Synthetic oligodeoxynucleotide purification by capping failure sequences with a methacrylamide phosphoramidite followed by polymerization

Article abstract of DOI:10.1039/c3ra46986g

Oligodeoxynucleotide (ODN) purification was achieved by capping failure sequences with a polymerizable methacrylamide phosphoramidite during automated synthesis, polymerizing the failure sequences into an acrylamide gel after cleavage and deprotection, and extraction of full-length sequences with water. The details regarding the technology including the capping efficiency of four polymerizable phosphoramidites, optimal capping time, diffusion speeds of ODN from gels with different cross-linking ratios to solution, and the efficiency of ODN extraction from gel were investigated. In addition, the technology was tested for purification of a long sequence and purification on larger scales. We also found that polymerization of failure sequences in a centrifuge tube in air did not affect purification results. Finally, we provided additional evidence that ODNs are stable under radical polymerization conditions by complete digestion of ODN followed by reversed-phase HPLC analysis of nucleosides. The Royal Society of Chemistry.

Full text of DOI:10.1039/c3ra46986g

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Synthetic oligodeoxynucleotide purification by
capping failure sequences with a methacrylamide
phosphoramidite followed by polymerization†
Cite this: RSC Adv., 2014, 4, 8746
a
b
a
a
*
Durga Pokharel, Yinan Yuan, Suntara Fueangfung and Shiyue Fang
Oligodeoxynucleotide (ODN) purification was achieved by capping failure sequences with a polymerizable
methacrylamide phosphoramidite during automated synthesis, polymerizing the failure sequences into an
acrylamide gel after cleavage and deprotection, and extraction of full-length sequences with water. The
details regarding the technology including the capping efficiency of four polymerizable
phosphoramidites, optimal capping time, diffusion speeds of ODN from gels with different cross-linking
ratios to solution, and the efficiency of ODN extraction from gel were investigated. In addition, the
technology was tested for purification of a long sequence and purification on larger scales. We also
found that polymerization of failure sequences in a centrifuge tube in air did not affect purification
results. Finally, we provided additional evidence that ODNs are stable under radical polymerization
conditions by complete digestion of ODN followed by reversed-phase HPLC analysis of nucleosides.
Received 24th November 2013
Accepted 17th January 2014
DOI: 10.1039/c3ra46986g
www.rsc.org/advances
deletion sequences. For a typical 20-mer ODN synthesis, they
usually consist of 30–60% ODN content depending on the scale
Introduction
of synthesis, quality of reagents and solvents, and carefulness of
the synthesizer operator to minimize exposure of reagents and
solvents to air.6 Because failure sequences have very similar
physical properties as the desired full-length sequences, this
class of impurities is challenging to remove. The second type of
impurities is small organic molecules from protecting groups.
These molecules are neutral, and therefore are easy to remove
from the negatively charged full-length sequences. The third
type impurities include deletion sequences, addition
sequences, and sequences resulted from depurination as well as
other modications during synthesis, cleavage and depro-
tection. Deletion sequences can be resulted from incomplete
capping, and incomplete de-tritylation. Addition sequences can
be resulted from premature de-tritylation. Deletion and addi-
tion sequences are most difficult to remove. Fortunately, they
only exist in minute quantities in crude ODN. In ODN produc-
tion, it is best to tune synthesis, cleavage and deprotection
conditions to avoid the third type impurities. As a result, the
major task of ODN purication usually is the removal of failure
sequences from the full-length sequences.
Applications such as total gene synthesis require the production
of large numbers of short ODNs within a short time with high
efficiency.1 An ideal method to achieve this is through high
throughput production. The high interest in using oligonucle-
otides (ONs) as therapeutic agents to cure human diseases
generates a need for large quantities of ONs for preclinical
research, clinical trial and patient use. This requires to develop
technologies for large scale production of ON.2 Currently, high
throughput and large scale syntheses of ON are possible due to
the commercialization of oligonucleotide microarray technolo-
gies,3 high throughput automated solid phase synthesizer,1c,4
and large scale synthesizer.5 However, the products contain
impurities, which must be removed before the applications. The
impurities can be classied into three types. The rst type is the
truncated failure sequences generated in each synthetic cycle
due to incomplete coupling of nucleoside phosphoramidite
monomer. These failure sequences are capped usually with
acetic anhydride to prevent them from growing in subsequent
synthetic cycles and becoming the more difficult-to-remove
Current methods for ODN purication include gel electro-
phoresis, ion exchange HPLC, trityl-on reversed-phase (RP)
HPLC, RP cartridge chromatography, hydrophobic RP chro-
matography,7 uorous affinity purication,6,8 biotin–avidin
based affinity purication9 and reaction-based solid phase
extraction.10 The limitations of these methods in large scale
ODN purication have been discussed earlier.11 For applica-
tions in high throughput purication, gel electrophoresis and
all chromatographic methods are not practical or highly
aDepartment of Chemistry, Michigan Technological University, 1400 Townsend Drive,
Houghton, Michigan 49931, USA. E-mail: shifang@mtu.edu
bSchool of Forest Resources and Environmental Science, Michigan Technological
University, 1400 Townsend Drive, Houghton, Michigan 49931, USA
† Electronic supplementary information (ESI) available: General methods; effects
of old capping agent, radical scavenger, harsher deprotection conditions, low
concentration capping solution, and air on purication; 1H, 13C and 31P NMR
spectra; mass spectra; HPLC proles; peak areas in HPLC proles of
nucleosides from digested ODN; recipes of polymerization solutions; and ODN
synthesis cycles. See DOI: 10.1039/c3ra46986g
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expensive, affinity purication methods may be expensive, and failure sequences by polymerization ODN purication techno-
to our best knowledge, reaction-based solid phase extraction logy, however, the capping agent is replaced with the polymer-
methods have not been well developed.
izable methacrylamide phosphoramidite 4, and the resulting
Recently, we communicated our preliminary results on the material is depicted with 5. Aer oxidation and detritylation
development of a new ODN purication technology based on steps, 5 becomes 6. The synthesis is continued and the capping
catching failure sequences by polymerization.11b Using this failure sequences with 4 is performed in each cycle.
method, the failure sequences in crude ODN could be simply
Aer cleavage and deprotection, there are mainly three
removed by co-polymerizing them into an insoluble polymer, entities in the crude product, the failure sequences that contain
and pure ODN could be obtained by simple extraction followed a methacrylamide group (7), the full-length sequences that do
by n-BuOH precipitation.12 The method does not require any not contain a methacrylamide group (8), and small organics
chromatography, and purication is achieved with simple from protecting groups. The crude product is subjected into
manipulations such as mixing, shaking and extraction. As a radical polymerization conditions in the presence of additional
result, it is potentially useful for large scale and high polymerization monomer (e.g. N,N-dimethylacrylamide) and a
throughput ODN purication. In this article, we further report cross-linker (e.g. N,N0-methylenebis(acrylamide)). The failure
our detailed studies of the technology.
sequences are incorporated into an insoluble polymer (9). The
full-length sequences (8) and small organics remain in solution
and are extracted with water. The small organics are neutral and
soluble in n-BuOH, while the full-length sequences are anionic
and insoluble in n-BuOH. The two are separated by precipita-
tion of ODN 8 with n-BuOH from a solution in concentrated
NH4OH (Scheme 1).
