Modification of oligodeoxynucleotides by on-column Suzuki cross-coupling reactions

Article abstract of DOI:10.1039/c8cc01360h

The on-column functionalization of oligodeoxynucleotides via base-free Suzuki cross-coupling reactions is reported herein. These cross-coupling reactions were carried out with various boronic acids and either full-length modified oligonucleotides containi

Full text of DOI:10.1039/c8cc01360h

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DOI: 10.1039/C8CC01360H
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Modification of oligodeoxynucleotides by on-column Suzuki cross-
coupling reactions†
Maria Ejlersen,a Chenguang Lou,a Yogesh S. Sanghvi,b Yitzhak Torc and Jesper Wengela*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
On-column functionalization of oligodeoxynucleotides by base-free conjugation (e.g. triazole linkers or phosphine oxides) that
Suzuki cross-coupling reactions is reported herein. These cross- might interfere with biological function.
couplings were carried out with various boronic acids and either Pd-catalyzed cross-coupling reactions can be used for
full-length modified oligonucleotides containing one or more 2’- nucleobase modifications of nucleosides, nucleotides and ONs,
deoxy-5-iodouridine (5IdU) monomer(s) or on oligonucleotide most commonly in the 5-position of pyrimidines and the 6- or 8-
fragments immediately after incorporation of 5IdU. Five different position of purines.11-13 The first report of Pd-catalyzed
functionalities were coupled to oligonucleotides containing one or reactions with ONs was a Sonogashira cross-coupling used for
three attachment points.
on-column postsynthetic modification of a DNA strand singly
modified with 2’-deoxy-5-iodouridine (5IdU).14 The same
method was used to introduce 1-pyrenyl15,16 and a spin-label17-
The need for modified DNA and RNA oligonucleotides has
increased rapidly due to advances in biotechnology and
biomedical research. Multiple methods for modification of
oligonucleotides (ONs) have been developed and the
modifications are commonly introduced either by automated
ON synthesis with modified phosphoramidites1 or by enzymatic
incorporation using modified nucleoside triphosphates.2 Both
methods often require long multistep syntheses for monomer
preparations and not all modifications are tolerated or
compatible with the required reagents and conditions used for
elongation of ONs.3,4 Some of these disadvantages can be
overcome by site-specific postsynthetic modification of ONs.
When using this approach, a stable precursor is incorporated
into the ON which can subsequently be functionalized
employing various conjugation chemistries.5 Commonly used
methods for conjugation is Cu-catalyzed6 or Cu-free7 Huisgen
cycloaddition, Staudinger ligation,8 thiol conjugation9 or amide
bond formation.10 These methods can be efficient but many
also form structurally rather complex products upon
19
to both purine and pyrimidine bases within an ON. The
Sonogashira coupling was later used for on-column reactions
with full-length ONs modified with 5IdU to introduce 23
different functionalities,20 spin-labels21 and modified Twisted
Intercalating Nucleic Acids.22 More recently, the Stille cross-
coupling was used for multiple reactions with full-length RNA
strands on-column while also testing different protecting
groups for the 2ʹ-hydroxy group.23 The Stille coupling was
performed with both 5ʹ-terminal and intrastrand incorporations
of 5-iodouridine and 2-iodoadenosine into either poly-U or
poly-A RNA strands.23 The Stille coupling was also used for on-
column modifications in good yields to 5IdU and 2’-deoxy-5-
iodocytosine (5IdC) incorporated into DNA strands.24
Contrary to the Sonogashira and Stille couplings, reports of
Suzuki cross-coupling reactions have been limited to
deprotected DNA strands in solution. Suzuki couplings have
thus been used to modify 5IdU and 5IdC with a photoswitchable
group25 and various sensitive functional groups.26 The Suzuki
coupling has also been used for arylation of 8-Br-2’-
deoxyguanosine thereby synthesizing functionalized DNA
strands not accessible by conventional solid-phase synthesis.27
The mild and versatile nature of Suzuki coupling reactions
makes it a good candidate for on-column modifications of ONs.
