO4, filtered, and concentrated. The crude product was dissolved in pyridine (70 mL), followed by the addition of DMTrCl (2.37 g, 7.0 mmol). After 14 h, the solution was diluted with MeOH and CHCl3, and washed with a saturated aqueous solution of NaHCO3. The aqueous solutions were backextracted with CHCl3, and all of the collected organic solution was dried over Na2SO4, filtered, and concentrated. The crude was purified by silica gel column chromatography (neutral silica gel, CH2Cl2−MeOH−pyridine (99:0.5:0.5−94.5:5:0.5, v/v/v)) to afford 10 (3.47 g, 5.0 mmol, 71%). 1 H NMR (300 MHz, CDCl3) δ 8.23 (d, J = 7.5 Hz, 1H), 7.42− 7.37 (m, 3H), 7.32−7.20 (m, 9H), 7.00 (d, J = 7.2 Hz, 1H), 6.92− 6.84 (m, 6H), 6.23 (t, J = 5.7 Hz, 1H), 5.14 (s, 2H), 4.50−4.40 (br, 1H), 4.10−4.03 (m, 1H), 3.82−3.78 (m, 9H), 3.56−3.37 (m, 2H), 2.70−2.60 (m, 1H), 2.30−2.18 (m, 1H), 2.10−2.07 (br, 1H). 13 C{1H} NMR (75 MHz, CDCl3) δ162.2, 159.8, 158.5, 155.3, 152.2, 144.1, 135.4, 135.3, 130.1, 130.0, 129.9, 128.9, 128.1, 127.9, 127.1, 127.0, 113.9, 113.2, 94.8, 87.2, 86.7, 86.4, 70.7, 67.6, 62.7, 55.2, 55.1, 41.9. FAB-HRMS: calcd for C39H40N3O9 [M + H]+, 694.2759; found, 694.2763. 3′,5′-Bis-O-(tert-butyldimethylsilyl)-N4 -((4-methoxybenzyl)oxycarbonyl)-2′-deoxyadenosine: 12. Compound 11 (7.19 g, 15 mmol) was dried by repeated coevaporation with pyridine, toluene, and CHCl3, and then dissolved in 1,2-dichloroethane (150 mL). CDI (3.89 g, 24 mmol) was added to the solution and the mixture was stirred for 17 h at reflux. 4-Methoxybenzyl alcohol (3.0 mL, 24 mmol) was added, and then the solution was stirred for 20 h at reflux. The solution was then cooled down to room temperature and washed with a saturated aqueous solution of NaHCO3. The aqueous solution was then backextracted with CH2Cl2, and all of the collected organic solution was dried over Na2SO4, filtered, and concentrated. The crude was purified by silica gel column chromatography (neutral silica gel, hexane−AcOEt (2:1, v/v)) to afford 12 (7.26 g, 75 mmol, 75%). 1 H NMR (300 MHz, CDCl3) δ 8.75 (s, 1H), 8.41 (s, 1H), 8.27 (s, 1H), 7.38 (d, J = 6.6 Hz, 2H), 6.89 (d, J = 6.6 Hz, 2H), 6.47 (t, J = 4.8 Hz, 1H), 5.23 (s, 2H), 4.63−4.59 (m, 1H), 4.05−4.01 (m, 1H), 3.89−3.75 (m, 5H), 2.67−2.60 (m, 1H), 2.48−2.40 (m, 1H), 0.92− 0.87 (m, 18H), 0.12−0.04 (m, 12H). 