Results and discussion
The working principle of the catching failure sequences by
polymerization ODN purication method is detailed in
Scheme 1. The entity 1 represents two of many n-mer ODNs on
solid support (usually controlled pore glass, CPG) at the end of
nth synthetic cycle, at which time the 50-end DMTr (4,40-dime-
thoxytrityl) group is removed (each cycle contains four steps:
Capping efficiency of four methacrylamide phosphoramidites
and optimal capping time
coupling, capping, oxidation and detritylation). Coupling 1 with Previously, we used the methacrylamide phosphoramidite 10 as
excess phosphoramidite monomer 2 in the presence of an the reagent for capping failure sequences.11b In the present
activator to generate (n + 1)-mer is usually highly efficient, but it study, we synthesized three more (11–13, Fig. 1). The capping
is inevitable for small amount of n-mer to fail the coupling and efficiency of them and 10 were evaluated. Compared with 10,
therefore generating failure sequences. The entity 3 represents compound 11 has an ether oxygen atom in the linker, which
one failure and one success sequences on solid support. may increase its solubility in polar organic solvents such as
Repeated coupling of 3 with 2 to complete the reaction is not a acetonitrile and have better reaction kinetics during capping.
good option due to the expense of 2 and potential side reactions Compound 12 has two ether oxygen atoms in the linker and the
such as premature detritylation. However, if the failure linker is longer. These may offer benets for reaction kinetics in
sequences were carried on to the next synthetic cycles, they both capping and polymerization steps. Compound 13 has an
would become deletion sequences, which are highly chal- even longer linker, and in the linker, there is an amide bond. It
lenging to remove during purication. As a result, they are was expected that the compound exist as a solid at room
usually capped with acetic anhydride. For using our catching temperature and therefore its purication and storage be more
Scheme 1 The catching failure sequences by polymerization ODN purification method.
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Fig. 1 Structures of polymerizable capping phosphoramidites.
convenient. Compound 11 was simply synthesized from amino
alcohol 14 in two steps in excellent yields (Scheme 2). The
intermediate compound 15 is known,13 but we used a different
procedure for its preparation (see Experimental section).
Capping phosphoramidites 12,13 were prepared from 16
(ref. 14) and 18,15 respectively using the same procedures for
preparing 11. The intermediate compound 17 was known,16 and
19 is new and was fully characterized.
In our initial studies, during automated ODN synthesis,
capping failure sequences was performed aer the oxidation
step that oxidizes the newly formed phosphite triester bonds in
the success sequences.11b As a result, aer capping, another
oxidation step was needed to oxidize failure sequences. In the
present study, we planned to simplify the process by omitting
the rst oxidation step and to oxidize the success and failure
sequences in one single step. In addition, reduction of capping
delivery time and the concentration of capping solutions were
also planned. To test feasibility, the 20-mer ODN 20 was
synthesized using a 0.15 M acetonitrile solution of 11 for
capping. Aer the coupling steps, without oxidation, the
capping solution was delivered to the synthesis column along
with the activator 1H-tetrazole. The delivery time and manner
were about the same as in the coupling step except that the
reagents were delivered for two more times, and aer each
delivery a waiting step was added. More specically, the
reagents were delivered to the synthesis column for a total of
four times (2.0 seconds, 1.5 seconds ꢀ 3). Aer each delivery the
Fig. 2 Labeling of ODNs.
reagents remained in the column for 90 seconds (Cycle 1 in
ESI†). These capping steps consumed less capping phosphor-
amidite compared with those used in our initial studies, which
were 2.5 seconds, 1.5 seconds ꢀ 3 with 0.2 M solution.11b Aer
the capping steps, the column was washed with acetonitrile,
and the synthesis was allowed to proceed as normal. The nal
detritylation, cleavage and deprotection were also carried out
under commonly used conditions. The crude product, which
was labeled as 20(c11l) (ODN labeling is summarized in Fig. 2),
was analyzed with RP HPLC (trace a, Fig. 3).
The remaining crude 20(c11l) was subjected to radical
acrylamide polymerization conditions widely used for the
preparation of gels in electrophoresis experiments (Scheme 3).
Briey, the crude ODN, which mainly contained the full-length
sequences 20, the failure sequences 21 and small organic
molecules from protecting groups, was mixed with a pre-
prepared polymerization solution containing the polymeriza-
tion monomer N,N-dimethylacrylamide and the cross-linker
N,N0-methylenebis(acrylamide). Polymerization was then initi-
ated under a nitrogen atmosphere with ammonium persulfate
and N,N,N0,N0-tetramethylethylenediamine (TMEDA). The
failure sequences were copolymerized into the acrylamide
gel 22. The full-length sequences (20) and small organics
remained in solution, and were extracted with water. Aer
Scheme 2 Synthesis of polymerizable phosphoramidites.
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Table 1 ODN purity and recovery yielda
ODN
Purity
Recovery yield
20(c10l)
20(c11l)
20(c12l)
20(c13l)
20(c10s)
20(c11s)
20(c12s)
20(c13s)
23
89%
93%
99%
100%
100%
100%
98%
94%
92%
94%
100%
92%
93%
72%
81%
82%
86%
95%
70%
67%
73%
88%
20(c11ls)
20(c11dci)
a
Purity was calculated from the areas of ODN and impurity peaks in
HPLC prole. Yield was calculated by dividing the area of ODN peak
in HPLC prole of puried sample by that in the prole of crude sample.
Fig. 3 RP HPLC profiles of ODN 20(c11l). (a) Crude ODN. (b) ODN
purified with catching by polymerization.
repeated with the capping conditions being changed from
phosphoramidite and activator delivering time of 2.0 seconds,
1.5 seconds ꢀ 3, and waiting time of 90 seconds aer each
delivery to delivering time of 2.0 seconds, 1.5 seconds ꢀ 2, and
waiting time of 30 seconds (Cycle 2 in ESI†). Aer cleavage and
deprotection, the four crude samples 20(c11s), 20(c10s),
20(c12s) and 20(c13s) were puried using the same procedure as
described above. The puried ODNs were analyzed with RP
HPLC, and good purity and recovery yields were found (Table 1).
Because the delivery time of capping reagents was almost the
same as that of nucleoside phosphoramidite monomers in
typical coupling steps (2.0 seconds, 1.5 seconds), further
shortening of capping time was not pursued. All the four
capping phosphoramidites (10–13) performed equally well, and
therefore any of them could be used for the catching by poly-
merization technology. However, compounds 12,13 are rela-
tively more expensive to make. Compound 13 did not exist as a
solid or foam at room temperature as we hoped, and moreover,
it was found more prone to oxidation during purication. As a
result, compounds 10,11 are preferred capping reagents for the
catching failure sequences by polymerization purication
technology.
Scheme
sequences.
