Synthesis and hybridization properties of sugar-modified oligonucleotides

Article abstract of DOI:10.1002/1522-2675(20011219)84:12<3643::AID-HLCA3643>3.0.CO;2-Q

L-Threoninol-derived acyclic nucleotide monomers were prepared and incorporated into oligonucleotides at preselected positions via phosphoramidite chemistry. Hybridization properties of these modified oligonucleotides with the corresponding natural oligom

Full text of DOI:10.1002/1522-2675(20011219)84:12<3643::AID-HLCA3643>3.0.CO;2-Q

Helvetica Chimica Acta Vol. 84 (2001)
3643
Synthesis and Hybridization Properties of Sugar-Modified Oligonucleotides
by Ashwani K. Sharma, Pradeep Kumar, and Kailash C. Gupta*
Nucleic Acids Research Laboratory, Centre for Biochemical Technology, Mall Road, Delhi University Campus,
Delhi-110007, India (E-mail: kcgupta15@hotmail.com; fax.: ‡91117667471; tel.: ‡91117666156)
l-Threoninol-derived acyclic nucleotide monomers were prepared and incorporated into oligonucleotides
at preselected positions via phosphoramidite chemistry. Hybridization properties of these modified oligo-
¡
nucleotides with the corresponding natural oligomers were studied, and their vis-a-vis comparison with serinol-
modified oligonucleotides was made. Stability of the modified oligomers against nuclease in human serum and
snake venom phosphodiesterase (SVPD) was examined.
Introduction. The ability of oligonucleotides to form a double helix by binding to
single-stranded nucleic acids when there is a complementary nucleotide sequence
makes them useful as diagnostic probes and as tools in molecular genetics [1 4].
Therefore, biological processes involving these nucleic acids can be affected by addition
of the respective oligonucleotides. This particular property of modified oligonucleo-
tides makes them useful in biotechnology and, more recently, in medicinal chemistry to
an extent that their use as antisense oligonucleotides have been shown to modulate
(usually inhibit) the expression either at the transcriptional or translational stage.
Consequently, these molecules are being considered as new-generation pharmaceut-
icals. However, natural oligonucleotides suffer from some limitations, viz., the
instability against nucleases, low stability of the duplex formed with medium-chain-
length oligonucleotides, and lack of penetration through lipophilic cell membranes. To
overcome these obstacles, modifications are generally introduced into synthetic
oligonucleotides to make them effective as antisense oligonucleotides. An oligo-
nucleotide consists of bases, sugar residues, and the internucleotide backbone, and all of
these are amenable to modification. Therefore, one or more than one type of modification
can be introduced into synthetic oligonucleotides. A number of successful modifica-
tions or replacements of phosphoramidates [5], methylphosphonate [6], and peptide
nucleic acids (PNA) [7] have been shown to produce oligonucleotides that complement
DNA and RNAwith similar or high stability while maintaining the sequence specificity.
Recently, an alternative approach, involving the replacement of sugar-containing
nucleotides with acyclic nucleotide analogs derived from serinol [8 10] and phenyl-
serinol [11], was developed; it allows the formation of an intramolecular H-bond
between the amide NH of the acetamide tether and the phosphate O-atom to adopt a
constrained conformation. These monomers, when incorporated into oligonucleotides,
may have an influence stronger than a single H-bond and favor the formation of
competent nucleic acid. Furthermore, the H-bond could neutralize the negative charge
of the phosphodiester backbone, and this neutralization is expected to enhance the
uptake of the oligonucleotide, rendering it potentially more useful as a therapeutic

3644
Helvetica Chimica Acta Vol. 84 (2001)
agent. Incorporation of an acyclic l-serinol-modified analog at the 3' terminus of an
oligonucleotide remarkably increased the resistance to 3'-exonucleases but disfavored
duplex formation with a natural component. But, in the case of acyclic phenylserinol-
modified analogs, the DNA duplex and triplex formations were not hampered. This
implies that the side chain at the b-position of the amino alcohols plays a significant role
in stabilizing the duplex.
Encouraged by these results, we decided to use threoninol in place of serinol or
phenylserinol. On comparing the structure of serinol with phenylserinol, we find that an
H-atom in serinol is replaced by a Ph group, which imparts much stability to the duplex.
