Design and Synthesis of New Acid Cleavable Linkers for DNA Sequencing by Synthesis

Article abstract of DOI:10.1080/15257770.2014.945647

A new kind of acid sensitive tetrahydrofuranyl (THF) linker was synthesized and then reacted with 5-(6)-carboxytetramethylrhodaminesuccinimidyl ester (5(6)-TAMRA, SE), followed by di(N-succinimidyl) carbonate (DSC) and modified 2′-deoxyuridine triphosphate (dUTP); the final product, as a reversible terminator for DNA sequencing by synthesis (DNA SBS), was given obtained and confirmed by ¹H-NMR, ³¹P-NMR, and HRMS with purity of up to 99%. The synthesized dye-labeled terminator incorporated into DNA strand successfully, and the fluorophore was cleaved completely under acidic conditions. The preliminary results encourage us to explore more acid-sensitive linkers for DNA SBS to increase the cleavage efficiency under weakly acidic conditions.

Full text of DOI:10.1080/15257770.2014.945647

This article was downloaded by: [McGill University Library]
On: 20 November 2014, At: 02:33
Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered
office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Nucleosides, Nucleotides and Nucleic
Acids
Publication details, including instructions for authors and
subscription information:
http://www.tandfonline.com/loi/lncn20
Design and Synthesis of New Acid
Cleavable Linkers for DNA Sequencing by
Synthesis
Min Jianga, Daonian Tangc, Xiaodong Zhaob, Qing Lic, Yuan Zhuangc,
Xiaofei Weia, Xiaowei Lib, Yazhi Liuc, Xin-Yan Wuc, Zhifeng Shaoab,
Bing Gongde & Yu-Mei Shena
a Shanghai Center for Systems Biomedicine, Key Laboratory
of Systems Biomedicine and Bio-ID Center, Shanghai Jiao Tong
University, Shanghai, China
b School of Biomedical Engineering, Shanghai Jiao Tong University,
Shanghai, China
c Key Laboratory for Advanced Materials and Institute of Fine
Chemicals, East China University of Science and Technology,
Shanghai
d College of Chemistry, Beijing Normal University, Beijing, China
e Department of Chemistry, University at Buffalo, State University of
New York, Buffalo, NY, USA
Published online: 05 Nov 2014.
To cite this article: Min Jiang, Daonian Tang, Xiaodong Zhao, Qing Li, Yuan Zhuang, Xiaofei Wei,
Xiaowei Li, Yazhi Liu, Xin-Yan Wu, Zhifeng Shao, Bing Gong & Yu-Mei Shen (2014) Design and Synthesis
of New Acid Cleavable Linkers for DNA Sequencing by Synthesis, Nucleosides, Nucleotides and Nucleic
Acids, 33:12, 774-785, DOI: 10.1080/15257770.2014.945647
To link to this article: http://dx.doi.org/10.1080/15257770.2014.945647
PLEASE SCROLL DOWN FOR ARTICLE
Taylor & Francis makes every effort to ensure the accuracy of all the information (the
“Content”) contained in the publications on our platform. However, Taylor & Francis,
our agents, and our licensors make no representations or warranties whatsoever as to
the accuracy, completeness, or suitability for any purpose of the Content. Any opinions
and views expressed in this publication are the opinions and views of the authors,
and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content
should not be relied upon and should be independently verified with primary sources
of information. Taylor and Francis shall not be liable for any losses, actions, claims,

proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or
howsoever caused arising directly or indirectly in connection with, in relation to or arising
out of the use of the Content.
This article may be used for research, teaching, and private study purposes. Any
substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing,
systematic supply, or distribution in any form to anyone is expressly forbidden. Terms &
Conditions of access and use can be found at http://www.tandfonline.com/page/terms-
and-conditions