3 Purification of ODN 20 by polymerization of failure
evaporation of volatiles and heating with concentrated NH4OH
briey, the ODN was precipitated with n-BuOH.11b The puried
20(c11l) was analyzed with RP HPLC (trace b, Fig. 3) and the
Diffusion speed of ODN from gel and extraction efficiency
purity was found to be 93%. The recovery yield of the purica- The diffusion speed of ODN from the acrylamide gel to solution
tion process was estimated to be 93% by dividing the area of the is an important factor to determine the efficiency of ODN
peak at 19 minutes in trace b by that in trace a in Fig. 3. The extraction. The purication technology is most useful if the
results indicate that the new agent 11 with the simplied diffusion speed is high and ODN can be extracted with high
capping procedure is capable of capping failure sequences efficiency and recovery yield. Our common sense was that gels
completely. We also tested the capping agents 10, 12 and 13 with higher cross-linking ratio [the mole ratio of N,N0-methyl-
under the same new ODN synthesis and catching by polymeri- enebis(acrylamide) over N,N-dimethylacrylamide] would result
zation purication conditions. All of them were found equally in lower diffusion speed and extraction efficiency. Gels with
suitable for the application. The RP HPLC proles for ODNs lower cross-linking ratio were expected to give better results.
20(c10l), 20(c12l) and 20(c13l) are shown in ESI.† Their recovery However, if the cross-linking ratio is too low, the gel may be too
yields and purity are shown in Table 1.
so and therefore difficult to handle. We were interested to nd
In order to see if capping time could be further shortened a compromised point where the most common 20-mer ODNs
and at the same time to distinguish the efficiency of the four could be extracted with acceptable efficiency while the gel is
capping reagents, the above ODN synthesis procedure was hard enough to be easily handled. From experiments on ODN
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purication with polyacrylamide gel electrophoresis, it is Aer three extractions, the value lowered to 0.4% (entry 4). The
known that gels with a cross-linking ratio of 1 : 25 are reason- results in Tables 2 and 3 indicate that ODN 20(c11s) can be
ably hard and 20-mer ODNs can be extracted from them with efficiently extracted from gel that has a cross-linking ratio of
acceptable efficiency and yields. We therefore decided to start 1 : 25 with water for three times in a total of 6 h.
our experiments with this cross-linking ratio.
To nd out if a gel with lower cross-linking ratio could offer
A portion of crude ODN 20(c11s), which contained 21, was higher diffusion speed and better extraction efficiency, the
subjected to polymerization using a polymerization solution above experiments were repeated with the exception of using a
that had a mole ratio of cross-linker over monomer of 1 : 25 (see polymerization solution that had a cross-linking ratio of 1 : 50.
details in Experimental section). Water was added to the gel, As shown in Tables 2 and 3, the diffusion speed and extraction
and small aliquots of supernatant were taken out every 10 min efficiency were not better, which was contrary to our prediction.
and the concentrations of ODN were determined with UV Instead, the data showed a slightly slower diffusion speed and
measurement. As shown in Table 2, aer 10 min, the concen- lower extraction efficiency although the differences were small
tration of ODN in the supernatant had reached about 40% (e.g. 39.3% vs. 39.9% at 10 min entry 1, and 91.2% vs. 94.3% at
(entry 1) of the value at 2 h, at which time the diffusion was close 100 min entry 10, Table 2). Because gels with a higher cross-
to equilibrium. For the concentration to reach 90% of equilib- linking ratio gave slightly better results, we repeated the
rium value, about 90 min were required (entry 9). To nd out experiments with a cross-linking ratio of 1 : 15 (Tables 2 and 3).
how many times were needed to extract all or most ODN from According to the trend, the diffusion speed and extraction effi-
gel, aer 2 h, the supernatant was removed completely (entry 12, ciency should become better, which was indeed the case
Table 2 and entry 1, Table 3). To the gel, water was added again, although the difference was small again. For example, within
and aer 2 h, the supernatant was removed and its concentra- 10 min, the concentration of ODN in the supernatant had
tion was determined with UV measurement (entry 2, Table 3). reached about 45% of equilibrium value (entry 1). For the
The extraction and UV measurement were repeated for three concentration to reach 90% of equilibrium value, about 80 min
additional times. As shown in Table 3, aer two extractions, were required (entry 8). For extraction efficiency, aer three
only about 3% extractable ODN remained in the gel (entry 3). extractions, no more ODN was detectable in additional extracts
Table 2 Monitoring ODN diffusion from gels with different cross-linking ratios to supernatanta
Time
(min)
ODN Con.b
% of
ODN Con.b
% of
ODN Con.b
% of
ODN Con.b
% of
ODN Con.b
% of
Entry
(ng mLꢁ1
)
ODNb,c
(ng mLꢁ1
)
ODNb,c
(ng mLꢁ1
)
ODNb,c
(ng mLꢁ1
)
ODNb,c
(ng mLꢁ1
)
ODNb,c
1
2
3
4
5
6
7
8
10
20
30
40
50
60
70
80
90
47.7
53.3
54.7
58.6
64.6
65.1
66.4
67.6
68.6
70.9
71.7
72.3
66.0
73.7
75.7
81.1
89.3
90.0
91.8
93.5
94.9
98.1
99.2
100
29.9
37.8
43.4
48.6
50.9
54.6
56.0
58.0
59.6
61.2
62.0
62.9
47.5
60.1
69.0
77.3
80.9
86.8
89.0
92.2
94.8
97.3
98.6
100
32.7
40.9
49.5
53.4
56.7
59.2
64.2
64.6
67.2
68.0
70.6
72.6
45.0
56.3
68.2
73.6
78.1
81.5
88.4
89.0
92.6
93.7
97.3
100
27.1
36.0
42.0
46.9
50.5
53.3
56.1
59.7
59.9
64.0
66.2
67.9
39.9
53.0
61.9
69.1
74.4
78.5
82.6
87.9
88.2
94.3
97.5
100
25.8
34.7
37.7
42.8
47.0
50.0
53.3
56.0
58.6
59.8
65.4
65.6
39.3
52.9
57.5
65.2
71.7
76.2
81.3
85.4
89.3
91.2
99.7
100
9
10
11
12
100
110
120
a
Crude ODN containing 20 and 21 was copolymerized into gel. Water was added to extract 20. The concentration of 20 in the supernatant was
b
monitored with UV over a two-hour period. In supernatants of gel with cross-linking ratio of 1 : 2, 1 : 7, 1 : 15, 1 : 25, 1 : 50, respectively.
c
Calculated by dividing ODN concentration at a specic time by that aer 120 min.
Table 3 Extraction efficiency of ODN from gela
Time
(h)
ODN Con.b
% of
ODN Con.b
% of
ODN Con.b
% of
ODN Con.b
% of
ODN Con.b
% of
Entry
(ng mLꢁ1
)
ODNb,c
(ng mLꢁ1
)
ODNb,c
(ng mLꢁ1
)
ODNb,c
(ng mLꢁ1
)
ODNb,c
(ng mLꢁ1
)
ODNb,c
1
2
3
4
5
2
4
6
8
10
72.3
9.5
2.8
0.0
0.0
85.5
11.2
3.3
0.00
0.00
62.9
10.3
3.1
0.0
0.0
82.4
13.5
4.1
0.0
0.0
72.6
18.1
3.3
0.0
0.0
77.2
19.3
3.51
0.0
67.9
23.5
2.6
0.4
0.3
71.7
24.8
2.8
0.4
0.3
65.6
22.3
13.2
2.1
63.3
21.5
12.7
2.0
0.0
0.4
0.4
a
The experiments in Table 1 were continued by removing the supernatant aer 2 h. Water was added to the gels again, and supernatants were
b
removed aer 2 h. The extractions were repeated every 2 h for three additional times. In supernatants of gel with cross-linking ratio of 1 : 2,
1 : 7, 1 : 15, 1 : 25, 1 : 50, respectively. Calculated by dividing ODN concentration in each extract by that of the total amount of ODN extracted
c
from the gel in the same volume of water.