13C{1H} NMR (75 MHz, CDCl3) δ 159.4, 152.3, 151.2, 150.6, 149.4, 141.2, 133.1, 130.1, 128.1, 127.3, 122.2, 113.6, 113.4, 87.7, 84.3, 71.7, 67.1, 64.1, 62.4, 54.9, 40.6, 25.6, 25.5, 18.0, 17.7, −5.0, −5.1, −5.7, −5.8. FAB-HRMS: calcd for C31H50N5O6Si2 [M + H]+, 644.3294; found, 644.3298. 5′-O-(4,4′-Dimethoxytrityl)-N4-((4-methoxybenzyl)oxycarbonyl)2′-deoxyadenosine: 13. Compound 12 (7.26 g, 11 mmol) was dissolved in THF (55 mL), followed by the addition of a 1 M TBAF solution in THF (55 mL). After 1 h, the solution was diluted with AcOEt (300 mL). The solution was then concentrated to half the volume to reduce the THF content, and then washed using a saturated aqueous solution of NaHCO3. The aqueous solution was then backextracted with AcOEt, and all of the collected organic solution was dried over MgSO4, filtered, and concentrated. The crude product was dissolved in pyridine (110 mL), followed by the addition of DMTrCl (4.48 g, 13.2 mmol). After 18 h, the solution was diluted 7979


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C{1H} NMR (75 MHz, CDCl3) δ 163.8, 163.7, 159.0, 158.7, 150.2, 144.2, 135.5, 135.3, 153.2, 130.1, 130.0 (d, 3JPC = 3.7 Hz), 128.1, 127.9, 127.1, 127.0, 126.8, 113.7, 113.6, 113.2, 113.2, 111.1, 86.8, 85.4, 85.3, 84.5, 82.2 (d, 2JPC = 9.3 Hz), 72.7 (d, 2JPC = 12.4 Hz), 67.3 (d, 3JPC = 3.1 Hz), 62.8, 55.2, 50.2, 47.2 (d, 2JPC = 34.0 Hz), 40.1, 28.0, 26.5, 25.9 (d, 3JPC = 3.7 Hz), 20.7, 11.7. 31P{1H} NMR (162 MHz, CDCl3) δ 156.1. FAB-HRMS: calcd for C43H46N3NaO9P+ [M + Na]+, 802.2864; found, 802.2874. Rp-dCMCbz Monomer: (Rp)-16. The same procedure as the synthesis of (Rp)-15 was applied for compound 10 (1.02 g, 1.47 mmol) and (4S,5R)-14 (2.94 mmol). The crude product was purified by silica gel column chromatography (NH-silica gel, toluene−AcOEt (7:3, v/v) containing 0.1% triethylamine) to afford (Rp)-16 (0.65 g, 0.70 mmol, 47%). 1 H NMR (400 MHz, CDCl3) δ 8.25 (d, J = 7.2 Hz, 1H), 7.40− 7.13 (m, 14H), 6.91−6.76 (m, 9H), 6.25 (t, J = 5.2 Hz, 1H), 5.68 (d, J = 6.0 Hz, 1H), 5.13 (s, 2H), 4.87−4.81 (m, 1H), 4.17−4.13 (m, 1H), 3.83−3.76 (m, 13H), 3.60−3.52 (m, 1H), 3.47 (d, J = 2.8 Hz, 2H), 3.22−3.13 (m, 1H), 2.82−2.75 (m, 1H), 2.38−2.32 (m, 1H), 1.67−1.57 (m, 2H), 1.22−1.15 (m, 1H), 1.01−0.94 (m, 1H). 13 C{1H} NMR (75 MHz, CDCl3) δ 162.0, 159.9, 158.9, 158.6, 152.1, 144.3, 144.1, 135.4, 135.3, 130.2, 130.2, 130.0, 128.2, 127.9, 127.0, 127.0, 126.7, 114.0, 113.6, 113.2, 94.4, 86.9, 86.7, 85.4, 82.0 (d, 2JPC = 9.3 Hz), 71.2 (d, 2JPC = 12.3 Hz), 67.7, 67.4 (d, 3JPC = 3.1 Hz), 61.8, 55.3, 55.2, 55.1, 53.1, 50.2, 47.2 (d, 2JPC = 34.6 Hz), 41.0, 28.0, 25.9 (d, 3JPC = 3.7 Hz), 20.7. 