Independent generation of C5′-nucleosidyl radicals in thymidine and 2′-deoxyguanosine

Article abstract of DOI:10.1021/jo062518c

(Chemical Equation Presented) The synthesis of the C5′ tert-butyl ketone of thymidine 1a and 2′-deoxyguanosine 2 is achieved by reaction of 5′-C-cyano derivatives with tert-butyl lithium followed by acid hydrolysis. The 5′R configuration is assigned by X-ray crystal structure determination of an opportunely protected derivative of 1a. The (5′S)-isomers of both nucleosides are not stable, and a complete decomposition occurs in the reaction medium. The photochemistry of 1a and 2 effectively produced the thymidin-5′-yl radical and the 2′-deoxyguanosin-5′-yl radical, respectively. In the thymidine system, the C5′ radical is fully quenched in the presence of a physiological concentration of thiols. In the 2′-deoxyguanosine system, the C5′ radical undergoes intramolecular attack onto the C8-N7 double bond of guanine leading ultimately to the 5′,8-cyclo-2′-deoxyguanosine derivative. The cyclization of the 2′-deoxyguanosin-5′-yl radical occurs with a rate constant of ca. 1 × 10⁶ s^-1 and is highly stereoselective affording only the (5′S)-diastereomer.

Full text of DOI:10.1021/jo062518c

Independent Generation of C5′-Nucleosidyl Radicals in Thymidine
and 2′-Deoxyguanosine
Antonio Manetto,†,‡ Dimitris Georganakis, Leondios Leondiadis, Thanasis Gimisis,*
§
§,|
,§
‡
‡
,†
Peter Mayer, Thomas Carell, and Chryssostomos Chatgilialoglu*
ISOF, Consiglio Nazionale delle Ricerche Via P. Gobetti 101, 40129 Bologna, Italy, Department of
Chemistry and Biochemistry, Ludwig-Maximilians UniVersity Munich, 81377 Munich, Germany, MS and
Dioxin Analysis Laboratory, IRRP, National Centre for Scientific Research ‘Demokritos’, Athens 153 10,
Greece, and Department of Chemistry, UniVersity of Athens, Panepistimiopolis, 15771 Athens, Greece
gimisis@chem.uoa.gr; chrys@isof.cnr.it
ReceiVed December 8, 2006
The synthesis of the C5′ tert-butyl ketone of thymidine 1a and 2′-deoxyguanosine 2 is achieved by
reaction of 5′-C-cyano derivatives with tert-butyl lithium followed by acid hydrolysis. The 5′R configuration
is assigned by X-ray crystal structure determination of an opportunely protected derivative of 1a. The
(5′S)-isomers of both nucleosides are not stable, and a complete decomposition occurs in the reaction
medium. The photochemistry of 1a and 2 effectively produced the thymidin-5′-yl radical and the 2′-
deoxyguanosin-5′-yl radical, respectively. In the thymidine system, the C5′ radical is fully quenched in
the presence of a physiological concentration of thiols. In the 2′-deoxyguanosine system, the C5′ radical
undergoes intramolecular attack onto the C8-N7 double bond of guanine leading ultimately to the 5′,8-
cyclo-2′-deoxyguanosine derivative. The cyclization of the 2′-deoxyguanosin-5′-yl radical occurs with a
6
-1
rate constant of ca. 1 × 10 s and is highly stereoselective affording only the (5′S)-diastereomer.
4
-6
Introduction
radiations
or treated chemically by highly oxidizing radical
7
species. They have also been identified in mammalian cellular
DNA in vivo, where their level is enhanced by conditions of
oxidative stress. The difficulty of repair and the amenability
to mutation render these lesions biologically significant and the
study of their formation necessary.
tions, the fate of the C5′ radical is not well understood. All the
Hydrogen abstraction from the 2-deoxyribose moiety of DNA
produces carbon-centered radicals whose fate depends upon the
8
,9
1
environment. The accessibility of the C-H bond in sugar
1
0-12
moieties determines the preferential site of attack even by the
highly reactive HO radicals. In B-DNA, the H5′ and H5′′ are
Under aerobic condi-
•
the most exposed ones, and therefore, the preference for
abstraction of these hydrogens is higher.2,3 Hydrogen abstraction
(4) Dirksen, M. L.; Blakely, W. F.; Holwitt, E.; Dizdaroglu, M. Int. J.
Radiat. Biol. 1988, 54, 195-204.
from the 5′-position of purine nucleosides can lead to 5′,8-
cyclonucleosides. They have been observed among the decom-
position products of DNA, when it is exposed to ionizing
(
5) Dizdaroglu, M.; Jaruga, P.; Rodriguez, H. Free Radical Biol. Med.
2001, 30, 774-784.
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istry 2002, 41, 3703-3711.
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(
(
†
‡
§
|
daroglu, M.; Gates, K. S. J. Am. Chem. Soc. 2003, 125, 11607-11615.
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P. J. J. Biol. Chem. 2001, 276, 36051-36057.
Consiglio Nazionale delle Ricerche.
Ludwig-Maximilians University Munich.
National & Kapodistrian University of Athens.
National Centre for Scientific Research ‘Demokritos’.
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(
Springer-Verlag: Berlin, 2006; pp 366-371.
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1
0.1021/jo062518c CCC: $37.00 © 2007 American Chemical Society
Published on Web 04/11/2007
J. Org. Chem. 2007, 72, 3659-3666
3659

Manetto et al.
SCHEME 1. Synthesis of the Ketones 1a and 1ba
FIGURE 1. Photolabile precursors of C5′ radicals.
a
Conditions: (i) TBDMS-CN, LiOEt, THF, 0 °C, 30 min, 82%; (ii)
tBuLi, THF, -78 °C, 2-5 min, 43% (5a), 38% (5b); (iii) for 1a, THF/
H2O/2 N HCl (40:20:1), 3 h, 88%; (iv) for 1b, CDCl3, 30 min, 70%. R )
tert-butyldimethylsilyl (TBDMS). T ) thymine.
proposed intermediates are based on rationalization of the prod-
ucts observed in DNA degradation by the neocarzinostatin
chromophore (NCS-chromophore) in the presence of gluta-
thione,1,13,14
containing a light-dependent flavin electron injector in the loop
region of the hairpin are found to capture electrons with
quantitative formation of the corresponding debrominated
oligonucleotides, similar to the analogous 8-bromo-2′-deox-
although related studies with metalloporphyrins
have also been reported.2
,15
Moreover, strand breakage was
observed to be base-selective, preferentially occurring (75%)
on the thymidine unit.
2
6
The chemistry of carbon-centered radicals resulting from
hydrogen atom abstraction from the sugar moieties has been
the subject of many recent studies. Selective generation of these
species is mainly obtained by photoreactive precursors using
nucleosides or oligonucleotides (ODNs). Indeed, generation of
a single radical species on duplex ODNs provides a powerful
tool for elucidating the role of reactive intermediates in the
formation of nucleic acid lesions. For example, C1′ and C4′
yguanosine derivatives.
In the present paper, we report the synthesis of 5′-keto
derivatives as photolabile precursors for selective generation
of the 5′-nucleosidyl radical. In particular, compounds 1a and
2 having thymine or guanine as the base are chosen to evaluate
the occurrence of a C5′ radical attack on the pyrimidine or purine
moieties (Figure 1).
positions have been studied in detail by photolysis of the
Results and Discussion
corresponding tert-butyl ketones.1
6-20
Photolabile precursors of
Synthesis of Thymidine 5′-tert-Butyl Ketone 1a. The
synthesis of 1a is shown in Scheme 1. Specifically, the aldehyde
C5′ radicals are missing.
It was recently found that 8-bromo-2′-deoxyadenosine cap-
tures electrons and rapidly loses a bromide ion to give the
corresponding C8 radical. This intermediate abstracts intramo-
lecularly a hydrogen atom from the C5′ position affording
selectively the 2′-deoxyadenosin-5′-yl radical. This allowed us
for the first time to verify that the C5′ radical attacks intramo-
lecularly the double bond of the base moiety, and this occurs
2
7-30
3
was converted in the two 5′-isomers of 4 with a yield of
2%. The crude product consisted of a 3:2 mixture of two
8
isomers, 4a and 4b. After separation by silica gel chromatog-
raphy, the two diastereomers were obtained as pure white foams
and were fully characterized, although from the data it was not
possible to assign the configuration at the 5′ position, even by
2
D NMR experiments. During the R-cyanohydrin formation,
5
-1
with a rate constant of kc )1.6 × 10 s to form, after oxidation,
under not perfectly anhydrous conditions or in the presence of
an excess of LiOEt, the 5′-hydroxyl position resulted unpro-
tected. In this case, an additional step for the in situ 5′-TBDMS
protection was required by classical methods (TBDMS-Cl,
AgNO3, imidazole).
