Convergent solution phase synthesis of chimeric oligonucleotides by a 2+2 and 3+3 phosphoramidite strategy

Article abstract of DOI:10.1071/CH09298

A chimeric oligonucleotide tetramer and hexamer were synthesized by the phosphoramidite approach using a 2+2 and 3+3 strategy, respectively. The concept of convergent synthesis provides an efficient route toward the synthesis of longer chimeric oligonucle

Full text of DOI:10.1071/CH09298

Full Paper
CSIRO PUBLISHING
www.publish.csiro.au/journals/ajc
Aust. J. Chem. 2010, 63, 227–235
Convergent Solution Phase Synthesis of Chimeric
Oligonucleotides by a 2+2 and 3+3
Phosphoramidite Strategy
Chih-Hau Chen,A Wei-Yu Chen,A Yu-Chie Chen,A Ming-Juan Lee,B
Chyuan-Der Huang,B Kaushik Chanda,A and Chung-Ming SunA,C
ADepartment of Applied Chemistry, National Chiao Tung University, Hsinchu 300-10, Taiwan.
BScinoPharm Taiwan Ltd, Tainan 741-44, Taiwan.
CCorresponding author. Email: cmsun@mail.nctu.edu.tw
A chimeric oligonucleotide tetramer and hexamer were synthesized by the phosphoramidite approach using a 2+2 and
3+3 strategy, respectively. The concept of convergent synthesis provides an efficient route toward the synthesis of longer
chimericoligonucleotides, suchassmallinterferingRNAoligonucleotideswithoutthepollutionof n − 1orshorterfailures.
This methodology offers an efficient and economical way to scale-up the synthesis of high purity oligonucleotides for
clinical trials and commercial uses.
Manuscript received: 19 May 2009.
Manuscript accepted: 27 October 2009.
Introduction
the past two decades. Bonora et al.[9] used poly(ethylene gly-
col) as a polymer support to prepare oligonucleotides of up to
20-mer. This approach can increase production yield to 100 µm
with unavoidable intermediate product loss during the repeated
precipitation and filtration steps.Vasseur[10] developed polymer-
supported reagents to assist the synthesis of tri-nucleotides with
10 mmol productivity. This strategy is relatively economical
and environmentally friendly because the polymer-supported
reagents can be recycled. However, the oligonucletide synthe-
sis in this approach is only up to trimers and final products
may be polluted by H-phosphonate diester resulting from excess
phosphoramidite. Damha and coworkers[11] used an ionic tag
as a soluble support for oligoribonucleotides synthesis on a
1 g scale. Furthermore, the dimer phosphoramidites are some-
times used as building blocks to prepare chemically modified
oligoribonucleotides.[12]
Since Watson and Crick[1] first constructed the model of
DNA, scientists have devoted themselves to revealing the
mysteries of human life. Organic chemists, in particular,
have tried to create artificial oligonucleotides to mimic natu-
ral oligonucleotides for medicinal applications. Consequently,
many oligonucleotide synthesis strategies have been developed
during recent decades such as the phosphotriester method,[2]
the phosphite triester method,[3] the phosphoramidite method,[4]
and the H-phosphonate method.[5] The importance of oligo-
nucleotides was emphasized again by the understanding of RNA
interference mechanism in Caenorhabditis elegans by Fire, a
Nobel Prize winner in 2006.[6] Furthermore, the first oligo-
nucleotide drug, Vitravene, which is the first antisence drug to
treat cytomegalovirus (CMV) retinitis in people with AIDS was
approvedbytheFoodandDrugAdministration(FDA)in1998.[7]
CALAA-01 is a nanoparticle-containing non-chemically mod-
ified small interfering RNAs developed for the treatment of
cancer and is now in phase I clinical trials.[8] Because of these
advanced milestones, many drug companies started to focus on
the research of novel antisense oligonucleotide sequences for
treating different diseases and to try to amplify the production
of oligonucleotides for therapeutics and diagnostic demands.
