Synthesis of the first tellurium-derivatized oligonucleotides for structural and functional studies

Article abstract of DOI:10.1002/chem.200900774

We report here the first synthesis of Te-nucleoside phosphoramidites and Te-modified oligonucleotides. We protected the 2′-tellurium functionality by alkylation and found that the Te functionality is compatible with solid-phase synthesis and that the Te o

Full text of DOI:10.1002/chem.200900774

DOI: 10.1002/chem.200900774
Synthesis of the First Tellurium-Derivatized Oligonucleotides for Structural
and Functional Studies
Jia Sheng, Abdalla E. A. Hassan, and Zhen Huang*[a]
Abstract: We report here the first syn-
thesis of Te-nucleoside phosphorami-
dites and Te-modified oligonucleotides.
We protected the 2’-tellurium function-
ality by alkylation and found that the
Te functionality is compatible with
solid-phase synthesis and that the Te
oligonucleotides are stable during de-
protection and purification. In addi-
tion, the redox properties of the Te
functionalities have been explored. We
found that the telluride and telluoxide
DNAs are interchangeable by redox re-
actions. At elevated temperature, the
Te-DNA can also be site-specifically
fragmented oxidatively or reductively
when 2’-TePh functionality is present,
whereas elimination of the nucleobase
is observed in the presence of 2’-TeMe.
Moreover, the stability of the DNA du-
plexes derivatized with the Te function-
alities has been investigated. Our Te
derivatization of nucleic acids provides
a
novel approach for investigating
DNA damage as well as for structure
and function studies of nucleic acids
and their protein complexes.
Keywords: DNA damage · oligonu-
cleotides · phosphoramidites · tellu-
rium · X-ray crystallography
Introduction
logical molecules. However, tellurium was successfully incor-
porated into proteins in 1989 through Te-resistant fungi
Replacement of the sulfur in methionine with selenium has
been widely used for protein-structure determination by X-
ray crystallography; here selenium serves as a scattering
grown in the presence of tellurite on a sulfur-free
medium.[22] Later, Te-methionine derivatization for protein-
structure determination was investigated and developed.
Boles and co-workers reported the expression of tellurome-
thionine (Te-Met) dihydrofolate reductase in 1994,[23] and
the Te-incorporation technique was further optimized to ef-
ficiently introduce Te-Met into several proteins.[24] Further-
more, the Te-protein stability under X-ray radiation and the
isomorphism of the Te proteins were confirmed, and more
tellurium chemistry has been developed.[25–27]
center
for
multiwavelength
anomalous
dispersion
(MAD).[1–3] Inspired by this revolution in protein-structure
determination, our research group has pioneered and devel-
oped selenium-derivatized nucleic acids (SeNA) for struc-
ture determination through atom-specific replacement of
oxygen with selenium.[4,5] Recently selenium has been intro-
duced at several different positions of DNAs and RNAs, in-
cluding the 5’,[6] 2’,[7–12] and 4’[13–14] positions of the ribose, the
phosphate backbone,[15–17] and the nucleobases.[18–20]
Moreover, the electronic and atomic properties of telluri-
um are ideal for generating clear isomorphous signals just
by using a normal in-house X-ray diffraction facility (CuKa
l=1.54 ꢀ). It was demonstrated that a synchrotron facility
is not necessary in order to collect X-ray diffraction data for
phase and structure determination of proteins derivatized
with the Te functionality. This Te-derivatization strategy is a
promising alternative to the conventional heavy-atom-soak-
ing procedure for multiple isomorphous replacements
(MIR).[28] Our success in the selenium derivatization of nu-
cleic acids has naturally encouraged us to develop tellurium-
derivatized nucleic acids (TeNA) for structure and function
studies. Herein we report the first synthesis of Te-derivat-
ized oligonucleotides (Scheme 1) and the investigation of
the chemical properties of TeNA.
As a chalcogen, tellurium follows sulfur and selenium in
Group VI of the periodic table. Tellurium has a larger
atomic radius (1.35 ꢀ) than selenium (1.17 ꢀ) and sulfur
(1.04 ꢀ). Probably due to its metallic properties and weak
covalent bonds with carbon and hydrogen,[21] no tellurium
functionality has so far been discovered in any natural bio-
[a] Dr. J. Sheng, Dr. A. E. A. Hassan, Prof. Z. Huang
Department of Chemistry, Georgia State University
50 Decatur Street, Atlanta, GA 30303-3083 (USA)
Fax : (+1)404-413-5505
E-mail: huang@gsu.edu
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.200900774.
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Chem. Eur. J. 2009, 15, 10210 – 10216

FULL PAPER
phenyltelluride nucleophile to attack the 2’-position from
the a face and giving a satisfactory yield. The generated key
intermediates 4a and 4b were successfully converted to the
corresponding phosphoramidites 5a and 5b in high yields by
following the standard protocol.[8] In contrast, it is difficult
to reduce dimethylditelluride efficiently with sodium boro-
hydride, even at an elevated temperature, probably due to
the relatively higher reactivity of methyltelluride. Further-
more, at the elevated temperature required for Te function-
alization, enol ether 6, created by nucleobase 1’,2’ elimina-
tion, was observed as the major product (Scheme 3). When
Scheme 1. Selenium- and tellurium-derivatized methionine and oligonu-
cleotide.