2
1-23
a 5′,8-cyclo-2′-deoxyadenosine as the final product. The
analogous sequence of 8-bromo-2′-deoxyguanosine does not
operate because the electron adduct undergoes fast protonation
-
at C8 ejecting Br and affording the one-electron oxidized 2′-
deoxyguanosine transient species.2 Interestingly, the 8-bromo-
4,25
With a mixture of 4a and 4b, the addition of tert-butyl lithium
2
′-deoxyadenosine moieties in a series of DNA hairpins
(tBuLi) was carried out at -78 °C, leading, after quenching
the reaction with acidic cold water, to a single product in 55%
yield, identified as the tert-butyl ketone 1a. Its configuration
(5′R) was later assigned by X-ray crystal structure determination
of the corresponding 3′-acetate (vide infra). The product yield
together with the absence of the (5′S)-isomer 1b indicated that
the addition reaction products exhibit different stability when
originating from either diastereomer 4a or 4b. To further
investigate their reactivity, each isomer was used for the addition
(
11) Brooks, P. J.; Wise, D. S.; Berry, D. A.; Kosmoski, J. V.; Smerdon,
M. J.; Somers, R. L.; Mackie, H.; Spoonde, A. Y.; Ackerman, E. J.;
Coleman, K.; Tarone, R. E.; Robbins, J. H. J. Biol. Chem. 2000, 275,
2
2355-22362.
12) Kuraoka, I.; Robins, P.; Masutani, C.; Hanaoka, F.; Gasparutto, D.;
Cadet, J.; Wood, R. D.; Lindahl, T. J. Biol. Chem. 2001, 276, 49283-
9288.
(
4
(
(
(
13) Goldberg, I. H. Acc. Chem. Res. 1991, 24, 191-198.
14) Chatgilialoglu, C.; O’Neill, P. Exp. Gerontol. 2001, 36, 1459-1471.
15) Pratviel, G.; Pitie, M.; Bernadou, J.; Meunier, B. Angew. Chem.,
Int. Ed. Engl. 1991, 30, 702-704.
(
16) Chatgilialoglu, C.; Gimisis, T. Chem. Commun. 1998, 1249-1250.
(24) Chatgilialoglu, C.; Caminal, C.; Guerra, M.; Mulazzani, Q. G.
Angew. Chem., Int. Ed. 2005, 44, 6030-6032.
(
17) Emanuel, C. J.; Newcomb, M.; Ferreri, C.; Chatgilialoglu, C.
J. Am. Chem. Soc. 1999, 121, 2927-2928.
18) Chatgilialoglu, C.; Ferreri, C.; Bazzanini, R.; Guerra, M.; Choi, S.
Y.; Emanuel, C. J.; Horner, J. H.; Newcomb, M. J. Am. Chem. Soc. 2000,
(25) Chatgilialoglu, C.; Caminal, C.; Altieri, A.; Vougioukalakis, G. C.;
Mulazzani, Q. G.; Gimisis, T.; Guerra, M. J. Am. Chem. Soc. 2006, 128,
13796-13805.
(
1
5
1
2
22, 9525-9533.
19) Tronche, C.; Goodman, B. K.; Greenberg, M. M. Chem. Biol. 1998,
, 263-271.
20) Meggers, E.; Michel-Beyerle, M. E.; Giese, B. J. Am. Chem. Soc.
998, 120, 12950-12955.
21) Chatgilialoglu, C.; Guerra, M.; Mulazzani, Q. G. J. Am. Chem. Soc.
003, 125, 3839-3848.
22) Flyunt, R.; Bazzanini, R.; Chatgilialoglu, C.; Mulazzani, Q. G.
J. Am. Chem. Soc. 2000, 122, 4225-4226.
23) Jimenez, L. B.; Encinas, S.; Miranda, M. A.; Navacchia, M. L.;
Chatgilialoglu, C. Photochem. Photobiol. Sci. 2004, 3, 1042-1046.
(26) Manetto, A.; Breeger, S.; Chatgilialoglu, C.; Carell, T. Angew.
Chem., Int. Ed. 2006, 45, 318-321.
(
(27) Montevecchi, P. C.; Manetto, A.; Navacchia, M. L.; Chatgilialoglu,
C. Tetrahedron 2004, 60, 4303-4308.
(
(28) Chen, M. Y.; Patkar, L. N.; Lu, K. C.; Lee, A. S. Y.; Lin, C. C.
Tetrahedron 2004, 60, 11465-11475.
(
(29) Tronchet, J. M. J.; Kovacs, I.; Dilda, P.; Seman, M.; Andrei, G.;
Snoeck, R.; De Clereq, E.; Balzarini, J. Nucleosides Nucleotides Nucleic
Acids 2001, 20, 1927-1939.
(
(
(30) Angeloff, A.; Dubey, I.; Pratviel, G.; Bernadou, J.; Meunier, B.
Chem. Res. Toxicol. 2001, 14, 1413-1420.
3660 J. Org. Chem., Vol. 72, No. 10, 2007

Thymidine and 2′-Deoxyguanosine C5′ Radicals
SCHEME 2. aDeprotection-Protection Strategy for the
Synthesis of 8
FIGURE 2. Configuration of the major cyanohydrin 4a and imine
a. R ) TBDMS. T ) thymine.
5
a
Conditions: (i) TBAF, MeOH, reflux, 5 h, 60%; (ii) TBAF, THF,
SCHEME 4. Synthesis of 5′-tert-Butyl Ketone 2aa
-
15 °C, 24 h, 55%; (iii) Ac2O, Py, rt, 24 h, 82%. R ) TBDMS. T )
thymine.
SCHEME 3. Generation of the C5′ Radical from Photolysis
of 1a and 6a
a
Conditions: (i) EDC, pyridine, CF3CO2H, toluene/DMSO (2:1), 95%;
(ii) TMSCN, ZnI2, CH2Cl2, 5 h, 88%; (iii) TBDMSCl, imidazole, DMF,
overnight, 25%; (iv) tBuLi, THF, -78 °C, 5 min, 32%; (v) CH3CO2H/
THF/H2O (3:1:1), 1 h, 78%.
a
R′SH ) BuSH for 1a or GSH for 6; R ) TBDMS for 10. R ) H for
1
1. T ) thymine.
priority change of the substituents as a consequence of the
Cahn-Ingold-Prelog priority rules (CIP rules) (Figure 2).
Photochemistry in the Thymidine System. Compounds 1a
and 6 were used as precursors of the C5′ radical. UV irradiation
under identical conditions and a careful monitoring was carried
out by TLC, together with H NMR analysis of the final
1
products. This led to the identification of two intermediate imine
products 5a and 5b (Scheme 1), fully characterizable, using
acetone-d6 as the NMR acid-free solvent, from the crude
products, after water quenching and workup of the crude tBuLi
addition. The imine 5b readily decomposed under slightly acidic
aqueous conditions or in weakly acidic solvents such as CDCl3,
when used as the NMR solvent. The corresponding ketone (1b)
cannot be isolated, as it also slowly decomposes to unidentified
fragments. A complete decomposition of the mixture containing
(1000 W Xe-lamp, 320 nm cut-off filter) of 1a in the presence
of a hydrogen donor such as 2.5 mM 1-butanethiol in MeCN
and of 6 in the presence of 2.5 mM gluthathione (GSH) in water,
under deaerated conditions, led quantitatively to the formation
of thymidine 10 or 11, respectively (Scheme 3). Figure 3 shows
the analysis by reversed-phase HPLC of aliquots taken during
the photolysis of 1a (0.12 mM in MeCN) in the presence of
1-butanethiol (2.5 mM) for 30 min. Under these conditions, the
5
b and 1b also occurred in acidic solution (THF/H2O/2 N HCl,
half-life of 1a was calculated to be t1/2 ) 6.6 min and no
cyclization product was observed. The resulting C5′ radical 9
could be obtained either by Norrish type I photocleavage or by
initial formation of an acyl radical that decarbonylates with a
40:20:1). On the other hand, acidic hydrolysis conditions applied
to the imine 5a led to its slow but quantitative conversion to
the stable ketone 1a.
The full deprotection of bissilylether 1a led to the ketone 6
as described in Scheme 2. Under specific conditions, selective
deprotection of the ketone 1a in the 3′ position was also possible,
providing the ketone 7.31 This compound was then protected to
the 3′-acetate 8, and single crystals, suitable for X-ray crystal-
lography, were obtained from a saturated solution in ethyl
acetate. One of the two independent molecules (8) present in
the crystal asymmetric unit is reported in the Supporting
Information. Both of the molecules are in the 5′R configuration,
and in both, the furanose rings are C2′endo puckered, as evidenced
by the Cremer and Pople puckering parameters (φ ≈ 70° for
5
6
-1 34
rate constant in the range of 10 -10 s . A physiological
concentration of thiol (e.g., 2.5 mM of GSH) is able to trap the
C5′ radical preventing subsequent reactions, such as the in-
35
tramolecular attack onto the C6-C5 double bond of thymine.
