Synthesis and hybridizing properties of P-stereodefined chimeric [PS]-{DNA:RNA} and [PS]-{DNA:(2′-OMe)-RNA} oligomers

Oxathiaphospholane derivatives of 2′-OMe-ribonucleosides and 2′-O-TBDMS-ribonucleosides (MN-OTP and TN-OTP, respectively; nucleobase protected) were synthesized and separated into pure P-diastereomers. X-ray analysis showed the RP absolute configuration of the phosphorus atom in the fast-eluting diastereomer of TA-OTP. The fast- and slow-eluting P-diastereomers of MN-OTP and TN-OTP were used in the solid-phase synthesis of phosphorothioate dinucleotides (MNPST and NPST, respectively), which were subsequently hydrolyzed with RP-selective phosphodiesterase svPDE and SP-selective nuclease P1 to determine the absolute configuration of the phosphorus atoms. P-Stereodefined phosphorothioate ([PS]) 10-mer chimeric oligomers [PS]-{DNA:(2′-OMe)-RNA} and isosequential [PS]-{DNA:RNA} containing two MNPS or NPS units were synthesized. Melting experiments performed for their complexes with Watson–Crick paired DNA matrix showed that MNPS or NPS units decrease the thermal stability of the duplexes (ΔTm = −0.5 ÷ −5.5 °C per modification) regardless of the absolute configuration of the P-atoms. When the (2′-OMe)-RNA matrix was used an increase in Tm was noted in all cases (ΔTm = +1 ÷ +7 °C per modification). The changes in thermal stability of the duplexes formed by [PS]-chimeras with DNA and (2′-OMe)-RNA matrices do not correlate with the absolute configuration of the phosphorus atoms.


Introduction
In the last ve decades, many DNA analogs have been chemically synthesized, mainly to improve their stability against nucleases and in this way to ensure long-lasting recognition of the target nucleic acid. This increased stability was expected to produce new probes useful for example for controlling gene expression or biochemical manipulations. To some extent, this goal has been achieved by the introduction of phosphorothioate analogs of DNA ([PS]-DNA), 1 although nonspecic binding to proteins 2 and immunological side effects 3,4 initially hindered their application in vivo.
[PS]-RNA oligomers were obtained shortly aer using phosphoramidite or H-phosphonate monomers with sulfurization. Note: interestingly, it has been shown that [PS]-DNA probes in the absence of transfecting agents enter mammalian cells more efficiently than DNA. 5 An explanation based on the dynamic covalent exchange of phosphorothioates with cellular thiols and disuldes has recently been published. 6 Some drugs containing [PS]-DNA and [PS]-RNA-derived units have been approved by the FDA and other agencies, and several others are in advanced clinical trials. 7 Noteworthy, in 2007 phosphorothioation of DNA was discovered in bacteria, 8 and in 2019, a DNA phosphorothioation-based antiviral system was discovered in archaea. 9 The identied chimeric phosphate:phosphorothioate compounds ([PO]: [PS]) contained the phosphorothioate phosphorus atoms only of R P absolute conguration. 10 In 2020, the R P phosphorothioate modication was detected in RNA isolated from prokaryotes and eukaryotes. 11 Although the phosphorothioate internucleotide bond is isoelectronic with the natural phosphate moiety, the properties of [PS]-DNA differ from natural DNA ([PO]-DNA) in several aspects, mainly due to the different steric requirements of the sulfur atom, the altered affinity for metal ions ("so" sulfur vs. "hard" oxygen), and unsymmetrical distribution of the negative charge in the diester phosphorothioate anion. 12 Depending on the intended application, these altered properties may be considered either disadvantageous or benecial, but in the vast majority of biology-related experiments, one should consider the stereochemistry of the internucleotide phosphorothioate moieties. Since the substitution of sulfur for a nonbridging oxygen atom creates a stereogenic center, for example, a [PS]-oligonucleotide decamer synthesized by a non-stereocontrolled method exists as a mixture of 2 9 ¼ 512 P-diastereomers, and the content of a single diastereomer is less than 0.2%. Since each Pdiastereomer can interact with a biological target (usually with xed chirality) in slightly different ways, the mixtures of hundreds of P-diastereomers may be useless for detecting the interactions of interest. To overcome this problem, stereocontrolled synthetic methods have been developed. [13][14][15] Among them, the approach utilizing so called oxathiaphospholane monomers (OTP; developed in this laboratory) 13,16 has yielded the most P-stereodened oligomers so far, and some of them showed interesting P-stereodependent properties in biochemical studies. [17][18][19][20][21] So far, the OTP method has mainly used separated P-diastereomers of 3 0 -O-(2-thio-4,4-pentamethylene-1,3,2oxathiaphospholane) derivatives of 2 0 -deoxyribonucleosides (1, 5 -) have been used in the synthesis of P-stereodened phosphoroselenoate analogs of DNA. 22 In 1996, a set of four monomers 3 (X ¼ O-TBDMS, R ¼ H) was synthesized and chromatographically separated into Pdiastereomers. 23 They were used in the solid-phase synthesis of dinucleoside phosphorothioates N PS N, which were obtained in 66-98% yields. However, the separation of fast-and slow-eluting monomers was very difficult and inefficient. In other attempts to synthesize P-stereodened [PS]-RNA, the synthesis of up to 10-mers was achieved using a method based on nucleoside-3 0 -Ooxazaphospholidine monomers with the 2 0 -O-TBDMS protecting group. 24 Good coupling yields (97-99%) were observed, but stereoselectivity was not perfect ($96 : 4). A diastereoselectivity $98 : 2 was observed when oxazaphospholidine monomers with 2 0 -O-(2-cyanoethoxymethyl) group were used. 25 This discovery prompted our attempt to develop a fully stereospecic method for the synthesis of In this report, we present the synthesis of OTP monomers 2 (X ¼ OMe, M N-OTP, Chart 1) and 3 (X ¼ O-TBDMS, T N-OTP) and their separation into pure P-diastereomers, as well as the result of the successful crystallographic analysis of detritylated 3a (B 0 ¼ Ade Bz ). The P-diastereomerically pure monomers 2 and 3 were used for the synthesis of chimeric dinucleotides 8a and 8b, respectively (Scheme 1), and the absolute conguration of the P atoms was determined enzymatically. Melting curves were recorded to determine the thermal stability of duplexes formed by P-stereodened chimeric