The required excess of reagents for heterogeneous on-column
derivatization is easily removed by thorough washings of the
column before deprotection from the solid support thus
simplifying purification. The Suzuki and Stille couplings generate
equivalent C-C bonds, but the Suzuki coupling offers a desirable
a.Biomolecular Nanoscale Engineering Center, Department of Physics, Chemistry
and Pharmacy, University of Southern Denmark, DK-5230 Odense M, Denmark.
b.Rasayan Inc. 2802, Crystal Ridge Road, Encinitas, California, 92024-6615, USA.
c. Department of Chemistry and Biochemistry, University of California, San Diego, La
Jolla, California 92093-0358, USA.
* To whom correspondence should be sent: Tel: +45 65502510, e-mail:
jwe@sdu.dk.
†Electronic Supplementary Information (ESI) available: Details on Suzuki cross-
coupling reactions, oligonucleotide synthesis, purification and analysis (RP-HPLC
and MALDI-TOF MS); copies of RP-HPLC curves, MALDI-TOF mass spectra. See
DOI: 10.1039/x0xx00000x
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Journal Name
ONa 5'-dUITA CG
CG
a
non-toxic alternative to the organotin compounds employed in
the Stille couplings. The Suzuki coupling does, however, require
base for activation of the organoboron reagent which may lead
to a stability issue relating to the base labile succinyl linker
between the ON and the support.
View Article Online
DOI: 10.1039/C8CC01360H
ONb 5'-TAdUI
ON1 5'-GTG AdUIA TGC
ON2 5'-GdUIG AdUIA dUIGC
O
O
I
R
b
NH
NH
Cross-coupling
N
O
N
O
O
O
O
O
O
N
dUI
dUS
We have sought to modify the 5-position of 5IdU with various
aromatic moieties including phenyl, furyl, thienyl, pyrenyl and
3-(dansylamino)phenyl groups as such modified pyrimidines are
fluorescent.28-30 Such nucleosides emit in the visible range and
the sensitivity provided by fluorescence-based tools can be
used for confirmation of a putative reaction, even if only very
low amounts of cross-coupling product are generated.31,32
Additionally, modifications at the 5-position of pyrimidine bases
of an antisense ON offers modulation of target affinity.33
Herein we report the introduction of aromatic moieties in the
5-position of uracil bases via incorporation of 5IdU into solid-
support bound ONs and subsequent on-support Suzuki cross-
coupling reactions. Up to five different functionalities were
introduced in a single position by cross-coupling with the full-
length ON or by individual cross-couplings immediately after
incorporation of an 5IdU nucleotide. Both methods were also
used to introduce three identical functionalities to the same
ON. The latter method was moreover used to introduce three
different functionalities to the same ON allowing for easy
modification of ONs in a sequential fashion.
Initially, model Suzuki cross-couplings with unprotected 5IdU
nucleoside and 2-furylboronic acid or 2-thienylboronic acid
were carried out to assess any potential side reactions, and to
find the optimal coupling partner for reactions with the
corresponding ONs (Scheme 1).
The cross-coupling reactions using conditions adapted from
Hopkins et al.34 were successful but proceeded in low yields,
giving 1a and 1b in 25% and 35% yield (Scheme 1), respectively,
while the major product was the dehalogenated 2’-
deoxyuridine. 2-Thienylboronic acid gave the higher yield and
the least by-products and was therefore chosen as the coupling
partner for initial reactions with ONs. No optimization of the
conditions was carried out as the local environment for on-
column reactions with ONs are very different from reactions in
solution, as the former typically require a higher excess of
reagents to compensate for the heterogeneous reaction
conditions.14,19
Cleavage &
deprotection
Method 1
R
O
O
NH
B
O
HO
O
O
O
Elongation
of ON
dUS
O
N
O
N
O
O
I
R
NH
O
NH
Elongation,
Cleavage &
Deprotection
Cross-coupling
Elongation
Method 2
O
HO
HO
O
O
dUI
dUS
O
O
Fig. 1
Schematic illustration of on-column Suzuki cross-coupling. Blue spheres
represent the CPG solid-support; ONs are drawn in red; dUI represents the 5IdU
nucleotide where dUS represents a cross-coupling product. (a) ON sequences used for
Suzuki cross-coupling reactions; (b) Generel representation of the two methods used for
on-column Suzuki cross-coupling reactions. Method 1: Cross-coupling reaction carried
out after synthesis of the full-length ON; Method 2: Cross-coupling reaction carried out
immediately after incorporation of the 5IdU nucleotide. Reagents and conditions:
Pd(OAc)2, TPPTS, boronic acid, Tris buffer (50 mM, pH 8.50)/MeCN (3:2 v/v, 1 mL), 70 °C,
4 h/8 h.