Nucleosides, Nucleotides and Nucleic Acids, 33:774–785, 2014
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2014.945647
DESIGN AND SYNTHESIS OF NEW ACID CLEAVABLE LINKERS
FOR DNA SEQUENCING BY SYNTHESIS
Min Jiang,1 Daonian Tang,3 Xiaodong Zhao,2 Qing Li,3 Yuan Zhuang,3
Xiaofei Wei,1 Xiaowei Li,2 Yazhi Liu,3 Xin-Yan Wu,3 Zhifeng Shao,1,2
Bing Gong,4,5 and Yu-Mei Shen1
1Shanghai Center for Systems Biomedicine, Key Laboratory of Systems Biomedicine and
Bio-ID Center, Shanghai Jiao Tong University, Shanghai, China
2School of Biomedical Engineering, Shanghai Jiao Tong University, Shanghai, China
3Key Laboratory for Advanced Materials and Institute of Fine Chemicals, East China
University of Science and Technology, Shanghai
4College of Chemistry, Beijing Normal University, Beijing, China
5Department of Chemistry, University at Buffalo, State University of New York, Buffalo, NY,
USA
A new kind of acid sensitive tetrahydrofuranyl (THF) linker was synthesized and then reacted
2
with 5-(6)-carboxytetramethylrhodaminesuccinimidyl ester (5(6)-TAMRA, SE), followed by di(N-
succinimidyl) carbonate (DSC) and modified 2ꢁ-deoxyuridine triphosphate (dUTP); the final prod-
uct, as a reversible terminator for DNA sequencing by synthesis (DNA SBS), was given obtained
and confirmed by 1H-NMR, 31P-NMR, and HRMS with purity of up to 99%. The synthesized dye-
labeled terminator incorporated into DNA strand successfully, and the fluorophore was cleaved com-
pletely under acidic conditions. The preliminary results encourage us to explore more acid-sensitive
linkers for DNA SBS to increase the cleavage efficiency under weakly acidic conditions.
Keywords Triphosphate analogues; fluorescent base analogues; synthetic methodology
INTRODUCTION
Haemophilus influenza was the first free-living organism to have its en-
tire genome sequenced in the late 1990s.[1] Since then, the Human Genome
Project (HGP) has cost more than $2 billion and has taken about a decade to
Received 2 December 2014; accepted 12 July 2014.
Address correspondence to Yu-Mei Shen, Shanghai Center for Systems Biomedicine, Key Laboratory
of Systems Biomedicine and Bio-ID Center, Shanghai Jiao Tong University, Shanghai 200240, China; E-
mail: yumeishen@sjtu.edu.cn, and Bing Gong, College of Chemistry, Beijing Normal University, Beijing
100875, China or Department of Chemistry, University at Buffalo, State University of New York, Buffalo,
NY 14260, USA. E-mail: bgong@buffalo.edu
774

New Acid Cleavable Linkers for DNA Sequencing by Synthesis
775
be completed. The workhorse technology behind this project was the Sanger
sequencing method.[2] Developed by Frederick Sanger and colleagues in
1977, it has been the most widely used sequencing method for almost
30 years.[3] This method is based on synthesizing DNA on a single-stranded
template while incorporating chain terminators randomly.[4] Despite the
fact that improvements to this method were made in the 1990s,[5,6] the
difficulty in achieving high-throughput and the complexity involved in the
automation are still the associated challenges.[7]
To address the limitations of current DNA-sequencing techniques, a
series of accurate and high-throughput technologies have been explored
and, in particular, Sequencing by Synthesis (SBS) approaches have been
investigated and implemented.[8] The cleavable linker used to attach a flu-
orophore to 2’-deoxynucleotides plays a determinant role in the success
of SBS. In 2008, G.Turcatti reported a new class of reduction-cleavable re-
versible terminator in which the disulfide linker was explored as a chemically
cleavable moiety.[8] S. Balasubramanian developed an excellent method of
sequencing by synthesis based on fluorescently tagged NTPs for which the
fluorescent dye and the azidomethyl protection were from the 3ꢁ-group
by Staudinger reactions.[8] In addition, photocleavable (PC),[9–13] metal-
assisted cleavable,[14–16] and some other kinds of cleavable linkers[17,18] were
synthesized and served as reversible terminators successfully in DNA SBS. To
our knowledge, acid-cleavable linkers in DNA SBS have never been reported
so far.
2-Tetrahydrofuranyl (THF) ethers and 2-tetrahydropyranyl (THP)
ethers are widely used in organic synthesis as protecting groups because
of their easy installation and stability with most nonacidic reagents.[19,20] In
addition, THF ethers have been shown to be more labile than THP ethers
under acidic conditions.[21] In this study, we present the synthesis of a novel
acid-cleavable reversible terminator dUTP-THF-5(6)TAMRA based on the
THF linker (Figure 1) and its preliminary application in DNA SBS. The
FIGURE 1 Structure of dUTP-THF-5(6)TAMRA.

