COMMUNICATIONS
[13] Analysis by HPLC was performed with an Adsorbosphere SAX
column (5 m, 4.6 Â 250 mm) with a gradient from 140 mm to 320 mm
potassium phosphate buffer, pH 3.5, over 20 min, followed by a 5-min
wash with 500 mm potassium phosphate buffer, pH 3.5.
[14] Substrate 5 was prepared as described previously: H. Chen, G.
Agnihotri, Z. Guo, N. L. S. Que, X. H. Chen, H.-w. Liu, J. Am. Chem.
Soc. 1999, 121, 8124 ± 8125.
Oxathiaphospholane Approach to the
Synthesis of P-Chiral, Isotopomeric
Deoxy(ribonucleoside phosphorothioate)s and
Phosphates Labeled with an Oxygen Isotope**
Â
Piotr Guga, Krzysztof Domanski, and Wojciech J. Stec*
[15] 6: 1H NMR (500 MHz, 2H2O): d 1.11 (d, 3J(H,H) 6.0 Hz, 3H;
5-Me hydrated form), 1.15 (d, 3J(H,H) 6.0 Hz, 3H; 5-Me keto form),
1.37 (s, 3H; 3-Me hydrated form), 1.45 (s, 3H; 3-Me keto form), 1.82
(s, 3H; 5''-Me), 1.92 (m, 2H; 2-H hydrated form), 2.23 ± 2.32 (m, 2H;
2'-H), 2.40 (m, 2H; 2-H keto form), 4.01 ± 4.10 (m, 3H; 5-H hydrated
form, 4'-H, 5'-H), 4.47 ± 4.53 (m, 1H; 3'-H), 4.72 (q, 3J(H,H) 6.0 Hz,
1H; 5-H keto form), 5.49 (m, 1H; 1-H hydrated form), 5.64 (m, 1H;
Introduced by Eckstein, phosphorothioate analogues of
nucleotides have become an indispensable tool for studying
the metabolism of nucleic acids.[1] Standard chemical methods
for the synthesis of oligo(deoxyribonucleoside phosphoro-
thioate)s (PS-Oligos) provide a mixture of 2n diastereoiso-
mers, where n is the number of phosphorothioate linkages.[2]
The enzymatic synthesis of stereodefined PS-Oligos is limited
to the preparation of (all-RP)-oligomers because of the
stereoselectivity of available DNA and RNA polymerases.
The first method for stereocontrolled chemical synthesis of
PS-Oligos was elaborated in our group,[3] and several alter-
native methods were recently reported.[4, 5] Stereodefined PS-
Oligos were used for studying the mode of action of several
bacterial and human enzymes[6±8] and the stereodependent
avidity of PS-Oligos toward complementary DNA or RNA.[9]
However, the presence of a sulfur atom affects the properties
of internucleotide bonds, mostly due to the different steric
requirements of sulfur atoms (P S vs P O bond length),
different affinity towards metal ions, and changes in the
distribution of the negative charge in the phosphorothioate
anion.[10] Therefore, the hydration pattern of PS-Oligos is
different from that of natural oligonucleotides,[11] and this
obstructs the evaluation of kinetic data of ªrescue effectsº of
thiophilic metal ions, and makes analysis of direct or water-
mediated contacts between metal ions and phosphate groups
much more difficult. These inconveniences could be avoided
by using P-chiral isotopomeric phosphates.[12] Here we
describe the synthesis of stereodefined oligo(deoxyribonu-
cleoside [18O]phosphorothioate)s (PS18O-Oligos) and oligo-
(deoxyribonucleoside [18O]phosphate)s (P18O-Oligos), in
which both of the nonbridging oxygen atoms of the inter-
nucleotide bond were replaced by S and 18O, or one of them
was replaced by 18O, respectively. Oligonucleotides containing
a single P-chiral [16O,18O] internucleotide bond were first used
by Eckstein[13] in studies on Eco RI endonuclease. Stereo-
defined P18O-Oligos can be used to investigate the interaction
of particular oxygen atoms with other molecules or metal ions,
given analytical methods that allow the isotopic effect to be
measured with satisfactory accuracy.[14]
3
1-H keto form), 6.24 (t, J(H,H) 4.2 Hz, 1H; 1'-H), 7.61 (s, 1H; 6''-
H); 13C NMR (75 MHz, 2H2O, hydrated form): d 11.6, 12.0, 23.5,
38.3, 40.9 (d, 3J(C,P) 7.5 Hz; C-2), 65.3 (d, 2J(C,P) 5.6 Hz; C-5'),
68.1, 70.7, 71.8, 84.7, 85.0 (d, 3J(C,P) 7.5 Hz; C-4'), 94.0 (d, 2J(C,P)
5.3 Hz; C-1), 94.3, 111.6, 137.2, 151.6, 166.5. The ratio of the hydrated
form to the keto form is approximately 3:1.
[16] The HPLC assay used for determining the kinetic parameters was
performed on an Adsorbosphere SAX column (5 m, 4.6 Â 250 mm),
which was eluted isocratically with 50 mm potassium phosphate
buffer, pH 3.5. The peak integrations of (S)-adenosylmethionine and
(S)-adenosylhomocysteine were used to determine the product
conversion.