where reactions at the terminal position renders steric factors
negligible. Suzuki cross-couplings were carried out with the 5-
mer strands retained on the columns whereafter the ONs were
cleaved from support, deprotected and subjected to analysis by
RP-HPLC and MALDI-MS. However, a significantly lower amount
of the test ONs were isolated after cross-coupling than expected
from a 0.2 µmol ON synthesis, and the amounts were
insufficient for HPLC analysis. The presence of the thiopheno-
modification was therefore verified by a positive fluorescence
signal. The low amount of the ON product after cleavage from
support and deprotection was ascribed to cleavage of the base
labile linker between the solid support and the ON during cross-
coupling. Davis et al.26 reported successful base-free Suzuki
cross-couplings with DNA strands in buffer systems which
inspired us to optimize the conditions for on-column Suzuki
cross-couplings by substituting the medium composed of base
and MQ H2O for a tris(hydroxymethyl)aminomethane (TRIS)
buffer. Cross-couplings with Test ONa and ONb were repeated
using these optimized conditions. Full conversion of the two
test ONs into cross-coupled products (Table 1) was observed by
HPLC analysis with no noticeable formation of by-products (Fig.
S17 and S19, ESI†).
The first reactions with ONs were carried out with two 5-mer
DNA-type strands containing all canonical bases and 5IdU
positioned either at the 5’-terminal (ONa) or internally (ONb) to
identify the scope and possible limitations of the approach (Fig.
1a and Table 1). The internal position mimics longer sequences
Following the initial successful cross-coupling reactions, 9-mer
DNA-type ONs with all canonical bases and one or three 5IdU
modifications were used for the following cross-coupling
reactions. The reactions were carried out by two different
methods (Fig.1b): a) Method 1 in which cross-couplings were
carried out with the full-length ON, and b) Method 2 in which
cross-couplings were carried out immediately after
incorporation of 5IdU followed by elongation of the ON. Cross-
couplings by Method 2 were thus always carried out with an
“end-positioned” nucleotide thereby making the iodinated
nucleotide more accessible. The substrate scope was explored
by reaction of full-length ON1 with 2-thienylboronic acid
(synthesis of ON3(a)), phenylboronic acid (synthesis of ON4), 5-
methyl-2-thienylboronic acid pinacol ester (synthesis of ON5),
pyrene-1-boronic acid (synthesis of ON6(a)) and
O
N
O
N
I
X
NH
NH
O
O
HO
HO
O
O
i
X = O
X = S
1a
1b
OH
OH
Scheme 1
Suzuki cross-coupling with unprotected 5IdU. Reagents and
conditions: 2-Furylboronic acid or 2-thienylboronic acid, Pd(OAc)2, TPPTS, MQ
H2O/MeCN (3:2 v/v), 80 °C, 16 h.
