The 4-oxopentyl group as a labile phosphate/thiophosphate protecting group for synthetic oligodeoxyribonucleotides

Article abstract of DOI:10.1016/S0040-4039(01)01094-2

An efficient and economical method for the solid-phase synthesis of oligodeoxyribonucleotides and their phosphorothioate analogues is described. The method entails the use of the 4-oxopentyl group for phosphate/thiophosphate protection. Post-synthesis removal of the protecting group is easily and rapidly achieved under mild conditions at ambient temperature using either pressurized gaseous amines or concentrated ammonium hydroxide.

Full text of DOI:10.1016/S0040-4039(01)01094-2

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 5635–5639
The 4-oxopentyl group as a labile phosphate/thiophosphate
†
protecting group for synthetic oligodeoxyribonucleotides
a
a,‡
a
b
Andrzej Wilk, Marcin K. Chmielewski, Andrzej Grajkowski, Lawrence R. Phillips and
a,
Serge L. Beaucage *
a
FDA-CBER, 8800 Rockville Pike, Bethesda, MD 20892, USA
b
National Cancer Institute, Frederick, MD 21701, USA
Received 30 May 2001; accepted 15 June 2001
Abstract—An efficient and economical method for the solid-phase synthesis of oligodeoxyribonucleotides and their phosphoroth-
ioate analogues is described. The method entails the use of the 4-oxopentyl group for phosphate/thiophosphate protection.
Post-synthesis removal of the protecting group is easily and rapidly achieved under mild conditions at ambient temperature using
either pressurized gaseous amines or concentrated ammonium hydroxide. © 2001 Elsevier Science Ltd. All rights reserved.
2
The recent manufacturing of an anti-viral oligodeoxyri1-
bonucleotide phosphorothioate on a kilogram scale
underscores the potential therapeutic value of synthetic
oligonucleotides against infectious diseases and various
types of cancer in humans. This remarkable achieve-
ment strongly challenges the current methods used to
synthesize oligonucleotides. Of critical importance, the
synthetic methods must not lead to chemical modifica-
tion of the therapeutic oligonucleotides that would
adversely affect potency, and must also be economical
to ensure a reasonable cost per dose of these drugs.
thermolytic deprotection properties. Moreover, the 4-
oxopentyl group can be cleaved completely from
oligonucleotides upon exposure to pressurized ammo-
4
nia/methylamine gas or concentrated ammonium
hydroxide. Given the versatility with which oligonucle-
otides carrying 4-oxopentyl groups for phosphate/thio-
phosphate protection can be deprotected, we now
report the syntheses of deoxyribonucleoside phospho-
ramidites 4a–d, and the use of these compounds in the
solid-phase preparation of an oligodeoxyribonucleotide
(
20-mer) and its phosphorothioate analogue.
The synthesis of 4a–d is illustrated in Scheme 1 and
During the course of our search for cost-effective phos-
phate/thiophosphate protecting groups, several possibil-
ities attracted our attention. For example, we found the
begins with the preparation of the phosphinylating
i-Pr2N
O
2
-(N-formyl-N-methyl)aminoethyl group to be ade-
(i-Pr2N)2PCl
P
CH3
OH
quate for this purpose and performed the synthesis of
i-Pr2N
O
H3C
C6H6
oligodeoxyribonucleotides, such as dT18 and d(AG) ,
O
10
2
1
2
using this group for phosphate protection. The 2-(N-
B1
DMTrO
B1
DMTrO
O
O
2/1H-tetrazole
CH2Cl2
i-Pr2N
O
OH
P
O
O
1
3
b
c
d
a
B = Thymin-1-yl
CH3
1
B = 4-N-Benzoylcytosin-1-yl
B = 6-N-Benzoyladenin-9-yl
B = 2-N-Isobutyrylguanin-9-yl
1
4a-d
1
Scheme 1. Synthesis of the deoxyribonucleoside phospho-
ramidites 4a–d.
