Solution-Phase Synthesis of Branched Oligonucleotides with up to 32 Nucleotides and the Reversible Formation of Materials

Article abstract of DOI:10.1002/ejoc.201700686

A linear solution-phase synthesis of branched oligonucleotides with adamantane as core has been developed. The method uses conventional phosphoramidites only, achieves chain assembly without chromatography of intermediates, and overcomes the low reactivit

Full text of DOI:10.1002/ejoc.201700686

A Journal of
Accepted Article
Title: Solution-Phase Synthesis of Branched Oligonucleotides with up
to 32 Nucleotides and the Reversible Formation of Materials
Authors: Clemens Richert, Alexander Schwenger, and Nicholas
Birchall
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201700686
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European Journal of Organic Chemistry
10.1002/ejoc.201700686
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Solution-Phase Synthesis of Branched Oligonucleotides with up
to 32 Nucleotides and the Reversible Formation of Materials
Alexander Schwenger, Nicholas Birchall, and Clemens Richert*
Abstract:
A
linear solution-phase synthesis of branched
phase synthesis that avoid or reduce the high cost and limited
scalability of solid-phase syntheses are being developed.
[24]
[25-27]
oligonucleotides with adamantane as core has been developed. The
method uses conventional phosphoramidites only, achieves chain
assembly without chromatography of intermediates, and overcomes
the low reactivity of adamantane-1,3,5,7-tetraol as core. The
assembly of four-arm hybrids with up to 32 nucleotides total was
performed, with monodisperse products of up to 10 kDa in size.
Overall yields of 20 % over 19 steps (hexamer arms) and 11 % over
Further, there is active research on new approaches for
preparing trimer building blocks, where part of the chain
[
28-30]
assembly is performed in solution.
Although branched
intermediates with DNA chains linked to soluble supports have
been reported, the largest products of such syntheses reported
in the recent literature contain no more than 5-9 nucleotides
[
25-27]
25
steps (octamer arms) of HPLC-purified compounds were
total,
demonstrating how difficult it is to prepare large
obtained. The adamatane-based hybrids show more DNA-
dominated assembly properties than their brethrens with larger
lipophilic cores. Reversible formation of macroscopic amounts of
materials through hybridization was achieved, both for self-
complementary systems and two-hybrid systems with two non-
selfcomplementary DNA sequences.
oligonucleotides without immobilization.
Our interest in DNA-based materials has focused on
branched oligonucleotides that form nanoporous three-
dimensional networks through hybridization. This is an active
[
31]
area of research,
in which compounds prepared by solid-
are also used for subsequent enzymatic
We had observed that, due to their ability to undergo
[
32]
phase methods
[33]
steps.
multivalent hybridization, synthetic tetrahedral DNA hybrids
consisting of an organic core and four DNA arms can form
materials in dilute aqueous solution. Sequences as short as CG
Introduction
dimers can act as "zippers" and bring about the assembly
Synthetic oligodeoxynucleotides have become important
[34]
process at a high concentration of divalent cations.
We then
[
1-3]
building blocks in nanotechnology.
A number of functional
found that the propensity to form materials in aqueous solution
nanomaterials have been prepared from modified, rather than
natural oligonucleotides, in order to benefit from structural traits
increases when constructs with six arms and rigid cores are
[35]
used.
But, when the number of arms was increased to eight
[
4-6]
not found in linear DNA.
To be useful for applications in the
and the size of the rigid and lipophilic core increased, the
sequence dependence of the assembly process was lost,
field of materials, macroscopic quantities of oligonucleotides are
called for. To produce such quantities is a challenge for nucleic
acid chemists.
indicating that it was no longer dominated by Watson-Crick base
[36]
pairing.
This prompted us to shift our attention to branched
For linear oligonucleotides, several modes of synthesis
DNA hybrids with a smaller, entirely aliphatic core, hoping to
build materials with larger pore size without losing the ability to
steer the assembly process through choice of the sequence and
length of the DNA arms.
[
7]
have been described.
Initially, solution-phases syntheses
were used exclusively, based on phosphodiester or
[
8-11]
phosphotriester intermediates,
combined with enzymatic
[
12]
ligation to assemble the final sequence. With the advent of
these labour-intensive approaches
Here we report the solution-phase synthesis of DNA
hybrids based on adamantane-1,3,5,7-tetraol. The synthesis of
these hybrids was performed entirely in solution, based on
inexpensive, commercial phosphoramidite building blocks.
Hybrids with up to 32 nucleotides were obtained in 200 mg
batches with no more than a single chromatographic step at the
very end of the syntheses. The resulting hybrids were found to
form three-dimensional networks reversibly, both when a single
type of hybrid with self-complementary DNA arms was used and
when a combination of two hybrids with sequences that are
complementary to each other were used.
[13-15]
solid-phase synthesis,
were all but abandoned. Three methods were optimized to
obtain oligodeoxynucleotides on a solid support, namely the
[
16]
[17]
phosphotriester method,
the H-phosphonate method,
and
[
[
18,19]
the phosphoramidite or phosphite triester method.
Optimization of the phosphoramidite method continues,
including approaches to reduce protecting group usage.
Still, solid-phase chain assembly dominates the field.
20,21]
[
22,23]
Large-scale solid-phases syntheses are costly and
producing bulk quantities of oligonucleotides via this approach is
problematic for applications other than medicinal chemistry or
nanotechnology. As a consequence, new methods for solution-
Results and Discussion
[]
Dipl.-Chem. A. Schwenger, M.Sc. Nicholas Birchall, Prof. C. Richert
Institut für Organische Chemie, Universität Stuttgart
As shown in Figure 1, the group of hybrids to be prepared
included compounds with dimer arms on a phenolic core (1),
homooligomer arms on an aliphatic core (2, 3), mixed-sequence
arms on the same core (4), and self-complementary dimer,
tetramer, hexamer, and octamer arms on the aliphatic
7
0569 Stuttgart (Germany)
Fax: (+ 49) 711-685-64321
E-mail: lehrstuhl-2@oc.uni-stuttgart.de
http://chip.chemie.uni-stuttgart.de
Supporting information for this article is given via a link at the end of
the document.
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European Journal of Organic Chemistry
10.1002/ejoc.201700686
FULL PAPER
adamantane core (5-8). Both self-complementary and non-self-
complementary sequences were chosen to allow for modulation
of hybridization strength in processes forming three-dimensional
networks.
The challenges in preparing longer DNA hybrids in solution
are significant, if one wants to start from inexpensive,
commercial phosphoramidites that have been optimized for
solid-phase synthesis. One challenge is to handle richly-
functionalized, labile biomacromolecules. The intermediates of
solution-phase syntheses are polar mixtures of diastereomers
that readily lose their cyanoethyl protecting groups, all but
precluding conventional chromatography on supports like silica.
Further, simultaneous couplings at the termini of all arms of the
branched constructs call for high-yielding reactions, a problem
[
37,38]
known from dendrimer chemistry.
Finally, the purification of
the final products can be challenging, as increasingly complex
mixtures are being formed that have the ability to assemble into
higher order structures, even in protected state.
Figure 1. Structures of target molecules; a) with tetrakis(p-hydroxyphenyl)adamantane core (TPA), and b) with adamantanetetraol core (TOA).
Since our earlier work had shown that large branching elements
[
36]
or "cores" can interfere with rule-based self-assembly,
we
focused on hybrids with small cores. The first target molecule
contained a tetrakis(p-hydroxyphenyl)adamantane (TPA) core,
[35,36]
which is considerably smaller than those studied earlier,
but
features the phenols as reactive nucleophiles, for which much of
[
39]
our earlier solution-phase work had been optimized.
At the beginning of our study, the favored assembly
method was block condensation of the core with CG dimer
"
zipper" arms via H-phosphonate chemistry (Scheme 1a). The
next step in shrinking the core led to 1,3,5,7-adamantanetetraol,
often referred to as 1,3,5,7-tetrahydroxyadamantane (TOA) in
our laboratory. Here, the hydroxy groups to which the DNA arms
were to be attached were tertiary, aliphatic alcohols, which are
much less nucleophilic and considerably more sterically
hindered than para-substituted phenols.
[
40]
The syntheses of adamantane tetraol (9) and tetrakis(4-
hydroxyphenyl)adamantane (10, TPA) started from 1,3,5,7-
tetrabromoadamantane, which is accessible from adamatane
Scheme 1. Synthetic strategies for the assembly of branched oligonucleotide
hybrids with organic cores.
[
40,41]
itself via four-fold bromination.
The tetrabromide was then
whose four-fold Friedel–Crafts alkylation
with anisole, catalyzed by TfOH, and subsequent ether cleavage
with BBr furnished 10 in 32% overall yield (Scheme 2).
[
40,42]
With the two cores in hand, we then synthesized DNA
hydrolyzed to 9,
hybrids. We initially attempted to prepare both 1 and 5 using our
[
36,39]
solution phase H-phosphonate method.
dimer building block 11,
Preparation of
3
[
39]
proceeded uneventfully. Couplings
involved 3 equiv of the dimer H-phosphonate per alcohol of the
cores, diphenyl chlorophosphate (DPCP) as condensing agent,
and a solvent mixture of pyridine/CH CN (4:1) at -40 °C,
3
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European Journal of Organic Chemistry
10.1002/ejoc.201700686
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followed by oxidation with aqueous iodine in pyridine, aqueous
[39]
workup, detritylation and hydrolysis/aminolysis.
