Oxathiaphospholane approach to the synthesis of oligodeoxyribonucleotides containing stereodefined internucleotide phosphoroselenoate function

Article abstract of DOI:10.1021/ol051302e

(Chemical Equation Presented) 5′-O-DMT-deoxyribonucleoside-3′- O(2-selena-4,4-pentamethylene-1,3,2-oxathiaphospholane) monomers, derivatives of dA, dC, dG, and T, can be resolved into pure P-diastereomers by silica gel column chromatography. They have bee

Full text of DOI:10.1021/ol051302e

ORGANIC
LETTERS
2005
Vol. 7, No. 18
3901-3904
Oxathiaphospholane Approach to the
Synthesis of Oligodeoxyribonucleotides
Containing Stereodefined Internucleotide
Phosphoroselenoate Function†
Piotr Guga, Anna Maciaszek, and Wojciech J.Stec*
Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences,
Department of Bioorganic Chemistry, Sienkiewicza 112, 90-363 L-o´dz´, Poland
wjstec@bio.cbmm.lodz.pl
Received June 3, 2005
ABSTRACT
5′-O-DMT-deoxyribonucleoside-3′-O-(2-selena-4,4-pentamethylene-1,3,2-oxathiaphospholane) monomers, derivatives of dA, dC, dG, and T, can
be resolved into pure P-diastereomers by silica gel column chromatography. They have been used for DBU-promoted, either solution- or
solid-phase synthesis of P-stereodefined phosphoroselenoate analogues of oligodeoxyribonucleotides. Fast- and slow-eluting monomers are
precursors of phosphoroselenoate internucleotide linkage of RP and SP absolute configuration, respectively.
The role of selenium in biological systems is of increasing
interest.1 Analogues of proteins and oligonucleotides, with
sulfur or oxygen atoms replaced with selenium, have been
evaluated as research tools for studies of interactions with
metal ions, and selenium-labeled biopolymers can be ef-
fectively analyzed by X-ray crystallography because of
multiwavelength anomalous dispersion (MAD).2 Here we
report on the stereocontrolled chemical synthesis of phos-
phoroselenoate oligonucleotides with internucleotide func-
tion(s) of predetermined sense of P-chirality. To date, a phase
transfer approach was developed to introduce the selenium
functionality in nucleosides at 5′-positions.3 Alternatively,
a SeMe group can be incorporated at the C2′-ribo position
of nucleoside, although this modification enforces the C3′-
endo structure.4,5 Labeling of oligonucleotides with a sele-
nium atom at internucleotide functions, yielding phospho-
roselenoate analogues of DNA (PSe-Oligo, 1), basically does
not affect conformation of the sugar ring.
† Dedicated to Professor Przemyslaw Mastalerz on the occasion of his
80th birthday.
(1) Flohe, L.; Andreesen, J. R.; Brigelius-Flohe, R.; Maiorino, M.; Ursini,
F. IUBMB Life 2000, 49, 411-420.
Diribonucleotide phosphoroselenoates were synthesized in
solution by Ogilvie in 1980.6 Solid-phase syntheses of PSe-
Oligos were performed using a phosphoramidite or H-
phosphonate approach with “selenation” of the PIII interme-
(2) Du, Q.; Carrasco, N.; Teplova, M.; Wilds, C. J.; Egli, M.; Huang, Z.
J. Am. Chem. Soc. 2002, 124, 24-25.
(3) Carrasco, N.; Ginsburg, D.; Du, Q.; Huang, Z. Nucleosides Nucle-
otides 2001, 9, 1723-1734.
10.1021/ol051302e CCC: $30.25
© 2005 American Chemical Society
Published on Web 08/04/2005