0
040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)01094-2

5
636
A. Wilk et al. / Tetrahedron Letters 42 (2001) 5635–5639
reagent 2. Typically, a solution of anhydrous diiso-
propylamine (19.6 mL, 140 mmol) is added dropwise
under an argon atmosphere, over a period of 30 min, to
Given that the 4-oxopentyl phosphate/thiophosphate
protecting group is easily removed from 5a,c under
mild conditions, the deoxyribonucleoside phospho-
ramidites 4a–d are then applied to the solid-phase syn-
a cold solution (5°C) of PCl (1.75 mL, 20 mmol) in dry
3
5
15
benzene (100 mL). The cold suspension is then allowed
thesis of an oligonucleotide (20-mer) and its fully
9a,15
to stir at ambient temperature and the formation of
phosphorothioated analogue
to further demonstrate
bis-(N,N-diisopropylamino)chlorophosphine is moni-
the utility and convenience of this protecting group.
Upon completion of the synthesis, the oligonucleotide
is released from the CPG support and fully deprotected
by treatment with either pressurized ammonia gas (ꢀ10
3
1
tored by P NMR spectroscopy. Upon completion of
6
the reaction (2 days), 5-hydroxy-2-pentanone (1) (2.43
mL, 24 mmol) is added by syringe to the stirred suspen-
3
1
sion. P NMR analysis of the reaction mixture indi-
bar, 25°C, 12 h) or concentrated NH OH (55°C, 10 h).
4
cates that bis-(N,N-diisopropylamino)chlorophosphine
The crude oligonucleotide is analyzed ‘DMTr-on’ and
(
l 135.9 ppm in C D ) is converted to the correspond-
10
P
6
6
‘
DMTr-off’ by RP-HPLC to estimate the overall yield
ing phosphordiamidite 2 (l 123.5 ppm in C D ) within
P
6
6
of the synthetic oligonucleotide. Purity is determined by
RP-HPLC comparison with an oligonucleotide identi-
cal in sequence that was synthesized from commercial
2
h at 25°C. The suspension is filtered and the filtrate is
evaporated under reduced pressure to afford a yellow
oil which is used without further purification in the
preparation of the phosphoramidites 4a–d (Scheme 1).
2
-cyanoethyl deoxyribonucleoside phosphoramidites.
Fig. 1A shows that the overall yield of the ‘DMTr-on’
0-mer deprotected under gas-phase conditions is 93%,
2
Crude phosphordiamidite 2 (980 mL, ꢀ3 mmol) is then
based upon areas of all relevant integrated RP-HPLC
peaks, thereby indicating an average yield of greater
than 99% for each coupling step. The RP-HPLC profi-
les of the ‘DMTr-off’ 20-mer deprotected by contact
with pressurized ammonia gas (Fig. 1B) and concen-
added by syringe under an inert atmosphere to a stirred
7
solution of the deoxyribonucleoside 3a–d (2 mmol) in
7
dry CH Cl (10 mL). Sublimed 1H-tetrazole (120 mg,
2
2
1.6 mmol) is added portionwise under a positive pres-
sure of argon to the solution. The reaction is monitored
by TLC until 4a–d is no longer produced. Triethyl-
amine (1 mL) is added to the solution, which is then
evaporated under reduced pressure to give a gum. The
material is purified by silica gel chromatography to8
afford pure 4a–d in yields ranging from 68 to 80%.
trated NH OH (Fig. 1C) show that the purity of the
4
B1
HO
HO
B
O
O
The dinucleoside phosphotriesters 5a,c are then pre-
pared under standard conditions by mixing 4a,c (0.2 M
CH3
OH
CH3
B1
O
P
O
P
H2N
O
O
i or ii
in MeCN), 1H-tetrazole (0.45 M in MeCN), and either
X
O
X
6
5
%-unprotected thymidine or 5%-unprotected N -benzoyl-
%-deoxyadenosine (0.2 mmol) covalently attached to
O
O
B
2
O
O
long chain alkylamine controlled pore glass (LCAA-
CPG). The resulting dinucleoside phosphite triester
intermediates are oxidized or sulfurized by treatment
with 0.02 M aqueous iodine or 0.05 M 3H-1,2-ben-
zodithiol-3-one 1,1-dioxide in MeCN, respectively.
