Synthesis of C3′ modified nucleosides for selective generation of the C3′-deoxy-3′-thymidinyl radical: A proposed intermediate in LEE induced DNA damage

Article abstract of DOI:10.1021/jo300045m

DNA damage pathways induced by low-energy electrons (LEEs) are believed to involve the formation of 2-deoxyribose radicals. These radicals, formed at the C3′ and C5′ positions of nucleotides, are the result of cleavage of the C-O phosphodiester bond throu

Full text of DOI:10.1021/jo300045m

Article
pubs.acs.org/joc
Synthesis of C3′ Modified Nucleosides for Selective Generation of the
C3′-Deoxy-3′-thymidinyl Radical: A Proposed Intermediate in LEE
Induced DNA Damage
Suaad A. S. Audat,† CherylAnn Trzasko Love,‡ Buthina A. S. Al-Oudat,†
and Amanda C. Bryant-Friedrich*,†
†Department of Medicinal and Biological Chemistry, The University of Toledo, 2801 W. Bancroft Street, Toledo, Ohio 43606, United
States
‡Department of Chemistry, Oakland University, 2200 N. Squirrel Road, Rochester, Michigan 48309, United States
S
* Supporting Information
ABSTRACT: DNA damage pathways induced by low-energy
electrons (LEEs) are believed to involve the formation of 2-
deoxyribose radicals. These radicals, formed at the C3′ and C5′
positions of nucleotides, are the result of cleavage of the C−O
phosphodiester bond through transfer of LEEs to the
phosphate group of DNA oligomers from the nucleobases. A
considerable amount of information has been obtained to
illuminate the identity of the unmodified oligonucleotide products formed through this process. There exists, however, a paucity
of information as to the nature of the modified lesions formed from degradation of these sugar radicals. To determine the identity
of the damage products formed via the 2′,3′-dideoxy-C3′-thymidinyl radical (C3′dephos sugar radical), phenyl selenide and acyl
modified sugar and nucleoside derivatives have been synthesized, and their suitability as photochemical precursors of the radical
of interest has been evaluated. Upon photochemical activation of C3′-derivatized nucleosides in the presence of the hydrogen
atom donor tributyltin hydride, 2′,3′-dideoxythymidine is formed indicating the selective generation of the C3′dephos sugar radical.
These precursors will make the identification and quantification of products of DNA damage derived from radicals generated by
LEEs possible.
Scheme 1. Proposed Pathway of LEE Induced Formation of
INTRODUCTION
■
C3′ and C5′-Deoxyribose Radicals in DNA10
The negative biological effects of ionizing radiation have been
largely attributed to damage caused at its most significant
biological target, DNA.1−4 Thus, the genotoxic effect of
secondary low-energy electrons (LEEs) on living cells has
been a topic of considerable investigation as it relates to
radiobiology.5−7 LEEs (<30 eV) are produced at high levels
through the transfer of energy from ionizing radiation to
cellular constituents (∼3 × 104/MeV).8,9 The elucidation of the
mechanistic pathways involved in DNA−LEE interactions has
included the use of nucleobases, sugar derivatives, oligonucleo-
tides, and plasmid DNA and has revealed that at a molecular
level, LEEs attach to all DNA components (sugar, phosphate,
and base) to deliver neutral and anionic radicals.10−12 It has
been established through these investigations that LEEs can
induce both single and double strand breaks as well as base
release. It is believed that following the capture of these
electrons by the nucleobases, they are transferred to the
phosphate moiety of the DNA (1)13 generating a transient
radical anion leading to C−O σ bond homolytic cleavage at the
3′ and/or 5′ positions. The result is the formation of sugar
radicals 2 and 5 along with the corresponding phosphorylated
oligonucleotides 3 and 4 (Scheme 1).10
formation of not only 3′ and 5′-phosphorylated DNA oligomers
3 and 4 but also oligonucleotides terminated with unidentified
sugar fragments.10 Additional evidence for the formation of
reactive intermediates 2 and 5 was obtained by the
identification of dephosphorylated C3′-sugar radicals (C3′dephos
)
The irradiation of short DNA oligomers in the condensed
phase, followed by chemical analysis using HPLC, indicated the
Received: January 24, 2012
Published: April 2, 2012
© 2012 American Chemical Society
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in ESR studies of argon ion irradiated and γ-irradiated hydrated
DNA.14 Moreover, dephosphorylated C3′ and C5′ radicals were
reported in DNA irradiated with low-LET radiation.15 These
techniques do not, however, lend themselves to the
determination of the structure of the final DNA modifications
that result from the occurrence of the 2-deoxyribose radicals
proposed as a result of DNA exposure to LEEs. Therefore,
investigation of the reactivity and fate of these reactive
intermediates should further elucidate the mechanisms involved
in DNA damage by these low energy electrons.
Organic chemistry has often been used to obtain stable
photolabile precursors for the independent generation of
reactive intermediates in nucleic acids.16−20 Acyl and phenyl
selenide derivatives of modified nucleosides and oligonucleo-
tides have been utilized as precursors for site-specific generation
of several radical-based intermediates, including those originat-
ing at the sugar−phosphate backbone of DNA.21 With the goal
to identify LEE induced nucleic acid derived products, as well
as to characterize radicals related to 2, we have synthesized 3-
deoxy-C3′-acyl-2-deoxyribose (6) and 3′-deoxy-C3′-acyl (7)
and selenophenyl (8) modified thymidines (Figure 1). We have
RESULTS AND DISCUSSION
■
To study the chemistry of sugar radicals related to 2, several
different precursors were considered. Previous studies demon-
strated the utility of both acyl modified and phenyl selenide
containing substrates in the photochemical generation of
nucleoside and nucleotide radicals in a site-specific manner.21
Acyl-substituted nucleosides have shown their utility as
precursors specifically for the site-specific generation of 2-
deoxyribose radicals.23−27 The ketone moiety absorbs light at
wavelengths outside the range, which induces other reactions in
nucleotides and subsequently undergoes Norrish type I
photocleavage. This photochemical process delivers an alkyl
radical and an acyl radical that undergo decarbonylation to
deliver a second alkyl radical. t-Butyl ketones are particularly
attractive as radical precursors because of the fact that the t-
butyl radical diffuses away from the site of radical generation
without participating in secondary reactions and is produced in
low levels.28
Alkyl phenyl selenides undergo facile homolytic cleavage
under similar conditions to deliver alkyl radicals with
dissociation energies of the Se−alkyl bond in the range of
∼60 kcal/mol as demonstrated by formation of the ethyl radical
from diethylselenide.29 The synthesis and purification of these
highly stable substrates is also straightforward. In addition to
these factors, the use of this moiety as a photochemical radical
precursor has facilitated the study of several DNA sugar and
base radicals with radical generation occurring at suitable
wavelengths.23
Synthesis of a Photochemical Precursor for ESR
Studies. Our initial synthetic approach to acyl modified
nucleic acid monomers involved the synthesis of modified
ribose derivatives as glycosyl donors to be utilized in
Figure 1. Radical precursors for the site-specific photochemical
generation of species related to the dephosphorylated C3′-deoxy-3′-
thymidinyl radical.
