Difluorophosphonylated Allylic Ether Moiety as a 2′-Modification of

Letter Cite This: Org. Lett. XXXX, XXX, XXX−XXX

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Difluorophosphonylated Allylic Ether Moiety as a 2′-Modification of RNA-Type Molecules: Synthesis, Thermal, and Metabolic Studies ́ i Legay,† Cyril Lebargy,† Emmanuel Pfund,*,†,§ Christelle Dupouy,*,‡,§ Sonia Rouanet,‡ Rem Jean-Jacques Vasseur,‡ and Thierry Lequeux† †

Normandie Université, Laboratoire de Chimie Moléculaire et Thioorganique, UMR 6507, ENSICAEN, UNICAEN, CNRS, 6 Bd du Maréchal Juin, 14050 Caen, France ‡ Institut des Biomolécules Max Mousseron, UMR 5247, Université Montpellier, CNRS, ENSCM, 34060 Montpellier, France

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S Supporting Information *

ABSTRACT: The first synthesis of oligonucleotides incorporating URF, a uridine modified with a difluorophosphonylated allylic ether onto the 2′-position, is described. Fluorinated homouridylates and miR-342-3p analogues are efficiently prepared. UV-melting experiments and enzymatic degradation studies indicate this new series of fluorinated oligonucleotides exhibit good and thermal metabolic stability as well as an increased lipophilicity. Comparison with oligonucleotides containing 2′-Oallyluridine instead of URF reveals improvement of these chemical properties is related to the presence of the difluoromethylphosphonate group.

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thermal and metabolic stability as well as interesting results in antisense medicinal chemistry (Figure 1).2c In this case, structural studies showed the thermal stability was assigned to the ribose C3′-endo sugar conformation while the resistance toward nucleases was related to a specific hydration bridging both 2′-oxygen atoms from the methoxyethyl group with the 3′-phosphate oxygen atom.6 In spite of

ith the continued progress of molecular biology, new therapeutic approaches using nucleic acids such as micro-RNA or small interfering RNA have emerged and represent important targets for a wide range of diseases.1 These different strategies are based on the design of oligonucleotides (ONs) having a complementary sequence to a specific mRNA to down-regulate genes. To be efficient as biopharmaceuticals, synthetic ONs must have several properties, including thermal stability of RNA duplex at 37 °C, resistance toward nucleases, and efficient and specific delivery. However, natural ONs are rapidly metabolized in biological systems due to their sensibility toward nucleases. To overcome these main limitations, several chemical modifications were considered leading to the conception of ON analogues with good biological activities.2 The formation of a thermally and enzymatically stable RNA duplex is a key step in gene silencing using RNA molecules. It has been shown that short RNA/RNA duplexes are thermally more stable than their analogues DNA/DNA,3 while RNA strands are metabolically less stable than DNA. Thus, several research groups have been interested in the modification of the 2′-position of nucleosides in order to access new series of ONs structurally close to RNA with a metabolic stability similar to DNA.2c However, design of such modified ONs is always challenging since the increase of nuclease resistance, obtained thanks to the presence of a sterically demanding 2′-alkyl ether,4 is inversely related to the duplex stability.5 In contrast, the wellknown 2′-O-methoxyethyl (2′-O-MOE) ONs I display both © XXXX American Chemical Society

