Stereocontrolled solid-phase synthesis of oligonucleoside H-phosphonates by an oxazaphospholidine approach

Article abstract of DOI:10.1002/anie.200804408

(Chemical Equation Presented) Stereodefined oligonucleoside H-phosphonates were synthesized on a solid support using diastereopure nucleoside 3′-O-oxazaphospholidine monomers. Several stereodefined backbone-modified analogues were obtained with the oligonucleoside H-phosphonates as precursors (see scheme; B^PRO = protected nucleobase, DMTr = 4,4′- dimethoxytrityl, Th = thymin-1-yl, TfO^- = triflate).

Full text of DOI:10.1002/anie.200804408

Communications
DOI: 10.1002/anie.200804408
Asymmetric Synthesis
Stereocontrolled Solid-Phase Synthesis of
Oligonucleoside H-Phosphonates by an
Oxazaphospholidine Approach**
Naoki Iwamoto, Natsuhisa Oka, Terutoshi Sato, and Takeshi Wada*
Angewandte
Chemie
496
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2009, 48, 496 –499

Angewandte
Chemie
Substitution of a nonbridging oxygen atom of a phosphate
diester internucleotide linkage is one of the conventional
methods used to confer additional properties and functions on
oligonucleotides. The resultant P-modified oligonucleotide
analogues possess the additional properties and functions
(e.g., stability to nucleases, cell membrane permeability)
while still retain those inherent to the parent oligonucleotides
(e.g., solubility in water, ability to work as substrates for
enzymes). As a result, these analogues have wide applications
as probes for enzymatic reactions, nucleic acid drug candi-
dates, etc.[1]
One of the most important features of these oligonucle-
otide analogues is their chiral phosphorus atoms. Since the
target molecules of these oligonucleotides are, in most cases,
chiral (DNA, RNA, proteins, etc.), their properties and
functions are theoretically dependent on the configurations
around the phosphorus atoms.[2] However, only limited data
on this dependence is available because of the lack of
oligonucleotide analogues stereodefined at the P center; the
exceptions are oligonucleoside phosphorothioates[2–5] and
methylphosphonates.[2c,6]
tertiary carbon center adjacent to the oxygen atom of the
phosphite. Although conversions of phosphite internucleoti-
dic linkages into H-phosphonates have been reported under
acidic conditions, their stereochemical pathways have not
been clarified.[8]
The concept is outlined in Table 1. We have designed
nucleoside derivatives of 3’-O-oxazaphospholidine with two
substituents at the 5 position of the oxazaphospholidine ring
Table 1: Synthesis of diastereopure dithymidine phosphite intermediates
4a–c and stereospecific conversion of 4c into dithymidine H-phospho-
nate (Rp)-5.
We focused on oligonucleoside H-phosphonates as key
compounds to overcome this limitation. Diastereopure dinu-
cleoside H-phosphonates, which were separated by column
chromatography, have been converted into a variety of P-
modified dinucleoside phosphate analogues in a stereospe-
cific manner,[7] thus indicating that oligonucleoside H-phos-
phonates stereodefined at the P center would work as
precursors for oligonucleotide analogues stereodefined at
the same center. However, the stereocontrolled synthesis of
H-phosphonate diesters has never been achieved. We sought
to develop a method to synthesize oligonucleoside H-
phosphonates stereodefined at the P center by using nucleo-
side 3’-O-oxazaphospholidine derivatives as monomer units.
The results of this study are described herein.
We have previously reported a method to synthesize
oligonucleoside phosphorothioates stereodefined at the P
center by using nucleoside 3’-O-oxazaphospholidine deriva-
tives as monomer units.[5] In this method, a diastereopure
phosphite internucleotide linkage is generated in every
synthetic cycle, and then sulfurized to a phosphorothioate
linkage. We hypothesized that the phosphite intermediate
stereodefined at the P center could be converted into the
corresponding H-phosphonate diester by an E1 reaction of
the P-protonated species, if the phosphite intermediate had a
Entry
Product
R
R’
Yield [%][a]
d.r.[a]
92:8
98:2
>99:1
1
4a
4b
4c
Ph
Me
Ph
Ph
Me
Me
65
>99
>99
2[b]
3
[a] Determined by 31P NMR spectroscopy. [b] The d.r. value of oxaza-
phospholidine 1b was 98:2. TBDMS=tert-butyldimethylsilyl, TBDPS=
tert-butyldiphenylsilyl, TfOÀ =triflate, Th=thymin-1-yl.
(1a–c). The proline-derived bicyclic oxazaphospholidine
rings[4b] were adopted based on our recent results, which
illustrated that configurationally stable nucleoside 3’-O-
oxazaphospholidine monomers could be obtained by using a
proline-derived bicyclic oxazaphospholidine ring to give
excellent diastereoselectivity.[9] The oxazaphospholidine
derivatives 1a–c were obtained from 5’-O-(TBDPS)thymi-
dine and 2-chlorooxazaphospholidines in 64–74% yield with
the diastereoselectivity ranging from 98:2 to greater than
99:1.[10]
The derivatives 1a–c were condensed with 3’-O-
(TBDMS)thymidine 2 in the presence of a low-nucleophilic
acidic activator N-(cyanomethyl)pyrrolidinium triflate
(CMPT; 3).[5] Analysis by 31P NMR spectroscopy showed
that the reaction of 1a with 2 was not complete after
10 minutes, probably as a result of the steric bulk and
electron-withdrawing effect of the Ph substituents at the
5 position (Table 1, entry 1). In contrast, 1b and c were
quantitatively converted into the desired dinucleoside phos-
[*] N. Iwamoto, Prof. Dr. N. Oka, T. Sato, Prof. Dr. T. Wada
Department of Medical Genome Sciences
Graduate School of Frontier Sciences, The University of Tokyo
Bioscience Building 702, 5-1-5 Kashiwanoha
Kashiwa, Chiba 277-8562 (Japan)
Fax: (+81)4-7136-3612
E-mail: wada@k.u-tokyo.ac.jp
[**] We thank Prof. Kazuhiko Saigo (University of Tokyo) for helpful
suggestions. This research was supported by grants from the
Ministry of Education, Culture, Sports, Science, and Technology
(MEXT) Japan and the Futaba Electronics Memorial Foundation
(N.O.).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200804408.
Angew. Chem. Int. Ed. 2009, 48, 496 –499
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
497

