Two-step synthesis of a 5-azidothymidine building block for the assembly of oligonucleotides for triazole-forming ligations

Article abstract of DOI:10.1055/s-0032-1317605

A two-step synthesis converting thymidine into a phosphotriester building block of 5-azido-5-deoxythymidine in 60% overall yield is presented. The building block was used to assemble an oligonucleotide with an azido group at its 5-terminus, which underwen

Full text of DOI:10.1055/s-0032-1317605

LETTER
▌2923
letter
Two-Step Synthesis of a 5′-Azidothymidine Building Block for the Assembly of
Oligonucleotides for Triazole-Forming Ligations
Two-Step Synthesis of a 5′-Azidothymidine Building Block
Hassan Said,a Claudia Guttroff,a Afaf H. El-Sagheer,b,c Clemens Richert*a
a
Institut für Organische Chemie, Universität Stuttgart, Pfaffenwaldring 55, 70569 Stuttgart, Germany
Fax +49(0)71168564321; E-mail: lehrstuhl-2@oc.uni-stuttgart.de
b
School of Chemistry, University of Southampton, Highfield, Southampton SO17 1BJ, UK
c
Chemistry Branch, Department of Science and Mathematics, Faculty of Petroleum and Mining Engineering, Suez Canal University,
Suez 43721, Egypt
Received: 18.09.2012; Accepted after revision: 19.10.2012
for 2′-azido-2′-deoxynucleosides14 and 3′-azido-2′,3′-de-
Abstract: A two-step synthesis converting thymidine into a phos-
oxythymidine.9 To the best of our knowledge, no building
photriester building block of 5′-azido-5′-deoxythymidine in 60%
block of a 5′-azido-2′,5′-dideoxynucleoside for phosphot-
riester-based chain assembly has been reported. The lack
overall yield is presented. The building block was used to assemble
an oligonucleotide with an azido group at its 5′-terminus, which un-
derwent ligation–cycloaddition, producing a strand with PCR-com- of such a building block complicates preparation of the
patible linkage in high yield.
azido component for PCR-compatible triazole-linked li-
gation products.6
Key words: azides, phosphotriester chemistry, click reaction, oli-
gonucleotide, ligation
Here we report a convenient two-step synthesis of 5′-azi-
dothymidine-3′-O-(2-chlorophenyl)monophosphate (2),
starting from thymidine, and its use for the assembly of a
Progress in the field of synthetic biology has been limited
by the inability to synthesize DNA of great length.1 The
most common approach for the synthesis of long stretches
of genetic information is the solid-phase synthesis of oli-
godeoxynucleotides and subsequent ligation of these oli-
gonucleotides to the desired sequences, followed by
polymerase-based formation of complementary strands
and amplification.2–5 Ligation reactions that rely on 1,3-
dipolar cycloaddition between an azido-terminated oligo-
nucleotide and a propargyl-terminated reaction partner
have recently come into focus because they are robust,
high yielding, and compatible with polymerase chain re-
action (PCR) amplification.6,7 This methodology has
greater biological relevance than approaches involving
other CLICK reactions.8,9
5′-azido-terminal oligonucleotide in high yield, based on
a final phosphotriester coupling step. The deprotected oli-
gonucleotide readily underwent a template-directed liga-
tion to a 3′-propargyl-terminated strand via copper-
catalyzed dipolar cycloaddition.
Scheme 1 shows the synthesis of 2. One-step regioselec-
tive conversion of the 5′-hydroxyl group into an azide15
was followed by phosphorylation under conditions simi-
lar to those used by Sproat and Gait13 and more recently
by Micura and co-workers.14 After column chromatogra-
phy, 2 was obtained in 60% overall yield. Coupling of 2
to support-bound oligonucleotide 3 gave full length oligo-
nucleotide 4 that was released from the support and fully
deprotected after removal of the chlorophenyl protecting
group (Scheme 2).