Hybrid 1 with
its phenolic core was thus obtained in 38% overall yield after
cartridge purification. The chromatogram of the crude showed
an intense peak for the product, together with the expected side-
products, including the symmetrical pyrophosphate formed by
dimerization of the H-phosphonate (Figure 2).
Figure 2. Representative RP-HPLC traces of crude products from the
synthesis of: (a) 1 via H-phosphonate coupling, (b) 5 via H-phosphonate
coupling, and (c) 5 via linear assembly with phosphoramidite monomers.
Product peaks are labeled with arrows.
One problem we encountered was solubility. Both acetonitrile
and dioxane as solvent gave almost no coupling because the
core did not dissolve well enough. Addition of dry DMF gave a
homogeneous solution and near-quantitative coupling. In the
optimized method, all other components of the reaction mixture
were dissolved in DMF and the tetrazole solution (0.45 M in
Scheme 2.
Using the same methodology, hybrid 5 was obtained in less than
10% yield, and its chromatographic purification was difficult.
Figure 2b shows the HPLC chromatogram of the crude. In
addition to other side products, incomplete coupling products
with one, two or three DNA arms were detected, even after long
reaction times. This suggested that the tertiary alcohols of 9 are
too unreactive for coupling to H-phosphonate 11.
CH
room temperature for 30 min and then stored overnight at 5 °C.
The subsequent oxidation used t-BuOOH. After aqueous
workup, the fully protected DNA hybrids were isolated by
precipitation with MTBE, starting from a concentrated CH Cl
2
3
CN) was added last. The reaction mixture was shaken at
We assumed that phosphoramidites would be more
reactive. Further, we suspected that the reactivity of monomers
would be higher than that of dimers. To avoid the costly methyl-
2
solution. Subsequent washing of the solid with methanol was
important for obtaining pure hybrids. The solubility in MeOH is
higher for pyrimidine hybrids, though, so that excessive washing
can lead to loss of material. Detritylation with dichloroacetic acid
in CH Cl was followed by quenching with MeOH. Concentrating
2 2
in vacuo and precipitations with MTBE then gave
mononucleotide hybrids 13a-t in near-quantitative yield (purines)
[
43]
protected monomers used earlier, we limited ourselves to the
more common cyanoethyl phosphoramidites and used linear
syntheses (Scheme 1b). For this, one of the four monomer
phosphoramidites 12a-t was coupled directly to the core.
or satisfactory yields (pyrimidines), without
chromatography (Table 1).
a
need for
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European Journal of Organic Chemistry
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Table 1. Synthesis of DNA hybrids 13a-t, starting from 9 and monomer phosphoramidites.
[
a]
a
Monomer
Product
Equiv. 12
Equiv tetrazole
Ratio DMF/CH
3
CN
time (h)
Yield of crude (%)
1
1
2g
2g
13g
13g
7
8
1.5
1.2
1:1.9
1:1.7
19
48
50
95
1
2t
13t
13t
7
6.5
1.5
1.5
1:2.3
1:1.4
22
21
65
68
12t
1
1
2c
2c
13c
13c
7
8
1.5
1.5
1:1.2
1:1.5
25
44
54
61
1
1
2a
2a
13a
13a
8
8
1.5
1.5
1:1.3
1:1.1
24
24
78
quant.
[
a]
See Figures S3-6 (Supporting Information) for typical NMR spectra.
The preferred protocol that emerged for the first coupling
to 9 involves 2 equivalents phosphoramidite per hydroxy group,
.2-1.5 equivalents of activator and a reaction time of 12-48 h,
increased to 81% when 2.25 equiv phosphoramidate and 2.9
equiv tetrazole were used, the DMF content was lowered to 37%,
and the coupling time was increased to 2 d.
1
depending on the phosphoramidite (Table 1). Just 1.5-1.75
equiv phosphoramidite per OH group gave an average yield of
62 %. More than 1.5 equiv of tetrazole over phosphoramidites
led to significant amounts of 'overreacted' products, containing
more than four nucleotides, most probably due to partial loss of
DMT groups and subsequent coupling to the exposed 5'-
alcohols.
Long coupling times increase the likelihood of
detritylation, but are also important for achieving near-
quantitative conversion. In many cases where near-quantitative
coupling occurred, visible crystals of diisopropylammonium
tetrazolide (DIPAT) appeared after 7 h (13a) or 24 h (13g),
helping to visually confirm successful couplings under our
reaction conditions.
Neither of the mononucleotide hybrids (13a-t) required
further purification for use in the next coupling cycle. When a
sample of 13g, featuring the most stable acyl protecting group
4
among the nucleobases, was fully deprotected with NH OH and
analyzed via MALDI-TOF and HPLC, a purity of approx. 90%
was found (Figures S13 and S32, SI). A large-scale synthesis
with 300 mg of 9 and 10 g of 12g gave 2.8 g of 13g, a yield of
95%.
With hybrids 13a-t in hand, we proceeded to synthesizing
hybrid 5 with CG dimer arms (Scheme 3). For this, detritylated
hybrid 13g was reacted with phosphoramidite 12c. Two equiv of
the phosphoramidite and three equiv tetrazole per arm in
3
CH CN/DMF (1:1.2) for one day gave crude 5 in 66% yield after
coupling, oxidation and detritylation. With 70% DMF in the
coupling mixture, the yield dropped by 30%. But, the yield
Scheme 3.
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European Journal of Organic Chemistry
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Deprotection with aqueous ammonia then gave a crude in
which 5 as the dominant component (Figure 2c), and HPLC
purification led to the pure hybrid in 67% overall yield for the
four-step sequence of coupling, oxidation, detritylation, and
basic deprotection. So, compared to the synthesis via H-
phosphonate dimer 11 (Figure 2b), a much higher yield resulted.
Encouraged by these results, we then prepared
homooligomers 2 and 3. In the first part of Table 2 (entries 1-7),
selected results for thymidine-containing oligomer 2 are shown.
For each step but the last, two different coupling conditions are
shown to highlight how changes in the reaction conditions affect
the yield of crude of intermediates 15-17. Overall, it again
emerged that the excess of phosphoramidite and tetrazole, the
DMF content, and the reaction time are important factors for
achieving high yields. For the last coupling cycle, producing the
protected precursor of 2, we used 11 equiv, which is still less
than the 15-25 equiv typically used for solid-phase DNA
phosphoramidite, and adjusting the DMF content, yields of 82-
90% for the four-fold extension were obtained on every stage of
chain extension process (intermediates 22-24). A good balance
of the amount of tetrazole, the reaction time and the DMF
content was obtained with the conditions shown in entries 13-15
of Table 2. After full deprotection, hexamer hybrid 4 was
isolated in 84 % yield for the last extension cycle and 15% (62%
per arm) overall yield for the 19 step route.
Finally, we undertook the synthesis of hybrids 6, 7, and 8
with self-complementary DNA arms.
This synthesis was
considered the most difficult, not just because up to 32
nucleotides had to be appended to the core, but also because
the propensity of the all-CG sequences to self-assemble is high,
and even protected intermediates have the ability to form large
base-paired structures, complicating synthesis and purification.
The last section of Table 2 (entries 16-22) gives an overview of
the coupling conditions and yields.
syntheses on conventional DNA synthesizers.
After full
The coupling to dimer hybrid 25 was carried out with 7.5 equiv of
deprotection and HPLC, 2 was obtained in 54% yield or 86% per
3
12g, 1.4 equiv tetrazole, and a DMF/CH CN mixture of 1:1.2,
DNA arm for the last coupling cycle.
yielding hybrid 25 in near-quantitative yield. For the coupling
cycle producing trimer hybrid 26, the equivalents of 12c were
increased, and the reaction time extended, resulting in a yield of
75% (93% per arm). The coupling rounds leading to 27 and 28
were carried out similarly, but with a coupling time of 2 d. Again,
the pyrimidine coupling was slightly lower yielding. The sixth
coupling cycle used 11 equiv of 12g and slightly more DMF in
the solvent mixture to reduce self-assembly and to obtain a
homogeneous solution. The seventh and eighth extension steps
were carried out with 1.2 equiv of tetrazole to avoid loss of DMT
groups. Crude heptamer 30 was obtained in a yield of 90%, and
the subsequent coupling used a 1:1 mixture of DMF/CH
ensure a homogeneous solution. Conventional deprotection
with hot aqueous ammonia gave tetramer hybrid (GCGC) TOA
6) in 55% from 26 and 25 % overall yield for 13 steps after
3
CN to
4
(
HPLC purification. Figure 4 shows chromatogram and MALDI-
TOF mass spectrum of this hybrid.
Figure 3. MALDI-TOF mass spectra of (a) pentamer hybrid 2 and (b) hexamer
hybrid 4.
Figure 3a shows a mass spectrum of the pentamer hybrid.
The overall yield of 2, with its 20 nucleotides, was 10% (56% per
arm) over 16 steps. Hybrid d(A) (3) was prepared similarly, as
5
detailed in Table S1 (Supporting Information), and was obtained
in 26% overall yield after HPLC purification. The higher yield
may again be due to solubility and thus greater ease in isolating
intermediates through precipitation.
