A novel reagent for the chemical phosphorylation of oligonucleotides

Article abstract of DOI:10.1016/j.tetlet.2003.10.167

A novel phosphoramidite reagent was developed to convert terminal hydroxyl groups of oligonucleotides into phosphate monoesters. The reagent's appearance as a solid foam is advantageous for its manipulation and handling in solid phase synthesis and improves its thermal stability.

Full text of DOI:10.1016/j.tetlet.2003.10.167

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 321–324
A novel reagent for the chemical phosphorylation
of oligonucleotides
Michael Leuck,* Kurt E. Vagle, J. Shawn Roach and Andreas Wolter
Proligo Biochemie GmbH Hamburg, Georg-Heyken-Str. 14, D-21147 Hamburg, Germany
Received 4 September 2003; revised 23 October 2003; accepted 28 October 2003
Abstract—A novel phosphoramidite reagent was developed to convert terminal hydroxyl groups of oligonucleotides into phosphate
monoesters. The reagentꢀs appearance as a solid foam is advantageous for its manipulation and handling in solid-phase synthesis
and improves its thermal stability.
ꢀ 2003 Elsevier Ltd. All rights reserved.
Various applications of oligonucleotides in molecular
biology, such as gene construction,1 ligase chain ampli-
fication reactions,2 ligase-mediated genotyping3 and the
conjugation of ligands to oligonucleotides,4 require the
presence of a 50-terminal phosphate group. 50-Phos-
phorylated oligonucleotides are commonly prepared
through solid-phase synthesis wherein the phosphate
groupis attached to the oligonucleotide in the last
of the coupling efficiency in the last cycle, and if reagent
3 is used, the cartridge-aided purification of the DMT-
ON oligonucleotide after the deprotection reaction on a
reversed phase resin, is employed as the second phos-
phate protecting group.
One disadvantage of both reagents 2 and 3 is their
appearance as viscous oils, which causes difficulties in
the filling and handling of the reagents and in the visual
verification of their complete dissolution in the solvent
of the coupling reaction, especially when the reagents
are applied in amber bottles. Reagent 3 also requires
prolonged coupling and detritylation times and is
incompatible with the commonly employed capping
reagents of the oligonucleotide synthesis cycle. These
problems were addressed by the design and the synthesis
coupling cycle with
a speciality phosphoramidite
reagent. Commercially available reagents, such as 25 and
36 (Fig. 1) contain two base-labile phosphate protecting
groups, which are removed during the alkaline depro-
tection of the oligonucleotide to give the desired
50-phosphate monoester. The cyanoethyl group is usu-
ally applied as the first phosphate protecting group. A
DMT-containing group, which allows the measurement
Figure 1. Novel phosphoramidite reagent 1 and commercially available reagents 2 and 3 employed for the 50-phosphorylation of oligonucleotides.
Keywords: Oligonucleotides; Chemical phosphorylation.
* Corresponding author. Tel.: +40-7970-2212; fax: +49-40-7970-2100; e-mail: mleuck@proligo.com
0040-4039/$ - see front matter ꢀ 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2003.10.167

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M. Leuck et al. / Tetrahedron Letters 45 (2004) 321–324
of the novel phosphoramidite 1. The structure of reagent
1 is based on two cyanoethyl protecting groups, wherein
one of the cyanoethyl groups is substituted in the
a-position with a DMT-protected thymidine moiety that
confers solidity on the product and allows the moni-
toring of the coupling reaction through the Vis-spec-
troscopic measurement of the dimethoxytrityl cation in
acidic solution. It can be expected that the a-substituted
cyanoethyl groupwould be cleaved more raipdly
through base-induced b-elimination than the unmodi-
fied cyanoethyl groupdue to the positive inductive effect
of the substituent.
tion can be easily monitored by visual inspection. This
property of reagent 1 is particularly valuable as it avoids
the tedious handling of very viscous liquids, which is a
common feature of other phosphorylation reagents. The
solid reagent can be stored at 4 ꢁC for prolonged peri-
ods.
