Novel reagents for terminal phosphorylation and thiophosphorylation of synthetic oligonucleotides

Article abstract of DOI:10.1016/S0040-4039(01)00834-6

Two novel phosphoramidite building blocks and a solid support that allow an efficient solid-phase phosphorylation or thiophosphorylation of synthetic oligonucleotides were developed. The utility of these synthetic tools was demonstrated in the preparation of 5′- or 3′-thiophosphorylated oligonucleotides, which were subsequently labeled at the termini with fluorescent reporters.

Full text of DOI:10.1016/S0040-4039(01)00834-6

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 4769–4773
Novel reagents for terminal phosphorylation and
thiophosphorylation of synthetic oligonucleotides
Andrei P. Guzaev* and Muthiah Manoharan
Department of Medicinal Chemistry, Isis Pharmaceuticals, 2292 Faraday Ave., Carlsbad, CA 92008, USA
Received 28 April 2001; accepted 16 May 2001
Abstract—Two novel phosphoramidite building blocks and a solid support that allow an efficient solid-phase phosphorylation or
thiophosphorylation of synthetic oligonucleotides were developed. The utility of these synthetic tools was demonstrated in the
preparation of 5%- or 3%-thiophosphorylated oligonucleotides, which were subsequently labeled at the termini with fluorescent
reporters. © 2001 Elsevier Science Ltd. All rights reserved.
Terminal thiophosphate (PS) group provides a conve-
nient site for regiospecific conjugation of synthetic
oligonucleotides to a variety of ligands bearing an
electrophilic group.1,2 However, extensive application of
this technique has been hampered by the lack of
efficient methods for the terminal thiophosphorylation
of synthetic oligonucleotides. Although convenient
methods for the 5%-3–5 and 3%-phosphorylation5,6 are well
documented in the literature, those dealing with the
introduction of the terminal PS group are limited. The
release of the terminal thionophosphomonoester
employs either reductive7 or oxidative1 conditions that
unnecessarily complicate the deprotection of oligonu-
cleotides. Besides, both approaches suffer from substan-
tial desulfurization of the PS moiety, which results in
the corresponding oligonucleotide 5%- or 3%-phosphate
(PO) in ca. 15% yield.1 The subsequent removal of this
side product by HPLC purification is not trivial.
Novel phosphoramidite reagents were synthesized as
depicted in Scheme 1. Compound 38 was treated with
4,4%,4%%-trimethoxytrityl chloride (TMT-Cl) in Py/diox-
ane to give 4, which was isolated on a silica gel column
in 67.5% yield.9 This was converted to the 2-cyanoethyl
protected phosphoramidite 1a under the standard con-
ditions.10 To obtain 1b, compound 4 was first treated
with [(i-Pr)2N]2P–Cl under basic conditions; a bisamid-
ite obtained was treated with (9H-fluorene-9-
yl)methanol (Fm-OH) and 1H-tetrazole under the
reported conditions.11,12 Compounds 1a and 1b were
isolated by column chromatography in 86 and 68%
yield, respectively. The phosphoramidite 1a was stable
at 4°C for more than 2 months. To assure similar
stability of 1b, traces of triethylamine retained in the
product from the purification step should be removed
by repeated co-evaporation with toluene.
O
TMTO
EtO
CN
1a: R =
1b: R =
OEt
We report here a convenient approach for the terminal
phosphorylation and thiophosphorylation of synthetic
oligonucleotides using phosphoramidite building blocks
1a and 1b and a solid support 2. In the case of
thiophosphorylation, the final deprotection under stan-
dard conditions results in the desired products with
only minor desulfurization (1–1.5%). The synthesized
oligonucleotides were successfully converted to their
fluorescent conjugates by reacting with iodoacetamide
derivatives of fluorescent dyes, fluorescein and pyrene.
O
O
OR
P
N
iPr
1a,b
iPr
(Fm)
O
TMTO
EtO
OEt
O
2
O
O
O
Keywords: nucleic acids; phosphorylation; phosphoramidites; solid-
phase synthesis; supported reagents/reactions; labelling.