Communications
Table 2: Solid-phase synthesis of P-modified dinucleoside phosphate
analogues (10 + 11) by H-phosphonates 9.[a]
phite intermediates 4b and c, respectively, without any loss of
diastereopurity (Table 1, entries 2 and 3). The resultant
phosphites 4b and c were treated with 1% anhydrous
trifluoroacetic acid (TFA) in CH2Cl2 to remove the tert-
alkyl group derived from the chiral auxiliary by an E1 reac-
tion. Although the conversion of 4b was not complete,[11] 4c
was quantitatively converted into the corresponding H-
phosphonate derivative (Rp)-5 without loss of diastereopur-
ity. 31P NMR analysis showed that a signal appeared at d =
7.7 ppm (1JP, H = 711.4 Hz), which is characteristic of an H-
phosphonate diester. The faster conversion of 4c than that of
4b can be attributed to the stabilization of the released tert-
carbocation by the phenyl group.
Next, a solid-phase synthesis of dinucleoside H-phospho-
nates by using the above method was investigated. 5’-O-
(DMTr)nucleoside 3’-O-oxazaphospholidines (Rp)- or (Sp)-6
(Table 2) were stereoselectively synthesized according to the
procedure outlined for 1c in modest to good yields (43–83%,
d.r. > 99:1).[12] Dinucleoside H-phosphonates 9 were man-
ually synthesized and attached to controlled pore glass
(CPG), and then converted into a variety of P-modified
dinucleoside phosphate analogues.[7] After deprotection of
the nucleobases and cleavage from the support, the resultant
crude products were analyzed by reversed-phase HPLC
(Table 2). The analysis clearly showed that the desired P-
modified dinucleoside phosphate analogues (10 + 11) were
successfully synthesized with excellent diastereopurity
(d.r. ranging from 98:2 to > 99:1). On the basis of the
stereochemical outcomes reported in the literature (H-
phosphonate to phosphorothioate or boranophosphate =
retention of configuration,[7b,d] H-phosphonate to phosphor-
amidate = inversion of configuration,[7c] condensation of oxa-
zaphospholidines promoted by CMPT= inversion of config-
uration [5]), we assigned the stereochemical outcome for the
conversion of the phosphites 8 into the H-phosphonates 9 as
occurring with retention of configuration, which is consistent
with the E1 mechanism.[13]
Entry Monomer BPRO[b]
unit
X
Isomers Yield [%][c]
10/11[c]
1
2
3
4
5
6
7
8
(Sp)-6
(Sp)-6
(Sp)-6
(Sp)-6
(Sp)-6
(Sp)-6
(Sp)-6
(Sp)-6
(Sp)-6
(Rp)-6
(Rp)-6
(Rp)-6
(Rp)-6
(Rp)-6
(Rp)-6
(Rp)-6
(Rp)-6
(Rp)-6
Th
SÀ
SÀ
SÀ
>99:1
>99:1
>99:1
>99:1
>99:1
>99:1
1:99
1:99
2:98
>1:99
>1:99
>1:99
>1:99
>1:99
>1:99
99:1
96
95
94
97
90
86
95
93
95
95
96
95
96
89
84
95
93
93
Cyac
Addmf
Guce,pac SÀ
Th
Th
Th
À
BH3
CH2OH
NH2
NHPr
Th
Th
Th
9
NH(CH2)2NMe2
10
11
12
13
14
15
16
17
18
SÀ
SÀ
SÀ
Cyac
Addmf
Guce,pac SÀ
À
Th
Th
Th
Th
Th
BH3
CH2OH
NH2
NHPr
99:1
NH(CH2)2NMe2 >99:1