Release of the dinucleoside phosphotriesters from
LCAA-CPG is effected by pressurized gaseous amines,
O
OH
9
CPG
6a
6
X = O, B = Thy
c
X = S, B = Ade
or concentrated NH OH at 25°C. Analysis of the crude
1
4
5a X = O, B = Thy
10
dinucleotides by reversed-phase (RP) HPLC reveals,
to our surprise, that the 4-oxopentyl phosphate protect-
ing group is completely removed from 5a within 15
min, 90 min, or 4 h when using methylamine gas (ꢀ2.5
bar), ammonia gas (ꢀ10 bar), or concentrated
1
Bz
5c
X = S, B = Ade
HO
B
O
1
1
NH OH, respectively. A proposed mechanism for the
4
CH3
N
O
P
conversion of 5a,c to 7a,c is shown in Scheme 2.
−
O
X
+
The reaction of ammonia with the ketone function of
O
B
O
(major)
the phosphate/thiophosphate protecting group in 5a,c
12
most likely leads to an hemiaminal intermediate 6a,c.
8
This intermediate may then undergo rapid cyclodeester-
ification to produce 7a,c with the concomitant forma-
tion of 2-methyl-1-pyrroline (8) as the major
OH
a,c
7
13
deprotection side-product. This deprotection pathway
is consistent with the removal of 4-aminobutyl or 4-N-
methylaminobutyl phosphate/thiophosphate protecting
Scheme 2. Proposed mechanism for the phosphate/thiophos-
phate deprotection of 5a,c. Reagents and conditions: (i)
ammonia gas (ꢀ10 bar, 25°C); (ii) conc. NH OH, 25°C.
14
groups reported earlier under similar conditions.
4

A. Wilk et al. / Tetrahedron Letters 42 (2001) 5635–5639
5637
oligonucleotide synthesized from 4a–d is equivalent to,
if not better than, that of the same 20-mer prepared
from 2-cyanoethyl deoxyribonucleoside phosphoramid-
ites (Fig. 1D).
In order to assess whether deprotection of the 4-
oxopentyl phosphate protecting group (under the con-
ditions described above) results in nucleobase
modification, the crude 20-mer is subjected to enzy-
matic hydrolysis catalyzed by snake venom phosphodi-
16
esterase and bacterial alkaline phosphatase.
RP-HPLC analysis of the enzymatic digest shows that
the crude 20-mer is completely hydrolyzed to the corre-
sponding nucleosides without detectable modification
1
7
of the nucleobases (see Fig. 2).
The fully phosphorothioated 20-mer oligonucleotide
analogue (synthesized from 4a–d) is also completely
deprotected by pressurized ammonia gas and is
obtained in yields comparable to that of the unmodified
Figure 2. RP-HPLC analysis of the crude 20-mer d(ATCCG-
TAGCTAAGGTCATGC) synthesized from 4a–d, depro-
tected by NH (ꢀ10 bar, 12 h, 25°C), and digested by snake
3
venom phosphodiesterase and bacterial alkaline phosphatase
(12 h, 37°C). Identities of the RP-HPLC peaks from left to
right are as follows: dC, dG, dT, dA and benzamide (ꢀ17
min) when compared to authentic commercial samples.
2
0-mer as judged by RP-HPLC analysis of the crude
3
1
oligonucleotide product (data not shown). P NMR
analysis of the crude phosphorothioated 20-mer is pre-
Figure 1. RP-HPLC profiles of a crude 20-mer synthesized
from 4a–d. A: 5%-DMTr-d(ATCCGTAGCTAAGGTCATGC)
31
Figure 3. 121 MHz P NMR spectra of crude 20-mer
deprotected by NH (ꢀ10 bar, 12 h, 25°C). B and C: d(ATC-
3
d(APSTPSCPSCPSGPSTPSAPSGPSCPSTPSAPSAPSGPSGPSTPSCPS-
APSTPSGPSC) in aqueous solvents. A: 20-mer synthesized
from commercial 2-cyanoethyl deoxyribonucleoside phospho-
ramidites and deprotected by concentrated NH OH (55°, 10
h). B: 20-mer prepared from 4a–d and deprotected by NH3
(ꢀ10 bar, 12 h, 25°C).
CGTAGCTAAGGTCATGC) deprotected by NH (ꢀ10 bar,
3
2
5°C, 12 h) and by concentrated NH OH (55°C, 10 h),
4
respectively. D: d(ATCCGTAGCTAAGGTCATGC) synthe-
sized from 2-cyanoethyl deoxyribonucleoside phosphoramid-
4
ites and deprotected by concentrated NH OH (55°C, 10 h).
