Synthesis of a novel ester analog of nucleic acids bearing a serine backbone

Article abstract of DOI:10.1016/j.bmcl.2006.02.076

A novel analog of nucleic acids bearing an optically active serine ester backbone, serine-based nucleobase-linked polyester (SNE), was synthesized. Monomers containing a thymine base were synthesized from l- and d-serines. Furthermore, reaction conditions

Full text of DOI:10.1016/j.bmcl.2006.02.076

Bioorganic & Medicinal Chemistry Letters 16 (2006) 2933–2936
Synthesis of a novel ester analog of nucleic acids
bearing a serine backbone
Asako Murata and Takeshi Wada*
Department of Medical Genome Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Bioscience Bldg. 702,
5
-1-5 Kashiwanoha, Kashiwa, Chiba 277-8562, Japan
Received 25 January 2006; revised 27 February 2006; accepted 28 February 2006
Available online 22 March 2006
Abstract—A novel analog of nucleic acids bearing an optically active serine ester backbone, serine-based nucleobase-linked poly-
ester (SNE), was synthesized. Monomers containing a thymine base were synthesized from L- and D-serines. Furthermore, reaction
conditions were thoroughly examined for the ester bond formation by using a new phosphonium-type condensing reagent on a solid
support without racemization. The release of the dimer from the resin was also investigated using a new type of linker, which could
be cleaved under neutral conditions.
ꢀ
2006 Elsevier Ltd. All rights reserved.
The base pairing of nucleic acids is one of the most
important events of molecular recognition in nature,
which guarantees the storage, transfer, and expression
of genetic information in living systems. The highly
specific molecular recognition through the pairing of
nucleobases has become increasingly important in
diagnostic and therapeutic applications. Moreover,
chiral recognition plays a critical role in most biolog-
ical functions such as enzyme catalysis and ligand–
receptor interactions. Recent years, the synthesis of
peptide nucleic acids (PNAs), nucleic acid analogs
based on an achiral polyamide backbone, and their
biological studies as well as diagnostic applications
B1
B2
B3
O
O
NH O NH O NH
O
O
O
O
O
Figure 1. The structure of serine-based nucleobase-linked polyester
SNE).
(
linkages of PNA and of the phosphodiester linkages
of nucleic acids by ester linkages is expected to change
the chemical and physiological properties of these
molecules, particularly solubility in water and back-
bone flexibility. Furthermore, the SNE has a shorter
1
were evaluated.
5
helical pitch compared with that of PNA, which is
Even though a wide variety of PNA analogs have
been reported to date, polyester analogs of nucleic
acids have never been reported because of the absence
of the synthetic strategy. Therefore, we focused our
attention on the synthesis of a novel polyester analog
of nucleic acids bearing an optically active serine
backbone (serine-based nucleobase-linked polyester:
identical for the hybridization with complementary
nucleic acids. In addition, helicity induced on SNE
oligomers by the chirality of the backbone might ex-
press an ability to recognize the chirality of other mol-
ecules. Because ester linkages are labile to strong acids
or bases, compared with the amide linkages, the devel-
opment of a new method for the synthesis of SNE
2
SNE) (Fig. 1) as a simple model of nucleic acids, a
3
6
oligomers is of great importance.
potential primordial nucleic acid, and a candidate
4
for therapeutic agents. The replacement of the amide
Here, we describe the synthesis of optically pure mono-
mer units containing a thymine base and the optimiza-
tion of the reaction conditions for an ester bond
formation on a solid support without racemization as
well as for the release of a dimer from the solid support
without the cleavage of the ester bond.
Keywords: Nucleic acid analog; Polyester; Serine; Solid-phase
synthesis; Racemization; Condensing reagent; PNA.
*
Corresponding author. Tel.: +81 4 7136 3612; fax: +81 4 7136
612; e-mail: wada@k.u-tokyo.ac.jp
3
0
960-894X/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.02.076

