Nucleoside H-boranophosphonates: a new class of boron-containing nucleotide analogues

Abstract

A study on the synthesis of nucleosideH-boranophosphonates, a new class of nucleotide analogues having a P→BH₃ and a P–H group, via condensation of the corresponding nucleosides with H-boranophosphonate derivatives is described.

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Chemical modifications of natural nucleotides and oligonucleotides by replacing the non-bridging oxygen atoms of their phosphate groups with other elements or substituents (e.g., sulfur atom) have been widely used, especially for the development of nucleic acid-based drug candidates.^1,2Nucleoside boranophosphates, in which a non-bridging oxygen atom of the phosphate group is replaced with a BH₃ group, are one of the most promising candidates owing to their significant stability to nucleases and high lipophilicity, which may save the need for an elaborate delivery system.³ Their low cytotoxicity has also been suggested.⁴ In addition, oligonucleoside boranophosphates have shown promising RNA interference activity,⁵ and may also be useful as target-specific ¹⁰B carriers for boron-neutron capture therapy (BNCT).⁶

These studies on the nucleoside boranophosphates have generated interest in other nucleotide analogues containing a P→BH₃ group. However, only a limited number of such analogues are available^7,8 with the methods developed for the synthesis of nucleoside boranophosphates.^9–11

To expand the availability of the boron-containing nucleotide analogues, we designed a novel nucleotide analogue having a P→BH₃ and a P–H group (nucleosideH-boranophosphonate 1, Fig. 1) with the expectation that it would work as a versatile precursor of a variety of boron-containing P-modified nucleotide analogues via the activation of the P–H function by bases or transition metals followed by reactions with electrophiles.¹² The P→BH₃ moiety may also work as a modifiable site via deboronation and subsequent reaction with electrophiles, which would expand the availability of P-modified nucleotide analogues.^12a,13

Fig. 1 Strategy for synthesis of various P-modified oligonucleotide analogues 2 and 3 by substitution of P–H and P–B bonds of oligonucleoside H-boranophosphonates 1, respectively. B = nucleobase.

To the best of our knowledge, there are only two reports on the synthesis of H-boranophosphonate derivatives.^12c,14 Centofanti reported the synthesis of dimethyl H-boranophosphonate from dimethyl phosphonite decades ago.¹⁴ In addition, the synthesis of silylH-boranophosphonate derivatives and their applications as precursors of alkylboranophosphonates were reported by Montchamp et al. very recently.^12c However, it is difficult to apply these methods to the synthesis of more functionalized molecules, such as nucleotide analogues.

We anticipated that an H-boranophosphonate derivative having at least one free P–O⁻ function would undergo condensation reactions with the hydroxy group of nucleosides to give the nucleosideH-boranophosphonates under mild reaction conditions. Based on this idea, we synthesized inorganicH-boranophosphonate as a monopyridinium salt (Scheme 1, 7). Phosphinic acid4 was treated with N,O-bis(trimethylsilyl)benzamide¹⁵ to afford bis(trimethylsilyl) phosphonite 5, which was then boronated by treatment with BH₃·THF. Montchamp et al. have also synthesized bis(trialkylsilyl) H-boranophosphonates and alkyl trialkylsilyl H-boranophosphoates via the corresponding phosphonite derivatives, though the bis(TMS) derivative 5 was not used for further applications due to its instability. In contrast, we used the silylation as a transient protection and the intermediate 6 was desilylated by treatment with MeOH and pyridine to synthesize 7. The resultant pyridinium H-boranophosphonate 7 was stable to air oxidation and water, and can be stored for at least several months at −30 °C without decomposition.

Scheme 1 Synthesis of pyridinium H-boranophosphonate 7.

Pyridinium H-boranophosphonate 7 was applied to the synthesis of nucleosideH-boranophosphonate derivatives. Firstly, nucleoside 5′-H-boranophosphonates were synthesized by condensation of 7 with appropriately protected nucleosides having a 5′-hydroxy group (8a–e, Table 1). When the reaction was carried out under conditions similar to those used for the boranophosphorylation of nucleosides,^11a the isolated yields of the desired thymidine 5′-H-boranophosphonate 9a were low mainly due to the formation of a 5′,5′-dinucleoside H-boranophosphonate diester (entries 1 and 2). In sharp contrast, condensations promoted by Bop-Cl in pyridine afforded the desired nucleoside 5′-H-boranophosphonates 9a–e in excellent yields without observable side-reactions (entries 3–7).

