Multistep DNA-templated reactions for the synthesis of functional sequence controlled oligomers

Article abstract of DOI:10.1002/anie.201002721

Biomimetic: A strand displacement mechanism was designed to permit DNA-templated synthesis of functional oligomers of arbitrary length (see scheme). Key features of the mechanism are that successive coupling reactions take place in near-identical environm

Full text of DOI:10.1002/anie.201002721

Communications
DOI: 10.1002/anie.201002721
DNA-Controlled Synthesis
Multistep DNA-Templated Reactions for the Synthesis of Functional
Sequence Controlled Oligomers**
Mireya L. McKee, Phillip J. Milnes, Jonathan Bath, Eugen Stulz, Andrew J. Turberfield,* and
Rachel K. OꢀReilly*
Maintaining a high level of control of the order of reactions is
a key goal in synthetic chemistry. The most common strategy
is to divide the construction of the desired molecule into a
sequence of isolated reaction steps, using protecting group
chemistry with purification and isolation of intermediate
products. In contrast, multistep synthesis of biomolecules is
achieved naturally in a single solution by selective catalysis
and by controlled modulation of the effective concentrations
of particular reactants. Natureꢀs approach avoids the need for
complex protecting group chemistries even when multiple
reactive species are present simultaneously. Examples of
natural templated synthesis include ribosomal and non-
ribosomal peptide synthesis and polyketide synthases.
Many advances have been made towards this ideal using
the concept of DNA-templated synthesis (DTS). The reac-
tivities of chemical groups attached to oligonucleotide
adapters can be controlled by holding them in close proximity
by means of DNA hybridization, increasing the effective
molarity of the reactive species and thus accelerating the
reaction.[1] The rate enhancement can be sufficient to ensure
that cross-reactions with other molecules, present in the same
solution but not connected by hybridized oligonucleotide
adapters, can be neglected.[2] However, the potential of these
methods for the synthesis of sequence-controlled functional
oligomers of significant length has not yet been fully realized.
In this study we investigate a DTS mechanism that has the
potential to allow oligomer synthesis without imposing a
length restriction.
be controlled by stepwise addition of reactive adapters
followed by purification (removing spent adapters) at each
step,[3,4] and by controlling stepwise changes in the secondary
structure of the template by increasing the temperature,
bringing successive groups into proximity with the reactive
site.[5] A limitation of these methods is that every new DNA
adapter is increasingly separated from the reactive end of the
template strand, potentially decreasing the reaction efficiency
as the number of steps increases.[7] This problem can be
reduced by adding a short, constant region, complementary to
the end of the template strand, to each adapter such that all
oligonucleotide adapters bind both to the end of the template
and to their specific binding site (forming a loop).[8] However,
the single-stranded portions of the template and DNA
adapters can fold into undesired secondary structures, frus-
trating this designed interaction and reducing the DNA-
templated reactivity.[9] DTS has also been implemented using
chemically modified DNA strands that self-assemble into
each of the arms of multibranched junctions.[6] This method
has the advantage that the reactive groups are all located at
the center of the junction, providing a constant reaction
environment. However, the number of branches in the
junction determines the number of building blocks that can
be used to build the oligomer, restricting its size.
Here we present a strategy for the synthesis of ordered
functional oligomers by means of sequential DNA-templated
reactions, using a strand displacement system that provides
the same reaction environment at each step. The general
mechanism is depicted in Scheme 1. Oligonucleotide adapters
have one of two complementary binding sequences and are
functionalized at the 5’ or 3’ end such that, when consecutive
adapters are annealed, they hybridize to each other to bring
their reactive groups into close proximity. Each adapter also
includes a unique “toehold” domain used to remove it from
the active complex, once its reaction is complete, by addition
of a fully complementary “remover” DNA strand. The
chemistry used is such that the growing oligomeric product
is transferred to the incoming adapter; this stepwise oligomer
growth is reminiscent of the natural peptide and polyketide
syntheses. After removal of the spent adapter, the active
adapter bearing the growing chain can hybridize and react
with the next adapter strand, allowing the cycle to be
repeated. A drawback to this approach is that the sequence
of the product oligomer is determined by the sequence in
which reactants are added, not by a template. This excludes
potential applications, such as molecular evolution, which rely
on retention of a DNA-coded sequence record attached to the
product.[1,10] An advantage is that the configuration of the
DNA adapters bearing reactants, and the local environment
Multistep DTS can make use of a linear template or DNA
multibranched junction to encode the sequence of the desired
product.[3–6] With a linear template, sequential reactions can
[*] Dr. M. L. McKee,[+] Dr. J. Bath, Prof. A. J. Turberfield
University of Oxford, Department of Physics, Clarendon Laboratory
Parks Road, Oxford, OX1 3PU (UK)
Fax: (+44)1865-272-400
E-mail: a.turberfield@physics.ox.ac.uk
Dr. P. J. Milnes,[+] Dr. R. K. O’Reilly
Department of Chemistry, University of Warwick
Gibbet Hill Road, Coventry, CV4 7AL (UK)
Fax: (+44)2476-521-276
E-mail: rachel.oreilly@warwick.ac.uk
Dr. E. Stulz
School of Chemistry, University of Southampton
Highfield, Southampton, SO17 1BJ (UK)
[+] These authors contributed equally to this work.