The present study was undertaken to reveal the effect of other substituents on serinol.
l-Threoninol was selected for two reasons: i) its preparation is straightforward, and ii)
it resembles serinol in structure as just one H-atom is replaced by a Me group.
Moreover, we assume that the extra Me group in the side chain of the threoninal moiety
will favor the stabilization of the duplex at least as well as the corresponding serinol-
derived nucleotide. Thus, we report here on l-threoninol-derived phosphoramidite
monomers and their incorporation in synthetic oligonucleotides at preselected sites (3'-
or 5'-terminus, or in the middle of the chains). The modified oligomers were then used
to study various properties, such as hybridization with natural oligomers and stability
against snake-venom phosphodiesterase (SVPD) and in human serum etc. Results
were also compared with the corresponding serinol-derived oligonucleotides.
Results and Discussion. Syntheses of phosphoramidite monomers (synthons) of l-
threoninol and serinol were carried out as depicted in the Scheme. l-Threoninol (1a)
was prepared from l-threonine by a published procedure [12], and serinol (1b) was
commercially available. l-Threoninol-derived phosphoramidite 5a and serinol-derived
phosphoramidite 5b were obtained in almost 35% yields starting from the correspond-
ing diols 1a and 1b, respectively. The synthesis strategy involved the following steps:
selective protection of the primary-amino group in the amino-diol 1 by a CF3CO group
( ! 2) and subsequent protection of one of the OH functions by the 4,4'-dimethoxy-
trityl ((MeO)2Tr) group yielded 3 in 65 68% yield. Then the CF3CO group was
removed selectively by treatment with aqueous NH4OH solution, and the crude
product was submitted to the reaction with 1-(carboxymethyl)thymine. After silica-gel
chromatography, the thymine derivatives 4 were obtained in 64 65% yield and
characterized by MALDI-TOF (Fig. 1,a and Fig. 2,a) and NMR. Phosphitylation of 4
with 2-cyanoethyl diisopropylphosphoramidochloridite resulted in the desired syn-
thons 5 in 85 88% yield.
Oligonucleotide synthesis was performed by means of phosphoramidite chemistry.
The l-threoninol- and serinol-modified reagents 5 were introduced at the 5'-terminus
and other preselected sites of the oligonucleotides. These reagents were found to be as
efficient as the normal nucleoside phosphoramidites during the coupling reaction.
Unlike the 5'-terminus, the 3'-terminus of the oligonucleotide is not available for any
manipulation during solid-phase synthesis, as it is anchored on the polymer support.
Therefore, the prederivatized supports 7a and 7b were employed to introduce the
modification at the 3'-terminus. These supports were functionalized via 6a,b, essentially
according to a procedure [13] reported from this laboratory, with a loading in the range
35 40 mmol/g support.

Helvetica Chimica Acta Vol. 84 (2001)
3645
Scheme. Preparation of Acyclic Phosphoramidites.
i) CF2COOEt, dioxane. ii) (MeO)2TrCl, pyridine. iii) Aq. NH4OH soln. iv) T-CH2COOH, EDC,
N-hydroxysuccinimide. v) Phosphitylation. vi) Succinic anhydride, N,N-dimethylpyridin-4-amine (DMAP),
Et3N. vii) LCAA-CPG, Ph3P, DMAP, BrCCl3.
Fig. 1. MALDI-TOF Mass spectra of a) compound 4a and b) oligomer d(xTT TTT TTT TTT TTT TTT TT) (I)
The three 3'-terminal modified oligomers II, V, and IX, a number of other modified
oligonucleotides, I, III, IV, VI VIII, and X XII, along with the standard d(TTT TTT
TTT TTT TTT TTT TT) (XIII), d(AAA AAA AAA AAA AAA AAA AA) (XIV),
d(AAG AAG AAA AAG A) (XV), and d(TTC TTC TTT TTC T) (XVI) were
synthesized (see Table1). The modified as well as normal oligonucleotides were
purified by fast protein liquid chromatography (FPLC; Mono-Q anion exchange
column) and their purity ascertained analytically by FPLC and MALDI-TOF MS
(Fig. 1,b and Fig. 2,b).