Traditionally, oligonucleotides were synthesized using the
phosphoramidite method by a solid phase automated synthesizer
on a small scale with 0.1–10 µmol quantities.A small quantity of
oligonucleotides is suitable for screening, biological testing, and
preclinical trials, but not enough for clinical trials and commer-
cialization. Another disadvantage is that solid phase synthesis
often needs excess reagents to force the reaction to completion
and limits this strategy for low cost production. Researchers have
proposed many scale-up and purification methodologies during
Results and Discussion
This study presents a novel convergent solution phase approach
to synthesize oligonucleotides using the phosphoramidite
method. Convergent2+2and3+3syntheticroutescanminimize
the number of synthesis steps and produce a high quality prod-
uct that does not suffer from the pollution of n − 1 failures. We
first synthesized dimer-5ꢀ-OH and dimer phosphoramidite, and
then coupled these two fragments into the tetramer. The tetramer
also can be transformed into tetramer-5ꢀ-OH and tetramer phos-
phoramidite. These two moieties are joined to form an octamer
after the coupling reaction. Hexamers, dodecamers, and longer
oligonucleotides can be efficiently synthesized in the same
manner (Fig. 1). Therefore, it is promising that the solution
phase synthesis of oligonucleotides is most amenable to scale-up
production.
© CSIRO 2010
10.1071/CH09298
0004-9425/10/020227

228
C.-H. Chen et al.
Tetramer-5Ј-OH
Dimer-5Ј-OH
Dimer
Octamer
Tetramer
ϩ
ϩ
Dimer phosphoramidite
Tetramer phosphoramidite
Hexamer-5Ј-OH
Trimer-5Ј-OH
Trimer
Dodecamer
Hexamer
ϩ
ϩ
Trimer phosphoramidite
Hexamer phosphoramidite
Fig. 1. The concept of convergent solution phase synthesis of chimeric oligonucleotides.
Table 1. Different coupling reagents were tested for levulinylation
O
O
NH
O
NH
Coupling reagent
DMTO
O
N
ϩ
HO
DMTO
N
O
O
O
O
O
O
OH
O
2
1
Condition
A
Coupling reagent
Solvent
CH2Cl2
Yield [%]
0
N
C N
N
N
N
N
B
CH2Cl2
0
P
N
N
O
PF6
C
D
E
CH3CN/dioxane
Dioxane
0
98
91
Cl
N
I
N
N
N
C N
NH
Cl
Dioxane
C
N
Beginning with a simple case, the 2+2 synthesis of
oligonucleotides was started from a 10 g scale. This study
develops the knowledge of oligonucleotide synthesis for
subsequent, more challenging tasks. The 3ꢀ-hydroxy group
of thymidine 1 was first protected with levulinic acid
and dicyclohexylcarbodiimide (DCC) as a coupling reagent.
However, we failed to the protect 3ꢀ secondary alco-
hol with levulinic acid even under refluxing conditions.[13]
Other coupling reagents have been tested in this coupling
reaction, including PyBop, Mukaiyama reagent, 1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide (EDC), and 1-ethyl-3-
(3ꢀ-dimethylaminopropyl)carbodiimide hydrochloride (EDCI)
(Table 1).[14] Excellent results were obtained when EDC
(yield 98%) or EDCI (yield 91%) (conditions D and E) were
employed.[15] All the reactions depicted herein were monitored
1
by TLC, H and 31P NMR spectroscopy, and matrix-assisted

Convergent Solution Phase Synthesis of Chimeric Oligonucleotides
229
O
HN
N
N
N
DMTO
N
O
O
P
OTBDMS
O
T
O
O
O
NC
O
O
4
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10
0
ppm
O
HN
N
N
N
DMTO
N
O
O
OTBDMS
O
P
O
T
O
O
O
NC
O
O
5
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10
0
ppm
Fig. 2. Progress of the oxidation monitored by 31P NMR spectroscopy.
Table 2. Reaction conditions of phosphite oxidation
laser desorption ionization (MALDI) mass spectra. After lev-
ulinylation, three additional signals appeared in the H NMR
1
O
O
spectrum (2.75, 2.50, and 2.15 ppm, as shown in the Acces-
sory Publication). Detritylation of 2 with 3% trichloroacetic acid
in dichloromethane afforded 5ꢀ-OH thymidine 3. Three sets of
HN
N
HN
N
N
N
N
N
N
N
DMTO
DMTO
1
proton signals disappeared in the H NMR spectrum (7.5–7.2,
O
O
Oxidation
6.9, and 3.75 ppm) after detritylation. Condensation of 3 with
phosphoamidite rA in the presence of activator, 5-(benzylthio)-
1H-tetrazole, resulted in the phosphite triester 4, which was then
further oxidized to phosphate triester 5 subsequently. After con-
densation, the crude product was purified with neutral silica gel
and the column was also deactivated with 3% pyridine in hexane.