* Corresponding author. Tel.: (760) 603 2445; fax: (760) 929 0036;
e-mail: aguzaev@isisph.com
H
N
O
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)00834-6

4770
A. P. Guzae6, M. Manoharan / Tetrahedron Letters 42 (2001) 4769–4773
In order to synthesize the solid support 2, 4 was treated
with diglycolic anhydride13 in pyridine/dioxane to give
a monoester 5 in quantitative yield (Scheme 1). This
was coupled to long chain alkylamine controlled pore
glass essentially as described previously14 to give the
solid support 2 loaded at 81 mmol g−1.15
was carried out on the solid support 2 (Scheme 3). The
solid support-bound 6 and 18 were assembled using the
conventional oxidation with I2 solution. Oligonucle-
otide phosphorothioates 7 and 19 were synthesized
using 3H-1,2-benzodithiol-3-one 1,1-dioxide as a sulfur-
transfer agent.16 To synthesize oligonucleotides 8 and
20, the sulfurization was carried out within the last and
the first elongation cycles, respectively, while all inter-
nucleosidic linkages were oxidized with t-BuOOH (10%
in MeCN; 10 min).
The utility of 1a, 1b and 2 as oligonucleotide phosphor-
ylation reagents was next studied. In the case of 5%-
phosphorylation, protected oligonucleotides 6–8 were
first assembled in a standard manner on 1 mmol scale
using 0.1 M solutions of 1a and 1b in MeCN for the
last coupling step (Scheme 2). To achieve the 3%-phos-
phorylation, the synthesis of oligonucleotides 18–20
When the syntheses were complete, the solid support-
bound material was deprotected with concentrated
aqueous ammonium hydroxide under the standard con-
O
O
TMTO
O
TMTO
TMT-Cl
HO
HO
OEt
OEt
3
i for 1a
ii for 1b
EtO
OEt
EtO
OEt
Py / Dioxane
O
O
O
P
O
O
OH
R
1a,b
4
N
iPr
iPr
CN
iii
O
1a: R =
1b: R =
O
TMTO
EtO
OEt
O
TMT =
O
5: R1 = OH
O
O
O
iv
R1
2: R1 =
N
H
(Fm)
O
O
Scheme 1. (i) [(i-Pr)2N]2P–O(CH2)2CN, 1H-tetrazole, MeCN (1 h, rt); (ii) 1, [(i-Pr)2N]2P–Cl, (i-Pr)2NEt, CH2Cl2 (45 min, −70 to
20°C); 2, Fm-OH, 1H-tetrazole (2 h, rt); (iii) diglycolic anhydride, Py, dioxane; (iv) 1, 2,2%-dithiobis(5-nitropyridine), Ph3P/
DMAP/MeCN/1,2-dichloroethane; 2, long chain alkylamine CPG.
OR
6a,b-8a,b
Oligonucleotide
P
Protected Oligo; P=Y
CN
TMTO
O
O
O
ii
a: R =
X
synthesis
EtO2C CO2Et
O
b: R = Fm
i
O
P
X
O P O
O
9-11: R1 = TMT
12-14: R1 = H
0.1M aq Piperidine
30 min
R1O
EtO2C CO2Et
O
O
OH
OH
Oligo; P=Y
Oligo; P=Y
AcOH/
H2O
X
15-17
Scheme 2. i: Concentrated NH3−H2O; ii: 1, Cl2HCCOOH (3% in CH2Cl2, 45 s); 2, concentrated NH3−H2O. 6, 9, 12, 15:
Oligo=T12, X=Y=O; 7, 10, 13, 16: Oligo=5%-ATGCAT2CTGC5A2G2A-3%, X=Y=S; 8, 11, 14, 17: Oligo=5%-
ATGCAT2CTGC5A2G2A-3%, X=S, Y=O.
O
X
O
O P X
O
Oligonucleotide
synthesis
NH3-H2O
Protected oligo; P=Y
NC
DMTO
O P O
O
RO
2
Oligo; P=Y
EtO2C CO2Et
O
18-20
21-23: R = DMT
24-26: R = H
AcOH/
H2O
Scheme 3. Oligo=5%-TGCATC5AG2C2AC2AT-3%; 18, 21 and 24: X=Y=O; 19, 22 and 25: X=Y=S; 20, 23 and 26: X=S, Y=O.