Results and discussion
The mechanistic principle of the OTP method The synthesis of OTP monomers 2 and 3 and the mechanistic principle of the coupling step are shown in Scheme 1. It has been documented 26 that the condensation does not follow a stereoinvertive S N 2P mechanism but is a stereoretentive process. The initial attack of a nucleoside 5 occurs from the side opposite to the most electronegative atom bonded to the phosphorus center (the oxygen atom in the oxathiaphospholane ring of 2 or 3, marked in blue; Scheme 1) to form a trigonal bipyramidal intermediate I1. Subsequently, a pseudorotation process furnishes the intermediate I2, in which the thioalkyl leaving group occupies the axial position, followed by cleavage of the P-S bond to form the triester intermediate I3. The condensation process concludes with the elimination of epis-ulde. Finally, deprotection of the resulting intermediates 6 and   29 Unfortunately, oligomers of the latter two series were obtained in low yields due to unexpected side reactions and unfavorable conformational factors.
In the present studies, a crystal suitable for X-ray analysis was obtained from the 5 0 -OH deprotected fast-eluting monomer 3a (B 0 ¼ Ade Bz ). Detritylation was carried out in anhydrous acetonitrile using a sodium hydrogen sulfate suspension deposited on silica gel. 31 The crystal structure was resolved ( Fig. 1) with the renement parameter R ¼ 0.0563 and ESI. † The  crystal data and the data acquisition and renement parameters are listed in Table S1 (ESI †). It should be noted that despite the presence of a 2 0 -oxygen atom, the (normally strong) anomeric effect is not decisive in this case and the sugar ring exists in the C2 0 -endo conformation ( Fig. 2), perhaps due to the steric requirements of the bulky TBDMS group. Crystallization experiments with all other DMT-labeled as well as detritylated OTP derivatives were unsuccessful.