[**] This work was supported by EPSRC IDEAS Factory grants EP/
F056605/1, EP/008597/1, and linked grants
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201002721.
7948
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adapter contains only a phosphine modification whereas the
last contains only the aldehyde moiety. This chemistry allows
for transfer of a building block attached to the ylide of one
DNA adapter to the aldehyde of the next adapter with
simultaneous cleavage from the first (Scheme 3).
Scheme 3. Transfer of the first building block to the second DNA
adapter. In the next step, the unreacted ylide moiety of the second
adapter will be used to couple to the aldehyde of the third adapter.[12]
The bifunctionality of the DNA adapter enables ordered
multistep transfer and the internal para-phenyl spacing unit
minimizes intramolecular Wittig reaction (Scheme 2c).[6]
There is no need for addition of auxiliary reagents, depro-
tection, or the purification of intermediates during DTS. The
strand displacement system keeps the distance between
reacting groups constant, even as the oligomeric chain
grows in length. Importantly, the adapter presented here
includes a variable side chain, introduced as the side chain of
an amino acid during synthesis, which permits the introduc-
tion of additional functionality (Scheme 2d).
The synthesis of the bifunctional ylide–aldehyde adapter
(Scheme 4) was achieved using a p-phenylenediamine as the
spacing unit and amino acids to introduce the variable side
chain. As proof of principle, l-alanine (Ala) and l-phenyl-
alanine (Phe) were incorporated to demonstrate the prepa-
ration of olefin oligomers with a controlled sequence of side
chains. Synthesis proceeded by coupling an Fmoc amino acid
to N-Boc-p-phenylenediamine (1, 5),[13] followed by Fmoc
deprotection (2, 6).[14] The masked aldehyde moiety (Sche-
me 2e) was introduced by reacting the amine with diacetyl-l-
tartaric anhydride, followed by basic removal of the acetyl
protecting groups (3, 7).[15] Unmasking of the aldehyde moiety
was later achieved by exposure to 50 mm NaIO4, 500 mm
NaOAc, pH 3.5 (see Supporting Information).
Scheme 1. Strand displacement mechanism for oligomer synthesis.
Hybridization brings reactive chemical groups into close proximity
allowing chemical group transfer from the first to the second adapter.
Subsequent strand exchange using remover strand 1 displaces the first
strand as a waste product. This cycle is repeated with different DNA
adapters to achieve sequential synthesis. The final product can be
isolated from the reaction mixture by using a biotin-tagged remover
strand.
for each reaction, is the same at each step, so the reactivity of
an incoming group should be independent of the number of
preceding coupling steps and the oligomer length limited only
by the intrinsic yield of a single step.
To test this strand displacement system, a series of olefin
4-mers were synthesized using three sequential DNA-tem-
plated Wittig reactions. This coupling chemistry was chosen
because of its robustness in aqueous solution[11] and because it
has been successfully used to synthesize triolefins using
phosphine modified oligonucleotides.[5] The design of the
DNA adapters used in this work is shown in Scheme 2. All of
the reactive oligonucleotides strands are modified, through a
terminal amine, with a bifunctional adapter containing both a
phosphine ylide (Scheme 2b) and an aldehyde moiety (Sche-
me 2e).[5] The first and last adapters are exceptions: the first
Boc deprotection was achieved using TFA to give the free
amine (4, 8).[16] Amino modified DNA strands S1–S3 (see
Supporting Information) were coupled with diphenylphos-
phine benzoic acid post-synthetically using standard peptide
coupling reagents.[17] To triphenylphosphino strands S2 and S3
was added an in situ mix of amine (4, 8) and N-succinimidyl
iodoacetate to give ylide–aldehyde DNA adapters S2-ALA,
S2-PHE, and S3-PHE (Scheme 5). The first monofunctional-
ized adapter was formed by reacting triphenylphosphino S1
Scheme 2. Design of functional DNA adapter unit. a) DNA adapter for
template control of Wittig reaction, b) triphenylphosphosphonium
(ylide moiety), c) para-phenyl spacing unit (to minimize intramolecular
Wittig reaction), d) function unit (amino acid), e) aldehyde.
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Scheme 4. Synthesis of ylide–aldehyde DNA precusors 4 and 8.