Figure 1. 2′-O-MOE and 2′-fluorinated oligonucleotides. Received: May 14, 2019

A

DOI: 10.1021/acs.orglett.9b01689 Org. Lett. XXXX, XXX, XXX−XXX

Letter

Organic Letters the important role of fluorine atoms in medicinal chemistry in terms of stability, lipophily, and activity,7 2′-fluorinated ribonucleic acids have been sparsely studied and are listed in Figure 1.8 Except for 2′-deoxy-2′-trifluoromethylthio (2′SCF 3 ) VII and 2′-O-(4-CF 3 -triazol-1-yl)methyl (2′OMTCF3) VIII,8b−e the other 2′-fluorinated ONs showed improved thermal RNA/RNA duplex stability compared to their corresponding unmodified duplexes. Furthermore, compared to 2′-O-MOE I, fluorinated analogues 2′-Ofluoroethyl (2′-O-FET) II and 2′-O-trifluoroethyl (2′-OTFE) III exhibited an increased RNA affinity with a similar metabolic stability.8i In addition to an improvement of both thermal and metabolic stability, the presence of fluorine atoms could also facilitate cellular uptake without the use of transfection agent. This phenomenon, called gymnosis,9 was previously reported with 2′-fluoro arabino oligonucleotides V.10 All of these specific features make 2′-fluorinated ONs widely used in antisense technologies and considered as valuable promising therapeutics.8a As a consequence, the design of new series of fluorinated ONs still needs to be continued to discover the ideal modification being able to combine all the required properties for an optimal biological response. In this context, we describe in this paper the synthesis and physicochemical studies of new RNA-type ONs (2′-O-AlCF2P) incorporating URF, a uridine modified with the (E)-difluorophosphonylated allylic ether moiety on the 2′position (Figure 2). The difluorophosphonylated allylic ether

a more contrasted metabolic stability.8i,11 In our case, besides having a positive effect on cellular uptake, we hypothesized the presence of the bulky difluorophosphonate group, known as a stable phosphate surrogate,12 could also increase both thermal and metabolic stability of ONs by favoring the C3′-endo conformation and a specific hydration in the 3′-phosphate vicinity as was the case with 2′-O-MOE I.6 To evaluate the role of this fluorinated group, ONs (2′-O-Al) containing 2′-Oallyluridine UAL8i,11 will be also considered in this study (Figure 2). To access to ONs containing URF, the synthesis of the unknown phosphoramidite 6 was first envisaged with a particular focus on the straightforward introduction of the (E)-difluorophosphonylated allylic ether (Scheme 1). Inspired by recent reports on the synthesis of trifluoromethylated allyl ether via an elimination reaction of the corresponding alkyl iodide,13 in addition to our previous work on the preparation of various iododifluoromethylphosphonates,14 we envisaged introducing the difluorophosphonylated allylic ether moiety in a two-step process involving a group-transfer radical reaction followed by an elimination. By this synthetic plan, phosphoramidite 6 was obtained in four steps starting from protected 2′O-allyluridine 1 (Scheme 1). Indeed, iododifluoromethylphosphonate 2 was first reacted with 115 in the presence of sodium dithionite as initiator to afford 3 in 74% yield as a nonseparable equimolar mixture of both diastereoisomers. As previously reported in the literature, an elimination reaction could be realized with DBU as base.13 We also found this reaction could be efficient when conducted with 2.4 equiv of TBAF. Under these conditions, deprotection of silylated ethers also occurred, leading to nucleoside 4 as a single isomer in good yield with an excellent selectivity (E/Z = 99/1). The structure of the major stereoisomer was determined by NOEselective 1D experiments in which a strong NOE effect was observed for H-9 after H-7 excitation supporting an Econfiguration of the carbon−carbon double bond (Figure 3a). Phosphoramidite 6 was finally obtained on a gram scale from 4 after selective protection of 5′-position with a dimethoxytrityl (DmTr) group and 3′-phosphitylation using a standard procedure.

Figure 2. Chemical structures of ONs studied. URF = 2′-Odifluorophosphonylated allylic ether uridine UAL = 2′-O-allyluridine.

moiety was chosen to increase the physicochemical properties of 2′-O-allyl ONs that usually exhibit good thermal stability but

