Synthesis of a nucleoside phosphorodithioate analogue responsive to microenvironmental changes through chiral induction

Article abstract of DOI:10.1016/j.tetlet.2012.12.028

We have synthesized a 2′-aminomethyl branched-chain sugar nucleoside phosphorodithioate from 2,2′-anhydro uridine and subjected the material to a subsequent cyclization reaction under aqueous conditions using bi-functional linkers. The rate of the cycliza

Full text of DOI:10.1016/j.tetlet.2012.12.028

Tetrahedron Letters 54 (2013) 1080–1083
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Synthesis of a nucleoside phosphorodithioate analogue responsive
to microenvironmental changes through chiral induction
Hisao Saneyoshi a, Takushi Mashimo b, Ken Hatano b, Yoshihiro Ito a, , Hiroshi Abe a,b,
⇑
⇑
a Nano Medical Engineering Laboratory, RIKEN Advanced Science Institute, 2-1, Hirosawa, Wako, Saitama 351-0198, Japan
b Division of Material Science, Graduate School of Science and Technology, Saitama University, 255 Shimo-Ohkubo, Sakura-ku, Saitama 338-8570, Japan
a r t i c l e i n f o
a b s t r a c t
We have synthesized a 20-aminomethyl branched-chain sugar nucleoside phosphorodithioate from 2,20-
anhydro uridine and subjected the material to a subsequent cyclization reaction under aqueous condi-
tions using bi-functional linkers. The rate of the cyclization reaction was dependent on the leaving group
on the bi-functional linkers. The generation of a chiral phosphorous peak from the achiral precursor, as
indicated by 31P NMR, was identified as a good indicator for potentially probing the local and global fea-
tures of the DNA structure.
Article history:
Received 19 October 2012
Revised 6 December 2012
Accepted 10 December 2012
Available online 25 December 2012
Keywords:
Phosphorodithioate
Chirality
Ó 2012 Elsevier Ltd. All rights reserved.
Nucleotide
Phosphorous NMR
DNA
Introduction
The chirality of a substance can play an essential role during its
molecular recognition and structural formation processes.1 Bio-
macromolecules in particular, such as nucleic acids, possess a vari-
ety of different substituents on their sugar–phosphate backbone
that contribute to the formation of their typically helical structures
(A-form and B-form duplexes), as well as some more unusual
structures that are influenced by different binding molecules.2 Nu-
cleic acids themselves can also provide chiral environments to
small molecules and work as molecular catalysts for a verity of dif-
ferent organic transformations, including the Diels–Alder, aldol,
and nucleophilic substitution reactions. Stereo selective syntheses
can be successfully accomplished using DNA based catalysis be-
cause the chiral environments created by the structural features
of DNA effectively facilitate these transformations.3 Furthermore,
the chiral nucleic acid analogues, phosphorothioate DNA/RNA have
been the subject of considerable attention as nucleic acid thera-
peutics because of their high levels of nuclease resistance.4 The chi-
rality of the phosphorous atom has a significant influence on the
stability and sensitivity of the nucleic acid duplex to nucleolytic
enzymes such as phosphodiesterase.5 The chiral recognition of
such enzymes is a particularly selective process and the stereose-
lective synthesis of phosphorothioate DNA has been developed to
address which of the isomers is active and which one is not.6 Phos-
Figure 1. Alkylation of phosphorodithioate, with the resulting chirality acting as an
indicator of the microenvironment.
phorodithioate DNA itself was developed more than two decades
ago as a novel DNA analogue and showed significant levels of
nuclease resistance and HIV reverse transcriptase (RT) inhibition.7
From a structural perspective, and in similar to natural phosphodi-
esters, the phosphorodithioate possesses no chirality on their
phosphorous atom. Alkylation of the phosphorodithioate on the
sulfur atom, however, leads to the generation of chirality on the
phosphorous atom, as shown in Figure 1. We were interested in
developing an understanding of chiral generation reactions that
are microenvironment dependent. At the fundamental level, our
idea relies on the expectation that we can use these transforma-
tions to act as reporter reactions to probe the microenvironment
⇑
Corresponding authors. Tel.: +81 48 467 4979; fax: +81 48 462 9300.
E-mail addresses: y-ito@riken.jp (Y. Ito), h-abe@riken.jp (H. Abe).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.12.028