Conditions: a) HBTU, DMF, 98% (1), 95% (5); b) cyclohexylamine,
CH2Cl2, DMF, 95% (2), 95% (6); c) (+)-O,O-diacetyl-l-tartaric anhy-
dride, CH2Cl2, 08C; d) NaOH, MeOH, 78% over 2 steps (3), 74% over
2 steps (7); e) TFA, CH2Cl2, 100% (4), 100% (8). HBTU=O-(benzo-
triazol-1-yl)tetramethyluronium hexafluorophosphate; TFA=trifluoro-
acetic acid; Boc=tert-butoxycarbonyl; Fmoc=9-fluorenylmethoxycar-
bonyl.
strand with 5-(iodoacetamido) fluorescein to give fluorescent
ylide DNA S1-FAM; the fluorescent tag was chosen to
facilitate monitoring of the transfer reaction. The last adapter
was monoaldehyde-functionalized by reacting 4-formylben-
zoic acid N-hydroxysuccinimide ester with amino-modified
strand S2 or S4 to give S2-BAL or S4-BAL, respectively.
Table 1 shows characterization of the complete oligonucleo-
tide adapters.
Table 1: ESI-MS characterization of DNA Wittig adapters.
DNA adapter
Calculated
mass [Da]
Observed
mass [Da]
S1-FAM
S2-BAL
10195.9
9487.3
10193.9
9486.1
S2-ALA
9994.8
9992.6
S2-ALA-Ald
S2-PHE
S2-PHE-Ald
S2-FAM
S3-PHE
9918.7
10070.9
9994.8
10032.9
10201.9
10125.8
12453.2
9917.7
10068.7
9993.7
10031.4
10199.7
10124.7
12452.2
Scheme 5. Synthesis of olefin 4-mers using strand exchange mecha-
nism.
only when the aldehyde moiety of S2-ALA was unmasked or
when adapter S2-BAL was used (yields between 61 to > 95%
depending on reaction conditions, Supporting Information).
Little or no transfer was observed between adaptors with non-
S3-PHE-Ald
S4-BAL
The overall group transfer efficiency between oligonu-
cleotide adapters was initially determined by synthesizing
olefin dimers in a single coupling step using oligonucleotides
S1-FAM and S2-BAL, S2-ALA or S2-ALA-Ald (see Sup-
porting Information). The transfer reactions were carried out
in 0.1m TAPS (N-tris(hydroxymethyl)methyl-3-aminopropa-
nesulfonic acid), 1m NaCl pH 8.5 (2 h incubation at room
temperature). The desired products were observed (Table 2)
Table 2: ESI-MS of olefin oligonucleotide products.
Olefin product
Calculated
mass [Da]
Observed
mass [Da]
S2-BAL-FAM
S2-ALA-FAM
S4-BAL-PHE-ALA-FAM
S4-BAL-PHE-PHE-FAM
9858.3
10290.1
13419.2
13495.3
9857.2
10289.1
13417.9
13493.9
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Angewandte
Chemie
complementary sequences (Supporting Information). The
limiting factor of the Wittig transfers is the potential oxidation
of the ylide adaptors as a side reaction.[5] This can be
minimized by lowering reaction times, which is possible as
olefin products were observed in as little as 5 min reaction
time (Supporting Information).
Any number of bifunctional oligonucleotide adapters
could be used in additional reaction cycles to make longer
oligomers. Here, two further coupling steps were introduced
using S2-ALA or S2-PHE and S3-PHE for the synthesis of
two different olefin 4-mers. After each transfer reaction (2 h
mechanism are that successive coupling reactions take place
in near-identical environments, independent of the number of
coupling steps, and that purification is only necessary in the
last synthesis step. This mechanism could be applied to other
coupling chemistries that effect transfer between adapters
(rather than simple ligation), for example, peptide bond
formation.[5] This technique provides an opportunity to
prepare long, functional oligomers using DNA-templated
synthesis.
incubation per step) the spent adapter bearing an unreactive Experimental Section
All oligonucleotides were supplied by Integrated DNA Technologies
phosphine oxide was displaced by addition of its comple-
mentary remover strand. The reaction was terminated by the
addition of the final monoaldehyde-functionalized S4-BAL
adapter. This strand was designed to be longer than other
DNA adapters to facilitate analysis by PAGE. The final
product was isolated from waste products by using a
biotinylated remover strand, fully complementary to the
final adapter, and streptavidin-coated magnetic beads
(Scheme 6). Densitometric analysis showed 72% overall
Inc. (USA); see Supporting Information for sequences, a detailed
description of DNA adapter synthesis, experimental procedures used,
and further characterization data.
Received: May 5, 2010
Revised: July 29, 2010
Published online: September 10, 2010
Keywords: biomimetic synthesis · DNA · oligomers ·
.
template synthesis · Wittig reaction
[1] Z. J. Gartner, D. R. Liu, J. Am. Chem. Soc. 2001, 123, 6961 –
6963.
