Synthesis and hybridization properties of an oligonucleotide consisting of 1′,4′-anhydro-2′,5′-dideoxy-2′- (thymin-1-yl)-D-altritol

Article abstract of DOI:10.1002/1522-2675(200205)85:5<1479::AID-HLCA1479>3.0.CO;2-P

The novel oligonucleotide analogue 7, consisting of 1′,4′-anhydro-2′,5′-dideoxy-2′- (thymin-1-yl)-D-altritol (4), residues was synthesized by the phosphoramidite approach on an automated DNA synthesizer. The phosphoramidite building block 6 was obtained b

Full text of DOI:10.1002/1522-2675(200205)85:5<1479::AID-HLCA1479>3.0.CO;2-P

Helvetica Chimica Acta Vol. 85 (2002)
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Synthesis and Hybridization Properties of an Oligonucleotide Consisting of
1',4'-Anhydro-2',5'-dideoxy-2'-(thymin-1-yl)-d-altritol
by Zhu Guan, Xiao-Bin Tian, Liang-Ren Zhang*, Ji-Mei Min, and Li-He Zhang
National Research Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100083, China
The novel oligonucleotide analogue 7, consisting of 1',4'-anhydro-2',5'-dideoxy-2'-(thymin-1-yl)-d-altritol
(4), residues was synthesized by the phosphoramidite approach on an automated DNA synthesizer. The
phosphoramidite building block 6 was obtained by phosphitylation of the corresponding isonucleoside 5.
Oligoisonucleotide 7 contains an extended phosphodiester linkage with a higher flexibility. Oligoisonucleotide 7
was studied with respect to hybridization properties, enzymatic stability, and CD spectra. It exhibits a high
stability towards snake-venom phosphodiesterase and an acceptable hybridization to complementary single-
stranded DNA and RNA.
Introduction. Much effort to enhance the biological activity of oligonucleotides as
inhibitors of gene expression has focused on the improvement of their stability to
nuclease digestion. This problem has been approached in several ways including
alteration of the phosphate and sugar moieties in the oligonucleotides [1][2]. Most of
the latter modifications contain a five-membered sugar ring closely resembling the
natural deoxyribose [3 5]. Currently, one drug based on the antisense strategy has
been approved by the Food and Drug Administration (FDA), and clinical evaluation
of many other antisense phosphorothioate oligodeoxynucleotides and DNA ¥ RNA
oligomer chimeras is underway [6]. Oligonucleotides incorporating hexose nucleoside
analogues were reported to possess significantly increased stability towards phospho-
diesterases thereby retaining hybridization properties [7]. Recently, LNA (locked
nucleic acid) was introduced, a modification where the backbone of the oligonucleotide
consists of a conformationally restricted nucleotide with a 2'-O,4'-C-methylene bridge.
Such nucleic acids have shown unprecedented helical thermal stability when hybridized
to their complementary DNA and RNA [8].
We are interested in the synthesis and hybridization properties of oligonucleotides
incorporating isonucleosides. Isonucleosides are a new class of nucleoside analogues in
which the nucleobase is linked to a ribose position other than C(1'). We have reported
the syntheses of 1',4'-anhydro-2'-deoxy-2'-nucleobase-l-arabinitol 1 and 1',4'-anhydro-
2'-deoxy-2'-nucleobase-d-arabinitol 2 from d- or l-xylose (Fig. 1) [9][10]. Oligonu-
cleotides bearing the isonucleosides 1 or 2 show increased stability towards snake-
venom phosphodiesterase (SVPDE), but only oligonucleotides built up from the
isonucleoside 2 retained their hybridization property [11]. To investigate whether
extension of the phosphodiester linkage formed could improve the hybridization
properties, we synthesized 1',4'-anhydro-2',5'-dideoxy-2'-nucleobase-d-altritol 3 in
which an additional methylene group is introduced at the 5'-position of compound 2
[12]. We report here the synthesis of a homo-oligonucleotide consisting of 1',4'-

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anhydro-2',5'-dideoxy-2'-(thymin-1-yl)-d-altritol (4) residues and the study on its
duplex stability when it is hybridized with complementary single DNA and RNA
strands.
Fig. 1. Isonucleosides 1
3
Results and Discussions.
The building block 5, suitable for solid-phase
oligonculeotide synthesis, was obtained starting from 1',4'-anhydro-2',5'-dideoxy-2'-
(thymin-1-yl)-d-altritol (4), which was prepared earlier in our laboratory from d-
glucose [12]. Compound 4 was protected with a dimethoxytrityl group ((MeO)2Tr) to
give 5 which was phosphitylated to the phosphoramidite 6 by standard procedures in
good yield (Fig. 2). The oligonucleotide analogue 7, fully based on 6, was assembled in
an automated DNA synthesizer on a 1-mmol scale. For synthetic convenience, the
synthesis started with commercially available thymidine-loaded controlled pore glass
(CPG). The synthesis followed the standard protocol, except for a prolonged coupling
time of 200 s to ensure adequate coupling yields. The coupling efficiency was
‡
determined by measuring the release of (MeO)2Tr : 66% of total incorporation yield
was obtained. HPLC Analysis of the crude product showed that a uniform oligomer
was formed (Fig. 3). The composition of oligomer 7 was confirmed by MALDI-TOF
mass spectrometry.
Fig. 2. Oligonucleotide 7 with isonucleoside residues and the synthetic intermediates 4
6