2934
A. Murata, T. Wada / Bioorg. Med. Chem. Lett. 16 (2006) 2933–2936
1
1
pentafluorophenyl ester 4. After removal of the TMS
groups, the hydroxy group was protected with an
MMTr group. Finally, the resulting compound was con-
verted to the corresponding benzyltrimethylammonium
salt to give the monomer unit (L)-3 in 97% total yield.
The monomer unit (D)-3 was synthesized in a similar
manner. In these cases, the optical purities of (L)-3
and (D)-3 were estimated to be 98% ee and 97% ee,
O
NH Cl
i), ii)
3
T
HO
OEt
OH
1
O
T
T
iii)
HN
O
HN
O
O NR4
MMTrO
OEt
MMTrO
9
O
O
respectively.
(L)-2
(L)-3
R N = Benzyltrimethylammonium
Next, we investigated reaction conditions for an ester
bond formation on a solid support and the effect of
the conditions on racemization (Scheme 3). Similar to
N-acylated amino acids, the monomer unit 3 would
be susceptible to racemization, compared with N-car-
bamated amino acids. Therefore, a coupling reaction
without racemization should be developed to obtain
optically pure SNE oligomers. To evaluate the
coupling efficiency for the ester bond formation, a
4
Scheme 1. Reagents and conditions: (i) DCC, Et
3
2 2
N, CH Cl , DMF, rt,
1
2 h; (ii) MMTr-Cl, pyridine, rt, 12 h (87%, two steps); (iii) benzylt-
rimethylammonium hydroxide, methanol, H O, 0 ꢁC, 1 h (92%). The
2
monomer unit (D)-3 was synthesized in a similar manner.
Thymine monomers were prepared from L- and D-serines
8
7
as outlined in Scheme 1. (Thymin-1-yl)acetic acid 1 was
condensed with (L)-serine ethyl ester in the presence of
1
2
novel linker developed by our group, which could
be cleaved by treatment with a phosphine reagent
under reductive and neutral conditions, was attached
to an aminomethylated highly cross-linked polystyrene
0
N,N -dicyclohexylcarbodiimide (DCC) and then the
hydroxy group was protected with a monomethoxytrityl
MMTr) group. The resulting ester (L)-2 was hydrolyzed
by treatment with benzyltrimethylammonium hydroxide
to give the monomer unit (L)-3 in good yield. The
monomer unit (D)-3 was synthesized in a similar manner.
However, the optical purities of the monomer units
were found to significantly decrease to 66% ee for (L)-3
(
1
3
(HCP) support. The loading amount of the linker
was estimated by an MMTr cation assay (24–
26 lmol/g).
The coupling reaction of the monomer unit (L)-3 (0.1 M)
with the hydroxy group of the linker was performed by
9
and (D)-3. Therefore, we decided to synthesize the
monomer units by another route outlined in Scheme 2.
1
4
using PyNTP (0.2 M) as a coupling reagent in the pres-
ence of a base or nucleophilic catalyst (0.3 M), and the
coupling efficiency was verified by an MMTr cation
Pertrimethylsilylated serine, prepared by a method
1
0
similar to the reported procedure, was treated with
an equimolar amount of anhydrous methanol to remove
the amino-protecting group selectively. The crude (L)-5
was then acylated with the thyminyl acetic acid
H
N
O
O
O
TFA
N
MMTrO
N3
O
O
i)
Pfp =
T
T
F
OH
OPfp
F
F
NO2
N PF6—
1
4
N P N
F
H
3
F
N
O
(PyNTP)
O
O
+
(L)-3, base or catalyst
NH2
OH
ii), iii)
NH2
OTMS
HO
N3
MMTr +
CH3CN, rt, time
HO
TMSO
O
O
L)-5
H
N
(
T
O
T
TFA
O
O
HN
O
O
iv)
v)
HN
O
OTMS
MMTrO
T
N3
TMSO
O
O
H
N
(
L)-6
O
MePPh , H O
2
2
T
T
O
mercaptoethanol
O
HN
HO
O
O
HN
O
OH
vi)
HN
O
O NR4
N3
dioxane
HO
MMTrO
+ MMTr +
O
O
L)-7
O
T
(
(L)-3
H
O
H
H
N
R4N = Benzyltrimethylammonium
HN
O
O
+
HO
N
HO
O
O
Scheme 2. Reagents and conditions: (i) DCC, DMF, rt, 17 h (37%); (ii)
TMS-Cl, CH Cl Cl , rt; (iv) 4, CH Cl
, 60 ꢁC, 1.5 h; (iii) MeOH, CH
rt, 12 h; (v) TFA, CH Cl , methanol, rt, 1 h; (vi) MMTr-Cl, pyridine,
O
2
2
2
2
2
2
,
2
2
chiral RP-HPLC analysis
Scheme 3. The ester bond formation on a HCP support.
rt, 13 h (97%, four steps). The monomer unit (D)-3 was synthesized in a
similar manner.