We then turned to mixed sequence hybrids. First, hexamer
4
was prepared, which contains three of the four different
nucleobases (A/C/T). Starting from 13a, dimer intermediate 21
was obtained in 90-98 % yield (first coupling cycle with 12c in
Table 2, entries No. 8-9). Through increasing the amount of the
phosphoramidite by 0.25 equiv/arm with every extension cycle,
lowering the activator content to 1.2 equiv over the
Figure 4.
(GCGC) TOA (6). (a) HPLC trace of purified product (C18-phase, gradient of
-25% CH CN in 10 mM TEAA buffer, at 55 °C column temperature, λdet 260
nm). (b) MALDI-TOF mass spectrum after HPLC purification.
Analytical data for self-complementary tetramer hybrid
4
1
3
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European Journal of Organic Chemistry
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Table 2. Results of the synthesis of (TTTTT)
4
TOA (2), (TCCTCA)
4
TOA (4), and (GCGCGCGC)
4
TOA (8) via linear, phosphoramidite-based chain assembly.
[
a]
Entry No.
Educt
Monomer
Product
Equiv
Equiv
tetrazole
Ratio
time
(h)
Yield of
crude (%)
12
DMF/CH
3
CN
1
2
13t
13t
12t
12t
(T*T*)
(T*T*)
4
TOA (15)
TOA (15)
7
8
1.5
1.5
1:1.6
1:1.3
22
23
50
73
4
1
2t
12t
2t
12t
3
4
15
15
(T*T*T*)
(T*T*T*)
4
TOA (16)
TOA (16)
8
9
1.3
1.3
1.7:1
1:1.2
32
47
72
91
4
1
5
6
16
16
(T*T*T*T*)
(T*T*T*T*)
4
TOA (17)
TOA (17)
9
10
1.3
1.3
1:1.2
1:1.3
50
48
23
57
4
[
b]
4
(T*T*T*T*T*) TOA (2p)
11
1.3
1:1.3
48
55
12t
7
17
[
c]
4
(TTTTT) TOA (2)
38
Bz Bz
8
9
13a
13a
12c
12c
(C
(C
A
)
4
TOA (21)
TOA (21)
9
8
1.3
1.2
1:1
1:1.4
72
48
90
98
Bz Bz
A
)
4
Bz Bz
1
1
0
1
21
21
12t
12t
(T*C
(T*C
A
)
)
4
TOA (22)
TOA (22)
9
10
1.2
1.3
1:1.4
1:1.3
48
39
87
90
Bz Bz
A
4
Bz
Bz Bz
1
1
1
2
3
4
22
22
23
12c
12c
12c
(C T*C
A
)
4
TOA (23)
TOA (23)
10
10
11
12
1.1
1.2
1.2
1.2
1:1.4
1:1.4
1:1.4
1:1.4
69
48
48
48
78
89
82
84
Bz
Bz Bz
(C T*C
A
)
4
Bz Bz
Bz Bz
(C
C
Bz Bz
T*C
A
)
4
TOA* (24)
Bz Bz
[b]
(
4
T*C C T*C A ) TOA* (4p)
15
24
12t
[
c]
(TCCTCA)
4
TOA (4)
23
iBu Bz
(
G
(
C
)
4
TOA (25)
7.5
1.4
1:1.2
26
99
64
16
13c
12g
[
d]
[c]
4
GC) TOA (25d)
Bz iBu Bz
1
1
1
2
2
7
8
9
0
1
25
26
27
28
29
12c
12g
12c
12g
12c
(C
G
C
)
4
TOA (26)
TOA (27)
8
9
1.3
1.3
1:1.2
1:1.2
44
48
75
82
iBu Bz iBu Bz
(
G C G C
)
4
[
c]
c]
(
GCGC)
4
TOA (6)
55
Bz iBu Bz iBu Bz
(C
G
C
G
C
)4TOA (28)
10
1.3
1.3
1:1.2
1:1.1
48
48
70
iBu Bz iBu Bz iBu Bz
11
85
(
G
C
G
C
G
C
)
4
TOA (29)
TOA (7)
[
(
GCGCGC)
4
77
Bz iBu Bz iBu Bz iBu Bz
(C
G
C
G
C
G
C
)4TOA (30)
12
1.2
1.2
1:1.4
1:1
48
48
90
iBu Bz iBu Bz iBu Bz iBu Bz
[b]
13
(G
C
G
C
G
C
G
C
4
) TOA (8p)
80
22
30
12g
[
c]
5
7
(
4
GCGCGCGC) TOA (8)
[
a]
[b]
Asterisk denotes thymidine nucleotide with protected backbone; Protecting group-bearing nucleobase and with protected backbone.
The letter 'p' denotes
[
c]
[d]
protected hybrid. Yield of fully deprotected hybrid after HPLC purification.
The letter 'd' denotes fully deprotected hybrid.
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European Journal of Organic Chemistry
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FULL PAPER
[36,44]
For hexamer hybrid 7, the yield of protected crude was
depends on the structure of the non-DNA part.
We
8
5% (96% coupling yield per arm) and 20% for the overall chain
expected the TOA hybrids to show hybridization properties that
are more strongly dominated by the DNA strands than the
assembly process. The protected form of octamer hybrid 8 was
obtained in 16% after 24 steps, and fully deprotected 8 with its
[36]
hybrids with large hydrophobic cores reported recently.
32 nucleotides in 11% yield after HPLC. Figure 5 shows the
Several DNA hybrids with aromatic cores have been found to
form nanoporous material irreversibly upon addition of
HPLC chromatogram, a MALDI-TOF mass spectrum and an
NMR spectrum of 8. The NMR spectrum was acquired at
elevated temperature, as the strong propensity for self-assembly
manifested itself in the room temperature spectrum through
broad signals.
[
34,35,39]
magnesium salts.
hybrid 25 (GC) TOA (25d) showed no melting transition in
buffered solution containing 150 mM NaCl. Upon addition of
MgCl , a very modest amount of a precipitate was formed below
0 °C, which fully re-dissolved at room temperature (see Figure
S53c of the SI).
Figure 6 shows UV-melting curves for the all-GC hybrids 6,
and 8. In each case, the sample was fully denatured with
NaOH at 90 °C, followed by neutralization with acetic acid,
addition of triethylammonium actetate buffer, NaCl and MgCl
The deprotected version of dimer
4
2
a)
1
7
1
0
20
30
40
[min]
2
.
Upon cooling and subsequent re-heating, only weak transitions
were observed for tetramer hybrid 6 (Figure 5a). Hexamer
hybrid 7 gave stronger and more cooperative UV-transitions,
with clear hysteresis between cooling and heating curves, and
b)
M-2H]2-
[
5
049
[
1
M-H]-
0095
m
formal T values of 35 and 57 °C. This suggested that large
structures were forming that were slow to dissociate on the time
scale of the experiment. Further, a well visible precipitate
formed upon cooling, which all but fully dissolved upon re-
heating. For hybrid 8 with its strongly pairing octamer arms,
sharp and cooperative transitions were measured, with formal
melting points of 45 and 63 °C. Cooling was accompanied by an
apparent hypochromicity of 50%, which was again the result of
decreased absorbance base stacking, loss of soluble material
through precipitation, and scattering of light by the now turbid
mixture. Also, the material fully redissolved upon heating,
despite the high concentration of magnesium ions in the solution.
4
000
6000
8000
10000
m/z
c) H6 (C), H8 (G)
H1′, H5 (C)
8
.0
7.6
7.2
6.8
6.4
6.0 ppm
Figure 5. Analytical data for purified octamer hybrid (GCGCGCGC)
a) HPLC chromatogram (same conditions as in Figure 4a). (b) MALDI-TOF
mass spectrum after single-stage HPLC, and (c) low-field region of H NMR
spectrum of 8 (0.02 mM solution in D O, 700 MHz, 70°C).
4
TOA (8).
(
The melting point of the linear control duplex (GCGCGCGC)
9 °C under the same buffer conditions (Figure S62, Supporting
Information).
2
is
1
5
2
The assembly of molecules or particles with multiple DNA
strands on their surface through Watson-Crick base pairing
Figure 6. UV-Monitored assembly and disassembly results for self-complementary DNA hybrids 6, 7 and 8, measured at 10, 8 or, 5 μM hybrid concentration,
respectively, in 10 mM TEAA buffer, pH 7.0, 150 mM NaCl and 100 mM MgCl , detected at 260 nm. Cooling curves (open circles) and heating curves (filled
2
circles) were acquired at 0.5 °C/min. For additional melting curve data, including curves acquired under other salt conditions, see Figure S54-S58 in the
Supporting Information.
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European Journal of Organic Chemistry
10.1002/ejoc.201700686
FULL PAPER
Reversible formation of a material was also observed for the
mixture of the homooligomer hybrids 2 and 3. Figure 7 shows
the UV profiles, together with photographs of cuvettes containing
nanoporous, as they can take up small molecules efficiently, and
in doing so can produce three-dimensional lattices with
photoactive chromophores.
concentrated solutions of the hybrids with (dT)
5
and (dA)
5
arms
on the TOA core. In the presence of MgCl
2
, a voluminous
precipitate formed upon cooling. Still, even here the solid fully
re-dissolved upon warming to room temperature (25 °C).