2 | J. Name., 2012, 00, 1-3
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Table 1 On-column modification of a 9-mer DNA strand by Suzuki cross-coupling with
one modification site. dUS represents the modified nucleotide
(Fig. 1a and Table 2). This corresponds to requiringVaietwhAirrtdicleoOf ntlhinee
nucleotides in the ON to undergo reaction. Cross-coupling of
DOI: 10.1039/C8CC01360H
ON2 with 2-thienylboronic acid using Method 1 was carried out
without increasing the amount of reagents relative to the
previous couplings as they were already in great excess. The RP-
HPLC spectrum for crude ON8(a) showed three major peaks
with very similar retention times (< 1.0 min span on IE-HPLC;
separation was possible by RP-HPLC and all three peaks were
individually isolated (Fig. S30, ESI†)). Common for the three
isolated product ONs were successful cross-coupling reactions
in two positions combined with either substitution (8%),
dehalogenation (7%) or no reaction (11%) in the third position
(note that the positional order has not been determined). The
isolated yield for ONs where the third cross-coupling was
unsuccessful (18%) is twice that of the desired ON8(a) with
three successful substitutions (8%). Attempted optimization of
conditions was performed by extending the reaction time from
4 to 8 h and by employing double cross-couplings. Extension of
the reaction time did not lead to isolation of the desired product
ON8(b) in a higher yield but instead a lower yield of the product
in which no reaction had occurred in the third position was
observed (from 11% to 5%, Table 2). Employing double cross-
couplings led to a significant increase in isolated yield to 18% for
ON8(c) but a significant amount of dehalogenated ON was still
observed (5%). The large increase in yield with double couplings
suggests that the catalyst becomes less effective over time,
possibly due to influx of oxygen.
Method 2 was also used to obtain triple thienyl-modified
ON8(d) requiring pausing of the ON synthesis three times in
total. Two major peaks were observed in the crude RP-HPLC
chromatogram and both were independently isolated (Fig. S33,
ESI†). One peak corresponded to the desired ON8(d) (22% yield)
and another to dehalogenation of the third position (4% yield).
By using Method 2, significantly lower amounts of the
dehalogenated ON were observed together with disappearance
of the peak corresponding to the ON for which no reaction at
the third position had occurred (Table 2). This suggests that
steric issues likely negatively impact the outcome of three cross-
couplings within the same ON. The success of triple
modifications when employing Method 2 enables further
expansion of the methodology by using a different boronic acid
for each sequential cross-coupling reaction.
ON #
A
B
3(a)
3(b)
4
Sequence
Modification
2-Thienyl
2-Thienyl
2-Thienyl
2-Thienyl
Phenyl
5-Methyl-2-thienyl
Pyrenyl
Pyrenyl
3-(Dansylamino)phenyl
3-(Dansylamino)phenyl
Method
Yield (%)a
b
5ʹ- dUSTA CG
1
1
1
2
1
1
1
2
1
2
-
-
5ʹ- TAdUS CG
b
5ʹ-GTG AdUSA TGC
5ʹ-GTG AdUSA TGC
5ʹ-GTG AdUSA TGC
5ʹ-GTG AdUSA TGC
5ʹ-GTG AdUSA TGC
5ʹ-GTG AdUSA TGC
5ʹ-GTG AdUSA TGC
5ʹ-GTG AdUSA TGC
29
38
29
21
7
27
28
48
5
6(a)c
6(b)c
7(a)
7(b)
aIsolated yields for full-length product. bProduct not isolated for determination of
yield for test sequences. Tris buffer (50 mM, pH 8.50)/DMF (2:3 v/v, 1 mL) were
used to dissolve pyrene-1-boronic acid; the concentration of pyrene-1-boronic acid
was also halved due to the solubility issue.
c
3-(dansylamino)phenyl boronic acid (synthesis of ON7(a)) by
using Method 1 (Fig. 1a and Table 1). Notably, Suzuki cross-
couplings with some boronic acids can generate significant
amounts of dehalogenated product, and it has been shown that
using the pinacol esters of these boronic acids can push the
reaction towards cross-coupling.26 RP-HPLC spectra for crude
ONs (ON3(a), ON4, ON5 and ON7(a)) following reaction and
deprotection/cleavage from the solid support showed no or a
tiny peak corresponding to the starting ON1 indicating
complete conversion into product ONs for reactions with all
four boronic acids (Fig. S22, Fig. S24-S25 and S28, ESI†).
