Phosphine Oxide: Synthesis and Incorporation Into Short DNA Oligomers

Heterocycl. Commun. 2015; 21(5): 303–314

Barbara Nawrot*, Olga Michalak, Barbara Mikołajczyk and Wojciech J. Stec Acyclic analogs of nucleosides based on tris(hydroxymethyl)phosphine oxide: synthesis and incorporation into short DNA oligomers

DOI 10.1515/hc-2015-0173 are widely applied in cell and molecular biology as Received August 14, 2015; accepted September 1, 2015 gene expression inhibitors in antisense or RNAi strategies [16–20], and are useful tools in enzymatic studies of Abstract: Tris-(hydroxymethyl)phosphine oxide (THPO) to nucleases, topoisomerases or transferases [2, 21, 22]. Ben- a certain extent resembles a part of 2′-deoxyribofuranose, eficially, certain analogs of oligonucleotides intended to although it exists in an acyclic form only and the oxygen be used in antisense or RNAi strategies exert enhanced atom at the THPO phosphorus center provides additional affinity towards target messenger RNA, or increased hydration site or acceptor of hydrogen bonds. After proper nucleolytic stability. However, there are persisting prob- protection of hydroxyl groups, THPO was functionalized lems in therapeutic applications of oligonucleotides, such with nucleobases and converted into phosphoramidite as poor cellular uptake or off-target effects, so there is still monomers suitable for incorporation into growing oligo- a need for novel nucleic acids analogs free of those short- nucleotide chains within the solid phase synthesis pro- comings. In this frame, we previously developed a new tocol. The resultant THPO-DNA analogs show reduced DNA analog containing an acyclic unit (originated from affinity to complementary DNA strands, and are resistant bis(hydroxymethyl)phosphinic acids, BHPA, 2) replaced towards snake venom and calf spleen exonucleases. for the deoxyribose ring [23, 24]. The nucleobase in 2 could Keywords: acyclic DNA analog; modified oligonucleo- occupy the position identical to that in a natural nucleotide; oligonucleotide analog; oligonucleotide synthesis; side or a more distant one, depending on the number of tris(hydroxymethyl)phosphine oxide. methylene units n (Figure 1). BHPA related analogs were also obtained in the form of a double-anionic derivative 3 deprived of the nucleobases [23, 24]. The BHPA-DNA analogs are slightly cytotoxic towards HUVEC and HeLa Introduction cells, and are resistant to nucleolytic degradation. The nucleoside analogs, where the 3′-, 4′- and 5′-carbon atoms In principle, oligonucleotide analogs are modified within of the original sugar moiety are represented by either the the internucleotide bonds (e.g. phosphorothioates [1, BHPA or tris(hydroxymethyl)phosphine oxide (THPO) 2], and phosphorodithioates [3], methylphosphonates moiety, have been already synthesized and their antiviral [4], boranophosphates [5], methylboranophosphonates properties investigated [25]. Hypothetically, introduction [6], phosphoramidates/phosphorothioamidates [7], and of a nucleobase to THPO would also create a DNA-like methylphosphoramidates [8]), heterocyclic bases (e.g. unit able to form the Watson-Crick hydrogen bonds with 2-thiouracil [9], 6-thioguanine [10]) or within the sugar a nucleoside in a complementary DNA or RNA strand. moiety (e.g. 2′-O-alkyl [11], LNA [12], 2′-F [13–15]). They Moreover, the DNA oligomers modified with the THPO units would contain more flexible sugar-phosphate backbone and additional hydration sites (the acceptor oxygen

*Corresponding author: Barbara Nawrot, Department of Bioorganic atoms at the THPO phosphorus centers) compared to the Chemistry, Centre of Molecular and Macromolecular Studies, natural DNA. In this paper, we present an approach for the Polish Academy of Sciences, Sienkiewicza 112, 90-363 Lodz, Poland, synthesis of suitably protected phosphoramidite deriva- e-mail: [email protected] tives of THPO-related acyclic nucleosides and their use Olga Michalak: Pharmaceutical Research Institute, 8 Rydygiera as monomers for synthesis of short DNA oligomers of the Street, 01-793 Warsaw, Poland structure . Structural characteristics of the acyclic THPO- Barbara Mikołajczyk and Wojciech J. Stec: Centre of Molecular and 4 Macromolecular Studies, Polish Academy of Sciences, DNA analogs, as well as their hybridization properties and Sienkiewicza 112, 90-363 Lodz, Poland stability against selected exonucleases are described. 304 B. Nawrot et al.: Acyclic DNA analogs

one molar equivalent of 4,4′-dimethoxytrityl chloride O B O B O O B O X O- (DMT-Cl) in pyridine at room temperature (Scheme 1). The P P P O (CH2)n O O O-DMT derivative 6 was obtained in 51% yield after silica O O O O O- O- O- O- gel chromatography. The second hydroxyl group in 6 was P P P P O O O O protected with tert-butyldimethylsilyl (TBDMS) moiety upon treatment with one molar equivalent of TBDMS-Cl 1 2 3 4 (in the presence of a four-fold molar excess of imidazole) Figure 1 A DNA unit (1), bis(hydroxymethyl)phosphinic acid (BHPA) furnishing compound 7A (R = TBDMS). Compound 6 was based acyclic analog of natural DNA (2, X = O or N), its abasic also treated with benzoyl chloride (Bz-Cl) in pyridine to double-anionic form (3), and a tris-(hydroxymethyl)phosphine oxide yield the O-DMT-O′-Bz derivative 7B (R = Bz). When 5 was (THPO) based analog (4). allowed to react with a two-fold molar excess of DMT-Cl, O,O′-di-DMT derivative 7C (R = DMT) was obtained in good yield.

Results and discussion Coupling of the protected THPO derivatives with nucleobases Synthesis of protected THPO-acyclic nucleoside phosphoramidites 11 Compound 7A was used as a starting material for the

PPh3/DIAD promoted coupling (the Mitsunobu reac- Protection of two hydroxyl groups in THPO tion conditions) [26] with N3-benzoyl-thymidine [27], N6-benzoyl-adenine [28, 29], or N2-isobutyryl-O6- For selective protection of only one hydroxyl group, diphenylcarbamoyl-guanine [30, 31]. The first reaction tris(hydroxymethyl)phosphine oxide 5 was treated with gave the derivative 8a (BPG = N3-Bz-Thy) in 95% yield

HO DMTO DMTO DMTO i ii iii O P OH O P OH O P OH O P OTs OH OH OR ODMT 5 6 7A R=TBDMS 9 7B R=Bz iv 7C R=DMT

O NHBz DMTO PG Me O P B PG NBz N vi B : v ODMT N O N O 8g

a b DMTO DMTO vii BPG BPG NHBz ODpc O P O P OR OH N N N N 8a,c,d R=TBDMS 10a-f N N N N NHiBu 8b R=DMT 8a' R=Bz viii c d DMTO O PG O NH2 O P B Me N NH NH N O NC N O P N N O N NHiBu N O 11b,c,e,f

e f g

Scheme 1 Transformation of tris(hydroxymethyl)phosphine oxide into acyclic nucleoside phosphoramidites.

Conditions: (i) 1 eq. DMT-Cl/pyridine, 20 h, room temperature (RT); (ii) for 7A: TBDMS-Cl, imidazole, CH3CN, 48 h, RT; for 7B: BzCl, pyridine,

24 h, RT; for 7C: 2 eq. DMT-Cl/pyridine, 20 h, RT; (iii) DMAP, p-TsCl/CH2Cl2, 2 h, 0–5°C; (iv) cytosine, NaH, DMF, 60°C, 3 h; (v) BzCl, pyridine,

24 h, RT; (vi): N3-benzoylthymine, N6-benzoyladenine or N2-isobutyryl-O6-diphenylcarbamoylguanine, PhP3, DIAD, THF, 48 h; (vii): for

8a,c,d: 1 m (t-Bu)4NF in THF, 24 h, RT; for 8a′: 28% aq. NH4OH, 24 h; for 8b: 1 M p-TsOH/MeOH, TLC control, pyridine; (viii) 2-cyanoethyl-N,N- diisopropylchlorophosphoramidite, CH2Cl2 , RT. B. Nawrot et al.: Acyclic DNA analogs 305

Table 1 Spectral characteristics and yields of THPO derivatives 8, 10 and 11.

