A new approach for pyrophosphate bond formation starting from phosphoramidite derivatives by use of 6-trifluoromethyl-1-hydroxybenzotriazole- mediated O-N phosphoryl migration

Article abstract of DOI:10.1016/j.tetlet.2003.11.110

A new method for pyrophosphate bond formation in the solid phase was developed by use of phosphoramidite derivatives, which were found to be readily converted by reaction with 6-trifluoromethyl-1-hydroxybenztriazole via an O-N phosphoryl rearrangement into pentavalent phosphotriester intermediates. These intermediates proved to react smoothly with not only phosphomonoesters but also phosphodiesters to give protected pyrophosphate derivatives which, in turn, could be easily deprotected to give the desired pyrophosphate derivatives.

Full text of DOI:10.1016/j.tetlet.2003.11.110

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 979–982
A new approach for pyrophosphate bond formation starting from
phosphoramidite derivatives by use of 6-trifluoromethyl-1-
hydroxybenzotriazole-mediated O–N phosphoryl migration
Akihiro Ohkubo,a,b Katsufumi Aoki,a Kohji Seiob,c and Mitsuo Sekinea,b,
*
aDepartment of Life Science, Tokyo Institute of Technology, 4259 Nagatsuta, Midoriku, Yokohama 226-8501, Japan
bCREST, JST (Japan Science and Technology Corporation), Nagatsuta, Midoriku, Yokohama 226-8501, Japan
cDivision of Collaborative Research for Bioscience and Biotechnology, Frontier Collaborative Research Center, Nagatsuta,
Midoriku, Yokohama 226-8501, Japan
Received 25 September 2003; revised 19 November 2003; accepted 21 November 2003
Abstract—A new method for pyrophosphate bond formation in the solid phase was developed by use of phosphoramidite deriv-
atives, which were found to be readily converted by reaction with 6-trifluoromethyl-1-hydroxybenztriazole via an O–N phosphoryl
rearrangement into pentavalent phosphotriester intermediates. These intermediates proved to react smoothly with not only phos-
phomonoesters but also phosphodiesters to give protected pyrophosphate derivatives which, in turn, could be easily deprotected to
give the desired pyrophosphate derivatives.
Ó 2003 Elsevier Ltd. All rights reserved.
A variety of pyrophosphate compounds such as nucleo-
tide coenzymes and sugar nucleotides1 play very
important roles in biological reactions. The synthesis of
such compounds and their analogues has been exten-
sively reported.1 From the chemical point of view,
however, there are three disadvantages in the previous
methods2 for pyrophosphate bond formation: (1) Most
of the reactions require much-prolonged periods of time
such as 10–24 h. (2) The starting materials such as
phosphorimidazolidates3 and phosphoromorpholidates4
exhibit inherent high polarity and instability so that the
purification and preservation of them is rather difficult.
(3) Solvents except highly polar solvents such as water
and DMF are not applicable to this reaction because of
poor solubility of the starting materials. As a result, the
isolated yield of target compounds is usually low.
tives.5 A preliminary mechanistic study revealed that the
conversion was not a simple oxidation, but proceeded
via the rearrangement of the phosphite triesters, as
exemplified in Scheme 1.5 Because the resulting benzo-
triazol-1-yl phosphate derivatives exhibit high reactivity
toward various nucleophilic functions, they can be used
as new active intermediates for the synthesis of pyro-
phosphate compounds by the reaction with phosphate
nucleophiles. In this paper, we report a new strategy for
pyrophosphate bond formation using benzotriazol-1-yl
phosphate as a key intermediate. Because the interme-
diates can be prepared in situ from the chemically stable
and lipophilic phosphoramidite compounds, our ap-
proach must be promising as a new strategy to overcome
the above-mentioned drawbacks of conventional pyro-
phosphate synthesis methodology. The usefulness of our
new strategy0 was demonstrated by0 the synthesis of
0
0
0
0
0
In connection with our continuous studies on the
development of new methods for the chemical synthesis
of oligodeoxyribonucleotides, we have recently reported
the facile oxidative conversion of several tervalent
phosphite benzotriazol-1-yl esters into the correspond-
ing pentavalent benzotriazol-1-yl phosphate deriva-
T3 pp5 T, dA3 ppT, dC3 pp5 T and dC3 pp5 T on polymer
supports.
In order to analyze in more detail the O–N phosphoryl
rearrangement mediated by HOBt, dimethyl N,N-di-
isopropylphosphoramidite 1 was chosen as a model
compound. When the phosphoramidite derivative 1 was
activated by use of 5 equiv of HOBt, the starting mate-
rial immediately changed to the corresponding phos-
phite intermediate 2a (31P NMR: 146.6 ppm), which, in
* Corresponding author. Tel.: +81-45-924-5706; fax: +81-45-924-5772;
e-mail: msekine@bio.titech.ac.jp
0040-4039/$ - see front matter Ó 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2003.11.110