Scheme 1. Synthesis of Difluorophosphonylated Phosphoramidite 6a

a

Reaction conditions: (a) (iPrO)2(O)PCF2I 2 (2.0 equiv), NaHCO3 (4.0 equiv), Na2S2O4 (8.0 equiv), MeCN/H2O (2/1), rt, 6 h, 74%; (b) TBAF (2.4 equiv), THF, rt, 4 h, 77%; (c) DmTrCl (3.0 equiv), NEt3 (3.3 equiv), pyridine, rt, 15 h, 79%. (d) 2-cyanoethyl-N,Ndiisopropylchlorophosphoramidite (3.0 equiv), EtN(iPr)2 (4.0 equiv), CH2Cl2, rt, 6 h, 82%. B

DOI: 10.1021/acs.orglett.9b01689 Org. Lett. XXXX, XXX, XXX−XXX

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Organic Letters

HPLC) (ON 8−13) or ion-exchange HPLC (IEX-HPLC) (ON 15−18) and characterized by MALDI-TOF mass spectrometry (Table S1 and Figures S10−S19). These results describe the first examples of ONs containing the difluorophosphonate moiety. As previously reported,20 ON lipohilicity was evaluated by RP-HPLC (Figure 4). Fluorinated ON 10, having a much

Figure 3. NOE-selective 1D experiments for compound 4: (a) irradiation of H-7, (b) irradiation of H-1′.

The effect of the 2′-difluorophosphonylated allyl ether on the sugar conformation of nucleosides 4−6 in solution was evaluated by 1H NMR. In these cases, compounds 4, 5 (3JH1′−H2′ = 1.9 Hz), and 6 (3JH1′−H2′ = 2.5 Hz) favor a C3′-endo conformation in agreement with the literature where a typical C3′-endo sugar pocket affords a 3JH1′−H2′ value around 2 Hz against 8 Hz for a C2′-endo conformation.16 In addition, the percentage of C3′-endo was estimated using the relationship % C3′-endo = 100 − [(3JH1′−H2′ × 100)/10.1].8b,17 By this method, the percentage of C3′-endo was around 80% for nucleosides 4 and 5 and 75% for phosphoramidite 6. These results were confirmed by NOE-selective 1D experiments with diol 4 in which a NOE effect was observed for H-2′ when H-1′ was excited due to a spatial proximity of these two protons (Figure 3b). Having in hand a large amount of phosphoramidite 6, its oligomerization using automated DNA synthesizer was next studied. First, we designed several 2′-O-modified homouridylates with one modification in the middle of the sequence (ON 8), three modifications spread out over the sequence (ON 9), and 10 modifications (ON 10). Fluorinated 21-mer ONs incorporating one and three URF spread out over the sequence were also considered (ON 15 and 16). These latter correspond to the sense strand of miR-342-3p that functions as a tumor suppressor.18 The comparison was performed with the same modified sequences containing 2′-O-allyluridine (ONs 11, 12, 17, and 18). For the homouridylates models, syntheses were realized on a 0.2 μmol scale for ONs 8, 9, 11, and 12 and 0.5 μmol scale for ONs 10 and 13. The synthesis of the modified 21-mer ONs 15−18 were performed on a 1 μmol scale. ONs were synthesized using the 2′-O-AlCF2P uridine phosphoramidite 6, the 2′-O-allyluridine phosphoramidite, and commercially available 2′-O-pivaloyloxymethyl (PivOM) ribonucleosides phosphoramidite as the 2′-OH precursor by using commercially available controlled pore glass (LCAA-CPG) linked to the first nucleoside through a 3′-O-succinyl linker.19 ON elongation was carried out upon following a published automated RNA synthetic procedure with a 180 s coupling step and 5-benzylmercaptotetrazole (BMT) as the activator. A double coupling step of 180 s was required for the modified phosphoramidite 6. After elongation, cyanoethyl groups (CNE) were first deprotected by DBU, and then an ammonia treatment removed acyl and 2′-O-PivOM groups and released oligonucleotides from the solid support. 19 The crude oligonucleotides were purified by reversed-phase HPLC (RP-