Compound R 31P NMR FAB MS (m/z) Yield (%) δ (ppm) [M+H]+ [M-H]-

8a TBDMS 38.52 912.8d 758.5 92a 8a′ Bz 40.87 921.5d 767.5 95a 8b DMT 41.15 942.2 940.3 44b 8c TBDMS 42.02 778.5 776.4 51 8d TBDMS 40.89 955.2 953.2 90

10a 38.40 – 653.5 38

10b 45.15 640.3 638.2 60

10c 42.39 664.3 662.0 84

10d 36.92 841.5 839.2 51

10e 45.46 551.6 549.0 60c, 41c

10f 43.64 646.4 644.3 40

11b 152.54; 152.34; 152.03f NDe ND 45 39.76; 39.64; 39.46; 39.33g

11c 151.88; 151.72; 151.57f ND ND 77 151.41; 38.10; 37.77g

11e 150.95; 150.71; 150.64;f ND ND 70 37.20; 37.02; 36.90; 36.72g

11f 151.91; 151.59; 151.27;f ND ND 41 39.54; 39.22; 38.99; 38.68g a b c The product was in ca. 30% contaminated with Ph3PO. The combined yield of transformations 9→8g→8b. The yields of two transformations: 8a→10e – 60%, and 8a′→10e – 41%. dThe [M+153]+ ion was registered for 8a and 8a′ in a complex with m-nitrobenzyl alcohol (NBA) matrix molecule (molecular mass 153). eND – Not determined. fResonances at δ > 150 ppm correspond to the phosphorus atom in the phosphoramidite part of the molecule. gResonances at δ < 40 ppm correspond to the phosphorus atom in the THPO part of the molecule.

(Table 1). Similarly effective was the condensation of 7B OMe MeO O H with the same nucleobase, which produced 8a′. NHBz NH2 HN N N The coupling of 7A with N6-benzoyl-adenine was DMTO 5 N H H 8 2 6 2 O 2' 4 H O N N N H DMTO N O H 1' more challenging, since two products (most likely regioi- O P 2' 1' H O P H P H somers) at ca. 1:1 ratio were obtained. Chromatographic 3' H H O H OTBDMS ODMT separation delivered the required N9-adenine derivative HO 8c 8g 10e 8c (fast-eluting, BPG = N6-Bz-Ade) in 51% yield. Its structure was confirmed by a 2D NMR analysis in a hetero- Figure 2 The detected spin-spin coupling (by a HMBC 2D NMR nuclear multiple bonds coherence (HMBC) experiment, experiment) between hydrogen atoms at C1′ in the THPO moiety and since the spin-spin coupling between the hydrogen atom selected neighboring carbon atoms in 8c, 8g, and 10e. at C1′ in the THPO moiety and C4 and C8 atoms of adenine confirmed the presence of the P-C-N9 linkage (Figure 2). The reaction of 7A with N2-isobutyryl-O6- compound 7 and the required compound 8b was not diphenylcarbamoylguanine furnished two products at obtained, probably because of insufficient acidity of the a 95:5 ratio (as determined by 31P NMR), and the major N1-H function. Therefore, cytosine was treated with NaH product (efficiently isolated in 90% yield by silica gel in DMF to generate anion, which was allowed to react chromatography) was the required N9-derivative 8d (BPG = with O,O′-bis-DMT-O″-tosyl-derivative 9 (obtained from N2-iBu-O6-Dpc-Gua) the structure of which was confirmed 7C by routine tosylation with tosyl chloride) furnishing as previously described [25]. a 95:5 mixture of regioisomers. The required N1-substi- Unfortunately, under the Mitsunobu reaction con- tuted regioisomer 8g (BPG = Cyt) was isolated by silica gel ditions, N4-benzoylcytosine did not react with any column chromatography and its structure was confirmed 306 B. Nawrot et al.: Acyclic DNA analogs by 2D HMQC and HMBC analysis, as shown previously silica gel chromatography the resulting 10b was obtained [25] (Figure 2). It was further benzoylated at the exoamine in 60% yield. function by treatment with benzoyl chloride in pyridine to yield 8b (BPG = N4-Bz-Cyt). Phosphitylation of derivatives 10

Deprotection of one hydroxyl group in derivatives 8 Phosphitylation of 10b,c,e,f was carried out in anhydrous dichloromethane using 2-cyanoethyl-N,N-diisopropyl- In the next step, a set of derivatives 8, suitably protected chlorophosphoramidite (1.2 eq.) in the presence of diiso- at the base moieties, were deprotected under various con- propylethylamine (3.0 eq.) at room temperature, followed ditions, depending on a protecting group to be removed by silica gel column chromatography. The correspond- to yield compounds possessing one OH function free and ing acyclic nucleoside amidites 11b,c,e,f (Scheme 1) were another one a DMT-protected (10). The TBDMS group of obtained in 41-77% yields (Table 1). Their 31P NMR spectra 8a,c,d was removed by treatment with 1.2 molar excess of contain two groups of signals with the chemical shifts TBAF reagent in THF, giving rise to 10a,c,d, respectively δ in a range of 37–39 ppm (phosphine oxide moieties) (Table 1). However, the treatment with TBAF partially and > 150 ppm (phosphoramidite groups). From the ste- removed the N3-Bz group in 10a, so a minor compound reochemical point of view, each compound 11 comprises

10e was also obtained and chromatographically isolated. of four diastereomers (RPSP, RPRP, SPRP, SPSP) and 3–4 reso- The structure of 10e (Figure 2) was confirmed by 2D HMBC nance lines are observed in each group of signals, depend- analysis and possible correlation contacts between the ing on differences in chemical shifts. hydrogens at C1′ and neighboring atoms were visible. The NOESY spectrum showed the NOE contacts between the hydrogens at C2′ and hydrogen atoms of the DMT group. Synthesis of chimeric oligomers containing Deprotection of 8d with TBAF produced also a minor the THPO motif compound 10f, resulting from the removal of the N6-Dpc group. The O′-Bz group of the THPO moiety in 8a′ was Phosphoramidite derivatives of THPO-based acyclic removed by treatment with 28% aqueous NH4OH, but this nucleosides 11b, 11c, 11e and 11f, together with commer- treatment also quantitatively removed the N3-Bz group, cial phosphoramidites of suitably protected thymidine, thus 10e was obtained as a sole product. One DMT pro- 2′-deoxycytidine, 2′-deoxyadenosine and 2′-deoxyguano- tecting group from bis-DMT derivative 8b was removed by sine were used for the automated solid phase synthesis TLC-controlled treatment with 1 m p-TsOH/MeOH, termi- of chimeric DNA oligomers 12-21 (Table 2, the lower-case nated by addition of pyridine. After solvents removal and italicized letters a, c, g and t represent the modified

Table 2 Mass spectrometry and chromatographic characteristics of chimeric DNA oligomers possessing tris(hydroxymethyl)phosphine oxide units (THPO), marked by lower-case italicized letters.

a No. Oligomer sequence 5′→3′ Molecular mass MALDI-TOF m/z RP-HPLC, Rt (min) DMT-on DMT-off