Reagent 1 was applied in the preparation of the 50-
phosphorylated oligonucleotides 10–12 (Table 1). The
automated solid-phase syntheses of the oligonucleotides
were performed on 1 lmol scale employing a 0.1 M
solution of phosphoramidite 1 in acetonitrile in the last
coupling step. A standard synthesis cycle protocol
(DMT-ON) was used with a coupling time of 90 s.
Cleavage of the oligonucleotides from the support was
accomplished by treatment with concd ammonia at
room temperature for 1 h. Removal of the protecting
groups was conducted through heating of the resulting
ammonia solutions at 55 ꢁC overnight. Anion exchange
HPLC and MALDI–TOF MS analysis of crude oligo-
nucleotides 10–12 confirmed the successful and nearly
quantitative phosphorylation of the oligonucleotides
(Table 1).
Phosphoramidite reagent 1 was prepared from the
commercially available 50-O-DMT-thymidine 4 in four
steps (Scheme 1). In the first step, 4 was converted into
the 4-nitrophenyl carbonate 5 (62%) by treatment with
4-nitrophenyl chloroformate in pyridine.7;8 The car-
bonate 5 was then added, under high dilution, to a
mixture of 1,3-diaminopropane and triethylamine in
CH2Cl2 to generate the carbamate 6 in 69% yield.9
Saponification of the chiral ester 7 was accomplished by
treatment with 1 M NaOH solution. After neutralization
with phosphate buffer the crude acid 8 was coupled with
amine 6 in aqueous solution by treatment with 1-(3-
dimethylaminopropyl)-3-ethylcarbodiimide hydrochlo-
ride (EDC) and 1-hydroxybenzotriazole hydrate (HOBt)
following the Nozaki procedure.10;11 The resulting
alcohol 9 was converted into the phosphoramidite
reagent 1 by treatment with chloro-(2-cyanoethyl) di-
isopropylaminophosphane and N,N-diisopropylethyl-
amine (DIPEA).12 Reagent 1 was obtained in 63% yield
and appears as a colorless solid foam that can be easily
manipulated and weighed into bottles in small portions.
It dissolves readily in acetonitrile, the most common
solvent for phosphoramidite couplings, and its dissolu-
A second synthesis of the 50-phosphorylated oligo-
nucleotide 10 was performed as described above in
DMT-OFF mode. The column effluents from the last
and the last but one detritylation stepand from the
respective washing steps with acetonitrile were collected
and photometrically analyzed for the orange dimeth-
oxytrityl cation at 480 nm. A coupling efficiency for
phosphoramidite reagent 1 of 97.8% was inferred.
Finally, the required reaction time for the complete
removal of the phosphate protective groups was
investigated. The 50-phosphorylated oligonucleotide 10
was prepared as above in DMT-ON mode. Aliquots of
Scheme 1. Synthesis of chemical phosphorylation reagent 1. Reagents and conditions: (a) 4-nitrophenyl chloroformate, pyr, CH2Cl2, rt, 16 h, 62%;
(b) 1,3-diaminopropane, Et3N, CH2Cl2, rt, 16 h, 69%; (c) 1 M NaOH, rt, 2 h; (d) EDC, HOBt, phosphate buffer, DMF, THF, rt, 16 h, 64%;
(e) iPr2NP(Cl)OCH2CH2CN, DIPEA, CH2Cl2, rt, 3 h, 63%.