980
A. Ohkubo et al. / Tetrahedron Letters 45 (2004) 979–982
5 equiv
N
N
N
N
N
MeO
MeO
O
MeO
P
MeO
MeO
N
O
R
N
R
P
P
R
OH
P
N
R
N
R
N
rearrangement
MeO
N
O
N
N
OH
N
O N
MeO
MeO
N
1
2a; R = H
2b; R = CF3
3a; R = H
3b; R = CF3
4a; R = H
4b; R = CF3
Scheme 1.
turn, was rapidly converted to the phosphotriester
intermediate 4a (31P NMR: 0.8 ppm), as shown in
Scheme 1. In the 31P NMR spectrum of the mixture
obtained after 5 min, a minor peak of the hydrolyzed
product having a P–H bond was observed at 11.8 ppm.
Because the phosphite intermediate 2a could be
observed, the rearrangement was thought to be the rate-
determining step of the overall reaction. It is likely that
the initial rearrangement product 3a reacts with another
HOBt molecule so promptly that we could not observe
3a in the 31P NMR spectrum of the mixture 4a. This
rearrangement of the phosphite intermediate 2a pro-
ceeded to a degree of 90% after 5 min, as shown in
Figure 1A. Completion of this reaction required 15 min.
In the case of 6-trifluoromethyl-HOBt (tfHOBt), the
rate of the rearrangement increased and the phosphor-
amidite unit was completely converted into compound
4b within 5 min, as shown in Figure 1B.
product 9. The simultaneous removal of the cyanoethyl
(CE) and DMTrO(CH2)2SO2(CH2)2 (DESE) groups
from 9 was performed as follows: Treatment of the resin
9 with 3% trichloroacetic acid in CH2Cl2 followed by the
successive treatments with 10% DBU in BSA–pyridine
(1:1, v/v)10 and Et3N–MeOH (1:1, v/v) gave the phos-
phomonoester 10 on the HCP resin. The condensation
of the in situ-generated reactive phosphotriester 6 with
the HCP resin 10 was carried out at room temperature
for 15 min. After the cyanoethyl group and the DMTr
group were removed from the resulting pyrophosphate
derivative 11 via a two-step procedure, the nucleotidic
materials released from the resin by the action of concd
ammonia were analyzed by H0PLC. As shown in Figure
0
2A, the desired product T3 pp5 T 12 was obtained in 81%
yield (HPLC) as the exclusive product except for a
0
minor peak at 8.6 min of p5 T 13, which resulted from
failure of the condensation. Even if the reaction time for
the condensation was extended to 60 min, the coupling
efficiency was unchanged (81%), as shown in Figure 2B.
This result indicates that the condensation for the
pyrophosphate bond formation does not require more
than 15 min. Because previous methods for pyrophos-
phate bond formation required much-prolonged periods
of time of more than 12 h, it should be noted that the
reaction time was considerably reduced by using our
method.
To demonstrate the usefulness of such a final product of
the phosphotriester type as an efficient phosphorylating
reagent for th0e pyrophosphate bond formation, the
0
synthesis of T3 pp5 T was carried out by reaction of a
thymidine 30-phosphotriester intermediate 6, which was
prepared in situ from the thymidine 30-phosphoramidite
derivative 5 by the tfHOBt-mediated reaction, with two
different phosphate species, that is, the phosphomono-
ester 10 and the phosphodiester 16, as described in
Methods A and B, respectively, of Scheme 2.
Method B involves the following new devices to obtain
the phosphodiester intermediate 16. The 50-phosphory-
lation of a T-loaded HCP resin 14 having a silyl-type
linker recently reported by us11 was similarly carried out
to give the fully protected 50-phosphorylated product 15.
In order to selectively remove one of the two 50-phos-
phate protecting groups, the resin 15 was treated suc-
cessively with 10% DBU in CH3CN and 3% TCA in
CH2Cl2. Since this operation contained no silylating
reagents, one of the two phosphate protecting groups,
that is, 2-cyanoethyl and HOCH2CH2SO2CH2CH2
(HESE), could be removed to give the phosphodiester
16 where the latter might remain as the substituent ÔRÕ
predominantly over the former. This is because the
cyano group serves as a stronger electron-withdrawing
group than the HOCH2CH2SO2 group so that the 2-
cyanoethyl group might be more rapidly removed than
the HOCH2CH2SO2CH2CH2 group. Generally, phos-
phodiester compounds have been recognized to be inert
to conventional phosphorylating reagents such as
phosphorimidazolidates and phosphoromorpholidates,
particularly in the liquid-phase synthesis. However, it
In Method A, the 50-phosphitylation of a T-loaded
highly cross-linked polystyrene (HCP) resin 76 contain-
ing a succinate linker7 was carried out by use of
DMTrO(CH2)2- SO2(CH2)2OP(NiPr2)(OCH2CH2CN) 88
that is, a 50-phosphorylating in the presence of benzim-
idazolium triflate9 (BIT). The successive treatment with
0.1 M I2 in pyridine–H2O gave the 50-phosphorylated
Figure 1. 31P NMR spectra of the crude mixtures obtained after the
phosphoramidite 1 was activated by HOBt (panel A) and tfHOBt
(panel B) at room temperature for 5 min.

