Large scale, liquid phase oligonucleotide synthesis by alkyl H- phosphonate approach

Article abstract of DOI:10.1016/S0968-0896(99)00287-4

A new approach to the liquid phase synthesis of oligonucleotide is described, it is based on oxidative coupling using alkyl H-phosphonate synthon and polyethylene glycol (PEG₅₀₀₀) as a soluble support. Nucleoside alkyl H-phosphonate undergoes o

Full text of DOI:10.1016/S0968-0896(99)00287-4

Bioorganic & Medicinal Chemistry 8 (2000) 337±342
Large Scale, Liquid Phase Oligonucleotide Synthesis by Alkyl
H-phosphonate Approach
Kamlesh J. Padiya and Manikrao M. Salunkhe*
Department of Chemistry, The Institute of Science, 15-Madam Cama Road, Mumbai 400 032, India
Received 3 June 1999; accepted 21 October 1999
AbstractÐA new approach to the liquid phase synthesis of oligonucleotide is described, it is based on oxidative coupling using
alkyl H-phosphonate synthon and polyethylene glycol (PEG5000) as a soluble support. Nucleoside alkyl H-phosphonate undergoes
oxidative coupling in presence of NBS. The use of polyethylene glycol as a soluble polymeric support preserves some convenient
features of the solid phase synthesis with new interesting advantages. This liquid phase method appears e€ective in terms of speed
and coupling yield and can be evaluated for the production of large amount of oligonucleotide (100 mM). # 2000 Elsevier Science
Ltd. All rights reserved.
Introduction
unit for the synthesis of oligonucleotides. A major
emphasis of our research has been to develop an alter-
native synthetic strategy with a minimum number of
Synthetic oligonucleotides are moving out of the
research laboratories and into practical biochemical
1
5
steps and aimed at high yield and better purity. In
view of this, we reinvestigate the use of nucleoside alkyl
H-phosphonate as a synthon for oligonucleotide syn-
1
±3
application.
The possible use of oligonucleotide and
4
their analogues in chemotherapy has recently made
their large scale synthesis a matter of considerable
importance. Although the demand for relatively large
quantities of material has so far been met mainly by the
scaling-up of solid phase synthesis, we believe that if
speci®c oligonucleotide sequence becomes licensed as a
drug and multikilogram quantities of it are required,
liquid phase synthesis is likely to become the method of
choice. Over past years the liquid phase method has
been proposed as an alternative to the well known solid
phase synthesis of oligonucleotide. It utilizes, as a handle
for the growing chains, a polymeric support (PEG)
subsequently soluble in the reaction media, the polymer
is freed from the unreacted reagent and soluble by pro-
thesis.2
0,21
Nucleoside alkyl H-phosphonate undergoes
0
oxidative coupling with unprotected 5 hydroxyl group
in presence of N-bromosuccinimide (NBS) as an acti-
vating agent.
Results and Discussion
The oxidative coupling of nucleosides is a relatively new
methodology,1
6,17
and in continuation of our work in
we report here the nucleoside 3 -
DNA chemistry,1
0
alkyl H-phosphonate (1) as a versatile starting material
for oligonucleotide synthesis via oxidative coupling. 5 -
5,18
0
5
±7
0
duct by crystallization.
Since the technique avoids
O-Acetyl thymidine-3 -benzyl-H-phosphonate has been
any heterogeneity due to insolubility of support, scaling
up of the process can be reasonably foreseen.
described by Todd19 for the synthesis of dithymidine,
however, it has not been generally used for the synthesis
of oligonucleotides in solution or in solid phase.
On other hand the great potential of the new thera-
peutic methodologies based on oligonucleotide deriva-
tives justi®es any e€ort aimed at the development of an
innovative system of production. At present nucleoside
0
Nucleoside 3 -alkyl H-phosphonates are stable com-
pounds which form as a by-product during the pre-
paration of nucleoside alkyl phosphoramidites. For
the present studies nucleoside-alkyl H-phosphonates
were prepared conveniently from the corresponding
phosphoramidites (Scheme 1), and yields of the pro-
ducts are shown in Table 1. These compounds have not
generally been found useful in oligonucleotide synthesis.