Unfortunately, azido-terminal oligonucleotides cannot be
prepared via phosphoramidite building blocks because the
potential for Staudinger reduction between the phospho-
rus(III) center and the azido group that makes such build-
ing blocks labile. Two approaches exist that overcome
this complication rely on solid-phase synthesis for oligo-
nucleotide chain assembly. One approach involves on-
support, two-step conversion of the 5′-terminal hydroxyl
group of the pre-assembled oligonucleotide into an
azide.10 On-support conversion has also been used for the
preparation of 5-azido-2′-deoxyuridine, where the azido
group is attached to the nucleobase.11 The other approach
uses a phosphorus(V) building block containing the azido
group. Azido phosphodiester building blocks for phos-
photriester-based chain extension12,13 have been described
O
O
N
O
N
NH
NH
NH
N3
N
O
HO
N3
O
O
O
i
ii
O
O
O
O
O
P
OH
OH
O
Et3NH
1
Cl
2
Scheme 1 Synthesis of 5′-azidothymidine building block 2. Re-
agents and conditions: (i) Ph3P, NaN3, CBr4, DMF, 24 h, 78%; (ii) N-
methylimidazole, 1,2,4-triazole, Et3N, 2-chlorophenyl phosphorodi-
chloridate, in THF, 77%.
SYNLETT 2012, 23, 2923–2926
Advanced online publication: 16.11.2012
DOI: 10.1055/s-0032-1317605; Art ID: ST-2012-B0788-L
© Georg Thieme Verlag Stuttgart · New York
Figure 1 shows MALDI–TOF mass spectra of crude oli-
gonucleotide 5 prepared by the conventional, two-step de-
rivatization of the full length oligonucleotide, as described
0
9
3
6
-
5
2
1
4
1
4
3
7
-
2
0
9
6

2924
H. Said et al.
LETTER
O
N
NH
N3
O
O
HO
O
N
O
P
ABz
ABz
CBz
T
GiBu
ABz
GiBu
GiBu
O
O
NH
O
N3
1. MSNT,
1-methylimidazole
O
O
A
A
C
T
+
2. syn-2-nitrobenzaldoxime,
1,1,3,3-tetramethylguanidine
O
O
O
P
3. full deprotection
with NH4OH
O
G
A
G
G
Et3NH
Cl
O
2
O
OH
3
4
Scheme 2 Coupling of chlorophosphate building block 2 to oligonucleotide 3 on controlled pore glass, followed by two-stage deprotection
in reference 10, and of slightly longer oligonucleotide 4 with evidence for the successful ligation of such an oligo-
prepared by the present methodology, using 2. The phos- nucleotide.16–19 We expect that a similar approach can be
photriester-based coupling, followed by two-step deprot- adopted to prepare the 5′-azido building blocks with the
ection proceeded smoothly, producing 4 with minimal remaining three nucleobases (A, C, and G). Syntheses of
amounts of side products.
5′-azido-2′,5′-dideoxynucleosides are known.15
With azido-terminal oligonucleotide 4 in hand, we then
proceeded to ligating this oligonucleotide to 3′-propargyl-
terminated oligonucleotide 6 on DNA 23-mer 7 as tem-
plate (Figure S3 in the Supporting Information). As ex-
pected, the 1,3-dipolar cycloaddition proceeded quickly,
giving ligation product 8 in near-quantitative yield, as de-
termined by gel electrophoresis (Figure 2).
Acknowledgment
The authors thank Prof. T. Brown for a review of the manuscript.
This work was supported by DFG (grant No. RI 1063/8-2 to C.R.)
and BBSRC (sLOLA grant No BB/J001694/1 ‘Extending the
boundaries of nucleic acid chemistry’).
In conclusion, we have presented a convenient two-step
synthesis of a thymidine building block for solid-phase
synthesis of 5′-azido-terminal oligonucleotides, together
Supporting Information for this article is available online at
http://www.thieme-connect.com/ejournals/toc/synlett.SnuIpofoiprmntgirStanoIuifpog
r
t
iornat
Figure 1 MALDI–TOF mass spectra of crude 5′-azido-terminal oligonucleotides. (a) Sequence 5, as prepared by on-support conversion of the
5′-terminal alcohol into the corresponding azide,10 followed by deprotection, and (b) sequence 4, prepared by chain extension with 2, followed
by two-stage deprotection.