The eluent was also prepared with 1% pyridine to preserve the
acid-sensitive phsophite triester bond.
A small amount of phosphite trimester 4 was decomposed in
silica gel during purification which caused the moderate yield.
The signal of phosphite triester appeared at 140 ppm in the 31P
NMR spectrum after condensation, and it was shifted to 0 ppm
after oxidation (Fig. 2). Three different reaction conditions were
investigated in the oxidation reaction as described in Table 2.
Optimal results were obtained with I2 in wet tetrahydrofuran
(THF) as an oxidative reagent and only extraction is enough
to yield a pure product without further complicated column
purification.[14] Furthermore, products obtained from condi-
tion F and G must be purified by column chromatography,
which is easily decomposed in silica gel during the purifica-
tion. The detritylation of 5 was successfully conducted in 5%
p-toluenesulfonic acid (Scheme 1). The common trichloroacetic
acid was not chosen as a detritylation reagent here because it may
O
P
O
OTBDMS
O
P
O
OTBDMS
O
O
T
O
T
O
O
O
O
NC
NC
O
O
O
O
5
4
Condition Reagent
Solvent
CH3CN
Yield [%]
F
G
H
80% TBHP in DTBP
5.5 M TBHP in decane CH3CN
0.2 M I2 1/4 pyridine/wet THF
15
18
89
destroy the P–O bonds. Prior to transfer of the 3ꢀ position protect-
ing group to levulinyl group, the deprotection of the 3ꢀ position
was examined with 3ꢀ methyl succinate-protected Ao=pT dimer.
After reaction with hydrazine hydrate, only the adenine
deprotection product was obtained and the 3ꢀ protecting group
remained intact. Compound 5 was delevulated with hydrazine
hydrate and the precursor 7 for the dimer phosphoramidite was
obtained (Scheme 1).

230
C.-H. Chen et al.
O
N
O
N
O
N
NH
NH
NH
DMTO
O
O
EDC, DMAP
Dioxane
TCA
DMTO
O
HO
O
O
O
OH
CH2Cl2
O
O
1
O
O
+
O
O
O
3
94%
HO
2
98%
O
O
O
HN
HN
N
N
N
N
N
DMTO
DMTO
N
N
N
O
O
0.2 M I2
5-(Benzylthio)-1H-tetrazole
O
P
OTBDMS
O
P
OTBDMS
Phosphoramidite rA, CH3CN
4:1 wet THF:pyridine
O
T
O
O
T
O
O
O
O
O
O
NC
NC
O
O
O
O
4
73%
87%
5
5% p-TSA
7:3 CH2Cl2:MeOH
Hydrazine hydrate
4:1 pyridine:acetic acid
HO
ABz
O
DMTO
ABz
O
O
OTBDMS
O
P
O
T
O
O
P
OTBDMS
O
O
NC
O
O
T
O
O
O
NC
O
OH
96%
6
7
96%
Scheme 1. Synthesis of 5ꢀ-OH dimer 6 and 3ꢀ-OH dimer 7.
The proposed mechanism (Scheme 2) began with the
hydrazine attack on the ketone of the levulinyl group. The
other amine of the hydrazine cleaved the ester bond of
5, which led to the formation of 7 and 6-methyl-4,5-
dihydropyridazin-3(2H)-one (ESI+: 113 m/z). Dimer phospho-
ramidite 8 was synthesized under two different conditions.
Basic conditions were proceeded with chloro(2-cyanoethoxy)-
(diisopropylamino) phosphine and disopropylethylamine. In an
alternative approach, tetraisopropylphosphorodiamidite and 5-
(benzylthio)-1H-tetrazole were employed (Table 3, conditions I
and J). The low yields of these methods were due to the sensi-
tivity of phosphoramidite to the weakly acidic silica gel during
column purification.The 31P signal of phosphoramidite was seen
at around 150 ppm after phosphitylation (Fig. 3). Dimer 5ꢀ-OH
6 was condensed with dimer phosphoamidite 8 to generate the
tetramer phosphite triester 9 and 5ꢀ-OH tetramer 11 was obtained
after oxidation and detritylation (Scheme 3).