12 T9tT9 5720 5721 23.74 23.16

13 T8tttT8 5730 5732 23.60 23.11

14 T9aT9 5730 5729 21.40 22.91

15 T8aaaT8 5760 5758 21.26 22.81

16 T9gT9 5746 5751 23.04 19.56

17 T8gggT8 5808 5809 22.57 19.05

18 T9cT9 5706 5713 22.14 19.12

19 T9ccT8 5697 5696 21.96 20.61 20 atttaattat 3077 3076 20.00/20.62 18.69/18.90 21 ataattaaat 3097 3095 18.71/19.47 17.19/17.01 aRP-HPLC purification was performed on a C18 column (4.6 × 250 mm, ThermoQuest) with a linear gradient of buffer A (0.1 m triethylammonium bicarbonate, pH 7.5) and buffer B (40% acetonitrile in 0.1 m triethylammonium bicarbonate, pH 7.5), flow rate 1 mL/min; a gradient of B for DMT-on 15–100%, DMT-off 0–100%, over 30 min. B. Nawrot et al.: Acyclic DNA analogs 307 acyclic units bearing Ade, Cyt, Gua or Thy nucleobases, and mass spectrometry analysis showed identical m/z respectively) [32]. The syntheses were performed on a values (Table 2). Moreover, all four detritylated samples succinyl-linked LCAA CPG solid support using an ABI exhibited identical electrophoretic mobility in 20% pol- 394 synthesizer (Applied Biosystems Inc., Foster City, CA, yacrylamide/7 m urea gel (data not shown). Therefore, USA). The only modification made in the manufacturer’s we concluded that each pair consists of molecules of the protocol was a prolonged coupling time (up to 600 s) same sequence, which differ in absolute configuration applied for the monomers 11. For monomers 11c and 11e of the P-stereogenic centers present in the THPO acyclic the coupling efficiency (determined by the DMT-cation nucleotide first from the 5′-end. Similar observations assay) was in the range of 98–99%, while for 11b and 11f were noted in earlier works on P-chiral, 5′-DMT protected the efficiency dropped below 90%. oligo(nucleoside phosphorothioate)s [37]. Perhaps for After synthesis of 12-19, standard cleavage from the the same reason, a relatively small difference in the solid support and deprotection of phosphate groups and chromatographic mobility was also seen after removal of nucleobases was done (28% aqueous NH4OH, 55°C, 16 h). the DMT group (Table 2). This assumption is supported Notably, in the case of fully modified decamers 20 and by reports that short oligo(nucleoside phosphorothio- 21, containing only the THPO-based units a, c, g, or t), ate)s (trimers, tetramers and pentamers) were chroma- an LCAA-CPG support with a universal linker was used tographically separated into diastereomeric species but [33], for which the synthesis started with a phosphora- the efficiency of this process strongly depended upon the midite monomer 11e. The assembled oligomers were then sequence of nucleobases and composition of the buff- cleaved from the support with a 20% ethanolic solution of ered eluent [38]. gaseous ammonia (55°C, 4 h), followed by the treatment with 28% aq. NH4OH/40% aq. MeNH2 (room temperature, 16 h) [34, 35]. Physicochemical characterization of To purify the oligomers, a two-step RP-HPLC (DMT-on/ oligomers 12-21 DMT-off) was applied [36]. The DMT-tagged oligomers (isolated during the first step) were detritylated with Hybridization properties of THPO-chimeric DNA oligomers 50% aq. acetic acid and purified in the DMT-off form. The respective retention times (Rt) are given in Table 2. Illustra- Thermal stability of complexes formed by oligomers 12-19 tive RP-HPLC profiles for 5′-T9gT9-3′ (16) in its DMT-on and with complementary single stranded DNA and RNA oli- DMT-off forms are shown in Figure 3. The structures of all gomers (THPO/DNA and THPO/RNA, respectively) and oligomers listed in Table 2 were confirmed by MALDI-TOF that of the duplex 20/21 was determined by UV-monitored mass spectrometry. thermal melting measurements and the results (the Tm Interestingly, the RP-HPLC profiles recorded for DMT- values) are given in Table 3. DNA/DNA and DNA/RNA 20 and DMT-21 showed two closely eluted main peaks of duplexes isosequential with the investigated duplexes similar intensity (Figure 3C). These four products (two were used as the reference complexes. pairs: DMT-20-fast and DMT-20-slow; DMT-21-fast and The values of the Tm parameter for duplexes DMT-21-slow) were collected separately and detritylated. formed by chimeric oligomers 12–19 with DNA and RNA Interestingly, within each pair of fully deprotected prod- templates are lower than those of the reference non- ucts the RP-HPLC retention times were almost identical modified duplexes (ΔTm* = -4.2 ÷ -7.2°C). This distinctly

AB5′-DMT-T9gT9-3′ 5′-T9gT9-3′ C 5′-DMT-ataattaaat-3′ 4000 1000 1000 Fast Slow 2000 500 500

0 0 0

020 40 020 40 020 40 Min Min Min

Figure 3 Reverse-phase HPLC profiles for purification of 16 in its DMT-on (A) and DMT-off form (B), and for DMT-21 oligomer (C). 308 B. Nawrot et al.: Acyclic DNA analogs

Table 3 Tm and ΔTm* values for complexes of chimeric oligomers 12-19 with complementary DNA and RNA strands and for the duplex 20/21, measured in 10 mm Tris-HCl, pH 7.4, 10 mm MgCl2, 100 mm NaCl.

No. Oligomer sequence 5′→3′ Tma (°C) ΔTm* (°C) Tma (°C) ΔTm* (°C)

THPO/DNA DNA/DNA THPO/RNA DNA/RNA

12 T9tT9 45.0 51.0 -6.0 34.4 41.4 -7.0

13 T8tttT8 35.5 51.0 -5.2 28.0 41.4 -4.5

14 T9aT9 45.9 50.4 -4.5

15 T8aaaT8 38.3 50.8 -4.2

16 T9gT9 45.6 52.8 -7.2

17 T8gggT8 44.3 56.8 -4.2

18 T9cT9 45.4 52.3 -6.9

19 T9ccT8 38.5 53.5 -7.5 20/21 9.9b 17.4 -0.4c

The ΔTm* is a difference between the Tm found for THPO/DNA or THPO/RNA duplex and a corresponding reference DNA/DNA or DNA/RNA duplex, respectively, calculated per one THPO-modified unit. aThe measurement error ±1.0°C. bNo DNA oligomer added. cCalculated for the total of 20 (2 × 10) modified units.

lower affinity of the investigated DNA analogs towards the complementary strands probably results from their ) higher flexibility in comparison to natural DNA mol- -1

cm 1 ecules, which brings the loss of entropy [39, 40]. It has -1 been shown that a rigid structure of DNA analog (like e.g. 220 230 240 250 260 270 280 290 300 310 320 LNA) provides better affinity to a complementary RNA strand [41], although one has to keep in mind that the –3

conformationally flexible peptide nucleic acid analogs Delta epsilon (M (PNA) exhibit extremely high affinity toward their DNA and RNA complements [42]. Fully THPO-modified 10-mers 20 and 21 were designed –7 Wavelength (nm) as complementary to each other. Interestingly, the thermal stability of a 20/21 duplex is by ca. 8°C lower than that Figure 4 The CD spectra of the duplexes: 20/21 (circles), of the reference non-modified DNA/DNA complex (Tm = 20/5′-d(ATAATTAAAT)-3′ (triangles) and 21/5′-d(ATTTAATTAT)-3′ 9.9°C vs. 17.4°C), although in terms of the decrease per a (crosses), as well as single stranded THPO-oligomers 20 and 21 (solid line) (2 μm duplex in a 10 mm Tris-HCl, pH 7.4, 10 mm MgCl , modified unit the loss of stability is, formally, rather small 2 100 mm NaCl buffer). (ΔTm* = -7. 5 °C/20 = -0.4°C). This low stability of 20/21 may be attributed to the fact, that the THPO based acyclic nucleosides 10 are obtained on a non-stereoselective way Circular dichroism analysis of duplexes formed by as mixtures of two P-enantiomeric forms. This feature is oligomers 20 and 21 transferred into the phosphoramidite monomers 11, thus the decamers 20 and 21 are synthesized as the mixtures The helical structures of duplexes formed by 20 and 21 with of 210 diastereomers, while each diastereomer may exert complementary DNA templates, as well as the structure of different hybridization properties. Of course, the pres- the homo-THPO duplex 20/21, were examined by circular ence of 210 stereoisomers of 20 and of the same number of dichroism spectroscopy (CD) (Figure 4). The CD spectra for stereoisomers of 21 makes the whole hybridizing system 20 and 21 alone indicate that these single-stranded oligom- extremely complicated. However, the assumption that ers do not adopt any ordered helical structure. To analyze P-diastereomeric inhomogeneity is a major factor decreas- the duplexes, the 1:1 equimolar mixtures of complemen- ing the thermal stability of 20/21 is supported by the fact tary oligomers (in 10 mm Tris-HCl, pH 7.4, 10 mm MgCl2, 100 that similarly flexible GNA (glycol nucleic acids) obtained mm NaCl buffer) were annealed by slow cooling from 95°C, form enantiomerically pure acyclic glycol nucleosides down to the room temperature. The CD spectra recorded forms quite stable double stranded structures, compared for 20/5′-d(ATAATTAAAT)-3′ and 21/5′-d(ATTTAATTAT)-3′ to the respective ds DNA duplexes [43]. duplexes as well as for the 20/21 duplex exhibit positive B. Nawrot et al.: Acyclic DNA analogs 309