Synlett 2012, 23, 2923–2926
© Georg Thieme Verlag Stuttgart · New York

LETTER
Two-Step Synthesis of a 5′-Azidothymidine Building Block
2925
(14) (a) Aigner, M.; Hartl, M.; Fauster, K.; Steger, J.; Bister, K.;
Micura, R. ChemBioChem 2011, 12, 47. (b) Fauster, K.;
Hartl, M.; Santner, T.; Aigner, M.; Kreutz, C.; Bister, K.;
Ennifar, E.; Micura, R. ACS Chem. Biol. 2012, 7, 581.
(15) (a) Bannwarth, W. Helv. Chim. Acta 1988, 71, 1517.
(b) Mag, M.; Engels, J. W. Nucleic Acids Res. 1989, 17,
5973.
(16) 5′-Azido-5′-deoxythymidine (1): The preparation of
compound 1 used a slight modification of the protocol
described in reference 15, using NaN3 instead of LiN3.
Briefly, triphenylphosphine (3.21 g, 12.24 mmol, 1.2 equiv),
NaN3 (1.95 g, 30.04 mmol, 3 equiv) and CBr4 (4.01 g, 12.09
mmol, 1.2 equiv) were added to a solution of thymidine
(2.48 g, 10.23 mmol, 1 equiv) in anhyd DMF (40 mL). The
reaction mixture was stirred at r.t. for 24 h under argon.
When TLC (CH2Cl2–MeOH, 9:1) showed complete
conversion, the reaction mixture was treated with sat.
NaHCO3 solution (50 mL). After extracting with CHCl3 (3 ×
50 mL), the organic solution was washed once with H2O and
twice with brine (2 × 100 mL), followed by drying over
Na2SO4. The reaction mixture was filtered and the solvents
were evaporated. The crude product was purified by column
chromatography on silica, eluting with a step gradient of
MeOH in CH2Cl2 (0–6%) to give compound 1 (2.14 g, 78%).
The spectroscopic data were in agreement with the
literature.15
5′-Azidothymidine-3′-O-(2-
chlorophenyl)monophosphate (2): 1,2,4-Triazole (0.757 g,
10.3 mmol, 5.5 equiv) was dissolved in anhyd THF (23 mL)
and treated with Et3N (1.3 mL, 9.3 mmol, 5 equiv). To this,
2-chlorophenyl phosphorodichloridate (800 μL, 4.62 mmol,
2.5 equiv) was added, immediately leading to a white
precipitate. The suspension was stirred at 20 °C for 30 min.
Then, a solution of 5′-azido-5′-deoxythymidine (0.5 g, 1.87
mmol, 1 equiv) and 1-methylimidazole (600 μL, 7.42 mmol,
4 equiv) in anhyd THF (8 mL) was added, and the reaction
mixture was stirred at r.t. for 45 min under Ar. When TLC
(CH2Cl2–MeOH, 9:1) showed complete conversion, the
reaction was quenched with H2O (400 μL) and Et3N (1.5
mL). The solvents were evaporated, and the residue was
dissolved in CH2Cl2 (50 mL) and sat. NaHCO3 (50 mL). The
product was extracted in CH2Cl2 (3 × 100 mL). The
combined organic layers were washed with brine (3 × 100
mL) and dried over Na2SO4. After evaporation of the
solvents, the crude product was purified by column
chromatography on silica (CH2Cl2–MeOH–Et3N, 98:1:1 for
packing the column), eluting with a step gradient of 0% to
9% MeOH–0.5% Et3N to give 2 (0.803 g, 77%) as an
orange-colored foam.