In the foregoing content, we have achieved the solution phase
2+2 chimeric oligonucleotide synthesis, but purifying the prod-
uct remained a barrier. As the length of the oligonucleotides
increased, the products were more easily decomposed by the
silica gel. Despite the low yield from the products decompos-
ing on the column, this problem can be solved by using other
purification methods such as extraction or using cellulose or
polystyrene as purification media. The total yield for the three
steps sequence including 2+2 coupling, iodine-induced oxida-
tion, and tetramer detritylation was improved to 81% just by
extraction and filtration without column purification. The more
challenging solution phase 3+3 oligonucleotide synthesis began
with the condensation of 6 with phosphoamidite rG to obtain
the trimer phosphite triester 12. In a similar manner, phos-
phite triester 12 was oxidized to form phosphate triester 13,
which immediately underwent detritylation to generate 5ꢀ-OH
trimer 14. The 3ꢀ-levulinyl group of 13 was also removed by
hydrazine hydrate (Scheme 4). Trimer 3ꢀ-OH 15 was trans-
formed into trimer phosphoramidite 16 by reacting 15 with the
same phosphitylation reagents described previously. Conden-
sation of the 5ꢀ-OH trimer 14 and trimer phosphoramidite 16
provided the hexamer phosphite triester 17, which was oxidized
and detritylated to form 5ꢀ-OH hexamer 19 (Scheme 5).
Conclusion
In summary, the synthesis of oligonucleotide tetramer and
hexamer through the solution phase 2+2 and 3+3 strat-
egy has been achieved. This convergent synthesis established
a practical method for the large-scale synthesis of oligo-
nucleotides. It reduced the synthetic steps for constructing
longer oligonucleotides and reduced the time needed to reach

Convergent Solution Phase Synthesis of Chimeric Oligonucleotides
231
O
O
O
HN
N
HN
N
HN
N
N
N
N
N
N
N
N
N
DMTO
DMTO
DMTO
N
O
O
O
O
P
O
P
OTBDMS
O
P
OTBDMS
OTBDMS
O
T
O
O
T
O
O
T
O
O
O
O
H
H
O
O
O
H
N
O
O
OϪ
H
NC
NC
NC
NH2
ϩ
N
NH2
O
O
O
H2N NH2
O
O
O
5
O
O
N
O
N
HN
N
HN
N
HN
N
N
N
N
N
N
N
DMTO
ϪH2O
N
DMTO
DMTO
O
O
O
O
P
O
OTBDMS
O
O
P
OTBDMS
O
P
OTBDMS
O
O
T
O
O
T
HN
N
O
O
T
O
ϩ
O
O
O
NC
O
O
NC
NC
H
O
Nϩ
N
N
OH
OϪ
H
O
NH2
7
(MS: 113 m/z)
Scheme 2. Mechanism of delevulinylation of dinucleotide 5.
Table 3. Phosphitylation in acidic and basic condition
O
O
HN
N
N
N
HN
N
N
DMTO
N
N
N
O
DMTO
Phosphitylation
O
O
P
OTBDMS
O
O
T
O
OTBDMS
O
O
O
P
O
T
NC
O
O
P
O
NC
CN
OH
N
O
7
8
Condition
I
Reagent
Solvent
CH2Cl2
Time [h]
6
Yield [%]
41
2-Cyanoethyl N,N,Nꢀ,Nꢀ-tetra-isopropylphosphordiamidite,
5-(benzylthio)-1H-tetrazole
J
2-Cyanoethyl N,N-diisopropyl-chlorophosphoramidite,
diisopropylethylamine
THF
1
58
targeted molecules. Moreover, this methodology avoided the
products from the pollution of n − 1 or shorter failures and it
is applicable for scale-up production to fit clinical trials and
commercialization.
standard. 13C and DEPT NMR spectra were recorded at 75 MHz
(Bruker DRX-300) and the chemical shifts were measured from
the solvent peak (CD2HCN in CD3CN) as an internal standard.
31P NMR spectra were recorded at 65 MHz and the chemical
shifts were measured from 85% H3PO4 as an external standard.
Acetonitrile was distilled from CaH2 and stored under nitrogen.
TLC was performed on Merck Kieseigel 60 F254 precoated glass
plates. Column chromatography was performed with silica gel
SiliaFlash, G60 (Silicycle Co. Ltd). The MALDI time-of-flight
(TOF) mass spectrometry was carried out by use of a Bruker
Daltonics Biflex III (Leipzig, Germany).