Vorbruggen type couplings to nucleobases.30 Even though
̈
this approach did not successfully lead to the nucleoside radical
precursors desired, it did allow us to develop a model substrate
to facilitate the identification of species related to 2 in argon-ion
beam irradiated DNA using ESR spectroscopy.14 For these
investigations 3-acetyl-2,3-dideoxyribose (14) was synthesized
(Scheme 2). Studies performed by Mann et al.31 proved that
the photoaddition of alcohols to unsaturated cyclic esters
occurs regiospecifically as well as face-selectively at the β-
carbon of butenolides such as 9. Subsequent work demon-
strated that these conditions could also successfully be utilized
for the introduction of cyclic acetals at the same position. On
the basis of these observations, we decided to introduce a
methyl ketone, in its protected ketal form, at the β-position of
had an interest for quite some time in reactive species
generated at the 2-deoxyribose moiety of nucleic acids.20,22 In
pursuit of our interests in C3′-radical species, we here introduce
the synthesis of analogues 6−8 as precursors of radical species
related to 2. Our laboratory is the first to investigate this radical
utilizing synthetically obtained radical precursors. Comparison
of these substrates as well as their overall suitability as
precursors of the 3′-radical of interest is presented.
Scheme 2. Synthesis of Precursor 14
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Scheme 3. Synthesis of Ketone 19
Scheme 4. Synthesis of Precursor 7b
Scheme 5. Synthesis of Precursors 8a and 8b
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Scheme 6. Photochemical Generation of 28 from Precursors 7b and 8a,b in the Presence of Tributyltin Hydride
butenolide 9 under the above-described conditions. Methyl
ketones also participate in Norrish type I photocleavage to
generate nucleic acid radicals, however with lower efficiency
than their t-butyl counterparts.22 A 2-methyl-1,3-dioxolane
substituent was introduced at position 4 of compound 9
through photochemical addition of the corresponding ketal
(10) in the presence of benzophenone (11, 87% yield).
Following reduction of the resulting lactone (12) with DIBAL-
H (97% yield), acidic hydrolysis delivered the desired ketone
(14) in 75% yield (Scheme 2).
Compound 14 was then utilized for experimental compar-
ison to determine the presence of a proposed C3′dephos sugar
radical in argon-ion beam irradiated DNA. The successful use
of this substrate in the generation of the radical of interest has
been reported elsewhere.14
Synthesis of C3′-Modified Thymidines as Photo-
chemical Precursors. As mentioned, our attempts to form
C3′-modified nucleosides through the coupling of modified
ribose derivatives to nucleobases were unsuccessful. We
therefore decided to turn our attention to the use of intact
nucleosides as starting materials for our syntheses. Preliminary
results, obtained from the above-described ESR studies in
which the highly diffusible and extremely reactive methyl
radical was generated, as well as the known efficiency of radical
generation by t-butyl ketones via Norrish type I photocleavage,
lead us to the synthesis of pivaloyl ketones and phenyl selenide
derivatives as precursors of the C3′-nucleoside radical. The
demonstrated efficiency of both classes of substrates in the
generation of nucleoside radicals warrants the synthesis of both
types of precursors. Additionally, to investigate the relationship
between the stereochemistry of the starting material and radical
initiated product formation, both the alpha and beta isomers of
the modified nucleosides were synthesized (Schemes 3, 4, and
5).
Consequently, commercially available thymidine was con-
verted to compound 15 according to previously published
synthetic methods.32 Through hydroboration−oxidation, al-
kene 15 was converted to primary alcohol 16 as exclusively the
β-isomer (Scheme 3). Oxidation of the primary alcohol using
the Dess−Martin periodinane at low temperature delivered
only the β-isomer of aldehyde 17 in 100% yield. Upon
treatment of 17 with t-butyllithium in the presence of CeCl3,
alcohols 18 were obtained in good yield (67%) as a
diastereomeric mixture. Subsequent Dess−Martin oxidation
delivered the desired ketone (19) as the β-isomer in 96% yield
(Scheme 3). The absolute configuration of these substrates was
confirmed through NOESY NMR analysis.
To obtain α-isomer 7b, an alternative synthetic approach was
necessary (Scheme 4). Methods developed by Mesmaeker et
al.33 made it possible for us to obtain exclusively 21 from t-
butyldiphenylsilyl protected C3′-styrylthymidine (20) via
oxidative cleavage. Using the same synthetic strategy as
employed for the β-isomer, the desired t-butyl alcohol was
obtained from 21 as a diastereomeric mixture of isomers (22),
however in very low yield (13%). Extension of the reaction
time to 8 h, however, increased the yield of 22 to 30% (Scheme
4). These results indicate that 17 is much more reactive to
nucleophilic addition than α-isomer 21 under the conditions
employed. This decrease in reactivity is likely due to steric
hindrance at the lower face of the nucleoside. Dess−Martin
oxidation of 22 provided the desired ketone (23) in 100% yield.