Figure 8. Photographs of samples of hybrids 7 and 8 prior to and after uptake
of ethidium bromide (EB) from aqueous solution. a) Materials formed from
aqueous solution at a 0.3 mM concentration of either hybrid in 10 mM TEAA
2
buffer, 150 mM NaCl, 100 mM MgCl , after annealing from 85 to 4°C in 12 h,
viewed at ambient light. b) The same samples after aspirating the mother
liquor, and adding a 3 mM solution of ethidium bromide in water. c) The
hybrid material after aspirating the EB solution. d) Picture of the same
samples as in c) under UV-illumination (254-365 nm). See the Supporting
Information for further experimental details.
Figure 7. Reversible formation of a material from hybrids with homopolymer
sequences. (a) Thermal association and melting profiles of
containing a mixture of (TTTTT) TOA (2) and (AAAAA) TOA (3) at 2 μM hybrid
concentration and cooling/heating rates of 0.5 °C/min (10 mM TEAA buffer, pH
, 150 mM NaCl). Blue symbols: cooling and heating curve in magnesium-
free buffer; black and red symbols: buffer plus 100 mM MgCl (cooling: black
a solution
4
4
7
Table 3. Encapsulation of ethidium bromide into hybrid material formed by 5,
2
7
or 8.
crosses, heating: red circles). Cooling and heating curves were measured
immediately after each other. (b) Photographs of 2 mM hybrid solutions at
Efficiency of material
formation (%)
Molar ratio hybrid/guest
Hybrid
[a]
[b]
in material
5
°C, after annealing from 85 °C and storing at 5 °C for 5 h. The compound
(CG)
4
TOA (5)
TOA (7)
90
87
86
1 : 1.9
1 : 7.8
1 : 8.9
numbers of hybrids in each sample are noted above the tubes. (c) Same
samples as in (b), but after 1 h at 25 °C. For additional control experiments
performed under melting curve conditions (2 µM hybrid concentration), see
Figures S59-S61 of the Supporting Information.
(
GCGCGC)
GCGCGCGC)
4
(
4
TOA (8)
[
a]
Percentage of hybrid that formed a solid from a 300 µM solution of the
hybrid in buffer (10 mM TEAA, 150 mM NaCl) containing 100 mM MgCl , after
annealing from 85 to 4°C in 12 h. Uptake from a 3 mM solution of ethidium
2
[
b]
Finally, we performed exploratory experiments on the
uptake of small molecules into the material formed by TOA
hybrids with self-complementary DNA arms. For this, 300 µM
bromide (EB), as determined by UV/Vis-spectrophotometry.
solutions of 5, 7, or 8 in buffer solution were treated with MgCl
2
and assembly of materials was induced by heating to 85 °C,
followed by cooling to 4 °C over the time course of 12 h. The
materials thus formed (Figure 8a) were washed with assembly
buffer and then treated with a 3 mM solution of ethidium bromide
in water. Either of the hybrids assembled with similar efficiency
Conclusions
Synthetically, our results show that solution-phase
syntheses of branched oligonucleotide hybrids with arms long
enough for stable duplex formation, are feasible, even if four
strands are assembled in parallel. Unlike solid-phase synthesis,
our optimized protocol uses long reaction times and a modest
excess of phosphoramidites of approx. 1.5-3 equiv per chain.
Handling loss, poor solubility, and 'overcoupling' caused by
detritylation are the most significant challenges during chain
extension that can be overcome by a proper choice of solvents
and amount of activator. Specifically, we have found that
increasing the amount of phosphoramidite by 0.25 equivalents
(
Table 3), but the amount of intercalator taken up post-assembly
from the aqueous solution mirrored the length of the DNA arms,
with 8 taking up close to the eight equivalents expected for
intercalation between every other base pair (nearest neighbor
exclusion principle). Further, the ethidium bromide guest was
brightly fluorescent (Figure 8d), strongly suggesting that the
chromophores were indeed intercalated and not aggregated.
Together this data suggest that the biohybrid materials are
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European Journal of Organic Chemistry
10.1002/ejoc.201700686
FULL PAPER
after each coupling cycle, increasing the DMF content in the
coupling mixture up to 50% for long sequences, and reducing
the tetrazole concentration to no more than 1.2 equiv over the
phosphoramidite for long coupling times, all help to achieve high
yields. With this, branched oligonucleotides with up to 32
nucleotides total are obtained without chromatography during
chain assembly in an easy-to-scale up procedure that avoids
supports. The results show that a wide range of different
sequences can be produced with minimal set-up costs, as no
dimer building blocks have to be prepared.
combined organic layers were concentrated in vacuo, the residue was
coevaporated three times from toluene and redissolved in CH
2 2
Cl (5 mL),
followed by precipitation with MTBE (45 mL) and isolation by
centrifugation.
dissolving in CH
remaining solid, which was washed with CH
This precipitation was repeated twic, followed by
Cl (5 mL). The supernatant was separated from any
Cl (2× 2 mL). Evaporation
2
2
2
2
yielded a crude that was used for deprotection. The protected hybrid
was dissolved in CH Cl (20 mL) with H O (30 µL, 1.7 mmol), followed by
2
2
2
2 2
addition of dichloroacetic acid (DCA, 6% in CH Cl , 26 mL). After 15 min,
methanol (15 mL) was added. The solution was then concentrated, and
the detritylated hybrid was isolated by precipitation with MTBE (30 mL)
2 2 3
and centrifugation, redissolving in CH Cl /CH OH (5 mL, 3/1, v/v) and
With
the
adamantane
tetraol
core,
branched
precipitation with MTBE (30 mL). This process was repeated twice.
Remaining protecting groups were removed by treating with ammonium
hydroxide (25%, 10 mL) for 5 h at 55 °C. Evaporation yielded crude 1
oligonucleotides behave more DNA-like than the known hybrids
with more lipophilic cores. Even with long DNA sequences as
arms, the TOA hybrids assemble into materials reversibly,
without being trapped in kinetically blocked, insoluble or
irreversible assemblies. These properties bode well for the
formulation of biomacromolecules in the three-dimensional
networks thus formed. Studies on new inclusion materials are
under way in our laboratories.
(216 mg). Cartridge purification on Sep-Pak C18 with a gradient of
CH CN (0-25%) in 10 mM NH Ac buffer, led to elution of 1 at 8% CH CN.
3
4
3
The title compound was obtained in a yield of 29.1 mg (9.8 μmol, 38%).
A small sample was purified by HPLC, using a gradient of CH
in 35 min) in 10 mM TEAA buffer, with elution of 1 at t
3
CN (1-30%
= 16 min.
R
-
128 32 52 8
MALDI-TOF-MS m/z calcd for C110H N O P [M-H] 2977, obsd 2978.
Extension Cycle (General Protocol A). The following is the chain
Bz
extension cycle leading to (A
)
4
TOA (13a).
This protocol is
Experimental Section
representative. The specifics of each individual coupling are given below,
for each intermediate or target compound. A sample of tetraol 9 (40 mg,
0.2 mmol) was mixed with the phosphoramidite (12a, 1.37 g, 1.5 mmol, 8
equiv), and molecular sieves (3 Å, 10 beads) were added. The mixture
was dried for 2 h at 45 °C and 0.001 mbar. The flask was flushed with
argon and sealed with a septum. Then, DMF (5 mL) and tetrazole
General. All HPLC purifications were performed on a Nucleosil C18
column (5 μm, 250 x 4.6 mm) from Macherey-Nagel (Düren, Germany).
A mixture of CH CN and triethylammonium acetate buffer (TEAA, 0.01 M,
3
pH 7) was used as eluant, with a flow rate of 1 mL/min and a column
temperature of 55 °C and detection at 260 nm. MALDI-TOF mass
solution (5.3 mL of a commercial 0.45 M solution in CH CN, known as
3
spectra were measured in the linear negative mode with
a
"activator solution" for DNA synthesis, 12 equiv) were added. The flask
matrix/comatrix mixture of 2,4-6-trihydroxyacetophenone/diammonium
citrate. UV-melting curves were measured at pH 7, with a det of 260 nm
and a cooling/heating rate of 0.5°C/min.
was placed in an ultrasonic bath for two minutes until the components
were fully dissolved. The flask was placed on an orbital shaker for 30
min and then stored at 5 °C for 24 h. Then, tert-butyl hydroperoxide (216
µL, 5.5 M in decane, 0.24 mmol, 12 equiv) was added. The reaction
1
(
,3,5,7-Tetrakis(4-hydroxyphenyl)adamantane (10). A solution of BBr
317 µL, 0.32 mmol) in CH Cl (3 mL) was cooled to -60°C, and a
solution of 1,3,5,7-tetrakis(4-methoxyphenyl)adamantane (14.8 mg, 26
µmol) in CH Cl (1 mL) was added dropwise. The mixture was stirred for
h and then allowed to warm to 25 °C, followed by stirring for 19 h. The
mixture was then treated with CH OH (10 ml), followed by drying in
vacuo. This step was repeated twice. The crude product was washed
with CH Cl (5 mL), and the residue was dried in vacuo. Purification via
column chromatography (silica gel, 10 g, CH Cl /MeOH, 10/1, v/v)
yielded 11 mg (21 µmol, 81%) of the title compound (10) as an off-white
3
2 2
mixture was allowed to react for 15 min at 5 °C. After addition of CH Cl
(20 mL), the mixture was washed with phosphate buffer (20 mL, 0.2 M,
pH 7). The aqueous phase was removed and back-extracted with
2
2
[
40]
CH
filtered and concentrated in vacuo. The residue was coevaporated twice
from MeOH, dissolved in a minimal amount of CH Cl (approx. 10 mL),
followed by precipitation with MTBE (45 mL) and separation by
centrifugation. The sequence of dissolving and precipitation was
2 2 2 4
Cl (3×15 mL). The combined organic layers were dried over Na SO ,
2
2
3
3
2
2
2
2
repeated three times. The solid thus obtained was treated with MeOH (5
mL) for 5 min in an ultrasonic bath, followed by centrifugation (5 min).