Experimental
General
1H NMR spectra were recorded at 300 MHz (Bruker DRX-300)
and the chemical shifts were measured from the solvent peak
as an internal standard (CD2HCN in CD3CN) as an external

232
C.-H. Chen et al.
DMTO
A
O
O
OTBDMS
O
P O
O
T
O
NC
OH
7
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10
0
ppm
DMTO
A
O
O
OTBDMS
O P O
O
T
O
NC
O
P
NC
O
8
170
160
150
140
130
120
110
100
90
80
70
60
50
40
30
20
10
0
ppm
Fig. 3. Progress of the phosphitylation monitored 31P NMR spectroscopy.
Synthesis of 5ꢀ-O-Dimethoxytrityl-3ꢀ-O-levulinoyl
thymidine (2)
0◦C for 1 h. After reaction completion, solvent was removed and
the crude product was subjected to column purification. First,
33% hexane in ethyl acetate was used as eluent to remove the
byproduct and then 100% ethyl acetate was used to elute product
3. The product was obtained as a white foam in 94% yield. δH
(300 MHz, CD3CN): 7.64 (s, 1H), 6.22 (t, J 7.0, 1H), 5.26 (d, J
1.6, 1H), 4.03 (d, J 2.2, 1H), 3.75 (d, J 2.1, 2H), 2.77 (t, J 6.2,
2H), 2.53 (t, J 6.5, 2H), 2.36–2.25 (m, 2H), 2.14 (s, 3H), 1.85
(s, 3H). δC (DEPT, 75 MHz, CD3CN): 208.0 (Cq), 173.2 (Cq),
165.1 (Cq), 151.7 (Cq), 137.2 (CH), 118.3 (Cq), 111.3 (Cq),
87.7 (CH), 85.6 (CH), 75.7 (CH), 62.7 (CH2), 38.3 (CH2), 37.7
(CH2), 29.8 (CH3), 28.7 (CH2), 12.5 (CH3).
To
a
solution of 5ꢀ-O-dimethoxytritylthymidine (10.0 g,
18.4 mmol) in dioxane (132 mL), levulinic acid (4.2 g,
36.0 mmol), EDC (5.8 g, 36.0 mmol), and dimethylaminopyri-
dine (DMAP, 0.22 g, 1.8 mmol) were added. After stirring for
2.5 h, TLC analysis (5% MeOH/DCM) indicated complete con-
version of the starting material. The solvent was evaporated
by vacuum, and the residue was dissolved in dichloromethane
(200 mL). After washing of the organic phase with water, 10%
KHSO4, and 10% NaHCO3 (three times), the organic phase was
dried (MgSO4), filtered, and concentrated to give a foam (2),
which was used directly in the next reaction. δH (300 MHz,
CD3CN): 10.02 (br, 1H), 7.58 (s, 1H), 7.49 (d, J 7.5, 2H), 7.43–
7.21 (m, 7H), 6.9 (d, J 8.7, 4H), 6.35 (t, J 6.4, 1H), 5.46 (s, 1H),
4.13 (s, 1H), 3.77 (s, 6H), 3.63 (s, 2H), 3.42 (dd, J 19.0 and
7.7, 2H), 2.76 (t, J 6.2), 2.54 (t, J 6.4, 2H), 2.54–2.36 (m, 2H),
2.14 (s, 3H), 1.50 (s, 3H). δC (DEPT, 75 MHz, CD3CN): 207.3
(Cq), 178.2 (Cq), 164.6 (Cq), 159.2 (Cq), 151.2 (Cq), 145.3 (Cq),
136.2 (CH), 136.1 (Cq), 135.9 (Cq), 130.5 (CH), 128.5 (CH),
128.4 (CH), 127.5 (CH), 117.8 (Cq), 113.7 (CH), 111.2 (Cq),
87.1 (Cq), 84.8 (CH), 84.1 (CH), 75.4 (CH), 67.1 (CH2), 55.4
(CH3), 54.8 (Cq), 37.9 (CH2), 37.5 (CH2), 29.4 (CH3), 28.3
(CH2), 11.8 (CH3).