776
M. Jiang et al.
reversible terminator can be incorporated into a DNA strand faithfully by
DNA polymerase, and the fluorescent dye cleaved completely within 25 min-
utes under acidic conditions.
RESULTS AND DISCUSSION
The synthesis of (5S)-5-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydr-
ofuran-2-ol (5) was carried out in a four-step reaction procedure starting
from l-glutamic acid, according to published procedures.[22–26] The known
compound 5 was made in 22% overall yield as a mixture of diastereoisomers,
and was used for the next step directly. Then, treatment of compound 5 with
excess 2-bromoethanol and catalytic Amberlyst A-15 in dichloromethane un-
der reflux delivered the desired ethers 6 in good yield. The two isomers for
compound 6 were separated by silica gel chromatography column and sub-
jected to1H-1H COSY and NOE study. De-protection of the silyl group, indi-
vidually, by using tetra-butylammoniumfluoride (TBAF) in THF furnished
7, followed by nucleophilic substitution to generate the new cleavable linker
8 (Scheme 1).
To evaluate the cleavability under acidic conditions, compound 9
was synthesized from commercial 5(6)-TAMRA, SE(ꢀ), and the linker 8
(Scheme 2 ).[27]
SCHEME 1 Synthesis of the acid cleavable linker.

New Acid Cleavable Linkers for DNA Sequencing by Synthesis
777
SCHEME 2 Synthesis of model compound 9 and expected mechanism of cleavage.
With the compound 9 (both of trans and cis isomers) in hand, cleavage
tests were performed on a small scale (3 mg) and analyzed by HPLC, where
the UV-visible compounds were detected (Figure 2). After treatment with cit-
ric acid/Na2HPO4 buffer (pH = 2.25) at 45◦C for 20 minutes, both trans and
cis isomers for compounds 9 were cleaved completely into the compound 13,
and this was confirmed by HR-MS. By comparison, the configuration nature
of the compound 9 has little effect on the efficiency of cleavage (Table 1).
Accordingly, we chose trans-((2S,5S)-5-(2-aminoethoxy)tetrahydrofuran-2-
yl)methanol (9-2) as the starting material to evaluate the cleavage of the
dye-labeled THF linker under acidic condition.
Conditions: yiliteSinoChrom ODS-BP C18 column
5
μm,
4.6 mm×250 mm, flow rate 0.6 mL/minute, gradient 0 minute 100%
A, 0% B → 5 minutes 90% A, 10% B→30 minutes 50%A, 50% B→50
minute 0%A, 100% B (A: H2O, B: Methanol).

778
M. Jiang et al.
SCHEME 3 Synthesis of dUTP-THF-5(6)TAMRA.
Trans-((2S,5S)-5-(2-aminoethoxy)tetrahydrofuran-2-yl)methanol (9-2)
was treated with DSC in acetonitrile to furnish the mixed succinimide car-
bonate (10) and used in the next step without further purification. The 5-(3-
amino-1-propynyl)-2ꢁ-deoxyuridine 5ꢁ-triphosphate [dUTP(AP3)] (11) was
prepared from the commercially available 5-iodo-2’-deoxyuridine according
to the Huang method.[28] The last step was conducted in Na2CO3/NaHCO3
buffer (0.1 M, pH 8.7).[29] The dUTP-THF-5(6)TAMRA(12) was purified
with RP-HPLC on a 250 × 9.4 mm C18 column to obtain the pure prod-
uct (retention time = 32 minutes) in yield of 33%. The fluorescent nu-
1
cleotide (12) was characterized by H NMR, ESI-HRMS, and 31P NMR
spectrum.
FIGURE 2 HPLC chromatogram of cleavage experiment and HR-MS of cleavage product. (Color figure
available online).