M. Leuck et al. / Tetrahedron Letters 45 (2004) 321–324
323
Table 1. 50-Phosphorylated oligonucleotides 10–12 prepared with the phosphoramidite reagent 1
Oligonucleotide
AX-HPLCa
Purity (%)
MALDI–MS
Found
OD260
Rt (min)
Calculated
50-p-d(TTT TTT TTT T) 10
50-p-d(CTC TCA GCG AGC CTC AA) 11
50-p-d(TTG AGG CTC GCT GAG AG) 12
15.62b
9.93c
9.63c
90.8
83.1
67.6
3059.9
5192.3
5343.9
3065.4
5188.7
5328.8
79.1
125.7
116.4
a Anion exchange HPLC conditions: Dionex DNAPac PA100 column (4 · 250 mm) eluting with a linear gradient at 85 ꢁC with a flow rate of 1.5 mL/
min, detection at k ¼ 260 nm, buffer A ¼ 25 mM trizma hydrochloride/1 mM EDTA/10%CH3CN, pH 7.5, buffer B ¼ 25 mM trizma hydrochloride/
1 mM EDTA/10%CH3CN/1 M NaCl, pH 7.5.
b Gradient 1: 10–46% buffer B in 22.00 min.
c Gradient 2: 36–80% buffer B in 22.00 min.
poured into stirred hexanes (500 mL) to give a colorless
precipitate that was filtered and dried to provide 4.35 g
(62%) of the carbonate 5 as a colorless powder.
the controlled pore glass (CPG) support containing the
oligonucleotide were treated with concd ammonia
solution or with AMA solution (concd ammonia/40% aq
methylamine 1/1, v/v) using various temperatures and
times. Anion exchange HPLC analysis of the ammonia
solutions indicated that complete deprotection was
achieved within 2 h in concd ammonia at 55 ꢁC or within
15 min in AMA solution at 65 ꢁC.
9. The carbonate 5 (4.15 g, 5.85 mmol) was dissolved in
CH2Cl2 (15 mL) and added dropwise to a stirred and ice-
cooled solution of 1,3-diaminopropane (2.16 g, 29.3 mmol)
and triethylamine (4.1 mL, 29.3 mmol) in CH2Cl2 (50 mL).
The mixture was stirred at room temperature overnight,
then diluted with CH2Cl2 (100 mL) and washed with aq
satd NaHCO3 solution (5 · 100 mL) and brine (100 mL).
The organic phase was dried over Na2SO4, filtered and
concentrated to provide a yellow foam, which was purified
via flash chromatography eluting with CH2Cl2/EtOH/
Et3N 80:15:5 (v/v/v) to give 2.60 g (69%) of the amine 6 as
a colorless amorphous solid. Rf 0.08 (CH2Cl2/EtOH/Et3N
80:15:5, v/v/v). 1H NMR (CD3CN, 300 MHz): d 7.50–7.41
(m, 3H), 7.38–7.25 (m, 7H), 6.90 (d, J ¼ 8:8 Hz, 4H), 6.26
(dd, J ¼ 5:9, 2.6 Hz, 1H), 6.10–6.04 (m, 1H), 5.35–5.29 (m,
1H), 4.08 (d, J ¼ 2:6 Hz, 1H), 3.85 (br s, 2H), 3.78 (s, 6H),
3.40 (dd, J ¼ 10:5, 3.4 Hz, 1H), 3.31 (dd, J ¼ 10:5, 2.9 Hz,
1H), 3.16 (q, J ¼ 6:5 Hz, 2H), 2.65 (t, J ¼ 6:7 Hz, 2H),
2.50–2.30 (m, 2H), 1.57 (quintet, J ¼ 6:7 Hz, 2H), 1.42 (s,
3H). 13C NMR (CD3CN, 75 MHz): d 164.0, 159.0, 156.1,
150.8, 145.0, 136.0, 135.8, 130.3, 128.3, 128.2, 127.3, 113.4,
110.9, 86.9, 84.4, 84.2, 75.2, 64.0, 55.2, 38.7, 38.3, 37.6,
32.2, 11.4.
In conclusion, the novel phosphoramidite 1, prepared in
five steps from a common thymidine intermediate, is
employed as an efficient reagent for the 50-phosphoryl-
ation of oligonucleotides. The reagentꢀs appearance as a
solid foam is advantageous for its manipulation and
handling in solid-phase synthesis and improves its
thermal stability.