The final step in the acquisition of radical precursors 7a and
7b involves the deprotection of the 5′-hydroxyl. In the case of
19, this was attempted with TFA/H2O at low temperature.30
The result was not the expected precursor, but decomposition
of the starting material. Attempts were made with other
reagents classically used to deprotect silylethers; however, none
of these led to the formation of 7a. We are currently
investigating other alternatives to facilitate the acquisition of
this compound. The treatment of 23 with TBAF in THF
resulted in removal of the t-butyldiphenylsilyl group to deliver
precursor 7b in 44% yield (Scheme 4).
Phenyl selenide radical precursors 8a and b were synthesized
in a straightforward manner using known literature procedures
(Scheme 5).34 These substrates were previously synthesized
employing 5′-trityl protection instead of the dimethoxytrityl
groups reported herein. β-3′-Selenophenyl-C3′-deoxythymidine
(8a)34 was obtained through nucleophilic substitution of the
mesyl group by the phenyl selenide ion followed by
detritylation to deliver the phenyl selenide radical precursor
in 91% yield. Analogously, dimethoxytrityl protected 27 was
obtained through nucleophilic attack of the phenyl selenide
anion, at the α-face of anhydrothymidine35 26 in good yield
(89%). Subsequent detritylation with 80% acetic acid at 95 °C
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Figure 2. Reverse phase HPLC analyses of the photolyzates resulting from (a) 7b and (b) 8b (green). In each experiment, controls (red), standard
29 along with radical precursors 7b or 8b (blue) were injected separately.
afforded known 8b34 in 85% yield (Scheme 5). The related
trityl protected derivative was obtained in slightly lower yield
using the same procedures.
CONCLUSIONS
■
Collectively, the results described herein demonstrate that the
C3′dephos thymdinyl radical (28) can be generated in nucleosides
Photochemical Generation of the 2′,3′-Dideoxy-C3′-
thymdinyl Radical from Radical Precursors. To determine
the suitability of radical precursors 7b and 8a,b for generation
of 28, we probed their efficiencies through photochemical
experiments in the presence of the hydrogen atom donor
tributyltin hydride (Scheme 6). The reduction of 28 through
rapid hydrogen atom donation should lead to the formation of
2,3-dideoxythymidine (29) as the sole product. Anaerobic
photolysis (λ ≥ 320 nm) of the three available precursors did
indeed deliver modified nucleoside 29 as the sole product.
Photochemical conversion of precursor 7b to radical 28 was
performed in a 1:1 mixture of acetonitrile/water containing an
excess of tributyltin hydride. Photolysis of 8a and 8b was
performed under similar conditions. However, in the case of 8a
the addition of THF (0.01%) to the solvent mixture was
required because of solubility issues related to the precursor.
Analysis of the photolyzates using reverse phase HPLC was
easily accomplished using independently synthesized standard
29.36,37 The identity of the reduction product was additionally
confirmed using ESI-MS. Figure 2 shows representative
chromatograms obtained through analysis of the mixtures
obtained from photolysis of α-3′-pivaloylthymidine 7b (Figure
2a) and α-3′-phenylselenylthymidine 8b (Figure 2b).
through exposure of precursors 7b, 8a, and 8b to UV light of
≥320 nm. As these substrates will be further utilized in the
synthesis of oliogonucleotide precursors of 2, several factors are
important in the determination of the most suitable precursor
to pursue. The accessibility of the precursors is a significant
consideration. It is clear that precursors 8a and 8b are much
less synthetically challenging to obtain because of the high
yielding transformations at each step and the overall length of
the synthesis when compared to precursors 7a and 7b.
As mentioned earlier, pivaloyl-modified thymidine 7b
undergoes a Norrish type I photocleavage liberating two alkyl
radicals upon loss of carbon monoxide, one of which is our
radical of interest (28). The other radical partner, the t-butyl
radical, is not believed to participate in the formation of
secondary products in such processes in oligonucleotides.28
Approximately 62% of 7b is converted to 29 after 1 h of
photolysis with no detection of side products. These results
support the use of 7b as a viable precursor of radical 2.
In the case of the phenyl selenide precursors 8a,b, the radical
of interest is generated by photochemical scission of the PhSe−
C bond to initiate a chain reaction that involves reaction with
nBu3SnH. This reaction is very efficient in the case of 8a,b
delivering reduction product 29 in high yield (59% for 8a and
92% for 8b) as the sole product after 15 min of photolysis. A
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11.2, 2.4), 3.91 (1H, dd, J = 11.6, 2.0), 3.99 (4H, m), 4.52 (1H, m);
13C (CDCl3, 400 MHz) δ −5.6, 18.2, 21.5, 25.8, 31.2, 44.1, 64.8, 65.0,
difference in stereochemistry at the C3′-position in the phenyl
selenides has an impact on photochemical conversion as
indicated by the lower yield when 8a is the precursor. This
observation is likely due to the difference in accessibility of the
phenyl selenide moiety to the tin radical needed to propagate
this radical chain reaction. Studies in DNA oligomers will be
performed under more physiological conditions employing
thiol trapping agents as well as molecular oxygen to determine
the ultimate fate of radical 28. Even though the studies herein
indicate that the radical of interest can be generated from
precursors 8a and b, their full utility will have to be determined
in the absence of nBu3SnH.
Of additional concern in the design of precursors 8a,b is their
ability to withstand the oxidative conditions required to obtain
phosphate linkages from phosphites or H-phosphonates during
automated DNA synthesis. RNA polymers containing alkyl
selenide moieties were obtained through automated RNA
synthesis using oxidative conditions identical to those found in
DNA synthesis.38 In these studies it was reported that exposure
of the alkyl selenide containing oligomer to 20 mM I2 for 20 s
led to the formation of 2−5% selenoxide when the modification
was near the 3′-terminus of the oligonucleotide. Our initial
investigations into the synthesis of oligomers containing 8a,b
using the H-phosphonate method do not indicate the
formation of the corresponding selenoxides using this method-
ology (data not shown). Collectively, these results indicate that
both 7 and 8 have the potential to be efficient precursors of the
radical of interest.