A. Ohkubo et al. / Tetrahedron Letters 45 (2004) 979–982
981
OCH2CH2CN
DMTrO(CH2)2SO2(CH2)2O
P
(CE)
NiPr2
(DESE)
O
N
8
20 equiv
100 equiv
NH
O
DMTrO
O
N
N
O
HO(CH2)2SO2(CH2)2O
N
NH
O
+
O
O
P
5 min
N
F3C
(HESE)
P
OH
N
HO
O
CEO
O
p5'T
13
-
O
tfHOBt
5
OH
O
O
N
NH
O
NH
O
N
DMTrO
Method A
DMTrO
O
O
O
O
O
N
O
O
O
N
P
P
O
Bttf
O
NH
O
O
P
O
N
CEO
CEO
O
NH
O
O
O
O
O
NH
NH
O
HESEO
O
P
e, d, g
O
-
T3'pp5'T
12
HO P
O
6
N
O
O
DMTrO
O
e, f
a-d
CEO
O
-
O
O
O
CH3CN - NMP ( 9:1, v/v )
O
O
O
O
O
11
O
O
O
r.t., 15 min
or
O
NH
O
NH
NH
NH
O
7
9
10
r.t., 60min
NH
O
N
DMTrO
Method B
O
O
N
O
N
O
O
O
O
NH
O
O
NH
O
P
NH
O
O
P
O
N
CEO
DESEO
P O
O
N
O
O
DMTrO
NH
O
RO
O
O
O
P
CEO
-
RO
O
Si
O
iPr
6
O
h, d, i
O
Si
iPr
O
Si
h
T3'pp5'T
12
iPr
a-d
O
Si
iPr
iPr
CH3CN - NMP ( 9:1, v/v )
r.t., 15 min
O
iPr
iPr
O
O
iPr
NH
O
NH
NH
NH
17
16
14
15
Scheme 2. (a) 3% CCl3COOH, CH2Cl2, rt, 30 s; (b) 8 (20 equiv) BIT (40 equiv) CH3CN, rt, 1 min; (c) 0.1 M I2, pyridine–H2O (9:1, v/v), rt, 2 min;
(d) 3% CCl3COOH, CH2Cl2, rt, 30 s; (e) 10% DBU, pyridine–bis(trimethylsilyl)acetamide (1:1, v/v), rt, 10 min; (f) Et3N–MeOH (1:4, v/v), rt, 20 s;
(g) concd NH3, rt, 40 min; (h) 10% DBU, CH3CN, rt, 1 min; (i) 1 M TBAF, THF, rt, 1 h.
were detected. The silyl-type linker in place of the suc-
cinate linker was used in Method B because it was
reported that treatment of succinate linker-containing
resins with DBU in the absence of BSA causes serious
hydrolysis of the succinate ester linkage via a DBU-
mediated intramolecular cyclization of this linker.10b;12
0
0
Thus, by use of Method B, T3 pp5 T could be isolated in
81% yield and characterized by LC-MS.13
0
0
0
0
Furthermore, we synthesized d[C3 pp5 T], d[A3 pp5 T],
0
0
and d[G3 pp5 T] to demonstrate the generality of our
new method. In the synthesis of these dimers, the pro-
tocol of Method A was used except for the prolonged
ammonia treatment (12 h) for the deprotection and
the release from HCP resin since the commercially
available N-protected phosphoramidite units, 4-N-
acetyldeoxycytidine-30-O-phosphoramidite, 6-N-phen-
oxyacetyldeoxyadenosine-30-O-phosphoramidite, and
2-N-p-isopropylphenoxyacetyl-deoxyguanosine-30-O-phos-
phor-amidite. The efficiency of these condensations for
Figure 2. The amino-exchange HPLC profiles of the mixtures released
from the resins 11 and 17 obtained after condensation of 6 with 10 and
16, respectively. A: Method A, reaction time 15 min; B: Method A,
reaction time 60 min; C: Method B, reaction time 15 min.
150min were ascertain to be as high as that in synthesis of
0
was surprisingly found that the in situ-generated phos-
phate benzotriazol-1-yl ester 6 underwent smooth cou-
pling with the phosphodiester species 16 as shown in
Figure 2C, which suggested excellent coupling efficiency
(94%). We strongly feel that it is necessary for us to
reconsider our stereotype of general pyrophosphate
bond formations. In addition to this finding, it should be
also emphasized that the existence of the hydroxyl group
of the HOCH2CH2SO2CH2CH2 residue attached to the
50-phosphate group on the resin 16 did not affect the
pyrophosphate bond formation. Actually, no byprod-
ucts arising from phosphorylation of the hydroxyl group
T3 pp5 T0, as shown in Figure 3. T0he i0solated yields of
0
0
0
d[C3 pp5 T], d[A3 pp5 T], and d[G3 pp5 T] were 83, 79,
and 84%, respectively, and characterized by LC-MS.14
In conclusion, we have proposed a new convenient
strategy for the formation of pyrophosphate bonds by
the use of readily available phosphoramidite derivatives
and demonstrated the usefulness of our new pyro-
phosp0horylation0 strategy as evi0 denced by the synthesis
0
0
0
0
0
of T3 pp5 T, C3 pp5 T, A3 pp5 T, and G3 pp5 T. One
benefit of our method is that the reaction time can be
considerably reduced compared with the previous