diate.7 In all instances, the products consist of a mixture of
P-diastereoisomers.8 Diastereomerically pure dinucleoside
phosphoroselenoates9 and DNA hexamers containing a single
internucleotide phosphoroselenoate linkage10 were obtained
by HPLC resolution of diastereomeric mixtures. Recently,
enzymatic synthesis of stereodefined PSe-Oligo has been
published, based on the use of both SP and RP diastereomers
of TTPRSe and DNA polymerase.11 It has been found that
stereorandomal PSe-Oligos have a diminished hybridization
capability7b to complementary DNA and RNA templates, as
compared with both the unmodified and phosphorothioate
oligomers. Nonetheless, selenium-labeled biopolymers are
useful probes for their structural and functional analysis. We
synthesized P-stereodefined phosphoroselenoate oligodeox-
yribonucleotides by modification of our method for synthesis
of oligo(nucleoside phosphorothioate)s,12,13 which employs
diastereomerically pure 5′-O-DMT-nucleoside-3′-O-(2-thio-
4,4-pentamethylene-1,3,2-oxathiaphospholane) monomers 2
(X ) S, Scheme 1). The mechanism of the condensation
for preparation of P-stereodefined PSe-Oligos. Earlier, the
2-selena-1,3,2-oxathiaphospholane derivative of 5′-O-DMT-
thymidine (3a, B′ ) Thy, R ) H) allowed for the synthesis
of thymidyl dinucleoside phosphoroselenoate.14 Within the
present work, two sets of deoxyribonucleoside monomers
3a (see Table 1S, Supporting Information) and 3b (B )
Table 1. Yield and Chromatographic and Spectroscopic
Properties of Separated Monomers 3b
1
yielda FAB-MSb yield
δ 31P NMR JP,Se
nucleobase
CytBz
[%]
m/z
[%]
Rfc
[ppm]d
[Hz]
56
886.5
31
31
20
40
23
31
20
30
0.64
0.62
0.65
0.63
0.59
0.57
0.53
0.51
99.61
100.04
99.06
99.73
99.25
100.05
99.90
100.44
946
945
945
945
944
944
947
947
AdeBz
59
60
45
910,5
797,4
1087.8
Thy
GuaiBu,DPC
a Yield of isolated mixture of both diastereomers, calculated over starting
5′-O-DMT-N-protected nucleosides. b Calculated value (for 80Se) m/z 887,
911, 798, 1088, respectively. Technical parameters: Cs+, 13 keV, matrix-
3-nitrobenzyl alcohol, negative ions mode. c HP TLC plates; ethyl acetate/
butyl acetate 2:1 v/v (dABz, dCBz) or butyl acetate:benzene 1:1 v/v (T,
dGiBu,DPC) were used to elute the silica gel columns and to develop the
Scheme 1
1
plates. d 200 MHz (for H), CD3CN.
AdeBz, CytBz, GuaiBu,DPC, and Thy; Table 1) were synthesized
as depicted in Scheme 1. Either mercaptoethanol or (1-
sulfanylcyclohexyl)-methanol15 were reacted with PCl3 to
obtain the phospitylating reagents 4a or 4b, respectively.16
The details of conversion 4b f 5 f 3b are provided in
Supporting Information.
In the 31P NMR spectra recorded for 3a and 3b, the
resonances in the range of 99-100 ppm accompanied by
satellite doublets resulting from the direct 31P-77Se spin-
spin coupling (1JP,Se ) 945-954 Hz) were found. Unfortu-
nately, attempts at chromatographic separation of P-diaste-
step (Scheme 1S, Supporting Information) suggested that the
synthesis of pure P-diastereomers of nucleoside-3′-O-(2-