Figure 2 Results of ligation assays. (a) Denaturing polyacrylamide
gel (20% PAGE) of samples of the ligation, as imaged by UV-shad-
owing. Lane 1: 200 pmol of each of oligonucleotides 4 and 6 (educts),
and template strand 7; lane 2: 200 pmol of ligation mixture after 1 h;
lane 3: 200 pmol of ligation mixture after 2 h; (b) MALDI–TOF mass
spectrum of reaction mixture of the template-directed cycloaddition
between 5′-azide oligonucleotide 4 with oligonucleotide 6 on tem-
plate 7 to produce ligation product 8. Assignment: [M – H]– of 4 =
2785 g/mol and [M – 2 H]2– of template strand 7 = 3529 g/mol; liga-
tion product 8 (M = 4904 g/mol) and [M – 2 H]2– of ligation product
at 2452 g/mol.
References
(1) Reese, C. Org. Biomol. Chem. 2005, 3, 3851.
(2) Dillo, P. J.; Rosen, C. A. BioTechniques 1990, 9, 298.
(3) Stemmer, W. P. C.; Crameri, A.; Ha, D.; Brennan, T. M.;
Heyneker, L. Gene 1995, 164, 49.
(4) Engels, J. W. Angew. Chem. Int. Ed. 2005, 44, 7166.
(5) Gibson, D. G.; Young, L.; Chuang, R. Y.; Venter, J. C.;
Hutchison, C. A.; Smith, H. O. Nat. Methods 2009, 6, 343.
(6) El-Sagheer, A. H.; Sanzone, A. P.; Gao, R.; Tavassoli, A.;
Brown, T. Proc. Natl. Acad. Sci. U.S.A. 2011, 108, 11343.
(7) El-Sagheer, A. H.; Brown, T. Chem. Commun. 2011, 47,
12057.
(8) Kumar, R.; El-Sagheer, A.; Tumpane, J.; Lincoln, P.;
Wilhelmsson, L. M.; Brown, T. J. Am. Chem. Soc. 2007,
129, 6859.
(9) El-Sagheer, A. H.; Brown, T. J. Am. Chem. Soc. 2009, 131,
3958.
1H NMR (300 MHz, DMSO-d6): δ = 1.15 (t, 3J = 7.3 Hz, 9
H, CH3CH2NH), 1.79 (s, 3 H, thymidine-Me), 2.23 (m, 2 H,
2′-H, 2′′-H), 3.03 (q, 3J = 7.3 Hz, 6 H, CH3CH2NH), 3.54 (m,
2 H, 5′-H, 5′′-H), 4.04 (m, 1 H, 4′-H), 4.62 (m, 1 H, 3′-H),
6.14 (t, 3J = 6.6 Hz, 1 H, 1′-H), 6.93 (t, 3J = 7.8 Hz, 1 H,
ArH), 7.18 (t, 3J = 7.2 Hz, 1 H, ArH), 7.35 (d, 3J = 7.9 Hz, 1
H, ArH), 7.51 (s, 1 H, 6-H), 7.58 (d, 3J = 7.8 Hz, 1 H, ArH),
9.5 (br s, 1 H, CH3CH2NH), 11.35 (s, 1 H, NH). 31P NMR
(121.5 MHz, DMSO-d6): δ = –6.87. MS (ESI–; MeOH–H2O,
1:1): m/z calcd for C16H16ClN5O7P [M–H]–: 456.05, found:
456.02.
(10) (a) Miller, G. P.; Kool, E. T. Org. Lett. 2002, 4, 3599.
(b) Miller, G. P.; Kool, E. T. J. Org. Chem. 2004, 69, 2404.
(11) Bayer, C.; Wagenknecht, H. A. Chem. Commun. 2010, 46,
2230.
(12) Letsinger, R. L.; Lunsford, W. B. J. Am. Chem. Soc. 1976,
98, 767.
(17) Synthesis of 5′-Azide Oligonucleotide (4): Azido building
block 2 was coupled to the 5′-terminus of the fully protected
oligonucleotide attached on controlled pore glass. The
immobilized octamer oligodeoxynucleotide on cpg (3, 10
mg, approx. 0.2 μmol loading, DMT-off state), had been
purchased from Biomers Inc. (Ulm, Germany), where it had
been assembled via conventional automated DNA synthesis.