Figure 5 MALDI-TOF mass spectrometry analysis of the products of the enzymatic hydrolysis of oligomer 13 with snake venom phosphodiesterase (PDE I, panel A) and with calf spleen phosphodiesterase (PDE II, panel B). The time of incubation is indicated above the consecutive plots.

Cotton effects at λ = 276 nm, and negative Cotton effects not shown). After 60 min of the reaction B, accumulation at λ = 253 nm. All three spectra are characteristic for the of the oligonucleotide 5′-TtttT8-3′ (a band at m/z 3602) was B-type DNA duplex helical structures, with the most observed, so the reaction stopped at the last natural nucle- ordered structure of the 20/21 duplex. Interestingly, in CD otide upstream of the modification site. Thus, the THPO spectra of 20/21 and 21/5′-d(ATTTAATTAT)-3′ the two isoel- modified unit inserted within the DNA chain is recognized liptic points are present at similar wavelengths (at 235 and by both 3′- and 5′-exonucleases tested. Consequently, the 265 nm), while the CD spectrum of 20/5′-d(ATAATTAAAT)-3′ use of the THPO units at the 3′- and 5′-ends of therapeu- very much differs from those previously mentioned, espe- tic nucleic acids will make them resistant towards cellular cially at the short-wave region (220–250 nm, no isoelliptic exonucleases and will improve their pharmacokinetics. point below 260 nm). This difference is quite surprising Similar abasic phosphinic acid ‘clamps’(BHPA, 3) were because at first glance 20 and 21 differ by only two nucle- already successfully applied in in vitro experiments for obases (4 × Ade, 6 × Thy vs. 6 × Ade, 4 × Thy). protection of deoxyribozymes (directed towards the HIV-1 viral RNA sequence) in HIV-1 infected cells (unpublished results), and of deoxyribozymes designed to cleave bcr-

Stability of the oligomer 13 (5′-T8tttT8-3′) towards selected abl mRNA fragments [44]. 5′- and 3′-exonucleases

Chimeric oligomer 13 (5′-T8tttT8-3′), possessing three THPO- thymine units (t) located in the central part of the homothy- Conclusions midine nonadecamer, was tested as a substrate for 3′- and 5′-exonucleases, i.e. for snake venom phosphodiesterase A series of novel DNA analogs was obtained by incorpora- (svPDE or PDE I) and for calf spleen phosphodiesterase tion of the tris(hydroxymethyl) phosphine oxide (THPO) (csPDE, PDE II), respectively. The digestion products were derived residues (acylic nucleoside analogs) into an oligo- identified by MALDI-TOF MS analysis (Figure 5). From the nucleotide chain. Structural flexibility and loss of entropy samples of 13 treated with PDE I (reaction A) or PDE II make these chimeric oligomers unable to form thermally (reaction B) small aliquots were taken after 15, 45 and 60 stable duplexes with their complementary RNA and DNA min, or after 10, 30 and 90 min, respectively. strands. However, the duplex formed by two fully modi- Both sets of spectra show the ladders of products, fied complementary THPO-oligomers adopts a helical which differ by m/z 304, corresponding to the products of structure (by CD analysis) and its thermal stability is only consecutive removal of PT (with PDE I) or TP (with PDE II) slightly lower than that of its isosequential DNA duplex. from the parent oligonucleotide 13 (m/z 5732). After 45 min Oligomers with the THPO units are stable toward two 3′- of the reaction A, accumulation of the oligonucleotide 5′- and 5′-exonucleases tested (hydrolysis stops at the modifi-

T8ttt-3′ (a band at m/z 3298) was observed. No further deg- cation site), and thus can be used to increase the stability radation of this product was observed over next 6 h (data of therapeutic oligonucleotides in body fluids. 310 B. Nawrot et al.: Acyclic DNA analogs

(hydroxymethyl)phosphine oxide 7C was obtained in 50% yield Experimental (3.11 g, 5 mmol) by chromatographic separation on a silica gel col- 1 umn (CHCl3/MeOH, 100:0→98:2, v/v). H NMR (200 MHz, CDCl3): δ Synthesis of an O-DMT derivative of THPO (6) 7.72–6.51 (m, 26H, aromatic protons), 4.02 (d, J = 4.7 Hz, 2H), 3.90–

3.80 (m, 2H), 3.77 (s, 6H, 2 × OCH3), 3.76 (s, 6H, 2 × OCH3), 3.54 (dd, J = = 31 δ To a solution of 10 g (0.07 mol) of tris(hydroxymethyl)phosphine 5.5 Hz, J 12.4 Hz, 2H); P NMR (200 MHz, CDCl3): 43.87; FAB MS + + oxide (5) in pyridine (100 mL) 4,4′-dimethoxytrityl chloride (DMT-Cl) (m/z): 767.3 [M+Na] , 897.6 [M+153] . (24 g, 0.07 mol) was added and the mixture was stirred for 20 h at room temperature. Pyridine was evaporated, the residue was (three times) dissolved in ethanol and evaporated to dryness. This step was Synthesis of an (N2-isobutyryl-O4-diphenylocarbamoyl- repeated with toluene. The desired product 6 was obtained in 51% guanine) derivative of 7A (8d) yield (16 g, 0.036 mol) by chromatographic separation on a silica gel 1 column (CHCl3/MeOH, 100:0→97:3, v/v, and 0.1% vol. of Et3N). H A suspension of N2-isobutyryl-O6-diphenylocarbamoyl-guanine NMR (200 MHz, CDCl3): δ 7.41–6.74 (m, 13H, aromatic protons), 4.19 (163 mg, 0.39 mmol) in anhydrous THF was heated under reflux for (m, JP-H = 3 Hz, 4H, CH2OH), 3.74 (s, 3H, OCH3), 3.73 (s, 3H, OCH3), 3.64 31 20 min and cooled to room temperature. Then 7A (166 mg, 0.3 mmol), (d, JP-H = 6 Hz, 2H, CH2ODMT); P NMR (CDCl3): δ 46.51; MS FAB (m/z): + triphenylphosphine (165 mg, 0.63 mmol) and DIAD (diisopropyl 544.3 [M+102 (Et3NH)] . azodicarboxylate) (124 μL, 0.63 mmol) were added (argon atmosphere, daylight protected). After 48 h the solvent was evaporated. The desired ((N2-isobutyryl-O4-diphenylocarbamoyl-guanine)- Synthesis of an O-DMT-O′-TBDMS derivative of THPO (7A) 9-methyl)(DMT-oxymethyl)(tert-butyldimethylsilyloxymethyl)phosphine oxide (8d) was obtained in 90% yield (257 mg, 0.26 mmol) by