References and Notes
1. (a) Wosnick, M. A.; Barnett, R. W.; Vicentini, A. M.;
Erfle, H.; Elliott, R.; Sumner-Smith, M.; Mantei, N.;
Davies, R. W. Gene 1987, 60, 115–127; (b) Carter, J. B.;
Pulaski, S. P.; Theriault, N. Y. BioTechniques 1988, 6,
470–474.
10. Nozaki, S. Chem. Lett. 1997, 1–2.
11. Ethyl (R)-4-cyano-3-hydroxybutanoate
7
(0.702 g,
2. Barany, F. PCR Meth. Appl. 1991, 1, 5–16.
3. Landegren, U.; Kaiser, R.; Sanders, J.; Hood, L. Science
1988, 241, 1077–1080.
4. (a) Chu, B. C. F.; Wahl, G. M.; Orgel, L. E. Nucl. Acids
Res. 1983, 11, 6513–6529; (b) Fedorova, O. S.; Savitskii,
A. P.; Shoikhet, K. G.; Ponomarev, G. V. FEBS Lett.
1990, 259, 335–337.
5. (a) Horn, T.; Urdea, M. S. Tetrahedron Lett. 1986, 27,
4705–4708; (b) Horn, T.; Allen, J. S.; Urdea, M. S.
Nucleosides Nucleotides 1987, 6, 335–340.
€
6. (a) Guzaev, A.; Salo, H.; Azhayev, A.; Lonnberg, H.
Tetrahedron 1995, 51, 9375–9384; (b) Guzaev, A.;
4.47 mmol) was treated with 1 M NaOH (9 mL, 9 mmol)
and the resulting mixture was stirred at room temperature
for 2 h. 1.5 M NaH2PO4 (9 mL) was added and the pH of
the solution was adjusted to 5.7 with concd HCl. After
addition of HOBtÆH2O (70 mg, 0.46 mmol) and DMF
(9 mL) the mixture was treated with a solution of the amine
6 (1.35 g, 2.09 mmol) in THF (4 mL). The pH was again
adjusted to 6.0 with diluted HCl (concd HCl/water 1:10, v/
v) and EDC (0.801 g, 4.18 mmol) was added. The reaction
mixture was stirred overnight, then diluted with water
(100 mL) and extracted with EtOAc (3 · 50 mL). The
organic phase was dried over Na2SO4 and concentrated.
The crude product was purified via flash chromatogra-
phy eluting with EtOAc/hexanes/acetonitrile 70:25:5 (v/v/
v) to provide 1.01 g (64%) of alcohol 9 as a colorless foam.
€
Lonnberg, H. Tetrahedron 1999, 55, 9101–9116.
7. A similar reaction to prepare compound 5 is described by
Li, H.; Miller, M. J. Tetrahedron Lett. 2000, 41, 4323–
4327.
Rf
0.43
(EtOAc/hexanes/EtOH
70:25:5,
v/v/v)
8. 50-O-DMT-thymidine 4 (5.02 g, 9.22 mmol) was co-eva-
porated with pyridine (2 · 20 mL), dissolved in pyridine
(25 mL) and treated dropwise under ice cooling with a
solution of 4-nitrophenyl chloroformate (1.95 g,
9.68 mmol) in CH2Cl2 (25 mL). The mixture was stirred
overnight at room temperature. The solution was concen-
trated to dryness and the remainder was redissolved in
CH2Cl2 (20 mL). This solution was added slowly to
vigorously stirred diethyl ether (150 mL). The resulting
