Solid-Phase Synthesis of Phosphorothioate Oligonucleotides Using Sulfurization Byproducts for in Situ Capping

Article abstract of DOI:10.1021/acs.joc.8b01553

Oligonucleotides containing phosphorothioate (PS) linkages have recently demonstrated significant clinical utility. PS oligonucleotides are manufactured via a solid-phase chain elongation process in which a four-reaction cycle consisting of detritylation, coupling, sulfurization, and failure sequence capping with Ac₂O is repeated. In the capping step, uncoupled sequences are acetylated at the 5′-OH to stop the chain growth and control the levels of deletion, or (n-1), impurities. Herein, we report that the byproducts of commonly used sulfurization reagents react with the 5′-OH and cap the failure sequences. The standard Ac₂O capping step can therefore be eliminated, and this 3-reaction cycle process affords a higher yield and higher or comparable overall purity compared to the conventional 4-reaction synthesis. This improvement results in reducing the number of reactions from ～80 to ～60 for the synthesis of a typical length 20-mer oligonucleotide. For every kilogram of an oligonucleotide intermediate synthesized, > 500 L of reagents and organic solvents is saved, and the E-factor is decreased to <1500 from ～2000.

Full text of DOI:10.1021/acs.joc.8b01553

Subscriber access provided by University of South Dakota
Article
Solid-Phase Synthesis of Phosphorothioate Oligonucleotides
Using Sulfurization Byproducts for in situ Capping
Jimin Yang, Jessica A Stolee, Hong Jiang, Li Xiao, William F
Kiesman, Firoz D Antia, Yannick A Fillon, Austen Ng, and Xianglin Shi
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b01553 • Publication Date (Web): 04 Sep 2018
Downloaded from http://pubs.acs.org on September 11, 2018
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 25
The Journal of Organic Chemistry
1
2
3
4
5
SolidꢀPhase Synthesis of Phosphorothioate Oligonucleotides Using Sulfurization
6
7
8
Byproducts for in situ Capping
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Jimin Yang, Jessica A. Stolee,† Hong Jiang,† Li Xiao, William F. Kiesman, Firoz D. Antia,
Yannick A. Fillon, Austen Ng, Xianglin Shi*
Antisense Oligonucleotide Development and Manufacturing; †Analytical Development, Biogen,
115 Broadway, Cambridge, MA 02142
Dedicated to Professor Yuqun Chen of Shanghai Institute of Organic Chemistry, Chinese
Academy of Sciences, for her 88th birthday
1
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 2 of 25
1
2
3
4
5
6
Table of Content Graphic
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Abstract: Oligonucleotides containing phosphorothioate (PS) linkages have recently
demonstrated significant clinical utility. PS oligonucleotides are manufactured via a solidꢀphase
chain elongation process in which a fourꢀreaction cycle consisting of detritylation, coupling,
sulfurization, and failure sequence capping with Ac2O is repeated. In the capping step uncoupled
sequences are acetylated at the 5′ꢀOH to stop the chain growth and control the levels of deletion,
or (nꢀ1), impurities. Herein, we report that the byꢀproducts of the commonly used sulfurization
reagents react with the 5′ꢀOH and cap the failure sequences. The standard Ac2O capping step
can therefore be eliminated and this 3ꢀreaction cycle process affords higher yield and higher or
comparable overall purity compared to the conventional 4ꢀreaction synthesis. This improvement
results in reducing the number of reactions from ~80 to ~60 for synthesis of a typical length 20ꢀ
2
ACS Paragon Plus Environment

Page 3 of 25
The Journal of Organic Chemistry
1
2
3
4
5
6
mer oligonucleotide. For every kilogram of an oligonucleotide intermediate synthesized, > 500
L of reagents and organic solvents are saved and the Eꢀfactor is decreased to < 1500 from ~2000.
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Introduction
Therapeutic oligonucleotides, such as antisense oligonucleotides (ASOs)1 and short interfering
RNAs (siRNAs),2 are designed to form doubleꢀstranded structures with target RNAs by Watsonꢀ
Crick complementary base pair interactions. Formation of the complexes can result in either
modulation of RNA splicing or degradation of target mRNAs via pathways involving RNase H
(for ASOs) or RNAꢀInduced Silencing Complex (for siRNAs), leading to disruption of
downstream production of target proteins. By these mechanisms, oligonucleotides potentially
can be used to treat disease caused by dysregulated gene expression.1ꢀ2 Therefore, many
oligonucleotides are being investigated as therapeutic agents in clinical trials for a variety of
indications.1ꢀ3 Some have proven to be useful therapeutic agents. For example, nusinersen, a
spliceꢀswitching ASO, has recently been approved by regulatory agencies to treat patients with
spinal muscular atrophy (SMA).4,5
Scheme 1. Oligonucleotides with PO and PS Backbones
O
Base
O
O
Base
Base
O
O
O
O
P
R
O
P
O
P
R
R
O
O
O
HS
O
O
Base
Base
Base
O
O
O
OH
OH
O
O
P
R
O
P
O
P
R
R
O
O
Base
HS
O
HS
O
Base
Base
O
O
O
O
OH
O
O
R
O
O
R
R
PS backbone
PO and PS mixed backbone
PO backbone
R= H, F, OMe, OCH2CH2OMe (MOE)
3
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 4 of 25
1
2
3
4
5
6
7
8
The ability of an oligonucleotide to form a doubleꢀstranded complex with a specific
region of RNA is largely determined by its base sequence. However, chemical modifications on
the ribose, for example the replacement of 2′−OH with a O(CH2)2OMe (MOE) group, or the
introduction of sulfur into the interꢀnucleotide linkages can have a significant impact on both the
potency and pharmacokinetic properties of the oligonucleotide (Scheme 1).1ꢀ3, 6
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Modification of interꢀnucleotide linkage, such as substitution of one of the nonꢀbridging
oxygen atoms in the phosphodiester (PO) linkage with a sulfur atom to form a phosphorothioate
(PS) linkage, was originally introduced to improve the stability of oligonucleotides against
nucleases and was recently shown to enhance the cellular uptake and trafficking.6ꢀ7
Unsurprisingly, the majority of oligonucleotide drugs in clinical studies contain PS linkages.3, 6ꢀ7
Interestingly, sequenceꢀspecific phosphorothioate insertion has been reported in naturally
occurring systems, such as bacteria genomes, with mechanisms and functions under
investigation.8ꢀ10 Therefore, study of the chemistry and biological functions of this class of
compounds is of significant interest.
PS oligonucleotides are synthesized using a solidꢀphase process similar to that used for
the synthesis of PO oligonucleotides,11ꢀ12 however the oxidation is substituted with a
sulfurization reaction (Scheme 2).13ꢀ14 In the process, the nucleosides are sequentially attached
to a solidꢀsupport resin. Four reactions are employed to attach each nucleotide. Scheme 2 shows
the reactions required to attach the second nucleotide and the following discussion applies to the
addition of every nucleotide. The detritylation reaction removes the dimethoxy trityl (DMT)
protecting group of the solidꢀsupport resin bound nucleotide (1) to generate free 5′ꢀOH in
compound 2. The incoming phosphoramidite 5 couples with 2 to generate the phosphite ester
4
ACS Paragon Plus Environment