Synthesis of 2′-Deoxyguanosine 5′-tert-Butyl Ketone 2. The
synthesis of 2 is shown in Scheme 4. The intermediate
36,37
5′-aldehyde
was prepared by applying a modification of the
33
38
Moffatt oxidation procedure on the starting alcohol 12. It
appeared as a complicated mixture of three possible diastere-
omeric hemiacetals that form by hydration of the 5′-aldehyde,
when in contact with an aqueous solution. When the above
mixture of diastereomeric hemiacetals was allowed to react, in
3
2
both molecules). The C2′endo puckered form, predominant in
solution,33 is also maintained in the solid state of these
molecules. Since the 5′-stereocenter was not involved in the
deprotection-protection steps, we could extrapolate the assign-
ment of the same 5′R configuration to the ketone 1a and a 5′S
configuration to the imine 5a and cyanohydrin 4a, due to a
39,40
the presence of TMSCN and ZnI2 in dry dichloromethane
containing 4 Å molecular sieves, it was converted into a pair
(
34) Chatgilialoglu, C.; Crich, D.; Komatsu, M.; Ryu, I. Chem. ReV. 1999,
99, 1991-2069.
35) Navacchia, M. L.; Manetto, A.; Montevecchi, P. C.; Chatgilialoglu,
(
C. Eur. J. Org. Chem. 2005, 4640-4648.
(
31) The availability of 7 offers a pathway for accessing the correspond-
(36) Kers, A.; Szabo, T.; Stawinski, J. J. Chem. Soc., Perkin Trans. 1
1999, 2585-2590.
ing phosphoramidites, required for modified oligonucleotide synthesis.
Nevertheless, it is apparent that the secondary hydroxyl at the 5′-position
will require modified conditions for protection, deprotection, and reactivity.
Similar problems have been tackled by other researchers, and alternatives
to the DMT group, e.g., levulinic acid, have been reported (see: Romieu,
A.; Gasparutto, D.; Cadet, J. J. Chem. Soc., Perkin Trans. 1 1999, 1257-
(37) Brown, P.; Richardson, C. M.; Mensah, L. M.; O’Hanlon, P. J.;
Osborne, N. F.; Pope, A. J.; Walker, G. Bioorg. Med. Chem. 1999, 7, 2473-
2485.
(38) Flockerzi, D.; Schlosser, W.; Pfleiderer, W. HelV. Chim. Acta 1983,
66, 2069-2085.
1
263).
(39) Evans, D. A.; Carroll, G. L.; Truesdale, L. K. J. Org. Chem. 1974,
39, 914-917.
(
32) Cremer, D.; Pople, J. A. J. Am. Chem. Soc. 1975, 97, 1354-1358.
(33) Jardetzky, C. D. J. Am. Chem. Soc. 1962, 84, 62-66.
(40) Hon, Y. S.; Yan, J. L. Tetrahedron 1998, 54, 8525-8542.
J. Org. Chem, Vol. 72, No. 10, 2007 3661

Manetto et al.
SCHEME 5. Products and Proposed Mechanism of
a
Formation in the Photolysis of tert-Butyl Ketone 2
FIGURE 3. Time-dependent HPLC chromatogram of 1a’s conversion
to 10 during photolysis. Normalized peaks.
a
R ) TBDMS.
of diastereomers. No products were observed when KCN and
2
starting material together with ca. 20% of N -isobutyroylguanine
present in the product mixture. To optimize the reaction
conditions, we attempted several variations but we were never
able to obtain better yields. For example, addition of CuI,44 to
mediate the strength of the organometallic reagent, led to
prolonged reactions and diminished yields. Once more, the imine
1
8-C-6 were used in the presence of TBDMSCl for the trapping
41
of the cyanohydrin produced. The diastereomeric mixture of
cyanohydrins proved unstable and reverted back into the
aldehyde when column chromatography purification or storage
was attempted. For this reason, the crude product was im-
mediately protected with TBDMSCl and was then further
purified. A chromatographically inseparable mixture of protected
R-cyanohydrins was isolated from the column in 52% yield
1
4 exhibited remarkable stability and could be purified through
column chromatography without any major loss of material. A
characteristic absorption at 186 ppm in the C NMR spectrum
was assigned to the CdNH carbon. Nevertheless, a slow
transformation was observed during C NMR in CDCl3,
indicating an acid-catalyzed hydrolysis. Mild aqueous acid
treatment (CH3CO2H/THF/H2O ) 4:1:1) of 14 led to a good
yielding (78%) conversion to 2 (Scheme 4). The new product
was fully characterized, and HRMS was in agreement with the
proposed transformation. Attempts to deprotect both TBDMS
groups of 2 were not successful. For example, NH4F/MeOH or
TBAF‚SiO2 led to decomposition with glycosidic bond scission
1
3
(Scheme 4). When this mixture was treated with diethyl ether,
a single diastereomer 13 precipitated in crystalline form in 25%
yield. The filtrate containing the other diastereomer together
with other impurities could not be further purified. A third
nucleosidic product was present, isolable in yields up to 30%.
After considerable experimentation, the product was character-
ized as a 5′-O-tert-butyldimethylsilylimidazolyl aminal arising
from the reaction of unreacted aldehyde under the TBDMSCl
protection conditions. The chemistry and reactivity of this
1
3
4
2
interesting intermediate have been reported elsewhere.
4
5
and formation of the aglycon.
Although NOESY experiments did not provide any useful
contacts for assignment purposes also in the case of 13, as in
the thymidine series, the (5′S) stereochemistry was assigned to
the crystalline protected cyanohydrin isolated. Evidence came
Photochemistry in the 2′-Deoxyguanosine System. A
variable-intensity 1000 W Xe lamp equipped with a UV-A filter
was utilized in the photolysis studies. Photolysis of a THF
solution of 2, in a pyrex vial, in the presence of excess tert-
1
from the anisotropy effect observed for the 2′â-H in the H NMR
2
spectrum, due to the nearby situated multiple bond of the nitrile.
In this geometry, this proton experiences the negative side of
butyl thiol, under deaerated conditions, at 110 mW/cm local
intensity, led to the complete consumption of the starting
material, within 60 min, at analytical conditions (0.5 mL, 13
mmol) and partial conversion at higher concentration (2 mL,
27 mmol). PLC purification of the reaction mixture produced
the reduced protected 2′-deoxyguanosine 15 and a new product
that was characterized, by comparison with literature data,4
as the protected (5′S)-5′,8-cyclo-2′-deoxyguanosine 16. The
stereochemistry of the 5′-carbon was inferred from the J5′,4′ )
the nitrile anisotropy cone and, as observed, is shifted by 0.72
ppm to a lower field than the 2′R-H.43 Further evidence
supporting our assignment came from the observed reactivity
of the isolated diastereomer (vide infra), which associates it with
the corresponding thymidine diastereomer (vide supra).
In the key step of the synthesis, fast tBuLi addition to 13, in
THF at low temperature, provided after aqueous workup the
imine 14 in 32% yield (Scheme 4). The reaction was never left
to completion as prolonged reaction times increased decomposi-
tion and base release. After a 5 min reaction, and the above-
reported yields of products, there was ca. 40% of unreacted
6,47
1
46
6.2 Hz value, in the H NMR spectrum, as has been described.
No other diastereomeric cyclic product could be detected in the
reaction mixture.
(
44) Hwang, J. T.; Greenberg, M. M. J. Am. Chem. Soc. 1999, 121,
(
41) Rawal, V. H.; Rao, J. A.; Cava, M. P. Tetrahedron Lett. 1985, 26,
275-4278.
42) Gimisis, T.; Arsenyan, P.; Georganakis, D.; Leondiadis, L. Synlett
003, 1451-1454.
43) Although the diastereomer of 13 could not be isolated in the deoxy
4311-4315.
4
(45) In the presence of BF3.Et2O, only the 3′-TBDMS group was
chemoselectively removed, thus offering a means of accessing the corre-
sponding phosphoramidite.31
(
2
(
(46) Romieu, A.; Gasparutto, D.; Cadet, J. Chem. Res. Toxicol. 1999,
12, 412-421.
(47) Navacchia, M. L.; Chatgilialoglu, C.; Montevecchi, P. C. J. Org.
Chem. 2006, 71, 4445-4452.
series, we were able to isolate both diastereomers in the ribo series (results
not presented), whereas an anisotropy effect was observed in only one of
the two diastereomers.
3662 J. Org. Chem., Vol. 72, No. 10, 2007

Thymidine and 2′-Deoxyguanosine C5′ Radicals
In the proposed mechanism for the above observed reaction,
photolysis of the tert-butyl ketone 2 generates the 2′-deoxygua-
nosin-5′-yl radical 17, which is partitioned between two
competitive reaction channels, i.e., a bimolecular reduction
leading to 2′-deoxyguanosine 15 and an intramolecular cycliza-
tion onto the 8-position of the purine base leading to radical 18
in relation to restricted conformations of duplex DNA. The
photostability of the imines (5a or 14) and their ability to
hydrolyze in acidic media to photolabile ketones also make these
transformations suitable for biotechnological applications.5
Toward this goal, these systems are currently under investiga-
tion.