New Acid Cleavable Linkers for DNA Sequencing by Synthesis
779
TABLE 1 The pyrolysis results at pH 2.25 (45◦C)
Time (min)
Comp 9-1/Cleavage conversion (%)
Comp 9-2/Cleavage conversion (%)
1
3
5
7
17.87
58.10
80.84
90.98
95.63
97.75
98.63
99.27
99.82
13.09
55.91
84.02
94.65
98.02
99.18
99.55
99.78
100
9
11
13
15
20
To use the dUTP-THF-5(6)TAMRA(12) for DNA sequencing, it is critical
that the nucleotide analogue be incorporated faithfully into a DNA strand
during the strand-extension reaction and be cleaved efficiently under op-
timized conditions. To this end, we performed a DNA extension assay by
using 12 as the substrate in solution, which was further subjected to cleav-
age reaction. The reaction products were analyzed by polyacrylamide gel
electrophoresis (Figure 3). As shown in Figure 3, autoradiogram results il-
lustrated that dUTP-THF-5(6)TAMRA(12) is can be incorporated into the
DNA strand (Lane 1). The fluorophore group could be partially cleaved
under pH 1.6 within 5 minutes and completely removed within 25 minutes.
In conclusion, we have successfully developed a novel acid-cleavable
linker for DNA SBS. The dUTP-THF-5(6)TAMRA (12) was synthesized suc-
cessfully based on the novel linker of THF in, overall, 10 steps. In our prelim-
inary experimental effort for DNA sequencing by synthesis, this reversible
terminator was shown to be an excellent substrate for DNA polymerase and
can be incorporated faithfully into DNA strand. Moreover, the DNA exten-
sion product can be completely cleaved at pH 1.6 within 25 minutes whereas
DNA duplexes were harmed seriously in acidic conditions. The results of
this preliminary research promotes the exploration of more acid-sensitive

780
M. Jiang et al.
FIGURE 3 Autoradiogram of DNA incorporation and cleavage (up), GR fluorescent staining of DNA
incorporation and cleavage (down).
linkers for DNA sequencing by synthesis, and further results will be reported
in the future.
EXPERIMENTAL SECTION
(((2S,5R)-5-(2-Bromoethoxy)tetrahydrofuran-2yl)methoxy)
(tert-butyl)dimethylsilane (6-1); (((2S,5S)-5-(2-bromoethoxy)
tetrahydrofuran-2yl)methoxy)(tert-butyl)dimethylsilane (6-2)
To a solution of 5 (232 mg, 1 mmol) in DCM (10 mL) was added
bromoethanol (250 mg, 2 mmol) and Amberlyst-15 (50 mg). The mixture
was stirred at reflux for 2 hours; then, the Amberlyst-15 was filtered and the
solvent was removed under vacuum. The residue was purified by column

New Acid Cleavable Linkers for DNA Sequencing by Synthesis
781
chromatography (eluted with 5% ethyl acetate in petroleum ether) to give
6-1 and 6-2 as a yellow oil.
6-1 (66 mg, 20% yield):1H NMR (CDCl3, 400 MHz): δ 5.13 (d, J =
4.0 Hz, 1H), 4.11–4.14 (m, 1H), 3.92–3.97 (m, 1H), 3.68–3.75 (m, 2H),
3.57–3.61 (m, 1H), 3.44–3.50 (m, 2H), 1.93–2.01 (m, 3H),1.78–1.80 (m,
1H), 0.90 (s, 9H), 0.07 (s, 6H); 13C NMR (CDCl3, 100 MHz): δ 104.36, 81.26,
67.21, 67.19, 32.82, 30.97, 26.24, 25.93, 18.36, −5.25, −5.28; HRMS: calc for
C13H27O3SiBrNa [M+Na]+361.0811, found 361.0836.
6-2(85 mg, 25% yield): 1H NMR (CDCl3, 400 MHz): δ 5.18 (d, J = 4.8 Hz,
1H), 4.16–4.19 (m, 1H), 3.92–3.96 (m, 1H), 3.73–3.76 (m, 1H), 3.61 (d, J =
4.4 Hz, 2H), 3.46–3.50 (m, 2H), 1.89–2.08 (m, 3H), 1.69–1.73 (m, 1H), 0.89
(s, 9H), 0.06 (d, J = 2.0 Hz, 6H); 13C NMR (CDCl3, 100 MHz): δ 104.67,
79.02, 67.29, 65.41, 32.08, 31.10, 25.93, 25.31, 18.36, −5.26, −5.31; HRMS:
calc for C13H27O3SiBrNa [M+Na]+ 361.0811, found 361.0835.
(2S,5R)-5-(2-bromoethoxy)tetrahydrofuran-2-yl)methanol (7-1)
To a solutionof 6-1 (100 mg, 0.3 mmol) in 5 mL of THF was added
TBAF (0.6 mL, 0.6 mmol).The mixture was stirred at room temperature
for 4 hours. Then, the solvent was removed under vacuum and the residue
was purified by column chromatography (eluted with 20% ethyl acetate in
1
petroleum ether) to give 7-1as a colorless oil (60 mg, 88% yield): H NMR
(CDCl3, 400 MHz): δ 5.15 (d, J = 4.4 Hz, 1H), 4.26–4.32 (m, 1H), 3.99–4.02
(m, 1H), 3.74–3.81 (m, 2H), 3.55 (dd, J = 5.2, 12.0 Hz, 1H), 3.49 (t, J =
6.0 Hz, 2H), 1.92–2.07 (m, 4H); 13C NMR (CDCl3, 100 MHz): δ 104.74, 81.55,
67.93, 65.61, 33.25, 30.79, 24.33; HRMS: calc for C7H13BrO3Na [M+Na]+
246.9946, found 246.9938.
(2S,5S)-5-(2-bromoethoxy)tetrahydrofuran-2-yl)methanol (7-2)
The procedure is same as 7-1 to yield 7-2 as a colorless oil (60 mg, 83%
yield): 1H NMR (CDCl3, 400 MHz): δ 5.19 (d, J = 4.8 Hz, 1H), 4.20–4.26 (m,
1H), 3.93–3.96 (m, 1H), 3.69–3.76 (m, 2H), 3.44–3.52 (m, 3H), 1.95–2.06
(m, 3H), 1.64–1.68 (m, 1H); 13C NMR (CDCl3, 100 MHz): δ 104.59, 78.62,
67.34, 64.78, 32.41, 30.94, 24.87; HRMS: calc for C7H13BrO3Na[M+Na]+
246.9946, found 246.9929.
(2S,5R)-5-(2-aminoethoxy)tetrahydrofuran-2-yl)methanol (8-1)
To a solution of 7-1 (136 mg, 0.6 mmol) was added ammonium hydroxide
(2 mL). The mixture was stirred at 40◦C for 6 hours, then the solvent was
removed under vacuum to give 8-1 as a colorless oil (90 mg, 93%). The
1
product was used for the next step without further purification: H NMR
(CDCl3, 400 MHz): δ 5.15 (d, J = 4.0 Hz, 1H), 4.22–4.23 (m, 1H), 3.97–4.02
(m, 2H), 3.89 (dd, J = 2.4, 12.0 Hz, 1H), 3.67 (dd, J = 4.8, 12.0 Hz, 1H),