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(entry 4, Table 3). We then further increased the cross-linking by catching failure sequences by polymerization as we did for
ratio to 1 : 7. The trends of diffusion speed and extraction effi- shorter sequences. RP HPLC analysis showed that the ODN had
ciency changes were the same. Within 10 min, the concentra- good purity and recovery yield (Table 1 and ESI†). The identity of
tion of ODN in the supernatant had reached 48% of equilibrium puried ODN was conrmed with ESI-MS (ESI†).
value (entry 1). For the concentration to reach 90% of equilib-
rium value, about 70 min were needed (entry 7). When the cross-
linking ratio was increased to 1 : 2, within 10 min, the
concentration reached 66% (entry 1), and the time for reaching
90% was reduced to 60 min (entry 6). These results consistently
show that the diffusion speed and ODN extraction efficiency
increase with increased cross-linking ratio of gel. This is
signicant for the technology because gels with higher cross-
linking ratios are harder and therefore easier to handle.
It was quite surprising to nd that the ODN diffusion speed
and extraction efficiency increase with cross-linking ratio. We
had expected the contrary because gels with higher cross-link-
ing ratio have smaller pores and ODN molecules would be more
difficult to travel through them. Our explanation of the observed
results is that the ODN molecules were not totally in the gel even
though this seemed impossible because the gel with other
contents including water and ODN looked like homogenous.
Instead, when the polymers grew, ODN molecules were expelled
or partially expelled out from the polymer matrix. With lower
cross-linking ratio when the pores in polymer were larger, a
higher portion of ODN molecules was in the polymer matrix, or
all ODN molecules were not in the polymer matrix, but they
could diffuse into it with relatively high rates. These resulted in
seemingly lower diffusion speeds of ODN from gels to solution
and lower extraction efficiency. With higher cross-linking ratio
when the pores in the polymer were smaller, a lower portion of
ODN molecules was in the polymer matrix, or all ODN mole-
cules were not in the polymer matrix, and they could only
diffuse into it with low rates. These resulted in seemingly higher
diffusion speeds of ODN from gels to solution and higher
extraction efficiency.
Purication at larger scales
Aer investigation of the effect of several additional factors such
as capping agent storage time, radical scavenger, harsher
deprotecting conditions, lower concentration of capping solu-
tion, polymerization in air (see ESI†), we decided to test the
technology for larger scale purication. For this goal, ODN 20
was synthesized on a 1 mmol scale. The synthesis conditions were
only slightly different from those using shortened capping time
described earlier, which was used for 0.2 mmol synthesis. The
standard 1 mmol synthesis cycle provided by manufacturer of the
synthesizer was modied. The steps for capping with acetic
anhydride were replaced with those for capping with polymer-
izable phosphoramidite. The latter was achieved by delivering
the solution of 11 in acetonitrile and activator to the synthesis
column in the same manner as in the coupling steps except that
an additional delivery was added and a waiting of 30 seconds was
added aer each delivery (Cycle 3 in ESI†). At the end of
synthesis, DMTr group was removed, and cleavage and depro-
tection were performed. The crude ODN was puried by catching
failure sequences by polymerization using the same procedure
described earlier. The only differences were that the 1 mmol ODN
was puried in one batch, the volume of the polymerization
solution was increased to 10 times of that used in the general
0.05 mmol purication procedure, and the volume of extraction
water was slightly larger. The ODN, which is labeled as 20(c11ls),
was found to have good purity and recovery yield with RP HPLC
(Table 1 and ESI†). It is remarkable that even though the puri-
cation scale was increased from 0.05 mmol to 1 mmol, which was
20 times, increasing the volume of polymerization solution to 10
times was sufficient to catch all the failure sequences.
We further performed three 1 mmol syntheses simulta-
neously under conditions that are more close to actual large
scale ODN production. Specically, 1H-tetrazole is considered
as an explosive and not recommended for large scale synthesis.
Therefore, 4,5-dicyanoimidazole (DCI) was used as activator. To
save phosphoramidite monomers, their concentration was
lowered from 0.1 M to 0.05 M. A 0.1 M solution of 11 in aceto-
nitrile was used for capping. With these modications, ODN 20
was synthesized under otherwise the same conditions for the
synthesis of 20(c11ls). According to trityl assay and RP HPLC
analysis of crude ODN, the synthesis yields were not signi-
cantly different from synthesis under conditions without the
above modications. The 3 mmol ODN was puried in one batch
under the same conditions described for purication of
20(c11ls) and is labeled as 20(c11dci). RP HPLC analysis indi-
cated that the ODN was highly pure and had excellent recovery
yield (Table 1 and ESI†).
Long sequence purication
For long sequence ODN purication, the catching failure
sequences by polymerization method requires signicant
amount of polymerizable capping phosphoramidite, which is
more expensive than acetic anhydride or phenoxyacetic anhy-
dride capping agents used in catching full-length sequences by
polymerization method.11a We therefore recommended the
catching full-length sequences by polymerization method for
long sequence purication.11b,17 However, it is still interesting to
test the suitability of the catching failure sequences method for
ODN purication because it may be more convenient in certain
applications such as high throughput purication. The 61-mer
ODN 23, which is a portion of the HIV protease gene, was
selected as the target. The synthesis was carried out on a
0.2 mmol scale using the procedure with shortened capping
time. Polymerizable phosphoramidite 11 was used as the
capping agent. In the coupling step, aer nishing phosphor-
amidite and activator delivery, an additional waiting of 30
ODN stability under radical polymerization conditions
seconds was added to increase yields (Cycle 2 in ESI† with A concern on the catching by polymerization purication
additional waiting aer coupling). The crude ODN was puried technologies, which involves copolymerization of ODN into
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polymer under free radical conditions, is the potential damage
of ODN under such conditions. Polymerization of ODN into
polyacrylamide gels under radical conditions is known in the
literature.18 However, to our best knowledge, detailed study of
ODN stability in the presence of free radicals has not been
reported. Certainly, RP HPLC analysis of ODNs puried with the
catching by polymerization technologies by comparing with
authentic ODN strongly supports that ODNs can survive such
conditions. Another evidence of stability comes from mass
spectrometry, which consistently gave molecular mass consis-
tent with calculated values.11 Further, we also subjected the
nucleosides guanosine, adenosine, thymidine and cytidine to
free radical acrylamide polymerization conditions, extracted
them from the polymer, and analyzed them with RP HPLC. No
damaged nucleoside was detected. Among the four nucleosides,
guanosine oxidation is more common, and the oxidation
product is 8-oxo-guanosine. We compared the guanosine from
the polymer with authentic 8-oxo-guanosine by co-injection into
RP HPLC, the two were found to have different retention times,
and no 8-oxo-guanosine was detected in the sample from pol-
ymer.11a However, concerns on ODN stability may still remain
because this is so important an issue for purication, especially
for ODN drug purication. For RP HPLC analysis, if only one of
the 20 bases of a 20-mer ODN is damaged, and the physical
properties of the damaged nucleobase are not changed signi-
cantly, the analysis may not be able to differentiate damaged
ODN from authentic ODN. For mass spectrometry analysis, if
only a minute portion of ODN is damaged, the method may not
be sensitive enough for detection. For the experiments to test
stability of nucleosides under radical conditions, the reactivity
of nucleosides in an ODN may not be the same as individual
nucleosides. As a result, all the methods we used to support
ODN stability under radical conditions are not conclusive.