7-60
(Scheme 5). The high stereoselectivity of the cyclization reaction
can be explained by the steric hindrance between the 5′-O-
Experimental Section
TBDMS substituent and the guanine moiety in analogy with
the adenine system.2
1,35,47
The cyclization occurs via a chair
(5′R)- and (5′S)-5′-Cyano-3′,5′-di-O-(tert-butyldimethylsilyl)-
thymidine (4a,b). A solution of aldehyde 32
7-30
(600 mg, 1.70
transition state leading to (5′S,8R)-18 followed by rearomati-
zation of the guanine moiety by reaction with the thiyl radical.
Under the conditions in which the photolysis was performed
mmol) in dry THF (6.0 mL) was added slowly to a mixture of
LiOEt (85 µL of 1 M THF, 0.085 mmol) and TBDMS-CN (311
mg, 2.20 mmol) in dry THF (4.0 mL) at 0 °C (ice-bath) under an
(i.e., in the presence of an excess of tBuSH), the rate constant
N
2
atmosphere. The reaction mixture was stirred at room temper-
ature until all starting material was consumed (30-50 min). After
workup (EtOAc/water/brine), the organic layer was dried over Na
SO and concentrated. The resulting yellow oil was purified on
of the unimolecular path can be measured by applying free-
radical clock methodology.4
8,49
Using 0.27 M tBuSH, the
2
-
concentration of thiol during the reaction remained essentially
constant (pseudo-first-order conditions) and the following
relation (eq 1) is obeyed. A kH/kc ) 5.0 was obtained as an
average of two independent experiments at ca. 30 °C. Assuming
4
silica (from 20% to 30% of EtOAc in i-hexane) leading to two
main fractions: a major isomer, 5′-cyano-thymidine 4a (376 mg,
0.76 mmol, 45%), and a minor isomer, 5′-cyano-thymidine 4b (308
mg, 0.62 mmol, 37%), both as white foams for a total yield of
82% for the pure compounds. H NMR (400 MHz, CDCl
6
-1 -1
a kH ) 5 × 10 Μ
s
for the reaction of secondary R-alkoxy
1
50
3
), isomer
carbon radical 17 with tBuSH, the cyclization rate constant
of kc ) 1 × 10 s can be estimated.51
6
-1
4a: δ ) 8.30 (s, 1H, NH), 7.46 (d, 1H, H6, J6-Me ) 1.3 Hz), 6.45
dd, 1H, H1′, J1′-2′a ) 9.3 Hz, J1′-2′b ) 5.6 Hz), 4.64 (d, 1H, H5′,
5′-4′ ) 4.0 Hz), 4.54 (dt, 1H, H3′, J3′-2′b ) 5.2 Hz, J3′ 4′ ) 1.4
Hz), 4.02 (dd, 1H, H4′, J4′-5′ ) 4.0 Hz, J4′-3′ ) 1.4 Hz), 2.25-
.13 (m, 2H, H2′), 1.93 (d, 3H, CH -5, JMe-6 ) 1.2 Hz), 0.94 (s,
9H, BuSi), 0.90 (s, 9H, BuSi), 0.24 (s, 3H, CH Si), 0.20 (s, 3H,
(
J
-
[
15]/[16] ) (k /k )[tBuSH]
(1)
H
c
2
3
t
t
Conclusion
3
1
CH
MHz, CDCl
H6, J6-Me ) 1.3 Hz), 6.27 (dd, 1H, H1′, J1′-2′a ) 8.2 Hz, J1′-2′b
6.0 Hz), 4.65 (d, 1H, H5′, J5′-4′ ) 3.4 Hz), 4.48 (dt, 1H, H3′,
J3′-2′b ) 6.1 Hz, J3′-4′ ) 3.1 Hz), 4.02 (dd, 1H, H4′, J ) 3.3
3
Si), 0.11 (s, 3H, CH
3 3
Si), 0.10 (s, 3H, CH Si). H NMR (400
We have disclosed a synthetic sequence for the preparation
of (5′R)-tert-butyl ketones 1 and 2. Our results demonstrate that
their photolysis affords selectively the corresponding C5′ radical.
The nature of the base plays an important role in the fate of the
C5′ radical. In the presence of a physiological concentration of
alkanethiol, the thymidin-5′-yl radical is efficiently reduced,
whereas the 2′-deoxyguanosin-5′-yl radical adds intramolecularly
to the C8-N7 double bond of the guanine moiety with a rate
3
), isomer 4b: δ ) 8.13 (s, 1H, H3 (NH)), 7.25 (d, 1H,
)
4′-5′
Hz, J4′-3′ ) 3.3 Hz), 2.29-2.23 (ddd, 1H, H2
b
′, J2′b-2′a ) 13.4 Hz,
J
J
1
CH
3
(
(
1
2′b-1′ ) 6.0 Hz, J2′b-3′ ) 2.9 Hz), 2.19-2.12 (m, 1H, H2
b
′,
3
2′a-2′b ) 13.4 Hz, J2′a-1′ ) 8.1 Hz), 1.93 (d, 3H, CH -5, JMe-6 )
t
t
.2 Hz), 0.96 (s, 9H, Bu-Si), 0.91 (s, 9H, Bu-Si), 0.26 (s, 3H,
-Si), 0.20 (s, 3H, CH -Si), 0.14 (s, 3H, CH -Si), 0.13 (s,
H, CH ), isomer 4a: δ ) 163.2
-Si). 13C NMR (100.6 MHz, CDCl
C), 150.2 (C), 135.0 (CH), 118.7 (C), 111.7 (C), 87.3 (CH), 84.6
CH), 72.3 (CH), 62.2 (CH), 40.2 (CH ), 25.7 (CH ), 25.6 (CH ),
8.2 (C), 17.9 (C), 12.5 (CH ), -4.6 (CH ), -4.8 (CH ), -5.2
(CH ), -5.3 ppm (CH ). 13C NMR (100.6 MHz, CDCl ), isomer
6
-1
3
3
3
constant of ca. 1 × 10 s , nearly an order of magnitude faster
than the analogous 2′-deoxyadenosin-5′-yl radical.2
1
3
3
Our findings furnish a molecular basis for forthcoming
experiments involving the incorporation of these photolabile
precursors in oligonucleotides and their application in DNA
damage mechanistic studies.52-54 In the absence of O2, the C5′
radical is partitioned between cyclization and the repair reaction
by hydrogen abstraction from glutathione. In the presence of
molecular oxygen, O2 trapping leads to the corresponding
peroxyl radical and eventually to a strand break with formation
2
3
3
3
3
3
3
3
3
4b: δ ) 163.0 (C), 149.9 (C), 135.4 (CH), 117.7 (C), 111.4 (C),
87.5 (CH), 85.5 (CH), 71.2 (CH), 62.8 (CH), 39.9 (CH ), 25.6
CH ), 25.6 (CH ), 18.2 (C), 17.8 (C), 12.4 (CH ), -4.6 (CH ),
4.8 (CH ), -5.0 (CH ), -5.1 (CH ). HR-ESI-MS isomer 4a: for
[M + H] , calcd 496.2658; found 496.2630. HR-
2
(
3
3
3
3
-
3
3
3
+
+
C
23
H
42
N
3
O
5
Si
ESI-MS isomer 4b: for C23
found 496.2661. R 4a (EtOAc/i-Hex 2:3) ) 0.58. R
i-Hex 2:3) ) 0.68.
5′S)-3′,5′-Di-O-(tert-butyldimethylsilyl)-5′-(2,2-dimethylpro-
2
+
+
H
42
N
O
3 5
Si
2
[M + H] , calcd 496.2658;
4b (EtOAc/
of a 5′-aldehyde, 3′-formyl phosphate, and trans-1,4-dioxo-2-
butene.5
5,56
By taking into account the thiol and molecular
f
f
oxygen concentrations, the above partitions will be evaluated
(
panimidoyl)thymidine 5a. To a solution of 4a (300 mg, 0.60
mmol) in THF (12 mL) at -78 °C was slowly added tert-BuLi
(
(
(
48) Griller, D.; Ingold, K. U. Acc. Chem. Res. 1980, 13, 317-323.
49) Newcomb, M. Tetrahedron 1993, 49, 1151-1176.
(
1.5 M, 1.5 mL, 2.4 mmol). After 2 min of vigorous stirring at
50) Tronche, C.; Martinez, F. N.; Horner, J. H.; Newcomb, M.; Senn,
-
78 °C, the reaction was quenched by water addition and allowed
to warm to room temperature. The mixture was diluted with EtOAc,
and the organic phases were then washed with water until neutrality
M.; Giese, B. Tetrahedron Lett. 1996, 37, 5845-5848.
(51) In recent studies of 8-bromo-2′-deoxyguanosine photolysis or 2′-
deoxyguanosine radiolysis, the cyclization of the fully deprotected 2′-
deoxyguanosin-5′-yl radical in aqueous media has been observed (Chat-
gilialoglu, C.; Bazzanini, R.; Jimenez, L. B.; Miranda, M. A., unpublished
results).