0
8±12
0
3
nate
-phosphoramidite
and nucleoside 3 H-phospho-
are commonly used as the starting nucleotidic
1
3,14
*
6
Corresponding author. Tel.: +91-22-281-6750; fax: +91-22-281-
750; e-mail: mmsalunkhe@hotmail.com
0968-0896/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(99)00287-4

3
38
K. J. Padiya, M. M. Salunkhe / Bioorg. Med. Chem. 8 (2000) 337±342
0
The condensation of 1 (0.11 mmol) with a 3 -O-acetyl
nucleoside (0.1 mmol), in the presence of a coupling
agent (NBS (0.12 mmol)/I (0.12 mmol)/CC1 (excess))
2
4
at room temperature in CH CN:triethylamine (4:1 v/v)
3
gave dinucleoside phosphate (3) as the only detectable
product (Scheme 2). The yields of 3 depends on the
coupling agent used, with NBS, 85% yield of 3 was
obtained within 5 min. When I (0.12 mmol) was used as
2
a coupling agent reaction was completed in about 10
1
min with a 74% yield of 3. The UV, H NMR, and
HPLC analyses indicated that the heterocyclic moiety
and all protecting groups (DMTr, -COR, -OCH ,
3
31
-
OCH CH CN) remained una€ected. P NMR shows
2 2
a singlet at d=2.21 indicating that the product is a
phosphotriester and not a phosphitetriester. Addition-
ally, the reverse phase HPLC elution time of the pro-
duct matches with the authentic sample prepared by the
solid phase phosphoramidite method.
Scheme 1. Rˆ-CH
3 2 2
or -CH CH CN; B=nucleoside bases.
Table 1. Yield of nucleoside-alkyl H-phosphonate
Sl no.
B
R
% Yield
1
1
1
1
1
1
1
a
b
b
d
e
f
T
C
A
G
C
G
A
CH
CH
CH
CH
2
2
2
2
CH
CH
CH
CH
2
2
2
2
CN
CN
CN
CN
92
90
87
85
95
89
87
A plausible mechanism is shown in Scheme 3, where by
4
mediate that reacts immediately with the free hydroxyl
group of a nucleoside to give the desired product. The
mechanism has been studied by 31P NMR (XZ=NBS)
and 4 was found to be the only intermediate.
is expected to be formed as a highly reactive inter-
CH
CH
CH
3
3
3
g
To set up the synthetic protocol for the PEG supported
synthesis of oligonucleotides via b-CE-ethyl H-phos-
phonates. The e€ects of di€erent coupling agents, sol-
vents and bases on coupling eciency were studied by
preparing a dimer d(AT). The results are shown in
They have been reported to be completely resistant to
activation by powerful condensing agent such as diphe-
nyl phosphorochloridites, in the presence or absence of
N-methylimidazole, 2,4,6-triisopropylbenzenesulfonyl
chloride or 2,4,6-triisopropylbenzenesulfonyl tetra-
zolides. Attempted reaction of these compounds with an
Table 2. When I was employed as a coupling agent the
2
complete removal of the reagent becomes dicult due
to its high anity for PEG. The four dimer d(TT),
d(AT), d(GT) and d(CT) were then prepared using NBS
0
unprotected 5 -hydroxyl group did not produce detect-
0
2
0,21
able amounts of phosphitetriester.
Nucleoside 3 -
alkyl H-phosphonates have been used for the synthesis
of oligonucleotides containing an interphosphoramidate
as a coupling agent, in the CH CN:TEA system. The
3
in¯uence of the excess of these compounds and NBS,
and time of the reaction were carefully studied; parti-
cular attention was paid to the minimum amount of
reagent required for the maximum yield. From these
studies 98±99% yield in the phosphate bond formation
2
2
group. Although these compounds are stable, we
observed that they become highly reactive in the pre-
sence of an activating agent like I or NBS, and undergo
2
oxidative coupling (Scheme 2).
0
Scheme 2. XZ=coupling agent, NBS or I
2
or CCl
4
; R=-CH
3
or -CH
2
CH
2
CN; R =-OAc or -PEG; B
1
and B
2
are nucleoside bases.

K. J. Padiya, M. M. Salunkhe / Bioorg. Med. Chem. 8 (2000) 337±342
339
Scheme 3.
was observed by 2.5 times excess of alkyl H-phosphonate
and 5 times excess of NBS as judged by UV analysis
The DMT absorption values have shown coupling e-
ciency of 98%. Finally deprotection, separation from
PEG, and puri®cation by NAP column a€orded dec-
amer 92% homogenous to HPLC (Fig. 2a). The overall
yield of decamer was 75%. The product has been char-
acterized by PAGE and HPLC elution time using the
authentic sample of oligomer having the same sequence
and prepared independently by solid phase method
using phosphoramidite approach (Fig. 2b). Hydrolysis
(snake venom phosphodiesterase and alkaline phos-
phatase) shows no base modi®cation or wrong linkages
as the oligomer a€orded dA, dC, dG and dT in the
predicted amount.