Results and Discussion
Our previous studies of nucleic acids derivatized with seleni-
um at the 2’-position of uridine and thymidine have revealed
that 2’-Se modification does not cause significant perturba-
tion of DNA and RNA structures.[8,9] Thus, we decided to
first synthesize the 2’-Te-nucleoside phosphoramidites (5a
and 5b in Scheme 2) and the 2’-Te-DNAs. 5’-DMTr-2,2’-an-
Scheme 3. Elimination reactions resulting from the 2’-TeMe functionality.
a) MeTeTeMe, NaBH4; b) AcOAc, Et3N; c) MeTeTeMe, NaBH4.
we protected the 3’-hydroxyl groups of 3a and 3b by acety-
lation to prevent their deprotonation during the Te function-
alization, to our surprise, 2’,3’ elimination was observed,[29]
which generated the olefin analogues 8a and 8b, the precur-
sors of the antiviral drug d4T.
Due to the difficulty of efficient dimethylditelluride re-
duction, a stronger reducing reagent (such as LiAlH4) was
used (Scheme 4). However, only trace amounts of the de-
sired compound 9 were observed. In order to minimize by-
product formation, we decided to run the telluride incorpo-
ration at a lower temperature (08C). Furthermore, we used
crown ether ([12]crown-4) to chelate the lithium ions in
order to enhance the methyltelluride reactivity. Fortunately,
we were able to obtain the desired product in 47% yield,
whereas the 1’,2’-eliminated product was still the major by-
product. The characteristic chemical shift of the Te-methyl
group was observed at ꢀ21.2 ppm in the 13C NMR spectrum.
Moreover, the tellurium isotope distributions in the HRMS
spectra indicated the incorporation of the Te functionality.
The generated key intermediates 9a and 9b were successful-
ly converted to the corresponding phosphoramidites 10a
and 10b in high yields by following the standard protocol.[8]
These synthesized Te-phosphoramidites were incorporat-
ed into oligonucleotides by solid-phase synthesis, generally
Scheme 2. Synthesis of the 2’-Te-uridine and 2’-Te-thymidine phosphor-
ACHTUNGTRENNUNGamidites (5a and 5b) and the Te-DNAs. a) PhTeTePh, NaBH4; b) 2-cya-
noethyl, N,N-diisopropyl chlorophosphoramidite; c) solid-phase synthesis.
hydrouridine 3a and thymidine 3b (DMTr=4,4’-dimethoxy-
trityl), which we had synthesized previously,[8,10] were chosen
as the starting materials. The tellurium functionalities were
introduced by an SN2 reaction after the commercially avail-
able tellurium reagents (dimethylditelluride and diphenyldi-
telluride) had been reduced. Like in the methylselenide in-
corporation, we initially tried to incorporate the correspond-
ing Te by using dimethylditelluride. However, due to the
synthetic challenges caused by the 1’,2’- and 2’,3’-elimina-
tions when using dimethylditelluride, we decided to incorpo-
rate the Te by using diphenylditelluride (Scheme 2). It
turned out it is easy to reduce diphenylditelluride with
sodium borohydride in dry THF at room temperature. The
ring-opening reaction went well at 508C, thus allowing the
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Z. Huang et al.
Figure 1. HPLC analysis of the Te-DNA [5’-DMTr-G(2’-TePh-dU)GTA-
CAC-3’]. This analysis was performed on a Zorbax SB-C18 column (4.6ꢂ
250 mm) with a linear gradient from 100% buffer A (20 mm triethylam-
monium acetate (TEAAc), pH 7.1) to 100% buffer B (50% acetonitrile
in 20 mm TEAAc) over 20 min. The retention time of the full-length Te-
DNA is 20.7 min.
Scheme 4. Synthesis of the 2’-Te-uridine and 2’-Te-thymidine phosphor-
ACHTUNGTRENNUNGamidites (10a and 10b) and the Te-DNAs. a) MeTeTeMe, LiAlH4,
Table 1. MALDI-TOF analysis of 2’-Te modified oligonucleotides.
[12]crown-4, THF; b) 2-cyanoethyl, N,N-diisopropyl, chlorophosphorami-
dite; c) solid-phase synthesis.
DNA sequence
Formula
MS [MꢀH]+
AHCTUNGTRENGN(UN calcd)
G(2’-TeMe-dU)GTACAC
ATGG(2’-TeMe-dU)GCTC
G(2’-TePh-dU)GTACAC
ATGG(2’-TePh-dU)GCTC
GCG(2’-TePh-dU)ATACGC
G(2’-TePh-dU)GTACAC
ATGG(2’-TePh-dU)GCTC
CT(2’-TePh-dU)CTTGTCCG
CT(2’-TeMe-dU)CTTGTCCG
C78H99N30O46P7Te
C88H112N32O54P8Te
C83H101N30O46P7Te
C93H114N32O54P8Te
C102H125N38O58P9Te
C84H103N30O46P7Te
C94H116N32O54P8Te
C111H140N32O69P10Te
C106H138N32O69P10Te
2536.1 (2536.2)
2856.3 (2856.4)
2599.4 (2599.3)
2919.0 (2918.5)
3218.0 (3216.7)
2611.8 (2612.3)
2933.3 (2932.5)
3464.5 (3465.4)
3400.5 (3401.8)
in >95% coupling yield, by using 5-(benzylmercapto)-1H-
tetrazole (5-BMT) as the activator. We found that the bulky
2’-Te moieties did not significantly affect the coupling reac-
tion and that these 2’-Te-modified phosphoramidites could
achieve almost quantitative coupling even with a standard
25 seconds of coupling time. The Te-oligonucleotides were
synthesized in the DMTr-on mode by using ultramild phos-
phoramidites and were cleaved with K2CO3 at room temper-
ature for 8 h.[18] To reduce the partially oxidized Te-DNAs, a
diborane treatment was performed right after the deprotec-
tion step. As an example, the HPLC profile of a crude 2’-
TePh-dU DNA with DMTr-on is showed in Figure 1. In ad-
dition, we determined the Te-coupling yield to be 95%.