2
and finally with brine. Anhydrification on MgCl and subsequent
(
52) Gimisis, T.; Cismas, C. Eur. J. Org. Chem. 2006, 1351-1378.
(57) Cecchini, S.; Masson, C.; La Madeleine, C.; Huels, M. A.; Sanche,
L.; Wagner, J. R.; Hunting, D. J. Biochemistry 2005, 44, 16957-16966.
(58) Greenberg, D. A.; Jin, K. L. Nature 2005, 438, 954-959.
(59) Galanski, M.; Baumgartner, C.; Meelich, K.; Arion, V. B.; Fremuth,
M.; Jakupec, M. A.; Schluga, P.; Hartinger, C. G.; Von Keyserlingk,
N. G.; Keppler, B. K. Inorg. Chim. Acta 2004, 357, 3237-3244.
(60) Ulbrich, K.; Etrych, T.; Chytil, P.; Pechar, M.; Jelinkova, M.; Rihova,
B. Int. J. Pharm. 2004, 277, 63-72.
(53) Neeley, W. L.; Essigmann, J. M. Chem. Res. Toxicol. 2006, 19,
4
91-505.
(
(
54) Pratviel, G.; Meunier, B. Chem.-Eur. J. 2006, 12, 6018-6030.
55) Chen, B. Z.; Bohnert, T.; Zhou, X. F.; Dedon, P. C. Chem. Res.
Toxicol. 2004, 17, 1406-1413.
56) Chen, B. Z.; Vu, C. C.; Byrns, M. C.; Dedon, P. C.; Peterson,
L. A. Chem. Res. Toxicol. 2006, 19, 982-985.
(
J. Org. Chem, Vol. 72, No. 10, 2007 3663

Manetto et al.
rotary evaporation of the solvent lead to 144 mg (0.27 mmol, 43%)
of yellow oil, analyzed as crude product. H NMR (400 MHz,
(5′S)-3′,5′-Di-O-(tert-butyldimethylsilyl)-5′-(tert-butylcarbon-
yl)thymidine 1b. A portion of 10 mL of CDCl was added to the
3
1
acetone-d
1
1
6
): δ ) 9.99 (s, 1H, NH), 7.51 (bs, 1H, H6), 6.28 (dd,
H, H1′, J1′-2′a ) 8.9 Hz, J1′-2′b ) 5.5 Hz), 4.78 (d, 1H, H5′, J )
.5 Hz), 4.53 (d, 1H, H3′, J ) 5.5 Hz), 4.21 (bs, 1H, H4′), 2.25-
.11 (m, 2H, H2′), 1.90 (d, 3H, CH -5, J ) 1.1 Hz), 1.25 (s, 9H,
oil of the crude product containing 5b (100 mg, 0.20 mmol), and
the solution was stirred for 30 min at room temperature. The
1
reaction was followed by H NMR. The hydrolysis of 5b to 1b
2
3
was complete in 30 min. Stirring of this solution for a longer time
led to the degradation of the product of reaction with the
replacement of the NMR peaks of 1b by many unknown peaks.
CDCl3 was removed by rotary evaporation followed by high
vacuum, and the obtained yellow oil (78 mg, 70%) was dissolved
t
t
t
Bu-CO), 1.02 (s, 9H, Bu-Si), 0.94 (s, 9H, Bu-Si), 0.24 (s, 3H,
-Si), 0.18 (s, 3H, CH -Si), 0.17 (s, 3H, CH -Si), 0.10 (s,
H, CH ): δ ) 185.8 (q,
-Si). 13C NMR (100.5 MHz, acetone-d
CdNH), 165.5 (C), 152.0 (C), 136.5 (CH), 112.0 (C), 89.3 (CH),
CH
3
3
3
3
3
6
8
2
-
Si
6.0 (CH), 73.1 (CH), 70.2 (CH), 42.5 (CH
7.4 (CH ), 27.1 (CH ), 19.9 (C), 19.6 (C), 13.7 (CH
), -3.6 (CH ), -3.6 (CH ). HR-ESI-MS for C27
[M + H] , calcd 554.3440; found 554.3420. R (EtOAc/i-
2
), 39.8 (C), 30.2 (CH
3
),
in acetone-d6 for NMR characterization. H NMR (400 MHz,
1
3
3
3
), -3.3 (CH
3
),
acetone-d ): δ ) 10.03 (s, 1H, NH), 7.32 (s, 1H, H6), 6.27 (bdd,
6
3.5 (CH
3
3
3
H
52
N
3
O
5
-
1H, H1′, J ) 7.3 Hz), 5.00 (d, 1H, H5′, J5 ) 2.5 Hz), 4.72 (m,
′-4′
+
+
2
f
1
H, H3′), 4.13 (bt, 1H, H4′, J ) 2.5 Hz), 2.15 (m, 2H, H2′), 1.85
Hex 2:3) ) 0.36.
5′R)-3′,5′-Di-O-(tert-butyldimethylsilyl)-5′-(tert-butylcarbo-
nyl)thymidine 1a. A portion of 10 mL of an acidic solution (THF/
O/2 N HCl, 40:20:1) was added to the imine 5a (100 mg, 0.20
t
t
(bs, 3H, CH
3
-5), 1.25 (s, 9H, Bu-CO), 0.97 (s, 9H, Bu-Si), 0.89
t
(
(s, 9H, Bu-Si), 0.13 (s, 3H, CH -Si), 0.12 (s, 3H, CH -Si), 0.09
3
3
13
(s, 3H, CH -Si), 0.08 (s, 3H, CH -Si). C NMR (150.8 MHz,
3
3
H
2
acetone-d ): δ ) 214.4 (C), 152.4 (C), 147.2 (C), 136.9 (CH), 111.9
6
mmol), and the mixture was stirred for 3 h at room temperature.
The phases were then separated after dilution with 10 mL of EtOAc,
and the organic layer was washed with water until neutrality and
(C), 88.7 (CH), 85.2 (CH), 76.7 (CH), 72.2 (CH), 45.3 (C), 41.8
(CH ), 28.6 (CH ), 27.4 (CH ), 27.2 (CH ), 19.9 (C), 19.4 (C), 13.5
2
3
3
3
(
CH
3
), -3.1 (CH
3
), -3.2 (CH
3
), -3.3 (CH
3
3
), -3.5 (CH ). HR-
finally with brine. Anhydrification on MgCl
evaporation of the solvent yielded an orange oil. Purification on
silica gel (from 1% to 3% of MeOH in CHCl ) provided the tert-
butyl ketone 1a as a yellow oil (98 mg, 0.18 mmol, 88%). H NMR
400 MHz, acetone-d ): δ ) 9.89 (s, 1H, NH), 7.85 (d, 1H, H6,
J ) 1.2 Hz), 6.30 (dd, 1H, H1′, J1′-2′a ) 9.1 Hz, J1′-2′b ) 5.4 Hz),
2
and subsequent rotary
+
+
ESI-MS for C27
H
50
N
2
NaO
6
Si
2
[M + Na] , calcd 577.3100; found
-
-
5
5
77.3068. HR-ESI-MS for C27
89.2901; found 589.2945. R (EtOAc/i-Hex 2:3) ) 0.76.
5′R)-5′-(tert-Butylcarbonyl)thymidine 6. To a solution of 3′,5′-
R)-TBDMS-ketone 1a (170 mg, 0.3 mmol) in MeOH (10 mL)
2 6
H50ClN O Si
2
[M + Cl] , calcd
3
f
1
(
(
6
(
was added a solution of TBAF (1 M) in THF (1 mL, 1.0 mmol).