4

5
638
A. Wilk et al. / Tetrahedron Letters 42 (2001) 5635–5639
sented in Fig. 3, and demonstrates that the unwanted
Aldrich, respectively. These are kept overnight over
P O in a dessicator under high vacuum prior to use.
production of desulfurized material (l ꢀ0 ppm) is
P
2
5
1
8
31
minimal (<2%) whether the 4-oxopentyl group or the
-cyanoethyl group is used for thiophosphate protec-
8. Compound 4a: 80%; P NMR (121 MHz, C D ): l
6 6
2
148.38, 149.04 ppm; FAB-HRMS: calcd for
+
tion during synthesis.
(C H N O P+Na) 798.3496, found 798.3541. Com-
42
54
3
9
31
pound 4b: 78%; P NMR (121 MHz, C D ): l 148.87,
6
6
Thus, the data presented herein support the use of the
1
49.16 ppm; FAB-HRMS: calcd for (C H N O P+
48 57 4 9
4-oxopentyl group for phosphate/thiophosphate protec-
+
31
Na) 887.3761, found 887.3682. Compound 4c: 72%;
P
tion in the synthesis of alkylation-free oligodeoxyri-
bonucleotides. The deoxyribonucleoside phosphora-
midites 4a–d are easily prepared and are coupled in
high efficiency in the stepwise solid-phase synthesis of
oligodeoxyribonucleotides. As a phosphate protecting
group, the 4-oxopentyl group is more versatile than the
NMR (121 MHz, C D ): 148.61, 148.85 ppm;
l
6
6
+
FAB-HRMS: calcd for (C H N O P+Na) 911.3873,
49
57
6
8
31
found 911.3881. Compound 4d: 68%; P NMR (121
MHz, C D ): l 148.03, 148.503 ppm; FAB-HRMS:
6
6
+
calcd for (C H N O P+Na)
893.3980, found
46
59
6
9
893.3965.
2
-cyanoethyl group in that it is completely removed
from oligonucleotides under neutral conditions (<1 h at
0°C in an aqueous buffer at pH 7.0). Such deprotec-
9
. Purchased from Glen Research or prepared according
to: (a) Iyer, R. P.; Phillips, L. R.; Egan, W.; Regan, J.
B.; Beaucage, S. L. J. Org. Chem. 1990, 55, 4693–4699.
9
tion conditions should be ideal for oligonucleotides
carrying nucleobases and/or reporter groups that are
base-sensitive. In regard to thiophosphate protection,
the 4-oxopentyl group is as versatile as the 2-cyanoethyl
group as they are both rapidly removed when treated
with pressurized gaseous amines (NH or CH NH ) or
(
b) Regan, J. B.; Phillips, L. R.; Beaucage, S. L. Org.
Prep. Proc. Int. 1992, 24, 488–492.
1
1
0. RP-HPLC analyses are performed using a 5 mm Supel-
cosil LC-18S column (4.6 mm×25 cm) and a linear gra-
dient of 1% MeCN/min starting from 0.1
M
3
3
2
triethylammonium acetate (pH 7.0) pumped at a flow
rate of 1 mL/min.
concentrated ammonium hydroxide. Considering the
simple and economical syntheses of 4a–d, these phos-
phoramidites are suitable for the large-scale preparation
of therapeutic oligonucleotides that is necessary for
clinical studies. It is also likely that the 4-oxopentyl
group will be useful for phosphate/thiophosphate pro-
tection in the synthesis of oligoribonucleotides and their
phosphorothioated analogues.
1. Comparatively, the removal of an ethyl phosphate pro-
tecting group from DNA oligonucleotides is reported to
proceed to the extent of only ꢀ10% when exposed to
concentrated NH OH for at least 36 h at 25°C; see:
4
Koziolkiewicz, M.; Wilk, A. In Methods in Molecular
Biology; Agrawal, S., Ed. Protocols for Oligonucleotides
and Analogs; Humana Press: Totowa, 1993; Vol. 20, pp.
207–224.
Acknowledgements
12. March, J. Advanced Organic Chemistry—Reactions,
Mechanism, and Structure, 4th ed.; John Wiley & Sons:
New York, 1992; p. 897.