To a solution of 2 g (4.5 mmol) of 6 in acetonitrile (60 mL), imidazole chromatographic separation on a silica gel column (CHCl3/MeOH, 1 (1.2 g, 18 mmol) was added, followed by TBDMS-Cl (0.68 g, 4.5 mmol), 100:0→95:5, v/v). H NMR (200 MHz, CDCl3): δ 8.21 (s, 1H, H-8), 7.81– and the mixture was stirred for 20 h at room temperature. Then, 6.78 (m, 23H, aromatic protons), 4.75 ppm (d, 2H, JP-H = 6.7 Hz, CH2N),

triethylamine (5 mL) was added and the solvent was evaporated. The 4.15 (dd, 2H, J = 2.5 Hz, J = 5.5 Hz, CH2Si), 3.75 (s, 3H, OCH3), 3.74 desired (DMT-oxymethyl)(tert-butyldimethylsilyloxymethyl)(hydrox- (s, 3H, OCH3), 3.57 (d, 2H, JP-H = 6.2 Hz, CH2ODMT), 2.95–2.81 (m, 1H, ymethyl)phosphine oxide 7A was obtained in 45% yield (1.1 g, CH(CH3)2), 1.22 (d, 6H, J = 6.8 Hz, 2 × CH3), 0.76 (s, 9H, 3 × CH3), -0.03 31 2.0 mmol) by chromatographic separation on a silica gel column (s, 3H, Si-CH3), -0.05 (s, 3H, Si-CH3); P NMR (CDCl3): δ 40.89; FAB MS 1 + - (CHCl3/MeOH, 100:0→95:5, v/v). H NMR (200 MHz, CDCl3): δ 7.4 6 – (m/z): [M+H] 955.2, [M-H] 953.2.

6.80 (m, 13H, aromatic protons), 4.14–4.07 (m, 4H, CH2OTBDMS), 3.78

(s, 3H, OCH3), 3.77 (s, 3H, OCH3), 3.62–3.53 (m, 2H, CH2ODMT), 0.81 31 (s, 9H, 3 × CH3), 0.06 (s, 3H, Si-CH3), 0.02 (s, 3H, Si-CH3); P NMR + + Synthesis of an N3-benzoyl-thymine derivative of 7A (8a) (CDCl3): δ 44.55; MS FAB (m/z): 579.4 [M+Na] , 709.3 [M+153] .

To a solution of N3-benzoyl-thymine (110 mg, 0.48 mmol) in anhydrous THF, 7A (200 mg, 0.37 mmol), triphenylphosphine Synthesis of an O-DMT-O′-Bz derivative of THPO (7B) (204 mg, 0.77 mmol) and DIAD (152 μL, 0.77 mmol) were added (argon atmosphere, daylight protected). After 48 h the solvent was To a solution of 3.5 g (7.8 mmol) of 6 in pyridine (50 mL) benzoyl chlo- evaporated. The desired product [(N3-benzoyl-thymine)-1-methyl] ride (1.1 g, 7.8 mmol) was added and the mixture was stirred for 16 h at (DMT-oxymethyl)( tert-butyldimethylsilyl-oxymethyl)phosphine room temperature. Pyridine was evaporated, the residue was (three oxide (8a) was obtained in 92% yield (260 mg, 0.34 mmol) by times) dissolved in ethanol and evaporated to dryness. This step was chromatographic separation on a silica gel column (CHCl3/MeOH, repeated with toluene. The desired (DMT-oxymethyl)(benzoyl-oxy- 100:0→95:5, v/v). 1 methyl)(hydroxymethyl)phosphine oxide 7B was obtained in 49% H NMR (200 MHz, CDCl3): δ 7.91–6.33 (m, 19H, aromatic pro- yield (2.1 g, 3.9 mmol) by chromatographic separation on a silica gel tons, H6), 5.08–4.91 (m, 2H, CH2N), 4.05 (d, 2H, J = 5.4 Hz, CH2Si), 3.78 1 column (CHCl3:MeOH, 100:0→95:5, v/v). H NMR (CDCl3): δ 8.12–6.73 (s, 3H, OCH3), 3.77 (s, 3H, OCH3), 3.61 (d, 2H, J = 6.3 Hz, CH2ODMT),

(m, 18H, aromatic protons), 4.90–4.77 (m, 2H), 4.24 (s, 2H), 3.79–3.73 1.93 (d, J = 1 Hz, CH3), 0.77 (s, 9H, 3 × CH3), 0.01 (s, 3H, CH3), -0.02 31 31 + (m, J = 7 Hz, J = 3 Hz, 2H), 3.75 (s, 3H, OCH3), 3.74 (s, 3H, OCH3); P (s, 3H, CH3); P NMR (CDCl3): δ 40.87; FAB MS (m/z): [M+154] 921.5, + ‑ NMR (CDCl3): δ 42.40; FAB MS (m/z): 699.2 [M+153] . [M-H] 76 7. 5 .

Synthesis of a O,O′-di-DMT derivative of THPO (7C) Synthesis of N6-benzoyl-adenine derivative of 7A (8c)

To a solution of 1.4 g (10 mmol) of tris(hydroxymethyl)phosphine To a solution of N6-benzoyl-adenine (115 mg, 0.48 mmol) in anhy- oxide (5) in pyridine (150 mL) 4,4′-dimethoxytrityl chloride (DMT-Cl) drous THF, 7A (200 mg, 0.37 mmol), triphenylphosphine (204 mg, (6.8 g, 20 mmol) was added and the mixture was stirred for 20 h 0.77 mmol) and DIAD (152 μL, 0.77 mmol) were added (argon at room temperature. Pyridine was evaporated, the residue was atmosphere, daylight protected). After 48 h the solvent was evapo- (three times) dissolved in ethanol and evaporated to dryness. This rated. The desired product [(N6-benzoyl-adenine)-9-methyl](DMT- step was repeated with toluene. The desired (bis-(DMT-oxymethyl)) oxymethyl)(tert-butyldimethylsilyl-oxymethyl)phosphine oxide (8c) B. Nawrot et al.: Acyclic DNA analogs 311

1 was obtained in 51% yield (148 mg, 0.19 mmol) by chromatographic on a silica gel column (CHCl3/MeOH, 100:0→90:10, v/v). H NMR separation on a silica gel column (CHCl3/MeOH, 100:0→95:5, v/v). (500 MHz, CDCl3): δ 7.32–6.66 (m, 27H, aromatic protons, H6), 5.59 (d, 1 H NMR (200 MHz, CDCl3): δ 8.93 (s, 1H, NH), 8.76 (s, 1H, H-2), J = 7.0 Hz, 1H, H-5), 4.12 (d, J = 5.6 Hz, 2H, CH2N), 3.81 (dd, J = 8.5 Hz,

8.32 (s, 1H, H-8), 7.96–6.68 (m, 18H, aromatic protons), 4.82 (d, 2H, J = 12.5 Hz, CH2ODMT), 3.73 (s, 6H, 3 × OCH3), 3.72 (s, 6H, 3 × OCH3), 3.52 31 J = 6.5 Hz, CH2N), 4.08 (dd, J = 5 Hz, J = 14 Hz, 1H, CH2Si), 4.02 (dd, (dd, J = 5.0 Hz, J = 12.5 Hz, 2H, CH2ODMT); P NMR (CDCl3): δ 41.92; 13 J = 6.5 Hz, J = 14 Hz, 1H, CH2Si), 3.78 (s, 3H, OCH3), 3.77 (s, 3H, OCH3), C NMR (125 MHz, CDCl3): δ 165.8 (C4), 156.2 (C-2), 145.2 (C6), 95.1 (C5),

3.60–3.59 (m, 2H, CH2ODMT), 0.80 ( s, 9H, 3 × CH3), 0.01 (s, 3H, CH3), 88.4 (d, JCP = 12 Hz, C-Ph), 58.6 (d, JCP = 83 Hz, CH2O), 44.3 (d, JCP = 67 31 + + -0.02 (s, 3H, CH3); P NMR (CDCl3): δ 42.02; FAB MS (m/z): [M+H] Hz, CH2N), 55.1 (CH3O), 143.6–113.3 (36C, 6 × Ph); FAB MS (m/z): [M+H] 778.5, [M-H]- 776.4. 838.2, [M-H]- 836.5.