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Fig. 3. HPLC Profile (detection at 260 nm) of crude oligomer 7
The enzymatic stability of oligomer 7 was analyzed by monitoring the hyperchromic
effect upon addition of SVPDE (3'-exonuclease) to a buffered solution of the oligomer.
The result indicated that no significant change of UV absorbance occurred within
30 min, while regular dT14 gave a time-dependent increase of absorbance. This means
that oligomer 7 consisting of isonucleoside residues is resistant to degradation by 3'-
exonuclease.
The hybridization property of oligomer 7 with complementary dA14 was inves-
tigated in a thermal denaturation study. A stable duplex of oligomer 7 with dA14 was
formed with a slightly reduced Tm value of 28.88, i.e., DTm ˆ À0.518 per modification, as
compared to the Tm of 35.58 for the control duplex dT14 ¥ dA14. A more stable duplex
was formed when 7 was hybridized with the complementary RNA strand polyA
(Table).
Table. Melting Temperature of Duplexes Formed in 0.14m NaCl, 0.01m Na2HPO4, and 1.0 mm EDTA at pH 7.2
7 ¥ dA14
7 ¥ polyA
dT¥ dA14
35.5
Tm [8]
28.8
31.5
As we described previously, the oligonucleotide consisting of isonucleoside 2 can
form a duplex with its complementary single strand. The hyperchromicity value for this
duplex was much less than for the control duplex dT14 ¥ dA14, reflecting poor base
stacking within the duplex [11]. Oligomer 7 has an extended phosphodiester linkage,
and the backbone is more flexible compared to the oligomer consisting of isonucleoside
2. It was expected that the perturbation of the base stacking in the oligomer consisting
of 2 could be compensated by the higher backbone flexibility in 7.
To study the global conformation of the duplex, the CD spectra of a duplex
composed of oligomer 7 and the complementary dA14 and polyA were measured. The
spectrum of the control duplex dT14 ¥ dA14 showed a positive Cotton effect at 218nm
and a negative Cotton effect at 248nm ( Fig. 4). The shape of the spectrum of duplex 7 ¥
dA14 was very similar to that of the control duplex. On the other hand, the CD spectrum
of duplex 7 ¥ polyA showed a very strong positive Cotton effect at 265 nm which

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Fig. 4. CDSpectra of duplexes of oligonucleotide 7 consisting of isonucleoside residues with its complementary
~
&
sequences: 7 ¥ dA14 (Â), 7 ¥ polyA ( ), and dT14 ¥ dA14 ( )
indicated that an A-like conformation was formed in solution. Oligomer 7 can form
duplexes with both DNA and RNA complementary strands which indicates that the
extended phosphodiester linkage in oligomer 7 renders the sugar moiety more flexible
to fit the required A or B conformation of the duplex.
Wengel and co-workers have synthesized the thymidine analogs 8 and 9 in which a
hydroxymethyl group was inserted at the 3'-position of the sugar moiety (Fig. 5). It was
found that the homo-oligomer containing 8 or 9 increased the stability toward SVPDE
and hybridized with a complementary strand with a slightly reduced Tm value [14][15].
Kofoed et al. exploited a comparative Tm between normal oligonucleotides and
oligonucleotides incorporating analogs 10 or 11 for the formation of duplexes with a
complementary hairpin sequence [16]. In this context, it is significant that oligomer 7
has desirable features such as resistance to cellular nucleases and the ability to form a
stable duplex with the A-DNA conformation.
Fig. 5. Some methylene-expanded nucleosides
Experimental Part
General. The 1',4'-anhydro-2',5'-dideoxy-2'-(thymin-1-yl)-d-altritol (4) was synthesized by the protocol
described earlier. dA14 was purchased from Beijing AuGCT Biotechnology. PolyA, diisopropylethylamine
(iPr2EtN), and snake-venom phosphodiesterase (SVPDE) were purchased from Sigma. The 2-cyanoethyl
diisopropylphosphoramidochloridite and thymidine-loaded controlled pore glass (CPG) were purchased from
Applied Biosystems. All solvents were dried and distilled prior to use. Evaporations were carried out under
reduced pressure and a bath temp. of < 458. Column chromatography (CC): silica gel (200 300 mesh; Qingdao
Chemical Co.). Optical rotations: Perkin-Elmer 243B polarimeter. UV Spectra: Pharmacia LKB-Biochrom-
4060 spectrophotometer. NMR Spectra: Varian VXR-300 or Bruker DPX-400 instrument; d in ppm, J in Hz. MS
and MALDI-TOF-MS: ZAB-HS, KYKY-ZHP-5, and LSI-1700 (Linear Scientific Inc.) instruments; m/z.