782
M. Jiang et al.
3.38–3.44 (m, 1H), 3.19–3.25 (m, 1H), 1.97–2.05 (m, 3H), 1.81–1.87 (m,
1H); 13C NMR (CD3OD, 100 MHz): δ 105.02, 81.38, 64.01, 63.49, 39.78,
32.68, 24.20; HRMS: calc for C7H16NO3 [M+H]+ 162.1130, found 162.1128.
(2S,5S)-5-(2-aminoethoxy)tetrahydrofuran-2-yl)methanol (8-2)
The procedure is as same as that of 8-1, and finally yielded compound
1
8-2 as a colorless oil (94 mg, 97% yield): H NMR (CD3OD, 400 MHz): δ
5.20–5.22 (m, 1H), 4.17–4.23 (m, 1H), 3.87–3.95 (m, 1H), 3.64–3.78 (m, 1H),
3.56–3.61 (m, 1H), 3.47–3.53 (m, 1H), 3.15 (t, J = 4.8 Hz, 1H), 1.91–2.10 (m,
3H), 1.64–1.70 (m, 1H); 13C NMR (CD3OD, 100 MHz): δ 104.72, 79.11, 63.87,
62.95, 39.53, 31.54, 24.86; HRMS: calc for C7H16NO3 [M+H]+ 162.1130,
found 162.1135.
2-(6-(dimethylamino)-3-(dimethyliminio)-3H-xanthen-9-yl)-4-((2-
(((2R,5S)-5-(hydroxymethyl)tetrahydrofuran-2-yl)oxy)ethyl)
carbamoyl)benzoate (9-1)
To a solution of 8-1 (15 mg, 0.019 mmol) in DMF (0.5 mL) was added
5(6)-TAMRA, SE (10 mg, 0.019 mmol) in DMF (0.5 mL) and Et3N (40μL,
0.285 mmol). The mixture was protected from light and stirred at room
temperature for 3 hours. Then, the solvent was removed under vacuum
and the residue was purified by TLC (eluted with 10% dichloromethane
1
in methanol) to give 9-1 as a red solid (10 mg, 96%): H NMR (CD3OD,
400 MHz): δ 8.13 (d, J = 8.0 Hz, 1H), 8.08 (dd, J = 1.6,8.0 Hz, 1H), 7.73 (d,
J = 1.6 Hz, 1H), 7.25 (dd, J = 1.6, 9.6 Hz, 2H), 6.99 (dd, J = 2.0, 9.2 Hz,
2H), 6.89 (d, J = 2.4 Hz, 2H), 5.10 (d, J = 1.6 Hz, 1H), 4.07–4.11 (m, 1H),
3.78–3.85 (m, 1H), 3.46–3.57 (m, 5H), 3.26 (s, 12H), 1.87–1.95 (m, 3H),
1.68–1.76 (m, 1H); HRMS: calc for C32H36N3O7 [M+H]+ 574.2553, found
574.2531; calc for C32H35N3O7Na[M+Na]+ 596.2373, found 596.2340.
2-(6-(dimethylamino)-3-(dimethyliminio)-3H-xanthen-9-yl)-4-((2-
(((2S,5S)-5-(hydroxymethyl)tetrahydrofuran-2-yl)oxy)ethyl)
carbamoyl)benzoate (9-2)
The procedure is the as same as that of 9-1 to yield 9-2 as a red solid
(8 mg, 73% yield):1H NMR (CD3OD, 300 MHz): δ 8.06–8.16 (m, 2H), 7.70
(s, 1H), 7.26 (d, J = 9.6 Hz, 2H), 7.00 (dd, J = 2.1,9.6 Hz, 2H), 6.92 (d, J
= 2.1 Hz, 2H), 5.16–5.17 (m, 1H), 4.07–4.11 (m, 1H), 3.76–3.84 (m, 1H),
3.40–3.65 (m, 5H), 3.28 (s, 12H), 1.83–2.02 (m, 3H), 1.60–1.61(m, 1H);
HRMS: calc for C32H36N3O7 [M+H]+ 574.2553, found 574.2534; calc for
C32H35N3O7 Na [M+Na]+ 596.2373, found 596.2363.