Synthesis of an N3-benzoylthymine derivative of 7B (8a′) Synthesis of an O,O′-bis-DMT-N4-benzoylcytosine derivative of THPO (8b) To a solution of N3-benzoyl-thymine (110 mg, 0.48 mmol) in anhydrous THF, 7B (202 mg, 0.37 mmol), triphenylphosphine (204 mg, To a solution of (cytosine-1-methyl)[bis(DMT-oxymethyl)]phosphine 0.77 mmol) and DIAD (152 μL, 0.77 mmol) were added (argon atmos- oxide (8g) (350 mg, 0.42 mmol) in anhydrous pyridine (20 mL, cooled phere, daylight protected). After 48 h the solvent was evaporated. to 0°C) benzoyl chloride (240 μL, 2.09 mmol) was added dropwise. The desired product ((N3-benzoylthymine)-1-methyl)(DMT-oxyme- The mixture was kept at room temperature for 16 h and then water thyl)(tert-butyldimethylsilyl-oxymethyl)phosphine oxide (8a′) was (12 μL) and 28% aqueous NH OH (12 μL) were added. After 0.5 h obtained in 90% yield (253 mg, 0.33 mmol) by chromatographic sepa- 4 the solvent was evaporated under reduced pressure. The residue ration on a silica gel column (CHCl /MeOH, 100:0→95:5, v/v). 3 was diluted with dichloromethane (100 mL) and washed with satu- 1H NMR (200 MHz, CDCl ): δ 7.93–6.73 (m, 24H, aromatic protons 3 rated aqueous NaHCO . The organic layer was dried with MgSO and + H6); 4.88 (dd, J = 5 Hz, J = 14 Hz, 1H, CH′N), 4.75 (dd, J = 4 Hz, J = 3 4 evaporated to dryness. The desired [(N4-benzoylcytosine)-1-methyl] 14 Hz, 1H, CH″N), 4.58 (dd, J = 4 Hz, J = 16 Hz, 1H, CH″OBz), 4.13 (dd, [bis(DMT-oxymethyl)]phosphine oxide (8b) was obtained in 74% J = 6 Hz, J = 16 Hz, 1H, CH′OBz), 3.82–3.71 (m, CH ODMT), 3.75 (s, 3H, 2 yield (289 mg, 0.31 mmol) by chromatographic separation on a silica OCH ), 3.74 (s, 3H, OCH ), 1.90 (d, J = 1 Hz, CH ); 31P NMR (CDCl ): δ 3 3 3 3 gel column (CHCl :MeOH, 100:0→90:10, v/v). - + 3 38.52; FAB MS (m/z): [M-H] 758.5, [M+153] 912.8. 1 H NMR (200 MHz, CDCl3): δ 8.62–6.73 ppm (m, 33H, aro-

matic protons, H6, H5); 4.40 (d, J = 7 Hz, 2H, CH2N), 3.8–3.7 (m, 2H,

CH2ODMT), 3.74 (s, 6H, 2 × OCH3), 3.73 (s, 6H, 2 × OCH3), 3.60 (dd, J = 31 Synthesis of an O,O′-di-DMT-O″-tosyl derivative of THPO 6.4 Hz, J = 12.8 Hz, 2H, CH2ODMT); P NMR (CDCl3): δ 41.15; FAB MS (9): Conversion of 7C to 9 (m/z): [M+H]+ 942.2, [M-H]- 940.3.

To a solution of 7C (400 mg, 0.53 mmol) and DMAP (410 mg, 3.36 mmol), in anhydrous methylene chloride (50 mL, cooled to 0°C) General procedure for removal of TBDMS protecting p-toluenesulfonic chloride (303 mg, 1.59 mmol) was added. The mix- group in 8a,c,d,f: synthesis of 10a,c-f ture was kept at 0–5°C for 2 h and then was diluted with dichloromethane (100 mL) and washed with saturated aqueous NaHCO3. The organic layer was dried with MgSO and evaporated to dryness. The desired [bis To a solution of 8 (8a,c,d,f, 200 mg, ≈ 0.25 mmol) in anhydrous THF 4 μ (DMT-oxymethyl)](p-toluenesulfonyloxymethyl)phosphine oxide 9 (3 mL), 1 m THF solution of tetra-n-butylammonium fluoride (311 L, was obtained in 40% yield (193 mg, 0.21 mmol) by chromatographic 0.31 mmol) was added. After 24 h the solvent was evaporated and the residue was dissolved in water and extracted with chloroform separation on a silica gel column using CHCl3 as an eluent. 1 (3 times). The organic layer was dried with MgSO4 and evaporated to H NMR (200 MHz, CDCl3): δ 7.69–6.66 (m, 20H, aromatic dryness. The products 10a,c-f were isolated by chromatography on a protons), 4.35 (d, J = 6 Hz, 2H, CH2OTs), 3.78 (s, 6H, 2 × OCH3), 3.77 31 silica gel column (CHCl3/MeOH, 100:0→90:10, v/v). (s, 6H, 2 × OCH3), 3.70–3.52 (m, 4H, CH2ODMT), 2.43 (s, 3H, CH3); P - + NMR (CDCl3): δ 39.87; FAB MS (m/z): [M-H] 898.1, [M+Na] 922.5. [(N3-Benzoylthymine)-1-methyl](DMT-oxymethyl)(hydroxyme- 1 thyl)phosphine oxide (10a) Yield 39%; H NMR (200 MHz, CDCl3): δ 7.97–6.73 (m, 19H, aromatic protons, H6), 4.56 (dd, J = 15.5 Hz, J = ′ Synthesis of an O,O -di-DMT-cytosine derivative of 5.0 Hz, 1H), 4.11 (dd, J = 15.5 Hz, J = 6.0 Hz, 1H), 4.02–3.88 (m, 2H), THPO (8g) 3.79 (s, 3H, OCH3), 3.78 (s, 3H, OCH3), 3.71–3.67 (m, 2H), 1.92 (d, 3H, 31 - J = 1.1 Hz, CH3); P NMR (CDCl3): δ 38.40; FAB MS (m/z): [M-H] 653.5. To a solution of cytosine (101 mg, 0.91 mmol) in anhydrous DMF (30 mL) sodium hydride (22 mg, 0.91 mmol, 60% suspension in min- [(N6-Benzoyladenine)-9-methyl](DMT-oxymethyl)(hydroxyme- 1 eral oil) was added. The mixture was kept at room temperature for thyl)phosphine oxide (10c) Yield 84%; H NMR (200 MHz, CDCl3): 20 min and then a solution of 9 (672 mg, 0.75 mmol) in DMF (10 mL) δ 8.75 (s, 1H, H-2), 8.16 (s, 1H, H-8), 8.04–6.80 (m, 18H, aromatic pro- ° was added dropwise. The mixture was kept at 60 C for 3 h and the sol- tons), 4.91 (dd, J = 3 Hz, J = 16 Hz, 1H, CH2N), 4.77 (dd, J = 8.5 Hz, J = vent was evaporated under reduced pressure. The desired (cytosine- 16 Hz, 1H, CH2N), 3.88 (d, J = 4.0 Hz, 2H, CH2OH), 3.78 (s, 3H, OCH3),

1-methyl)[bis(DMT-oxymethyl)]phosphine oxide (8g) was obtained 3.77 (s, 3H, OCH3), 3.75–3.65 (m, J = 5.0 Hz, J = 7.3 Hz, 2H, CH2ODMT); 31 + - in 60% yield (376 mg, 0.45 mmol) by chromatographic separation P NMR (CDCl3): δ 42.39; FAB MS (m/z): [M+H] 664.3, [M-H] 662.0. 312 B. Nawrot et al.: Acyclic DNA analogs