[2] C. T. Calderone, J. W. Puckett, Z. J. Gartner, D. R. Liu, Angew.
Chem. 2002, 114, 4278 – 4282; Angew. Chem. Int. Ed. 2002, 41,
4104 – 4108.
[3] Z. J. Gartner, M. W. Kanan, D. R. Liu, J. Am. Chem. Soc. 2002,
124, 10304 – 10306.
[4] Z. J. Gartner, B. N. Tse, R. Grubina, J. B. Doyon, T. M Snyder,
D. R. Liu, Science 2004, 305, 1601 – 1605.
[5] T. M. Snyder, D. R. Liu, Angew. Chem. 2005, 117, 7545 – 7548;
Angew. Chem. Int. Ed. 2005, 44, 7379 – 7382.
[6] M. H. Hansen, P. Blakskjaer, L. K. Petersen, T. H. Hansen, J. W.
Hojfeldt, K. V. Gothelf, N. J. V. Hansen, J. Am. Chem. Soc. 2009,
131, 1322 – 1327.
[7] Z. J. Gartner, M. W. Kanan, D. R. Liu, Angew. Chem. 2002, 114,
1874 – 1878; Angew. Chem. Int. Ed. 2002, 41, 1796 – 1800.
[8] Z. J. Gartner, R. Grubina, C. T. Calderone, D. R. Liu, Angew.
Chem. 2003, 115, 1408 – 1413; Angew. Chem. Int. Ed. 2003, 42,
1370 – 1375.
Scheme 6. Resulting olefin 4-mers are easily purified after the final
reaction step. 20% Denaturing PAGE gel after Sybr Gold staining.
Lane 1: One-step reaction dimers; Lane 2: two-step reaction trimers;
Lane 3: three-step reaction 4-mers; Lane 4: 4-mer after streptavidin
magnetic bead purification (Supporting Information). In all cases
products remain conjugated to the final adapter (30 nucleotides,
except in the case of the 4-mer which was attached to a longer, 40-
nucleotide adapter to facilitate PAGE analysis).
[9] T. M. Snyder, B. N. Tse, D. R. Liu, J. Am. Chem. Soc. 2008, 130,
1392 – 1401.
[10] M. M. Rozenman, B. R. McNaughton, D. R. Liu, Curr. Opin.
Chem. Biol. 2007, 11, 259 – 268.
fluorescent group transfer (see Supporting Information)
from the initial adapter through the chain of transfer reactions
to the 4-mer olefin product S4-PHE-PHE-FAM and 41% for
S4-BAL-PHE-ALA-FAM. ESI analysis confirmed the for-
mation of the desired oligomers S4-BAL-PHE-ALA-FAM
and S4-PHE-PHE-FAM, respectively (Table 2). The fact that
a 4-mer with defined sequence can be obtained in good yields
indicates that even longer oligomers could be synthesized by
this mechanism.
In summary, we have developed a DNA-templated syn-
thesis mechanism that is designed to facilitate the synthesis of
oligomers with controlled sequences of subunits. We have
demonstrated selective and robust sequence-controlled for-
mation of two related olefin 4-mer sequences using templated
Wittig chemistry with bifunctional (ylide and aldehyde)
adapters. Each building block of the polyolefin product
incorporates a side chain which can be used to introduce
further chemical or physical functionality. Key features of the
[11] J. Dambachera, W. Zhaoa, A. El-Battaa, R. Annessa, C. Jianga,
M. Bergdahl, Tetrahedron Lett. 2005, 46, 4473 – 4477.
[12] Following Liu and co-workers, a trans geometry for the olefin is
assumed (Ref. [3,4] and B. E. Maryanoff, A. B. Reitz, Chem.
Rev. 1989, 89, 863 – 927).
[13] D. S. Holmes, R. C. Bethell, N. Cammack, I. R. Clemens, J.
Kitchin, P. McMeekin, C. L. Mo, D. C. Orr, B. Patel, I. L.
Paternoster, R. Storer, J. Med. Chem. 1993, 36, 3129 – 3136.
[14] T. J. Attard, N. M. OꢀBrien-Simpson, E. C. Reynolds, Int. J. Pept.
Res. Ther. 2009, 15, 69 – 79.
[15] J. Wagner, R. A. Lerner, C. F. Barbas III, Bioorg. Med. Chem.
1996, 4, 901 – 916.
[16] J. Hasserodt, K. D. Janda, Tetrahedron 1997, 53, 11237 – 11256.
[17] M. Caprioara, R. Fiammengo, M. Engeser, A. Jꢁschke, Chem.
Eur. J. 2007, 13, 2089 – 2095.
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