To obtain additional evidences on the stability of ODN under
radical acrylamide polymerization conditions, we used ion
exchange HPLC to analyze one of our samples puried with the
catching failure sequences by polymerization process. It was
expected that ion exchange HPLC have different resolution on
Fig. 4 RP HPLC profiles of nucleosides from ODN enzymatic diges-
tion essay. (a) Nucleosides from ODN 20(c11s), which was purified with
the catching by polymerization technique. (b) Nucleosides from
authentic ODN 20. (c) Nucleosides from ODN 24. (d) Nucleosides from
the mixture ODN 25. The peaks in a, b and d correspond to dC, dG, dT
and dA, respectively. Those in c correspond to dC, dG, 8-oxo-dG, dT
and dA, and 2-mercaptoethanol, respectively. The inconsistency of
retention times in c with those in a, b and d is caused by the use of a
different column.
potentially damaged ODNs than RP HPLC. To our satisfaction, observed (trace a). No additional peaks that correspond to any
we still did not found any damaged ODN, and only one peak was damaged nucleosides were detected. An authentic ODN 20,
observed (ESI†). We also tried to nd any impurity using RP which was from a commercial source and was puried with
HPLC by UV detecting at 210 nm instead of the usually used preparative RP HPLC, was also subjected into the same diges-
260 nm; again, only one peak was observed (ESI†). Further, we tion process. The resulting HPLC prole of nucleosides (trace b)
performed an ODN enzymatic digestion essay to detect any was exactly the same as that from 20(c11s). Another ODN (24),
damaged nucleosides.19 A portion of the ODN sample 20(c11s) which is 20 with one dG being replaced with 8-oxo-dG (Fig. 2),
puried with the catching failure sequences by polymerization was synthesized and puried by DMTr-on preparative RP HPLC
method was digested with Snake Venom Phosphodiesterase (see ESI† for crude and pure HPLC proles and MALDI-TOF
(SVP) into nucleotides, which were dephosphorylated to give MS), and subjected to digestion.20 The HPLC prole (trace c)
nucleosides by Bacterial Alkaline Phosphatase (BAP). The showed ve peaks besides a peak from 2-mercaptoethanol
neutral nucleosides were then separated from the highly polar indicating that the essay could detect potentially damaged
species including inorganic salts and enzymes in the digestion nucleosides. The relative retention times of the nucleosides
mixture by precipitation of the latter materials by ethanol from including 8-oxo-dG were consistent with literature values.19,20 To
a sodium acetate buffer. The nucleosides were soluble in address the concern that ODN damage may be dependent on
ethanol and remained in the supernatant. The polar species the secondary structures resulted from different sequences, a
were not soluble and were precipitated. The nucleosides were complex mixture of 20-mer ODN (25), which contained 419
analyzed with RP HPLC. As shown in Fig. 4, only four peaks, theoretical sequences, were synthesized using a mixture of the
which correspond to the four natural nucleosides, were four nucleoside phosphoramidite monomers. The synthesis
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procedure with a shortened capping time was used (Cycle 2 in atmosphere was added 14 (2.0 g, 19.0 mmol, 1.0 equiv.), freshly
ESI†), and the failure sequences were capped with meth- distilled CH2Cl2 (60 mL), and saturated Na2CO3 (60 mL). The
acrylamide phosphoramidite 11. The crude ODN was subjected mixture was cooled to 0 ꢂC, and methacryloyl chloride (1.86 mL,
to the general catching failure sequences by polymerization 19.0 mmol, 1.0 equiv.) was added via a syringe slowly with
process (ESI†). The resulting mixture of full-length sequences vigorous stirring. Aer addition, the reaction was allowed to
was subjected to the same SVP digestion, BAP dephosphoryla- proceed overnight while warming to rt gradually. The contents
tion, and RP HPLC analysis process (trace d, Fig. 4). Only four were then transferred into a separatory funnel. Aer removing
peaks were observed. To conrm that the peaks were from ODN the organic layer, the aqueous layer was extracted with CH2Cl2
and not from SVP and BAP, the essay process was repeated (50 mL ꢀ 3). The combined organic phase was dried over
without using any ODN. No nucleoside peaks were observed anhydrous Na2SO4, and ltered. Volatiles were removed under
(ESI†). The four peaks in traces a, b and d correspond to dC, dG, reduced pressure. The crude product was puried with ash
dT and dA, respectively. The areas of them are consistent with column chromatography (SiO2, CH2Cl2–CH3OH, 9 : 1) giving 15
calculated values (ESI†).19 These results provide strong evidence as a colorless oil (3.12 g, 18.0 mmol, 95%): Rf ¼ 0.7 (SiO2,
that ODNs with different sequences are stable under the radical CH2Cl2–CH3OH, 9 : 1).
acrylamide polymerization conditions. Further analysis to
detect any minute quantities of potentially damaged ODNs Phosphoramidite 11
under the radical conditions is beyond the scope of this study.
Future directions would be to use the puried ODNs in
molecular biology experiments.
An oven-dried round-bottomed ask containing 15 (0.5 g,
2.89 mmol, 1.0 equiv.) and a magnetic stirring bar was evacu-
ated and then relled with nitrogen. The evacuation and
nitrogen-lling cycle was repeated for three times. Freshly
Conclusions
distilled CH2Cl2 (5 mL) was added via a syringe forming a light
yellow solution. 2-Cyanoethyl-N,N,N0,N0-tetraisopropylphos-
phoramidite (1.01 mL, 3.18 mmol, 1.1 equiv.) and 1H-tetrazole
(0.45 M solution in CH3CN, 7.07 mL, 3.18 mmol, 1.1 equiv.)
were added slowly via syringes sequentially. Aer stirring at rt
for 2 h, the reaction mixture was concentrated to dryness using
a ow of nitrogen. The residue was puried with ash column
chromatography (SiO2, hexanes–ethyl acetate–Et3N, 1 : 3 : 0.2)
to give 11 as a colorless oil (0.98 g, 2.62 mmol, 90%): Rf ¼ 0.7
(SiO2, hexanes–ethyl acetate–Et3N, 1 : 3 : 0.2); 1H NMR (400
MHz, CDCl3) d 6.29 (br s, 1H), 5.61 (s, 1H), 5.24 (s, 1H), 3.81–3.60
(m, 4H), 3.57–3.50 (m, 6H), 3.45–3.40 (m, 2H), 2.56 (t, J ¼ 6.4 Hz,
2H), 1.88 (s, 3H), 1.18–1.04 (m, 12H); 13C NMR (100 MHz,
CDCl3) d 168.6, 140.2, 119.6, 117.8, 71.2 (d, J ¼ 7.6 Hz), 69.8, 62.9
(d, J ¼ 16.7 Hz), 58.6 (d, J ¼ 19.7 Hz), 43.2 (d, J ¼ 12.1 Hz), 39.6,
24.8, 24.7 ꢀ 2, 24.66, 20.55, 20.47, 18.8; 31P NMR (162 MHz,
CDCl3) d 148.3.