New Acid Cleavable Linkers for DNA Sequencing by Synthesis
783
2-(6-(dimethylamino)-3-(dimethyliminio)-3H-xanthen-9-yl)-4-((2-
(((2S,5S)-5-(((((2,5-dioxopyrrolidin-1-yl)oxy)carbonyl)oxy)methyl)
tetrahydrofuran-2-yl)oxy)ethyl)carbamoyl)benzoate (10)
9-2 (6.6 mg, 0.012 mmol) and Et3N (15 μL, 0.096 mmol) were dis-
solved in CH3CN (anhy, 0.5 mL), and N,Nꢁ-disuccinimidyl carbonate (12 mg,
0.048 mmol) was added. After the reaction mixture was stirred for 4 hours at
room temperature, the solution was used directly for the next step without
purification.
dUTP-THF-5(6)TAMRA (12)
A solution of dUTP(AP3) (11) (11 mg, 0.012 mmol) in 0.1 M sodium
bicarbonate buffer (pH 9.16, 0.5 mL) was added to the solution of crude
10. The resulting reaction mixture was protected from light and periodically
vortexed for 2 hours. After lyophilization, the resulting residue was dissolved
in TEAA buffer (20 mM, pH 7.0, 1.0 mL) and purified with RP-HPLC on
a 250×9.4 mm C18 column to obtain the product (retention time = 32
minutes) as a triethylammonium salt. The product was then precipitated
from NaCl/ethanol to remove significant amounts of TEAA salts to give
sodium salt of 12 as a red solid (5 mg, 33%). Mobile phase: A, 20 mm TEAA
in water; B, methanol. Elution was performed with 100% A isocratic over 10
minutes, followed by a linear gradient of 0–50% Bfor 20 minutes and then
50% B isocratic over another 20 minutes at a flow rate of 4 mL/minute:
1HNMR (D2O, 400 MHz): δ8.32 (s, 1H), 7.71 (s, 1H), 7.62 (d, J = 8.0 Hz, 1
H), 7.25 (d, J = 9.6 Hz,2H), 6.93–7.05 (m, 3 H), 6.71 (s, 2H), 5.68–5.72 (m,
1 H),5.28 (d, J = 8.8 Hz,1H),4.47 (s, 1 H), 3.88–4.19 (m, 7 H), 3.68–3.85
(m, 4 H), 3.55–3.63 (m, 1 H), 3.25 (d, J = 8.8 Hz, 12 H), 2.23–2.34 (m, 1 H),
2.02–2.18 (m, 3 H), 1.89–1.96 (m, 1 H), 1.51–1.61 (m, 1H); 31P NMR (D2O,
162 MHz): −18.95, −10.37, −5.20; HRMS: calc for C45H50N6O22P3 [M-H]−
1119.2191, found 1119.2238.
FUNDING
We acknowledge the financial support provided by the National Natural
Science Foundation of China (81071250 and 91227109), Major Projects
in National Science and Technology, “Creation of Major New Drugs”
(2011ZX09501-001-05), and the US National Science Foundation (CHE-
1306326).
SUPPORTING INFORMATION AVAILABLE
Spectral data for 6-12(1H, 13C, and 31P NMR) and general experimental
procedures are available, free of charge, online.