3646
Helvetica Chimica Acta Vol. 84 (2001)
Fig. 2. MALDI-TOF Mass spectra of a) compound 4b and b) oligomer d(yTT TTT TTT TTT TTT TTT TT)
(VIII)
Oligonucleotide duplexes were constituted from unmodified oligomer d(AAA
AAA AAA AAA AAA AAA AA) (XIV) and modified I XI, oligomer XIII and
XIV, modified hetero oligomer XII and oligomer XV, and oligomer XVI and XV (see
Table 1). The stability of the duplex was measured by UV melting experiments, by
detection of the change in UVabsorbance at 260 nm in the temperature range 15 608.
Tm Values were confirmed by distinct peaks of first-derivative plots, and the data
Table 1. Sequences of Modified and Normal Oligonucleotides
Sequencea)
I
II
III
IV
V
VI
VII
VIII
IX
d(xTT TTT TTT TTT TTT TTT TT)
d(TTT TTT TTT TTT TTT TTT Tx)
d(TTT TTT TTT TTT TTT TTx xT)
d(TTT TTT TTT xTT TTT TTT TT)
d(xTT TTT TTT TTT TTT TTT Tx)
d(TTT TTT TTT TTT TTT TTT xT)
d(TxT TTT TTT TTT TTT TTT TT)
d(yTT TTT TTT TTT TTT TTT TT)
d(TTT TTT TTT TTT TTT TTT Ty)
d(TTT TTT TTT TTT TTT TTy yT)
d(TTT TTT TTT yTT TTT TTT TT)
d(TTC TTC TxT TTC T)
X
XI
XII
XIII
XIV
XV
XVI
d(TTT TTT TTT TTT TTT TTT TT)
d(AAA AAA AAA AAA AAA AAA AA)
d(AAG AAG AAA AAG A)
d(TTC TTC TTT TTC T)
a) x ˆ l-Threoninol-modified residue; y ˆ serinol-modified residue.

Helvetica Chimica Acta Vol. 84 (2001)
3647
obtained for control (unmodified) and modified duplexes are shown in Table 2. These
data establish that l-threoninol modifications introduced in oligonucleotides do not
hamper the duplex formation. A single l-threoninol or serinol modification at the 5'-
terminus decreased the duplex stability by a DTm of À 2.7 and À 3.78, respectively, per
modification and a corresponding single modification at the 3'-terminus resulted in a
DTm of À 3.2 and À 5.48, respectively, per modification. Two l-threoninol modifica-
tions at the n À 1 and n À 2 position from the 3'-terminus (see III) decreased the duplex
stability only by a DTm of À 1.28 per modification, while the same serinol modifications
(see X) resulted in a DTm of À 1.68 per modification. On the contrary, the insertion of a
l-threoninol residue in the middle of the oligomer (see IV) resulted in a DTm of À 5.58
per modification, while a DTm of À 9.78 was noticed in the case of the corresponding
serinol-modified oligomer (see XI). This clearly indicates that the l-threoninol-
modified oligonucleotides show a better hybridization behavior than the corresponding
serinol-modified oligonucleotides. On comparing the thermal stability of the duplexes
formed between the hetero oligomers XII and XVand between the unmodified hetero
oligomers XVI and XV, one can see that introduction of a l-threoninol residue in the
middle of the oligomers destabilizes the duplex by DTm À 11.58.
Table 2. UV Tm Data for Duplexes
S.No.
Duplex
Tm [8]
DTm /mod. [8]
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
I ¥ XIV
II ¥ XIV
40.0
39.5
40.2
37.2
40.9
40.0
40.6
39.0
37.2
39.4
33.0
29.5
42.7
41.0
À 2.7
À 3.2
À 1.2
À 5.5
À 1.8
À 2.7
À 2.1
À 3.7
À 5.4
À 1.6
À 9.7
À 11.5
III ¥ XIV
IV¥ XIV
V¥ XIV
VI ¥ XIV
VII ¥ XIV
VIII ¥ XIV
IX ¥ XIV
X ¥ XIV
XI ¥ XIV
XII ¥ XV
XIII ¥ XIV
XVI ¥ XV
As expected, the modification at 5'-or near the 5 '-terminus did not improve the
stability of the modified oligonucleotides against exonucleases (human serum) and
snake-venom phospodiesterase (SVPD). However, the 3'-or near-3 '-terminus-
modified oligonucleotides showed very good stability against exonucleases (human
serum) and against SVPD. Interestingly, the oligomer modified in the middle was
digested quickly up to the modified position and, from then on, was quite stable against
exonucleases and SVPD.