Several details of the catching failure sequences by polymeriza-
tion ODN purication technology were investigated. We found
that the four methacrylamide phosphoramidites 10–13 were
similarly effective for capping failure sequences in the purica-
tion technique. However, because 10 and 11 are easier to
prepare, they are the best choices. The studies on the diffusion
speeds of ODN from polyacrylamide gel to solution indicate that
extraction of ODN from gel is efficient. Normally, three extrac-
tions will recover more than 90% ODN. Contrary to our predic-
tion, ODN extraction efficiency is higher in the case of gels with
higher cross-linking. This is a favorable nding for the tech-
nology because gels with higher cross-linking are harder and
easier to work with. The technology was successfully applied for
the purication of a long sequence and purication at a larger
scale. Capping failure sequences with older methacrylamide
phosphoramidite and phosphoramidite solution containing a
radical scavenger were also studied and were found that they
have little adverse effects on the purication results. The more
commonly used exo-amino nucleobases protecting groups that
require harsher conditions to remove were tested in the ODN
purication method. They were found equally compatible with
the technology as the more labile phenoxyacetyl protecting
groups. Polymerization in air is more convenient than under a
nitrogen atmosphere. This is especially important for high
throughput purication. We demonstrated that it does not affect
purication results. Finally and most importantly, we provided
further evidences to support that ODNs are stable under free
radical acrylamide polymerization conditions. This study is most
important in the context of ODN drug purication.
Methacrylamide 17
The compound is known,16 but a different procedure, which was
the same as that for the synthesis of compound 15, was used for
its preparation. Reaction of compound 16 (ref. 14) (1.3 g,
8.71 mmol, 1.0 equiv.) with methacryloyl chloride (851 mL,
8.71 mmol, 1.0 equiv.) in saturated Na2CO3 (40 mL) and CH2Cl2
(40 mL) gave 17 as a colorless oil aer ash column chroma-
tography purication (SiO2, CH2Cl2–CH3OH, 9 : 1; 1.6 g,
7.37 mmol, 85%): Rf ¼ 0.6 (SiO2, CH2Cl2–CH3OH, 9 : 1).
Phosphoramidite 12
The same procedure for the synthesis of phosphoramidite 11 was
followed. Thus compound 17 (500 mg, 2.30 mmol, 1.0 equiv.),
2-cyanoethyl-N,N,N0,N0-tetraisopropylphosphoramidite (0.80 mL,
2.53 mmol, 1.1 equiv.) and 1H-tetrazole (0.45 M in CH3CN,
5.60 mL, 2.53 mmol, 1.1 equiv.) in CH2Cl2 (5 mL) gave compound
Experimental
Methacrylamide 15
The compound is known,13 but a different procedure was used 12 as a colorless oil aer ash column chromatography puri-
for its preparation. To a round-bottomed ask under a nitrogen cation (SiO2, hexanes–ethyl acetate–Et3N, 1 : 3 : 0.2; 0.85 g,
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2.04 mmol, 88%): Rf ¼ 0.7 (SiO2, hexanes–ethyl acetate–Et3N, for capping failure sequences with Ac2O were deleted. Aer the
1
1 : 3 : 0.2); H NMR (400 MHz, CDCl3) d 6.33 (br s, 1H), 5.61 (s, coupling steps, new steps for delivering 11 from the 5th bottle
1H), 5.22 (s, 1H), 3.76–3.70 (m, 4H), 3.58–3.49 (m, 10H), 3.43–3.38 position and 1H-tetrazole solution from the 9th position (i.e. the
(m, 2H), 2.56 (t, J ¼ 6.4 Hz, 2H), 1.86 (s, 3H), 1.10–1.07 (m, 12H); same activator for the coupling steps) were added. These steps
13C NMR (100 MHz, CDCl3) d 168.5, 140.2, 119.6, 117.9, 71.4 (d, J were the same as those in the coupling steps except that the
¼ 7.6 Hz), 70.7, 70.4, 69.8, 62.8 (d, J ¼ 18.2 Hz), 58.6 (d, J ¼ 18.2 base was from the 5th bottle, the reagents were delivered for two
Hz), 43.2 (d, J ¼ 12.1 Hz), 39.5, 24.8, 24.7 ꢀ 2, 24.66, 20.5, 20.4, additional times, and aer each delivery a waiting of 90 seconds
18.8; 31P NMR (162 MHz, CDCl3) d 149.5.
was added. More specically, the solutions of 11 and 1H-tetra-
zole were delivered to column for 2.0 seconds, 1.5 seconds ꢀ 3,
and aer each delivery, the reagents remained in the column for
90 seconds. Aer capping, steps for washing the column with
acetonitrile and oxidation were added (Cycle 1 in ESI†). The
synthesis was initiated using the above reagents and cycle on a
0.2 mmol scale. In the last synthetic cycle, the 50-DMTr group
was removed. Cleavage and deprotection were carried out on
the synthesizer with concentrated NH4OH (900 min ꢀ 4) at rt.
The synthesized ODN is labeled as 20(c11l). The same sequence
was synthesized under the same conditions using polymer-
izable phosphoramidites 10 and 12,13 as capping reagent giving
20(c10l), 20(c12l) and 20(c13l), respectively.
Methacrylamide 19
The same procedure for the synthesis of compound 15 was
followed. Reaction of 18 (ref. 15) (2.0 g, 8.54 mmol, 1.0 equiv.)
with methacryloyl chloride (0.84 mL, 8.54 mmol, 1.0 equiv.) in
CH2Cl2 (80 mL) and saturated Na2CO3 (60 mL) gave 19 as a
colorless oil (2.2 g, 7.28 mmol, 85%) aer ash column chro-
matography purication (SiO2, CH2Cl2–CH3OH, 9 : 1): Rf ¼ 0.4
(SiO2, CH2Cl2–CH3OH, 9 : 1); 1H NMR (400 MHz, CDCl3) d 6.94
(br s, 1H), 6.80 (br s, 1H) 5.58 (s, 1H), 5.19 (s, 1H), 3.53–3.44 (m,
6H), 3.43–3.32 (m, 4H), 3.30–3.24 (m, 4H), 2.19 (t, J ¼ 7.0 Hz,
2H), 1.81 (s, 3H), 1.74–1.66 (m, 2H); 13C NMR (100 MHz, CDCl3)
d 174.1, 169.1, 139.9, 120.1, 70.3, 69.92, 69.86, 61.7, 50.3, 39.6,
39.4, 33.4, 28.5, 18.7. HRMS (ESI, [M + H]+) m/z calcd for
Purication of ODN 20 by polymerization of failure sequences
– the general ODN purication procedure
C
14H27N2O5 303.1914, found 303.1918.