81.0, 109.5, 176.7. HRMS [M + H]+ calc. for C15H29O5Si 317.1784,
found 317.1785.
5-O-(tert-Butyldimethylsilyl)-3-C-(2-methyl-1,3-dioxolane)-
2,3-dideoxy-α- and β-D-pentofuranose(13). A solution of lactone
12 (0.31 g, 0.98 mmol) in anhydrous dichloromethane (3.0 mL) was
cooled to −78 °C, and diisobutylaluminum hydride (DIBAL-H, 0.93
mL) was added dropwise. After 5 h, methanol (0.16 mL) was added,
and stirring was continued at −78 °C for another 1.5 h. Ethyl acetate
(0.73 mL) and saturated NaHCO3 (0.16 mL) were added, and the
reaction was warmed to room temperature over a one-hour period.
The solution was dried over MgSO4, filtered through Celite, and
evaporated to dryness in vacuo to afford a clear oily residue. Yield: 0.30
1
g (97%); H NMR (CDCl3, 400 MHz) δ 0.06 (6H, s), 0.13 (6H, s),
0.89 (9H, s), 0.94 (9H, s), 1.30 (3H, s), 1.36 (3H, s), 1.90 (1H, dd, J =
13.8, 1.8), 2.01 (2H, m), 2.21 (1H, m), 2.53 (1H, m), 2.72 (1H, m),
3.62 (2H, m), 3.71 (1H, dd, J = 10.8, 3.6), 3.84 (1H, dd, J = 10.6, 2.2),
3.99 (8H, m), 4.18 (2H, m), 4.24 (1H, d, J = 9.2), 4.85 (1H, d, J =
11.2), 5.36 (2H, m); 13C (CDCl3, 400 MHz) δ −5.4, −5.2, 18.49, 18.5,
22.6, 22.9, 25.9, 26.0, 36.1, 38.4, 44.7, 46.9, 64.8, 64.91, 64.94, 65.6,
65.7, 79.6, 81.1, 81.2, 98.79, 98.8, 110.3, 110.5. HRMS [M − H]− calc.
for C15H29O5Si 317.1784, found 317.1785.
3-C-Acetyl-2,3-dideoxy-α- and β-D-pentofuranose (14). A
solution of lactone 13 (0.08 g, 0.25 mmol) in THF (4.29 mL) was
cooled to 0 °C, and 1 M HCl (0.69 mL) was added dropwise. The
reaction was warmed to room temperature, and stirring was continued
for 48 h. The reaction mixture was neutralized with Amberlyst A-21
ion-exchange resin, filtered, and the solution was evaporated to dryness
in vacuo to afford a grainy yellow oil. Purification by flash column
chromatography (9:1 dicholoromethane/methanol) afforded the
desired ketone as a yellow oil. Yield: 0.03 g (75%); 1H NMR
(CH3OD, 400 MHz) δ 4 isomers 1.79−2.20 (2H, m), 2.21−2.25 (3H,
m), 2.32−3.13 (1H, m), 3.63−4.15 (2H, m), 4.18−4.44, 4.64−4.68
(1H, m), 5.38−5.50 (1H, m); 13C (CH3OD, 600 MHz) δ 4 isomers
25.9, 27.4, 27.6, 27.7, 27.9, 29.4, 29.8, 30.1, 38.0, 38.3, 38.8, 38.9, 52.9,
53.4, 55.0, 56.7, 64.5, 65.1, 66.1, 66.6, 83.2, 92.0, 96.9, 99.6, 210.1,
210.4, 210.9, 211.6. HRMS [M + H]+ calc. for C7H13O4 161.0814,
found 161.0814.
The identification of sugar damage fragments induced by the
exposure of DNA to low energy electrons has been significantly
hindered by a lack of knowledge of their mechanisms of
formation. Radical precursors capable of generating one of the
intermediates observed in this process, the 2′,3′-dideoxy-C3′-
thymidinyl radical (28), will provide much needed information
on the structure of these lesions and will facilitate the
determination of the impact of these species on cellular
processes.
1-[(5-O-tert-Butyldimethylsilyl)-3-C-(hydroxymethyl)-2,3-di-
deoxy-β-D-threo-pentofuranosyl)]thymine (16). To a solution of
15 (0.94 g, 2.66 mmol) in anhydrous THF (5.4 mL) under nitrogen
was added BH3/1,4-oxathiane (0.33 mL of a 7.8 M solution in
oxathiane, 2.87 mmol) at room temperature. After cooling of this
mixture to 0 °C, a 2 M solution of NaOH (1.5 mL) was slowly added,
followed by the dropwise addition of 30% aqueous H2O2 (0.38 mL).
Stirring was continued for 1 h at room temperature. The reaction
mixture was poured into ice-water (68 mL) and extracted with
diethylether (80 mL). The combined organic phase was washed with
water (68 mL) and saturated aqueous NaHCO3 (2 × 68 mL), dried
(Na2SO4), and evaporated to dryness in vacuo. The product was
purified on a silica gel column (50:50 ethylacetate/pentane) to give 16
EXPERIMENTAL SECTION
■
General Methods. All reactions were carried out in oven-dried
glassware under an atmosphere of argon unless otherwise stated.
Anhydrous THF was dried over activated alumina. Column
chromatography was performed on an automated chromatography
system equipped with an in-line variable wavelength detector. TLC
was performed on precoated TLC plates of silica gel 60 F-254. All
nuclear magnetic resonance spectra were recorded on either a 400 or
600 MHz NMR in CDCl3 or CD3OD as solvent. All chemical shifts
are reported in parts per million downfield from TMS. Coupling
constants are given in Hertz. HPLC analysis was performed on a liquid
chromatography system using a C-18 5 μm column (4.6 × 250 cm).
Gradients: A stepwise gradient was applied. A: 50 mM TEAA buffer
pH = 7.0. B: acetonitrile; 0−20% B over 15 min, 20−75% B over 3
min, 75−90% B linearly over 4 min. Flow rate, 1.0 mL/min.