A. Murata, T. Wada / Bioorg. Med. Chem. Lett. 16 (2006) 2933–2936
Table 1. The coupling efficiency and the effect of the coupling
conditions on racemization of the monomer unit (L)-3
2935
a
Coupling yield (%)
b
Ee (%)
Entry
Base or catalyst
1
2
3
4
5
6
7
—
21
92
96
84
68
58
7
Pyridine
Lutidine
Collidine
DMAP
Quant
Quant
98
c
DTBMP
4-Methoxy
11
72
93
30
pyridine N-oxide
Quinoline
8
66
97
a
b
c
The yield was estimated by an MMTr cation assay.
The enantiomeric excess of 3 was monitored by a chiral RP-HPLC.
,6-Di-tert-butyl-4-methylpyridine.
2
assay (Table 1). All reactions were performed in acetoni-
trile at room temperature for 15 min. The residue at-
tached to the resin was released from the solid support
by treatment with 0.2 M methyldiphenylphosphine-
1
2
mercaptoethanol in aqueous dioxane. The released
products were dissolved in water and washed with chlo-
roform to remove phosphine oxide and excess phos-
phine. After concentration of the aqueous portion, the
products were analyzed by chiral reversed-phase
Figure 2. RP-HPLC profile of the crude dimer TT–SNE.
9
HPLC.
double coupling protocol was found to be the most
effective from the viewpoints of both the efficient
coupling and the acceptable degree of racemization
As shown in Table 1, the coupling yield was very low,
when the reaction was carried out without any base or
catalyst (entry 1). In contrast, the reaction proceeded
smoothly in the presence of an effective base or catalyst.
However, the degree of racemization increased by
increasing the basicity of the additive. As a result, the
best result was realized by using quinoline as a base;
the highest enantiomeric purity of the monomer unit
(entry 3).
In the next stage, the dimer TT–SNE was synthe-
sized on the solid support under the optimized con-
ditions and detached from the resin. The released
products were analyzed by reversed-phase HPLC
1
5
(
L)-3 was achieved. Then, the coupling conditions were
(
Fig. 2), and the desired dimer TT–SNE was ob-
further optimized using PyNTP/quinoline to realize
both the high coupling efficiency and the high optical
purity of the product.
served as a main peak. The result indicates that
the dimer was synthesized efficiently on the solid
support and successfully detached from the resin
without decomposition of the product. After purifi-
cation by RP-HPLC, the pure dimer TT–SNE was
obtained in 52% yield.
The concentration of the monomer unit (L)-3 and
reaction time for the ester bond formation were fur-
ther investigated (Table 2). Prolonged reaction time
increased the coupling yield, whereas the degree of
racemization increased (entry 2). In contrast, the
coupling efficiency was improved by increasing the
concentration of the monomer unit (L)-3 (0.2 M),
and the racemization was at a low level. Finally, a
In conclusion, optically pure monomers containing a
thymine base were synthesized, and coupling conditions
were optimized by using a new linker-attached solid sup-
port to form an ester bond with minimum racemization.
Finally, the corresponding dimer was synthesized under
the optimized conditions on the solid support and
detached under almost neutral conditions to give the
product without any decomposition. The solid-phase
synthesis of long oligomers containing four nucleobases
is now in progress.
Table 2. The coupling efficiency and the effect of the coupling
conditions on racemization using PyNTP/quinoline as reagents
b
Ee (%)
Entry Concentration of Reaction
monomer (M)
Coupling
a
yield (%)
time
1
2
3
4
5
0.1
0.1
0.1
0.2
0.2
15 min · 1
1 h
66
93
99
95
99
93
89
96
95
94
Acknowledgments
c
15 min · 2
15 min
30 min
This work was supported by a Grant-in-Aid for Scien-
tific Research from the Ministry of Education, Cul-
ture, Sports, Sciences, and Technology, Japan. We
are indebted to Professor K. Saigo for helpful
suggestion.
a
b
c
The yield was estimated by an MMTr cation assay.
The enantiomeric excess of 3 was monitored by a chiral RP-HPLC.
A double coupling protocol.

2936
A. Murata, T. Wada / Bioorg. Med. Chem. Lett. 16 (2006) 2933–2936
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2
rate of 1 ml/min.
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5
1
5. The HPLC analysis and purification of the dimer was
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6
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7
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+
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