2
2
1
solid: R
1.97 (s, 12 H), 6.74 (d, J = 8.7 Hz, 8 H), 7.27 (d, J = Hz, 8.7, 8 H);
NMR (75.5 MHz, CD OD) δ = 156.3, 142.5, 127.0, 115.9, 39.8; HRMS
f
2
= 0.48 (CH Cl
2
/MeOH 10/1, v/v); H NMR (300 MHz, CD
3
OD) δ
The supernatant was discarded, and the residue was dissolved in CH
(4 mL), followed by precipitation with MTBE (45 mL) and centrifugation.
The residue was dissolved in CH Cl (5 mL), separated from any
2 2
Cl
3
3
13
=
C
3
2
2
-
(
ESI-TOF) m/z calcd for C34
H O
32 4
503.2223, obsd [M-H] 503.2217.
remaining particles and the solution concentrated in vacuo to yield the
fully protected hybrid (720 mg). Then, the DMT groups were removed by
dissolving in CH
2 2 2
Cl (4 mL) and addition of H O (47 µL, 2.6 mmol) and
(
CG) TPA (1) To assemble 1 via the H-phosphonate block condensation
4
[
36,39]
dichloroacetic acid (6 mL, 6% in CH Cl ). After 10 min, the reaction was
2
2
method,
414 mg, 0.36 mmol, 14 equiv) and molecular sieves (3 Å, seven beads)
was dried for 2 h at 45 °C and 0.001 mbar, followed by flushing with
argon. A solution of pyridine in CH CN (3 mL, 4:1, v/v) was added, and
a mixture of 10 (26 μmol, 1 equiv), H-phosphonate dimer 11
quenched by addition of methanol (3 mL). The solution was then
concentrated by rotary evaporation to remove the dichloromethane, and
the detritylated hybrid was precipitated by addition of MTBE (15 mL).
The precipitate was isolated by centrifugation, redissolved in a minimal
(
3
the mixture was cooled to -40 °C. Then, diphenyl chlorophosphate (112
µL, 0.54 mmol) was added, and the mixture was stirred at -40 °C for 120
min. A solution of iodine in pyridine (541 µL, 1 M) was added, followed
after 1 min by H
at -40 °C and then for 30 min at room temperature. After addition of
CH Cl (15 mL), the solution was washed with aqueous sodium
thiosulfate (8 mL, 10%, w/w) and phosphate buffer (7 mL, 0.2 M, pH 7).
The aqueous phase was extracted with CH Cl (3 × 15 mL). The
2 2 3
volume of CH Cl /CH OH (1 mL, 4:1, v/v) and precipitated again by
addition of MTBE (15 mL). This process was repeated two more times to
give 13a (418 mg, 0.2 mmol, quant). The hybrid thus obtained was used
for the next coupling cycle without further purification.
2
O (32 µL, 1.8 mmol). The mixture was stirred for 10 min
2
2
Full Deprotection with Aqueous Ammonia (General Protocol B).
Fully deprotected hybrids were obtained by treating the DMT-free hybrid
2
2
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European Journal of Organic Chemistry
10.1002/ejoc.201700686
FULL PAPER
with ammonium hydroxide (30-25%, 1 mL per 10 mg) for 5 h at 55 °C.
(broad peaks, mixture of diastereomers); MALDI-TOF-MS m/z 3741 [M-
-
Then, excess ammonia was removed by passing a stream of N
2
onto the
4
H-CE] . (CG) TOA (5). The synthesis of 5 was performed according to
solution until the sample was odorless. The remaining solution was
lyophilized to yield the crude hybrid. The crude was then purified on a
General Protocol B, starting from 5p (40 mg, 10.5 µmol). One fraction
was purified by HPLC, using a gradient of CH CN (1-15% in 30 min), t
of 5 = 16.6 min. Alternatively, cartridge purification used a gradient of
CH CN in 10 mM NH Ac buffer, 0-20%. Hybrid 5 eluted at 1.5-5%, and
was obtained in an overall yield of 25 mg (9.4 μmol, 67%). MALDI-TOF-
3
R
3
Sep-Pak C18 cartridge, using a gradient of 0-25% CH CN in 10 mM
NH Ac buffer or by reversed-phase HPLC (C18 column, 250 × 20 mm),
4
3
4
3
using a gradient of 1-15% CH CN in 10 mM TEAA buffer at 55°C.
-
112 32 52 8
MS m/z calcd for C86H N O P [M-H] 2672, obsd 2673.
Bz
(
4
G ) TOA (13g). General Protocol A was employed, starting from 9
300 mg, 1.5 mmol), 12g (10 g, 12 mmol) in DMF (19 mL) and tetrazole
iBu Bz
(
(G
4
C ) TOA (25). Hybrid 25 was prepared following General Protocol
solution (0.45 M, 31.9 mL, 14.3 mmol) with 48 h coupling time at 5°C,
followed by oxidation with t-BuOOH (2.8 mL, 15.6 mmol), DMT
A, starting with coupling of 13c (377 mg, 0.19 mmol) and 12g (1.2 g, 1.4
mmol, 7.5 equiv) in DMF (3.2 mL) with tetrazole solution (3.8 mL, 1.7
mmol, 1.35 equiv) for 26 h at 5 °C, followed by oxidation with t-BuOOH
deprotection with CH
in CH Cl , 50 mL), and precipitations from CH
v/v) with MTBE (45 mL). Yield 2.85 g (1.42 mmol, 95%).
121.5 MHz, DMSO-d ): -7.58 (broad peak: mixture of
diastereomers); MALDI-TOF-MS m/z calcd for C78
2
Cl
2
(25 mL), water (336 μL) and DCA solution (6%
2
2
2
Cl /MeOH (4 × 5 mL, 3/2,
2
(389 µL, 2.2 mmol, 1.5 equiv), DMT removal with CH
(55 μL) and DCA solution (6% in CH Cl , 11 mL), and precipitations from
CH Cl /MeOH (5 mL, 3:2, v/v) with MTBE (45 mL). The detritylated
hybrid 25 was isolated in a yield of 715 mg (188 µmol, 99%). MALDI-
2 2
Cl (5 mL), water
31
P NMR
2
2
(
6
δ
=
2
2
-
H
100
N
(G)
24
O
32
P
4
[M-H]
TOA (14g).
Compound 14g was obtained from 13g using General Protocol B. Hybrid
4g was purified by HPLC, using a gradient of CH CN (1-15% in 45min),
-
-
-
2
008, obsd 2006 [M-H] and 1951 [M-H-CE] .
4
4
TOF-MS m/z 3737 [M-H-CE] . (GC) TOA (25d). Hybrid 25d was
prepared following General Protocol B, starting from 25 (180 mg, 47.5
µmol). An analytical sample was purified by HPLC, using a gradient of
1
3
-
t
R
(14g) = 21 min, MALDI-TOF-MS m/z calcd for C50
H
64
N
20
O
28
P
4
[M-H]
CH
purification used a gradient of CH
5d eluted from 1.6% to 5% CH
MALDI-TOF-MS m/z calcd for C H N O P [M-H] 2672, obsd 2672.
3
CN (1-15% in 50 min), t
R
= 21.5 min. Alterantively, cartridge
CN in 10 mM NH Ac buffer, 0-20%,
CN; yield 82 mg (30.6 μmol, 64%).
1516, obsd 1515.
3
4
2
3
-
Bz
(
C
)
4
TOA (13c). The synthesis followed General Protocol A, starting
from 9 (40 mg, 0.2 mmol) and 12c (1.33 g, 1.6 mmol, 8 equiv) in DMF
3.5 mL) and tetrazole solution (5.3 mL, 2.4 mmol, 1.5 equiv) for 44 h at
°C, followed by oxidation with t-BuOOH (0.4 mL, 2.4 mmol, 1.5 equiv),
DMT deprotection with CH Cl (5 mL), water (44 μL) and DCA solution
6% in CH Cl , 7 mL), and precipitations from CH Cl /MeOH (4 mL, 3:1,
86 112 32 52 8
(
5
4
(T*T*) TOA (15). The extension cycle leading to 15 was performed
according to General Protocol A, starting with cupling with 13t (476 mg,
292 µmol) and 12t (1.7 g, 2.3 mmol, 8 equiv) in DMF (6 mL) and tetrazole
solution (7.8 mL, 3.5 mmol, 1.5 equiv) for 23 h at 5 °C, followed by
oxidation with t-BuOOH (637 µL, 3.5 mmol, 1.5 equiv), DMT removal with
2
2
(
2
2
2
2
v/v) with MTBE (a 45 mL). Detritylated hybrid 13c was obtained in a
yield of 241 mg (121 µmol, 61%). MALDI-TOF-MS m/z calcd for
CH
and precipitations from CH
The detritylated hybrid 15 was obtained in a yield of 654 mg (214 µmol,
2
Cl
2
(15 mL), water (53 μL) and DCA solution (6% in CH
2 2
Cl , 16 mL),
-
-
-
C H N O P
86 92 16 32 4
[M-H] 1984, obsd 1984 [M-H] , 1931 [M-H-CE] .