Page 5 of 25
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
intermediate 3. The sulfurization converts the phosphite ester 3 into a phosphorothioate 4. The
last step in this process, step 4, is capping of the unreacted 2 with Ac2O. Repeating these four
reactions affords fullꢀlength oligonucleotide products in a variety of lengths.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 2. Schematic of Solidꢀphase Synthesis of PS Oligonucleotides by Conventional Four
Reaction Process
General conditions: 1. Cl2CHCO2H (DCA), PhCH3; 2. Dicyanoimidazole (DCI), CH3CN; 3.
PADS (6), picolineꢀCH3CN; 4. Ac2O, pyridine, CH3CN.
5
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 6 of 25
1
2
3
4
5
6
7
8
9
The capping reaction is used to control deletion impurities. It does not contribute to
chain elongation and would be unnecessary if the coupling reaction was complete. However, in
practice the coupling reactions do not proceed to completion and the capping step is employed to
convert the remaining 5′ꢀOH intermediate 2 into its acetate ester, a failure sequence, which can
be separated from the fullꢀlength oligonucleotide in purification. Without capping, a shorter
oligonucleotide impurity (generally known as (nꢀ1)) is formed and is difficult to separate from
the fullꢀlength product.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
The capping reaction is typically carried out with an excess of Ac2O (> 200 equiv relative
to the failure sequences). While effective in controlling (nꢀ1) impurities, side reactions caused
by the capping step lead to impurities15ꢀ16 and decrease product yield.17 The mechanism for the
yield decrease is still unclear. The impurities identified include nucleobase guanine15 and
adenine15ꢀ16 modified structures. Therefore, skipping the capping step in some cycles to improve
the yield and/or purity of ASOs has been reported in the patent literature.18 However, to our
knowledge, a systematic study examining the effects of eliminating the Ac2O capping step
during PS oligonucleotide manufacturing has not been reported. Herein, we report the results
from our investigation and propose a new failure sequence capping role played by the byproducts
of the sulfurization agents and summarize the environmental impact of the process change.
Results and Discussion
The impact of the capping step on the synthesis of a DMTꢀon 18ꢀmer 2′ꢀMOE ribose
phosphorothioate oligonucleotide 9 (Scheme 3) was examined using NittoPhase HL Unylinker
resin and 5′ꢀDMT phosphoramidites as the starting materials following a typical solidꢀphase
6
ACS Paragon Plus Environment

Page 7 of 25
The Journal of Organic Chemistry
1
2
3
4
Scheme 3. Structures of Oligonucleotide 9-14
5
6
7
8
9
DMTO
5'
3'
G Me
s
T
C
G
T Me
s
A
MeC
MeC
A
G
T
A
T
T
A
T
C
T
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
OH
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
9 Blue for 2' OCH2CH2OMe (MOE)
DMTO
5'
3'
MeC
MeC
MeC
A
MeC
MeC
A
G
A
A
G
T
T
G
T
G
A
A
G
T
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
OH
10
Black for 2' H
DMTO
5'
3'
MeC
MeC
MeC
A
MeC
G G
MeC
s
A
A
U
G
G
A
U
T
G
A
A
U
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
OH
11 Red for 2'-OMe, Black for 2' H
DMTO
5'
3'
G Me
s
MeC
MeC
MeC
A
MeC
G
A
A
G
G
A
C
T
T
G
G
T
G
G
A
A
A
T
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
OH
12
Blue for 2' OCH2CH2OMe (MOE), Black for 2' H
DMTO
5'
3'
MeC
MeC
MeC
A
MeC
MeC
G
A
A
A
U
U
T
A
U
U
G
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
OH
13
Green for cEt modification, Black for 2' H
DMTO
5'
3'
MeC
A
MeC
MeC
G
A
A
G
U
A
U
G
T
G
A
C
A
C
O
G
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
s
OH
14 Purple for 2'-F, Black for 2' H
O
Base
s for PS linkage
O
=
O
Base
H
Me
O
O
O
HS
O
R
O
P
O
13
cEt nucleotide in
O
P
SH
O
NH2
N
NH2
N
O
N
O
N
Base =
NH2
N
H3C
H3C
N
NH
NH
N
NH
NH2
N
O
N
O
N
O
O
N
N
G
N
A
MeC
C
T
U
synthesis protocol.13, 19 The syntheses were carried out under similar conditions, except in the 4ꢀ
reaction cycle synthesis an Ac2O capping step was included; but in the 3ꢀreaction cycle synthesis
the capping step was excluded but the sulfurization reagent was recirculated through the column
(Figure 1). The two most widely used sulfurization reagents in the manufacturing
7
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 8 of 25
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 1. The Reactions to Attach Each Nucleotide
of PS oligonucleotides, phenylacetyl disulfide (6)13,20 and 3ꢀaminoꢀ1,2,4ꢀdithiazoleꢀ5ꢀthione (7)
21 were used, because they afford the products in high yield and purity. After completion of the
syntheses, product was cleaved from the resin and the base protecting groups were deprotected
by ammonolysis. The 5′ DMTꢀon product was analyzed via reversedꢀphase (RP) UHPLCꢀUVꢀ
MS to determine the yield and purity of the product, and the level of (nꢀ1) impurities.
Figure 2 shows the RPꢀUHPLCꢀUV chromatograms of crude DMTꢀon 9 obtained from
syntheses with and without the Ac2O capping step, using 6 as the sulfurization agent. The small
peaks eluting before the main product peaks include the deprotected capped failure sequences.
Identities of the failure sequences were confirmed by high resolution mass spectrometric
(HRMS) analysis. As the figure shows, the two syntheses resulted in crude product with the
same failure sequences and in similar quantity.
8
ACS Paragon Plus Environment

