Further optimization of detritylation in solid-phase oligodeoxyribonucleotide synthesis

Article abstract of DOI:10.1080/15257770.2010.537291

Various conditions for optimum detritylation (i.e., the removal of 5′-O-trityl protecting groups) during solid-phase synthesis of oligodeoxyribonucleotides were investigated. Di- and tri-chloroacetic acids of variable concentrations were used to study the

Full text of DOI:10.1080/15257770.2010.537291

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Further Optimization of Detritylation in
Solid-Phase Oligodeoxyribonucleotide
Synthesis
Kha Tram a , Yogesh S. Sanghvi b & Hongbin Yan a
a Department of Chemistry , Brock University , St. Catharines,
Ontario, Canada
b Rasayan Inc. , Encinitas, California, USA
Published online: 22 Jan 2011.
To cite this article: Kha Tram , Yogesh S. Sanghvi & Hongbin Yan (2011) Further Optimization of
Detritylation in Solid-Phase Oligodeoxyribonucleotide Synthesis, Nucleosides, Nucleotides and Nucleic
Acids, 30:1, 12-19, DOI: 10.1080/15257770.2010.537291
To link to this article: http://dx.doi.org/10.1080/15257770.2010.537291
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Nucleosides, Nucleotides and Nucleic Acids, 30:12–19, 2011
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2010.537291
FURTHER OPTIMIZATION OF DETRITYLATION IN SOLID-PHASE
OLIGODEOXYRIBONUCLEOTIDE SYNTHESIS
Kha Tram,1 Yogesh S. Sanghvi,2 and Hongbin Yan1
1Department of Chemistry, Brock University, St. Catharines, Ontario, Canada
2Rasayan Inc., Encinitas, California, USA
Various conditions for optimum detritylation (i.e., the removal of 5ꢁ-O-trityl protecting groups)
2
during solid-phase synthesis of oligodeoxyribonucleotides were investigated. Di- and tri-chloroacetic
acids of variable concentrations were used to study the removal of the 4,4ꢁ-dimethoxytrityl (DMTr)
group. It was found that the DMTr group could be completely removed under much milder acidic
conditions than what are currently used for automated solid-phase synthesis. The 2,7-dimethylpixyl
(DMPx) is proposed as an alternative and more readily removable group for the protection of the 5ꢁ-
OH functions both in solid- and solution-phase synthesis. The improved detritylation conditions are
expected to minimize the waste and offer a protocol for incorporation of acid sensitive building-blocks
in oligonucleotides.
Keywords Detritylation; DMTr; pixyl; DMPx; solid-phase synthesis; oligonucleotides
INTRODUCTION
Since its introduction, 4,4ꢁ-dimethoxytrityl (DMTr 1; Figure 1) group[1]
has been used widely for the protection of 5ꢁ-OH functions in oligonu-
cleotide synthesis.[2] Its removal is conveniently effected by treatment with
a solution of di- and tri-chloroacetic acids. One possible side reaction that
could arise during detritylation is depurination.[3] This is of particular con-
cern when the sequence to be assembled contains nucleosides that are par-
ticularly prone to depurination, such as N 6-benzoyl-2ꢁ-deoxyadenosine and
to a lesser degree N 2-isobutyryl-2ꢁ-deoxyguanosine. Of particular note, some
modified nucleosides such as 8-aryl derivatives of 2ꢁ-deoxyguanosine are
known to be extremely prone to depurination under acidic conditions.[4]
Some of these modified nucleosides undergo depurination under acidic
Received 30 September 2010; accepted 1 November 2010.
The authors wish to thank the Natural Sciences and Engineering Research Council of Canada and
the Research Corporation for funding this work. The authors also thank Professor Colin B. Reese for
helpful discussions.
Address correspondence to Hongbin Yan, Department of Chemistry, Brock University, St.
Catharines, ON, L2S 3A1, Canada. E-mail: tyan@brocku.ca
12