General Procedures for the Condensation, Oxidation,
Detritylation, Delevulinylation, and Phosphitylation
Reaction
Condensation
5ꢀ-OH oligonucleotide (16.9 mmol, 1.0 equiv), phospho-
ramidite (22.0 mmol, 1.3 equiv), and activator, 5-(benzylthio)-
1H-tetrazol (84.5 mmol, 5.0 equiv) was added in a 500 mL
flask under nitrogen. Acetonitrile was added and the reac-
tion mixture was stirred for 30 min until reaction comple-
tion. Acetonitrile was removed by rotary evaporation and
the crude product was purified by column chromatogra-
Synthesis of 5ꢀ-Hydroxyl-3ꢀ-O-levulinoyl-thymidine (3)
To a solution of 5ꢀ-O-dimethoxytrityl-3ꢀ-O-levulinoyl-thymidine
(2) (11.6 g, 18.0 mmol) in dichloromethane (200 mL), 6%
trichloroacetic acid in dichloromethane (200 mL) was added at
phy (column was packed with 5% pyridine in hexane). 5ꢀ-
Bz
O-DMTr-rA
p
dT-3ꢀ-O-Lev (4): δP (65 MHz, CD3CN):
CNE
139.3, 139.3. 5ꢀ-O-DMTr-rABzpoCNEdTpCNErABz poCNEdT-3ꢀ-
O-Lev (9): δP (65 MHz, CD3CN): 140.0, 140.0, 139.8, 139.8,

Convergent Solution Phase Synthesis of Chimeric Oligonucleotides
233
5Ј-O-DMTr-rABzpoCNEdT-3Ј-OH
DMTO
ABz
7
O
Phosphitylation
O
P
OTBDMS
O
O
ABz
T
O
DMTO
NC
O
O
5Ј-OH-rABzpoCNEdT-3Ј-O-Lev
O
P
O
P
OTBDMS
O
5-(Benzylthio)-1H-tetrazole
O
O
T
6
ϩ
ABz
O
O
O
CH3CN
NC
NC
O
O
P
O
OTBDMS
O
NC
O
O
P
T
O
8
NC
O
41%
O
O
O
9
32%
DMTO
ABz
HO
ABz
O
O
O
OTBDMS
O
OTBDMS
O
O
O
O
P
P
T
O
T
O
NC
O
O
NC
O
P
O
P
0.2 M I2
O
O
5% p-TSA
O
O
O
ABz
ABz
O
4:1 wet THF:pyridine
7:3 CH2Cl2:MeOH
O
O
NC
NC
O
OTBDMS
O OTBDMS
O
O
O
O
P
P
T
O
T
O
O
O
NC
NC
O
O
O
O
O
O
92%
10
11
84%
Scheme 3. Synthesis of dimer phosphoramidite 8 and 5ꢀ-OH tetramer 11.
139.6, 139.6, 139.6, 139.4, −1.7, −1.8, −1.8, −1.9, −1.9,
−2.0, −2.0, −2.1, −2.1. m/z (MALDI TOF): Anal. Calc. for
(65 MHz, CD3CN): −1.7, −1.8, −2.0, −2.1, −2.3, −2.4, −2.5,
−2.5. 5ꢀ-O-DMTr-rGiBupoCNErABzpoCNEdT-3ꢀ-O-Lev (13): δP
(65 MHz, CD3CN): −1.9, −2.0, −2.1, −2.2. m/z (MALDI
TOF): Anal. Calc. for C83H102N14O24P2Si2: 1796.62. Found
C101H120N17O29P3 Si2 2183.72. Found 2206.75 (M + Na)+. 5ꢀ-
iBu
O-DMTr-rG
p
rABzpoCNEdT- 3ꢀ-O-Lev (12): δP (65 MHz,
CNE
CD3CN) 139.6, 139.5, 139.5, 139.4, −1.9, −2.0, −2.3,
−2.4. 5ꢀ-O-DMTr-rGiBupoCNErABzpoCNEdTpCNErGiBupoCNE
rABzpoCNEdT-3ꢀ-O-Lev (17): δP (65 MHz, CD3CN): 140.3,
140.3, 140.0, 140.0, 139.8, 139.7, 139.7, 139.1, −1.7, −1.9,
−1.9, −2.0, −2.1, −2.2, −2.2, −2.2, −2.4, −2.4.