3
1
(
Table 3). P NMR of d(AT) shows a singlet at d 2.21
which indicates the product to be phosphotriester and
not phosphitetriester; also the reverse phase HPLC elu-
tion time of the product matches with the authentic
sample prepared by the solid phase phosphoramidite
method (Fig. 1).
From all these studies a standard synthetic protocol for
synthetic cycle as shown in Table 4 was set up.
Synthesis of d(ACGGGCCCGT) (100 ꢀM)
The feasibility of the method was tested by the synthesis
of d(ACGGGCCCGT) using the method described.
The results outline a method whereby alkyl H-phos-
phonate can be used as synthon for large scale, liquid
phase synthesis of oligonucleotide. The synthons are
easily prepared via readily scalable procedure and
Table 2. Coupling eciency using di€erent coupling agent, for the
preparation of d(AT)
Coupling
agenta
Coupling eciency (%)
3
CH CN/Py CH CN/TEA
Table 3. E€ect of excess equivalent of NBS, and b-CE-H-phospho-
nate on coupling eciency
CH Cl
2
2
/Py
3
(
4:1 v/v)
(4:1 v/v)
(4:1 v/v)
Sl no.
Equivalent of
NBS
Equivalent of b
CEH-phosphonate
Coupling eciency
(%)
CCl
4
60b
72
62
75
96
69
79
99
I
2
1
2
3
4
5
10.0
5.0
5.0
2.5
2.5
10.0
5.0
2.5
5.0
2.5
98
98
98
96
95
NBS
90
a10 times excess of both the coupling agent and nucleoside, b-CE-H-
phosphonate were used.
b
4 2 2
CCl was used instead of CH Cl as a solvent.

3
40
K. J. Padiya, M. M. Salunkhe / Bioorg. Med. Chem. 8 (2000) 337±342
reduces one step per synthetic cycle as compare to
classical strategy. This is most important because
the weight reduction of about 1% of the total
amount has been observed during the repeated
precipitation and ®ltration step.
3
4
. Use of a cheap activating agent as compared to
1H-tetrazole used in the phosphoramidite method
is an additional advantage.
. The synthesis is performed in a homogenous solu-
tion where lower excess of reagent is required and
appreciable cost saving are possible.
5
6
. Large amounts of oligomer can be obtained from
one synthetic run.
. The synthesis is easily monitored by non-destruc-
tive spectrophotometric method.
Figure 1. Reverse phase HPLC of d(AT). (a) Synthesized on PEG
support by nucleoside b-CE-H-phosphonate approach. (b) Synthe-
sized on solid phase by phosphoramidite approach.
Experimental
Table 4. Oligonucleotide synthetic cycle
Polyethylene glycol monomethyl ether-5000 (Fluka) was
puri®ed by crystallization from dichloromethane
Step
Reagent
Quantity
Time
(
min)
(
KOH pellets. HPLC grade acetonitrile (E. Merck, Ger-
DCM): diethyl ether and dried under vacuum over
Detritylation
Coupling
3% DCA in DCM
b-CE-H-phosphonate
20 ml
2.5 equiv
15
5
Ê
many), was predried over activated 4 A molecular sieves
(
0.1M) in CH
NBS (0.5 M) in CH
Ac
3
CN
and distilled over CaH . 4-Dimethylaminopyridine
2
3
CN
5.0 equiv
1.0 mL
(
DMAP), dicyclohexylcarbodiimide (DCC), N-methyl-
Capping
2
O
3
2
,6-Lutidine
NMI
1.0 mL
1.0 mL
10.0 mL
imidazole (NMI) and 1-H-tetrazole were purchased
from Aldrich. All the nucleoside phosphoramidites were
commercially available from Glen research, USA.
Anhydrous diethyl ether was purchased from Spectro-
chem, India. All glass equipments were oven dried and
stored in a dessicator over KOH pellets.
3
in CH CN
NMR spectra were recorded on a Bruker AMX 500
spectrometer, HPLC were carried out on a Perkin±
Elmer, using RP C-18 column, with Et N:HOAc
3
(
pH=7) and CH CN gradient of 1%/min starting from
3
0
%, ¯ow rate 1 mL/min. Polyacrylamide gel electro-
phoresis (PAGE) was performed on a Hoefer instru-
ment. TLC were performed on a precoated silica gel
sheet 60 F254 (Merck, Germany).