Detritylation in Solid-Phase Oligonucleotide Synthesis
13
Ph
Ph OH
Ph
R
R
R
R
MeO
OMe
O
O
2a; R = H (Pixyl or Px)
b; R = Me (DMPx)
1 (DMTr)
3a; R = H
b; R = Me
FIGURE 1 DMTr and pixyl protecting groups and their precursors.
conditions up to 200 times more readily than deoxyriboguanosine, leading
to particular challenges in the chemical synthesis of oligonucleotides con-
taining these modified nucleosides.[5] These abasic sites, once formed, are
prone to chain cleavages under basic conditions, such as ammonolysis that
is used to deprotect the oligonucleotides. It is clearly of importance to en-
sure that complete detritylation does not lead to depurination during acidic
deprotection. Indeed, milder detritylation conditions, that is, use of weaker
acids in detritylation and shorter acid contact times are very likely to be
beneficial in the synthesis of such sequences. In this regard, dichloroacetic
acid (DCA; pKa = 1.5) is to be preferred over trichloroacetic acid (TCA;
pKa = 0.7).[6] In other cases, cation scavengers such as pyrrole,[7] alcohols,[8]
silanes, anisole, thioanisole, benzyl mercaptan, and ethane-1,2-diol,[9] have
been found to be useful in facilitating complete detritylation. It would be
desirable to establish the “minimal” acid strength and acid contact time that
are essential for complete detritylation during solid-phase oligonucleotide
synthesis. We previously reported that when 3% TCA was used in the detrity-
lation step, acid delivery time as short as 10 seconds (versus 110 seconds)
did not compromise the yield of full-length product significantly.[10] We now
report the effects of acid concentration and contact time on oligonucleotide
quality during solid-phase synthesis.
RESULTS AND DISCUSSION
A model study involving thymidine decamer (T10) was first undertaken.
This oligothymidylic acid model was chosen because thymidine has been
shown to be the most difficult nucleoside to detritylate compared to dC, dA
and dG.[11] Syntheses of the T10-mers on a 1.0 µmol scale were carried out
˚
on controlled pore glass (LCCA-CPG: 500 A, 33 µmol/g) with an ABI 3400
DNA synthesizer using the ABI protocol for 1 µmol DNA (see Table 1 for
cycle conditions). The pressure on the TCA bottle and the flow rate of acid
to the column were the same as the values that the instrument manufac-
turer recommends. Phosphoramidites were prepared as 0.1 M solutions in
anhydrous acetonitrile.
A solution of 3% TCA in dichloromethane (DCM) was used initially.
Acid delivery time was varied at 110, 70, 60, 50, 30, and 20 seconds. The T10

14
K. Tram et al.
TABLE 1 Solid-phase synthesis cycle conditions
Step
Condition
Detritylation
Washing
Coupling
Capping
Oxidation
Washing
Trityl
as appropriate
3 × 12 seconds with reverse flush (7 seconds) in between
20 seconds, 5-EtS-tetrazole (0.25 M solution in acetonitrile) as activator
12 seconds delivery followed by 6 seconds wait time
0.02 M I2 in THF/Pyridine/H2O
2 × 20 seconds with reverse flush (15 seconds) and block flush (3 seconds) in between
Off
products were cleaved and unblocked by treatment with aqueous ammonium
hydroxide for 16 hours at 55◦C. After lyophilization, the fully-unblocked
T10–mers were analyzed by anion-exchange chromatography on a DNAPac
PA-100 analytical column (4 × 250 mm) with a Dionex 3000 IC system. As can
be seen from Table 2, shortened detritylation time (20 seconds versus 110
seconds) with TCA did not lower the overall yield. However, when a weaker
acid solution, that is, DCA, was used for detritylation, marked differences
TABLE 2 Overall yields of full-length sequences as determined by anion-exchange HPLC
5ꢁ-O-protecting group: DMTr
Yields of full-length sequencec
DMPx
T10
d
Acid Acid concentration (%)a Detritylation time (s)b T10
Mixed 21-mer sequencee,f
TCA
DCA
3.0
3.0
20
30
110
20
30
40
50
60
110
30
40
50
60
70
30
50
110
88
–
87
86
87
–
–
–
86
86
90
–
–
84
87
88
–
88
73
82
87
92
92
89
62
77
81
89
90
1
–
86
88
89
89
88
–
1.5
0.5
incomplete
–
–
86
–
–
–
–
49
89
–
–
4
–
aAcid in dry dichloromethane, w/v for TCA and v/v for DCA.
bContinuous delivery to column.
cYields were determined by anion-exchange HPLC.
dESI-MS observed for T10-mer [M-H]−: 2978.7, C100H130N20O68P9− requires 2978.96.
eMixed 21-mer sequence: d(GCGTTTGCTCTTCTTCTTGCG).
fESI-MS observed for the 21-mer [M-H]−: 6360.0, C204H262N63O134P20− requires 6360.08.
–: did not perform the experiment.