1
as a clear colorless oil. Yield: 0.88 g (89%); H NMR (CDCl3, 400
MHz) δ 0.15 (6H, s, Si(CH3)2), 0.94 (9H, s, SiC(CH3)3), 1.81 (1H,
m, 2′-Hβ), 1.93 (3H, s, CH3), 2.44 (1H, m, 2′-Hα), 2.72 (1H, m, 3′-H),
3.34 (1H, t, J = 6.4 Hz, OH), 3.73−3.89 (3H, m, CH2OH, 5′-H), 4.00
(1H, m, 5′-H), 4.16 (1H, m, 4′-H), 6.06 (1H, dd, J = 8.2, 6.2 Hz, 1′-H),
7.48 (1H, s, 6-H), 9.44 (1H, br s, NH); 13C (CDCl3, 400 MHz) δ
−5.3, 12.8, 18.4, 26.0, 34.0, 42.6, 61.8, 62.6, 79.8, 84.3, 111.0, 135.6,
150.8, 164.2. HRMS [M + H]+ calc. for C17H31O5N2Si 371.2002,
found 371.2001.
1-[(5-O-tert-Butyldimethylsilyl)-3-C-(formyl)-2,3-dideoxy-β-
D-threo-pentofuranosyl)]thymine (17). A solution of 16 (0.83 g,
2.24 mmol) in CH2Cl2 (8 mL) was cannulated into a solution of
Dess−Martin periodinane (1.43 g, 3.37 mmol) in anhydrous CH2Cl2
(16 mL) at 0 °C. After the mixture was stirred overnight at room
temperature, diethyl ether (70 mL) was added, and the solution was
poured slowly into a solution of saturated NaHCO3 (45 mL)
containing Na2S2O3·5H2O (5.56 g). The organic layer was removed,
and the aqueous layer was extracted with diethyl ether (95 mL). The
5-O-(tert-Butyldimethylsilyloxymethyl)-4-C-(2-methyl-1,3-
dioxolane)-tetrahydrofuran-2-one (12). A pyrex test tube (16 ×
150 mm) was charged with protected butenolide 9 (0.40 g, 1.75
mmol), benzophenone (11) (0.32 g, 1.76 mmol), and 2-methyl-1,3-
dioxolane 10 (14.0 mL). After degassing for 1 h under a constant
stream of nitrogen, the stirred solution was irradiated for 2 h with a
500 W Hg arc source. The solution was then evaporated to dryness in
vacuo to afford a clear, pale yellow oil. Purification by flash column
chromatography (9:1 pentane/ethyl acetate) afforded the title
compound as a clear colorless oil. Yield: 0.48 g (87%); 1H NMR
(CDCl3, 400 MHz) δ 0.07 (3H, s), 0.08 (3H, s), 0.89 (9H, s), 1.30
(3H, s), 2.48 (1H, dd, J = 17.0, 3.4), 2.71 (2H, m), 3.67 (1H, dd, J =
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combined organic phase was washed with saturated NaHCO3 (95 mL)
followed by H2O (95 mL), dried (Na2SO4), and evaporated to dryness
in vacuo. The product was isolated as a white amorphous solid. Yield:
0.83 g (100%); 1H NMR (CDCl3, 600 MHz) δ 0.10 (6H, s,
Si(CH3)2), 0.91 (9H, s, SiC(CH3)3), 1.94 (3H, d, J = 1.2 Hz, CH3),
2.43 (2H, m, 2′-H), 3.27 (1H, m, 3′-H), 3.95 (2H, m, 5′-H), 4.40 (1H,
m, 4′-H), 6.14 (1H, dd, J = 7.2, 6.60 Hz, 1′-H), 7.44 (1H, d, J = 1.2 Hz,
6-H), 8.27 (1H, br s, NH), 9.85 (1H, s, CHO); 13C (CDCl3, 600
MHz) δ −5.5, 12.7, 18.3, 26.0, 31.4, 51.0, 61.8, 80.3, 83.8, 111.4, 135.4,
150.9, 164.2, 199.5. HRMS [M + H]+ calc. for C17H29O5N2Si calc.
369.1846, found 369.1846.
2.24 (1H, m), 2.45 (1H, d, J = 5.6 Hz), 2.71 (2H, m), 3.19 (1H, d, J =
5.2 Hz), 3.33 (1H, t, J = 4.99 Hz), 3.81, 3.88 (2H, 2 dd, J = 11.4, 2.8
Hz), 3.98 (1H, m), 4.07 (2H, m), 4.33 (1H, m), 6.13 (1H, t, J = 6.8
Hz), 6.23 (1H, t, J = 6.4 Hz), 7.36−7.69 (22H, m), 9.08 (1H, br s),
9.17 (1H, br s); 13C (CDCl3, 400 MHz) δ 12.3, 19.6, 26.6, 26.7, 27.2,
32.8, 35.8, 36.1, 38.5, 40.1, 40.6, 63.3, 66.0, 77.4, 82.2, 82.9, 84.3, 85.4,
85.6, 111.0, 128.03, 128.07, 128.12, 130.11, 130.15, 130.19, 130.24,
132.74, 132.96, 133.27, 133.34, 135.57, 135.69, 135.75, 135.81, 150.6,
150.7, 164.2. HRMS [M + Na]+ calc. for C31H42O5N2SiNa 573.2761,
found 573.2760.
1-[5-(tert-Butyldiphenylsilyl)-2,3-dideoxy-3-C-pivaloyl-β-D-
erythro-pentofuranosyl]thymine (23). A solution of 22 (0.17 g,
0.309 mmol) in anhydrous CH2Cl2 (1 mL) was added to a solution of
the Dess−Martin periodinane (0.2 g, 0.472 mmol) in anhydrous
CH2Cl2 (2.2 mL) at 0 °C. Stirring was continued at 0 °C for 15 min
and then at room temperature overnight. The reaction mixture was
diluted with diethyl ether (10 mL), poured into ice-cold aqueous
saturated NaHCO3 (6.2 mL) containing Na2S2O3.5H2O (0.77 g, 3.09
mmol), and stirred for 10 min. The organic layer was washed with
saturated NaHCO3, H2O, and saturated NaCl, dried over Na2SO4, and
the solvent was removed under reduced pressure to afford 23 as
1-[(5-O-tert-Butyldimethylsilyl)-3-C-(2,2-dimethyl-1-hydrox-
ypropyl)-2,3-dideoxy-β-D-threo-pentofuranosyl)]thymine (18).