Page 9 of 25
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 2. Stacked RPꢀUHPLC UV Chromatograms of Crude DMTꢀon 9 Synthesized with (Red
trace) and without (Black trace) the Ac2O capping step with 6 as the sulfurization agent.
Figure 3. Mass spectra shows the nꢀ1 impurities of Crude DMTꢀon 9 synthesized with (red) and
without (black) the Ac2O capping step. Full mass spectra are provided in SI (Figure S4). The
letter p indicates that the deletion includes the corresponding nucleotide.
9
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 10 of 25
1
2
3
4
5
6
7
8
9
Overlaid mass spectra in Figure 3 shows impact of removing the capping step on the
level of (nꢀ1) impurities. The three (nꢀ1) impurity peaks represent a sum of fourteen22 impurities
that resulted from omitting one of the four different nucleotide building blocks during the 18
synthesis cycles. For example, the nꢀp(MOE G) peak consists of two different structures with
the same mass. Compared with the parent compound (Scheme 3), one structure lacks 5th and the
other 1st or 2nd of the nucleotide from the 3′ꢀend, since deletion of 1st or 2nd MOE G results in the
same structure. Due to one mass unit difference, the nꢀp(MOE MeC) and nꢀp(MOE T) peaks are
not resolved and their sum is reported.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
These results show that two syntheses result in products with little difference in the levels
of total (nꢀ1) impurities arising from deletion of MOE G and MOE A, although the capping step
was designed to reduce these impurities. The peak for nꢀp(MOE MeC) + p(MOE T) is the sum of
nine different (nꢀ1) species due to deletion of one of the seven MOE T and four MOE MeC
nucleotides. The slight increase (see values in Table 1) in the 3ꢀreaction process might be
attributed to small process variation, including the detritylation step.23 Similar results were
obtained with 7 as the sulfurization agent as summarized in Table 1 (see SI, Figures S5 and S6).
The 3ꢀreaction cycle process also afforded ~5% higher yield and the same or better overall
purity.
Table 1. The DMTꢀon 9 Crude Product Yield, Purity, and nꢀ1 Impurities
Percentage (%)
4-reaction process
3-reaction process
6
6
7
nꢀp(MOE G)
nꢀP(MOE A)
0.49
0.26
0.70
92
0.45
0.31
0.80
92
0.48
0.31
0.74
94
nꢀp(MOE MeC) + p(MOE T)
MS Purity
UV Purity
92
94
95
Yield
71
75
77
10
ACS Paragon Plus Environment

Page 11 of 25
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
Five more DMTꢀon 20ꢀmer PS oligonucleotides 10−14 were synthesized using twenty
different phosphoramidites including those with 2’ꢀMOE24 and 2',4'ꢀconstrained 2'ꢀOꢀethyl25
modifications, common oligonucleotide building blocks (Scheme 3). Compounds 11−14 consist
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
of 2′ꢀ modified and 2′ꢀdeoxy ribose nucleotides, resembling the structures of gapmer
oligonucleotide compounds. Each of the nucleotides was synthesized four times using the same
3ꢀ and 4ꢀreaction synthesis conditions with 6, 7, or 8 as the sulfurization agents, respectively.
RPꢀUHPLCꢀUVꢀMS analysis data showed that 3ꢀreaction and 4ꢀreaction processes
afforded similar results for the failure sequences and (nꢀ1) impurities for oligonucleotide
compounds 10−14 (see SI Figures S7ꢀ16). Representative examples, the overlay of the zoomedꢀ
in mass spectra of the(nꢀ1) region of 10 synthesized by the 3ꢀ and 4ꢀreaction processes using 6,
7, and 8 as the sulfurization reagents, are shown in Figure 4. UV absorbance (or optical density
(OD)) per ꢀmol of compounds 10−14 obtained from 3ꢀreaction process is similar to or better
than that obtained from the 4ꢀreacton process. All these results demonstrate that the
conventional Ac2O capping step adds no value to control of (nꢀ1) impurities in the synthesis of
phosphorothioate oligonucleotides.
11
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 12 of 25
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 4. Overlays of the (nꢀ1) Impurities of 10 Synthesized Using the 3ꢀ and 4ꢀReaction
Process, in solid and dotted traces, respectively, using 6 (purple), 7 (navy blue), and 8 (black).
Mechanistic studies
We suspected that sulfurization reagent byproducts perform in situ capping reaction of
failure sequences (Scheme 4). Hanusek and coꢀworkers in studies of sulfurization mechanisms
Scheme 4. Potential in situ Capping Reactions of Failure Sequences in the Sulfurization Step
found that the byproducts of 7 and 8, thiocarbamoyl isothiocyanate or derived compounds, react
with EtOH efficiently.26, 27 This result led us to question whether a byproduct of 6 could react in
a similar manner with a free 5’ꢀOH. Careful review of data from synthesis of a PO/PS mixed
12
ACS Paragon Plus Environment