A. P. Guzae6, M. Manoharan / Tetrahedron Letters 42 (2001) 4769–4773
4771
ditions (2 h/rt for 6 or 6 h/55°C for 7, 8 and 18–20). For
6–8, this led to 9–11, which were purified by reverse-
phase HPLC and characterized. Oligonucleotides 9–11
were detritylated with 10% aqueous AcOH to give 12–14,
which were subsequently converted to 15–17 by a 30 min
treatment with 0.1 M aqueous piperidine. Alternatively,
6–8 were detritylated on the synthesizer and then depro-
tected with concentrated aqueous ammonium hydroxide
to give crude 15–17. The deprotection of 18–20 released
the oligonucleotides 21–23. The deprotection mixtures
and the final products were analyzed by reverse-phase
HPLC–ESMS and anion-exchange HPLC.
common 4,4%-dimethoxytrityl (DMT) group was used,
the detritylation of a non-nucleosidic moiety in analogs
of 27 on solid phase required treatment with TFA (2%
in CH2Cl2) and dichloroacetic acid (DCA; 3% in CH2Cl2)
for 40 s and 5 min, respectively.4–6 In contrast, when 2
and 6–8 were detritylated under the standard conditions
(3% DCA in CH2Cl2), the removal of the TMT protec-
tion was complete in 45 s, i.e. as fast as that of the DMT
group from a 5%-nucleoside in solid support-bound,
5%-DMT protected oligonucleotides. This assured the
compatibility of 1a, 1b and 2 with the conventional
protocol of the detritylation on automated synthesizers
and simplified their use in the routine preparation of
synthetic phosphorylated oligonucleotides.
The analysis revealed quantitative introduction of the
non-nucleosidic moiety to 9–11 and the terminal PO
residuesto15and24. Similarly, 3%-terminalthiophosphor-
ylation resulted in quantitative modification of oligonu-
cleotides to give 22 and 23 contaminated with a minor
amount of desulfurized oligonucleotides. To test the
feasibility of 1a, 1b and 2 for the terminal thiophosphor-
ylation, the extent of desulfurization was determined. As
shown by HPLC−ESMS, the content of mono-desulfur-
ized oligonucleotides in crude samples of 10, 11, 16, 17,
22 and 25 adhered to the limits characteristic for the
standard phosphorothioate synthesis (ca. 0.5–0.6% per
linkage). The extent of desulfurization was slightly higher
in 23 and 26 (ca. 1.5% with respect to the intact
oligonucleotides). This might reflect a partial desulfuriza-
tion resulting from the exposure of the 3%-terminal PS
moiety to 19 cycles of oxidation. In comparison with the
reported methods,1 these results demonstrate a dramatic
improvement in the purity of the oligonucleotides. More
importantly, the method reported here does not require
any additional postsynthetic procedures for the deprotec-
tion of the terminal PS group.
In contrast to detritylation on solid phase, the removal
of the DMT group from phosphate and base-deprotected
oligonucleotides 28 in solution using 80% aqueous AcOH
proceeded in a normal fashion.4,5 This prompted us to
verify whether the stability of the TMT group was
sufficient for the safe isolation of the TMT-On oligonu-
cleotides in high yields and purity. The greater lability of
the TMT group was indeed reflected in the fact that the
detritylation of 9–11 required only 10% aqueous AcOH
for 30 min. However, no detectable loss of the TMT
group was observed during the ammonolysis and subse-
quent evaporation and purification of 9–11. This suggests
that the TMT group displays sufficient stability required
for the safe processing of TMT-On oligonucleotides.
The presence of terminal PS groups in oligonucleotides
17 and 26, was further verified by alkylation with
fluorescent labeling reagents, iodoacetamides 29 and 30
(Scheme 4).1 The products, 31–34, demonstrated UV–vis
All terminally phosphorylated oligonucleotides, 15−17
and 24−26, were isolated by HPLC in yields typical for
the scale of synthesis employed (68% for 15 and 35−45%
for 16, 17 and 24−26). The high yields and purity of these
compounds indicated that the release of the terminal PO
and PS groups was most likely governed by the mecha-
nism and the kinetics reported for the earlier generation
of the phosphoramidite 1 and the solid support 2.4–6
O
I
R
H
N
N
H
O
O P S
O
17, 26
R
Oligo; P=O
29, 30
O
31-34
29, 31, 33: R =
OMe
MeO
CN
O
P
Protected Oligo
O
O
O
O
O
O
O
Ph
O
EtO2C CO2Et
27
30, 32, 34: R =
O
P
O2C
Oligo
DMTO
O
O
EtO2C CO2Et
28
Scheme
4. For
17,
31
and
32:
Oligo=5%-pS-
The practical utility of 1a, 1b and 2 was significantly
improved by using the TMT protection. When the more
ATGCAT2CTGC5A2G2A-3%; for 26, 33 and 34: Oligo=5%-
TGCATC5AG2C2AC2AT-pS-3%.