[(N2-Isobutyryl-O6-diphenylcarbamoylguanine]-9-methyl) General procedure for phosphitylation of compounds 10: (DMT-oxymethyl)(hydroxymethyl)phosphine oxide (10d) Yield synthesis of 11b,c,e,f 1 51%; H NMR (200 MHz, CDCl3): δ 7.42 (s, 1H, H-8), 7.39-6.73 (m, 23H, aromatic protons), 4.83 (d, JP-H = 5 Hz, 2H, CH2N), 4.45 ppm (dd, J = To a solution of 10 (0.15 mmol) in a mixture of CH CN (1 mL) and 9 Hz, J = 16 Hz, 1H, CH′OH), 4.22 (dd, J = 5 Hz, J = 16 Hz, 1H,CH″ OH ), 3 CH2Cl2 (1 mL), N,N-diisopropylamine (78 μL, 0.45 mmol) was added, 3.79 (s, 3H, OCH3), 3.78 (s, 3H, OCH3), 3.40 (dd, JP-H = 7.2 Hz, J = 13 Hz, followed by 2-cyanoethyl-N,N-diisopropyloaminochlorophosphite 1H, CH′ODMT), 3.25 (dd, JP-H = 4.1 Hz, J = 13 Hz, 1H, CH″ODMT), 31 (35 μL, 0.18 mmol). The reaction progress was monitored by TLC. 2.90–2.28 (m, 1H, CH(CH3)2), 0.98 (d, J = 4.6 Hz, 6H, 2 × CH3), P NMR + - After ca. 1 h the volatile components were evaporated. The desired (CDCl3): δ 36.91; FAB MS (m/z): [M+H] 841.5, [M-H] 839.2. products were isolated by chromatographic separation on a silica gel column (CHCl /MeOH, 100:0→98:2, v/v). (Thymine-1-methyl)(DMT-oxymethyl)(hydroxymethyl)phos- 3 phine oxide (10e) Yield 60% (combined for two steps); 1HNMR

(500 MHz, CDCl3): δ 7.42-6.14 (m, 14H, aromatic protons, H6), 4.34 (Thymine-1-methyl)](DMT-oxymethyl)(2-cyanoethoxy-N,N-

(dd, 2H, J = 5 Hz, J = 15.0 Hz, CH2N), 4.10 (dd, J = 3.6 Hz, J = 9.5 Hz, diisopropylaminophosphineoxymethyl)phosphine oxide (11e) 31 δ 2H, CH2OH), 3.79 (s, 3H, OCH3), 3.78 (s, 3H, OCH3), 3.61 (dd, 2H, J = Yield 70%; P NMR (CDCl3): 150.95, 150.71, 150.64, 37.20, 37.02, 36.90, 31 6.0 Hz, J = 12.8 Hz, CH2ODMT), 1.79 (d, 3H, J = 1.1 Hz, CH3); P NMR 36.72. 13 (CDCl3): δ 45.46; C NMR (125 MHz, CDCl3): δ 166.6 (C2), 160.4, 154.3

(18, 3 × Ph), 152.8 (C4), 143.0 (C6), 111.6 (C5), 89.9 (d, JCP = 11 Hz, C-Ph), [(N4-Benzoylcytosine)-1-methyl](DMT-oxymethyl)(2-cyanoeth-

59.2 (d, JCP = 81 Hz, CH2O), 58.3 (d, JCP = 80 Hz, CH2O ), 55.8 (CH3O), oxy-N,N-diisopropylaminophosphineoxymethyl)phosphine + 31 δ 44.4 (d, JC-P = 66 Hz, CH2N), 12.3 (CH3); FAB MS (m/z): [M+H] 551.6, oxide (11b) Yield 45%; P NMR (CDCl3): 152.54; 152.34, 152.03; [M-H]- 549.0. 39.76; 39.64; 39.46; 39.33.

[(N2-Isobutyrylguanine)-9-methyl](DMT-oxymethyl)(hydroxy- [(N6-Benzoyladenine)-9-methyl](DMT-oxymethyl)(2-cyanoeth- 1 methyl)phosphine oxide (10f) Yield 40%; H NMR (200 MHz, oxy-N,N-diisopropylaminophosphine-oxymethyl)phosphine CDCl3): δ 7.76 (s, 1H, H-8); 7.37–6.08 (m, 13H, aromatic protons), 31 oxide (11c) Yield 77%; P NMR (CDCl3): δ 151.88, 151.72, 151.57, = = 4.75–4.64 (d, 2H, J 7.0 Hz, J 16 Hz, CH2N), 4.08 (d, 2H, CH2OH), 151.41, 38.10, 37.77. 3.74 (s, 3H, OCH3), 3.73 (s, 3H, OCH3), 3.59 (s, 2H, CH2ODMT), 2.94–2.81 31 (m, 1H, CH(CH3)2), 1.14 (d, 6H, J = 6.7 Hz, 2 × CH3); P NMR (CDCl3): δ 43.64; FAB MS (m/z): [M+H]+ 646.4, [M-H]- 644.3. [(N2-Isobutyrylguanine)-9-methyl](DMT-oxymethyl) (2-cyanoethoxy-N,N-diisopropylaminophosphineoxymethyl) 31 phosphine oxide (11f) Yield 41%; P NMR (CDCl3): δ 151.91, 151.59, 151.27, 39.54, 39.22, 38.99, 38.68. Removal of the benzoyl protecting group in 8a′: synthesis of 10e

To a solution of 8a′ (200 mg, 0.26 mmol) in methanol (10 mL), 28% Synthesis of oligomers 12-21 aqueous NH4OH (10 mL) was added. After 24 h the solvent was evaporated and the residue was evaporated with ethanol and then with Chimeric THP-DNA oligomers 12-19 and homo-THPO oligomers 20 toluene (three times). The desired (thymine-1-methyl)(DMT-oxyme- and 21 were prepared on an ABI 394 synthesizer (Applied Biosys- thyl)(hydroxymethyl)phosphine oxide 10e was obtained in 41% yield tems Inc., Foster City, CA, USA) at a 1 μmol scale. Acetonitrile solu- (82 mg, 0.15 mmol) by chromatographic separation on a silica gel col- tions of phosphoramidites 11b, 11c, 11e and 11f, and of commercial umn (CHCl3/MeOH, 100:0→95:5, v/v). thymidine, 2′-deoxycytidine, 2′-deoxyadenosine and 2′-deoxy- guanosine phosphoramidites, at concentrations 0.08–0.12 m were used. For the monomers 11, a coupling time of 600 s was applied. Removal of the DMT protecting group in 8b: synthesis of The syntheses of oligomers 12-19 were performed on a thymidine succinyl-linked LCAA CPG solid support, and for oligomers 20 and 10b 21, the LCAA-CPG support with a universal linker was used. The

cleavage of oligomers 12-19 was done in 28% aqueous NH4OH over To a solution of 8b (170 mg, 0.18 mmol) in dichloromethane (5 mL), 1 m 16 h at 55°C. Fully modified oligomers20 and 21 were cleaved from methanolic solution of p-tolueneosulfonic acid (180 μL, 0.18 mmol) the support with a 20% ethanolic solution of ammonia (55°C, 4 h), was added. The reaction progress was monitored by TLC. The mix- followed by the treatment with 28% aq. NH4OH/40% aq. MeNH2 ture was quenched with pyridine (1 mL) and the volatile components (room temperature, 16 h). All oligomers were purified by a two-step were evaporated. The desired ((N4-benzoyl-cytosine)-1-methyl)(DMT- RP-HPLC (DMT-on/DMT-off). The DMT-tagged oligomers (isolated oxymethyl)(hydroxymethyl)phosphine oxide 10b was obtained in during the first step) were detritylated with 50% aq. acetic acid and 60% yield (69 mg, 0.11 mmol) by chromatographic separation on a purified in the DMT-off form. RP-HPLC purification was performed 1 silica gel column (CHCl3/MeOH, 100:0→95:5, v/v). HNMR (200 MHz, on a C18 column (4.6 × 250 mm, ThermoQuest) with a linear gradi-