Synthesis and enzymatic analysis of M N PS T and N PS T dinucleotides
Since X-ray analysis was successful only for the derivative of fasteluting 3a, enzymatic analysis was performed to complete the stereochemical assignment in the remaining 2 and 3. For this purpose, 4 pairs of P-diastereomeric diesters M N PS T (6a, X ¼ OMe) and 4 pairs of P-diastereomeric diesters N PS T (6b, X ¼ OH) were prepared from 3 0 -O-acetyl-thymidine (5, B 0 ¼ Thy, Scheme 1) and diastereomerically pure (fast-or slow-eluting) M N-OTP 2a-d or T N-OTP 3a-d, respectively. The base-labile protecting groups in intermediates 6 were removed with concentrated ammonia. Subsequently, products 7a (X ¼ OMe; derived from 2) were routinely detritylated with 50% AcOH aq . The DMT and TBDMS moieties in the freeze-dried products 7b (X ¼ O-TBDMS; derived from 3) were removed by treatment with 3HF Â NEt 3 . The MALDI TOF MS data for the resulting dinucleotides 8a and 8b are given in Table 3.
Small amounts (2-3 OD 254 units) of all 16 P-diastereomeric dinucleotides 8 were isolated using RP-HPLC and further treated with the svPDE and nP1 enzymes, which are R P -and S Pstereoselective, respectively. Subsequent RP-HPLC analysis revealed that all dinucleotides 8 obtained from the fast-eluting 2a-d and 3a-d were hydrolyzed with svPDE and not with nP1. Their counterparts obtained from the slow-eluting monomers were hydrolyzed with nP1 and not with svPDE. These results, together with the X-ray data collected for 3a, conrm the stereoretentive course of the condensation step.

Relative reactivity of M N-OTP and T N-OTP compared with dN-OTP
It has been reported previously that when monomers 1 were used, 92-94% yields were observed with single coupling, allowing 18-23 nt long PS-DNA oligomers to be assembled. 18 Previous experiments with the OTP derivatives of LNA nucleosides showed that double coupling was necessary to achieve an acceptable yield of the condensation step. 27 Using this modied protocol, chimeric [PS]-{DNA:LNA} oligomers were obtained with LNA units in every third position. § This lower reactivity was tentatively attributed to steric hindrance due to the C2 0 -O-CH 2 -C4 0 bridge near the reacting phosphorus center. However, unsatisfactory attempts to synthesize oligomers with 3 0 -amino-2 0 ,3 0 -dideoxy nucleosides ([NPS]-DNA) 29 and subsequent molecular modeling experiments indicated the importance of the deoxyribose ring conformation in the OTP monomers. Importantly, only the OTP derivative of 3 0 -amino-3 0 -deoxythymidine allowed the synthesis of [NPS]-DNA 8-mer, while none of the monomers with the other three nucleobases allowed more than four coupling steps. Therefore, competition experiments were performed to determine the relative reactivity of selected purine and pyrimidine monomers 2 and 3 compared   to the corresponding dN-OTP. For this purpose, the approximately equimolar mixtures 1/2 and 1/3 (measured by 31 P NMR) were reacted with thymidine bound to a solid support (10 mg of each OTP monomer was used, i.e., a 14-18-fold excess). The resultant M N PS T/dN PS T and T N PS T/dN PS T mixtures were detritylated, detached from the solid support, deprotected with concentrated ammonia (the TBDMS group in the T N PS T dinucleotides was not removed), and analyzed using RP-HPLC. The required standards were prepared independently. The results ( Table 4) generally show that the M N-OTP monomers studied were two to three times less reactive compared with the corresponding dN-OTP, as the selectivity ratio values (calculated by dividing the percent composition of the dinucleotide products by the percent composition of the OTP substrates) ranged from 0.36 to 0.55. Only for the slow-eluting M A-OTP the difference was almost negligible (Table 4, selectivity ratio 0.91, double underlined). The reactivity of T N-OTP was 5-12 times lower, with the lowest selectivity ratio value of 0.08 (Table 4, underlined). Reactivity comparable to M N-OTP was found only for the fasteluting T A-OTP (selectivity ratio 0.39). The generally low reactivity observed for 2 and 3 suggested that the synthesis protocol optimized for [PS]-DNA 34 and later modied for the synthesis of [PS]-LNA oligomers 27 would be inadequate. In addition, the widely varying selectivity ratios indicated that several factors might inuence reactivity, so that optimization of the protocol for all 16 diastereomers of M N-OTP and T N-OTP would be a lengthy process with uncertain prospects for success. An attempt to synthesize uniformly modied [PS]-( M A) 4 T, in which the most reactive slow-eluting M A-OTP (2a, see Table 4) was double-coupled (thymidine was bound to the support), yielded only a small amount of product. The results of the DMT cation assay, shown in Fig. 3 (green line), clearly indicate that although the rst coupling was carried out with 86% yield, the next three coupling steps were much less efficient with 74, 67 and 49% yield, respectively. The synthesis of In the synthesis of each oligomer, the dN-OTP monomer was replaced in two selected cycles by M N-OTP or T N-OTP carrying the same nucleobase, except for the T-OTP monomer, which was replaced by M U-OTP or T U-OTP congeners. The codes, specic sequences, and HR MS data for two synthesized reference [PS]-DNA and sixteen chimeric [PS]-oligomers are listed in Table 5. [Note: in codes such as UR or M US, the capital letters R or S denote [PS]-oligomers with phosphorus atoms with either R P or S P absolute conguration, respectively].
The DMT cation decay assay was performed for each synthesis, and calculations for the steps using M N-OTP or T N-OTP (double coupling was performed) gave the average coupling yields of 78 and 79%, respectively (Table S2, ESI †). Aer cleavage from the support and removal of the base-labile protecting groups, the DMT-labeled oligomers were puried Table 4 The composition (mol%, by 31 P NMR) of the mixtures of M N-OTP/dN-OTP and T N-OTP/dN-OTP used in the reaction with thymidine attached to the solid support, and the composition (mol%, by HPLC) of the resultant mixtures of dinucleotides M N PS T/dN PS T and T N PS T/dN PS T OTP substrates, dinucleotide products ab The dinucleotide products were detritylated before cleavage from the solid support. b In the adenosine containing products, the benzoyl protecting group was removed with concentrated ammonia. c The selectivity ratios are calculated by dividing % composition of products by % composition of substrates. d The TBDMS group in T N PS T dinucleotides was not removed.