After HPLC purification, the DMTr group was removed by
treatment with 3% trichloroacetic acid, and the oligonucleo-
tides were purified again by HPLC, followed by MALDI-
TOF MS analysis to confirm the integrity of these Te-DNAs
(Table 1 and Figure 2). To our surprise, both methyltelluride
and phenyltelluride functionalities are stable during the de-
protection and purification.
It is noteworthy that a few of the Te-DNAs are oxidized
to telluoxides during the solid-phase synthesis and purifica-
tion (Figure 2). Fortunately, the telluoxides can be reduced
back to the tellurides. Using the purified 2’-Te-DNAs, we
demonstrated that, at room temperature, telluride DNA can
be oxidized to telluoxide DNA by I2, and the telluoxide
DNA can be reduced back to the telluride DNA by B2H6
treatment (Scheme 5 and Figure 3). This provides a useful
approach to recover the oxidized Te-DNA during DNA syn-
thesis, and also allows investigation of the redox properties
of the Te-DNA. Interestingly, under heating (508C), we ob-
served a site-specific cleavage of the 2’-TePh DNA at the
Figure 2. MALDI-TOF MS analysis of the Te-DNA [5’-G(2’-TePh-dU)G-
TACAC-3’]. The peak with a mass of [M+16] indicates formation of the
telluoxide.
modification site in the presence of either B2H6 or I2
(Scheme 5 and Figure 4). This result is consistent with our
previous observation of the 2’,3’ elimination with the Te-nu-
cleosides.[29] This site-specific fragmentation provides a good
model for the study of DNA damage. Furthermore, in a 2’-
TeMe-modified DNA nonamer [5’-ATG G(2’-TeMe-dU)G
CTC-3’], the 1’,2’ base-elimination reaction was observed
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Tellurium-Derivatized Oligonucleotides
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Scheme 5. Redox and fragmentation of DNA oligonucleotides containing
2’-Te functionalities. a) B2H6 or I2, 508C; b) B2H6; c) I2; d) B2H6.
Figure 4. Cleavage of the 2’-TePh-modified DNA octamer [5’-G(2’-TePh-
dU)GTACAC-3’]. A) Before heating. Obs for C83H101N30O46P7Te: 2599.3
[MꢀH]+. B) After fragmentation with either I2 or B2H6 at 508C to hexa-
mer (5’-p-GTACAC-3’). Calcd for C58H74N23O36P6: 1856.2, [MꢀH]+;
obs:1856.3.
decreased by 158C (7.58C per modification), while the Tm of
the 2’-TeMe-DNA duplex decreased by 108C (5.08C per
modification) compared with the native duplex. This sug-
gests that short duplex of the self-complimentary DNA can
not accommodate these two bulky modifications well. When
introducing the Te functionalities to a non-self-complemen-
tary duplex [5’-C(2’-TeX-dU)T CTT GTC CG-3’ and 5’-
CGG ACA AGA AG-3’, X=Me or Ph], it was observed
that the melting temperatures of the native, 2’-TeMe, and 2’-
TePh duplexes were 44.0, 40.7, and 36.88C, respectively. The
difference between the native and 2’-TeMe-modified DNAs
is 3.38C. It is noteworthy that when the TeMe moiety is in-
troduced close to a terminal position, the Te modification is
relatively well accommodated (Figure 5). Since the termini
are usually dynamic and spacious, our result suggests that
the dynamics and available space allow a better accommo-
dation. Therefore, the perturbation caused by the 2’-Te func-
tionality is most likely a local event, and this bulky Te modi-
fication could be useful in investigating the formation of
DNA or RNA loops, bulges, and other secondary or 3D
structures.
Figure 3. Redox properties of the 2’-TePh-DNA octamer [5’-G(2’-TePh-
dU)GTACAC-3’]. A) Nonoxidized Te-DNA; B) oxidized Te-DNA after
I2 treatment; C) coinjection of the oxidized and Te-DNAs; D) oxidized
Te-DNA after B2H6 treatment.
(Scheme 5, and Figure S1 in the Supporting Information),
and the C=C double bond formed was further reduced by
B2H6. It is surprising that the 2’-TePh functionality generates
the fragmented product, whereas the 2’-TeMe functionality
creates the abasic product. We are in the process of further
studying our observations. These two Te-DNA modification
strategies can be useful in investigating site-specific DNA
cleavage and base damage.[30–32]
Due to the size of the Te atom, we anticipated that the in-
corporated Te functionalities at the 2’-position would per-
turb the DNA duplexes. In addition, we expected that the
2’-TePh moiety would cause more perturbation than the 2’-
TeMe. We investigated the Te-modified duplex stability by
measuring their UV melting temperatures. In the case of a
self-complementary duplex [5’-G(2’-TeX-dU)G TAC AC-
3’]2, X=Ph or Me, where double-Te modifications were in-
troduced, the melting temperatures dropped dramatically
(Figure S2). In the case of the 2’-TePh-DNA duplex, the Tm
Conclusion
In summary, we have synthesized for the first time Te-deriv-
atized nucleoside phosphoramidites and Te-oligonucleotides
with a high coupling yield. Several 2’-Te-modified oligonu-
cleotides were synthesized and characterized by HPLC and
MS. We have demonstrated that both of the 2’-TeMe and 2’-
TePh functionalities are compatible with the solid-phase
synthesis, deprotection, and purification. In addition, we
have revealed the redox properties of the Te functionalities
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Z. Huang et al.