Cerium chloride was dried as described by Kamiya.39 To a suspension
of dry CeCl3 (16.7 g, 44.8 mmol) in THF (105 mL) at −78 °C was
added a 1.6 M solution of t-butyllithium (28.0 mL, 44.8 mmol) in
pentane. This was allowed to stir for 1.5 h before a solution of 17 (0.83
g, 2.24 mmol) in THF (41 mL) was added dropwise at the same
temperature. The reaction stirred at this temperature for 3.5 h, after
which it was quenched by the addition of aqueous NH4Cl (200 mL)
and allowed to warm to room temperature. The product was then
extracted with CH2Cl2 (6 × 250 mL), the organic layer was dried over
Na2SO4, and the solvent was removed in vacuo. The crude product
was purified on a silica gel column (40% ethyl acetate in hexane) to
give 18 as a diasteromeric mixture of a clear colorless oil. Only one
isomer was successfully purified and characterized. Yield: 0.42 g
1
colorless foam. Yield: 0.17 g (100%); H NMR (CDCl3, 400 MHz) δ
1.11 (18H, s, SiC(CH3)3 and COC(CH3)3), 1.62 (3H, d, J = 0.8,
CH3), 2.24 (1H, m, 2′-Hβ), 2.43 (1H, m, 2′-Hα), 3.66 (1H, dd, J =
11.8, J = 2.6, 5′-H), 3.84 (1H, m, 3′-H), 4.09 (1H, dd, J = 12, J = 2, 5′-
H), 4.25 (1H, dt, J = 6.8, J = 2.4, 4′-H), 6.22 (1H, dd, J = 6.8 Hz, J =
5.2 Hz, 1′-H), 7.37−7.47 (6H, m, Ar), 7.53 (1H, d, J = 0.8, 6-H),
7.64−7.68 (4H, m, Ar), 8.88 (1H, br s, NH); 13C (CDCl3, 400 MHz)
δ 12.3, 19.6, 26.0, 27.3, 30.5, 39.0, 43.7, 44.9, 63.5, 83.8, 85.6, 111.1,
128.2, 130.3, 132.8, 135.6, 150.3, 164.1, 215.3. HRMS [M + Na]+ calc.
for C31H40O5N2SiNa 571.2604, found 571.2617.
1
(44%); H NMR (CDCl3, 400 MHz) δ 0.16 (6H, s), 0.94 (18H, 4s),
1.93 (3H, d, J = 1.2 Hz), 2.15 (1H, m), 2.27 (1H, m), 2.69 (1H, m),
3.47 (1H, d, J = 3.2), 3.60 (1H, d, J = 2.8 Hz), 3.90 (1H, m), 4.03 (2H,
m), 6.08 (1H, dd, J = 9.0, 5.8 Hz), 7.50 (1H, d, J = 1.2 Hz), 8.8 (1H,
br s); 13C (CDCl3, 400 MHz) δ −5.2, 12.8, 18.5, 26.0, 27.0, 31.5, 35.5,
40.8, 63.0, 75.9, 80.5, 83.9, 111.1, 135.9, 150.8, 163.9. HRMS [M +
H]+ calc. for C21H39O5N2Si 427.2628, found 427.2629.
1-(2,3-Dideoxy-3-C-pivaloyl-β-D-erythro-pentofuranosyl)-
thymine (7b). To a solution of 23 (0.15 g, 0.27 mmol) in THF (2.3
mL) was added a 1 M solution of TBAF in THF (0.41 mL) at room
temperature. Stirring was continued for 1 h. The solvent was removed
in vacuo, and the crude mixture was purified by column
chromatography with ethyl acetate to afford 7b as a colorless foam.
1-[(5-O-tert-Butyldimethylsilyl)-3-C-(2,2-dimethyl-1-oxo-
propyl)-2,3-dideoxy-β-D-threo-pentofuranosyl)]thymine (19).
To a solution of Dess−Martin periodinane (0.30 g, 0.71 mmol) in
anhydrous CH2Cl2 (3 mL) was cannulated a solution of 18 (0.20 g,
0.47 mmol) in CH2Cl2 (2 mL) at 0 °C. Stirring was continued at 0 °C
for 15 min and then at room temperature for 4 h. Diethylether (14
mL) was added, and the solution was poured slowly into a solution of
(1.16 g, 4.69 mmol) Na2S2O3·5H2O in saturated NaHCO3 (9 mL).
The organic phase was washed with saturated NaHCO3 (20 mL),
followed by H2O (20 mL) and saturated NaCl (20 mL), dried with
MgSO4, and evaporated to dryness in vacuo. The product was isolated
1
Yield: 0.04 g (44%); H NMR (CDCl3, 400 MHz) δ 1.18 (9H, s,
COC(CH3)3), 1.93 (3H, s, CH3), 2.44 (2H, m, 2′-H), 2.65 (1H, dd, J
= 6.2 Hz, J = 3.4, 5′-OH), 3.62 (1H, m, 3′-H), 3.97 (2H, m, 5′-H), 4.29
(1H, dt, J = 8 Hz, J = 2.4 Hz, 4′-H), 6.0 (1H, dd, J = 7.2 Hz, J = 4.4 Hz,
1′-H), 7.48 (1H, d, J = 0.8 Hz, 6-H), 8.67 (1H, br s, NH); 13C (CDCl3,
400 MHz) δ 12.8, 25.8, 38.7, 43.3, 45.2, 61.9, 84.7, 88.2, 110.9, 137.6,
150.4, 164.1, 215.8. HRMS [M + Na]+ calc. for C15H22O5N2Na
333.1426, found 333.1419.