0
side 3 -(2 cyanoethyl)/methyl H-phosphonate. 5 -O-(p,p -
0
0
Synthesis of 5 -O-(p,p -dimethoxytrityl)2 -deoxynucleo-
0
0
0
0
0
Dimethoxytrityl)2 -deoxynucleoside 3 -(2 cyanoethyl):
methyl N,N-diisopropylphosphoramidite (1 mmol), was
dissolved in CH CN (10 mL). 0.30 mL of H O and 0.10 g
Figure 2. Reverse phase HPLC of d(ACGGGCCCGT). (a) Synthe-
sized on PEG support by nucleoside b-CE-H-phosphonate approach.
(
b) Synthesized on solid phase by phosphoramidite approach.
3
2
of tetrazole were added, the mixture was stirred at rt
and the reaction was followed by TLC (solvent system:
ethyl acetate:petroleum ether (8:2)). After completion
of the reaction, solution was concentrated and the resi-
due was partitioned between DCM and water. The
organic phase was dried over anhydrous Na SO and
appears to be stable towards normal laboratory condi-
tion. A particularly useful feature as well is the obser-
vation that activation with NBS or I directly yield the
2
dinucleoside phosphate as the only detectable product.
2
4
evaporated to yield 1 (87±93%).
Conclusion
0 0 0
a. 5 -O-(p,p -Dimethoxytrityl) thymidine 3 -(2 cyano-
1
ethyl) H-phosphonate (yield=92%). H NMR (CD CN):
1
The described method o€ers the following advantages.
3
d 1.48 (s, 3H, CH ), 2.15 (t, 2H, CH CN), 2.60 (m, 2H,
3
2
1
2
. The reaction in the presence of an activating agent
is fast with good yield of coupling.
. The method is less time-consuming as it does not
require an additional oxidation step, thereby
H2
0
), 2.90±2.95 (m, 2H, H5
0
), 3.45±3.52 (m, 2H, CH O),
2
3.80 (s, 6H, CH ,OAr), 4.31±4.36 (m, 2H, H3
(t, 1H, H1
0
0
, H4
), 6.92 (d, 4H, J=8.5 Hz, protons ortho to
OCH of DMTr), 7.26±7.52 (m, 9H, Ar), 7,60 (s, 1H, H-6),
0
), 6.41
3
3

K. J. Padiya, M. M. Salunkhe / Bioorg. Med. Chem. 8 (2000) 337±342
341
6
(
.27 and 7.73 (d, 1H, H-P, J=730 Hz). 31P NMR
CD CN) d 7.19 (d, J=729 Hz).
Functionalisation of PEG
3
One gram of PEG was dehydrated by coevaporation
with anhydrous CH CN and dissolved in 10 mL of
0
0
0
0
1
b. 5 -O-(p,p -Dimethoxytrityl) 2 -deoxycytidine 3 -(2
1
3
0
0
0
cyanoethyl) H-phosphonate (yield=90%). H NMR
CD CN): d 2.61 (m, 1H, H ,
), 3.01 (m, 5H, H200
DCM. 0.6 mmol of 5 -O-DMT-2 -deoxynucleoside-3 -O-
succinate was dissolved in 5 mL of DCM and 0.33 mmol
of dicyclohexylcarbodiimide (DCC) was added under
(
CH CN, H
0
2
3
0
), 3.57 (m, 2H, CH O), 3.85 (s, 6H, CH O-
2
5
2
3
ꢀ
Ar), 4.36±4.43 (m, 1H, H4
H, H1
OCH of DMTr), 7.29±7.70 (m, 14H, Ar-H), 8.21 (d,
0
), 4.47 (m, 1H, H3
0
), 6.30 (m,
stirring at 0 C. After 15 min the solution was ®ltered,
1
0
), 6.98 (d, 4H, J=8.5 Hz, protons ortho to
poured into the PEG solution and 0.33 mmol of DMAP
was added. The ®nal solution was concentrated and left
to react for 24 h, at rt. The solution was ®ltered and the
3
3
1
1
H, H-6), 6.35 and 7.77 (d, 1H, J=710 Hz, H-P).
NMR (CD CN): d 7.16 (d, J=708 Hz).