The mixture was refluxed for 5 h and then stirred at room
temperature for 24 h. MeOH was then removed by reduced pressure,
and the crude product was purified on silica gel (EtOAc in i-hexane
from 30% to 90%). A white foam (61 mg, 0.19 mmol, 60%) was
isolated from the fractions and identified as 3′,5′-OH ketone 6 by
NMR and mass analysis. A less polar fraction was collected leading
to 10 mg (7.3% from the bis-protected ketone) of white powder
5
.17 (d, 1H, H5′, J ) 2.4 Hz), 4.61 (d, 1H, H3′, J ) 4.8 Hz), 4.57
(
d, 1H, H4′, J ) 2.2 Hz), 2.29-2.14 (m, 2H, H2′), 1.93 (d, 3H,
t
t
CH -5, J ) 1.1 Hz), 1.24 (s, 9H, Bu-CO), 0.96 (s, 9H, Bu-Si),
3
t
0
.95 (s, 9H, Bu-Si), 0.18 (s, 3H, CH
Si), 0.15 (s, 3H, CH -Si), 0.11 (s, 3H, CH
MHz, CDCl ): δ ) 8.12 (s, 1H, H3 (NH)), 7.85 (bs, 1H, H6), 6.32
dd, 1H, H1′, J1′-2′a ) 9.0 Hz, J1′-2′b ) 5.4 Hz), 4.86 (d, 1H, H5′,
3
-Si), 0.17 (s, 3H, CH
3
-
1
3
3
-Si). H NMR (600
3
(
J
5′-4′ ) 2.0 Hz), 4.47 (d, 1H, H3′, J3′-2′b ) 5.0 Hz), 4.44 (d, 1H,
analyzed by NMR and mass spectral analyses and identified as 3′-
H4′, J ) 1.5 Hz), 2.25 (dd, 1H, H2
b
′, J2b′-2′a ) 12.8 Hz, J2′b-1′
)
)
1
OH, 5′-TBDMS ketone 7. H NMR (600 MHz, CDCl
(
7
1
3
): δ ) 8.93
5
1
0
.4 Hz), 2.02 (bs, 3H, CH -5), 2.00-1.98 (m, 1H, H2a′, J2′a-2′b
2.9 Hz, J2′a-1′ ) 9.0 Hz, J2′a-3′ ) 4.9 Hz), 1.23 (s, 9H, Bu-CO),
3
bs, 1H, H3 (NH)), 7.36 (bs, 1H, H6), 6.26 (dd, 1H, H1′, J1′-2′a )
t
.5 Hz, J1′-2′b ) 6.2 Hz), 4.77 (d, 1H, H5′, J ) 1.6 Hz), 4.72 (dt,
H, H3′, J ) 5.7 Hz, J ) 2.7 Hz), 4.46 (m, 1H, H4′), 2.37-2.34
t
t
.92 (s, 9H, Bu-Si), 0.91 (s, 9H, Bu-Si), 0.11 (s, 6H, (CH
-Si), 0.02 (s, 3H, CH
-Si). 13C NMR (150.8
): δ ) 210.6 (C), 163.7 (C), 150.1 (C), 136.5 (CH),
10.7 (C), 88.1 (CH), 86.1 (CH), 75.2 (CH), 74.8 (CH), 43.1 (C),
1.5 (CH ), 27.5 (CH ), 25.8 (CH ), 25.8 (CH ), 18.4 (C), 18.1
), -4.5 (CH ), -4.6 (CH ), -4.8 (CH ), -5.5 (CH ).
HR-ESI-MS for C27 [M + H] , calcd 555.3280; found
55.3277. UV λmax ) 264 nm; ꢀ260 ) 1049 ( 64 M cm . R
EtOAc/i-Hex 2:3) ) 0.78.
5′R)-3′,5′-Di-O-(tert-butyldimethylsilyl)-5′-(2,2-dimethylpro-
3 2
) -
Si), 0.10 (s, 3H, CH
MHz, CDCl
1
4
3
3
(
b
ddd, 1H, H2 ′, J2b′-2′a ) 13.5 Hz, J2′b-1′ ) 5.9 Hz, J ) 2.9 Hz),
3
2
.23-2.18 (m, 1H, H2a′, J2′a-2′b ) 13.7 Hz, J2′a-1′ ) 7.2 Hz), 1.96
t 13
(bs, 3H, CH
3
-5), 1.27 (s, 9H, Bu-CO). C NMR (150.8 MHz,
2
3
3
3
CDCl
3
): δ ) 214.1 (q, C5′′), 163.7 (C), 150.4 (C), 135.5 (CH,
(C), 12.4 (CH
3
3
3
3
3
+
+
C6), 111.2 (q, C5), 86.3 (CH, C4′), 85.4 (CH, C1′), 73.3 (CH, C5′),
72.5 (CH, C3′), 42.9 (q, C6′′), 40.6 (CH , C2′), 27.1 (CH , C6′′-
51 2 6 2
H N O Si
-
1
-1
2
3
5
(
f
t
-
-
Bu), 12.6 (CH
calcd 325.1405; found 325.1409. R
5′R)-5′-O-(tert-Butyldimethylsilyl)-5′-(tert-butylcarbonyl)thy-
midine 7. An independent synthesis of 7 was achieved as follows:
70 mg (0.3 mmol) of ketone 1a was dissolved in THF (10 mL),
3
, C5-Me). HR-ESI-MS for C15
H N O
21 2 6
[M - H] ,
f
(EtOAc/i-Hex 1:1) ) 0.11.
(
(
panimidoyl)thymidine 5b. To a solution of 4b (300 mg, 0.60
mmol) in THF (12 mL) at -78 °C was slowly added tert-BuLi
1
(1.5 M, 1.5 mL, 2.4 mmol). After 2 min of vigorous stirring at
and TBAF (1 M in THF, 1 mL, 1.0 mmol) was added to this mixture
at low temperature (-15 °C) and stirred for 24 h at -15 °C. Under
these conditions, the relative yields after purification on silica gel
-
78 °C, the reaction was quenched by water addition and left to
warm to room temperature. The mixture was diluted with EtOAc,
and the organic phases were then washed with water until neutrality
(
EtOAc in i-hexane from 30% to 90%) of 6 and 7 were 10% and
and finally with brine. Anhydrification on MgCl
rotary evaporation of the solvent led to 125 mg (0.22 mmol, 38%)
2
and subsequent
1
55%, respectively. H NMR (600 MHz, DMSO-d ): δ ) 11.27 (s,
6
1
1H, H3 (NH)), 7.80 (bs, 1H, H6), 6.12 (dd, 1H, H1′, J1′-2′a ) 8.5
Hz, J1′-2′b ) 5.4 Hz), 5.07 (d, 1H, H5′, J ) 2.2 Hz), 4.41 (bs, 1H,
of yellow oil, analyzed as crude product. H NMR (400 MHz,
acetone-d ): δ ) 10.06 (s, 1H, NH), 7.34 (bs, 1H, H6), 6.21 (bdd,
H, H1′, J ) 7.2 Hz), 4.73 (d, 1H, H5′, J ) 1.4 Hz), 4.61 (m, 1H,
H3′), 4.04 (dd, 1H, H4′, J4′-3′ ) 3.4 Hz, J4′-5′ ) 1.3 Hz), 2.21-
6
1
H3′), 4.30 (d, 1H, H4′, J ) 4.8 Hz), 2.15 (dd, 1H, H2′, J2′b-2′a)
-13.2 Hz, J2′b-1′ ) 5.7 Hz), 1.93 (ddd, 1H, H2′, J2′a-2′b ) -13.8
Hz, J2′a-1′ ) 8.9 Hz, J2′a-3′ ) 5.4 Hz), 1.83 (bs, 3H, CH -5), 1.16
-Si), 0.03
): δ ) 210.8
(C), 163.2 (C), 149.7 (C), 135.4 (CH), 108.5 (C), 86.6 (CH, C4′),
84.7 (CH, C1′), 74.3 (CH, C5′), 71.9 (CH, C3′), 42.2 (CH , C2′),
26.6 (CH ), 25.1 (CH ), 17.5 (C), 11.8 (CH ), -5.3 (CH ), -5.9
(CH ). HR-ESI-MS for C21 Si [M + H] , calcd 441.2415;
found 441.2396. HR-ESI-MS for C21
calcd 475.2037; found 475.2078. R ) (EtOAc/i-Hex 1:1) ) 0.50.
t
2
1
0
.16 (m, 2H, H2′), 1.86 (bs, 3H, CH
3
-5), 1.27 (s, 9H, Bu-CO),
.00 (s, 9H, Bu-Si), 0.88 (s, 9H, Bu-Si), 0.14 (s, 3H, CH -Si),
.10 (s, 3H, CH -Si), 0.07 (s, 3H, CH -Si), 0.03 (s, 3H, CH
): δ ) 187.2 (q, CdNH),
65.0 (C), 156.9 (C), 137.0 (CH), 111.9 (C), 89.3 (CH), 88.2 (CH),
5.9 (CH), 70.5 (CH), 49.1 (CH ), 40.1 (C), 30.6 (CH ), 27.4 (CH ),
7.2 (CH ), 19.8 (C), 19.3 (C), 13.5 (CH ), -2.2 (CH ), -3.2 (CH ),
3.4 (CH ), -3.6 (CH ). HR-ESI-MS for C27 [M +
H] , calcd 554.3440; found 554.3431. R (EtOAc/i-Hex 2:3) ) 0.34.