1
as a white solid. Yield: 0.19 g (96%); H NMR (CDCl3, 400 MHz) δ
0.06 (6H, s, Si(CH3)2), 0.88 (9H, s, SiC(CH3)3), 1.16 (9H, s,
COC(CH3)3), 1.96 (3H, d, J = 0.8 Hz, CH3), 2.06 (1H, m, 2′-Hβ),
2.49 (1H, m, 2′-Hα), 3.68 (2H, m, 5′-H), 3.81 (1H, m, 3′-H), 4.20 (1H,
m, 4′-H), 6.14 (1H, dd, J = 8.0, 6.0 Hz, 1′-H), 7.51 (1H, d, J = 0.8 Hz,
6-H), 9.23 (1H, br s, NH); 13C (CDCl3, 400 MHz) δ −5.1, 12.9, 18.7,
26.0, 26.2, 36.7, 44.2, 45.0, 62.6, 80.8, 84.3, 111.4, 135.6, 150.8, 164.0,
214.1. HRMS [M + H]+ calc. for C21H37O5N2Si 425.2472, found
425.2473.
1-[5-(tert-Butyldiphenylsilyl)-2,3-dideoxy-3-C-(2,2-dimethyl-
1-hydroxypropyl)-β-D-erythro-pentofuranosyl]thymine (22).
Cerium chloride was dried as described by Kamiya.39 To a suspension
of dry CeCl3 (8.32 g, 22.34 mmol) in THF (52 mL) was added
dropwise 1.7 M t-butyllithium in pentane (13.14 mL, 22.34 mmol) at
−78 °C. The solution was allowed to stir at this temperature for 1.5 h.
Aldehyde 21 (0.55 g, 1.117 mmol) in THF (20 mL), which was
coevaporated with dry THF, was added over 25 min and allowed to
stir at the same temperature for 8 h, before being quenched by the
addition of of saturated NH4Cl (101 mL) at −78 °C. The reaction
mixture was extracted with dichloromethane (× 6), the organic layer
was dried over Na2SO4, filtered, and the solvent was removed under
reduced pressure to afford a brownish foam. The crude mixture was
purified by column chromatography with 1:1 ethyl acetate/hexane to
afford 22 as a diastereomeric mixture in the form of a colorless foam.
Yield: 0.17 g (30%); 1H NMR (CDCl3, 400 MHz) δ 0.88−0.92 (18H,
2s), 1.1 (18H, s), 1.61 (6H, s), 1.85 (1H, m), 2.02 (1H, d, J = 5.6),
1-[5-O-(4,4′-Dimethoxytrityl)-2,3-dideoxy-3-C-selenophen-
yl-β-D-threo-pentofuranosyl]thymine (25). To a cold solution (0
°C) of diphenylselenide (2.9 g, 9.3 mmol) in THF (116 mL) was
added LiAlH4 (0.26 g, 6.9 mmol) portion-wise under N2 (g). Upon
warming, the solution turned colorless. To this was added
methanesulfonate 24 (3.6 g, 5.8 mmol) in anhydrous THF (58
mL). The mixture was heated at reflux for 2 h and then allowed to
cool. The reaction mixture was quenched with methanol (58 mL), and
the solvent was removed in vacuo. The residue was treated with
saturated NH4Cl solution (116 mL) and extracted with ethyl acetate
(3 × 116 mL). The organic layer was washed with water (2 × 58 mL)
and brine (58 mL), dried over MgSO4, and concentrated. The crude
product was purified by silica gel column chromatography with ethyl
1
acetate/hexane (1:3) to afford a white foam. Yield: 2.4 g (78%); H
NMR (CDCl3, 400 MHz) δ 1.46 (3H, s), 2.37 (1H, m), 2.82 (1H, m),
3.48 (1H, dd, J = 10.4 Hz, J = 4.4 Hz), 3.62 (1H, dd, J = 10.8 Hz, J =
2.8 Hz), 3.79 (6 H, s), 3.85 (1H, q, J = 16.0 Hz, J = 7.6 Hz), 4.4 (1H,
m), 6.16 (1H, t, J = 6.6 Hz), 6.8−7.5 (18H, m), 7.72 (1 H, s), 9.47
(1H, bs); 13C NMR (CDCl3, 400 MHz) δ 11.9, 40.2, 41.3, 55.4, 65.1,
81.0, 84.1, 87.4, 111.1, 113.2, 113.2, 127.3, 128.0, 128.7, 129.5, 130.6,
130.6, 134.0, 135.3, 136.2, 144.2, 150.8, 158.8, 164.2. HRMS [M +
Na]+ calc. for C37H36O6N2SeNa 707.1636, found 707.1649.
1 - ( 2 , 3 - D i d e o x y - 3 - C - s e l e n o p h e n y l - β - D - t h r e o -
pentofuranosyl]thymine (8a).34 To compound 25 (1.19 g, 1.74
mmol) was added a solution of acetic acid/water (4:1 v/v), and the
3835
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The Journal of Organic Chemistry
Article
reaction mixture was heated to 85 °C for 1 h. After cooling to room
temperature, the solvent was removed in vacuo. The residue was
coevaporated with several portions of methanol. Precipitation of the
pure product was achieved by addition of dichloromethane followed
by ethyl acetate. The white solid was isolated by filtration. Yield: 0.65 g
(91%); 1H NMR (CD3OD, 400 MHz) δ 1.92 (3H, d, J = 0.8 Hz), 2.3
(1H, m), 2.8 (1H, m), 3.92 (2H, m), 4.05 (1H, q, J = 7.6 Hz), 4.36
(1H, m), 6.06 (1H, t, J = 6.6 Hz), 7.3 (3H, m), 7.54 (2H, m), 8.03
(1H, d, J = 0.8 Hz); 13C (CD3OD, 400 MHz) δ 12.7, 41.0, 42.2, 64.7,
71.7, 83.8, 86.0, 111.2, 128.9, 130.6, 131.4, 134.76, 138.6. HRMS [M +
Na]+ calc. for C16H18O4N2SeNa 405.0329, found 405.0305.
product was chromatographed on silica gel with 6% methanol in
dichloromethane to afford compound 29 as a white powder. Yield: 0.3
1
g (96%); H NMR (CDCl3, 400 MHz) δ 1.92 (3H, d, J = 0.8 Hz),
2.06 (3H, m), 2.4 (1H, m), 3.74 (1H, d, J = 12.0 Hz), 4.01 (1H, d, J =
12.0 Hz), 4.19 (1H, m), 6.12 (1H, dd, J = 6.8 Hz, J = 4.0 Hz), 7.53
(1H, d, J = 0.8 Hz), 8.72 (1H, bs); 13C (CDCl3, 400 MHz) δ 12.83,
25.34, 32.33, 63.71, 81.39, 86.39, 110.79, 136.48, 150.52, 163.96.