P
ꢀ
modi®ed polymer precipitated under stirring at 0 C
3
with an excess of diethyl ether (about 10 vol). It was
further puri®ed by crystallization from DCM/diethyl
ether. The -OH group have been esteri®ed in 70±75%
yield, which corresponds to 140±150 mM of nucleoside/g
of PEG, as determined by measuring the DMT absorp-
tion at 489 nm.
0 0 0 0
c. 5 -O-(p,p -Dimethoxytrityl) 2 -deoxyadenosine 3 -(2
1
cyanoethyl) H-phosphonate (yield=87%). H NMR
(
1
CD CN): d 3.02 (t, 2H, CH CN), 3.09 (m, 2H, H
0
),
.54±3.57 (m, 2H, H5 ), 3.59 (t, 2H, CH O), 3.91 (s, 6H,
3
2
2
3
0
2
CH O), 4.51 (m, 1H, H4
H, H1
0
0
), 4.59 (m, 1H, H3
), 6.98 (d, 4H, J=8.6 Hz, protons ortho to
OCH of DMTr), 7.36±7.62 (m, 14H, Ar-H), 8.56 (s,
0
), 6.76 (m,
3
1
The unreacted OH groups of PEG were acetylated by
reacting with 10 mL of acetonitrile solution containing
10% of acetic anhydride, 10% 2,6-lutidine and 10% N-
methylimidazole (v/v). The reaction mixture was kept at
room temperature under stirring for 3 min. The polymer
was precipitated from the ice-cooled solution with di-
ethyl ether, ®ltered and dried under vacuum over KOH
pellets.
3
1
H, H-8), 8.69 (s, 1H, H-2), 6.46 and 7.9 (d, 1H,
3
1
J=720Hz). P NMR (CD CN): d 7.06 (d, J=721Hz).
3
0 0 0 0
d. 5 -O-(p,p -Dimethoxytrityl)2 -deoxyguanosine 3 -(2
1
cyanoethyl) H-phosphonate (yield=85%). H NMR
1
(
2
CD CN): d 1.12 (d, 6H, 2CH ), 2.35 (m, 1H, CHCO),
3
3
.58±2.75 (m, 4H, CH CN, H2
0
), 3.26±3.47 (m, 4H,
), 3.75 (s, 6H, CH O-Ar), 4.17 (m, 1H, H4 ),
), 6.75 (d, 4H,
J=8.6 Hz, protons ortho to OCH of DMTr), 6.74±7.42
2
CH O, H
0
0
2
5
3
4
.24±4.30 (m, 1H, H3 ), 6.15 (m, 1H, H1
0 0
General procedure for oligonucleotide assembly
3
0
0
(
m, 9H, Ar-H), 7.73 (s, 1H, H-8), 6.22 and 7.67 (d, 1H,
Detritylation. One gram of 5 -DMT-nucleoside 3 -O-
PEG was dissolved in 10 mL of DCM. 10 ml of 6%
dichloroacetic acid (DCA) in DCM was added dropwise
under stirring. After 15 min the polymer was pre-
31
J=725 Hz, P-H). P NMR (CD CN): d 6.62 (d,
3
J=726 Hz).
0
0
0
ꢀ
1
e. 5 -O-(p,p -Dimethoxytrityl)2 -deoxycytidine 3-methyl
1
cipitated at 0 C, with diethyl ether (200 mL), washed
H-phosphonate (yield=95%). H NMR (CDCl ): d 2.38
m, 1H, H2
with diethyl ether and then ®ltered. The extent of
deblocking was controlled qualitatively by TLC (no
orange colour present with acidic spraying) and quanti-
tatively by UV analysis. If some DMT is still present,
the DCA treatment was repeated. The ®nal polymer was
recrystallized from DCM/diethyl ether and dried under
vacuum over KOH pellets.
3
(
2
0
), 2.90 (m, 1H, H200), 3.47±3.2 (m, 3H, H3
0
,
H5
H, H1
0
), 3.71 (m, 3H, CH OP), 4.34 (m, 1H, H ), 6.30 (m,
0
4
3
1
OCH , of DMTr, H-5), 7.23±7.51 (m, 14H, Ar-H) 6.12
0
), 6.86 (d, 5H, J=8.6 Hz protons ortho to
3
and 7.54 (d, 1H, J=710 Hz, P-H), 8.18 (dd, J=4.5 Hz
and 7.1 Hz, H-6), 8.8 (br, 1H, NHCO). 31P NMR
(
CDCl ): d 7.74 (d, J=710 Hz).