(HRMS) analysis was performed at Scripps (La Jolla, CA) and at the
Georgia State University Mass Spectrometry Center.
2’-Deoxy-2’-phenyltellanyl-5’-O-(4,4’-dimethoxytrityl)uridine (4a) and 2’-
deoxy-2’-phenyltellanyl-5’-O-(4,4’-dimethoxytrityl)thymidine (4b): A so-
lution of diphenylditelluride (0.2 g, 0.5 mmol) in THF (5 mL) was added
to a stirred suspension of NaBH4 (6.2 mg, 0.15 mmol) in anhydrous THF
(5 mL) under argon at 08C. Several drops of dry ethanol were added
until bubbles formed and the solution turned colorless. Starting material
3a or 3b (0.285 or 0.292 g separately, 0.5 mmol, dissolved in 5 mL of
THF) was added to this solution, and the reaction mixture was slowly
warmed up to room temperature and then to 508C over 3 h. The reaction
was monitored by TLC. The solvent was then evaporated. The residue
was dissolved in CH2Cl2 (20 mL) and washed with water (3ꢂ20 mL). The
CH2Cl2 solution was dried (MgSO4) and concentrated, and the crude
product was purified by silica gel column chromatography with 0–3%
methanol in CH2Cl2 to give compounds 4a and 4b as slightly yellow
1
solids. Yields: 4a: 310 mg (80%); H NMR (400 MHz, CDCl3, 258C): d=
3.45–3.46 (m, 2H), 3.82 (s, 3H), 3.92–3.95 (m, 1H), 4.24 (m, 1H), 4.54–
4.57 (m, 1H), 5.12 (d, J=8 Hz, 1H), 6.63 (d, J=9.2 Hz, 1H), 6.81–6.86
(m, 4H), 7.19–7.37 (m, 12H), 7.45 (d, J=8 Hz, 1H), 7.82 (m, 2H);
13C NMR (100 MHz, CDCl3, 258C): d=36.9, 55.3, 63.9, 85.5, 87.2, 91.6,
102.5, 109.6, 113.3, 127.2, 127.8, 128.1, 128.1, 128.7, 129.5, 130.1, 135.2,
140.2, 144.2, 150.2, 158.7, 162.7; HRMS: m/z: calcd for C36H34N2O7TeNa:
Figure 5. Normalized thermodenaturation curves of the DNA duplexes
[5’-C(2’-TeX-dU)T CTT GTC CG-3’ and 3’-CGG ACA AGA AG-5’; X=
Me or Ph]. The samples (1 mm duplex) were heated from 6 to 758C at a
rate of 0.58Cminꢀ1. The average Tm values (four measurements) are
44.0ꢁ0.1 (native), 40.7ꢁ0.1 (2’-TeMe), and 36.8ꢁ0.28C (2’-TePh).
1
759.1326 [M+Na]+, found: 759.1316. 4b: Yield: 292 mg (78%); H NMR
(400 MHz, CDCl3, 258C): d=1.21 (s, 3H), 3.35–3.52 (m, 2H), 3.81 (s,
6H), 3.98–4.01 (m, 1H), 4.22 (m, 1H), 4.55 (m, 1H), 6.69 (d, J=10 Hz,
1H), 6.81–6.84 (m, 4H), 7.16–7.35 (m, 15H), 7.83 (m, 2H), 8.17 (brs,
1H); 13C NMR (100 MHz, CDCl3, 258C): d=11.5, 36.8, 55.3, 63.9, 85.2,
87.2, 91.0, 109.5, 111.3, 113.3, 127.2, 128.0, 128.1, 128.6, 129.6, 130.1,
135.0, 135.2, 140.3, 144.2, 150.4, 158.8, 163.1; HRMS: m/z: calcd for
C37H36N2O7TeNa: 773.1477 [M+Na]+, found: 773.1475.
and the interchange of telluride and telluoxide DNAs by
redox reactions. We also found that the 2’-TePh DNA can
be reductively or oxidatively cleaved, whereas the nucleo-
base of the 2’-TeMe DNA can be eliminated by 1’,2’ elimi-
nation to create an abasic site. This Te-DNA system could
be useful in studying DNA fragmentation and nucleobase
damage.[32] Furthermore, in our UV-melting study of the 2’-
Te-modified DNA duplexes, as expected, we observed Tm
decreases for the Te-DNA duplexes. Since the Tm decreases
are dependent on the location of the Te modifications and
the size of the protecting groups, Te derivatization might be
a useful strategy for probing DNA and RNA polymerization
and catalysis.[33–35] Moreover, this Te derivatization of nucleic
acids has great potential in nucleic acid X-ray crystallogra-
phy as well as in structural and functional studies of nucleic
acid–protein complexes.