Melting experiments for canonical and hybrid duplexes
For the melting experiments, the oligomers were mixed with the equimolar amount of Watson-Crick-paired DNA matrix (Md) or mRNA matrix (Mm) (2.0 mM concentration each) in pH 7.2 buffer. The observed changes in UV absorbance followed the two-state mechanism and the recorded curves had the expected S-shape. An example set of melting curves is shown in Fig. 5.
Melting temperatures (compiled in Table 6) were calculated using the numerical tting method provided by the UV spectrophotometer manufacturer. The DT m values were calculated by subtracting the control T m from the values recorded for the duplexes tested.  Fig. 6, black bars), compared to the reference Bd/ Mm duplex (T m ¼ 51 C). The duplexes containing the M C PO or M U PO nucleotides were less stable than Bd/Mm and the destabilizing effect was strongest for M U (DT m ¼ À5 C). These results differ from those of Wengel and coworkers, who showed that one to three LNA nucleotides introduced into the DNA strand at either the adenine (5 0 -AGCACCAG) or thymine (5 0 -TGCTCCTG) residues increased the thermal stability of the corresponding {DNA:LNA}/RNA heteroduplexes in 110 mM Na + buffer. 33 For two introduced A-LNA or T-LNA units, DT m ¼ 10 and 16 C, respectively, were found. It can be argued that RNA matrices were used in these experiments, but Dolot and coworkers showed that crystals of DNA/RNA and DNA/ M RNA duplexes have very similar geometries. 34      were synthesized in good yield and effectively separated into pure P-diastereomers. The X-ray crystallographic analysis revealed the R P absolute conguration of the phosphorus atom in the fast-eluting diastereomer 3a (B 0 ¼ Ade Bz ). Using the R Pselective phosphodiesterase svPDE and the S P -selective Nuclease P1 it was found that in both monomer series the fastand slow-eluting P-diastereomers are precursors of dinucleotides with R P and S P absolute conguration of the phosphorus atoms, respectively. Importantly, no traces of P-epimerization were observed during coupling steps. P-