31P{1H} NMR (162 MHz, CDCl3) δ 156.7. FAB-HRMS: calcd for C51H54N4O11P+ [M + Na]+, [M + H]+, 929.3521; found, 929.3528. Sp-dCMCbz Monomer: (Sp)-16. The same procedure as the synthesis of (Rp)-15 was applied for compound 10 (1.01 g, 1.46 mmol) and (4R,5S)-14 (3.67 mmol). The crude product was purified by silica gel column chromatography (NH-silica gel, toluene−AcOEt (7:3, v/v) containing 0.1% triethylamine) to afford (Sp)-16 (0.55 g, 0.59 mmol, 40%). 1 H NMR (400 MHz, CDCl3) δ 8.36 (d, J = 7.6 Hz, 1H), 7.43− 7.16 (m, 14H), 6.94−6.84 (m, 9H), 6.24−6.20 (m, 1H), 5.68 (d, J = 6.4 Hz, 1H), 5.13 (s, 2H), 4.90−4.83 (m, 1H), 4.19−4.15 (m, 1H), 3.87−3.76 (m, 13H), 3.57−3.43 (m, 3H), 3.22−3.13 (m, 1H), 2.69− 2.63 (m, 1H), 2.41−2.32 (m, 1H), 1.69−1.60 (m, 2H), 1.26−1.17 (m, 1H), 1.04−0.95 (m, 1H). 13C{1H} NMR (75 MHz, CDCl3) δ 162.0, 159.8, 158.9, 158.6, 158.6, 154.8, 152.1, 144.3, 144.0, 135.4, 135.2, 130.2, 130.1, 130.0, 129.9 (d, 3JPC = 3.7 Hz), 128.1, 127.9, 127.0, 126.7, 114.0, 113.6, 113.6, 113.2, 94.4, 86.9, 86.5, 85.2, 85.1, 82.0 (d, 2JPC = 9.3 Hz), 70.3 (d, 2JPC = 11.1 Hz), 67.6, 67.4 (d, 3JPC = 3.8 Hz), 61.4, 55.2, 55.2, 55.1, 47.2 (d, 2JPC = 34.7 Hz), 41.0, 28.0, 25.9 (d, 3JPC = 3.7 Hz). 31P{1H} NMR (162 MHz, CDCl3) δ 156.0. FAB-HRMS: calcd for C51H53N4NaO11P+ [M + Na]+, 951.3341; found, 951.3349. Rp-dAMCbz Monomer: (Rp)-17. The same procedure as the synthesis of (Rp)-15 was applied for compound 13 (1.05 g, 1.47 mmol) and (4S,5R)-14 (2.94 mmol). The crude product was purified by silica gel column chromatography (NH-silica gel, toluene−AcOEt (8:2, v/v) containing 0.1% triethylamine) to afford (Rp)-17 (0.62 g, 0.65 mmol, 44%). 1 H NMR (400 MHz, CDCl3) δ 8.67 (s, 1H), 8.38−8.20 (br, 1H), 8.06 (s, 1H), 7.39−7.14 (m, 13H), 6.91−6.70 (m, 8H), 6.47 (t, J = 6.4 Hz, 1H), 5.76 (d, J = 6.4 Hz, 1H), 5.22 (s, 2H), 5.09−5.03 (m, 1H), 4.29−4.26 (m, 1H), 3.89−3.74 (m, 13H), 3.62−3.54 (m, 1H), 3.41−3.33 (m, 2H), 3.20−3.11 (m, 1H), 2.99−2.92 (m, 1H), 2.71− 2.65 (m, 1H), 1.68−1.59 (m, 2H), 1.26−1.16 (m, 1H), 1.03−0.94 (m, 1H). 