H. Saneyoshi et al. / Tetrahedron Letters 54 (2013) 1080–1083
1081
O
O
O
O
NH
NH
N
NH
N
N
MMTrO
MMTrO
O
O
N
MMTrO
O
O
O
c
O
N
Ref. 8
O
O
HO
a, b
5 steps 41%
HO
OH
HO
HO
OTs
HO
1
2
3
4
O
NH
O
O
NH
O
NH
O
N
MMTrO
S
N
N
MMTrO
MMTrO
O
O
O
O
d
e, f, g
h, i
S
P
O
NH
HO
NH
HO
N3
CF3
CF3
S
O
O
5
6
7
O
O
NH
O
NH
O
MMTrO
N
MMTrO
N
O
O
NH2
O
P
O
S
j, k
l
NH
O
O
NH
O
P
O
S
S
HS
*
NH
O
O
N
N
O
O
O
9
8
HO
HO
Scheme 1. Reagents and conditions: (a) OsO4, NMO, 2,6-lutidine, dioxane-H2O, rt, 3 h; (b) NaBH4, MeOH, ꢀ78 °C to 0 °C, 2 h, 60% over 2 steps; (c) TsCl, pyridine, 0 °C,
overnight, 72%; (d) NaN3, DMF, 100 °C, 2 h, rt, overnight, 96%; (e) PPh3, THF, 55 °C, 4 h; (f) NH4OH, rt, 1 h; (g) (CF3CO)2O, pyridine, CH2Cl2, rt, 1 h, 86% over 3 steps; (h) (–
SCH2CH2S–)P(iPr)2N, 1H-tetrazole, CH3CN, rt, 5 min; (i) elemental sulfur, sonication for 1 min and rt, 4 h, 92% over 2 steps; (j) 30-O-Ac-thymidine, DBU, CH3CN, rt, 5 min; (k)
NH4OH, rt, 9 h, 81% over 2 steps; (l) See Scheme 3.
and global shape of DNA in the presence of target molecules. In the
context of synthetic organic chemistry, the target molecule is ex-
pected to behave as a chiral ligand for the reporter reaction. Herein,
we report the synthesis of a nucleoside phosphorodithioate dimer
as a useful building block for the microenvironment dependent
synthesis of conformationally restricted nucleotides.
which was subsequently reacted with diisopropylamine in toluene
(instead of benzene, as described in the previous paper) to give the
target phosphoramidite derivative as shown in Scheme 2. The 30-
hydroxyl group of the nucleoside derivative 6 was phosphorylated
in the presence of 1H-tetrazole and sulfidated with elemental sul-
fur to give the trithio cyclic phosphate derivative 7. The subsequent
ring opening coupling reaction with the appropriately protected
thymidine derivative was performed with DBU to give coupling
product 8. 31P NMR showed a single peak indicating the presence
of a single isomer.
Next, we employed a tandem amidation–alkylation reaction
sequence to allow for subsequent modification of the sugar–
phosphate backbone, as in Scheme 3. Haloacetic acid N-hydroxy-
succinimide ester was selected as the bi-functional reagent for the
process. To test the reactivity of the material, three different types
of linkers were used for the modification, as shown in Scheme 3.
Using ESI-mass spectroscopic analysis,12 we established that the
amino group had reacted with the cross linker first followed by an
intramolecular alkylation reaction. The alkylation reaction was
In Scheme 1, the synthetic work started from the commercially
available 2,20-cyclouridine 1, which was converted into 20-vinyl
uridine 2 in 41% yield over 5 steps according to a procedure previ-
ously published by Sukeda.8 The 20-hydorxymethyl uridine deriva-
tive 3 was prepared according to a two stage procedure. Compound
2 was initially converted into the corresponding 20-formyl interme-
diate by reaction with a combination of OsO4, sodium periodate,
and 2,6-lutidine according to a one pot procedure.9 The 20-formyl
intermediate was found to be unstable and was therefore immedi-
ately reduced with NaBH4 in MeOH to give the 20-hydorxymethyl
uridine derivative 3. To introduce an azide group on branched side
chain, the primary hydroxyl group was tosylated to give compound
4, which was subsequently substituted with sodium azide in N,N-
dimethylformamide to give compound 5. The azide group was then
reduced with PPh3, and the resulting aza–ylide complex decom-
posed with NH4OH to give the 20-amino methyl modified nucleo-
side, which was acylated with trifluoroacetic anhydride to give
compound 6.
Cl
P
N
P
Cl
P
a
b
S
S
S
S
Cl
Cl
To complete the synthesis of phosphorodithioate, cyclic dith-
iophosphoramidite was employed as a phosphorylation reagent.10
The cyclic dithiophosphoramidite reagent was prepared according
to a literature procedure.11 Briefly, phosphorus trichloride was
treated with ethanedithiol and Et3N to give chlorodithiophosphite,
31P-NMR (CDCl3
168.3 (Lit11. 169.0)
)
31P-NMR (CDCl3
95.2 (Lit11. 95.6)
)
Scheme 2. Reagents and conditions: 1,2-ethanedithiol, Et3N, Et2O, rt, 20 h 73%; (b)
diisopropylamine, toluene, rt, 2 h, 81%.