Detritylation in Solid-Phase Oligonucleotide Synthesis
15
1 3% DCA, 110 s
3% DCA, 40 s
3% DCA, 20 s
2
3
mAU
WVL:260 nm
3
2
1
min
3.50
0.00
0.50
1.00
1.50
2.00
2.50
3.00
Retention Time [min]
FIGURE 2 Anion-exchange HPLC profiles of T10-mers. 1) Use of 3% DCA solution during detritylation
with 110 seconds continuous acid delivery furnished an 89% yield of full-length T10-mer; 2) use of 3%
DCA solution during detritylation with 40 seconds continuous acid delivery furnished an 87% yield of
full-length T10-mer; 3) use of 3% DCA solution during detritylation with 20 seconds continuous acid
delivery provided a 73% yield of full-length T10-mer. Eluent A: 0.25 M Tris (constant at 10%), pH 8.0;
eluent B: water; eluent C: NaCl (1.0 M). Gradient: convex gradient (curve 5) of 10% C to 55% in 20
minutes. Flow rate: 1.5 ml/min.
were observed (as shown in Figure 2). Thus, the times required for complete
detritylation when 3.0 and 1.5% DCA were used were 50 and 60 seconds,
respectively, and the use of 0.5% DCA did not lead to complete detritylation
even after 110 seconds continuous acid delivery (Table 2).
Subsequently, a similar comparison was carried out with a 21-mer
oligodeoxyribonucleotide mixed sequence [d(GCGTTTGCTCTTCTTC
TTGCG)]. As can be seen from Table 2 and Figure 3, about 86% yields
of full-length 21-mer product (which corresponds to an average stepwise
yield of about 99.2%) were achievable when milder detritylation conditions
were used.
From the comparisons shown above, it becomes apparent that if 3% TCA
solution is used during detritylation, DMTr can be removed in as little as 20
seconds, which is close to the time required for the acid to reach the column
in an ABI 3400 DNA synthesizer. Thus, use of a more acid-labile 5ꢁ-protecting
group is not necessary. However, if a weaker acid, for example, DCA, is used

16

Detritylation in Solid-Phase Oligonucleotide Synthesis
17
in detritylation, a more acid-labile 5ꢁ-protecting group would be desirable
in that it would further reduce the overall detritylation cycle time. For this
reason, the use of the 5ꢁ-O-2,7-dimethylpixyl (DMPx) protecting group 2b
(Figure 1) was investigated.
As a pixyl (Px 2a, Figure 1)[12] analogue,[13] DMPx (Figure 1)[14] was
shown to be more acid-labile than the DMTr protecting group. Furthermore,
the precursor reagent DMPx-OH 3b is cheaper than Px-OH 3a and is readily
prepared (Scheme 1) in very high yield[14] by treating di-p-tolyl ether 4 with
benzotrichloride 5a or benzoic acid 5b in the presence of zinc chloride and
phosphorus oxychloride.
Ph OH
R
i
+
O
O
5a; R = CCl3
b; R = COOH
4
6
SCHEME 1 Reagents and conditions: i) ZnCl2, POCl3, 80–95◦C, 1–2 hours.
The kinetic studies[15] were first conducted to determine the acid la-
bility of the DMTr, Px-, and DMPx-group in solution. In all the kinetic ex-
periments, 5ꢁ-protected-3ꢁ-O-acetylthymidine derivatives 7a–c (Figure 4)[16]
(0.025 M solution in DCM) were treated with 5 mol. equiv. of DCA in
the presence of 15 mol. equiv. of pyrrole at 0◦C. As expected, the results
demonstrated that acid-lability increases in the order of DMTr, Px, and
DMPx, as revealed by their half times of hydrolysis (Table 3).
When DMPx- was used as the 5ꢁ-protecting group in the synthesis of
T10-mers, it became apparent that shorter acid contact time could ensure
its complete removal (Table 2). As an example, when 0.5% DCA was used
as detritylating agent, a 50 seconds acid-delivery ensured complete removal
of DMPx, whereas only 4% of full-length T10 was obtained under the same
conditions when the DMTr protecting group was used.
O
NH
RO
7a; R = 1
b; R = 2a
c; R = 2b
N
O
O
OAc
FIGURE 4 Substrate for hydrolysis kinetic experiments.