13C{1H} NMR (75 MHz, CDCl3) δ 159.8, 158.9, 158.4, 158.4, 152.6, 150.9, 150.8, 149.4, 144.5, 141.4, 135.7, 135.6, 130.5, 130.2 (d, 3JPC = 3.7 Hz), 129.9, 129.9, 128.1, 127.7, 127.5, 126.8, 126.6, 122.4, 113.9, 113.6, 113.0, 86.4, 85.7, 84.8, 82.3 (d, 2JPC = 9.9 Hz), 73.3 (d, 2JPC = 11.1 Hz), 67.5, 67.5, 63.1, 55.2, 55.2, 55,1, 47.2 (d, 2JPC = 34.6 Hz), 39.1 (d, 3JPC = 4.3 Hz), 28.1, 25.9 (d, 3JPC = 3.8 Hz). 31P{1H} NMR (162 MHz, CDCl3) δ 155.0. FAB-HRMS: calcd for C52H53N6NaO10P+ [M + Na]+, 975.3453; found, 975.3459. Sp-dAMCbz Monomer: (Sp)-17. The same procedure as the synthesis of (Rp)-15 was applied for compound 13 (1.05 g, 1.46 13

with MeOH (50 mL) and CHCl3 (300 mL), and washed with a saturated aqueous solution of NaHCO3. The organic solution was dried over Na2SO4, filtered, and concentrated. The aqueous solution was then backextracted with CHCl3, and all of the collected organic solution was dried over Na2SO4, filtered, and concentrated. The crude was purified by silica gel column chromatography (neutral silica gel, CH2Cl2−MeOH−pyridine (98:1.5:0.5−96.5:3:0.5, v/v/v)) to afford 13 (5.08 g, 7.0 mmol, 64%). 1 H NMR (300 MHz, CDCl3) δ 8.74−8.70, 8.66 (s, 1H), 8.04 (s, 1H), 7.39−7.33 (m, 2H), 7.29−7.14 (m, 9H), 6.89−6.74 (m, 6H), 6.40 (t, J = 6.6 Hz, 1H), 5.20 (s, 2H), 4.73−4.67 (br, 1H), 4.16 (m, 1H), 3.79−3.72 (s × 2, 9H), 3.39 (d, J = 4.2 Hz, 2H), 3.09−2.96 (br, 1H), 2.87−2.76 (m, 1H), 2.56−2.47 (m, 1H), 1.65 (br, 1H). 13C{1H} NMR (75 MHz, CDCl3) δ 159.6, 158.3, 152.4, 151.0, 150.6, 149.3, 144.3, 141.3, 135.4, 130.3, 129.8, 127.9, 127.6, 127.3, 126.7, 122.0, 113.7, 112.9, 86.3, 86.3, 84.6, 71.9, 67.4, 63.5, 55.1, 55.0, 40.0. FABHRMS: calcd for C40H40N5O8 [M + H]+, 718.2871; found, 718.2880. Synthesis of Oxazaphospholidine Monomers. Compound (4S,5R)-14. (αR,2S)-7 (1.07 g, 5.16 mmol) was dried by repeated coevaporation with toluene and dissolved in toluene (3.0 mL). NMethylmorpholine (1.13 mL, 10.32 mmol) was added to the solution, and then the solution was added dropwise to a solution of phosphorus trichloride (0.45 mL, 5.16 mmol) in toluene (2.5 mL) at 0 °C. The mixture was warmed to room temperature and stirred for 2 h, and then filtered under argon atmosphere at −78 ° C. The filtrate was then concentrated to afford compound (4S,5R)-14 (1.28 g). This compound was used for phosphitylation without further purification. Compound (4R,5S)-14. The same procedure as the synthesis of (4S,5R)-14 was applied for (αS,2R)-7 (1.55 g, 7.48 mmol) to afford (4R,5S)-14 (1.99 g). This compound was also used for phosphitylation without further purification. Rp-T Monomer: (Rp)-15. 