Following purification, full-length ON3(a), ON4, ON5 and
ON7(a) were isolated in a yield of 29%, 29%, 21% and 28%,
respectively, in satisfactory purity as shown by HPLC analysis.
The yield for ON5 using the protected boronic acid was lower
than for reactions with unprotected boronic acids indicating a
slower or more unfavorable reaction likely due to steric factors
with the protected reagent (Table 1). The lower yields on
isolated ON6(a) relative to ON7(a) are not surprising, for the
mixed reagent solutions in the former quickly turned from light
yellow to dark after 0.5 h at 70 °C, indicative of strong side
reactions. This may also result partly from the lower
concentration of pyrene-1-boronic acid used in the case of
ON6(a) due to the solubility issue. The isolated yields for
ON3(a), ON4, ON5 and ON7(a) were comparable to the
reported yields for Suzuki cross-couplings of ONs in solution by
Jäschke25 but lower than reported by Davis also for in solution
cross-couplings.26 The isolated yields are, however, similar to
reports for on-column Sonogashira14,20 and Stille24 couplings.
We achieved almost complete conversion of dUI into dUS also
for cross-coupling using Method 2 with 2-thienylboronic acid,
pyrene-1-boronic acid and 3-(dansylamino)phenylboronic acid
(Fig. S23, Fig. S27 and S29, ESI†). ON3(b), ON6(b) and ON7(b)
were isolated in total yields of 38%, 27% and 48%, respectively,
which are significantly higher than those isolated of ON3(a)
ON6(a) and ON7(a) using Method 1 (Table 1). These results are
in agreement with Jäschke’s findings25 showing higher yields for
cross-couplings with terminal modifications than with internal
modifications. The successful couplings with one modification
prompted the expansion of the methodology to multiple
incorporations using 9-mer ON2 with three 5IdU modifications
Thus, an attempt of successively incorporating 2-thienyl (Th), 5-
methyl-2-thienyl (MeTh) and phenyl (Ph) afforded one major
product ON as observed from RP-HPLC (Fig. S34, ESI†) which
was isolated in a yield of 23% (ON9). Incorporation of different
boronic acids in the same sequence offers great potential for
easy but structurally diverse functionalization without the need
for synthesis of a series of phosphoramidites. Furthermore, on-
column conjugation offers the opportunity for introduction of
different functionalities to the same ON which is not possible by
the method for Suzuki cross-couplings in solution reported by
Jäschke25 and Davis.26
In summary, we demonstrated the first successful use of
base-free Pd-catalyzed Suzuki cross-coupling reactions as a
convenient strategy for postsynthetic on-column modification
of oligodeoxynucleotides. Cross-couplings were accomplished
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DOI: 10.1039/C8CC01360H
Table 2 On-column modification of a 9-mer DNA strand by Suzuki cross-coupling with three modification sites. dUs represents the modified nucleotide
ON #
8(a)
8(b)
8(c)
8(d)
9
5’-GdUSG AdUSA dUSGC
Time (h)
4
Method
Yield (%)a
Dehal. (%)b
No rx (%)b
3 × Th
3 × Th
3 × Th
1
1
1
2
2
8
8
18
22
23
7
8
5
4
-
11
5
5
-
8
2 × 4
3 × 4
3 × 4
3 × Th
1 × Ph, 1 × MeTh, 1 × Th
-
aIsolated yield of triply modified ON. bIsolated yield for ONs with dehalogenation or no reaction in one position; Th = 2-thienyl, MeTh = 5-methyl-2-thienyl, Ph = phenyl.
7
8
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5-iodouridine modified 9-mer was observed for cross-couplings
with five different boronic acids, and with the highest yield
observed when cross-coupling reactions were carried out
immediately after incorporation of the 2’-deoxy-5-iodouridine
monomer. Cross-couplings with on the full-length triply 2’-
deoxy-5-iodouridine modified 9-mer were more challenging,
but successful cross-couplings were obtained by employing
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design and synthesis of future oligonucleotides.
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Conflicts of interest
There are no conflicts to declare.
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