1082
H. Saneyoshi et al. / Tetrahedron Letters 54 (2013) 1080–1083
O
O
O
NH
O
NH
O
O
MMTrO
N
MMTrO
N
O
S
O
X
N
O
O
(1.5 equiv)
X= Cl, Br, I
O
P
NH2
O
NH
S
O
O
P
O
S
9
aq buffer-CH3CN
(1:1, v/v), temp
HS
*
NH
O
NH
O
N
O
N
O
O
O
8
HO
-X
HO
O
-NHS
31P-NMR (DMSO-d6)
NH
31P-NMR (DMSO-d6)
MMTrO
N
116.7
O
O
92.9, 91.8
NH
O
S
X
O
P
O
O
HS
NH
N
O
8'
O
HO
Scheme 3. Post synthesis cyclization under aqueous conditions using a bi functional linker.
found to be the rate determining step, with the rate being dependent
on the nature of the leaving group as shown in Figure 2. The differ-
ence in the reactivity of the linker revealed that chlorine performed
inefficiently as a leaving group, providing only 7% of the cyclized
product even after 24 h. In contrast, the bromo and iodo leaving
O
O
O
NH
O
X
MMTrO
HS
N
O
O N
O
(1.5 eq)
groups provided enhanced levels of reactivity to give the cyclized
13
O
species after only 6 h.
The diastereomeric ratio of the products
NH2
O
O
PBS buffer (pH 7.0)-CH3CN (1:1, v/v), rt, 2 mM
was found to be almost identical for each of the different leaving
groups. This selectivity was attributed to the chirality of the nucleo-
tide itself. The cyclization reaction was performed at different tem-
peratures and pHs to clarify the reactivity and the diastereomeric
ratio of the products. The results revealed that increasing the tem-
perature to 37 °C enhanced the rate of the reaction, with the reaction
reaching completion within 1 h. In contrast, the reaction proceeded
at a slower rate when it was conducted at the lower temperature of
4 °C, and required 24 h to reach completion. Taken together, these
results clearly indicated that the rate of the alkylation was con-
trolled by the temperature as well as the nature of the leaving group.
Interestingly, the temperature has no discernible impact on the dia-
stereomeric ratio. Variations in the pH of the reaction mixture had a
pronounced impact on the observed level of reactivity. Under acidic
conditions, the reaction did not proceed at all because the proton-
ation of the amino group on the nucleotide effectively subdued
any reactivity. Under the basic conditions, the reaction also failed
to provide any of the desired product probably because of the insta-
bility of the cyclized product 9 under the basic conditions. It is envis-
aged that future work conducted in the presence of target molecules,
such as complementary DNA/RNA or other binding molecules, will
reveal changes in the selectivity that will be dependent on the local
conformation. The most intriguing property of these compounds
was the characteristic NMR shift of phosphorodithioate triester that
allowed for the effective discrimination of the product peaks from
other phosphorous peaks such as those occurring in other natural
compounds containing phosphodiesters, phosphomonoesters,
phosphotriesters, pyrophosphates, and other phosphorothioates.
In conclusion, we have synthesized a 20-aminomethyl branched-
chain sugar nucleoside phosphorodithioate and subjected this
P S
O
NH
O
N
O
NH
MMTrO
HS
N
HO
O
O
H
O
N
O
P
O
S
ESI -Mass
Cl = 940.08
Br = 986.13
I = 1031.99
X
O
O
92.9 ppm
91.8 ppm N
HO
NH
O
Yield (%)(b)
(diastereomer ratio: 91.8,
92.9 ppm)
Entry
X
Time (h) Temp (oC) pH(a)
1
2
3
4
5
6
7
Cl
Br
I
24
6
rt
rt
7.4
7.4
7.4
7.4
7.4
5.0
9.0
7 (75:25)
58 (69:31)
63 (69:31)
75 (65:35)
51 (68:32)
0
6
rt
I
1
37
4
I
24
6
I
rt
I
6
rt
0
(a) pH 7.4 PBS buffer; pH 5.0 or 9.0 50 mM phosphate buffer
(b) Isolated yield
Figure 2. 31P NMR spectrum of the cyclic phosphorodithioate triester
9 and
cyclization conditions.