CDCl3): δ 8.09–6.69 (m, 20H, aromatic protons, H6, H5); 4.85 (d, J = ent of a buffer A (0.1 m triethylammonium bicarbonate, pH 7.5) and

15.2, 2H); 4.29 (dd, J = 9.8 Hz, J = 16 Hz, 2H); 3.74 (s, 3H, OCH3); 3.73 (s, a buffer B (40% acetonitrile in 0.1 m triethylammonium bicarbo- 31 3H, OCH3); 3.23–3.09 (m, 2H); P NMR (CDCl3): δ 45.15; FAB MS (m/z): nate, pH 7.5); a gradient for DMT-on analysis: 15–100% B, for DMT- [M+H]+ 640.3, [M-H]- 638.2. off: 0–100% B over 30 min, flow rate 1 mL/min. B. Nawrot et al.: Acyclic DNA analogs 313

Assays for enzymatic digestion of THPO-DNA Dedication: We dedicate this paper to the memory of oligomers 13 Dr. Kyo Watanabe, whom we sorely miss. We will keep him in our hearts as a respected scientist, a unique person, a Digestion of the chimeric oligomer 13 with snake venom and calf great colleague, and a very special friend of Poland and spleen phosphodiesterases (PDE I and PDE II, respectively) was per- the Polish people. formed as described earlier [8, 24]. Briefly, to a 1 μL aliquot of a 5 μm solution of oligonucleotide 13 diluted with water (3 μL), a solution of PDE I from Crotalus durissus (1 μL, EC 3.1.15.1, Boehringer Mannheim GmbH, Germany, 0.1 mU/μL) or PDE II from calf spleen (1 μL, EC 3.1.16.1, Sigma, St. Louis, MO, USA, 0.1 μg/μL) was added. The mix- References tures were incubated at 37°C. The 1 μL samples were withdrawn from the reaction mixture after 0, 5, 15 and 45 min (for PDE I) or after 0, 10, [1] Eckstein, F. Phosphorothioates, essential components of 30 and 60 min (for PDE II), and analyzed by MALDI-TOF MS. therapeutic oligonucleotides. Nucleic Acid Ther. 2014, 24, 374–387. [2] Nagarajan, R.; Kwon, K.; Nawrot, B.; Stec, W. J.; Stivers, J. T. Catalytic phosphoryl interactions of topoisomerase IB. Bio- MALDI-TOF mass spectrometry measurements chemistry. 2005, 44, 11476–11485. [3] Yang, X.; Sierant, M.; Janicka, M.; Peczek, L.; Martinez, C.; Samples (1 μL) withdrawn from the digestion reaction mixtures were Hassell, T.; Li, N.; Li, X.;Wang, T.; Nawrot, B. Gene silencing loaded on a sample plate, mixed with the matrix solution [1 μL of an activity of siRNA molecules containing phosphorodithioate 8/1 (v/v) mixture of 2,4,6-trihydroxyacetophenone (10 μg/mL in etha- substitutions. ACS Chem. Biol. 2012, 7, 1214–1220. nol) and diammonium citrate (50 μg/mL in water)] and left for crystalli- [4] Miller, P. S.; Cassidy, R. A.; Hamma, T.; Kondo, N. S. Studies on zation. MALDI-TOF spectra were recorded on a Voyager-Elite instrument anti-human immunodeficiency virus oligonucleotides that have (PerSeptive Biosystems, CT, USA) in a reflector mode, at a resolution of alternating methylphosphonate/phosphodiester linkages. 2000. The m/z negative ion peaks are shown in the spectra. Pharmacol. Ther. 2000, 85, 159–163. [5] Hall, A. H.; Wan, J.; Spesock, A.; Sergueeva, Z.; Shaw, B. R.; Alexander, K. A. High potency silencing by single-stranded Melting temperature (Tm) measurements boranophosphate siRNA. Nucleic Acids Res. 2006, 34, 2773–2781. [6] Krishna, H.; Caruthers, M. H. Solid-phase synthesis, thermal The Tm measurements were performed on a Cintra 40 instrument denaturation studies, nuclease resistance, and cellular uptake (GBC Australia). The samples (2 μm concentration of duplexes) were of oligodeoxyribonucleoside)methylborane phosphine-DNA prepared by hybridization of modified oligomers (in a 10 m Tris- chimeras. J. Am. Chem. Soc. 2011, 133, 9844–9854. HCl, pH 7.4, 10 mm MgCl , 100 mm NaCl buffer) with the complemen- 2 [7] Gryaznov, S. M. Oligonucleotide n3′→p5′ phosphoramidates tary single stranded DNA or RNA as listed in Table 3. Before melting, and thio-phoshoramidates as potential therapeutic agents. the duplexes were annealed from 95°C to 5°C at a temperature gradi- Chem. Biodivers. 2010, 7, 477–493. ent of 0.5°C/min. and kept at 5°C for 5 min. Melting was performed [8] Nawrot, B.; Boczkowska, M.; Wójcik, M.; Sochacki, M.; up to 86°C at a temperature gradient of 0.2°C/min. The melting tem- Kazmierski, S.; Stec, W. J. Novel internucleotide 3′-NH-P(CH ) peratures Tm were calculated using the first order derivative method. 3 (O)-O-5′ linkage. Oligo(deoxyribonucleoside methanephos- phonamidates); synthesis, structure and hybridization properties. Nucleic Acids Res. 1998, 26, 2650–2658. Circular dichroism measurements [9] Sipa, K.; Sochacka, E.; Kazmierczak-Baranska, J.; Maszewska, M.; Janicka, M.; Nowak G.; Nawrot, B. Effect of Samples of the duplexes 20/21, 20/5′-d(ATAATTAAAT)-3′ and base modifications on structure, thermodynamic stability, and 21/5′-d(ATTTAATTAT)-3′, as well as single stranded THPO-oligomers gene silencing activity of short interfering RNA. RNA 2007, 13, 20 and 21 were prepared at 2 μm concentration in a 10 mm Tris- 1301–1316.

HCl, pH 7.4, 10 mm MgCl2, 100 mm NaCl buffer. The spectra were [10] Nawrot, B.; Widera, K.; Wojcik, M.; Rebowska, B.; Nowak, G.; recorded on a Jobin Yvon CD6 dichrograf. Measurements were made Stec, W. J. Mapping of the functional phosphate groups in at room temperature using 0.5 cm path-length quartz cuvettes of the catalytic core of deoxyribozyme 10-23. FEBS J. 2007, 274, 1 mL capacity, 2 nm bandwidth and 1-2 s integration time. Each 1062–1072. spectrum was smoothed with a 9- or 15-point algorithm (included [11] Kraynack, B. A.; Baker, B. F. Small interfering RNAs containing in the manufacturer’s software, version 2.2.1) after averaging of at full 2′-O-methylribonucleotide-modified sense strands display least three scans. Argonaute2/eIF2C2-dependent activity. RNA. 2006, 12, 163–176. [12] Jepsen, J. S.; Wengel, J. LNA-antisense rivals siRNA for gene Acknowledgments: Financial support from the Statutory silencing. Curr. Opin. Drug Discov. Dev. 2004, 7, 188–194. [13] Ferrari, N.; Bergeron, D.; Tedeschi, A. L.; Mangos, M. M.; Funds of Centre of Molecular and Macromolecular Studies Paquet, L.; Renzi, P. M.; Damha, M. J. Characterization of of the Polish Academy of Sciences is gratefully acknowl- antisense oligonucleotides comprising 2′-deoxy-2′-fluoro- edged. The authors thank Dr. Piotr Guga for critical read- beta-D-arabinonucleic acid (FANA): specificity, potency, and ing of the manuscript and fruitful discussion. duration of activity. Ann. N.Y. Acad. Sci. 2006, 1082, 91–102. 314 B. Nawrot et al.: Acyclic DNA analogs

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