3
t
t
t
t
(s, 9H, Bu-CO), 0.87 (s, 9H, Bu-Si), 0.04 (s, 3H, CH
3
3
3
-
(s, 3H, CH
3
-Si). 13C NMR (150.8 MHz, DMSO-d
6
3
3
13
Si). C NMR (100.5 MHz, acetone-d
6
1
7
2
2
2
3
3
3
3
3
3
+
+
3
3
3
3
3
37 2 6
H N O
+
-
-
-
3
3
H
52
N
3
O
5
Si
2
2 6
H36ClN O Si [M + Cl] ,
+
f
f
3664 J. Org. Chem., Vol. 72, No. 10, 2007

Thymidine and 2′-Deoxyguanosine C5′ Radicals
Synthesis of (5′R)-3′-O-Acetyl-5′-O-(tert-butyldimethylsilyl)-
′-(tert-butylcarbonyl)thymidine 8. To a solution of 3′-OH, 5′-
3
(CH Cl/MeOH, 9:1) ) 0.44. To a stirred solution containing the
5
above cyanohydrin (1.50 g, 2.71 mmol) and imidazole (680 mg,
9.50 mmol) in dry DMF (15 mL), under an inert atmosphere, was
added TBDMSCl (1.22 g, 8.14 mmol), at room temperature. After
the initial gas evolution had subsided, the reaction was stirred
overnight and then diluted with EtOAc and washed with sat. aq
TBDMS-ketone 7 (73 mg, 0.2 mmol) in dry pyridine (2 mL) was
added 35 µL (0.38 mmol) of acetic anhydride. The mixture was
stirred at room temperature for 24 h. The solvent was removed by
rotary evaporation, and the residue was coevaporated three times
with 1 mL of toluene. The ketone 8 was obtained in 82% yield as
a white powder and was characterized. The 5′(R) configuration was
NaHCO
organic phase was filtered, condensed under a vacuum, purified
by column chromatography (CHCl /MeOH, 95:5), and precipitated
from an ether/hexane mixture to yield 13 (400 mg, 25%) as a white
3 2 4
and brine (each 50 mL). After drying over Na SO , the
assigned by resolution of the crystal obtained from a saturated
3
1
EtOAc solution of 8. H NMR (600 MHz, CDCl
3
): δ ) 8.06 (s,
1
1
J
5
(
H, H3 (NH)), 7.87 (d, 1H, H6, J ) 1.1 Hz), 6.30 (dd, 1H, H1′,
1′-2′a ) 9.2 Hz, J1′-2′b ) 5.4 Hz), 5.22 (d, 1H, H3′, J ) 5.9 Hz),
.08 (d, 1H, H5′, J ) 2.1 Hz), 4.51 (d, 1H, H4′, J ) 1.8 Hz), 2.48
dd, 1H, H2′, J2′b-2′a ) -13.8 Hz, J2′b-1′ ) 5.4 Hz), 2.13 (m, 1H,
H2′), 2.14 (s, 3H, Ac), 2.03 (d, 3H, CH -5, J ) 1.0 Hz), 1.21 (s,
H, Bu-CO), 0.92 (s, 9H, Bu-Si), 0.11 (s, 3H, CH -Si), 0.06
s, 3H, CH ): δ ) 210.6 (C),
-Si). 13C NMR (150.8 MHz, CDCl
71.0 (C), 163.5 (C), 150.1 (C), 136.0 (CH), 111.1 (C), 85.5 (CH),
5.5 (CH), 74.8 (CH), 43.1 (C), 37.6 (CH ), 27.4 (CH ), 25.8 (CH ),
1.0 (CH ), 18.4 (C), 12.5 (CH ), -4.5 (CH ), -5.4 (CH ). HR-
Si [M + H] , calcd 483.2521; found
solid. mp: 134-136 °C. H NMR (200 MHz, CDCl ): δ ) 12.05
3
(bs, 1H, NH), 9.41 (bs, 1H, NH), 7.92 (s, 1H, H8), 6.15 (dd, 1H,
H1′, J1′-2′a ) 9.6 Hz, J1′-2′b ) 5.0 Hz), 4.69 (d, 1H, H3′, J ) 5.2
Hz), 4.63 (d, 1H, H5′, J ) 4.2 Hz), 4.05 (dd, 1H, H4′, J4′-5′ ) 4.2
i
3
Hz, J4′-3′ ) 1.4 Hz), 2.61 (hpt, 1H, Pr), 3.01 (ddd, 1H, H2′R),
t
t
9
(
1
8
2
3
2.29 (dd, 1H, H2′â, J2′b-2′a ) -13.2 Hz, J2′b-1′ ) 5.2 Hz), 1.26,
i
3
3
1.23 (each s, 3H, Pr), 0.92 (d, 18H, SitBu), 0.21, 0.18 (each s, 3H,
SiMe), 0.14 (s, 6H, SiMe). 13C NMR (50 MHz, CDCl
): δ ) 178.7,
155.8, 148.4, 147.6 (each C), 138.2 (CH), 122.1, 119.0 (each C),
88.5, 85.0, 82.8, 72.4 (each CH), 43.4 (CH ), 36.6 (CH), 25.8
(6 × CH ), 19.0 (2 × CH ), 18.4, 18.0 (each C), -4.3, -4.9 (each
CH ). IR (NaCl) 2332 (CtN), 1713 (CdO), 1688 cm (CdO).
HR-ESI-MS for C27
589.2997. R (CH Cl
3
2
3
3
3
3
3
3
2
+
+
ESI-MS for C23
H
39
N O
2 7
3
3
-
-
-1
4
4
83.2502. HR-ESI-MS for C23
81.2376; found 481.2425. R (EtOAc/i-Hex, 2:3) ) 0.42.
X-Ray Crystal Structure Determination. X-ray data were
H
37
N
2
O
7
Si [M - H] , calcd
3
-
-
f
H
2
45
N
6
O
5
Si
2
[M - H] , calcd 589.2995; found
f
2
/MeOH, 9:1) ) 0.49.
collected at 200 K with a diffractometer equipped with a rotating
anode and graded multilayer X-ray optics to get monochrome Mo
KR radiation (λ ) 0.71073 Å). The structure was solved with direct
methods with SIR9761 and refined with SHELXL-97. Hydrogen
atoms were calculated in idealized positions riding on their parent
atoms. One of the tert-butyl groups is disordered; a split model
was applied with isotropic refinement of the split atoms. Data for
(5′S)-2-N-Isobutyryl-3′,5′-di-O-(tert-butyldimethylsilyl)-5′-
(2,2-dimethylpropanimidoyl)-2′-deoxyguanosine (14). To a solu-
tion of the nitrile 13 (450 mg, 0.76 mmol) in dry THF (4 mL), at
62
ï
-78 C, under an argon atmosphere, was added dropwise t-BuLi
(1.34 M in pentane, 2.84 mL, 3.8 mmol). After 5 min of vigorous
stirring, the bright red reaction mixture was quenched by addition
of methanol (1 mL) followed by CH COOH (0.5 mL) and H O (1
3
2
compound 8: C23
H
38
N
2
3
O
7
Si, fw ) 482.643, colorless block,
mL) and left to slowly warm to room temperature. Dilution with
EtOAc (30 mL), washing with H O, sat. aq NaHCO , and brine
0
.18 × 0.15 × 0.11 mm , monoclinic, P21 (No. 4), a ) 14.1327-
2
3
(
4) Å, b ) 10.9042(3) Å, c ) 17.7449(6) Å, â ) 93.3188(11)°,
2 4
(each 30 mL), drying over Na SO , filtering, and evaporating under
reduced pressure furnished the crude product that was further
3
V ) 2730.01(14) Å, Z ) 47 119 unique reflections, 593 parameters
were refined, R1 (I > 2σ(I)) ) 0.0621, wR2 (all reflections) )
purified by silica gel flash column chromatography (CHCl /MeOH,
3
0
.1774, S ) 1.062. Residual electron density between 0.763 and
97:3) to yield the imine 14 (160 mg, 32.4%) as an off-white foam,
-
3
-
0.316 e Å . Further details are available under the depository
together with unreacted starting material 13 (180 mg, 40%) and
1
number CCDC 618360 from the Cambridge Crystallographic Data
Centre.
2-N-isobutyroylguanine (38 mg, 20%). H NMR (200 MHz, CD -
2
Cl ): δ ) 11.95 (bs, 1H, NH), 10.20 (bs, 1H, NH), 8.00 (s, 1H,
2
(5′S)-2-N-Isobutyryl-3′,5′-di-O-(tert-butyldimethylsilyl)-5′-cy-
H8), 6.20 (dd, 1H, H1′, J1
) 6.3 Hz, J
1′-2′b
) 6.1 Hz), 4.72 (d,
′-2′a
ano-2′-deoxyguanosine (13). To a stirred solution of compound
1H, H5′, J ) 1.8 Hz), 4.49 (m, 1H, H3′), 4.03 (dd, 1H, H4′,
38
i
12
(2.40 g, 5.31 mmol), under an inert atmosphere, in dry toluene/
DMSO (2:1, 48 mL), at room temperature, were added solid EDC
3.03 g, 15.9 mmol), pyridine (1.48 mL, 18.3 mmol), and CF
COOH (0.20 mL, 2.65 mmol). After the last addition, the cleared
solution was stirred for 15 min, then diluted with CH Cl and washed
with water and brine (each 50 mL). After drying over Na SO , the
J
) 3.5 Hz, J
) 1.7 Hz), 2.72 (hpt, 1H, Pr), 2.29-2.35
4
′-3′
4′-5′
t
i
i
(m, 2H, H2′), 1.26 (s, 9H, Bu), 1.16 (s, 3H, Pr), 1.15 (s, 3H, Pr),
t
(
3
-
0.92, 0.88 (each s, 9H, Si Bu), 0.11, 0.09, 0.07, 0.04 (each s, 3H,
SiMe). 13C NMR (125 MHz, CDCl ): δ ) 186.4, 179.7, 156.2,
3
3
148.4, 148.2 (each C), 136.6 (CH), 121.2 (C), 87.8, 83.7, 72.5,
70.5 (each CH), 43.2 (CH ), 38.9 (C), 36.4 (CH), 29.4 (CH ), 26.2
2
4
2
3
organic phase was condensed under a vacuum and the well-dried
(6 × CH ), 19.3 (2 × CH ), 18.6, 18.3 (each C), -3.7, -4.3 (each
3
3
+
+
foamy residue (2.26 g, 95%) was applied directly to the next step.