Purication of 20(c11l) is described as an example. The ODN
solution from the synthesizer was divided equally into four
portions and dried in centrifuge tubes in a centrifugal vacuum
concentrator. One portion was dissolved in 150 mL water, 20 mL
was injected into RP HPLC to generate trace a (Fig. 3). The
remaining 130 mL solution was transferred into a 25 mL
2-necked round-bottomed ask containing a magnetic stirring
bar. The centrifuge tube was washed with water (40 mL ꢀ 3), and
the washes were transferred to the same ask. A polymerization
solution (250 mL, cross-linking ratio 1 : 25) was added via a
pipette under positive nitrogen pressure. The solution was
gently stirred under a nitrogen ow at rt. Aer 5 min, the
nitrogen ow was stopped but stirring was continued under a
nitrogen atmosphere. The polymerization reaction was then
initiated with (NH4)2S2O8 (10%, 5 mL) and N,N,N0,N0-tetrame-
Phosphoramidite 13
The same procedure for the synthesis of phosphoramidite 11
was followed. Thus compound 19 (0.5 g 1.65 mmol, 1.0 equiv.),
2-cyanoethyl-N,N,N0,N0-tetraisopropylphosphoramidite
(0.58
mL, 1.82 mmol, 1.1 equiv.) and 1H-tetrazole (0.45 M in CH3CN,
4.05 mL, 1.82 mmol, 1.1 equiv.) in CH2Cl2 (5 mL) gave
compound 13 as a colorless oil aer ash column chromatog-
raphy purication (SiO2, hexanes–ethyl acetate–Et3N, 1 : 3 : 0.2;
0.75 g, 1.49 mmol, 90%): Rf ¼ 0.65 (SiO2, hexanes–ethyl acetate–
1
Et3N, 1 : 3 : 0.2); H NMR (400 MHz, CDCl3) d 6.40 (br s, 1H),
6.22 (br s, 1H), 5.64 (s, 1H), 5.27 (s, 1H), 3.84–3.69 (m, 2H), 3.65–
3.41 (m, 14H), 3.40–3.32 (m, 2H), 2.58 (t, J ¼ 6.0 Hz, 2H), 2.24 (t,
J ¼ 7.4 Hz, 2H), 1.95–1.87 (m, 5H), 1.16–1.08 (m, 12H); 13C NMR
(100 MHz, CDCl3) d 172.8, 168.6, 140.2, 119.7, 118.0, 70.4, 69.9
(d, J ¼ 18.3 Hz), 62.9 (d, J ¼ 16.7 Hz), 58.4 (d, J ¼ 19.8), 58.5, 45.5
(d, J ¼ 6.0 Hz), 43.1 (d, J ¼ 10.6), 39.5, 39.3, 33.1, 27.3, 24.8 ꢀ 2,
23.11, 23.04, 20.6, 20.3, 18.8; 31P NMR (162 MHz, CDCl3) d 149.5.
thylethylenediamine (TMEDA,
5 mL), which were added
sequentially via a pipette and a syringe, respectively. A clear gel
was formed within 5 min and stirring stopped automatically.
The gel was allowed to stand for 1 h to ensure complete
polymerization.
The gel was broken into about 4 pieces with a clean spatula
or preferably a knife with a sharp blade that does not grind the
Synthesis of ODN 20 using polymerizable phosphoramidites
10–13 to cap failure sequences – the general ODN synthesis
procedure
The synthesis using 11 as the capping agent is described as an gel into ne pieces. To the ask, sufficient water that could
example. The phosphoramidite chemistry was used. CPG cover all the gel (ꢃ3 mL) was added. The mixture was stirred or
˚
(1000 A pore size) with a LCAA-succinyl ester linkage was used shaken gently at rt for 3 h. The supernatant was separated from
as the solid support. The phosphoramidite monomers used the gel. The gel was further extracted with water (3 mL ꢀ 3)
were Pac-dA-CE, Ac-dC-CE, i-Pr-Pac-dG-CE and dT-CE. For under the same conditions. Aer the volume was reduced in a
capping failure sequences, a 0.15 M solution of 11 in dry centrifugal vacuum concentrator, the extracts were ltered into
acetonitrile (distilled under nitrogen or from a commercial one 1.5 mL centrifuge tube that has a spin ltration function.
source) was placed on the 5th phosphoramidite position. The The extracts were evaporated into dryness. To the residue,
two bottles normally used for storing Ac2O capping reagents concentrated NH4OH (100 mL) was added. The tube was closed.
were le empty. A new synthetic cycle was created by copying Aer vortexing and centrifuging briey, it was kept for 25 min
ꢂ
the standard 0.2 mmol synthesis cycle into a new le. The steps on a heating block that was pre-heated to 80 C. The solution
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was then cooled to rt, and n-BuOH (900 mL) was added. The tube
was vortexed for 30 s and centrifuged for 8 min at 14.1 relative
centrifugal force. The supernatant was removed carefully with a
pipette. The residue was dried in a centrifugal vacuum
concentrator shortly, and then dissolved in 130 mL water. Aer
vortexing and centrifuging shortly, 20 mL was injected into RP
HPLC to generate trace b (Fig. 3). The recovery yield of the
catching by polymerization process was estimated to be 93% by
dividing the area of the peak at 19 min in trace b by that in trace
a (Fig. 3). Crude ODNs 20(c10l), 20(c12l) and 20(c13l) were
puried using the same procedure, and their recovery yields
and purity are in Table 1. RP HPLC proles are in ESI.†
Synthesis of long sequence ODN 23
The 61-mer ODN 23 was synthesized on a 0.2 mmol scale using
phosphoramidite 11 for capping failure sequences. The general
synthesis procedure with shorter capping time (Cycle 2 in ESI†)
was used except that a 30 seconds additional waiting step was
added aer the coupling steps to increase coupling yields.
Purication of ODN 23 by polymerization of failure sequences
The general ODN purication procedure was used. The RP
HPLC proles from crude and puried ODN 23 were generated
using a different buffer A that contained 10% urea under
otherwise the same conditions described in the General
methods section (ESI†). Recovery yield and purity are in Table 1.
The puried 23 was analyzed with negative ESI MS using
Waters' Tofspec-2E, calcd for C599H749N235O361P60 18875.1,
found 18875.1. Image of the MS is included in ESI.†
Synthesis of ODN 20 using polymerizable phosphoramidites
10–13 to cap failure sequences with shorter capping time
ODN 20 was synthesized again following the general ODN
synthesis procedure described earlier on a 0.2 mmol scale except
that the capping time was decreased from 2.0 seconds,
1.5 seconds ꢀ 3 deliveries and 90 seconds waiting aer each
delivery to 2.0 seconds, 1.5 seconds ꢀ 2 deliveries and 30 seconds
waiting aer each delivery (Cycle 2 in ESI†). The synthesized
ODNs are labeled as 20(c10s), 20(c11s), 20(c12s) and 20(c13s).