3
0
was coevaporated 3 times with anhydrous acetonitrile,
dried under high vacuum and dissolved in CH CN:TEA
3
0
Condensation. One gram of 5 -OH-nucleoside-3 -O-PEG
0
0
0
1
0
1
f. 5 -O-(p,p -Dimetboxytrityl)2 -deoxyadenosine 3 -methyl
H-phosphonate (yield=89%). H NMR (CDCl ): d 2.80
3
(
(
H4
m, 1H, H2
0
), 3.11 (m, 1H, H200), 3.45 (m, 2H, H5
s, 6H, CH O-Ar), 3.80 (m, 3H, CH O-P), 4.4 (m, 1H,
0
), 3.78
(1.5:1.0 v/v), to the solution were added 2.5 equiv of
alkyl H-phosphonate from a 0.1 M solution in CH CN
3
3
3
0
), 6.52 (m, 1H, H1
0
), 6.80 (m, 4H, J=8.5 Hz protons
and 5.0 equiv of NBS from a 0.5 M solution in CH CN.
3
ortho to OCH of DMTr), 7.00±7.50 (m, 9H, ArH),
3
The solution was stirred under nitrogen atmosphere for
5 min. The polymer was precipitated from the ice cooled
solution with 10 vol of ether and recrystallized from
31
6.17 and 7.55 (d, 1H, J=690 Hz, P-H). P NMR
(
CDCl ): d 7.87 (d, J=692 Hz).
3
CH CN:ether. The yield of the reaction was evaluated
3
0
methyl H-phosphonate (yield=87%). H NMR (CDCl ):
0
0
0
1
g. 5 -O-(p,p -Dimethoxytrityl) 2 -deoxyguanosine 3
1
spectroscopically from the DMT absorption, after a
TLC analysis to ascertain the complete removal of
reagents and by-products.
3
d 1.12 (d, 6H, 2CH ), 2.40 (m, 1H, CHCO), 2.8 (m, 2H,
3
H2
0
), 3.12 (m, 2H, H5
0
), 3.63 (m, 3H, CH OP), 3.78 (s,
3
0
by reacting with 10 mL of CH CN solution containing
6
6
H, CH OAr), 3.81 (m, 1H, H
3
0
), 4.24 (m, 1H, H3
0
),
), 6.81 (d, 4H, aromatic protons
Capping. The unreacted 5 -OH groups were acetylated
4
.16 (m, 1H, H1
0
3
ortho to CH O of DMTr, J=8.8 Hz), 7.15±7.36 (m,
3
10% of acetic anhydride, 10% of 2,6-lutidine and 10%
of N-methylimidazole (v/v). The reaction mixture was
kept at room temperature under stirring for 3 min. The
PEG was precipitated from the ice-cooled solution with
9
H, ArH), 7.82 (s, 1H, H-8), 6.05 and 7.47 (d, 1H,
3
1
J=710 Hz, P-H).
J=712 Hz).
P NMR (CDCl ): d 8.54 (d,
3

3
42
K. J. Padiya, M. M. Salunkhe / Bioorg. Med. Chem. 8 (2000) 337±342
diethyl ether, ®ltered and dried under vacuum over
KOH pellets.
6. Bonora, G. M.; Biancotto, G.; Mani, M.; Scremin, C. L.
Nucleic Acid Res. 1993, 21, 1213.
7. Bonora, G. M.; Scremin, C. L.; Colonna, F. P.; Garbesi, A.
Nucleic Acid Res. 1990, 18, 3155.
Deprotection and puri®cation. The ®nal PEG-oligo-
nucleotide (DMT-o€) was dissolved in 30% aqueous
8
1
9
4
1
. Beaucage, S. L.; Iyer, R. P.; Caruthers, M. H. Tetrahedron
992, 48, 223.
. Ramon, E.; Valeriy, S.; Caruthers, M. H. Tetrahedron 1990,
6, 721.
0. Dorman, M. A.; McBride, L. J.; Caruthers, M. H. Tetra-
ammonia and reaction mixture was allowed to stand at
ꢀ
60 C for 24 h after cooling the aqueous solution was
extracted three times with 25 mL of ether. The oligonu-
cleotide was separated from PEG by precipitation with
hedron 1984, 40, 95.
11. McBride, L. J.; Caruthers, M. H. Tetrahedron Lett. 1984,
25, 245.
3
an excess of methanol as described (where PEG is
soluble), and following centrifugation.
1
2
1
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