2’-Phenyltellanyl-5’-O-(4,4’-dimethoxytrityl)uridine 3’-O-(2-cyanoethyl)-
diisopropylamino phosphoramidite (5a) and 2’-phenyltellanyl-5’-O-(4,4’-
dimethoxytrityl)thymidine 3’-O-(2-cyanoethyl)diisopropylamino phos-
phoramidite (5b): Dry methylene chloride (2.5 mL), N,N-diisopropyl-
AHCTUNGERTGeNNUN thylamine (0.17 mL, 1.03 mmol, 1.5 equiv), and 2-cyanoethyl N,N-diiso-
propylchlorophosphoramidite (195 mg, 0.83 mmol, 1.2 equiv) were added
sequentially under argon to a flask (25 mL) containing 4a or 4b (500 or
510 mg separately, 0.68 mmol). The reaction mixture was stirred at
ꢀ108C in an ice-salt bath under argon for 10 min, then the bath was re-
moved. The reaction was complete after 45 min at room temperature (in-
dicated by TLC, 5% MeOH in CH2Cl2) and generated a mixture of two
diastereomers. The reaction was then quenched with NaHCO3 (2 mL,
sat.) and stirred for 5 min, and the product was extracted with CH2Cl2
(3ꢂ5 mL). The combined organic layers were washed with NaCl (10 mL,
sat.) and dried over MgSO4(s) for 20 min, followed by filtration. The sol-
vent was then evaporated under reduced pressure, and the crude product
was redissolved in CH2Cl2 (2 mL). This solution was added dropwise to
petroleum ether (200 mL) with vigorous stirring to generate a white pre-
cipitate. The petroleum ether solution was decanted carefully. The crude
product was again dissolved in CH2Cl2 (2 mL) and then loaded onto an
Al2O3 column (neutral) that was equilibrated with CH2Cl2/hexanes (1:1).
The column was eluted with a gradient of CH2Cl2!CH2Cl2/EtOAc (7:3).
After evaporation of the solvent and drying over high vacuum, pure 5a
and 5b (570 mg) were obtained as white foamy products (88–90% yield).
Experimental Section
General methods: Most solvents and reagents were purchased from
Sigma–Aldrich (St. Louis, MO; USA) or VWR (West Chester, PA;
USA) without further purification unless mentioned otherwise. Dimethyl
ditelluride was purchased from Organometallics (East Hampstead, NH;
USA). Triethylamine (TEA) and THF were dried over KOH(s) and
sodium metal, respectively, and distilled under argon. When necessary,
solid reagents were dried under high vacuum. Reactions with compounds
sensitive to air or moisture were performed under argon. Solvent mix-
tures are indicated as v/v ratios. Thin-layer chromatography (TLC) was
run on Merck 60 F254 plates (0.25 mm thick; Rf values in the text are for
the title products), and visualized under UV light or by a Ce-Mo staining
1
5a: H NMR (400 MHz, CDCl3, 258C): d=1.13–1.35 (m, 24H), 2.37, 2.68
(2t, J=6.4 Hz, 4H), 3.38–3.69 (m, 12H), 3.83 (s, 12H), 3.90–4.03 (m,
2H), 4.32, 4.38 (2m, 2H), 4.67–4.82 (m, 2H), 4.95, 5.00 (2d, J=8 Hz,
2H), 6.73, 6.75 (2d, J=5.8 Hz, 2H), 6.83–6.86 (m, 8H), 7.16–7.37 (m,
26H), 7.76 (brs, 2H), 7.81–7.85 (m, 4H); 13C NMR (100 MHz, CDCl3,
258C): d=20.13, 20.20, 20.44, 20.51, 24.50, 24.61, 24.69, 25.23, 33.85,
34.43, 43.36, 43.40, 43.49, 43.52, 55.29, 57.68, 57.87, 59.04, 59.23, 63.58,
63.73, 74.91, 75.09, 84.97, 87.29, 89.38, 111.69, 113.46, 117.29, 126.99,
127.81, 128.54, 130.21, 130.42, 135.22, 135.40, 135.44, 144.10, 150.37,
158.85, 163.34; 31P NMR (160 MHz, CDCl3, 258C): d=148.6, 149.2;
HRMS: m/z: calcd for C45H52N4O8PTe: 937.2579 [M+H]+, found:
solution (phosphomolybdate, 25 g; CeACHTNUTRGNE(UGN SO4)2·4H2O, 10 g; H2SO4, 60 mL,
conc.; H2O, 940 mL) with heating. Flash chromatography was performed
1
by using Fluka silica gel 60 (mesh size 0.040–0.063 mm). H and 13C spec-
1
tra were recorded on a Bruker-400 spectrometer (400 and 100 MHz). All
chemical shifts are given in ppm relative to tetramethylsilane, and all
coupling constants (J) are in Hz. High-resolution mass spectrum
937.2578. 5b: H NMR (400 MHz, CDCl3, 258C): d=1.10–1.42 (m, 24H),
2.34, 2.66 (2t, J=6.4 Hz, 4H), 3.35–3.69 (m, 12H), 3.83 (s, 12H), 3.92–
4.05 (m, 2H), 4.31, 4.37 (2m, 2H), 4.65–4.82 (m, 2H), 6.71, 6.74 (2d, J=
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Tellurium-Derivatized Oligonucleotides
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5.8 Hz, 2H), 6.84–6.86 (m, 8H), 7.16–7.37 (m, 26H), 7.77 (brs, 2H), 7.80–
7.84 (m, 4H); 13C NMR (100 MHz, CDCl3, 258C): d=11.69, 20.12, 20.22,
20.43, 20.53, 24.49, 24.60, 24.67, 25.24, 33.85, 34.43, 43.37, 43.40, 43.49,
43.53, 55.27, 57.67, 57.88, 59.05, 59.25, 63.57, 63.74, 74.92, 75.09, 84.97,
87.27, 89.38, 111.68, 113.45, 117.28, 126.98, 127.81, 128.54, 130.20, 130.39,
135.24, 135.38, 135.42, 144.11, 150.35, 158.87, 163.36; 31P NMR (160 MHz,
CDCl3, 258C): d=148.4, 149.3; HRMS: m/z: calcd for C46H53N4O8PTeNa:
973.2561 [M+Na]+, found: 973.2560.