Table 1 Synthesis of nucleoside 5′-H-boranophosphonates 9a–e by 5′-boranophosphonylation of nucleosides8a–e^a

Entry	8	B^PRO	R^2	R^3	Reagents and conditions	Yield of 9a–e (%)
a TEAB = triethylammonium bicarbonate; B^PRO = (protected) nucleobase; Th^bz = N^3-benzoylthymin-1-yl; DMTr = 4,4′-dimethoxytrityl; Piv-Cl = pivaloyl chloride; NT = 3-nitro-1H-1,2,4-triazole; Bop-Cl = bis(2-oxo-3-oxazolidinyl)phosphinic chloride; Pac = phenoxyacetyl.
1	a	Th^bz	H	DMTr	7 (1.5 equiv.), Piv-Cl (1.5 equiv.), iPr2NEt (3 equiv.), NT (1.5 equiv.), MeCN, rt, 3 h	39
2	a	Th^bz	H	DMTr	7 (2 equiv.), Bop-Cl (2 equiv.), iPr2NEt (4 equiv.), NT (2 equiv.), MeCN, rt, 2 h	43
3	a	Th^bz	H	DMTr	7 (1.2 equiv.), Bop-Cl (1.2 equiv.), Py, rt, 2 h	95
4	b	Th	H	DMTr	7 (1.6 equiv.), Bop-Cl (1.6 equiv.), Py, rt, 1 h	95
5	c	Th	H	Bz	7 (1.2 equiv.), Bop-Cl (1.2 equiv.), Py, rt, 30 min	92
6	d	Th	H	Pac	7 (1.2 equiv.), Bop-Cl (1.2 equiv.), Py, rt, 40 min	90
7	e	Ur	OPac	Pac	7 (2 equiv.), Bop-Cl (2 equiv.), Py, rt, 2 h	98

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Next, we attempted to synthesize fully-deprotected nucleoside 5′-H-boranophosphonates, which would be interesting as a new class of boron-containing nucleoside monophosphate analogues. Firstly, the detritylation of 3′-O-DMTr-thymidine 5′-H-boranophosphonate 9b was attempted by treatment with an 80% AcOH aqueous solution. However, decomposition of the H-boranophosphonate moiety (ca. 70%) was observed after 30 min (Table 2, entry 1). It is probably due to the reaction of the liberated DMTr⁺ with the BH₃ group of the H-boranophosphonate moiety as observed in the case of boranophosphates.^10b,11a,16 In contrast, the H-boranophosphonate monoesters were stable under conventional ammonolysis conditions, and the nucleoside 5′-H-boranophosphonates 10a,b were isolated in good yields (entries 2 and 3).

Table 2 Deprotection of nucleoside 5′-H-boranophosphonates 9b,d,e

Entry	B	9	R^2	R^3	Deprotection conditions	10	Yield of 10 (%)
a Decomposition of H-boranophosphonate moiety was observed.
1	Th	b	H	DMTr	80% AcOH, rt, 30 min	a	—^a
2	Th	d	H	Pac	sat. NH3–MeOH, rt, 50 min	a	71
3	Ur	e	OPac	Pac	sat. NH3–MeOH, rt, 3 h	b	68

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The reaction conditions optimized for the 5′-boranophosphonylation were also applicable to the synthesis of nucleoside 3′-H-boranophosphonates. The condensation of 5′-O-DMTr-nucleosides 11a,b with 7 in pyridine in the presence of Bop-Cl afforded the desired nucleoside 3′-H-boranophosphonates 12a,b in excellent yields (Table 3, entries 3 and 4), whereas the reactions promoted by Piv-Cl or Bop-Cl in MeCN in the presence of iPr₂NEt and NT resulted in low yields of 12 mainly due to the generation of a 3′,3′-dithymidine H-boranophosphonate derivative (entries 1 and 2).