2
Cl /MeOH (5 mL, 1:1, v/v) with MTBE (45 mL).
2
(
C) TOA (14c). Compound 14c was obtained from 13c using General
4
-
31
Protocol B. MALDI-TOF-MS m/z calcd for C46
H N O P
64 12 28 4
[M-H] 1355,
73%). P NMR (121.5 MHz, DMSO-d
mixture of diastereomers); MALDI-TOF-MS m/z 3004 [M-H-CE] .
TT) TOA (15d). General Protocol B was employed to obtaind 15d,
followed by purification via HPLC, using a gradient of CH CN (1-15% in
5 min), t = 26 min, MALDI-TOF-MS m/z calcd for C90 [M-
6
): δ = 2.5, -7.2, -7.8 (broad peaks;
-
obsd 1354.
(
4
4
(T*) TOA (13t). The synthesis followed General Protocol A, starting from
3
4
R
120 16 60 8
H N O P
9
(20 mg, 0.1 mmol) and 1t (483 mg, 0.65 mmol, 6.5 equiv) in DMF (1.5
-
H] 2632, obsd 2633.
mL) and tetrazole solution (2.16 mL, 0.97 mmol, 1.5 equiv) for 21 h at
°C, followed by oxidation with t-BuOOH (177 µL, 0.97 mmol, 1.5 equiv),
DMT deprotection with CH Cl (3 mL), water (27 μL) and DCA solution
6% in CH Cl , 3.5 mL), and precipitations from CH Cl /MeOH (5 mL, 3:1,
v/v) with MTBE (45 mL). Hybrid 13t was isolated in a yield of 110 mg
5
Bz Bz
2
2
4
(A A ) TOA (18). General Protocol A was used, starting from 13a (102
(
2
2
2
2
mg, 49 µmol) and 12a (378 mg, 441 µmol, 9 equiv) in DMF (1 mL) and
tetrazole solution (1.27 mL, 0.57 mmol, 1.3 equiv) for 47 h at 5 °C,
followed by oxidation with t-BuOOH (120 µL, 0.66 mmol, 1.5 equiv),
31
(67.5 µmol, 68%).
6
P NMR (121.5 MHz, DMSO-d ): δ = -7.48 (broad
peak: mixture of diastereomers);
MALDI-TOF-MS m/z calcd for
detritylation with CH
CH Cl , 4 mL), and precipitations from CH
MTBE (15 mL). Hybrid 18 was isolated in a yield of 169 mg (43 µmol,
7%). (AA) TOA (18d). The deprotected hybrid was obtained from 18
using General Protocol B. Crude hybrid was purified by HPLC, using a
2
Cl
2
(2.5 mL), water (11 μL) and DCA solution (6% in
-
-
-
C H N O P
62 80 12 32 4
[M-H] 1628, obsd 1628 [M-H] , 1574 [M-H-CE] .
2
2
2
Cl /MeOH (1 mL, 4:1, v/v) with
2
Bz
8
4
(
A
)
4
TOA (13a). General Protocol A was followed verbatim (vide supra),
and 13a was isolated in a yield of 418 mg (0.2 mmol, quant). MALDI-
-
-
gradient of CH CN (1-15% in 45 min), t = 24.4 min, MALDI-TOF-MS m/z
TOF-MS m/z calcd for C90
H
92
N
24
O
28
P
4
[M-H] 2080, obsd 2080 [M-H] ,
TOA (14a). General Protocol B was employed to
obtained 14a, followed by purification via HPLC, using a gradient of
CH CN (1-15% in 45 min) in 100 mM TEAA buffer, t = 37.5 min, MALDI-
[M-H] 1452, obsd 1452.
3
R
-
-
calcd for C90
H
112 40 44 8
N O P [M-H] 2704, obsd 2706.
2026 [M-H-CE] . (A)
4
Bz Bz
3
R
4
(C A ) TOA (21). Hybrid 21 was prepared following General Protocol
-
TOF-MS m/z calcd for C50
H
64
N
20
O
24
P
4
A, starting from 13a (1 g, 485 µmol) and 12c (3.2 g, 3.9 mmol) in DMF
7.3 mL) and tetrazole (10.4 mL, 4.68 mmol, 1.2 equiv.) for 48 h at 5 °C,
followed by oxidation with t-BuOOH (922 µL, 5.2 mmol, 1.3 equiv.), DMT
removal with CH Cl (4 mL), water (104 μL) and DCA solution (6% in
CH Cl , 8 mL), and precipitations. The detritylated hybrid 21 was
isolated in a yield of 1.8 g (480 µmol, 98 %). (CA) TOA (21d).
Compound 21d was obtained from 21 using General Protocol B, followed
by purification via HPLC, using a gradient of CH CN (1-15% in 45 min), t
20.8 min, MALDI-TOF-MS m/z calcd for C86
obsd 2606.
(
Bz iBu
(
C
G
4
) TOA (5p). The hybrid 5p, where "p" denotes the protected
form of 5, was prepared following General Protocol A via coupling of 13g
2.8 g, 1.4 mmol) and 12c (10.6 g, 12.8 mmol, 9 equiv) in DMF (22 mL)
2
2
(
2
2
and tetrazole solution (36.9 mL, 16.6 mmol, 1.3 equiv) for 48 h at 5 °C,
followed by oxidation with t-BuOOH (3.5 mL, 19.2 mmol, 1.5 equiv), DMT
4
deprotection with CH
in CH Cl , 70 mL), and precipitations from CH
with MTBE (45 mL). The detritylated hybrid was isolated in a yield of 4 g
2 2
Cl
(26 mL), water (229 μL) and DCA solution (6%
3
R
-
=
112 32 48 8
H N O P [M-H] 2608,
2
2
2
Cl /MeOH (5 mL, 3:2, v/v)
2
31
(
6
1.14 mmol, 81%). P NMR (121.5 MHz, DMSO-d ): δ = 2.71, -7.3, -7.9
This article is protected by copyright. All rights reserved.

European Journal of Organic Chemistry
10.1002/ejoc.201700686
FULL PAPER
Bz iBu Bz
Bz Bz Bz Bz
(
C
G
C
)
4
TOA (26). General Protocol A was used, starting from 25
(A
A
A
A
)
4
TOA (20). The extension cycle leading to 20 was
(
715 mg, 188 µmol) and 12c (1.25 g, 1.5 mmol, 8 equiv) in DMF (3.7 mL)
performed according to General Protocol A, starting with coupling with 19
(133 mg, 23 µmol) and 12a (215 mg, 250 µmol, 11 equiv) in DMF (579
µL) and tetrazole (724 µL, 0.32 mmol, 1.3 equiv) for 48 h at 5 °C,
followed by oxidation with t-BuOOH (68 µL, 0.37 mmol, 1.5 equiv),
and tetrazole (4.35 mL, 1.96 mmol, 1.3 equiv) for 44 h at 5 °C, followed
by oxidation with t-BuOOH (356 µL, 1.96 mmol, 1.5 equiv), detritylation
with CH
mL), and precipitations from CH
45 mL). Hybrid 26 could be isolated with a yield of 744 mg (137 µmol,
2
Cl
2
(3.5 mL), water (38 μL) and DCA solution (6% in CH
2 2
Cl , 13
2
Cl /MeOH (4 mL, 3:1, v/v) with MTBE
2
washing with MeOH (5 mL), DMT removal with CH
μL) and DCA solution (6% in CH Cl , 10 mL), and precipitations from
CH Cl /MeOH (2 mL, 2:1, v/v) with MTBE (10 mL). Hybrid 20 was
obtained in a yield of 130 mg (16 µmol, 73%). (AAAA) TOA (20d).
General Protocol was employed to obtained 20d, followed by
purification via HPLC, using a gradient of CH CN (1-15% in 45 min), t
208 80 84
0 min, MALDI-TOF-MS m/z calcd for C170H N O P16 [M-H] 5210,
2 2
Cl (2 mL), water (5
(
2
2
7
2
5%). (CGC)
6d followed by purification via HPLC, using a gradient of CH
= 24 min, MALDI-TOF-MS m/z calcd for C126
M-H] 3831, obsd 3832.
4
TOA (26d). General Protocol B was employed to obtained
2
2
3
CN (1-15%
4
in 45 min), t
[
R
H N O P
168 44 76 12
B
-
3
R
=
-
3
obsd 5211.
4
(T*T*T*) TOA (16). The synthesis followed General Protocol A, starting
from 15 (309 mg, 101 µmol) and 12t (678 mg, 0.9 mmol, 9 equiv.), in
DMF (2.1 mL) and tetrazole (2.6 µL, 1.18 mmol, 1.3 equiv) for 47 h at
iBu Bz iBu Bz
(G
C
G
4
C ) TOA (27). Hybrid 27 was prepared following General
5
°C, followed by oxidation with t-BuOOH (248 µL, 1.36 mmol, 1.5 equiv),
washing with MeOH (4 mL), DMT removal with CH Cl (3 mL), water (26
μL) and DCA solution (6% in CH Cl , 6 mL), and precipitations from
CH Cl /MeOH (4 mL, 2:1, v/v) with MTBE (10 mL). The detritylated
hybrid 16 was isolated in a yield of 414 mg (92 µmol, 91%). (TTT) TOA
16d). General Protocol B was employed to obtained 16d, followed by
Protocol A, starting from 26 (745 mg, 137 µmol) and 12g (1.03 g, 1.2
mmol, 9 equiv) in DMF (3 mL) and tetrazole (3.6 mL, 1.6 mmol, 1.3
equiv) for 48 h at 5 °C, followed by oxidation with t-BuOOH (336 µL, 1.85
2
2
2
2
2
2
mmol, 1.5 equiv), DMT removal with CH
DCA solution (6% in CH Cl mL), and precipitations from
CH Cl /MeOH (5 mL, 8:1, v/v) with MTBE (45 mL). The detritylated
hybrid 27 was isolated in yield of 807 mg (109 µmol, 82%).