4772
A. P. Guzae6, M. Manoharan / Tetrahedron Letters 42 (2001) 4769–4773
spectra consistent with the presence of appropriate
dyes. As evidenced by reverse-phase HPLC, treatment
of the conjugates 31–34 with concentrated aqueous
ammonium hydroxide did not result in the loss of the
reporter group. This suggests that the conjugation at
the terminal PS group results in the negatively charged
S,O-dialkyl phosphorothiolate moiety.
dried over Na2SO4 and evaporated. The residue was
purified on a silica gel column eluting with a gradient
from 0:95:5 to 30:65:5 ethyl acetate/hexane/triethylamine
(TEA) to give 1a (650 mg, 86.3%) as a colorless oil. HR
FABMS: found m/z 752.8309; C40H53N2O10P requires
752.8301. 1H NMR (CDCl3): l 7.40–7.25 (6H, m), 6.9–
6.65 (6H, m), 4.3–4.0 (6H, m), 3.77 (9H, s), 3.65 (2H, s),
3.8–3.4 (4H, m), 2.55 (2H, m), 1.40–1.00 (18H, m). 13C
NMR (CDCl3): l 168.6, 168.4, 158.3, 136.5, 130.0,
117.3, 113.0, 85.6, 63.9, 63.6, 62.1, 61.9, 61.4, 61.3, 61.1,
60.9, 55.1, 43.1, 42.9, 24.6, 24.4, 20.5, 14.2. 31P NMR
(CDCl3): l 147.4.
In conclusion, the use of the phosphoramidite building
blocks 1a and 1b and the solid support 2 improves the
methodology for the terminal phosphorylation of syn-
thetic oligonucleotides. Moreover, it allows a straight-
forward
preparation
of
thiophosphorylated
11. A solution of chloro bis[(N,N,-diisopropyl)amino]-
phosphite (827 mg, 3.1 mmol) in dry CH2Cl2 (10 mL)
was added dropwise to a mixture of 4 (1430 mg, 2.6
mmol) and N-ethyl-N,N-diisopropylamine (439 mg, 3.4
mmol) in dry CH2Cl2 (10 mL) under magnetic stirring
at −30°C. The reaction mixture was warmed to room
temperature, and the stirring was continued for 1 h.
Fm-OH (712 mg, 3.6 mmol) and 1H-tetrazole (0.45 M
in MeCN; 5.6 mL, 2.5 mmol) were added. The resulting
mixture was kept at rt for 2 h. Aqueous NaHCO3 (5%;
5 mL) was added, the emulsion was diluted with brine
(25 mL) and extracted with ethyl acetate (3×50 mL).
Extracts were washed with brine (3×25 mL), dried over
Na2SO4 and evaporated. The residue was dissolved in
toluene (50 mL), applied on a silica gel column, and
separated eluting with a step gradient from 0:99:1 to
25:74:1 ethyl acetate/hexane/TEA. The fractions were
evaporated, co-evaporated with dry toluene (5×100 mL),
and dried on an oil pump to give 1b (1548 mg, 68.1%)
oligonucleotides in high purity and their subsequent
site-specific conjugation with thiophilic reporter
groups.
References
1. (a) Alefelder, S.; Patel, B. K.; Eckstein, F. Nucleic Acids
Res. 1998, 26, 4983–4988; (b) Garc´ıa-Echeverr´ıa, C.;
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as a white solid foam. HR FABMS: found m/z
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283−308.
727.3486; C39H54NO10P requires 727.3485. 1H NMR
(CDCl3): l 7.8–7.5 (4H, m); 7.5–7.15 (10H, m); 6.9–6.7
(6H, m); 4.50–4.22 (2H, m); 4.20–4.05 (4H, m); 4.05–
3.85 (2H, m); 3.72 (9H, s); 3.80–3.40 (5H, m); 1.30–1.00
(18H, m). 31P NMR (CDCl3): l 147.4. HR FABMS:
found m/z 727.3486; C39H54NO10P requires 727.3485.