tion was monitored by TLC (5% CH3OH in CH2Cl2), it was found that
the yield reached a maximum after 4–5 h before dropping again due to
the generation of compound 6. The reaction was quenched by adding sa-
turated sodium chloride (10 mL), followed by addition of CH2Cl2
(10 mL). The organic solvent was further washed, dried over anhydrous
MgSO4, and removed under reduced pressure. The residue was then puri-
fied on a silica gel column (equilibrated with CH2Cl2), eluted with a
methanol/methylene chloride gradient (CH3OH in CH2Cl2, 0–3%) to
1
afford the pure foamy products 9a and 9b in 40–47% yield. 9a: H NMR
(R)-5-(4,4’-Dimethoxytrityloxymethyl)-2,3-dihydrofuran-4-ol (6): Dimeth-
yl ditelluride (0.05 mL, 0.3 mmol) was added under argon to a stirred sus-
pension of NaBH4 (12 mg, 0.3 mmol) in dry THF (5 mL). Several drops
of dry ethanol were then added until bubbles formed. The suspension
was heated to 508C, then a solution of starting material 3a (320 mg,
0.6 mmol) in THF was added. The mixture was heated for 3 h at this tem-
perature under argon. The solvent was then evaporated, and the residue
was dissolved in CH2Cl2 (20 mL) and washed with water (3ꢂ20 mL). The
CH2Cl2 layer was dried (MgSO4) and concentrated, and the residue was
purified by silica gel column chromatography with pure CH2Cl2 to give
compound 6 as white solid. Yield: 230 mg (92%); 1H NMR (400 MHz,
CDCl3, 258C): d=3.18–3.22 (m, 2H), 3.81 (s, 3H), 4.44–4.49 (m, 1H),
4.79–4.81 (m, 1H), 5.20–5.21 (m, 1H), 6.62–6.63 (m, 1H), 6.84–6.86 (m,
4H), 7.23–7.46 (m, 9H), 7.85 (d, J=7.6 Hz, 1H); 13C NMR (100 MHz,
CDCl3, 258C): d=55.2, 63.7, 76.2, 86.0, 88.3, 103.3, 113.1, 126.8, 127.8,
128.1, 130.1, 136.0, 144.8, 150.3, 158.5; HRMS: m/z: calcd for C26H25O5:
417.1702 [MꢀH]ꢀ, found: 417.1708.
2,2’-Anhydro-1-[2’-deoxy-3’-acetyl-5’-O-(4,4-dimethoxytrityl)-b-d-arabino
furanosyl]uracil (7a): Acetic anhydride (0.19 mL, 2 mmol) and then sev-
eral drops of triethylamine were added to a solution of 3a (320 mg,
0.6 mmol) in THF. The mixture was stirred at 508C for a further 45 min
(monitored by TLC, 5% MeOH in CH2Cl2) before being quenched with
methanol (4 mL). The solvent were removed under reduced pressure,
and the residue was redissolved in CH2Cl2 (40 mL). The suspension was
washed with sodium bicarbonate (sat., 2ꢂ15 mL) and sat. brine (2ꢂ
15 mL). The organic layer was dried over MgSO4(s) and concentrated
under reduced pressure, and the resulting residue was subjected to silica
gel chromatography (0–5% MeOH in CH2Cl2) to give 7a (291 mg, 85%)
as a white solid. 1H NMR (400 MHz, CDCl3, 258C): d=2.14 (s, 3H),
2.99–3.06 (m, 2H), 3.81 (s, 6H), 4.45 (m, 1H), 5.30–5.32 (m, 1H), 5.40
(m, 1H), 5.86 (d, J=7.6 Hz, 1H), 6.27 (d, J=5.6 Hz, 1H), 6.80–6.83 (m,
4H), 7.21–7.35 (m, 10H); 13C NMR (100 MHz, CDCl3, 258C): d=20.7,
55.3, 62.6, 77.0, 85.8, 86.3, 86.6, 90.4, 110.2, 113.3, 127.1, 128.0, 129.8,
135.2, 144.1, 158.6, 134.5, 159.1, 169.4, 171.2; HRMS: m/z: calcd for
C32H31N2O8: 571.2080 [M+H]+, found 571.2080.
(400 MHz, CDCl3, 258C): d=2.0 (s, 3H), 2.5 (s, 1H), 3.52 (dd, J=2.8,
7.2 Hz, 2H), 3.69 (t, J=4 Hz, 1H), 3.82 (s, 6H), 4.21–4.23 (m, 1H), 4.31–
4.36 (m, 1H), 5.40 (d, J=8.4 Hz, 1H), 6.33 (d, J=8.4 Hz, 1H), 6.86–6.88
(m, 4H), 7.26–7.39 (m, 9H), 7.77 (d, J=8.4 Hz, 1H), 8.38 (brs, 1H);
13C NMR (100 MHz, CDCl3, 258C): d=ꢀ21.2, 33.35, 55.29, 63.37, 73.75,
84.51, 87.29, 89.90, 102.73, 113.35, 127.28, 128.07, 130.08, 135.12, 139.77,
144.20, 150.23, 158.79, 162.64; HRMS: m/z: calcd for C31H31N2O7Te :
673.1194 [MꢀH]ꢀ, found: 673.1204. 9b: 1H NMR (400 MHz, CDCl3,
258C): d=1.43 (s, 3H), 1.99 (s, 3H), 3.47 (dd, J=1.6, 9.6 Hz, 2H), 3.70–
3.74 (m, 1H), 3.82 (s, 6H), 4.24–4.25 (m, 1H), 4.36–4.37 (m, 1H), 6.37 (d,
J=9.2 Hz, 1H), 6.85–6.87 (m, 4H), 7.26–7.40 (m, 9H), 7.60 (s, 1H), 8.03
(brs, 1H); 13C NMR (100 MHz, CDCl3, 258C): d=ꢀ20.8, 11.71, 33.11,
55.29, 63.86, 74.62, 84.64, 87.19, 89.90, 111.75, 113.32, 127.29, 128.09,
129.14, 135.20, 135.16, 144.21, 150.48, 158.82, 163.41; HRMS: m/z: calcd
for C32H33N2O7Te: 687.1350 [MꢀH]+, found: 687.1354.