Figure 4. Comparison of lypophilicities of ON 7, 10, and 13. RPHPLC chromatograms of (a) ON 7, (b) ON 13, and (c) ON 10. RPHPLC analysis conditions: column Accucore AQ, 50 × 4,6 mm, elution with a 10 min linear gradient of 0−70% of B (50 mM TEAAc, 80% ACN, pH 7) in eluent A (50 mM TEAAc, 1% ACN, pH 7). Column temperature 30 °C. λ 260 nm. Flow rate: 1.9 mL·min−1.

higher retention time compared to the unmodified ON 7 and its nonfluorinated analogue 13, exhibited an increased lipophilic character. That could facilitate cellular uptake of such ONs as already mentioned in the literature.21 The melting temperature study of each ON was next realized. After hybridization to their complementary unmodified RNA strands (rA12), thermal stability of the formed duplex was measured by UV-melting experiments and compared to the unmodified duplex (Table 1 and Figures S20 and S21). A single URF introduction in the sequence increases the thermal stability by 1.1 °C and by 3.4 °C with 3 incorporations of this fluorinated nucleoside (entries 1−3). This effect seems to be related to the presence of the difluorophosphonate function. In fact, a single introduction of the nonfluorinated allylic nucleoside UAL destabilizes the duplex, while three UAL incorporations raise the thermal stability by 1.5 °C only, versus 3.4 °C with the corresponding fluorinated oligonucleotide (entries 1, 3, 5, and 6). The same stabilization effect was obtained in the 21-mer ON series (entries 9−12). High duplex stabilization (ΔTm = +7.6 and +10.6 °C) was also observed with ONs 4 and 7 containing either 10 URF or 10 UAL (entries 1, 4, and 7). Concerning ON 7, our obtained results are consistent with those from the literature where 2′-O-allyloligonucleotides exhibit a good thermal stability when annealed with their complementary RNA strands.11 In addition, circular dichroism (CD) spectra were also recorded and revealed that URF incorporations do not affect the secondary structure of a standard A-shape RNA helix (Figures S22 and S23). The enzymatic stability of (UOH)10dTdT 7, (UAL)10dTdT 13, and (URF)10dTdT 10 was then studied toward 3′exonuclease and was monitored by RP-HPLC and MALDITOF mass spectrometry (Figures 5 and S24−S26). After C

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Organic Letters Table 1. UV-Melting Experiments of ONs with Their Complementary Strands rA12 for ONs 7−13 and 5′AGGGGUGCUAUCUGUGAUUGA-3′ for ONs 14−18 entry

ON

sequence 5′→3′a

Tmb (°C)

ΔTmc (°C)

1 2 3 4 5 6 7 8 9 10 11 12

7 8 9 10 11 12 13 14 15 16 17 18

UUUUUUUUUUdTdT UUUUUURFUUUUdTdT URFUUUURFUUUURFU dTdT (URF)10dTdT UUUUUUALUUUUdTdT UALUUUUALUUUUALU dTdT (UAL)10dTdT UCA AUC ACA GAU AGC ACC CCU URFCA AUC ACA GAU AGC ACC CCU URFCA AURFC ACA GAURF AGC ACC CCU UALCA AUC ACA GAU AGC ACC CCU UALCA AUALC ACA GAUAL AGC ACC CCU

14.6 15.7 18.0 22.2 11.3 16.1 25.2 76.7 77.7 77.3 75.9 76.9

+1.1 +3.4 +7.6 −3.3 +1.5 +10.6 / +1.0 +0.6 −0.8 +0.2

a URF = 2′-O-AlCF2P, UAL = 2′-O-Al. bTm values were obtained from UV-melting curves at 260 nm with 1.5 μM strand concentration in 10 mM sodium cacodylate, 100 mM NaCl, pH 7. Data are averages of two hybridization/melting cycles. Estimated errors in Tm = ± 0.5 °C. cΔTm is the difference in Tm relative to the unmodified duplex.