Page 13 of 25
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
backbone oligonucleotide (15) (Scheme 5), consisting of 4 PO and 15 PS linkages, lent some
support to the hypothesis that capping occurs in situ in the sulfurization step with 6 as the
sulfurization agent.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 5. Structure of PS/PO Mixed Backbone Oligonucleotide 15
In a 3ꢀreaction synthesis, in situ capping during sulfurization would take place when
installing a PS linkage, but no capping would occur when installing a PO linkage. As a result,
(nꢀ1) species associated with PS linkages would be expected to be similar in the 3ꢀreaction and
4ꢀreaction syntheses, but the (nꢀ1) species associated with PO linkages would be higher in the 3ꢀ
reaction process because no capping species would be present. In accord with these
expectations, the results (Figure 5) show that (nꢀ1) impurities arising from PS linkages are nearly
the same for both 3ꢀ and 4ꢀreaction synthesis processes, however, (nꢀ1) species, nꢀp(MOE A)P=O
,
nꢀp(MOE G)P=O, and nꢀp(MOE MeC)P=O, arising from PO linkages are higher in the 3ꢀreaction
synthesis (see SI Figures 17ꢀ18 for full RPꢀUHPLCꢀUVꢀMS chromatograms and MS spectra).
Multiple runs of synthesis of 15 confirmed this trend. These results are consistent with the high
level of total (nꢀ1) impurities (>10%) obtained from a synthesis of a full PO 20ꢀmer carried out
without the Ac2O capping step (results not shown) and the role of the capping step in full PO
oligonucleotide synthesis.
13
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 14 of 25
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 5. Overlaid mass spectra showing the (nꢀ1) impurities of 15 synthesized by the 3ꢀ and 4ꢀ
reaction processes in black and red. The levels of (nꢀ1) impurities are comparable at PS
linkages, but higher at PO linkages in the 3ꢀreaction process.
From lab scale results, the 3ꢀreaction cycle process on average affords ~9% higher yield
and the same or slightly higher overall purity of 15 than the 4ꢀreaction cycle process. Due to the
yield increase and improvement of overall purity, this 3ꢀreaction cycle process is also attractive
at least for the synthesis of PO/PS mixed backbone oligonucleotides with four or less PO
linkages. Due to reduction of the solvent usage roughly by 25% and increase of the yield, the Eꢀ
factor is improved significantly. For example, the synthesis of 1 kilogram of a typical 20ꢀmer
oligonucleotide drug candidate consumes ~2000 liters of CH3CN and PhCH3 and generates the
same amount of organic waste. This process improvement reduces the Eꢀfactor to ~1300 from
~2000 (Table 2).
Table 2. Yield, Solvent Usage and Waste Generation
4-reaction cycle
3-reaction cycle
~67
~76
Yield of DMTꢀON 15 (%)
Liters of Solvents / kg of
DMTꢀon 15
~2000
~1500
14
ACS Paragon Plus Environment

Page 15 of 25
The Journal of Organic Chemistry
1
2
3
4
5
Liters of Organic waste / kg of
DMTꢀon 15
~2000
~1300
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
To provide further support of the role of PADS byproduct in capping, the waste stream of
the PADS sulfurization step generated during the solidꢀphase synthesis of 15 was collected and
mixed with nꢀBuOH at 22 ºC. The esterification product PhCH2CO2Bu was isolated and its
structure was confirmed by NMR (Scheme 6). This result indicates that a reaction byproduct
with a structure PhCH2COꢀY (the structure of Y is unclear) is generated in PADS sulfurization
and can convert the 5′ꢀOH of failure sequences into phenyl acetate. In addition, PhCH2CONH2,
a product of ammonolysis of capped failure sequence (Scheme 5), was found present in the crude
reaction mixture of 15 by LCꢀMS analysis.
Scheme 6. Esterification of nꢀBuOH with Waste of 6 Sulfurization Reaction
Further evidence was obtained from a model NMR reaction of the sulfurization of
P(OMe)3, an analogue of intermediate (3) in the solidꢀphase synthesis (Scheme 2), in the
presence of nꢀBuOH, a 5′ꢀOH failure sequence analogue (Scheme 7). In this experiment, a
1
solution of 6 and 1ꢀmethylimidazole (NMI, 1.0 equiv) in CD3CN was aged until its H NMR
spectrum showed a similar composition to the aged 6 solution (CH3CN−3ꢀpicoline, 1:1, v/v)
used for solidꢀphase synthesis.13 Then, nꢀBuOH (1.0 equiv) and P(OMe)3 (1.0 equiv) were
15
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 16 of 25
1
2
3
4
5
6
7
8
9
added to the solution and 1H NMR spectra of the reaction mixture were taken at different time
points. The regions of the spectra including the signals of CH3CH2CH2CH2ꢀO of BuOH and
PhCH2CO2Bu are shown in Figure 6 (see SI Figures 19ꢀ22 for expanded spectra and spectra
from other NMR experiments).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 7. In Situ Conversion of nꢀBuOH to PhCH2CO2Bu in 6 Sulfurization Reaction
Figure 6. Partial NMR Spectra of the Reaction Mixture of Aged 6 and P(OMe)3 in the Presence
of nꢀBuOH
The spectra clearly show an increase in the intensity of PhCH2CO2Bu and decrease of nꢀ
BuOH signals over time. PhCH2CO2Bu was isolated and its structure was confirmed by NMR
(SI) from a larger scale reaction carried out under the same conditions. The sulfurization went to
completion in < 2 min as no increase of S=P(OMe)3 signals was observed (results not shown).
16
ACS Paragon Plus Environment