H. Saneyoshi et al. / Tetrahedron Letters 54 (2013) 1080–1083
1083
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material to a subsequent cyclization reaction using bi-functional
linkers. The current reaction involves the simple chiral generation
reaction of an achiral phosphorous atom. It is envisaged that the
reaction will be microenvironment dependent when conducted in
the presence of a target strand or other nucleic acid binding materi-
als such as distamycin antibiotics and intercalators. Furthermore,
this reaction could represent a new method for monitoring nucleic
acid hybridization and nucleic acid–ligand interaction by 31P NMR.
Further studies of this reaction in the presence of different oligonu-
cleotide strands are currently underway.
7. (a) Bjergårde, K.; Dahl, O. Nucleic Acids Res. 1991, 19, 5843–5850; (b) Marshall,
W. S.; Beaton, G.; Stein, C. A.; Matsukura, M.; Caruthers, M. H. Proc. Natl. Acad.
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Acknowledgments
8. Sukeda, M.; Ichikawa, S.; Matsuda, A. J. Org. Chem. 2000, 65, 8988–8996.
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H.S., H.A., and Y.I. were financially supported by Ministry of
Education, Culture, Sports, Science and Technology (MEXT) and
New Energy and Industrial Technology Development Organization
(NEDO). We are grateful for the support received from the Brain
Science Institute (BSI) Research Resource Center for mass spectrum
analysis.
11. Miller, G. P.; Silverman, A. P.; Kool, E. T. Bioorg. Med. Chem. 2008, 16, 56–64.
ꢀ
12. LRMS data for intermediate 80: Cl: calcd for (C42H44ClN5O12PS2
)
[MꢀH]ꢀ
940.18, found 940.07; Br: calcd for (C42H46BrN5O12PS2+) [M+H]+ 986.15, found
986.13; I: calcd for (C42H44IN5O12PS2ꢀ) [MꢀH]ꢀ 1032.12, found 1031.99.
13. Post cyclization reaction for the synthesis of
9 under aqueous conditions:
Supplementary data
Compound 8 (30 mg, 29
lmol) was dissolved in PBS–CH3CN (15 mL, 1:1, v/
v). The iodo acetic acid NHS ester (12 mg, 42
lmol) was then added and the
resulting mixture stirred at, rt for 6 h. The reaction mixture was diluted with
EtOAc and washed with H2O. The organic solution was dried (Na2SO4), filtered
and evaporated in vacuo. The residue was purified by column chromatography
eluting with CHCl3/MeOH (10:1) to give 9 (17 mg, 63%). 1H NMR (400 MHz,
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2012.
12.028.
DMSO-d6)
d 11.53–11.34 (3H, m), 7.62–7.26 (15H, m), 6.94–6.92 (2H, d,
J = 8.69), 6.23–6.21 (1H, m), 5.93 (1H, t, J = 9.65), 5.66–5.32 (2H, m), 4.80–4.24
(4H, m), 4.11–3.72 (5H, m), 3.51–3.42 (2H, m), 3.17–2.98 (2H, m), 2.24–2.08
(2H, m), 1.79 (3H, s); 31P NMR (DMSO-d6) d 92.9, 91.8; HRMS (ESI) calcd for
(C42H44N5NaO12PS2+) [M+Na]+ 928.2058, found 928.2066.
References and notes
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U.S.A. 2012, 109, E595–604; (b) Moriuchi, T.; Hirao, T. Acc. Chem. Res. 2010, 43,
1040–1051.