selena-1,3,2-oxathiaphospholane) 3 (X ) Se) should allow
reomers of the monomers 3a (B′ ) AdeBz, CytBz, GuaiBu
,
(4) Teplova, M.; Wilds, C. J.; Wawrzak, Z.; Tereshko, V.; Du, Q.;
Carrasco, N.; Huang, Z.; Egli, M. Biochimie 2002, 84, 849-858.
(5) Buzin, J.; Carrasco, N.; Huang, Z. Org. Lett. 2004, 6, 1099-1102.
(6) Ogilvie, K. K.; Nemer, M. J. Tetrahedron Lett. 1980, 21, 4145-
4148.
and Thy) on a silica gel column have failed. The “spiro”
monomers 3b (B′ ) CytBz, AdeBz, Thy) were much more
useful as we were able to separate amounts of 400-500 mg
on a single silica gel column (see Table 1). The guanosyl
monomer 3b (B′ ) GuaiBu) was resolved onto pure diaster-
eomers only after protection at the O6-site with diphenyl-
carbamoyl chloride (66% yield). The slow-eluting monomer
3b (B′ ) AdeBz, 95% diastereomeric purity) was used for
condensation with 3′-O-Ac-thymidine (5 equiv) in the
presence of DBU (1.05 equiv) in dry pyridine. The 31P NMR
spectrum recorded after 2 h showed the presence of
(7) (a) Stec, W. J.; Zon, G.; Egan, W.; Stec, B. J. Am. Chem. Soc. 1984,
106, 6077-6079. (b) Mori, K.; Boiziau, C.; Cazenave, C.; Matsukura, M.;
Subasinghe, C.; Cohen, J. S.; Broder, S.; Toulme, J. J.; Stein, C. A. Nucleic
Acid Res. 1989, 17, 8207-8219 (c) Bollmark, M.; Stawin´ski, J. Chem.
Commun. 2001, 771-772. (d) Stawin´ski, J.; Thelin, M. Tetrahedron Lett.
1992, 33, 7255-7258. (e) Holloway, G. A.; Pavot, C.; Scaringe, S. A.; Lu,
Y.; Rauchfuss, T. B. ChemBioChem 2002, 3, 1061-1065. (f) Potrzebowski,
M. J.; Błaszczyk, J.; Majzner, W. R.; Wieczorek, M. W.; Baraniak, J.; Stec,
W. J. Solid State Nucl. Magn. Reson. 1998, 11, 215-224. (g) Baraniak, J.;
Korczyn´ski, D.; Kaczmarek, R.; Stec, W. J. Nucleosides Nucleotides 1999,
18, 2147-2154.
(8) Stec, W. J.; Wilk, A. Angew. Chem. 1994, 106, 747-761; Angew.
Chem., Int. Ed. Engl. 1994. 33, 709-722.
1
resonances at 50.78 [95%, JP,Se ) 814 Hz] and 50.30 ppm
(9) Koziołkiewicz, M.; Uznan´ski, B.; Stec, W. J.; Zon, G. Chem. Scr.
1986, 26, 251-260.
(10) Wilds, C. J.; Pattanayek, R.; Pan, C.; Wawrzak, Z.; Egli, M. J. Am.
Chem. Soc. 2002, 124, 14910-14916.
(14) Misiura, K.; Pietrasiak, D.; Stec, W. J. J. Chem. Soc., Chem.
Commun. 1995, 613-614.
(11) Carrasco, N.; Huang, Z. J. Am. Chem. Soc. 2004, 126, 448-449.
(12) Stec, W. J.; Grajkowski, A.; Karwowski, B.; Kobylan´ska, A.;
Koziołkiewicz, M.; Misiura, K.; Okruszek, A.; Wilk, A.; Guga, P.;
Boczkowska, M. J. Am. Chem. Soc. 1995, 117, 12019-12029.
(13) Guga, P.; Okruszek, A.; Stec, W. J. in Topics in Current Chemistry;
Majoral, J. P.; Springer: Berlin, 2002; Vol. 220.
(15) Stec, W. J.; Karwowski, B.; Boczkowska, M.; Guga, P.; Koziołk-
iewicz, M.; Sochacki, M.; Wieczorek, M. W.; Błaszczyk, J. J. Am. Chem.
Soc. 1998, 120, 7156-7167.
(16) Guga, P.; Stec, W. J. In Current Protocols in Nucleic Acid
Chemistry; Beaucage, S. L.; Bergstrom, D. E.; Glick, G. D.; Jones, R. A.,
Eds.; John Wiley & Sons: Hoboken, NJ, 2003; pp 4.17.1-4.17.28.
3902
Org. Lett., Vol. 7, No. 18, 2005