Experimental section
1 H NMR and 31 P NMR spectra were acquired using Bruker instruments (AV-200, DRX-500, or Avance Neo 400; operating for 1 H at 200, 500, and 400 MHz, respectively). Chemical shi (d) values are in ppm, referenced to internal tetramethylsilane (TMS) or residual solvent protons for 1 H NMR and external 85% H 3 PO 4 for 31 P NMR.
High-resolution mass spectra (HR MS) were acquired using a Synapt G2 Si mass spectrometer (Waters Corporation, Milford, MA) equipped with an ESI source and a quadrupole time-of-ight mass analyzer. Measurements were performed in negative ion mode with the capillary and sampling cone voltages set at 2.7 kV and 20 V, respectively. The temperature of the source was 110 C. To ensure satisfactory accuracy, data were acquired in a centroid mode and readings were corrected during acquisition using leucine-enkephalin as an external reference (Lock-SprayTM) generating reference ions at m/z 554.2615 Da ([M-H] À ) in negative ESI mode. Data sets were processed using MassLynx 4.1 soware (Waters).
MALDI-TOF MS analyses of oligonucleotides were performed with negative ion detection using a Voyager Elite instrument (PerSeptive Biosystems Inc, Framingham, MA) operating in reector mode or an Axima Performance instrument (Shimadzu Biotech Corp., Japan) operating in linear mode.
TLC silica gel 60 plates F254 developed in CHCl 3 /MeOH, 9 : 1 (v/v), were used for routine analyses. HP TLC Silica gel 60 plates with a UV-F 254 indicator were used to evaluate the separability of the P-diastereomers of 2 and 3.
A Varian binary HPLC system (two PrepStar 210 pumps, 25 mL pump heads, one ProStar 320 UV/vis detector at 275 nm) was used for HPLC separation of the P-diastereomers of monomers 2 and 3. A Phenomenex Luna 5 mm silica column (100Å; 250 Â 10 mm; ow rate 5 mL min À1 ) was used in search of the appropriate conditions. Preparative separation was performed with a Pursuit XRs silica gel column (10 mm, 250 Â 21.2 mm) eluted at a ow rate of 25 mL min À1 . Gradient-grade HPLC solvents from Sigma-Aldrich, Baker, or ChemPur were used.
Routine UV spectra were recorded with a CINTRA 4040 spectrophotometer (GBC, Dandenong, Australia). UVmonitored melting experiments were performed at 260 nm in cuvettes with 1 cm path length using a UV-vis NIR spectrophotometer V-770 (Jasco, Japan) equipped with a 6 Â 1 Peltier thermal cell.
For thermal dissociation experiments, oligonucleotide samples were dissolved in pH 7.2 buffer containing 10 mM Tris-HCl, 100 mM NaCl, and 10 mM MgCl 2 . Annealing was performed from 85 C to 15 C with a temperature gradient of 1 C min À1 . Melting proles were recorded over a range of 15/ 85 C (0.5 C min À1 ). Melting temperatures (T m ) were calculated using the soware provided by Jasco. P-Stereodened PSCh oligonucleotides were synthesized manually at a 1 mmol scale, using the slightly modied previously published protocols. 16,35 The rst nucleoside unit was anchored to the solid support by a DBU-resistant -succinoyl linker [-C(O)CH 2 CH 2 C(O)--LCAA-CPG]. 36 Routine coupling steps were performed using 20 mg of the dN-OTP monomers 1. In the cycles where the monomers 2 or 3 were incorporated, double coupling was executed (20 mg + 20 mg) and in both steps the coupling time was extended to 20 minutes. The assembled DMT-labeled oligomers were cleaved from the support (25% NH 4 OH, r.t., 3 h) and the protecting groups from nucleobases were removed with 25% NH 4  Unmodied DNA and (2 0 -OMe)-RNA oligonucleotide matrices (Md and Mm, respectively) were synthesized on a Gene-World synthesizer (K&A Laborgeraete GbR, Schaaeim, Germany) and routinely isolated by a two-step RP-HPLC method.