[a] Reagents and reaction conditions; 1) 1m 3 in CH3CN, RT, 3 min;
2) 1% TFA/CH2Cl2, RT, 15 sec; 3) 10 wt% sulfur, CS2/pyridine/Et3N
(35:35:1, v/v/v), RT, 3 h; then conc. NH3, RT, 1–12 h (X=SÀ); BH3·SMe2-
N,O-bistrimethylsilylacetamide/DMF (1:1:8, v/v/v), RT, 15 min; then sat.
NH3/CH3OH, RT, 1 h (X=BH3À); 0.1m TMSCl/pyridine-1-methyl-2-
pyrrolidone (1:9, v/v), RT, 10 min; then HCHO (gas), RT, 30 min; then
PrNH2/CH3CN (1:4, v/v), RT, 30 min (X=CH2OH), sat. NH3, CCl4/1,4-
dioxane (4:1 v/v), 08C, 30 min (X=NH2), CCl4/RNH2 (9:1, v/v), RT, 1 h
(X=NHR). [b] Cyac =N4-acetylcytosin-1-yl, Addmf =N6-(dimethylamino)-
methyleneadenin-9-yl, Guce,pac =O6-cyanoethyl-N2-phenoxyacetylguanin-
9-yl. [c] Determined by HPLC analysis. DMTr=4,4’-dimethoxytrityl.
The method was then applied to the synthesis of
oligonucleoside H-phosphonates stereodefined at the P cen-
ter. A cycle consisting of a) condensation promoted by
CMPT, and b) treatment with 1% TFA/CH2Cl2 and Et3SiH
[14]
(1:1, v/v)
was repeated on a CPG to synthesize oligonu-
cleoside H-phosphonates stereodefined at the P center. The
resultant oligomers were converted into P-modified oligonu-
cleotide analogues. By using the cycle, (all-Rp)- and (all-Sp)-
[TS]9T, d[CSASGST], [TB]3T, and [TN]3T (subscript “S” =
phosphorothioate diester; “B” = boranophosphate diester,
“N” = N-[(2-dimethylamino)ethyl]-phosphoramidate) were
successfully synthesized (yields ranged from 56% to 92%
and were determined by reverse-phase HPLC analysis).[12]
Thus, we have succeeded in the first synthesis of oligonu-
cleoside H-phosphonates stereodefined at the P center and a
wide variety of P-modified oligonucleotides by the oxaza-
phospholidine method. Since these oligonucleotide analogues
(except for phosphorothioates) are otherwise difficult to
obtain, studies on their properties and applications will be
intriguing. In addition, the method is expected to facilitate
access to a variety of optically pure organophosphorus
compounds other than nucleic acid analogues, which are
also in high demand as biochemical probes, chiral discrim-
inating agents, ligands for catalytic reactions, etc.[15] Further
study on these issues is currently underway.
Received: September 6, 2008
Published online: November 3, 2008
Keywords: asymmetric synthesis · H-phosphonates ·
.
oligonucleotides · solid-phase synthesis
498
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Chemie
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[12] See the Supporting Information.
[13] The sterochemical outcome of the synthesis of the dinucleoside
hydroxymethyl-phosphonate from 9 was tentatively assigned as
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methylphosphonates.
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Angew. Chem. Int. Ed. 2009, 48, 496 –499
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