The financial assistance from the Department of Science and Technology, New Delhi, is gratefully
acknowledged.

3648
Helvetica Chimica Acta Vol. 84 (2001)
Experimental Part
1. General. All solvents and reagents employed in the present investigation were purified prior to their use.
LCAA-CPG, N-[3-(dimethylamino)propyl]-N'-ethylcarbodiimide (EDC) and thymine were from Sigma
Chemical Co., USA. CF3COOEt, N-hydroxysuccinimide (N-HOSu), and diisopropylethylamine were from
Aldrich Chemical Co., USA. Other chemicals and reagents were obtained from local suppliers and purified
before use. CC ˆ Column chromatography. Reversed-phase HPLC: Shimadzu-LC-4A system, UV detection at
254 nm, recording with a Shimadzu-C-R7A chromatopac. Anion-exchange fast protein liquid chromatography
(FPLC): Pharmacia FPLC system, two P-500 pumps, single-path UV monitor (UV-1) operating at 254 nm;
Mono-Q-HR-5/5 (10/10) column; elution with 1m NaCl buffer (pH 12). 1H-NMR Spectra: Bruker Avance-DPX-
300 at 300 MHz; d in ppm, J in Hz. MALDI-TOF-MS: Kompact SEQ (Kratos, UK).
2. N-{1-{[(4,4'-Dimethoxytrityl)oxy]methyl}-2-hydroxypropyl}-2,2,2-trifluoroacetamide (3a). To a suspen-
sion of 2-aminobutan-1,3-diol (1a; 55 mmol) in dry 1,4-dioxane, CF3COOEt (100 mmol, 10 ml) was added. The
mixture was stirred at r.t. for 48 h and then evaporated. The product 2a was dried by co-evaporation with anh.
pyridine (100 ml). (MeO)2TrCl (60 mmol) was added and the mixture stirred overnight. After evaporation and
removal of traces of pyridine by several co-evaporations with toluene, the syrupy material thus obtained was
taken up in AcOEt (250 ml), and the soln. was sequentially washed with sat. aq. NaHCO3 soln. (2 Â 100 ml), 5%
aq. citric acid soln. (2 Â 50 ml), and sat. NaCl soln. (1 Â 100 ml), dried (Na2SO4), and evaporated: 3a (65%).
N-{2-[(4,4'-Dimethoxytrityl)oxy]-1-(hydroxymethyl)ethyl}-2,2,2-trifluoroacetamide (3b). Similarly, 3b was
prepared starting from 2-aminopropan-1,3-diol (1b) in 68% yield.
3. 2-(1,3-Dihydro-5-methyl-2,4-dioxopyrimidin-1-yl)-N-{1-{[(4,4'-dimethoxytrityl)oxy]methyl}-2-hydroxy-
propyl}acetamide (4a). A soln. of 3a (5 mmol) in MeOH (20 ml) was treated with aq. NH4OH soln. at 58 for
2 h and then evaporated. The residue was dissolved in AcOEt, the soln. washed with sat. NaHCO3 soln. (2 Â
25 ml) and sat. NaCl soln. (1 Â 25 ml), dried (Na2SO4), and evaporated, and the oily mass (almost quant.) dried
by co-evaporation with DMF and finally dissolved in DMF (50 ml). After addition of N-hydroxysuccinamide
(5 mmol), 1-(carboxymethyl)thymine (ˆ2-(1,3-dihydro-5-methyl-2,4-dioxopyrimidin-1-yl)acetic acid;
5 mmol), and EDC (6 mmol), the mixture was stirred overnight. The soln. was evaporated, the residue
redissolved in AcOEt (75 ml), the soln. subsequently washed with aq. sat. Na2CO3 soln. (2 Â 25 ml), 5% aq.
citric acid soln. (2 Â 25 ml), and sat. NaCl soln. (1 Â 25 ml), dried (Na2SO4), and evaporated, and the crude
1
product purified by CC (gradient MeOH/CH2Cl2 containing 1% Et3N): 4a (65%). H-NMR (CDCl3): 1.1 (d,
1 Me); 1.89 (s, 1 Me); 3.22 3.4 (br. m, CHOH, CH2O[(MeO)2Tr]); 3.78 (s, 2 MeO); 3.96 4.38 (m, CHN,
CH2CO); 6.81 7.37 (m, 14 H, NH, (MeO)2Tr). MALDI-TOF-MS: 574.6.