13
This research is supported in part by an appointment to
the Postgraduate Research Participation Program at
the Center for Biologics Evaluation and Research
administered by the Oak Ridge Institute for Science
and Education through an interagency agreement
between the US Department of Energy and the US
Food and Drug Administration.
1
1
1
3. C NMR analysis of a model experiment aimed at
inducing cyclodeesterification of O-(4-oxopentyl)-O,O-
diethyl phosphate (see Ref. 19) to O,O-diethyl phos-
phate, when left standing in concentrated NH OH,
4
reveals the presence of five major signals exhibiting
chemical shifts (175.0, 60.7, 38.5, 22.6 and 19.3 ppm)
identical to those of an authentic sample of 8 (Aldrich)
in concentrated NH OH. These findings strongly sup-
4
port the deprotection mechanism proposed in Scheme 2.
4. (a) Wilk, A.; Srinivasachar, K.; Beaucage, S. L. J. Org.
Chem. 1997, 62, 6712–6713; (b) Wilk, A.; Grajkowski,
A.; Phillips, L. R.; Beaucage, S. L. J. Org. Chem. 1999,
References
1
2
3
4
. Guzaev, A. P.; Manoharan, M. J. Am. Chem. Soc. 2001,
1
23, 783–793.
64, 7515–7522; (c) Wilk, A.; Grajkowski, A.;
. Grajkowski, A.; Wilk, A.; Chmielewski, M. K.; Phillips,
L. R.; Beaucage, S. L. Org. Lett. 2001, 3, 1287–1290.
. Wilk, A.; Grajkowski, A.; Phillips, L. R.; Beaucage, S.
L. J. Am. Chem. Soc. 2000, 122, 2149–2156.
. Boal, J. H.; Wilk, A.; Harindranath, N.; Max, E. E.;
Kempe, T.; Beaucage, S. L. Nucleic Acids Res. 1996, 24,
Chmielewski, M. K.; Phillips, L. R.; Beaucage, S. L. In
Current Protocols in Nucleic Acid Chemistry; Beaucage,
S. L.; Bergstrom, D. E.; Glick, G. D.; Jones, R. A.,
Eds.; John Wiley & Sons: New York, 2001; pp. 2.7.1–
2.7.12.
5. Phosphoramidites 4a–d are kept overnight over P O in
3
115–3117.
2
5
a dessicator under high vacuum prior to being dissolved
in dry MeCN to a concentration of 0.2 M. The wait
time required for the coupling step and the oxidation
reaction is 150 s and 60 s, respectively.
5
. Diisopropylamine and benzene (Aldrich) are dried by
refluxing over CaH , distilled, and transferred under
2
anhydrous conditions to amber glass bottles containing
,
4 A molecular sieves.
6
7
. Purchased from Aldrich and used as received.
. The deoxyribonucleosides 3a–d and sublimed 1H-Tetra-
zole are obtained from Chem-Impex International and
16. Enzymatic digestion is performed as described in:
Scremin, C. L.; Zhou, L.; Srinivasachar, K.; Beaucage,
S. L. J. Org. Chem. 1994, 59, 1963–1966.

A. Wilk et al. / Tetrahedron Letters 42 (2001) 5635–5639
7. Incubation of thymidine, N -benzoyl-2%-deoxycytidine,
5639
4
1
1
molytically removed under neutral conditions (see Ref.
2). The use of pressurized gaseous amines is therefore
strongly recommended for the cleavage of 4-oxopentyl
groups from phosphorothioated oligonucleotides.
19. O-(4-Oxopentyl)-O,O-diethyl phosphate is synthesized as
reported for the preparation of O,O-diethyl-O-[4-[N-
methyl-N-(2,2,2-trifluoroacetyl)amino]butyl] phosphate
(see Ref. 14b) and is purified by silica-gel chromatogra-
phy prior to use.
6
2
N -benzoyl-2%-deoxyadenosine, and N -isobutyryl-2%-
deoxyguanosine with 8 under condition identical to those
used to evaluate nucleobase alkylation by acrylonitrile
(see Ref. 14b) was performed. No nucleobase modifica-
tion caused by 8 in a setting similar to that of large-scale
oligonucleotide deprotection was detected by RP-HPLC.
8. The extent of desulfurization is more pronounced when
4
-oxopentyl thiophosphate protecting groups are ther-
.