2’-Methyltellanyl-5’-O-(4,4’-dimethoxytrityl)uridine 3’-O-(2-cyanoethyl)-
diisopropylamino phosphoramidite (10a) and 2’-methyltellanyl-5’-O-(4,4’-
dimethoxytrityl)thymidine 3’-O-(2-cyanoethyl)diisopropylamino phos-
phoramidite (10b): Dry methylene chloride (2.5 mL), N,N-diisopropyl-
AHCTUNGERTGeNNUN thylamine (0.1 mL, 0.51 mmol, 1.5 equiv), and 2-cyanoethyl N,N-diiso-
propylchlorophosphoramidite (120 mg, 0.51 mmol, 1.5 equiv) were added
sequentially under argon to a flask (25 mL) containing 9a or 9b (230 or
234 mg, respectively, 0.34 mmol). The reaction mixture was stirred at
ꢀ108C in an ice-salt bath under argon for 10 min, then the bath was re-
moved. The reaction was completed after 45 min at room temperature
(as indicated by TLC, 5% MeOH in CH2Cl2) to generate a mixture of
two diastereomers. The reaction was then quenched with NaHCO3
(2 mL, sat.) and stirred for 5 min, and the product was then extracted
with CH2Cl2 (3ꢂ5 mL). The combined organic layers were washed with
NaCl (10 mL, sat.) and dried over MgSO4(s) for 20 min, followed by fil-
tration. The solvent was then evaporated under reduced pressure, and
the crude product was redissolved in CH2Cl2 (2 mL). This solution was
added dropwise to petroleum ether (200 mL) with vigorous stirring to
generate a white precipitate. The petroleum ether solution was then dec-
anted. The crude product was again dissolved in CH2Cl2 (2 mL) and then
loaded onto an Al2O3 column (neutral) that was equilibrated with
CH2Cl2/hexanes (1:1). The column was eluded with a gradient of methyl-
ene chloride and ethyl acetate (CH2Cl2!CH2Cl2/EtOAc 7:3). After sol-
vent evaporation and drying over high vacuum, 10a and 10b were ob-
tained as white foamy products (88–90% yield). 10a: 1H NMR
(400 MHz, CDCl3, 258C): d=0.85–1.38 (m, 24H), 1.98, 1.99 (2s, 6H),
2.41, 2.67 (2t, J=7.6 Hz, 4H), 3.42–3.69 (m, 12H), 3.83 (s, 12H), 3.89–
4.05 (m, 4H), 4.22, 4.30 (2m, 2H), 4.57–4.77 (m, 2H), 6.43, 6.55 (2d, J=
8.6 Hz, 2H), 6.80–6.92 (m, 8H), 7.16–7.45 (m, 18), 7.78, 7.79 (s, 2H);
13C NMR (CDCl3, 258C): d=ꢀ21.32, 19.16, 19.66, 20.88, 24.81, 24.44,
43.30, 43.46, 46.34, 47.53, 51.47, 51.85, 55.62, 57.03, 58.39, 62.52, 73.36,
73.67, 84.57, 87.35, 88.12, 103.39, 113.42, 117.28, 127.21, 127.94, 128.36,
130.19, 130.25, 135.17, 135.38, 139.53, 144.20, 150.42, 158.80, 163.19;
31P NMR (160 MHz, CDCl3, 258C): d=148.5, 149.2; ESI-TOF: m/z calcd
for C40H48N4O8PTe: 873.2272 [M+H]+, found: 873.2264. 10b: 1H NMR
(400 MHz, CDCl3, 258C): d=0.89–1.42 (m, 24H), 1.40 (2s, 6H), 2.10 (2s,
6H), 2.42, 2.69 (2t, J=7.8 Hz, 4H), 3.48–3.72 (m, 12H), 3.82 (s, 12H),
3.90–4.05 (m, 2H), 4.22, 4.31 (2m, 2H), 4.58–4.72 (m, 2H), 6.42 (d, J=
8.6 Hz, 2H), 6.79–6.92 (m, 8H), 7.20–7.45 (m, 18H), 7.62 (2s, 2H), 8.25
(brs, 2H); 13C NMR (100 MHz, CDCl3, 258C): d=ꢀ21.32, 4.58, 4.72,
20.13, 20.16, 20.48, 20.55, 22.52, 24.39, 24.47, 24.59, 24.68, 24.76, 24.83,
43.21, 43.32, 43.34, 43.41, 46.35, 46.42, 47.37, 47.42, 50.36, 55.32, 63.29,
63.33, 74.90, 75.09, 84.99, 87.28, 89.36, 111.70, 113.44, 117.29, 126.99,
127.83, 128.55, 130.20, 130.41, 135.23, 135.41, 135.44, 144.11, 150.38,
5’-O-(4,4-Dimethoxytrityl)-2’,3’-didehydro-2’,3’-dideoxyuridine (8a): Di-
methyl ditelluride (0.05 mL, 0.3 mmol) was added under argon to a
stirred suspension of NaBH4 (12 mg, 0.3 mmol) in anhydrous THF
(5 mL). Several drops of dry ethanol were then added until bubbles
formed. The suspension was heated to 508C, and a solution of 7 in THF
(170 mg, 3 mmol) was added. The reaction was finished after 3 h, as
monitored by TLC. The solvents were then evaporated, and the residue
was dissolved in CH2Cl2 and washed with water. The organic solution
was dried over MgSO4 and concentrated. The crude product was purified
by silica gel column chromatography with 0–3% methanol in CH2Cl2 to
1
give a 90% yield of 8a. H NMR (400 MHz, CDCl3, 258C): d=3.47–3.48