precipitate was filtered off and discarded. The filtrate was
1H NMR (CD3CN, 300 MHz): d 9.09 (br s, 1H), 7.50–
7.40 (m, 3H), 7.38–7.23 (m, 7H), 6.90 (d, J ¼ 8:8 Hz, 4H),
6.68–6.61 (m, 1H), 6.27 (dd, J ¼ 6:2, 2.3 Hz, 1H), 5.86
(t, J ¼ 6:2 Hz, 1H), 5.34–5.28 (m, 1H), 4.19 (quintet,
J ¼ 6:5 Hz, 1H), 4.12–4.08 (m, 1H), 3.78 (s, 6H), 3.41 (dd,
J ¼ 10:5, 3.4 Hz, 1H), 3.32 (dd, J ¼ 10:5, 2.9 Hz, 1H),
3.20 (quintet, J ¼ 6:4 Hz, 2H), 3.11 (dt, J ¼ 7:2, 6.4 Hz,
2H), 2.63 (dd, J ¼ 16:8, 5.0 Hz, 1H), 2.53 (dd, J ¼ 16:8,
6.2 Hz, 1H), 2.45–2.30 (m, 4H), 2.18 (s, 1H), 1.61 (quintet,
J ¼ 6:4 Hz, 2H), 1.43 (s, 3H). 13C NMR (CD3CN,

324
M. Leuck et al. / Tetrahedron Letters 45 (2004) 321–324
75 MHz): d 171.1, 163.8, 159.0, 156.0, 150.7, 145.1, 135.8,
phosphoramidite reagent 1 as a colorless foam. Rf 0.25
(EtOAc/CH2Cl2/acetonitrile 70:23:7, v/v/v) 1H NMR
(CD3CN, 300 MHz): d 9.11 (br s, 1H), 7.55–7.42 (m,
3H), 7.40–7.25 (m, 7H), 6.90 (d, J ¼ 9:1 Hz, 4H), 6.67–
6.60 (m, 1H), 6.27 (dd, J ¼ 6:3, 2.0 Hz, 1H), 5.90 (t,
J ¼ 6:2 Hz, 1H), 5.35–5.27 (m, 1H), 4.50–4.38 (m, 1H),
4.11–4.07 (m, 1H), 3.90–3.69 (m, 8H), 3.67–3.50 (m, 2H),
3.40 (dd, J ¼ 10:4, 3.7 Hz, 1H), 3.31 (dd, J ¼ 10:4, 3.1 Hz,
1H), 3.27–3.05 (m, 4H), 2.80–2.30 (m, 8H), 1.60 (quintet,
J ¼ 6:3 Hz, 2H), 1.44 (s, 3H) 1.25–1.15 (m, 12H).
13C NMR (CD3CN, 75 MHz): d 169.6, 164.0, 159.3,
156.2, 151.0, 145.3, 136.0, 135.9, 130.5, 128.5, 128.4, 127.5,
119.2, 117.8, 113.6, 111.1, 87.1, 84.6, 84.4, 75.4, 67.9, 67.6,
67.5, 67.3, 64.3, 59.0, 58.8, 55.4, 43.7, 43.6, 43.5, 42.3, 42.1,
38.2, 37.8, 36.5, 29.9, 25.2, 24.4, 24.3, 20.4, 11.7. 31P NMR
(CD3CN, 121.5 MHz): d 150.2, 150.1.
135.7, 130.3, 128.3, 128.2, 127.3, 118.3, 113.4, 110.8, 86.9,
84.4, 84.2, 75.3, 64.8, 64.0, 55.2, 41.5, 37.8; 37.5, 36.1, 29.5,
25.2, 11.4.
12. A solution of the alcohol 9 (0.950 g, 1.26 mmol) and N,N-
diisopropylethylamine (2.2 mL, 12.6 mmol) in CH2Cl2
(15 mL) was treated with chloro-(2-cyanoethyl) diisopro-
pylaminophosphane (0.312 g, 1.32 mmol) and stirred at
room temperature for 3 h. The reaction mixture was
concentrated to dryness and the remainder was redissolved
in EtOAc (100 mL). The resulting solution was extracted
with 10% aq Na2CO3 solution (2 · 50 mL) and brine
(50 mL). The organic phase was dried over Na2SO4 and
concentrated to dryness. The crude product was purified
via flash chromatography eluting with EtOAc/CH2Cl2/
acetonitrile 70:23:7 (v/v/v) to provide 0.758 g (63%) of the