(GCGC) TOA (6). Hybrid 6 was prepared following General Protocol B,
starting from 27 (90 mg, 12 µmol). The product 6 was purified by HPLC,
using a gradient of CH CN (1-25% in 60 min), with elution of hybrid 6 at
tR = 25 min, yield 34 mg (6.6 µmol, 55%). MALDI-TOF-MS m/z calcd for
2 2
Cl (2 mL), water (22 μL) and
4
2
2
, 8
(
2
2
purification via HPLC, using a gradient of CH
3
CN (1-15% in 45 min), t
R
-
a
(
16d) = 27 min, MALDI-TOF-MS m/z calcd for C130
172 24 88
H N O P12 [M-H]
4
3849, obsd 3849.
3
Bz Bz Bz
(
4
A A A ) TOA (19). General Protocol A was employed, starting from
-
C
162 208 64 100
H N O P16 [M-H] 5146, obsd 5145.
18 (129 mg, 32.5 µmol) and 12a (279 mg, 320 µmol, 10 equiv) in DMF
(
0.8 mL) and tetrazole solution (0.94 mL, 0.42 mmol, 1.3 equiv) for 47 h
at 5 °C, followed by oxidation with t-BuOOH (89 µL, 0.48 mmol, 1.5
equiv), washing with MeOH (5 mL), DMT deprotection with CH Cl (2 mL),
water (7 μL) and DCA solution (6% in CH Cl , 3 mL), and precipitations
from CH Cl /MeOH (1 mL, 4:1, v/v) with MTBE (10 mL). Hybrid 19 could
be isolated with a yield of 174 mg (29 µmol, 92%). (AAA) TOA (19d).
General Protocol was employed to obtained 19d followed by
purification via HPLC, using a gradient of CH CN (1-15% in 45 min), t
9 min, MALDI-TOF-MS m/z calcd for C130
Bz
Bz Bz
(C T*C
4
A ) TOA (23). The synthesis followed General Protocol A,
2
2
starting from 22 (1.5 g, 279 µmol) and 12c (2.3 g, 2.79 mmol, 10 equiv.)
in DMF (4.9 mL) and tetrazole (6.95 mL, 3.1 mmol, 1.2 equiv) for 48 h at
5 °C, followed by oxidation with t-BuOOH (650 µL, 3.6 mmol, 1.3 equiv.),
DMT deprotection with CH
(6% in CH Cl , 20 mL), and precipitations. Detritylated hybrid 23 was
obtained in a yield of 1.8 g (248 µmol, 89%). (CTCA) TOA (23d).
Compound 23d was obtained from 23 using General Protocol B, followed
by purification via HPLC, using a gradient of CH CN (1-15% in 45 min), t
27.8 min, MALDI-TOF-MS m/z calcd for C162H N O P16 [M-H] 4982,
212 52 112
2
2
2
2
4
2 2 2
Cl (5 mL) and H O (60 µL) and DCA solution
B
2
2
3
R
=
4
-
2
160 60 64
H N O P12 [M-H] 3957,
obsd 3958.
3
R
-
=
Bz Bz
obsd 4983.
(
4
T*C A ) TOA (22). The extension cycle leading to 22 was performed
according to General Protocol A, starting with coupling with 21 (1.4 g,
49 µmol) and 12t (2.3 g, 3.1 mmol, 9 equiv.) in DMF (5.9 mL) and
3
4
(T*T*T*T*T*) TOA (2p). The hybrid 2p, where "p" denotes the protected
tetrazole (8.4 mL, 3.7 mmol, 1.2 equiv) for 48 h at 5 °C, followed by
oxidation with t-BuOOH (743 µL, 4.1 mmol, 1.3 equiv.), DMT removal
form of 2, was prepared following General Protocol A involving coupling
of 17 (205 mg, 35 µmol) and 12t (284 mg, 0.38 mmol, 11 equiv.), in DMF
(0.8 mL) and tetrazole (1.1 mL, 0.5 mmol, 1.3 equiv) for 48 h at 5 °C,
followed by oxidation with t-BuOOH (104 µL, 0.57 mmol, 1.5 equiv),
with CH
mL), and precipitations. Hybrid 21 was obtained in a yield of 1.6 g (310
µmol, 87%). (TCA) TOA (22d). General Protocol B was employed to
obtained 22d, followed by purification via HPLC, using a gradient of
CH CN (1-15% in 45 min), t = 26.4 min, MALDI-TOF-MS m/z calcd for
12 [M-H] 3825, obsd 3822.
2 2 2 2
Cl (5 mL), water (66 μL) and DCA solution (6% in CH Cl , 20
4
washing with MeOH (4 mL), DMT removal with CH
μL) and DCA solution (6% in CH Cl , 3 mL), and precipitations from
CH Cl /MeOH (5 mL, 3:1, v/v) with MTBE (45 mL). The detritylated was
isolated in a yield of 119 mg (19 μmol, 55%). (TTTTT) TOA (2). The
2 2
Cl (2 mL), water (3
2
2
3
R
2
2
-
C
126
164
H N
40 76
O P
4
synthesis of 2 was performed according to General Protocol B, starting
from 2p (119 mg, 19 µmol), followed by purification via HPLC, using a
4
(T*T*T*T*) TOA (17). The synthesis followed General Protocol A,
gradient of CH
µmol, 38%). MALDI-TOF-MS m/z calcd for C210
3 R
CN (1-20% in 65 min), t (2) = 37 min, yield 83 mg (13.2
starting from 16 (314 mg, 70 µmol) and 12t (512 mg, 0.7 mmol, 10 equiv),
in DMF (1.6 mL) and tetrazole (2 mL, 0.9 mmol, 1.3 equiv) for 48 h at
-
276 40 144
H N O P20 [M-H]
6
283, obsd 6280.
5
°C, followed by oxidation with t-BuOOH (191 µL, 1 mmol, 1.5 equiv),
washing with MeOH (4 mL), DMT deprotection with CH Cl (2 mL), water
10 μL) and DCA solution (6% in CH Cl , 3 mL), and precipitations from
CH Cl /MeOH (3 mL, 3:1, v/v) with MTBE (20 mL). Hybrid 17 was
isolated in a yield of 253 mg (40 µmol, 57%). (TTTT) TOA (17d). The
2
2
Bz Bz Bz Bz Bz
(
2
2
(A
A
A
A
A
)
4
TOA (3p).
The extension cycle leading to 3p
2
2
performed according to General Protocol A, starting with coupling with 20
(100 mg, 13 µmol) and 12a (133 mg, 0.15 mmol, 12 equiv.), in DMF (0.36
mL) and tetrazole (0.5 mL, 0.25 mmol, 1.3 equiv) for 48 h at 5 °C,
followed by oxidation with t-BuOOH (42 µL, 0.23 mmol, 1.5 equiv),
4
synthesis of 17d was performed according to General Protocol B, starting
from 17 (30 mg, 5 µmol). An analytical sample was purified by HPLC,
using a gradient of CH
TOF-MS m/z calcd for C170
3
CN (1-15% in 45 min), t
R
-
(17d) = 35 min, MALDI-
washing with MeOH (5 mL), DMT deprotection with CH
(2 μL) and DCA solution (6% in CH Cl , 4 mL), and precipitations from
CH Cl /MeOH (1 mL, 4:1, v/v) with MTBE (5 mL). Hybrid 3p was isolated
in a yield of 83 mg (8.6 µmol, 67 %). (AAAAA) TOA (3). General
2 2
Cl (2 mL), water
224 32 116
H N O P16 [M-H] 5066, obsd 5066.
2
2
2
2
4
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European Journal of Organic Chemistry
10.1002/ejoc.201700686
FULL PAPER
Protocol B was employed to obtained 3, starting from 3p (83 mg, 8.6
4
was isolated in a yield of 449 mg (35 µmol, 90%). (CGCGCGC) TOA
µmol), followed by purification via HPLC, using a gradient of CH
0% in 65 min), with elution of hybrid 3 at t = 31 min., yield 38 mg (5.8
µmol, 45%). MALDI-TOF-MS m/z calcd for C210
3
CN (1-
(30d). Hybrid 30d was prepared following General Protocol B, starting
from 30 (64 mg, 5 µmol). An analytical sample was purified by HPLC,
2
R
-
256 100 104
H N O P
20 [M-H]
using a gradient of CH
3 R
CN (1-15% in 45 min), t (30d) = 29 min, MALDI-
-
6463, obsd 6463.
352 108 172
TOF-MS m/z calcd for C274H N O P28 [M-H] 8776, obsd 8777.