Page 17 of 25
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
The data obtained from this NMR experiment (Table 3) showed ~7% (0.07 equiv) nꢀBuOH was
converted to its phenyl acetate in ~ 2 min and ~ 0.5 equiv of the capping agent was generated
from 1.0 equiv of PADS. In practice about 3−4 equiv of PADS are used in the manufacturing
process; therefore ~ 1.0 equiv of the active capping byproduct is expected to be produced. Based
on the levels of (nꢀ1) impurities observed in the synthesis of 15, < 0.8% (0.008 equiv) of the 5′ꢀ
OH did not couple and needed to be capped. Therefore, the byproduct capping agent is ~ 125
equiv of the failure sequence. These results taken together show the proposed in situ capping
reaction is sufficient to control (nꢀ1) impurities and consistent with the results of synthesis 9−15.
It is expected 7 and 8 behave similarly.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Table 3. Conversion of n-BuOH in the Sulfurization Reaction
Time (min)
nꢀBuOH : PhCH2CO2Bua
2
93 : 7
30
80
180
360
84 : 16
75 : 25
61 : 39
50 : 50
a Ratios were calculated by integration of 1H δ 1.48ꢀ1.55 (m, 2H) for nꢀBuOH and δ 1.57ꢀ1.65
(m, 2H) for PhCH2CO2Bu.
Conclusion
We have demonstrated that solidꢀphase synthesis without Ac2O capping produces PS
oligonucleotides in comparable or higher purity and yield using the most common
oligonucleotide building blocks and sulfurization reagents 6, 7, and 8, due to an in situ capping
reaction performed by the sulfurization byproducts. Our results with 6 specifically show that the
in situ capping reaction is sufficient to control the (nꢀ1) impurities. The same conclusion is made
17
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 18 of 25
1
2
3
4
5
6
for 7 and 8 based on literature mechanistic results and our failure sequence and (nꢀ1) data for
compounds 9−12.
7
8
9
To our knowledge, this is the first work to recognize and demonstrate the capping effect
of the sulfurization byproducts and its application in the synthesis of phosphorothioate
oligonucleotides, which are of significant interest as therapeutic agents. Compared with the
decadeꢀold conventional 4ꢀreaction cycle process, this new manufacturing process provides the
following advantages: a) elimination of roughly 25% reactions in each synthesis, i.e. 19 out of 80
reactions in a synthesis of a typical 20ꢀmer oligonucleotide; b) obviation of the impurities15ꢀ16
and yield loss17 resulting from the Ac2O capping reaction, without compromising control of (nꢀ1)
impurities; c) significant reduction of Eꢀfactor.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Experimental
Oligonucleotide Synthesis. Synthesis of oligonucleotides was carried AKTA 100 solidꢀphase
synthesizer using Primer Support Unylinker (GE Healthcare Life Sciences) or NittoPhase HL
o
Unylinker (Kinovate Life Sciences) as the solid support at ambient temperature (~ 22 C) by
using the 3ꢀ or 4ꢀreaction processes. Columns used for the syntheses were either Fineline 35
oligonucleotide column or customꢀmade stainlessꢀsteel column (Φ 2 x 6.2 cm). The syntheses
were performed at 0.72, 1.1, or 2.2 mmol scales, which was determined by the loading of DMTꢀ
Unylinker on the solid support. The results from different scales are reproducible. All the
equivalents were relative to the DMTꢀUnylinker on the solid support. The 3ꢀreaction synthesis
for attaching each nucleotide consists of detritylation, coupling, and sulfurization. The
detritylation step was carried out using a 10% solution of dichloroacetic acid (~110 equiv for the
cycle 1, deprotection of DMT on Unylinker and ~ 80 equiv for the rest of the cycles) in toluene
18
ACS Paragon Plus Environment

Page 19 of 25
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
as the reagent with a typical reaction time ~ 4 min for cycle 1 and ~3 min for the rest of the
cycles. The coupling step was carried out by simultaneous delivering equal volume of a solution
of corresponding phosphoramidite (~ 1.5 equiv, 0.2 M in acetonitrile except for cEt
phosphoramidites, which were 0.15 M solutions in CH3CN−DMF (1:1, v/v)) and a solution of
4,5ꢀdicyanoimidazole (1.0 M in acetonitrile containing 0.1 M Nꢀmethylimidazole) and then
circulating the mixed solutions through the solid support in the column. The coupling reaction
time (delivery plus circulation) was ~ 5.0 min. For compound 13, the coupling reaction time was
~15 min. The sulfurization step was completed by delivery of one of the following solutions
followed by circulating the solutions through the column (total reaction time 6−20 min): (a) 0.2
M 6 in acetonitrile/3ꢀpicoline (1:1, v/v) (The 6 solution was aged for at least 12 h before use
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
13
according to the literature procedure); (b) 0.2 M 7 in pyridine;21 (c) 0.1 M 8 in pyridine
solution. For the synthesis of compound 15, the oxidation was performed by delivering 2.1
equiv. of I2 solution (0.05 M in pyridineꢀH2O, 9:1, v/v) to the column in 5 min. In the 4ꢀreaction
synthesis, the sulfurization reagents were flown through the synthesis column without circulation
and a capping step was performed after the sulfurization step. The capping step was performed
by delivery of the same amount of a solution of Nꢀmethyl imidazoleꢀpyridineꢀACN (2:3:5, v/v/v)
and a solution of acetic anhydride (~ 8 equiv) in ACN (1:4, v/v) to the solid support. The
reaction time for capping was ~ 1 min. All the other steps were carried out the same way as for
the 3ꢀreaction synthesis. After each reaction, the solid support was washed with acetonitrile.
After the synthesis was completed, the phosphorus protecting group was removed by
circulating a mixture of NEt3 and acetonitrile (1:1, v/v). Cleavage of the oligonucleotides from
the support and base deprotection were performed in ~30% NH4OH at 50−60 °C in sealed
pressure bottles to afford the crude DMTꢀon oligonucleotide products.
19
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 20 of 25
1
2
3
4
5
6
7
8
9
When a reference sample was available, the DMTꢀon product yield was obtained by the
UPLCꢀUVꢀMS analysis and reported. For other compounds that we do not have reference
standards, overlaid mass spectra are provided for MS purity comparison and optical density per
ꢀmol measured at 260 nm (OD260) and UV purity were reported for yield comparison. The
HRMS data of these compounds are also provided.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
UHPLC-UV-MS analysis. Sample preparation: A crude DMTꢀon sample (~100 mg, containing
~3.0 mg of the product in ~15% aqueous NH4OH) was evaporated under vacuum to dryness.
The residue was then dissolved in 0.01% triethylamine water solution (~30 mL). The sample
solution was analyzed by ionꢀpair reversed phase UHPLCꢀUVꢀMS. High resolution mass
spectra were acquired using a Waters Xevo QTof system with Lockꢀspray applied. UHPLC data
were collected with UV detection at 260 nm using Waters Acuity UPLC BEH C18 column (130
Å, 1.7 µm, 2.1 x 50 mm) and mobile phase A: 5 mM tributylamine acetate and 1 µM EDTA in
10% acetonitrile in water and B: 5 mM tributylamine acetate and 1 µM EDTA in 80%
acetonitrile in water; a linear gradient from 45ꢀ80% B in 3.56 min; flow rate 0.51 mL per minute.
The mass spectra were acquired with mass range covering the fourꢀcharged state species. The
UHPLC chromatograms and MS spectra of the product generated from 3ꢀ and 4ꢀreaction
processes were overlaid (normalized to the main product peak(s), unless stated otherwise) to
show the levels of the impurities, particularly (nꢀ1) impurities. The UHPLC chromatograms and
MS spectra of the product generated from 3ꢀ and 4ꢀreaction processes were smoothed (with the
exception of Figure 3) and overlaid (normalized to the main product peak(s), unless stated
otherwise) to show the levels of the impurities, particularly (nꢀ1) impurities.
20
ACS Paragon Plus Environment