5′-O-DMTr-thymidine (0.86 g, 1.58 mmol) was dried by repeated coevaporation with pyridine, toluene, and THF, and then dissolved in THF (8 mL). Triethylamine (1.5 mL, 11 mmol) was added and the mixture was cooled down to −78 °C. A 0.5 M THF solution of (4S,5R)-14 (9.5 mL) was added dropwise and gradually warmed to room temperature. After 2 h, the mixture was diluted with AcOEt (300 mL) and washed with a saturated aqueous solution of NaHCO3. The aqueous solution was then backextracted with AcOEt, and all of the collected organic solution was dried over Na2SO4, filtered, and concentrated. The crude product was purified by silica gel column chromatography (NH-silica gel, toluene−AcOEt (7:3, v/v) containing 0.1% triethylamine) to afford (Rp)-15 (0.44 g, 0.56 mmol, 36%). 1 H NMR (400 MHz, CDCl3) δ 9.00−8.70 (br, 1H), 7.59 (s, 1H), 7.42−7.13 (m, 11H), 6.89−6.74 (m, 6H), 6.45−6.41 (m, 1H), 5.67 (d, J = 6.4 Hz, 1H), 4.94−4.89 (m, 1H), 4.13 (q, J = 2.4 Hz, 1H), 3.81−3.74 (m, 10H), 3.60−3.53 (m, 1H), 3.49−3.36 (m, 2H), 3.20− 3.11 (m, 1H), 2.60−2.54 (m, 1H), 2.40−2.32 (m, 1H), 1.66−1.58 (m, 2H), 1.42 (s, 3H), 1.21−1.14 (m, 1H), 1.00−0.91 (m, 1H). 13 C{1H} NMR (75 MHz, CDCl3) δ 163.8, 158.9, 158.6, 158.6, 150.3, 144.3, 135.5, 135.3, 135.3 130.2, 130.1, 130.0, 128.1, 127.9, 127.0, 126.6, 113.7, 113.6, 113.2, 111.1, 86.8, 85.4, 84.6, 82.2 (d, 2JPC = 9.8 Hz), 73.2 (d, 2JPC = 12.7 Hz), 67.3 (d, 3JPC = 3.2 Hz), 62.9, 55.2, 50.1, 47.1(d, 2JPC = 34.5 Hz), 40.0 (d, 3JPC = 4.9 Hz), 28.0, 26.5, 25.9 (d, 3 JPC = 3.0 Hz), 20.6, 11.7, 11.6. 31P{1H} NMR (162 MHz, CDCl3) δ 155.6. FAB-HRMS: calcd for C43H46N3NaO9P+ [M + Na]+, 802.2864; found, 802.2866. Sp-T Monomer: (Sp)-15. The same procedure as the synthesis of (Rp)-15 was applied for 5′-O-DMTr-T (0.85 g, 1.57 mmol) and (4R,5S)-14 (4.71 mmol). The crude product was purified by silica gel column chromatography (NH-silica gel, toluene−AcOEt (7:3, v/v) containing 0.1% triethylamine) to afford (Sp)-15 (0.82 g, 1.05 mmol, 67%). 1 H NMR (400 MHz, CDCl3) δ 8.40−8.33 (br, 1H), 7.64 (s, 1H), 7.43−7.15 (m, 11H), 6.91−6.79 (m, 6H), 6.41 (t, J = 6.66 Hz, 1H), 5.63 (d, J = 6.4 Hz, 1H), 4.94−4.88 (m, 1H), 4.18 (q, J = 2.4 Hz, 1H), 3.86−3.76 (m, 10H), 3.56−3.48 (m, 2H), 3.39−3.34 (m, 1H), 3.20−3.13 (m, 1H), 2.50−2.43 (m, 1H), 2.39−2.31 (m, 1H), 1.68− 1.55 (m, 2H), 1.40 (s, 3H), 1.26−1.19 (m, 1H), 1.06−0.90 (m, 1H). 7980