Synthesis of oxathiaphospholane monomers 2 and 3a general procedure
Oxathiaphospholane monomers 2 and 3 were synthesized analogously to a general procedure published for the synthesis of standard OTP monomers. 34 Briey, a suitable nucleoside 4 (2 mmol, N-protected if necessary; Scheme 1) was dried overnight in a 25 mL round bottom ask with two necks (with a magnetic stirrer inside) at high vacuum (0.01 mm Hg). To the ask purged with dry argon, 10 mL of anhydrous pyridine was added using a gas-tight syringe. To the stirred solution, 0.36 mL of 2-chloro-4,4-pentamethylene-1,3,2-oxathiaphospholane (OTP-Cl, 0.51 g, 2.4 mmol) was added at room temperature for 5 minutes using a gas-tight syringe. Aer approximately 5 minutes, 0.2 g of dry elemental sulfur (approximately 6 mmol) was added, and stirring was continued at room temperature for 12 hours. Excess sulfur was ltered off, the solvent evaporated, and the residue dissolved in 2 mL of acid-free chloroform (distilled with pyridine). The crude product was applied to a silica gel column (glass tube 15 Â 3 cm, height of gel suspension layer -5 cm, 230-400 mesh) and the column was eluted with acid-free chloroform. Appropriate fractions (8-10 mL each, analyzed by TLC on silica gel 60 plates, Rf's are given in Table 1) were combined, and the solvent was evaporated under reduced pressure (15-20 mm Hg) at a water bath temperature not exceeding 30 C. Anhydrous toluene (5-6 mL) was then added and the solution was evaporated to dryness excluding moisture. Finally, the samples were dried under high vacuum (oil pump) for 12 hours and stored at À15 C. The yields of the isolated mixtures of P-diastereomers are given in Tables 1  and 2.

Detritylation of the fast-eluting diastereomer of 3a
To a solution of 120 mg (0.12 mmol) of fast-eluting isomer of 5 0 -O-DMT-2 0 -O-TBDMS-N 6 -Bz-adenosine-3 0 -O-(2-thio-4,4pentamethylene-1,3,2-oxathiaphospholane) in 2 mL of anhydrous acetonitrile a sodium hydrogen sulfate/silica gel reagent (50 mg) was added and the suspension was stirred at room temperature. The reaction progress was monitored by TLC using a 9 : 1 (v/v) chloroform : methanol mixture as an eluent (Rf of the substrate 0.57; Rf of the product 0.31). Aer 26 hours the reaction went to complete. The silica gel was ltered off and the solvent was evaporated in vacuo. The product was isolated by semipreparative HPLC (a silica gel column eluted with ethyl acetate) and evaporation at reduced pressure furnished white solid material (52 mg, 62%).

Details of X-ray data collection and reduction
Single colorless transparent plate-shaped crystals of the detritylated fast-eluting P-diastereomer of 3a were obtained by recrystallization from a mixture of ethyl acetate and methanol (4 : 1 v/v). A suitable crystal of 0.11 Â 0.08 Â 0.02 mm dimension was selected and mounted on a suitable support. During data collection the crystal was kept at a steady temperature T ¼ 100.00(10) K. The structure was solved with the XT structure solution program 37 using an intrinsic phasing solution method and Olex2 (ref. 38) as a graphical interface. The model was rened with ShelXL (version 2018/3; ref. 39), using the least squares minimization.
In solution synthesis of dinucleoside 3 0 ,5 0 -phosphorothioates M N PS T (8a) and N PS T (8b) To a sample of a P-diastereomerically pure (fast-or slow-eluting) oxathiaphospholane monomer 2 or 3 (5 mg, ca. 5 mmol) and 3 0 -O-Ac-thymidine (3 mg, ca. 11 mmol), 600 mL of dry acetonitrile and 5 mL of 1,8-diazabicyclo- [5.4.0]undec-7-ene (DBU, 1.1 molar equivalent over the OTP monomers) were added under dry argon. Aer 2 h the reaction was complete. The acetonitrile was removed under reduced pressure and the products 6 were treated (in a tightly closed vessel) with concentrated ammonia solution (1 mL) for 18 h either at 55 C (6a) or at 37 C (6b), except for B ¼ Ura (2 h at room temperature). The products 7a were routinely detritylated using 50% AcOH aq . In case of 7b, approximately 10% of the ammoniacal solution was evaporated to dryness and the DMT and TBDMS moieties were removed with 3HFxNEt 3 (150 mL, 17 h, room temperature) followed by evaporation under reduced pressure. The dimers 8 were isolated using RP-HPLC. Conditions: a C18 column, 250 Â 4.6 mm, 5 mm; elution at 1.0 mL min À1 with a gradient of 0.1 M TEAB, pH 7.3 to 40% CH 3 CN in 0.1 M TEAB over 20 minutes.

Conflicts of interest
There are no conicts to declare.