[17] Inductively coupled plasma (ICP) analysis for metal ions indicated the
presence of approximately 0.4 mol of ZnII per mole of TylC3.
However, the zinc does not appear to be important for activity, since
dialysis of the enzyme against 5 mm 1,10-phenanthroline for 4 d did
not reduce the activity, although ICP analysis indicated that approx-
imately half of the zinc was removed. Attempts to reconstitute the
enzyme with zinc also failed to increase the activity. Likewise, the
addition of MgII did not increase the activity of TylC3.
[18] Enediolates are common intermediates in many biotransformations.
For a few examples, see: a) R. V. J. Chari, J. W. Kozarich, J. Am.
Chem. Soc. 1983, 105, 7169 ± 7171; b) A. E. Johnson, M. E. Tanner,
Biochemistry 1998, 37, 5746 ± 5754; c) C. J. Jeffery, B. J. Bohnson, W.
Chien, D. Ringe, G. A. Petsko, Biochemistry 2000, 39, 955 ± 964.
[19] H. H. Bear, H. R. Hanna, Carbohydr. Res. 1982, 110, 19 ± 41.
[20] S. Hanessian, N. R. Plessas, J. Org. Chem. 1969, 34, 1035 ± 1044.
[21] 8: 1H NMR (300 MHz, 2H2O): d 1.10 (d, 3J(H,H) 6.6 Hz, 3H;
5-Me), 1.73 (s, 3H; 5''-Me), 1.86 (m, 1H; 2-Hax), 2.08 (m, 1H; 2-Heq),
2.18 (m, 2H; 2'-H), 3.98 (m, 4H; 3-, 3'- and 5'-H), 4.06 (dq, 3J(H,F)
21.6, 3J(H,H) 6.6 Hz, 1H; 5-H), 4.44 (m, 1H; 4'-H), 5.40 (m, 1H;
1-H), 6.16 (m, 1H; 1'-H), 7.57 (s, 1H; 6''-H); 13C NMR (75 MHz,
2H2O): d 11.6, 12.0, 23.5, 38.3, 40.9 (d, 3J(C,P) 7.5 Hz; C-2), 65.3 (d,
2J(C,P) 5.6 Hz; C-5'), 68.6 (dd, 1J(C,F) 31.7, 1J(C,F) 24.1 Hz;
C-4), 70.9, 84.8, 85.2 (d, 3J(C,P) 9.0 Hz; C-4'), 93.6 (d, 2J(C,P)
4.6 Hz; C-1), 111.6, 137.3, 151.6, 166.5; 19F NMR (282 MHz, 2H2O):
d -125.9 (d, 2J(F,F) 253 Hz), 128.3 (dd, 2J(F,F) 253, 3J(H,F)
21.2 Hz); 31P NMR (121 MHz, 2H2O): d 11.2 (d, 2J(P,P)
20.7 Hz),
12.1 (d, 2J(P,P) 20.7 Hz); HRMS (ESI) calcd for
C16H23F2N2O13P2 [M H] : 551.0649; found: 551.0669.
[22] The difluoromethylene moiety is a strongly electron-withdrawing
group which can stabilize the corresponding b-anion both by induction
and by negative hyperconjugation: B. E. Smart in Chemistry of
Organic Fluorine Compounds II (Eds.: M. Hudlicky, A. E. Pavlath),
American Chemical Society, Washington, 1995, pp. 979 ± 1010. Al-
though the pKa data of protons adjacent to the difluoromethylene
group are not available, examples of a-anion-induced b-fluoride
elimination in similar structures are well known: A. M. Kornilov, I. B.
Kulik, A. E. Sorochinsky, V. P. Kukhar, Tetrahedron Asymmetry 1995,
6, 199 ± 206; D. Schirlin, S. Baltzer, J. M. Altenburger, C. Tarnus, J. M.
Remy, Tetrahedron 1996, 52, 305 ± 318.
To obtain stereodefined PS18O-Oligos, we synthesized 5'-O-
DMT-nucleoside-3'-O-(2-thio-ªspiroº-4,4-pentamethylene-
Â
[*] Prof. Dr. W. J. Stec, Dr. P. Guga, K. Domanski
Centre of Molecular and Macromolecular Studies
Polish Academy of Sciences
Department of Bioorganic Chemistry
Â
Â
Sienkiewicza 112, 90-363 èodz (Poland)
[23] D. A. Johnson, H.-w. Liu in Comprehensive Natural Products Chem-
istry, Vol. 3 (Eds.: D. Barton, K. Nakanishi, O. Metho-Cohn), Elsevier,
Oxford, 1999, pp. 311 ± 366.
Fax : (48)42-6815483
[**] This work was financially supported by the State Committee for
Scientific Research (KBN, Poland, Grant 4P05F00617, to W.J.S.), and,
in part, by the Human Science Promotion Foundation (Japan, to H.
Takaku and W.J.S.).
610
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