Page 21 of 25
The Journal of Organic Chemistry
1
2
3
4
5
6
7
8
9
UV absorbance (optical density, OD) measurement. A crude DMTꢀon sample (1.0
mL, containing ~3.0 mg of product in ~15% aqueous NH4OH) was diluted in a 1000ꢀmL
volumetric flask with deionized water. The sample solution was filled in a quartz UV cell with a
nominal cell path length of 1.0 cm and UV absorbance was measured at 260 nm on a dualꢀbeam
UV spectrometer. Total OD of the crude DMTꢀon sample was calculated by multiplying the
UV absorbance by the dilution factor and total volume of the crude product solution. The values
were reported as OD per ꢀmol as the total oligonucleotide product yield. With the UV and MS
purity information data, comparison of the yield of DMTꢀon product from the 3ꢀ and 4ꢀreaction
cycle processes can be made. For 9 and 15, the yields were determined based on the UV and MS
purity using the corresponding reference standards.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
DMT-on phosphorothioate oligonucleotide 9. Yield 71% (4ꢀreaction cycle synthesis),
75% and 77% (3ꢀreaction cycle process with PADS and ADTT as the sulfurization reagents,
respectively).
DMT-on phosphorothioate oligonucleotide 10. Calcd for C221H273N78O99P19S19
6798.8434, found 6798.8769. OD/ꢁmol 4ꢀreaction cycle synthesis 147 (PADS), 142 (ADTT),
148 (DDTT); 3ꢀreaction cycle 155 (PADS), 147 (ADTT), 155 (DDTT). UV purity 4ꢀreaction
cycle synthesis 83% (PADS), 74% (ADTT), 78% (DDTT); 3ꢀreaction cycle process 78%
(PADS), 79% (ADTT), and 78% (ADTT).
DMT-on phosphorothioate oligonucleotide 11
.
Calcd for C228H287N78O109P19S19
7056.9021, found 7056.9228. OD/ꢁmol 4ꢀreaction cycle synthesis 150 (PADS), 149 (ADTT); 3ꢀ
reaction cycle synthesis 154 (PADS), 156 (ADTT). UV purity 4ꢀreaction cycle synthesis 81%
(PADS) and 78% (ADTT); 3ꢀreaction cycle process 79% (PADS) and 80% (ADTT).
21
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 22 of 25
1
2
3
4
DMT-on phosphorothioate oligonucleotide 12
.
Calcd for C251H333N78O119P19S19
5
6
7
8
7539.2112, found 7539.2916. OD/ꢁmol 4ꢀreaction cycle synthesis 159 (PADS), 150 (ADTT); 3ꢀ
reaction cycle synthesis 159 (PADS), 154 (ADTT). UV purity 4ꢀreaction cycle synthesis 79%
(PADS) and 81% (ADTT); 3ꢀreaction cycle process 79% (PADS) and 82% (ADTT).
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
DMT-on phosphorothioate oligonucleotide 13 with ADTT as the sulfurization agent
.
Calcd for C238H287N78O109P19S19 7176.9021, found 7176.9908. OD/ꢁmol 4ꢀreaction cycle
process 126; 3ꢀreaction cycle 129. UV purity 71% and 73% 4ꢀ and 3ꢀreaction cycle process,
respectively.
DMT-on phosphorothioate oligonucleotide 14. Calcd for C216H253F10N78O99P19S19
6908.6709, found 6908.7060. OD/ꢁmol 4ꢀreaction cycle synthesis 141 (PADS), 133 (ADTT); 3ꢀ
reaction cycle synthesis 141 (PADS), 138 (ADTT). UV purity 4ꢀreaction cycle synthesis 74%
(PADS) and 71% (ADTT); 3ꢀreaction cycle process 76% (PADS) and 73% (ADTT).
DMT-on phosphorothioate oligonucleotide 15 with PADS as the sulfurization agent.
Yield 67% (4ꢀreaction cycle synthesis), 76% (3ꢀreaction cycle process). UV purity for 4ꢀreaction
cycle synthesis 86%; for 3ꢀreaction cycle process 88%. Mass purity ~ 89% (4ꢀreaction cycle
synthesis), ~ 91% (4ꢀreaction cycle synthesis).
NMR Experiments. Esterification reaction of n-BuOH in the reaction mixture of
P(OMe)3 and PADS aged in the presence of 3-picoline (1.0 equiv). Procedure: To a NMR
tube were added phenylacetyl disulfide (PADS, 151.21 mg, 500.0 µmol), 3ꢀpicoline (46.56 mg,
500.0 µmol, 48.65 µL) and acetonitrileꢀd3 (1.00 ml) and the solution was held at 22 ºC for 12
1
hours. H NMR spectrum indicated the majority of PADS still remained, only a small portion
was converted into a new active sulfurization species. After addition of nꢀBuOH (37.06 mg,
22
ACS Paragon Plus Environment