(m, 2H), 3.82 (s, 3H), 4.97–4.98 (m, 1H), 5.06 (d, J=7.6 Hz, 1H), 5.89–
5.91 (m, 1H), 6.35–6.37 (m, 1H), 6.84–6.86 (m, 4H), 7.05 (d, J=2.0 Hz,
1H), 7.27–7.38 (m, 9H), 7.85 (d, J=7.6 Hz, 1H); 13C NMR (100 MHz,
CDCl3, 258C): d=55.4, 64.2, 86.1, 86.9, 89.6, 102.2, 113.2, 127.1, 127.8,
127.4, 129.1, 130.20, 150.6, 127.1, 134.6, 141.4, 150.6, 158.6, 163.2; HRMS:
m/z: calcd for C30H27N2O6: 511.1869 [MꢀH]+, found 511.1868.
2’-Deoxy-2’-methyltellanyl-5’-O-(4,4’-dimethoxytrityl)uridine (9a) and 2’-
deoxy-2’-methyltellanyl-5’-O-(4,4’-dimethoxytrityl)thymidine (9b): A so-
lution of LiAlH4 (0.55 mmol) in THF (1m) was added over 5 min to a so-
lution of dimethyl ditelluride (CH3TeTeCH3, 0.2 mL, 1.1 mmol) in THF
(10 mL) in an ice bath under argon. After the solution had turned slightly
yellow, [12]crown-4 (0.6 mmol, 0.1 mL) was added, and the mixture was
stirred at 08C for a further 20 min before starting material 3a or 3b (0.57
or 0.58 g, respectively, in 1.0 mmol THF) was added dropwise. The reac-
Chem. Eur. J. 2009, 15, 10210 – 10216
ꢁ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
10215

Z. Huang et al.
158.86, 163.35. 31P NMR (160 MHz, CDCl3, 258C): d=148.6, 149.5; ESI-
TOF: m/z calcd for C41H52N4O8PTe: 889.2585 [M+H]+, found: 889.2587.
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Synthesis of the Te-derivatized nucleic acids (TeNA): All DNA and
RNA oligonucleotides were synthesized chemically on a 1.0 mmol scale
by using an ABI3400 DNA/RNA synthesizer. The concentration of the
Te-modified phosphoramidites was less than that of conventional ones
(0.06m instead of 0.1m). Coupling was carried out with 5-(benzylmer-
capto)-1H-tetrazole (5-BMT) in acetonitrile (0.25m) as activator. All the
other native phosphoramidites were protected with ultramild deprotec-
tion on the nucleobases. The coupling time for the Te phosphoramidite
was set to 60 s. All the oligonucleotides were prepared in DMTr-on
mode. After synthesis, the beads were treated with K2CO3 (1 mL, 0.05m)
for 8 h, then the supernatant was neutralized with 1m HCl and treated
with borane in THF (1m, 5 mL) before HPLC analysis and purification.
The 5’-DMTr deprotection of DNAs was performed in a 3% trichloro-
acetic acid solution for 3 min, followed by petroleum ether extraction to
remove DMTr-OH.
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HPLC analysis and purification: The DNA oligonucleotides were ana-
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raphy (RP-HPLC) in both DMTr-on and DMTr-off mode. Purification
was carried out by using a 21.2ꢂ250 mm Zorbax, RX-C18 column at a
flow rate of 6 mLminꢀ1. Buffer A consisted of triethylammonium acetate
(TEAAc, 20 mm, pH 7.1), whereas buffer B contained 50% aqueous ace-
tonitrile in buffer A. Analysis was performed on a Zorbax SB-C18
column (4.6ꢂ250 mm) at a flow of 1.0 mLminꢀ1 with the same buffer
system. The DMTr-on oligonucleotides were eluted with up to 100% buf-
fer B in 20 min in a linear gradient, whereas the DMTr-off oligonucleo-
tides were eluted with up to 70% buffer B in a linear gradient over the
same period of time. The collected fractions were lyophilized and redis-
solved in a small amount of water. It was found to be better to adjust the
pH to 7.0 after the final purification.
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MgCl2 (10 mm). The solution was heated to 708C for 2 min, cooled slowly
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Acknowledgements
We thank Drs. Jozef Salon and Chandrasekaran Sekar for their discus-
sions. This work was supported by the GCC Distinguished Cancer Clini-
cians and Scientists Award.
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Received: March 25, 2009
Published online: August 18, 2009
10216
www.chemeurj.org
ꢁ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2009, 15, 10210 – 10216