to the oligonucleotide (URF)10dT that was confirmed by MALDI-TOF mass analysis (Figure 5c). In this later case, only one phosphodiester linkage between the two nonfluorinated thymidines (dT) was digested by the enzyme suggesting the 2′-fluorinated modification confers high RNA stability toward 3′-exonucleases. Finally, miRNA-342-3p analogues resulting from annealing of 21-mers 14, 16, and 18 with their corresponding complementary RNA strand were incubated in 10% human serum at 37 °C for 48 h. Their stability was monitored by gel electrophoresis, and their half-lives were determined (Figure S27 and Table S2). Indeed, the duplex containing three UAL modifications with a half-life of 289 min conferred a stability comparable with that of the unmodified one (t1/2 = 216 min). On the other hand, the duplex bearing three URF modifications exhibited a remarkably higher stability with a half-life of 671 min. These assays clearly indicate the metabolic stability was related to the difluorophosphonate function that could bring a specific hydration in the 3′phosphate linkage vicinity as observed with 2′-O-MOE oligonucleotides I.6 In conclusion, we have described the first synthesis of ONs incorporating URF, a uridine modified with a difluorophosphonylated allylic ether onto the 2′-position. Indeed, fluorinated homouridylates and miR-342-3p analogs were efficiently prepared. UV-melting experiments and enzymatic degradation studies clearly indicate this new series of fluorinated ON exhibit both good and thermal metabolic stability. By comparison with ONs containing the nonfluorinated UAL modification, we also demonstrated that improvement of these properties was related to the presence of the difluoromethylphosphonate group. Targeted fluorinated ONs also have an increased lipophilicity compared to their nonfluorinated analogues that was highlighted by RP-HPLC. All of these preliminary results in terms of thermal and metabolic as well as lipophilicity make these new 2′-fluorinated ONs valuable and promising candidates for RNA therapeutics. Introduction of the difluorophosphonylated allylic ether moiety onto the 2′-position of all four standard RNA nucleosides is in progress, and physicochemical studies, cellular uptake measurements of these new ONs, as well as their therapeutic application in a micro-RNA approach will be reported in due course.

Figure 5. Enzymatic stability of ONs toward SVPD. HPLC analyses of (a) ON 7, (b) ON 13, and (c) ON 10 incubated in the presence of SVPD after 15 min for ON 7 and 13 and after 120 min for ON 10. The insets show the MALDI-TOF spectra of (a) ON 7, (b) ON 13, and (c) ON 10 pure and incubated in the presence of SVPD.

incubation at 37 °C in the presence of snake venom phosphodiesterase (SVPD), unmodified ON 7 and ON 13 containing 10 UAL were degraded within 15 min (Figure 5a,b) as already mentioned with 2′-O-allyl-oligonucleotides.11 In contrast, after 120 min incubation with (URF)10dTdT 10, the HPLC chromatogram revealed one major peak corresponding D

DOI: 10.1021/acs.orglett.9b01689 Org. Lett. XXXX, XXX, XXX−XXX

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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.9b01689. Synthetic procedures and NMR spectra (1H, 13C, 19F, 31 P) for compounds 3−6. NOE-selective 1D NMR spectra for compound 4. HPLC chromatograms, MALDI-TOF MS spectra, UV-melting curves, and CD spectra for purified ONs 7−18; enzymatic stability studies (RP-HPLC, MALDI-TOF MS and gel electrophoresis) of ONs 7, 10, 13, 14, 16, and 18 (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Emmanuel Pfund: 0000-0003-1896-0109 Christelle Dupouy: 0000-0002-4508-838X Thierry Lequeux: 0000-0003-3783-4458 Author Contributions §

E.P. and C.D. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the excellence laboratory LabEx SYNORG (ANR-11-LABX-0029), the Conseil Régional de Normandie, and the European FEDER funding. Financial support from the Fondation ARC (Grant No. PJA 20131200099 to E.P.) is gratefully acknowledged.

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REFERENCES

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DOI: 10.1021/acs.orglett.9b01689 Org. Lett. XXXX, XXX, XXX−XXX