784
M. Jiang et al.
REFERENCES
1. Hall, N. Advanced sequencing technologies and their wider impact in microbiology. J. Exp. Biol.
2007, 210(9), 1518–1525.
2. Sanger, F.; Nicklen, S.; Coulson, A.R. DNA sequencing with chain-terminating inhibitors. P. Natl.
Acad. Sci. USA 1977, 74(12), 5463–5467.
3. Schuster, S.C. Next-generation sequencing transforms today’s biology. Nature 2008, 200(8), 16–18.
4. Sanger, F.; Air, G.M.; Barrell, B.G.; Brown, N.L.; Coulson, A.R.; Fiddes, J.C.; Hutchison, C.A.;
Slocombe, P.M.; Smith, M. Nucleotide sequence of bacteriophage [phi]X174 DNA. Nature 1977,
265(5596), 687–695.
5. Prober, J.; Trainor, G.; Dam, R.; Hobbs, F.; Robertson, C.; Zagursky, R.; Cocuzza, A.; Jensen, M.;
Baumeister, K. A system for rapid DNA sequencing with fluorescent chain-terminating dideoxynu-
cleotides. Science 1987, 238(4825), 336–341.
6. Smith, L.M.; Sanders, J.Z.; Kaiser, R.J.; Hughes, P.; Dodd, C.; Connell, C.R.; Heiner, C.; Kent, S.B.;
Hood, L.E. Fluorescence detection in automated DNA sequence analysis. Nature 1986, 321, 674–679.
7. Shendure, J.; Ji, H. Next-generation DNA sequencing. Nat. Biotech. 2008, 26(10), 1135–1145.
8. Turcatti, G.; Romieu, A.; Fedurco, M.; Tairi, A.-P. A new class of cleavable fluorescent nucleotides:
synthesis and optimization as reversible terminators for DNA sequencing by synthesis. Nucleic. Acids
Res. 2008, 36(4), e25.
9. Li, Z.; Bai, X.; Ruparel, H.; Kim, S.; Turro, N.J.; Ju, J. A photocleavable fluorescent nucleotide for
DNA sequencing and analysis. P. Natl. Acad. Sci. USA 2003, 100(2), 414–419.
10. Seo, T.S.; Bai, X.; Kim, D.H.; Meng, Q.; Shi, S.; Ruparel, H.; Li, Z.; Turro, N.J.; Ju, J. Four-color DNA
sequencing by synthesis on a chip using photocleavable fluorescent nucleotides. P. Natl. Acad. Sci.
USA 2005, 102(17), 5926–5931.
11. Meng, Q.; Kim, D.H.; Bai, X.; Bi, L.; Turro, N.J.; Ju, J. Design and synthesis of a photocleavable fluo-
rescent nucleotide 3’-O-allyl-dGTP-PC-Bodipy-FL-510 as a reversible terminator for DNA sequencing
by synthesis. J. Org. Chem. 2006, 71(8), 3248–3252.
12. Guo, J.; Yu, L.; Turro, N.J.; Ju, J. An integrated system for DNA sequencing by synthesis using novel
nucleotide analogues. Accounts Chem. Res. 2010, 43(4), 551–563.
13. Seo, T.S.; Bai, X.; Ruparel, H.; Li, Z.; Turro, N.J.; Ju, J. Photocleavable fluorescent nucleotides for
DNA sequencing on a chip constructed by site-specific coupling chemistry. P. Natl. Acad. Sci. USA
2004, 101(15), 5488–5493.
14. Bi, L.; Kim, D.H.; Ju, J. Design and synthesis of a chemically cleavable fluorescent nucleotide, 3’-
O-allyl-dGTP-allyl-bodipy-FL-510, as a reversible terminator for DNA sequencing by synthesis. J. Am.
Chem. Soc. 2006, 128(8), 2542–2543.
15. Ju, J.; Kim, D.H.; Bi, L.; Meng, Q.; Bai, X.; Li, Z.; Li, X.; Marma, M.S.; Shi, S.; Wu, J. Four-color DNA
sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators. P. Natl. Acad.
Sci. USA 2006, 103(52), 19635–19640.
16. Ju, J.; Kim, D.H.; Bi, L.; Meng, Q.; Li, X. Four-color DNA sequencing by synthesis using cleavable
fluorescent nucleotide reversible terminators. US7883869 B2, 2011.
17. Knapp, D.C.; D’Onofrio, J.; Engels, J.W. Fluorescent labeling of (oligo) nucleotides by a new fluoride
cleavable linker capable of versatile attachment modes. Bioconjugate Chem. 2010, 21(6), 1043–1055.
18. Knapp, D.C.; Serva, S.; D’Onofrio, J.; Keller, A.; Lubys, A.; Kurg, A.; Remm, M.; Engels, J.W. Fluoride-
cleavable, fluorescently labelled reversible terminators: synthesis and use in primer extension. Chem.-
Eur. J. 2011, 17(10), 2903–2915.
19. Greene, T.W.; Wuts, P.G. The Role of Protective Groups in Organic Synthesis. Protective Groups in
Organic Synthesis, Third Edition 2007, 1–16.
20. Eliel, E.L.; Nowak, B.E.; Daignault, R.A.; Badding, V.G. Reductions with metal hydrides. XIV. reduc-
tion of 2-tetrahydropyranyl and 2-tetrahydrofuranyl ethers. J. Org. Chem. 1965, 30(7), 2441–2447.
21. Kruse, C.G.; Poels, E.K.; Jonkers, F.L.; Van der Gen, A. Synthetic applications of cyclic .alpha.-chloro
ethers and thioethers. 4. Use of (thio)acetal esters as reagents for the protection of alcohols. Synthesis
of 2-tetrahydrothienyl ethers. J. Org. Chem. 1978, 43(18), 3548–3553.
22. Cai, X.; Chorghade, M.S.; Fura, A.; Grewal, G.S.; Jauregui, K.A.; Lounsbury, H.A.; Scannell, R.T.;
Yeh, C.G.; Young, M.A.; Yu, S. Synthesis of CMI-977, a potent 5-lipoxygenase inhibitor. Org. Process
Res. Dev. 1999, 3(1), 73–76.