1819.39 (M + Na)+. 5ꢀ-O-DMTr-rGiBupoCNErABzpoCNE
d
TpoCNErGiBupoCNErABzpoCNEdT-3ꢀ-O-Lev (18): δP (65 MHz,
CD3CN): −1.7, −1.8, −1.9, −2.0, −2.0, −2.1, −2.2, −2.2,
−2.2, −2.4, −3.0, −3.0, −3.1. m/z (MALDI TOF): Anal.
Calc. for C143H182N29O46P5Si4: 3308.06. Found 3331.51
(M + Na)+.
Oxidation
A
solution of 0.2 M I2 in 4/1 THF/pyridine was
Detritylation for Dimer or Longer Oligoribonucleotide
added to the phosphite trimester and the reaction mix-
ture was stirred for 10 min. The reaction was quenched
by extraction with 1 M Na2S2O3, 10% KHSO4, 10%
NaHCO3, and a mixture of brine/water (1/1), the organic
layer was collected and dried by MgSO4. After filtration
and solvent removal, product was obtained as a brown
foam. 5ꢀ-O-DMTr-rABzpoCNEdT-3ꢀ-O-Lev (5): δP (65 MHz,
CD3CN): −1.9, −2.1. m/z (MALDI TOF): Anal. Calc. for
C62H71N8O16PSi: 1242.45. Found 1265.00 (M + Na)+. 5ꢀ-
O-DMTr-rABzpoCNEdTpCNErABzpoCNEdT-3ꢀ-O-Lev (10): δP
To the solution of phosphate triester in 7/3 dichloromethane/
methanol at 0◦C, 10% p-toluenesulfonic acid in
dichloromethane/methanol (7/3) was added and the resulting
mixture was stirred for 30 min at 0◦C. After completion, water
was added and the resulting mixture was vigorously stirred for
10 min at 0◦C.A saturated NaHCO3 aqueous solution was added
and again, the reaction mixture was stirred strongly for 10 min
at 0◦C. The reaction mixture was then washed with saturated
NaHCO3 solution, 0.2 M Na2S2O3, and 0.5 M NaCl.The organic

234
C.-H. Chen et al.
DMTO
G
DMTO
G
O
O
O
P
O
P
OTBDMS
OTBDMS
AoϭpT-5Ј-OH
O
O
O
O
A
O
A
6
NC
O
O
NC
0.2 M I2
5-(Benzylthio)-1H-tetrazole
ϩ
4:1 wet THF:pyridine
O
P
O
P
OTBDMS
OTBDMS
CH3CN
O
O
O
O
Phosphoramidite rG
T
O
T
O
NC
O
NC
O
O
O
O
O
O
O
92%
13
66%
12
5% p-TSA
7:3 CH2Cl2:MeOH
Hydrazine hydrate
4:1 pyridine:acetic acid
HO
G
O
DMTO
G
O
P
OTBDMS
O
O
O
A
O
O
P
OTBDMS
NC
O
O
O
A
O
O
P
OTBDMS
O
NC
O
O
T
O
O
P
OTBDMS
NC
O
O
O
O
O
T
O
NC
O
O
OH
93%
14
79%
15
Scheme 4. Synthesis of 5ꢀ-OH trimer 14 and 3ꢀ-OH trimer 15.
DMTO
G
O
O
P
OTBDMS
O
O
A
O
NC
O
Phosphitylation
5-(Benzylthio)-1H-tetrazole
O
P
OTBDMS
GoϭpAoϭpT-3Ј-OH
15
GoϭpAoϭpT-5Ј-OH
ϩ
CH3CN, r.t., 1 h
O
O
14
O
T
NC
O
O
P
NC
O
N
16
49%
0.2 M I2
4:1 wet THF:pyridine
5% p-TSA
7:3 CH2Cl2:MeOH
GoϭpAoϭpT:pGoϭpAoϭpT-5Ј-OH
51%
GoϭpAoϭpToϭpGoϭpAoϭpT-5Ј-OH
r.t., 5 min
17
19
53%
Scheme 5. Synthesis of trimer phosphoramidite 16 and 5ꢀ-OH hexamer 19.
layer was separated and dried over anhydrous MgSO4. After
filtration the solvent was evaporated under rotary evaporation.
The crude product was purified by column choromatography
or ether precipitation. The product was obtained as a white
solid. 5ꢀ-OH-rGiBupoCNErABzpoCNEdT-3ꢀ-O-Lev (14): m/z
(MALDI TOF): Anal. Calc. for C62H84N14O22P2Si2: 1494.49.