Table 3 Synthesis of nucleoside 3′-H-boranophosphonates 12a,b by 3′-boranophosphonylation of nucleosides11a,b

Entry	11	B^PRO	Reagents and conditions	Yield of 12 (%)
1	a	Th^bz	7 (1.2 equiv.), Piv-Cl (1.5 equiv.), iPr2NEt (3 equiv.), NT (1.5 equiv.), MeCN, rt, 4 h	41
2	a	Th^bz	7 (1.2 equiv.), Bop-Cl (3 equiv.), iPr2NEt (6 equiv.), NT (3 equiv.), MeCN, rt, 1.5 h	50
3	a	Th^bz	7 (1.2 equiv.), Bop-Cl (1.2 equiv.), Py, rt, 1 h	95
4	b	Th	7 (2 equiv.), Bop-Cl (2 equiv.), Py, rt, 3 h	82

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The 5′-O-protected nucleoside 3′-H-boranophosphonates are potentially useful as monomer units to synthesize oligonucleotide analogues having an H-boranophosphonate diester backbone. As we mentioned above, such oligonucleotide analogues would work as precursors of a variety of backbone-modified oligonucleotide analogues through the P–H or P–B modifications. To investigate the potential of the H-boranophosphonates, a dithymidine H-boranophosphonate was synthesized from the 5′-O-DMTr-nucleoside 3′-H-boranophosphonate 12a. The 3′-H-boranophosphonate 12a was allowed to condense with 3′-O-DMTr-N³-benzoyl-thymidine 8a in MeCN in the presence of Bop-Cl and 2,2,6,6-tetramethylpiperidine (Scheme 2, reaction i). The reaction proceeded smoothly at rt. Although a partial decomposition during silica gelcolumn chromatography was observed, the dithymidine H-boranophosphonate diester13a was isolated in a modest yield.

Scheme 2 (i) Synthesis of dithymidine H-boranophosphonates 13a,b, (ii) 5′-O-detritylation of 13b and (iii) conversion of 13a into dithymidine boranophosphorothioate 15 by P–H modification. Reagents and conditions: (i) N³-benzoyl-3′-O-R³-thymidine (0.75 equiv.), Bop-Cl (2.5 equiv.), 2,2,6,6-tetramethylpiperidine (6 equiv.), MeCN, rt, 1 h; (ii) 3% dichloroacetic acid in CH₂Cl₂–Et₃SiH (3 : 1, v/v), rt, 1 min; (iii) S₈ (3 equiv.), Et₃N (3 equiv.), MeCN, rt, 3 h.

To explore the potential of the nucleosideH-boranophosphonates, we carried out the following two experiments. Firstly, a 3′-O-benzoyl-dithymidine H-boranophosphonate 13b was synthesized as a crude product in a similar manner and treated with 3% dichloroacetic acid in CDCl₃–Et₃SiH (1 : 1, v/v)^11b,16 for 5′-O-detritylation. In contrast to the detritylation of the H-boranophosphonate monoester 9b by treatment with 80% AcOH (Table 2, entry 1), which caused the decomposition of the H-boranophosphonate moiety, the 5′-O-DMTr group of 13b was quantitatively removed without decomposition of the product (Scheme 2, reaction ii). It indicates that a solid-phase synthesis of oligonucleoside H-boranophosphonates via condensation and 5′-O-detritylation is feasible.

Secondly, the possibility of the P–H modification of the H-boranophosphonate diester linkage was investigated by treating the dithymidine H-boranophosphonate 13a with S₈ in the presence of Et₃N under anhydrous conditions. A ³¹P NMR analysis of the reaction showed that 13a (δ_P 135.1, 133.7) was quantitatively converted into the corresponding dithymidine boranophosphorothioate 15 (δ_P 162.5, 161.0)^8a,b within 3 h at rt, and 15 was isolated in good yield (Scheme 2, reaction iii).

In conclusion, nucleosideH-boranophosphonates were synthesized via condensation of the corresponding nucleosides and H-boranophosphonate derivatives. The mild reaction conditions and high efficiency of this method are attractive for further applications to the synthesis of a broad spectrum of H-boranophosphonate derivatives. In addition, conversion of the P–H group of a dinucleoside H-boranophosphonate into a P–S^– group demonstrated that the H-boranophosphonate derivatives are potential precursors of a variety of boron-containing oligonucleotide analogues. Further studies on the synthesis of nucleosideH-boranophosphonates and their applications are in progress.

We thank Professor Kazuhiko Saigo (University of Tokyo) for helpful suggestions. This research was supported by a Grant-in-Aid for Young Scientists (B) (No. 20750127) from MEXT Japan (N.O.).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available: Experimental details and NMR spectra. See DOI: 10.1039/b901045a

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