New Acid Cleavable Linkers for DNA Sequencing by Synthesis
785
23. Pilli, R.A.; Victor, M.M.; de Meijere, A. First total synthesis of aspinolide B, a new pentaketide
produced by aspergillus o chraceus. J. Org. Chem. 2000, 65(19), 5910–5916.
24. Shiro, Y.; Kato, K.; Fujii, M.; Ida, Y.; Akita, H. First synthesis of polyoxin M. Tetrahedron 2006, 62(37),
8687–8695.
25. Hamamoto, H.; Suzuki, Y.; Takahashi, H.; Ikegami, S. A new solid-phase reaction system utilizing
a temperature-responsive catalyst: oxidative cyclization with hydrogen peroxide. Adv. Synth. Catal
2007, 349(17–18), 2685–2689.
26. Montagnat, O.D.; Lessene, G.; Hughes, A.B. Synthesis of azide-alkyne fragments for “Click” chemical
applications. Part 2. Formation of oligomers from orthogonally protected chiral trialkylsilylhomo-
propargyl azides and homopropargyl alcohols. J. Org. Chem. 2009, 75(2), 390–398.
27. Esser-Kahn, A.P.; Trang, V.; Francis, M.B. Incorporation of antifreeze proteins into polymer coatings
using site-selective bioconjugation. J. Am. Chem. Soc. 2010, 132(38), 13264–13269.
28. Caton-Williams, J.; Smith, M.; Carrasco, N.; Huang, Z. Protection-free one-pot synthesis of 2ꢁ-
deoxynucleoside 5ꢁ-triphosphates and DNA polymerization. Org. Lett. 2011, 13(16), 4156–4159.
29. Ludwig, J.; Eckstein, F. Rapid and efficient synthesis of nucleoside 5ꢁ-0-(1-thiotriphosphates), 5ꢁ-
triphosphates and 2ꢁ,3ꢁ-cyclophosphorothioates using 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-
one. J. Org. Chem. 1989, 54(3), 631–635.