2 × CH ). LR-ESI-MS: 649 [M + H] , 1297 [2M + H] , 209
3
1
+
-
-
H NMR (200 MHz, CDCl
3
): characteristic peaks at δ ) 9.76 (s,
(CH Cl /MeOH,
:1) ) 0.65. To a stirred solution containing the above foam (1.87
g, 4.16 mmol) and ZnI (70 mg) in dry CH Cl (14 mL), under an
[B + H] . HR-ESI-MS: for C H N O Si [M - H] , calcd
31
55
6
5
2
H, CHO), 7.79 (s, H, H8), 6.33 (m, H, H1′). R
9
f
2
2
647.3778; found 647.3757. R (CH Cl /MeOH, 9:1) ) 0.43.
f
2
2
(
5′R)-2-N-Isobutyryl-3′,5′-di-O-(tert-butyldimethylsilyl)-5′-
tert-butylcarbonyl)-2′-deoxyguanosine (2). A solution of the tert-
butyl imine 14 (50 mg, 0.077 mmol) in THF/CH CO H/H O (1:
:1, 3 mL) was stirred for 1 h at room temperature. Dilution with
2
2
2
(
inert atmosphere, was added dropwise TMSCN (1.35 mL, 10.12
mmol), at room temperature. After 5 h or upon disappearance of
starting material on TLC, the mixture was diluted with EtOAc and
3
2
2
4
EtOAc (10 mL), washing with H
2
O, sat. aq NaHCO
3
, and brine
washed with sat. aq NaHCO
over Na SO , the organic phase was condensed under a vacuum
and the well-dried product foam (1.74 g, 88%) was applied directly
3
and brine (each 50 mL). After drying
(
each 10 mL), drying over Na SO , filtering, and evaporating under
2 4
2
4
reduced pressure furnished the crude product that was further
purified by silica gel flash column chromatography (CHCl3/MeOH,
1
to the next step. H NMR (200 MHz, CDCl
3
): characteristic peaks
1
9
7:3) to yield the product 2 (39 mg, 78.1%) as a white foam. H
at δ ) 7.86 (s, H, H8â), 7.78 (s, H, H8R), 6.19 (m, 2H, H1′). R
f
NMR (200 MHz, CDCl ): δ ) 11.85 (bs, 1H, NH), 8.65 (bs, 1H,
3
NH), 8.25 (s, 1H, H8), 6.21 (dd, 1H, H1′, J1′-2′a ) J1′-2′b ) 6.0
(61) Altomare, A.; Burla, M. C.; Camalli, M.; Cascarano, G. L.;
Hz), 4.89 (d, 1H, H5′, J ) 2.6 Hz), 4.63 (m, 1H, H3′), 4.45 (dd,
Giacovazzo, C.; Guagliardi, A.; Moliterni, A. G. G.; Polidori, G.; Spagna,
i
1
2
H, H4′, J4′-3′ ) 3.3 Hz, J4′-5′ ) 2.6 Hz), 2.62 (hpt, 1H, Pr), 2.30-
R. J. Appl. Crystallogr. 1999, 32, 115-119.
i
i
.37 (m, 2H, H2′), 1.28 (s, 3H, Pr), 1.24 (s, 3H, Pr), 1.20 (s, 9H,
CO Bu), 0.93, 0.88 (each s, 9H, Si Bu), 0.13 (s, 6H, SiMe), 0.12,
(62) Sheldrick, G. M. SHELXL-97, 97-2 version; University of G o¨ ttin-
t
t
gen: Germany.
63) Nagatsugi, F.; Uemura, K.; Nakashima, S.; Maeda, M.; Sasaki, S.
Tetrahedron 1997, 53, 3035-3044.
0
.03 (each s, 3H, SiMe). 13C NMR (50 MHz, CDCl
3
): δ ) 210.3,
(
185.9, 156.4, 148.5, 143.7 (each C), 136.8 (CH), 121.1 (C), 90.6,
J. Org. Chem, Vol. 72, No. 10, 2007 3665

Manetto et al.
8
2
3.6, 80.9, 74.7 (each CH), 46.0 (C), 38.9 (CH
6.3, 26.1 (each 3 × CH ), 19.4 (2 × CH ), 18.5, 18.3 (each C),
3.9, -4.2 (each 2 × CH ). UV λ ) 253 (ꢀ ) 9973), 281 nm
2
), 36.4 (CH), 29.6,
(d, 1H, H1′, J ) 4.8 Hz), 5.16 (d, 1H, H5′, J ) 6.2 Hz), 4.85 (m,
i
3
3
1H, H3′), 4.56 (d, 1H, H4′, J ) 5.8 Hz), 2.60-2.85 (hpt, 1H, Pr),
-
3
2.45-2.65 (m, 1H, H2′R), 2.20-2.35 (m, 1H, H2′â), 1.27, 1.25
-
1
-1
-
i
t
t
(
ꢀ ) 7748 M cm ). HR-ESI-MS: for C31
H N O
54 5 6
Si
2
[M -
(each s, 3H, Pr), 0.96 (s, 9H, Si Bu), 0.88 (s, 9H, Si Bu), 0.28,
-
13
H] , calcd 648.3618; found 648.3603. R
.34.
5′S)-2-N-Isobutyryl-3′,5′-di-O-(tert-butyldimethylsilyl)-5′,8-
f
(CH
2
Cl
2
/MeOH, 9:1) )
0.27, 0.05, 0.04 (each s, 3H, SiMe). 16. C NMR (50 MHz,
0
CDCl
87.3, 85.2, 71.0, 63.4 (each CH), 41.0 (CH
(each 3 × CH ), 19.1 (2 × CH ), 18.5, 18.4 (each C), -3.9, -4.2
(each CH ). HR-ESI-MS: for C26
3
): δ ) 186.1, 158.4, 154.7, 150.3, 147.5, 111.8 (each C),
(
2
), 37.0 (CH), 26.1, 25.9
cyclo-2′-deoxyguanosine (16). To a solution of 2 (35 mg, 0.054
mmol) in THF (2 mL) in a 5-mL pyrex vial was added t-BuSH
3
3
-
3
), -4.7 (2 × CH
3
H
f 3
44
N
5
O
5
Si
2
-
(
30 µL, 0.54 mmol). After 15 min of argon bubbling, the solution
[M - H] , calcd 562.2886; found 562.2875. R
(CH Cl:MeOH,
was irradiated at 5 °C with an Xe lamp at 1000 W, delivering 110
9:1) ) 0.53.
2
mW/cm local intensity of UV-A radiation on the sample. After
6
0 min of irradiation, the solvent was evaporated under reduced
pressure and the residue was purified through a short path column
chromatography (CHCl ) to yield starting material 2 (15 mg, 43%),
Acknowledgment. This work was supported in part by the
European Community’s Marie Curie Research Training Network
under contract MRTN-CT-2003-505086 [CLUSTOXDNA].
A.M. thanks also the “Marco Polo” program (Alma Mater
Studiorum, University of Bologna).
3
the reduced 2′-deoxyguanosine 15 (8 mg, 27%), and the cyclized
title compound 16 (6 mg, 20%). 15.63 1H NMR (200 MHz,
CDCl ): δ ) 11.50 (bs, 1H, NH), 8.20 (bs, 1H, NH), 7.95 (s, H,
3
H8), 6.21 (dd, 1H, H1′, J1′-2′a ) J1′-2′b ) 6.6 Hz), 4.56 (m, 1H,
H3′), 3.96 (dd, 1H, H4′, J4′-3′ )7.2, J4′-5′ ) 3.6 Hz), 3.75 (d, 2H,
Supporting Information Available: 1H NMR spectra of 1a/b,
2
1
, 4a/b, 5a/b, 7, 8, 13, 14, and 16; COSY and HMQC spectra of
4; and X-ray crystallographic structure and data of 8. This material
H5′, J ) 3.6 Hz), 2.59 (hpt, 1H, CHMe
2
, J ) 7.2 Hz), 2.35-2.45
i
(
1
m, 2H, H2′), 1.28 & 1.25 (each d, 3H, Pr, J ) 7.2 Hz), 0.90 (s,
is available free of charge via the Internet at http://pubs.acs.org.
t
1
8H, 2 × Bu), 0.09, 0.07 (each s, 6H, SiMe
2
). 16. H NMR (200
MHz, CDCl
3
): δ ) 11.85 (bs, 1H, NH), 8.60 (bs, 1H, NH), 6.17
JO062518C
3666 J. Org. Chem., Vol. 72, No. 10, 2007