2-(1,3-Dihydro-5-methyl-2,4-dioxopyrimidin-1-yl)-N-{2-[(4,4'-dimethoxytrityl)oxy]-1-(hydroxymethyl)-
1
ethyl]acetamide (4b). Similarly, 4b was prepared starting from 3b in ca. 64% yield. H-NMR (CDCl3): 1.87 (s,
Me); 3.06 3.31 (br. m, CH2OH, CH2O[(MeO)2Tr]); 3.78 (s, 6 H, 2 MeO); 4.28 4.4 (m, CHN, CH2CO); 6.81
7.36 (m, 14 H, NH, (MeO)2Tr). MALDI-TOF-MS: 560.6.
4. 2-Cyanoethyl 2-{[2-(1,3-Dihydro-5-methyl-2,4-dioxopyrimidin-1-yl)-1-oxoethyl]amino}-3-[(4,4'-di-
methoxytrityl)oxy]-1-methylpropyl Diisopropylphosphoramidite (5a). To 4a (2 mmol) in dry dichloroethane
i
(15 ml), Pr2EtN (8 mmol) was added. The mixture was cooled in an ice bath and 2-cyanoethyl diisopropyl-
phosphoramidochloridite (3 mmol) was added dropwise through a glass syringe within 5 min with vigorous
stirring. The reaction was allowed to proceed at r.t. for 1 h (TLC monitoring). Dry MeOH (0.5 ml) was added,
the mixture was diluted with dichloroethane (50 ml), the soln. washed with 10% Na2CO3 soln. (2 Â 30 ml) and
sat. NaCl soln. (2 Â 30 ml), dried (Na2SO4), and evaporated, and the oily mass purified by CC (silica gel,
dichloroethane/AcOEt/Et3N 45 :45 :10): 5a (85%).
2-{[2-(1,3-Dihydro-5-methyl-2,4-dioxopyrimidin-1-yl)-1-oxoethyl]amino}-3-[(4,4'-dimethoxytrityl)oxy]-
propyl 2-Cyanoethyl Diisopropylphosphoramidite (5b). In the same manner, derivative 5b was prepared from
4b in 88% yield.
5. 4-{2-{[2-(1,3-Dihydro-5-methyl-2,4-dioxopyrimidin-1-yl)-1-oxoethyl]amino}-3-[(4,4'-dimethoxytri-
tyl)oxy]-1-methylpropoxy}-4-oxobutanoic Acid (6a). A mixture of 4a (1.0 mmol), DMAP (ˆ N,N-dimethyl-
pyridine-4-amine; 0.5 mmol), succinic anhydride (1.5 mmol), and Et3N (1 mmol) in 1,2-dichloroethene (5 ml)
was stirred at r.t. for 30 min and then monitored by TLC. After completion of the reaction, the mixture was
further diluted with 1,2-dichloroethene (50 ml) and the soln. washed with 5% aq. citric acid soln. (2 Â 25 ml) and
sat. NaCl soln. (2 Â 25 ml), dried (Na2SO4), and evaporated: 6a (quant.).
4-{2-{[2-(1,3-Dihydro-5-methyl-2,4-dioxopyrimidin-1-yl)-1-oxoethyl]amino}-3-[(4,4'-dimethoxytrityl)oxy]-
propoxy}-4-oxobutanoic Acid (6b). Similarly, 6b was prepared from 4b.

Helvetica Chimica Acta Vol. 84 (2001)
3649
6. Derivatized Polymer Supports 7a,b. A soln. of 6a or 6b (0.2 mmol), DMAP (0.4 mmol) and
bromotrichloromethane (0.2 mmol) in DMF (1 ml) was mixed with triphenylphosphine (0.2 mmol) dissolved
in DMF (0.5 ml), prior to the addition to long chain alkylamine controlled pore glass (500 mg). The suspension
was agitated for 30 min at r.t. Then the support was filtered through a sintered disc glass funnel. The support was
washed sequentially with DMF, MeOH, and Et2O (10 ml of each). The loading on the supports was in the range
of 35 40 mmol/g of support based on the trityl assay.