Found 1517.07 (M + Na)+. 5ꢀ-OH-rGiBupoCNErABzpoCNEd
TpoCNErGiBupoCNErABzpoCNEdT-3ꢀ-O-Lev (19): m/z
(MALDITOF):Anal. Calc. for C122H164N29O44P5Si4: 3005.93.
Found 3029.35 (M + Na)+.

Convergent Solution Phase Synthesis of Chimeric Oligonucleotides
235
Delevulinylation
References
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PAC196817030349
To the solution of phosphate triester in 4/1 pyridine/acetic
acid, hydrazine monohydrate (1.5 equiv) was added and reacted
for 2 h. After reaction completion, acetylacetone (2.0 equiv)
was added to quench the excess hydrazine, and the mixture
was stirred for 10 min. The solvents were removed under
vacuum and the residue was dissolved in dichloromethane
and extracted with water, 10% KHSO4 (two times), 10%
NaHCO3 (two times), and brine. After drying by MgSO4 and
filtration, the solvents were removed by rotary evaporation.
The crude product was purified by column chromatography
(column was packed with 3% pyridine in hexane). 5ꢀ-O-
DMTr-rABzpoCNEdT-3ꢀ-OH (7): m/z (MALDITOF):Anal. Calc.
for C57H65N8O14PSi: 1144.41. Found 1145.70 (M + H)+. 5ꢀ-
O-DMTr-rGiBupoCNErABzpoCNEdT-3ꢀ-OH (15): m/z (MALDI
TOF): Anal. Calc. for C78H96N14O22P2Si2: 1698.58. Found
1721.32 (M + Na)+.
(b) H. G. Khorana, Biochem. J. 1968, 109, 709.
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Basic Conditions for Phosphitylation
To a solution of 3ꢀ-OH oligonucleotides (1.0 equiv) in THF
under nitrogen at 0◦C, N,N-diisopropylethylamine (3.0 equiv)
and chloro(2-cyanoethoxy)(diisopropylamino) phosphine (1.5
equiv) were added and the reaction mixture was stirred for
1 h. After completion, the mixture was washed with 5%
aqueous NaHCO3 and dried over MgSO4. The crude prod-
uct was purified by column chromatography (column was
packed with 3% pyridine in hexane) and product was obtained
as yellow form. 5ꢀ-O-DMTr-rABzpoCNEdT-3ꢀ-O-poCNEN(i-Pr)2
(8): δP (65 MHz, CD3CN) 148.9, 148.9, 148.8, 148.7, −1.7,
−1.8, −1.9, −2.0. m/z (MALDI TOF): Anal. Calc. for
C66H82N10O15P2Si: 1344.52. Found 1345.60 (M + H)+. 5ꢀ-O-
DMTr-rGiBupoCNErABzpoCNEdT-3ꢀ-O-poCNEN(i-Pr)2 (16): δP
(65 MHz, CD3CN) 148.9 148.9, 148.9, 148.8, 148.8, 148.7,
148.6, 148.5, −1.7, −1.8, −1.8, −1.9, −1.9, −2.0, −2.0,
−2.1. m/z (MALDITOF):Anal. Calc. for C87H113N16O23P3Si2:
1898.69. Found 1921.66 (M + Na)+.
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doi:10.1016/S0040-4039(00)78935-0
Acidic Conditions for Phosphitylation
To a solution of 3ꢀ-OH oligonucleotides (1.0 equiv) in
anhydrous dichloromethane, 2-cyanoethyl tetraisopropylphos-
phorodiamidite (1.3 equiv), and 5-(benzylthio)-1H-tetrazol (0.4
equiv) were added at ambient temperature.After 5 h, the mixture
was washed with 5% aqueous NaHCO3 and dried over MgSO4.
The crude product was purified by column chromatography (col-
umn was packed with 3% pyridine in hexane) and product was
obtained as yellow form.
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kortenaar, D. V. Filippov, G. A. van der Marel, Org. Process Res. Dev.
2006, 10, 1238. doi:10.1021/OP060133Q
Acknowledgements
The authors are thankful to National Science Council of Taiwan (NSC) and
ScinoPharm® Taiwan for financial assistance and National Center for High-
Performance Computing for computer time and facilities.