(13) Sproat, B. S.; Gait, M. J. Am. Chem. Soc. 1976, 98, 3655.
© Georg Thieme Verlag Stuttgart · New York
Synlett 2012, 23, 2923–2926

2926
H. Said et al.
LETTER
The support was dried at 0.1 mbar for 1 h. A solution of 5′-
azido nucleotide 2 (8.5 mg, 15 μmol), previously co-
evaporated twice from anhydrous pyridine and dried at 0.1
mbar, was treated with 1-(2-mesitylenesulfonyl)-3-nitro-
1H-1,2,4-triazole (MSNT; 22.2 mg, 75 μmol) in anhydrous
pyridine (100 μL) at r.t. for 15 min. Then, 1-methyl-
imidazole (10 μL, 126 μmol) was added and the mixture was
transferred to a polypropylene cup containing the DNA-
bearing cpg. After 50 min, the supernatant was carefully
aspirated, and the cpg was washed with anhyd pyridine (3 ×
200 μL) and MeCN (5 × 200 μL). The support was treated
with a solution of syn-2-nitrobenzaldoxime (18 mg, 10
mmol) and 1,1,3,3-tetramethylguanidine (20 μL) in
dioxane–H2O (250 μL, 1:1). The mixture was left at r.t. for
16 h and the support was then washed with dioxane–H2O
(1:1; 5 × 200 μL). After drying at 0.1 mbar, the cpg was
treated with NH4OH (25% aq NH3, 500 μL) for 5 h at 55 °C.
The mixture was cooled to r.t. and excess NH3 was removed
by gently blowing a stream of nitrogen onto the surface until
the solution was odorless. The solution was lyophilized and
the oligonucleotide was purified by reversed-phase HPLC
with a step gradient of MeCN (0% for 5 min to 25% in 35
min, tR = 24 min) in 0.1 M triethylammonium acetate buffer
(pH 7.0). Elution was monitored by UV-absorption at λ =
260 nm.
(18) Synthesis of 3′-Alkyne Oligonucleotide 6: The
oligonucleotide was synthesized via standard
phosphoramidite synthesis. The first nucleoside 5′-O-(4,4′-
dimethoxytrityl)-3′-O-propargyl-5-methyldeoxycytidine
was attached to the solid support (33 μmol/g loading, AM
polystyrene, Applied Biosystems) according to the method
reported in reference 20. The support was transferred into a
DNA synthesizer column and the assembly of the sequence
was performed in the 3′- to 5′-direction, followed by
cleavage, deprotection, and HPLC purification.
(19) Template-Directed Ligation of Oligonucleotides:
Oligonucleotides 4, 6 and template strand 7 (0.5 nmol each)
were mixed, lyophilized and dissolved in 0.2 M NaCl (125
μL). For annealing, the solution was heated to 85 °C for 5
min and cooled to 4 °C in the course of 2 h. A solution of tris-
hydroxypropyltriazole21 (2.8 μmol in 0.2 M NaCl, 38 μL),
sodium ascorbate (4 μmol in 0.2 M NaCl, 8 μL) and
CuSO4·5H2O (0.04 μmol in 0.2 M NaCl, 4 μL) was added to
the oligonucleotides, and the reaction mixture was kept at 0
°C for 1 h and at r.t. for 1 h. Then, the sample was desalted
by gel filtration (Sephadex G-25 column) and analyzed by
MALDI–TOF–MS and 20% denaturing polyacrylamide gel
electrophoresis.
(20) El-Sagheer, A. H.; Brown, T. Proc. Natl. Acad. Sci. U.S.A.
2010, 107, 15329.
(21) Chan, T. R.; Hilgraf, R.; Sharpless, K. B.; Fokin, V. V. Org.
Lett. 2004, 6, 2853.
Synlett 2012, 23, 2923–2926
© Georg Thieme Verlag Stuttgart · New York

Copyright of Synlett is the property of Georg Thieme Verlag Stuttgart and its content may not be copied or
emailed to multiple sites or posted to a listserv without the copyright holder's express written permission.
However, users may print, download, or email articles for individual use.