18
K. Tram et al.
TABLE 3 Half times (t1/2) of hydrolysis of DMTr-, Px-, and DMPx protecting groups
t1/2 (s)
Substrate
Determined in this study
Literature values[17]
7a (DMTr-)
7b (Px-)
7c (DMPx-)
489
195
45
450
190
–
CONCLUSION
Using T10-mer as a model system, the minimal detritylation conditions for
the removal of DMTr in oligonucleotide synthesis were determined. These
conditions also allowed successful assembly of a mixed sequence. Use of
DMPx- as an alternative protecting group for the 5ꢁ-OH functions of nucleo-
sides further reduces the required acid contact time during oligonucleotide
synthesis. This feature may permit incorporation of acid labile nucleosides
into oligonucleotides. We also believe that shorter acidic deprotection cy-
cle described herein would lead to minimization of solvent waste generated
during oligonucleotide synthesis.
REFERENCES
1. Schaller, H.; Weimann, G.; Lerch, B.; Khorana, H.G. Studies on polynucleotides. XXIV. The stepwise
synthesis of specific deoxyribopolynucleotides. Protected derivatives of deoxyribonucleosides and
new syntheses of deoxyribonucleoside-3ꢁ phosphates. J. Am. Chem. Soc. 1963, 85, 3821–3827.
2. Seliger, H. Protection of 5ꢁ-hydroxy functions of nucleosides. In Current protocols in nucleic acid
chemistry, eds. S.L. Beaucage, D.E. Bergstrom, G.D. Glick, R.A. Jones, Wiley, New York, 2001; v1,
2.3.1–2.3.34.
3. Suzuki, T.; Ohsumi, S.; Makino, K. Mechanistic studies on depurination and apurinic site chain
breakage in oligodeoxyribonucleotides. Nucleic Acids Res. 1994, 22, 4997–5003.
4. Schlitt, M.K.; Sun, K.M.; Paugh, R.J.; Millen, A.L.; Navarro-Whyte, L.; Wetmore, S.D.; Manderville,
R.A. Concerning the hydrolytic stability of 8-aryl-2ꢁ-deoxyguanosine nucleoside adducts: implications
for abasic site formation at physiological pH. J. Org. Chem. 2009, 74, 5793–5802.
5. Richard Manderville, private communications.
6. Adams, S.P.; Kavka, K.S.; Wykes, E.J.; Holder, S.B.; Galluppi, G.R. Hindered dialkylamino nucleoside
phosphite reagents in the synthesis of two DNA 51-mers. J. Am. Chem. Soc. 1983, 105, 661–663.
7. Reese, C.B.; Serafinowska, H.T.; Zappia, G. An acetal group suitable for the protection of 2ꢁ-hydroxy
functions in rapid oligoribonucleotide synthesis. Tetrahedron Lett. 1986, 27, 2291–2294.
8. Habus, I.; Agrawal, S. Improvement in the synthesis of oligonucleotides of extended length by
modification of detritylation step. Nucleic Acids Res. 1994, 22, 4350–4351.
9. Ravikumar, V.; Andrade, M.; Mulvey, D.; Cole, D.J. Carbocation scavenging during oligonucleotide
synthesis. US Patent 5,510,476, 1996.
10. Millar, S.; Yan, H.; Sanghvi, Y.S.; Pon, R. Some observations on detritylation in solid-phase oligonu-
cleotide synthesis. Nucleic Acids Symp. Ser. 2008, 52, 311–312.
11. Krotz, A.H.; McElroy, B.; Scozzari, A.N.; Cole, D.L.; Ravikumar, V.T. Controlled detritylation of
antisense oligonucleotides. Org. Proc. Res. Dev. 2003, 7, 47–52.
12. Chattopadhyaya, J.B.; Reese, C.B. The 9-phenylxanthen-9-yl protecting group. J. Chem. Soc., Chem.
Commun. 1978, 639–640.

Detritylation in Solid-Phase Oligonucleotide Synthesis
19
13. Gaffney, P.R.J.; Liu, C.; Rao, M.V.; Reese, C.B.; Ward, J.G. Some substituted 9-phenylxanthen-9-yl
protecting groups. J. Chem. Soc., Perkin Trans 1, 1991, 1355–1360.
14. Song, Q.; Khammungkhune, S.; Ross, B.S.; Griffey, R.H. Substituted pixyl protecting groups for
oligonucleotide synthesis. PCT Int. Appl. WO 2005077966 A1. 2005.
15. Experiments were conducted under identical conditions as reported in reference 17.
16. The 5ꢁ-O-DMPx protected nucleoside building blocks 2a–c were prepared in the same fashion as
described in R.T. Day, D. Williams, P. Soriano, and Y.S. Sanghvi, Large-scale synthesis of 5ꢁ-O-pixyl
protected 2ꢁ-deoxynucleosides useful for oligonucleotide synthesis. Nucleos. Nucleot. Nucleic Acids
2005, 24, 1135–1138.
17. Reese, C.B.; Yan, H. Alternatives to the 4,4ꢁ-dimethoxytrityl (DMTr) protecting group. Tetrahedron
Lett. 2004, 45, 2567–2570.