Helvetica Chimica Acta Vol. 85 (2002)
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1',4'-Anhydro-2',5'-dideoxy-6'-O-(4,4'-dimethoxytriphenylmethyl)-2'-(thymin-1-yl)-d-altritol (5). Com-
pound 4 (300 mg, 1.17 mmol) was dissolved in anh. pyridine (20 ml), then dimethoxytrityl chloride (477 mg,
1.40 mmol) was added under stirring with ice-water cooling. The soln. was stirred at r.t. for 24 h. After
evaporation, the mixture was purified by CC (silica gel, AcOEt/CHCl3/Et3N 7:2 :1: 5 (640 mg, 97.8%). 1H-NMR
((D6)DMSO): 1.75 (s, MeÀC(5)); 1.95 (m, 2 HÀC(5')); 3.05 (t, J ˆ 6.6, 2 HÀC(6')); 3.63 (m, HÀC(4')); 3.72
(s, 2 MeO); 3.74 (d, J ˆ 7.5, 2 HÀC(1')); 3.91 (t, J ˆ 8.9, HÀC(3')); 4.76 (m, HÀC(2')); 6.86 7.40 (m, 14 ar-
‡
om. H). FAB-MS: 559 ([M ‡ H] ).
1'4'-Anhydro-2',5'-didehydro-6'-O-(4,4'-dimethoxytriphenylmethyl)-2'-(thymin-1-yl)-d-altritol 3'-O-(2-Cya-
noethyl) Diisopropylphosphoramidite (6). To a soln. of 5 (477 mg, 0.877 mmol) in anh. THF (7.0 ml), iPr2EtN
(0.5 ml, 2.85 mmol) was added under Ar. To this soln., 2-cyanoethyl diisopropyl phosphoramidochloridite
(0.46 ml, 1.9 mmol) was added slowly under ice-water cooling. After stirring at 08 for 10 min and at r.t. for
30 min, the mixture was quenched by addition of MeOH (1 ml). After stirring for 10 min, AcOEt (20 ml) was
added, the org. layer washed twice with 5% aq. NaHCO3 soln. (5.0 ml) followed by H2O (5 ml), dried (Na2SO4),
and evaporated, and the oily residue purified by CC (silica gel, petroleum ether/AcOEt/CH2Cl2 2 :1:1 (1%
Et3N)): 6 (649 mg, 96%). Colorless foam. 31P-NMR ((D6)DMSO): 150.7.
Solid-Phase Synthesis of Oligonucleotide 7. Oligonucleotide synthesis was carried out on a 1-mm scale with a
DNA synthesizer (model 391A; Applied Biosystems) applying regular phosphoramidite chemistry (DMT off).
Cleavage and deprotection of the oligonucleotide were performed in conc. aq. ammonia soln. at 508 for 24 h.
The oligomer was purified by HPLC (ZOBAX Oligo semi-prep. column (9.4 mm  250 mm), gradient elution
with eluants A (MeCN/0.02m NaH2PO4 1:4) and B (1.0m NaCl in eluant A), flow rate 1.0 ml/min). The product-
containing fraction was desalted by means of a Sephadex-G-15 column. The pure oligonucleotide was
lyophilized and stored at À208. Total incorporation yield: 66%. MALDI-TOF-MS: 4368.88 (calc. 4365.06).
Enzymatic Stability of Oligonucleotide 7. Oligonucleotide 7 (0.2 OD) in 1.0 ml of buffer soln. (0.1m, NaCl,
0.14 mm MgCl2, 0.1m Tris ¥ HCl, pH 8.6) was digested with 1.2 U of SVPDE at 378. During digestion, the increase
in absorbance at 260 nm was followed. The absorption vs. time curve of the digestion was plotted and the
hyperchromicity evaluated.
UV Melting Experiments. UV Melting experiments were recorded with a Pharmacia-LKB-Biochrom-4060
spectrophotometer. Oligomers were dissolved in a buffer soln. containing 0.14m NaCl, 0.01m Na2HPO4, 1.0 mm
EDTA (pH 7.2). The soln. containing oligonucleotide 7 at a concentration of 2 mm was mixed with an equimolar
amount of dA14 or equimolar per nucleotide amount of polyA. Samples were incubated at 808 for 5 min, slowly
cooled to 48, and kept at this temp. overnight. These samples were used for the thermal denaturation studies.
Thermally induced transitions of every mixture were monitored at 260 nm. Sample temp. was increased at 0.28/
min between 15 and 758.
CDSpectra . CD Spectra were measured at 208 with a J715 CD spectrophotometer (JAC) in thermostati-
cally controlled 1-cm cuvettes. The oligomers were dissolved and analyzed in buffer containing 10 mm
Na2HPO4, 0.14m NaCl, and 1.0 mm EDTA (pH 7.2), and at a oligonucleotide concentration of 4 mm or
equimolar per nucleotide amount.
The project was supported by the National Natural Science Foundation of China and the Ministry of Science
and Technology of China (G1998051103).
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Received December 3, 2001