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The Journal of Organic Chemistry mmol) and (4R,5S)-14 (3.67 mmol). The crude product was purified by silica gel column chromatography (NH-silica gel, toluene−AcOEt (8:2, v/v) containing 0.1% triethylamine) to afford (Sp)-17 (0.67 g, 0.70 mmol, 48%). 1 H NMR (400 MHz, CDCl3) δ 8.67 (s, 1H), 8.33−8.26 (br, 1H), 8.12 (s, 1H), 7.41−7.35 (m, 4H), 7.30−7.12 (m, 9H), 6.91−6.74 (m, 8H), 6.45 (t, J = 6.4 Hz, 1H), 5.71 (d, J = 6.4 Hz, 1H), 5.22 (s, 2H), 5.06−5.00 (m, 1H), 4.34−4.31 (m, 1H), 3.91−3.76 (s, 13H), 3.62− 3.52 (m, 1H), 3.48−3.34 (m, 2H), 3.22−3.13 (m, 1H), 2.94−2.88 (m, 1H), 2.63−2.58 (m, 1H), 1.69−1.58 (m, 2H), 1.26−1.19 (m, 1H), 1.06−0.98 (m, 1H). 13C{1H} NMR (75 MHz, CDCl3) δ 159.8, 158.9, 158.4, 152.6, 150.9, 150.8, 149.3, 144.4, 141.3, 135.6, 135.5, 130.5, 130.1 (d, 3JPC = 3.8 Hz), 130.0, 128.1, 127.8, 127.5, 127.0, 126.8, 126.7, 122.4, 113.9, 113.7, 113.6, 113.1, 86.5, 85.7, 85.6, 84.6, 82.2 (d, 2JPC = 9.9 Hz), 73.0 (d, 2JPC = 11.8 Hz), 67.5, 67.4 (d, 3JPC = 3.1 Hz), 62.9, 55.2, 55.2, 55.1, 53.0, 47.2 (d, 2JPC = 34.0 Hz), 39.6, 28.0, 26.0 (d, 3JPC = 3.1 Hz), 20.7. 31P{1H} NMR (162 MHz, CDCl3) δ155.4. FAB-HRMS: calcd for C52H54N6O10P+ [M + Na]+, 953.3634; found, 953.3643. Rp-dGtse Monomer: (Rp)-18. The same procedure as the synthesis of (Rp)-15 was applied for O6-tse-5′-O-DMTr-2′-deoxyguanosine (0.67 g, 1.00 mmol) and (4S,5R)-14 (3.95 mmol), except for the reaction was conducted at −78 °C. The crude product was purified by silica gel column chromatography (NH-silica gel, toluene−AcOEt (9:1, v/v) containing 0.1% triethylamine) to afford (Rp)-18 (0.34 g, 0.38 mmol, 37%). 1 H NMR (400 MHz, CDCl3) δ 7.69 (s, 1H), 7.43−7.13 (m, 11H), 6.92−6.72 (m, 6H), 6.32 (t, J = 6.0 Hz, 1H), 5.75 (d, J = 6.4 Hz, 1H), 5.08−5.00 (br, 1H), 4.65−4.53 (m, 4H), 4.26 (s, 1H), 3.88−3.70 (m, 9H), 3.64−3.53 (m, 1H), 3.42−3.29 (m, 2H), 3.20−3.13 (m, 2H), 3.00−2.90 (m, 1H), 2.57−2.50 (m, 1H), 1.68−1.55 (m, 2H), 1.26− 1.12 (m, 3H), 1.03−0.96 (m, 1H), 0.08 (s, 9H). 13C{1H} NMR (75 MHz, CDCl3) δ 161.3, 159.1, 158.9, 158.4, 153.4, 144.7, 137.6, 135.8, 135.8, 130.3 (d, 3JPC = 3.8 Hz), 130.1, 130.0, 128.1, 127.8, 127.3, 127.0, 126.7, 126.7, 116.3, 114.2, 113.7, 113.6, 113.1, 86.3, 85.4, 84.1, 82.2 (d, 2JPC = 9.3 Hz), 81.8, 73.8 (d, 2JPC = 11.8 Hz), 67.6 (d, 3JPC = 3.7 Hz), 67.0, 64.8, 63.2, 55.3, 55.2, 55.1, 53.1, 50.3, 47.9, 47.2 (d, 2 JPC = 34.1 Hz), 45.7, 39.1, 31.0, 29.7, 28.1, 26.5, 26.0 (d, 3JPC = 3.7 Hz), 20.7, 17.5, −1.4. 