Bz iBu Bz iBu Bz
iBu Bz iBu Bz iBu Bz iBu Bz
(
C
G
C
G
C
)
4
TOA (28). The extension cycle leading to 28 was
(G
C
G
C
G
C
G
C ) TOA (8p). The hybrid 8p, where "p"
4
performed according to General Protocol A, starting with coupling with 27
706 mg, 92 µmol) and 12c (736 mg, 915 µmol, 10 equiv) in DMF (2.1
mL) and tetrazole (2.6 mL, 1.19 mmol, 1.3 equiv) for 48 h at 5 °C,
denotes the protected form of 8, was prepared following General Protocol
A involving coupling of 30 (377 mg, 30 µmol) and 12g (322 mg, 383 µmol,
13 equiv) in DMF (1.5 mL) and tetrazole (1.5 mL, 0.56 mmol, 1.2 equiv)
for 48 h at 5 °C, followed by oxidation with t-BuOOH (104 µL, 0.7 mmol,
(
followed by oxidation with t-BuOOH (250 µL, 1.4 mmol, 1.5 equiv),
washing with MeOH (5 mL), DMT removal with CH
μL) and DCA solution (6% in CH Cl , 6 mL), and precipitations from
CH Cl /MeOH (1 mL, 5:3, v/v) with MTBE (10 mL). Hybrid 28 was
isolated in a yield of 608 mg (64 µmol, 70%). (CGCGC) TOA (28d). The
hybrid 28d was prepared following General Protocol B, starting from 28
90 mg, 9.5 µmol). An analytical sample was purified by HPLC, using a
gradient of CH CN (1-20% in 65 min), t = 30 min, MALDI-TOF-MS m/z
calcd for C198 20 [M-H] 6303, obsd 6308.
2
Cl
2
(4 mL), water (10
1.5 equiv), washing with MeOH (5 mL), DMT deprotection with CH
mL), water (4 μL) and DCA solution (6% in CH Cl , 4 mL), and
precipitations from CH Cl /MeOH (5 mL, 5:1, v/v) with MTBE (15 mL).
Hybrid 8p was isolated in yield of 350 mg (24 µmol, 80%).
(GCGCGCGC) TOA (8). General Protocol B was employed to obtained
8, followed by purification via HPLC, using a gradient of CH CN (1-15%
in 45 min), 8 t (8) = 34 min., yield 17.1 μmol, 57%. MALDI-TOF-MS m/z
calcd for C314
2 2
Cl (3
2
2
2
2
2
2
2
2
4
a
4
(
3
3
R
R
-
-
H
256
76 124
N O P
400 128 196
H N O P32 [M-H] 10093, obsd 10095.
Bz Bz
Bz Bz
(
4
C C T*C A ) TOA (24). General Protocol A was employed, starting
from 23 (1.6 g, 229 µmol, 1 equiv.) and 12c (2.1 g, 2.5 mmol, 11 equiv.)
in DMF (4.7 mL) and tetrazole (6.8 mL, 2.8 mmol, 1.2 equiv.) for 48 h at
Acknowledgements
5
°C, followed by oxidation with t-BuOOH (598 µL, 3.3 mmol, 1.3 equiv.),
detritylation with CH Cl (5 mL), H O (45 µL) and DCA solution (6 % in
CH Cl , 23 mL), and precipitations. Hybrid 24 was isolated in a yield of
.7 g (188 µmol, 82%). (CCTCA) TOA (24d). Compound 24d was
obtained from 24 using General Protocol B, followed by purification via
HPLC, using a gradient of CH CN (1-15% in 45 min), t = 30.8 min,
20 [M-H] 6139, obsd 6139.
The authors thank Shiliang He for discussions, Tanja Walter for
measuring melting curves, Peter Tremmel for the acquisition of
NMR spectra, and Helmut Griesser for a review of parts of the
manuscript. This work was supported by DFG (grant No. RI
2
2
2
2
2
1
4
3
R
1063/15-1 to C.R.).
-
260 64 124
MALDI-TOF-MS m/z calcd for C198H N O P
iBu Bz iBu Bz iBu Bz
Keywords: Oligonucleotides • DNA • solution-phase synthesis •
phosphoramidites • adamantane
(
G
C
G
C
G
4
C ) TOA (29). Hybrid 29 was prepared following
General Protocol A, starting with coupling of 28 (508 mg, 55.4 µmol) and
12g (511 mg, 609 µmol, 11 equiv) in DMF (1.5 mL) and tetrazole (1.7 mL,
0
.79 mmol, 1.3 equiv) for 48 h at 5 °C, followed by oxidation with t-
[1]
[2]
[3]
N. C. Seeman, Nature 2003, 421, 427-431.
BuOOH (166 µL, 0.9 mmol, 1.5 equiv), washing with MeOH (5 mL), DMT
removal with CH Cl (4 mL), water (11 μL) and DCA solution (6% in
CH Cl , 7 mL), and precipitations from CH Cl /MeOH (5 mL, 5:1, v/v) with
MTBE (15 mL). Detritylated hybrid 29 was isolated in a yield of 516 mg
47 µmol, 85%). (GCGCGC) TOA (7). Hybrid 7 was prepared following
General Protocol B, starting from 29 (68 mg, 6.2 µmol). Product 7 was
purified by HPLC, using a gradient of CH CN (1-20% in 60 min), with
elution of hybrid 7 at t = 28 min, yield 43 mg (5.6 µmol, 77%). MALDI-
TOF-MS m/z calcd for C238
B. Saccà, C. M. Niemeyer, Angew. Chem. Int. Ed. 2012, 51, 58-66.
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11198–11211.
2
2
2
2
2
2
[4]
F. A. Aldaye, A. L. Palmer, H. F. Sleiman, Science 2008, 321, 1795-
1799.
(
4
[5]
[6]
[7]
[8]
L. H. Tan, H. Xing, Y. Lu, Acc. Chem. Res. 2014, 47, 1881−1890.
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3
R
-
H
304 96 148
N O P24 [M-H] 7619, obsd 7623.
[9]
Bz Bz
Bz Bz
4
(T*C C T*C A ) TOA (4p). The synthesis followed General Protocol
A, starting from 24 (1.5 g, 173 µmol,) and 12t (1.6 g, 2.1 mmol, 12 equiv.)
in DMF (3.9 mL) and tetrazole (6.6 mL, 2.5 mmol, 1.2 equiv.) for 48 h at
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5
°C, followed by oxidation with t-BuOOH (492 µL, 2.7 mmol, 1.3 equiv.),
DMT deprotection with CH Cl (6 mL), H O (22 µL), and DCA (16 mL,
), and precipitations. Hybrid 4p was isolated in a yield of
.68 g (146 µmol, 84%). (TCCTCA) TOA (4). General Protocol B was
[12] K. L. Agarwal, H. Büchi, M. H. Caruthers, N. Gupta, H. G. Khorana, K.
Kleppe, A. Kumar, E. Ohtsuka, U. L. Rajbhandary, J. H. Van de Sande,
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2
2
2
6
1
2 2
% in CH Cl
4
employed to obtained 4, followed by purification via HPLC, using a
gradient of CH CN (1-15% in 45 min), t (4) = 34 min, yield 40 µmol, 23%.
MALDI-TOF-MS m/z calcd for C238H N O P24 [M-H] 7353, obsd 7357.
312 72 152
3
R
-
Bz iBu Bz iBu Bz iBu Bz
[16] M. J. Gait, H. W. D. Matthes, M. Singh, B. S. Sproat, R. C. Titmas,
Nucleic Acid Res. 1982, 10, 6243-6254.
(
3
C
G
C
G
C
G
4
C ) TOA (30). The extension cycle leading to
0 was performed according to General Protocol A, starting with coupling
with 29 (429 mg, 39 µmol) and 12c (390 mg, 468 µmol, 12 equiv) in DMF
1 mL) and tetrazole (1.4 mL, 0.56 mmol, 1.2 equiv) for 48 h at 5 °C,
followed by oxidation with t-BuOOH (128 µL, 0.7 mmol, 1.5 equiv),
washing with MeOH (5 mL), DMT removal with CH Cl (3 mL), water (7
μL) and DCA solution (6% in CH Cl , 5 mL), and precipitations from
CH Cl /MeOH (1 mL, 5:3, v/v) with MTBE (15 mL). Detritylated hybrid 30
[
[
[
17] B. C. Froehler, P. G. Ng, M. D. Matteucci, Nucleic Acids Res, 1986, 14,
399-5407.
18] M. D. Matteucci, M. H. Caruthers, J. Am. Chem. Soc. 1981, 103, 3185-
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19] M. H. Caruthers, Science 1985, 230, 281-285.
5
(
3
2
2
2
2
2
2
This article is protected by copyright. All rights reserved.

European Journal of Organic Chemistry
10.1002/ejoc.201700686
FULL PAPER
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European Journal of Organic Chemistry
10.1002/ejoc.201700686
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Oligonucleotide Synthesis
A. Schwenger, N. Birchall, C. Richert*
Page No. – Page No.
Solution-Phase Synthesis of
Branched Oligonucleotides with up to
32 Nucleotides and the Reversible
Formation of Materials
Text for Table of Contents
Table Contents Text
A protocol for the solution-phase synthesis of branched oligodeoxynucleotides was developed that can be scaled up readily
and that produces products with the ability to form materials through base pairing.
Key Topic
DNA Synthesis
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