Synthesis of ODN 20 on a larger scale
The general ODN synthesis procedure was followed except that
a different synthesis cycle was used. To create the new cycle
(Cycle 3 in ESI†), the standard 1 mmol synthesis cycle provided
by the manufacturer of the synthesizer was copied into a new
le. The steps for capping failure sequences with Ac2O were
deleted. Aer the coupling steps, new steps for delivering pol-
ymerizable phosphoramidite 11 (0.15 M acetonitrile solution)
from the 5th phosphoramidite bottle position and 1H-tetrazole
solution from the 9th position (i.e. the same activator for the
coupling steps) were added. These steps were the same as those
in the coupling steps except that the base was from the 5th
bottle, one additional delivery of reagents was added, and aer
each delivery a waiting of 30 seconds was added. More speci-
cally, the solutions of 11 and 1H-tetrazole were delivered to
column for 2.5 seconds ꢀ 3, and aer each delivery, the
reagents remained in the column for 30 seconds. Other modi-
cations of the cycle were the same as detailed in the general
synthesis procedure. The synthesis was performed using this
new cycle at 1 mmol scale following the conditions described in
the general synthesis procedure. The ODN is labeled as
20(c11ls).
Purication of ODN 20 synthesized using shorter capping
time by polymerization of failure sequences
The general ODN purication procedure described earlier was
used. The RP HPLC proles are in ESI.† The recovery yields and
purity are in Table 1.
Diffusion speed of ODN from acrylamide gel and extraction
efficiency
One fourth of the crude ODN 20(c11s) was subjected into the
radical acrylamide polymerization conditions in a 25 mL 2-
necked round-bottomed ask as described in the general ODN
purication procedure. The polymerization solution used in the
procedure had a 1 : 25 cross-linking ratio between the cross-
linker N,N0-methylenebis(acrylamide) and the polymerization
monomer N,N-dimethylacrylamide. Aer complete polymeriza-
tion, the gel was broken into several pieces with a clean spatula.
Water (3 mL) was added to the gel, and the mixture was stirred
gently with magnetic stirring bar. The aliquots of supernatant (5
mL each) were taken out via a pipette in every 10 min until 2 h.
The ODN concentrations in the aliquots were determined using a
UV-Vis spectrophotometer at 260 nm (Table 2). At 2 h, all the
remaining supernatant were taken out and transferred into clean
centrifuge tubes. The ODN concentration in the supernatant was
determined with UV. To the gel was added water (3 mL) again
and the mixture was gently stirred. Aer 2 h, the supernatant was
removed and the ODN concentration was determined with UV.
The extraction and ODN quantication process was performed
for three more times (Table 3). The ODN diffusion and extraction
Purication of 20(c11ls) by polymerization of failure
sequences
The general ODN purication procedure was followed. The 1
mmol ODN was puried in one batch. The volume of the poly-
merization solution was 10 times of that used in 0.05 mmol scale
purication described in the general procedure. Extraction of full-
length sequences from gel was achieved by water (4 mL ꢀ 4).
HPLC proles are in ESI.† Recovery yield and purity are in Table 1.
Synthesis of ODN 20 with DCI as activator
studies were repeated four more times using polymerization ODN 20 was synthesized under the same conditions described
solutions with a different cross-linking ratio. One with a ratio of for the synthesis of 20(c11ls) except for the following modi-
1 : 50 between N,N0-methylenebis(acrylamide) and N,N-dime- cations. DCI (0.25 M in acetonitrile) instead of 1H-tetrazole was
thylacrylamide, and the others 1 : 15, 1 : 7 and 1 : 2. The data used as the activator for coupling and capping. The concen-
were recorded in Tables 2 and 3.
tration of phosphoramidite monomers was lowered from 1.0 M
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to 0.05 M. Capping was performed using a 0.1 M acetonitrile chilled on dry ice again for 10 min. The tube was centrifuged for
solution of 11. The synthesis was performed in three 1 mmol 5 min, and the supernatant was transferred into a new 1.5 mL
columns simultaneously. The ODN is labeled as 20(c11dci).
centrifuge tube. Aer drying in a centrifugal vacuum concen-
trator, the nucleosides were dissolved in water (30 mL). The
sample (20 mL) was analyzed with RP HPLC under the condi-
tions described in the General section except that the gradient
system was changed to: solvent A (100%) for 5 min, solvent B (0–
10%) in solvent A over 30 min, followed by solvent B (10–100%)
in solvent A over 25 min. The resulting prole is shown in Fig. 4
(trace a). The same ODN digestion and RP HPLC analysis
procedure was applied to a control ODN (18 mg) from a
commercial source puried with preparative RP HPLC (trace b),
the mixture ODN 25 (33 mg, trace d), and a blank control (ESI).
The control ODN 24 (9 mg) was synthesized and digested
following known procedure (trace c).20
Purication of 20(c11dci) by polymerization of failure
sequences
The 3 mmol ODN was puried in one batch following the
procedure for the purication of 20(c11ls). The volumes of the
polymerization solution and extraction solutions were
increased proportionally (i.e. 3 times of those for the 1 mmol
20(c11ls) purication). The volumes of the polymerization
initiators and the reagents for ODN precipitation from NH4OH
solution by n-BuOH were not changed. The RP HPLC proles
from crude and puried ODN 20(c11dci) including one that was
generated by UV detection at 210 nm from the puried ODN are
included in ESI.† Recovery yields and purity are in Table 1.
Acknowledgements
Synthesis of mixture ODN 25
Financial support from US NSF (CHE-0647129 and CHE-
1111192), Michigan Universities Commercialization Initiative,
MTU Research Excellence Fund (REF-TC), MTU Chemistry
Department, MTU Biotech Research Center, and The Royal Thai
Government Scholarship (S. F.); the assistance from Mr Dean
W. Seppala (electronics) and Mr Jerry L. Lutz (NMR); and NSF
equipment grants (CHE-9512445 and CH-1048655) are all
gratefully acknowledged.
The general ODN synthesis procedure with a shorter capping
time was followed (Cycle 2 in ESI†). The synthesis was carried
out on a 0.2 mmol scale using polymerizable phosphoramidite
11 for capping failure sequences. To synthesize the mixture
ODN, the solution of Pac-dA-CE, Ac-dC-CE, i-Pr-Pac-dG-CE and
dT-CE in 1 : 1 : 1 : 1 mole ratio was used as the phosphor-
amidite monomers in the coupling steps in all synthesis cycles.
The cycle was executed 20 times to give the 20-mer ODN mixture
25. At the end of synthesis, the DMTr group was removed. The
ODN were cleaved and deprotected with concentrated NH4OH
at rt on synthesizer.
Notes and references
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The general ODN purication procedure was used to remove
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RP HPLC analysis
To a 1.5 mL centrifuge tube, which contained 18 mg ODN 20
puried by catching failure sequences by polymerization, was
added 29 mL master mixture solution containing 16.45 unit SVP
(Worthington Biochemical Corporation), 0.4 unit BAP (TaKaRa
Bio Inc.), and 1ꢀ of BAP buffer. The tube was vortexed and
centrifuged briey to mix thoroughly and to bring the master
mixture to the bottom of the tube. The mixture was then incu-
bated at 37 ꢂC for 24 h. The digested ODN was then prepared for
RP HPLC analysis following reported procedure.19 Briey,
sodium acetate buffer (3 M, 4 mL) and ethanol (100 mL) were
added into the tube. The tube was then vortexed shortly and
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relative centrifugal force, the supernatant was carefully trans-
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added ethanol (300 mL), and the contents were vortexed and
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