(5%), indicating virtually quantitative conversion into di-
nucleoside phosphoroselenoate with stereoselectivity close
to 100% (Figure 1S, Supporting Information). After depro-
tection, the APSeT dinucleotide was isolated (RP-HPLC) and
its molecular weight was confirmed with MALDI-TOF MS
(m/z 618).
Correlation of chromatographic mobility of fast- and slow-
3b with the respective RP and SP absolute configuration of
the resulting internucleotide phosphoroselenoate bond in
d(NPSeT) (N ) dA, dC, T, or dG) has been achieved
enzymatically using Rp-specific snake-venom phosphodi-
esterase (svPDE) and Sp-specific nuclease P1 (nP1)9 (Figure
2S, Supporting Information).
from the internucleotide bonds, the oligomers were detrity-
lated on the support under anhydrous conditions. After
cleaveage from the support and deprotection with concen-
trated NH4OH at 55 °C for 16 h, they were finally isolated
by RP-HPLC (Figure 4S). The products were collected when
absorption exceeded 20% of the height of main peaks,
yielding 6.5 and 5.5 OD units of 8a and 8b, respectively,
which were analyzed using MALDI-TOF MS. The molecular
ions centered at m/z 2077 confirmed their identity (Figure
1B). The signals around m/z 2015 (ca. 18% compared to the
desired product) has been attributed to the molecules with
one out of five selenium atoms along the chain, randomly
replaced with oxygen.
The fast-3b (B′ ) AdeBz, 20 mg) was used for elongation
at the 5′-end of d(AGCGGTCGGC) (6) (synthesized by
phosphoramidite approach using the DBU-resistant sarcosi-
nyl-succinoyl linker16 to yield 5′-O-DMT-d(APSeAGCG-
GTCGGC) (7) of RP absolute configuration. The HPLC
analysis of the DMT-tagged 6 (ca. 10% of the support was
taken out from the synthetic column to ensure accurate
analysis of the “core” oligomer to be elongated) and resulting
7 (Figure 3S, Supporting Information) showed that the
condensation process furnished PSe-Oligo in ca. 90% yield.
Subsequent HPLC purification followed by MALDI-TOF
MS analysis confirmed the identity of the product. The
molecular ion at m/z 3746 was accompanied by a signal at
m/z 3444, attributed to the detritylated (due to acidity of the
matrix used) oligomer (Figure 1A).
Another selenium-labeled oligomer of the sequence (SP)-
d(AACPSeTGC) (9) was obtained by synthesis of d(TGC)
by phosphoramidite approach (at 1-µmol scale), followed
by elongation with slow-3b (B′ ) CytBz, 20 mg) and two
consecutive couplings with 5′-O-DMT-deoxyadenosine-3′-
O-(2-oxo-4,4-pentamethylene-1,3,2-oxathiaphospholane) (10,
B′ ) AdeBz, Scheme 1). The monomer 10 was prepared in
almost quantitative yield from its 2-thio precursor 2 (X )
S, unresolved mixture of diastereomers) upon treatment with
5 molar equiv of SeO2 in dry acetonitrile.16 The product 9
was detritylated on the support, deprotected, and isolated by
RP-HPLC as described above, yielding 7.5 OD units. Its
identity was confirmed by MALDI-TOF MS (Figure 5S).
For assignment of melting temperatures of complexes of
stereodefined PSe-Oligos with complementary DNA, RNA,
and 2′-OMe-RNA templates, two stereoregular dodecamers
[All-RP]- and [All-SP]-PSe-dA12 (RP- and SP-10, respectively)-
were synthesized, isolated in the amounts of 15 and 19 OD
units, respectively, and analyzed by MALDI-TOF MS
(Figure 6S) and PAGE. Obtained melting data (collected in
Table 2) show interesting stereodependence of melting
Table 2. Melting Temperaturesa for Complexes of
Stereodefined [All-RP]-, [All-SP]-PSe-dA12 and dA12 with
Complementary DNA, RNA, and 2′-OMe-RNA Templates
template
b
b
c
form of dA12
T12
U12
(2′-OMe)U12
[All-Rp]-PSe
[All-Sp]-PSe
PO
22.2
32.5
35.4
29.5
19.1
23.6
58.1d
28.3
34.7
Figure 1. (A) MALDI-TOF MS analysis of 7 obtained from 6
using fast-3b (B′ ) AdeBz), after HPLC (DMT-on) purification.
(B) MALDI-TOF MS analysis of 8a, after HPLC purification.
a Buffer: 10 mM TRIS-Cl pH7.4, 100 mM NaCl, 10 mM MgCl2. dA12
oligonucleotide concentration, 2 µmol.Temperature gradient, 0.2 °C/min.
b PSe- or PO-dA12/template molar ratio, 1:1. c PSe- or PO-dA12/template
molar ratio, 1:2. d Similar unusually high thermal stability (Tm ) 53.3 °C)
was observed for phosphorothioate [All-Rp]-PS-dA12/(2′-OMe)U12; manu-
script in preparation.
The [All-RP]- and [All-SP]-PSe-d(TCTCAG) hexamers
(8a,b) possessing phosphoroselenoate linkages of RP or SP
absolute configuration at each internucleotide position,
respectively, were synthesized on solid support (0.5 µmol
scale, 10 mg of the appropriate monomer 3b per coupling)
using a protocol adapted from the synthesis of phospho-
rothioate analogues of DNA.12 Since model studies showed
that routine detritylation of the DMT-ON isolated oligomer
with 50% acetic acid resulted in massive loss of selenium
temperatures on the type of the template used, while relevant
melting curves have good S-shape indicating high cooper-
ativity of the transition (Figure 7S).
In summary, a novel, efficient, and stereocontrolled
method for the solid-support synthesis of phosphoroselenoate
analogues of oligodeoxyribonucleotides has been developed.
It allows for the synthesis of oligomers with any combination
Org. Lett., Vol. 7, No. 18, 2005
3903

of internucleotide phosphoroselenoate linkages of RP or SP
absolute configuration, as well as unmodified phosphate
bonds, which in general cannot be achieved using enzymatic
methods.
experimental details. 13C NMR and FAB MS characteristics
of monomers 3b. Scheme illustrating the mechanism of
condensation. 31P NMR spectrum of crude 5′-O-DMT-
d(ABzPSeT)-3′-O-Ac. Chromatograms for 6, 7, and 8a,b and
enzymic digestion of d(APseT), MALDI-TOF MS spectrum
for 9 and SP-10, melting curve for the complex of RP-10/
Acknowledgment. Financial support by the State Com-
mittee for Scientific Research (MNiI, Poland, Grant
3T09A07226 to P.G.) is gratefully acknowledged.
1
(2′-OMe)U12, H and 13C NMR spectra for monomers 3b.
This material is available free of charge via the Internet at
http://pubs.acs.org.
Supporting Information Available: Table containing
spectroscopic properties of monomers 3a. Text giving
OL051302E
3904
Org. Lett., Vol. 7, No. 18, 2005