Page 23 of 25
The Journal of Organic Chemistry
1
2
3
1
500.00 µmol, 45.75 µL), H NMR spectrum (T0) was taken. To the NMR tube was then added
P(OMe)3 (62.04 mg, 500.00 µmol, 58.97 µL) and a series of 1H NMR spectra were taken.
Esterification reaction of n-BuOH in the reaction mixture of P(OMe)3 and PADS
aged in the presence of NMI (1.0 equiv). The above NMR experiments were repeated using Nꢀ
methyl imidazole (NMI, 1.0 equiv) as the base for the aging. In the presence of NMI (1.0 equiv)
at 22 ºC in 12 h, the NMR spectrum of the PADS solution was very similar to that of the PADS
aged in acetonitrileꢀ3ꢀpicolne (1:1, v/v) at 22 ºC for 12 h and used for solid phase synthesis.
Therefore, the results from this experiment provided direct evidence of failure sequence capping
in the PADS sulfurization step of solid phase synthesis. Compound PhCH2CO2Bu was isolated
from a reaction mixture obtained under the same conditions as this NMR experiment, but at
larger scale. The 1H and 13C NMR data matched the literature data.
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Supporting Information
Procedures, characterization data: UHPLCꢀUV chromatograms and MS spectra of the DMTꢀon
oligonucleotides 9−15; 1HꢀNMR spectra of sulfurization and capping reaction experiments. This
material is available free of charge via the Internet at http://pubs.acs.org
AUTHOR INFORMATION
Corresponding Author
*Email: xianglin.shi@biogen.com
23
ACS Paragon Plus Environment

The Journal of Organic Chemistry
Page 24 of 25
1
2
3
4
5
6
7
8
9
ACKNOWLEDGMENTS. The authors are grateful to Dr. Daniel C. Capaldi, Dr. Isaiah
Cedillo, Dr. Christopher C. Coffin, Dr. Andrew K. McPherson, Dr. Max N. Moore, and Dr.
Anthony N. Scozzari, Ionis Pharmaceuticals Inc. for interesting and thoughtꢀprovoking
discussions. We also thank Dr. Ruiting Liang, Mr. Bing Guan, Dr. Xiaoye Su, Ms. Amy Moran,
and Mr. Richard Smart for the analysis of the samples; Dr. Boliang Dong and Dr. Jeanꢀ
Christophe Hus for the help with NMR experiments; and especially Dr. Michael Kaufman for his
support to this work.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
References
1.
Chan, J. H. P.; Lim, S.; Wong, W. S. F., ANTISENSE OLIGONUCLEOTIDES: FROM DESIGN TO
THERAPEUTIC APPLICATION. Clinical and Experimental Pharmacology and Physiology 2006, 33 (5ꢀ6), 533ꢀ
540.
2.
Gene Silencing. Mol Ther-Nucl Acids 2015, 4.
3. Khvorova, A.; Watts, J. K., The chemical evolution of oligonucleotide therapies of clinical utility. Nat
Biotech 2017, 35 (3), 238ꢀ248.
Lam, J. K. W.; Chow, M. Y. T.; Zhang, Y.; Leung, S. W. S., siRNA Versus miRNA as Therapeutics for
4.
5.
6.
Wadman, M., Antisense rescues babies from killer disease. Science 2016, 354 (6318), 1359ꢀ1360.
Dolgin, E., Call in the backup. Nat Med 2012, 18 (11), 1602ꢀ1606.
Eckstein, F., Phosphorothioates, Essential Components of Therapeutic Oligonucleotides. Nucleic Acid Ther
2014, 24 (6), 374ꢀ387.
7.
Crooke, S. T.; Wang, S.; Vickers, T. A.; Shen, W.; Liang, X.ꢀh., Cellular uptake and trafficking of
antisense oligonucleotides. Nat Biotech 2017, 35 (3), 230ꢀ237.
8.
Cao, B.; Chen, C.; DeMott, M. S.; Cheng, Q.; Clark, T. A.; Xiong, X.; Zheng, X.; Butty, V.; Levine, S. S.;
Yuan, G.; Boitano, M.; Luong, K.; Song, Y.; Zhou, X.; Deng, Z.; Turner, S. W.; Korlach, J.; You, D.; Wang, L.;
Chen, S.; Dedon, P. C., Genomic mapping of phosphorothioates reveals partial modification of short consensus
sequences. Nature communications 2014, 5, 3951.
9.
Cao, B.; Zheng, X. Q.; Cheng, Q. X.; Yao, F.; Zheng, T.; Babu, I. R.; Zhou, H. C.; Dedon, P.; You, D. L.,
In vitro analysis of phosphorothioate modification of DNA reveals substrate recognition by a multiprotein complex.
Sci Rep-Uk 2015, 5.
10.
Phosphorothioation of DNA in bacteria by dnd genes. Nature chemical biology 2007, 3 (11), 709ꢀ10.
11. Matteucci, M. D.; Caruthers, M. H., Synthesis of Deoxyoligonucleotides on a Polymer Support. J. Am.
Chem. SOC. 1981, 103 (11), 3185ꢀ3191.
12. Sierzchala, A. B.; Dellinger, D. J.; Betley, J. R.; Wyrzykiewicz, T. K.; Yamada, C. M.; Caruthers, M. H.,
Wang, L.; Chen, S.; Xu, T.; Taghizadeh, K.; Wishnok, J. S.; Zhou, X.; You, D.; Deng, Z.; Dedon, P. C.,
Solidꢀphase oligodeoxynucleotide synthesis: a twoꢀstep cycle using peroxy anion deprotection. J Am Chem Soc
2003, 125 (44), 13427ꢀ41.
13.
Krotz, A. H.; Gorman, D.; Mataruse, P.; Foster, C.; Godbout, J. D.; Coffin, C. C.; Scozzari, A. N.,
Phosphorothioate oligonucleotides with low phosphate diester content: Greater than 99.9% sulfurization efficiency
with "aged" solutions of phenylacetyl disulfide (PADS). Org Process Res Dev 2004, 8 (6), 852ꢀ858.
24
ACS Paragon Plus Environment