7. Oligonucleotide Synthesis and Deprotection. Oligonucleotide synthesis was carried out on a 0.2-mmol
scale with a Pharmacia-LKB Gene Assembler Plus according to the standard protocol [14]. A large number of
oligonucleotides, I XVI, unmodified or modified at preselected sites, were prepared (see Table 1) from
standard nucleoside phosphoramidites, from supports 7a,b for 3'-terminal modifications, and from 5a,b for
modifications at preselected sites other than the 3'-terminus. The cleavage of oligomers from the support and
their deprotection were achieved in a single step by treatment with 30% aq. NH4OH soln. at 558 for 16 h. The
fully deprotected oligomers were purified by ion-exchange FPLC.
8. MALDI-TOF-MS. The mass spectra of the purified oligomers were recorded in the negative linear mode.
The matrix used for monomer units was norharmane (10 mg/ml in 50% MeCN/H2O) and 3-hydroxypicolinic
acid (10mg/ml in 20% MeCN/H2O) for oligonucleotides along with diammonium citrate.
9. Melting Experiments. The melting experiments were carried out in a buffer system consisting of 10 mm
sodium phosphate, 100 mm NaCl, and 0.1 mm EDTA (pH 7.1), and containing equal concentrations of the two
complementary strands. The increase in absorbance at 260 nm as a function of time was recorded while the temp.
was raised from 15 to 608 in 0.58 increments.
10. Enzymatic Studies of the Modified Oligonucleotides. The stability of the oligodeoxyribonucleotides
containing the modified nucleosides at different positions towards snake-venom phosphodiesterase (SVPD)
and human serum (HS) was studied by polyacrylamide-gel electrophoresis after labeling the oligomers with 32
P
[15]. The cleavage of the oligonucleotides was studied over the time period 0 90 min. Aliquots after intervals
of 15 min were withdrawn and loaded onto the gel. After running, the gel was exposed onto the X-ray film and
the spots visualized after developing the autoradiogram.
REFERENCES
[1] G. Zon, Pharm. Res. 1998, 5, 539.
[2] B. J. Donich, Cancer Invest. 1991, 9, 185.
[3] S. T. Crooke, Annu. Rev. Pharmacol. Toxicol. 1992, 32, 329.
[4] C. Cazenave, C. Helene, in −Antisense Nucleic Acids and Proteins: Fundamentals and Applications×, Eds.
J. N. M. Mol and A. R. V. Krol, Marcel Dekker Press, New York 1991, p. 47.
[5] S. Gryaznov, K. J. Chen, J. Am. Chem. Soc. 1994, 116, 3143.
[6] T. Loschner, J. W. Engels, Nucleosides Nucleotides 1988, 7, 729.
[7] M. Egholm, O. Buchardt, P. E. Nielsen, R. H. Berg, J. Am. Chem. Soc. 1992, 114, 1895.
[8] K. S. Ramasamy, W. Seifert, BioMed. Chem. Lett. 1996, 6, 1799.
[9] R. Benhida, M. Devys, J. L. Fourrey, F. Lecubin, J.S. Sun, Tetrahedron Lett. 1998, 39, 6167.
[10] V. A. Efimov, M. V. Choob, A. A. Buryakova, O. G. Chakhmakhcheva, Nucleosides Nucleotides 1999, 18,
1425.
[11] V. S. Rana, V. A. Kumar , K. N. Ganesh, BioMed. Chem. Lett. 1997, 7, 2837.
[12] D. Shapiro, H. M. Flowers, E. Hecht, J. Org. Chem. 1957, 22, 461.
[13] P. Kumar, A. K. Sharma, P. Sharma, B. S. Garg, K. C. Gupta, Nucleosides Nucleotides 1996, 15, 879.
[14] −Pharmacia-LKB Gene Assembler Plus Manual×, Upsala, Sweden.
[15] R. Frank, H. Blocker, in −Chemical and Enzymatic Synthesis of Gene Fragments×, Eds. H. G. Gassen, A.
Lang, Verlag Chemie Press, Weinheim 1982, p. 225.
Received June 2, 2001