982
A. Ohkubo et al. / Tetrahedron Letters 45 (2004) 979–982
References and notes
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Figure 3. The anion-exchange HPLC profiles of the crude mixtures
from the synthesis by use of Method A. A: d[C3 pp5 T]; B: d[A3 pp5 T];
0
0
0
0
0
0
C: d[G3 pp5 T].
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methods. Another advantageous feature is that phos-
phoramidite derivatives for this reaction are more stable
than the previous starting materials such as phosphor-
imidazolidates and phosphoromorpholidates during
purification or preservation. More detailed studies of
the mechanism of this new pyrophosphorylation and the
high-throughput synthesis of sugar nucleotides in
the solution phase and 50-capped oligonucleotides on
polymer supports15 are now under way.
11. Kobori, A.; Miyata, K.; Ushioda, M.; Seio, K.; Sekine, M.
Chem. Lett. 2002, 16–17.
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13. ESI mass (M-H) calcd 625.10, found 625.13.
14. CppT ESI mass (M-H) calcd 610.10, found 610.39. AppT
ESI mass (M-H) calcd 634.11, found 634.45. GppT ESI
mass (M-H) calcd 650.10, found 650.39.
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Acknowledgements
This work was supported by a Grant-in-Aid for Scien-
tific Research from the Ministry of Education, Culture,
Sports, Science and Technology, Japan. This work was
also supported by CREST of JST (Japan Science and
Technology Agency).