Facile synthesis of oligonucleotide phosphoroselenoates

Article abstract of DOI:10.1021/ol702305v

Se-(2-Cyanoethyl)phthalimide was synthesized from di-(2-cyanoethyl) diselenide. This reagent was found to be an efficient selenium transfer reagent in the synthesis of selenophosphates. Thus, nucleotide H-phosphonate diesters that are formed in situ throu

Full text of DOI:10.1021/ol702305v

ORGANIC
LETTERS
2007
Vol. 9, No. 24
5103-5106
Facile Synthesis of Oligonucleotide
Phosphoroselenoates
Kha Tram, Xiaolu Wang, and Hongbin Yan*
Department of Chemistry, Brock UniVersity, St. Catharines, ON, L2S 3A1, Canada
tyan@brocku.ca
Received September 22, 2007
ABSTRACT
Se-(2-Cyanoethyl)phthalimide was synthesized from di-(2-cyanoethyl) diselenide. This reagent was found to be an efficient selenium transfer
reagent in the synthesis of selenophosphates. Thus, nucleotide H-phosphonate diesters that are formed in situ through the H-phosphonate
chemistry undergo quantitative reaction with Se-(2-cyanoethyl)phthalamide. The resulting Se-(2-cyanoethyl) oligonucleotide phosphoroselenoate
triesters are subsequently deprotected to give oligonucleotide phosphoroselenoate diesters in excellent yields.
Selenium derivatization has proved to be a useful approach
in solving the phase problem in X-ray crystallography.1 One
approach of selenium derivatization is to replace a nonbridg-
ing oxygen atom of the internucleotide linkages with
selenium.1e,g,i In the chemical synthesis of oligonucleotide
selenophosphate diesters, a general approach involves the
oxidative transfer of selenium to P(III) centers, such as
phosphite triesters2 and H-phosphonate diesters.3 However,
these transformations are usually not efficient due to the
relatively low reactivity of selenium toward P(III).
A few years ago, Reese and co-workers4 demonstrated the
synthesis of sulfur-transfer reagents and their application in
oligonucleotide synthesis using the modified H-phosphonate
approach.5 Their approach has been successfully used in
oligonucleotide synthesis, both of natural phosphates and
phosphorothioates.6 We therefore embarked on the investiga-
tion of a selenium transfer reagent that is efficient for the
synthesis of oligonucleotide phosphoroselenoates via the
modified H-phosphonate approach.
(1) (a) Sheng, J.; Jiang, J.; Salon, J.; Huang, Z. Org. Lett. 2007, 9, 749.
(b) Carrasco, N.; Ginsburg, D.; Du, Q.; Huang, Z. Nucleosides Nucleotides
Nucleic Acids 2001, 20, 1723. (c) Du, Q.; Carrasco, N.; Teplova, M.; Wilds,
C. J.; Egli, M.; Huang, Z. J. Am. Chem. Soc. 2002, 124, 24. (d) Teplova,
M.; Wilds, C. J.; Wawrzak, Z.; Tereshko, V.; Du, Q.; Carrasco, N.; Huang,
Z.; Egli, M. Biochimie 2002, 84, 849. (e) Wilds, C. J.; Pattanayek, R.; Pan,
C.; Wawrzak, Z.; Egli, M. J. Am. Chem. Soc. 2002, 124, 14910. (f) Buzin,
Y.; Carrasco, N.; Huang, Z. Org. Lett. 2004, 6, 1099. (g) Carrasco, N.;
Huang, Z. J. Am. Chem. Soc. 2004, 126, 448. (h) Carrasco, N.; Buzin, Y.;
Tyson, E.; Halpert, E.; Huang, Z. Nucleic Acids Res. 2004, 32, 1638. (i)
Carrasco, N.; Williams, J. C.; Brandt, G.; Wang, S.; Huang, Z. Angew.
Chem., Int. Ed. 2006, 45, 94.
(2) (a) Bollmark, M.; Stawinski, J. Chem. Commun. 2001, 771. (b)
Koziolkiewicz, M.; Uznanski, B.; Stec, W. J.; Zon, G. Chem. Scr. 1986,
26, 251. (c) Mori, K.; Boiziau, C.; Cazenave, C.; Matsukura, M.; Subasinghe,
C.; Cohen, J. S.; Border, S.; Toulme, J. J.; Stein, C. A. Nucleic Acids Res.
1989, 17, 8207. (d) Stec, w. J.; Zon, G.; Egan, W.; Stec, B. J. Am. Chem.
Soc. 1984, 106, 6077. (e) Baraniak, J.; Korczynski, D.; Kaczmarek, R.;
Stec, W. J. Nucleosides Nucleotides 1999, 18, 2147.
We first tested the reaction between Se-(2-cyanoethyl)-
diselenide 1, which is readily prepared using the literature
procedure,7 with diethylphosphite in pyridine (Scheme 1).
Consumption of diethylphosphite was followed by 31P NMR.
After the solution was stirred overnight at room temperature,
(3) (a) Lindh, I.; Stawinski, J. J. Org. Chem. 1989, 54, 1338. (b)
Stawinski, J.; Thelin, M. J. Org. Chem. 1994, 59, 130.
(4) Klose, J.; Reese, C. B.; Song, Q. L. Tetrahedron 1997, 53, 14411.
(5) Reese, C. B.; Song, Q. L. Bioorg., Med. Chem. Lett. 1997, 7, 2787.
(6) (a) Reese, C. B.; Song, Q. L. J. Chem. Soc., Perkin Trans. 1 1999,
1477. (b) Reese, C. B.; Yan, H. J. Chem. Soc., Perkin Trans. 1 2002, 2619.
(7) Logon, G.; Igunbor, C.; Chen, G.; Davis, H.; Simon, A.; Salon, J.;
Huang, Z. Synlett 2006, 1554.
10.1021/ol702305v CCC: $37.00
© 2007 American Chemical Society
Published on Web 11/01/2007