HRMS [M + Na]+ calc. for C10H14O4N2Na 249.0851, found 249.0846.
Standard Photolysis Conditions. A solution of either radical
precursor 8b/7b (1 mM in 1:1 CH3CN/H2O) or 8a (1 mM in
1:1:0.01 CH3CN/H2O/THF) was transferred to a quartz cuvette and
degassed by bubbling argon through the solution for 20 min. tri-
Butyltin hydride (1000 equiv) was added under an argon atmosphere.
The mixture was immediately photolyzed (8 for 15 min, 7b for 60
min) at 15 °C. The photolysis was performed using an 500 W high-
pressure mercury arc lamp fitted with an IR filter, focusing lens, and
320 nm cutoff filter. The irradiation mixture was analyzed directly
without workup by analytical HPLC with UV detection at 254 nm.
Products were identified by ESI-MS and by comparison with authentic
samples. Product yields were determined using standard curves.
1-[5-O-(4,4′-Dimethoxytrityl)-2,3-dideoxy-3-C-selenophen-
yl-β-D-erythro-pentofuranosyl]thymine (27). Diphenylselenide
(2.0 g, 5.00 mmol) was rendered anhydrous by repeated evaporation
with pyridine. The residue was dissolved under argon in anhydrous
THF (32 mL) and cooled to 0 °C. LiAlH4 (142.5 mg, 3.75 mmol) was
added portion-wise, and the solution was allowed to warm to room
temperature, resulting in the formation of a clear solution. To this was
added a THF (6.5 mL) solution of anhydrothymidine 26 (1.5 g, 3.2
mmol). The reaction mixture was heated under reflux for 2 h, allowed
to cool, and quenched with methanol (6.5 mL). The solvent was
removed under reduced pressure, and the residue was treated with
saturated NH4Cl solution (65 mL) and extracted with ethylacetate (3
× 65 mL). The organic layer was washed with water (2 × 33 mL) and
brine (33 mL), dried over MgSO4, and concentrated. The crude
product was purified by silica gel column chromatography CH2Cl2/
ASSOCIATED CONTENT
■
S
* Supporting Information
HPLC chromatograms. 1H, 13C, and 1H-NOESY NMR spectra.
Standard curves. This material is available free of charge via the
Internet at http://pubs.acs.org.
1
MeOH (98:2) to afford a white foam. Yield: 1.95 g (89%); H NMR
(CDCl3, 400 MHz) δ 1.39 (3H, d, J = 0.8 Hz), 2.52 (2H, m), 3.39
(1H, dd, J = 10.8 Hz, J = 3.2 Hz), 3.57 (1H, dd, J = 11.0 Hz, J = 2.4
Hz), 3.79 (6H, s), 3.85 (1H, q, J = 8.4 Hz), 4.05 (1H, dt, J = 8.4 Hz, J
= 2.4 Hz), 6.1 (1H, dd, J = 6.6 Hz, J = 3.6 Hz), 6.8−7.5 (18H, m), 7.72
(1H, d, J = 0.8 Hz), 9.35 (1H, bs); 13C NMR (CDCl3, 400 MHz) δ
12.0, 36.9, 40.9, 55.4, 62.2, 85.0, 85.8, 86.9, 110.8, 113.3, 127., 127.3,
128.1, 128.3, 128.7, 129.5, 130.3, 135.6, 135.8, 144.5, 150.5, 158.8,
164.3. HRMS [M + Na]+ calc. for C37H36O6N2SeNa 707.1636, found
707.1663.
AUTHOR INFORMATION
Corresponding Author
*E-mail: amanda.bryant-friedrich@utoledo.edu.
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
1-(2, 3-Dideoxy-3-C-se lenophe nyl- β-D -erythro-
pentofuranosyl)thymine (8b).34 A solution of water/glacial acetic
acid (1:4 v/v) was added to compound 27 (1.7 g, 2.5 mmol), and the
mixture was heated to 85 °C for 2 h. The reaction mixture was cooled
to room temperature, and the solvent was removed in vacuo. The
residue was coevaporated with methanol and purified on silica gel with
ethyl acetate−hexane (1:3) to afford a white foam. Yield: 0.92 g
We are thankful to the National Science Foundation (CHE-
512973 and 0848383), Oakland University, and the University
of Toledo for supporting this work. MALDI-ToF MS was
performed on instrumentation obtained through an NSF grant
(DBI-923184). We also thank Dr. Yong Wah Kim for his
assistance on 2D NMR analysis.
1
(85%); H NMR (CDCl3, 400 MHz) δ 1.9 (3H, d, J = 0.8 Hz), 2.27
(1H, t, J = 4.8 Hz), 2.45 and 2.56 (2H, each as m), 3.79 (2H, m), 4.02
(2H, m), 6.02 (1H, dd, J = 6.9 Hz, J = 3.8 Hz), 7.3−7.6 (6H, m), 8.5
(1H, bs); 13C NMR (CDCl3, 400 MHz) δ 12.8, 35.9, 40.0, 61.2, 85.7,
86.6, 110.9, 126.7, 128.9, 129.7, 135.9, 136.8, 151, 164. HRMS [M +
Na]+ calc. for C16H18O4N2SeNa 405.0329, found 405.0322.
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