Page 25 of 25
The Journal of Organic Chemistry
1
2
3
4
5
14.
Scozzari, D. C. C. a. A. N., Manufacturing and Analytical Procvesses for 2'ꢀOꢀ(2ꢀMethoxyethyl)ꢀModified
Oligonucleotides. In Second Edition Antisense Drug Technology, Principles, Strategies, and Applications Second
ed.; Crooke, S. T., Ed. CRC Press: Boca Raton, 2008; pp 401ꢀ434.
6
7
15.
Andrew A. Rodriguez ⇑, I. C., Brendan P. Mowery, Hans J. Gaus, Seetha S. Krishnamoorthy, Andrew K.
McPherson, Formation of the N2ꢀacetylꢀ2,6ꢀdiaminopurine oligonucleotide impurity caused by acetyl capping.
8
9
Bioorganic & Medicinal Chemistry Letters 2014, 24, 3243ꢀ3246.
16.
Rodriguez, A. A.; Cedillo, I.; McPherson, A. K., Conversion of adenine to 5ꢀaminoꢀ4ꢀpyrimidinylimidazole
caused by acetyl capping during solid phase oligonucleotide synthesis. Bioorganic & Medicinal Chemistry Letters
2016, 26 (15), 3468ꢀ3471.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
17.
Rodriguez, A., Problems Associated with Capping during Oligonucleotide Synthesis. TIDES Conference
2014, Rhode Island May 14, 2014
18.
Isaiah E. Cedillo, D. J., Phillip Michael Weaver. Method of preparing oligomeric compounds using
modified capping protocols. US20150218205 A1, 2012.
19.
Ravikumar, V. T.; Kumar, R. K.; Olsen, P.; Moore, M. N.; Carty, R. L.; Andrade, M.; Gorman, D.; Zhu,
X.; Cedillo, I.; Wang, Z.; Mendez, L.; Scozzari, A. N.; Aguirre, G.; Somanathan, R.; Bernees, S., UnyLinker: An
efficient and scaleable synthesis of oligonucleotides utilizing a universal linker molecule: A novel approach to
enhance the purity of drugs. Org Process Res Dev 2008, 12 (3), 399ꢀ410.
20.
Zacharia S. Cheruvallath, R. L. C., Max N. Moore, Daniel C. Capaldi, Achim H. Krotz, Patrick D.
Wheeler,; Brett J. Turney, S. R. C., Hans J. Gaus, Anthony N. Scozzari, Douglas L. Cole, and Vasulinga T.
Ravikumar, Synthesis of Antisense Oligonucleotides: Replacement of 3Hꢀ1,2ꢀBenzodithiolꢀ3ꢀone 1,1ꢀDioxide
(Beaucage Reagent) with Phenylacetyl Disulfide (PADS) As Efficient Sulfurization Reagent: From Bench to Bulk
Manufacture of Active Pharmaceutical Ingredient. Org Process Res Dev 2000, 4, 199ꢀ204.
21.
Tang, J.ꢀY.; Han, Y.; Tang, J. X.; Zhang, Z., LargeꢀScale Synthesis of Oligonucleotide Phosphorothioates
Using 3ꢀAminoꢀ1,2,4ꢀdithiazoleꢀ5ꢀthione as an Efficient SulfurꢀTransfer Reagent. Org Process Res Dev 2000, 4,
194ꢀ198.
22.
By deletion of one nucleotide, compound I can generate fourteen different (nꢀ1) structures. This is because
when consecutive nucleotides are the same, deletion of either produces the same structure of nꢀ1 impurity. For
example, deletion of first and second G at the 3' end (Scheme 3) gives nꢀ1 with the same structure.
23.
detritylated and then coupled with the phosphoramidite in the next cycle.
24. Martin, v. P., 38. Ein neuer Zugang zu 2’ꢀOꢀAlkylribonucleosiden und Eigenschaften deren
Oligonucleotide. HELVETICAH IMICAAC T 1995, 78, 486ꢀ504.
25. Punit P. Seth, G. V., Charles A. Allerson, Andres Berdeja, Hans Gaus, Garth A. Kinberger, Thazha P.
Inclomplete detritylation reaction also leads to nꢀ1 imurity, because the remaining DMT will likely be
Prakash, Michael T. Migawa, Balkrishen Bhat, and Eric E. Swayze, Synthesis and Biophysical Evaluation of 20,40ꢀ
Constrained 2'OꢀMethoxyethyl and 2',4'ꢀConstrained 2'OꢀEthyl Nucleic Acid Analogues. J. Org. Chem. 2010, 75,
1569ꢀ1581.
26.
Hanusek, J.; Russell, M. A.; Laws, A. P.; Jansa, P.; Atherton, J. H.; Fettes, K.; Page, M. I., Mechanism of
the sulfurisation of phosphines and phosphites using 3ꢀaminoꢀ1,2,4ꢀdithiazoleꢀ5ꢀthione (xanthane hydride). Organic
& Biomolecular Chemistry 2007, 5 (3), 478ꢀ484.
27.
Ponomarov, O.; Laws, A. P.; Hanusek, J., 1,2,4ꢀDithiazoleꢀ5ꢀones and 5ꢀthiones as efficient sulfurizing
agents of phosphorus(III) compounds ꢀ a kinetic comparative study. Organic & Biomolecular Chemistry 2012, 10
(44), 8868ꢀ8876.
25
ACS Paragon Plus Environment