Scheme 1. Reaction of Diethylphosphite with
Se-(2-Cyanoethyl)diselenide 1
Scheme 3. Synthesis of Se-(2-Cyanoethyl) Phosphoroselenoate
Triester
Se-(2-cyanoethyl)diethylphosphoselenoate triester 2 was
isolated by column chromatography. Treatment of this triester
2 with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) gave di-
ethylphosphoroselenoate diester 3 as a single product
(Scheme 1).
Although selenium transfer took place with Se-(2-cyano-
ethyl)diselenide 1, it is not efficient enough to be a useful
reagent in this regard. Therefore a more efficient selenium
transfer reagent was sought. In the past, N-phenylselenoph-
thalimide and N-phenylselenosuccinimide have been suc-
cessfully synthesized.8 However, synthesis of their Se-alkyl
analogues has been challenging due to the instability of these
compounds. With modification of the literature method,9 we
were able to synthesize Se-(2-cyanoethyl)phthalimide 4 from
Se-(2-cyanoethyl)diselenide 1 (Scheme 2). Thus, 2-cyano-
phosphonate 5 with 3′-O-phenoxyacetylthymidine 6 was
effected by pivaloyl chloride to form dinucleotide H-
phosphonate diester 7 (7.99 and 9.56 ppm, panel b, Figure
1), which reacted in situ with Se-(2-cyanoethyl)phthalimide
Scheme 2. Preparation of Se-Cyanoethylphthalimide 4
ethyl selenyl chloride, which was generated by treating di-
(2-cyanoethyl) diselenide 1 with sulfuryl chloride prior to
use, was allowed to react with potassium phthalimide to give
Se-(2-cyanoethyl)phthalimide 4 as a colorless solid in 65%
yield. This material appeared to be very sensitive to oxygen
and moisture. However, when stored at -10 °C in an
atmosphere of inert gas, such as nitrogen, it is stable over 1
month. It is noted that when preparing NMR solutions of
this compound, the procedure should be carried out in a glove
box, using deuterated chloroform dried and deoxygenated
by conventional procedures.
We then tested the ability of Se-(2-cyanoethyl)phthalimide
4 to react with H-phosphonate diesters 7 in the modified
H-phosphonate approach (Scheme 3). The coupling reaction
and selenium transfer reaction were followed by 31P NMR
(Figure 1). Thus, coupling of 5′-O-DMTr-thymidine H-
Figure 1. 31P NMR spectra of the reaction described in Scheme
3: (a) H-phosphonate 5; (b) 5 and 6 in the presence of pivaloyl
chloride (step a); and (c) after addition of selenium transfer reagent
1 (step b).
4 to form Se-(2-cyanoethyl)phosphoroselenoate triester 8
(19.71 and 20.74 ppm, panel c, Figure 1). The reaction was
found to be complete in less than 2 min. On a preparative
scale (1 mmol), Se-(2-cyanoethyl)phosphoroselenoate triester
(8) Nicolaou, K. C.; Claremon, D. A.; Barnette, W. E.; Seitz, S. P. J.
Am. Chem. Soc. 1979, 101, 3704.
(9) Kirby, G. W.; Trethewey, A. N. J. Chem. Soc., Chem. Commun. 1986,
1152.
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Org. Lett., Vol. 9, No. 24, 2007