1H NMR (CDCl3): l 7.8–7.5 (4H, m); 7.5–7.15 (10H,
m); 6.9–6.7 (6H, m); 4.50–4.22 (2H, m); 4.20–4.05 (4H,
m); 4.05–3.85 (2H, m); 3.72 (9H, s); 3.80–3.40 (5H, m);
1.30–1.00 (18H, m). 31P NMR (CDCl3): l 147.4.
8. Guzaev, A.; Lo¨nnberg, H. Synthesis 1997, 1281–1284.
9. Compound 3 (11.5 g, 55.0 mmol) was treated with
TMT-Cl (19.2 g, 52.0 mmol) in Py (16 mL) and dioxane
(100 mL) overnight and the solvent was evaporated. The
residue was dissolved in CH2Cl2 (500 mL), washed with
NaHCO3 (5% aq., 3×100 mL), brine (2×100 mL), dried
over Na2SO4 and evaporated. Purification on a silica gel
column using a step gradient of ethyl acetate (0–15%) in
toluene gave 4 (8.2 g, 67.5%) as an oil. HR MALDI
MS: calcd for C31H36O9 (M+H+) 553.2442, found
12. Guzaev, A. P.; Manoharan, M. J. Am. Chem. Soc. 2001,
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15. A mixture of 4 (1.11 g, 2.0 mmol), 1,4-dioxane-2,6-dione
(0.70 g, 6.0 mmol), Py (5 mL), and dioxane (5 mL) was
kept overnight at rt. The solvent was evaporated and
the residue was dissolved in ethyl acetate (50 mL). The
solution was washed with 2 M aq. triethylammonium
acetate (5×10 mL) and water (5×10 mL) and dried over
Na2SO4. The extract was evaporated to give crude
monoester 5 (1.54 g; triethylammonium salt) as a solid
foam in quantitative yield. Crude 5 (0.99 g, 1.28 mmol)
and Ph3P (0.40 g, 1.5 mmol) were dissolved in 1,2-
dichloroethane (5 mL). To this was added a solution of
2,2%-dithiobis(5-nitropyridine) (0.47 g, 1.5 mmol) and
DMAP (0.18 g, 1.5 mmol) in 1,2-dichloroethane (5 mL).
The mixture was shaken for 15 min and filtered. The
precipitate was washed on filter with 1,2-dichloroethane
1
553.2438. H NMR (CDCl3): l 7.29 (6H, d, J=8.8 Hz),
6.83 (6H, d, J=8.8 Hz), 4.26–4.11 (6H, m), 3.80 (9H, s),
3.62 (2H, s), 2.11 (1H, t, J=5.8 Hz), 1.24 (6H, t, J=7.0
Hz). 13C NMR (CDCl3): l 169.3, 158.5, 136.1, 129.8,
113.2, 86.0, 63.7, 61.9, 61.6, 60.6, 55.2, 14.1.
10. 1H-Tetrazole (0.45 M in MeCN, 1.1 mL, 0.5 mmol) was
added to 4 (553 mg, 1.0 mmol) and 2-cyanoethyl
N,N,N%,N%-tetraisopropyl phosphorodiamidite (332 mg,
1.1 mmol) in CH2Cl2 (5 mL). The solution was stirred
for 2 h, and NaHCO3 (5% aqueous, 5 mL) was added.
The mixture was diluted with brine (15 mL) and
extracted with CH2Cl2 (3×40 mL). The extracts were
.

A. P. Guzae6, M. Manoharan / Tetrahedron Letters 42 (2001) 4769–4773
4773
(5 mL), and the combined filtrates were added to alkyl-
amine CPG (5.98 g; 119 mmol g−1, 0.71 mmol). The
suspension was shaken for 1 h and filtered. The solid
support was washed with 1,2-dichloroethane (3×10 mL),
treated with Ac2O/N-methylimidazole/THF (10:10:80) for
30 min, washed with 1,2-dichloroethane (5×10 mL) and
MeCN (5×10 mL), and dried to give 2. An aliquot of 2 was
treated with TFA (2% in CH2Cl2), and the concentration
of the released TMT cation was determined colorimetri-
cally at 486 nm to give the loading of 81 mmol g−1
.
16. Iyer, R. P.; Phillips, L. R.; Egan, W.; Regan, J. B.;
Beaucage, S. L. J. Org. Chem. 1990, 55, 4693–4699.
.