31P{1H} NMR (162 MHz, CDCl3) δ 155.0. FAB-HRMS: calcd for C48H58N6O8PSi+ [M + H]+, 905.3818; found, 905.3826. Sp-dGtse Monomer: (Sp)-18. The same procedure as the synthesis of (Rp)-18 was applied for O6-tse-5′-O-DMTr-2′-deoxyguanosine (0.67 g, 1.00 mmol) and (4R,5S)-14 (3.95 mmol). The crude product was purified by silica gel column chromatography (NH-silica gel, toluene−AcOEt (9:1, v/v) containing 0.1% triethylamine) to afford (Sp)-18 (0.34 g, 0.38 mmol, 38%). 1 H NMR (400 MHz, CDCl3) δ 7.73 (s, 1H), 7.44−7.13 (m, 11H), 6.90−6.76 (m, 6H), 6.29 (t, J = 6.4 Hz, 1H), 5.70 (d, J = 6.0 Hz, 1H), 5.01 (m, 1H), 4.61−4.53 (m, 4H), 4.33−4.27 (m, 1H), 3.90−3.77 (m, 10H), 3.62−3.52 (m, 1H), 3.46−3.42 (m, 1H), 3.33−3.30 (m, 1H), 3.22−3.13 (m, 1H), 2.94−2.85 (m, 1H), 2.52−2.42 (m, 1H), 1.70−1.57 (m, 2H), 1.26−1.19 (m, 3H), 1.07−0.98 (m, 1H), 0.08 (s, 9H). 13C{1H} NMR (75 MHz, CDCl3) δ 161.3, 159.1, 158.9, 158.5, 158.4, 153.4, 144.6, 137.4, 135.7, 135.7, 132.2, 130.2 (d, 3JPC = 3.8 Hz), 130.0, 128.1, 127.8, 127.3, 126.9, 126.8, 126.7, 116.1, 114.2, 113.7, 113.6, 113.1, 86.3, 85.3, 85.2, 83.8, 82.2 (d, 2JPC = 9.3 Hz), 73.3 (d, 2JPC = 11.8 Hz), 67.4 (d, 3JPC = 3.1 Hz), 64.7, 63.1, 55.2, 55.2, 55.1, 53.0, 50.2, 47.2 (d, 2JPC = 34.6 Hz), 39.1, 39.0, 28.0, 26.5, 25.9 (d, 3JPC = 3.7 Hz), 20.6, 17.5, −1.5. 31P{1H} NMR (162 MHz, CDCl3) δ 155.4. FAB-HRMS: calcd for C48H58N6O8PSi+ [M + H]+, 905.3818; found, 905.3825.

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Procedures of enzymatic assays; UV melting analysis; and solid-synthesis HPLC profiles, UV melting curves, and NMR spectra of compounds (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Rintaro Iwata Hara: 0000-0003-2399-3431 Takeshi Wada: 0000-0001-7403-4033 Author Contributions

The manuscript was written through contributions of all authors. Funding

This work was supported by JST CREST (Grant number JPMJCR12L4) and JSPS KAKENHI (Grant Number 15H03839). Notes

The authors declare no competing financial interest.

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REFERENCES

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Stereocontrolled Synthesis of Boranophosphate DNA by an

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