8 was isolated in excellent yields (95%). The DMTr-
protecting group was readily removed by treatment with
dichloroacetic acid in the presence of pyrrole. These phos-
phoroselenoate triesters are stable compounds when stored
at 4 °C.
The Se-(2-cyanoethyl)phosphoroselenoate triester 9 was
characterized by NMR spectroscopy. The 31P NMR spectrum
indicated a mixture of two diastereoisomers, split by coupling
with adjacent selenium with a coupling constant of 490 Hz
Figure 3. C18 HPLC profile of triester 9 (see the Supporting
Information for the eluting program).
Figure 2. 31P NMR spectrum of Se-(2-cyanoethyl) phosphorose-
lenoate triester 9.
loss of selenium was observed. When ethyl acetate, which
is a moderate solvent for oxygen, was used instead, around
23% loss of selenium was recorded. However, when 1%
(Figure 2). The purity of the triester 9 is shown by the
reverse-phase HPLC profile (Figure 3).
Removal of the Se-cyanoethyl group from the Se-(2-
cyanoethyl)phosphoroselenoate triester 9 was effected with
DBU in the presence of trimethylchlorosilane (Scheme 4).
The phenoxyacetyl protecting group was completely removed
by incubation with concentrated aqueous ammonium hy-
droxide for 5 min at room temperature.10
Scheme 4. Unblocking of Dinucleotide Se-(2-Cyanoethyl)
Phosphoroselenoate Triester 9
After precipitation from diethyl ether containing 1%
mercaptoethanol, phosphoroselenoate diester 10 was obtained
as the only product as is indicated by the 31P NMR spectrum
in Figure 4. Coupling constants of 777 Hz were observed.
These phosphoroselenoate diesters, when dissolved in
solution, showed extreme sensitivity toward molecular
oxygen. Attempts to precipitate the product from organic
solvents was generally accompanied by loss of selenium,
which is indicated by the appearance of a new signal at
around 0 ppm. The extent of loss of selenium seemed to be
related to the capacity of the organic solvent to dissolve
molecular oxygen. Thus, when diethyl ether, which is one
of the best organic solvents for dissolving molecular
oxygen,11 was used to precipitate the product, up to 50%
mercaptoethanol was added to diethyl ether, loss of selenium
was completely suppressed, presumably because mercapto-
ethanol functioned as an oxygen scavenger.
The choice of protecting group for the hydroxyl of the
sugar ring and exocyclic amino group of base residues
seemed to be critical. Thus, when a levulinyl group was used
to protect the 3′-hydroxyl of thymidine, a concomitant loss
of 5% of selenium was observed during incubation with
aqueous ammonium hydroxide over 45 min. However,
because phenoxyacetyl can be completely removed in 5 min
(10) Reese, C. B.; Stewart, J. C. M. Tetrahedron Lett. 1968, 4273.
(11) Che, Y.; Tokuchi, K.; Ohsaka, T. Bull. Chem. Soc. Jpn. 1998, 71,
651.
Org. Lett., Vol. 9, No. 24, 2007
5105

In summary, we have demonstrated the synthesis of Se-
(2-cyanoethyl)phthalimide and its use in the synthesis of
phosphoroselenoate tri- and diesters. The purity of these
dinucleotide phosphoroselenoates 10 and 12 is illustrated by
the reverse-phase HPLC profiles as shown in Figure 5. This
Figure 4. 31P NMR spectrum of phosphoroselenoate diester 10.
Figure 5. C18 HPLC profiles on 10 (left) and 12 (right). See the
Supporting Information for the eluting program.
under the same conditions,10 exposure of the protected
substrate to oxygen is minimized so that loss of selenium
can be suppressed. This protecting group strategy was
extended to the synthesis of Tp(Se)C dimer phosphorosele-
noate diester 12 (Scheme 5) successfully.
approach may prove useful in the future preparation of
oligonucleotide phosphoroselenoates.
Acknowledgment. This research was supported by
awards from Research Corporation, Canada Foundation for
Innovation, and the Natural Science and Engineering Re-
search Council of Canada. The authors thank Tim Jones and
Razvan Simionescu for mass spectrometric and NMR
analysis, respectively.
Scheme 5. Unblocking of Tp(Se)C Dimer Phosphoroselenoate
11
Supporting Information Available: Experimental pro-
cedures and spectroscopic data for new compounds. This
material is available free of charge via the Internet at
http://pubs.acs.org.
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