A traceless aryl-triazene linker for DNA-directed chemistry

Article abstract of DOI:10.1039/c3ob27504c

DNA-directed synthesis of encoded combinatorial libraries of small organic compounds most often involves transfer of organic building blocks from one DNA strand to another. This requires cleavable linkers to enable cleavage of the link to the original DNA strand from which the building block is transferred. Relatively few cleavable linkers are available for DNA-directed synthesis and most often they leave an amino group at the organic molecule. Here we have extended the application of aryltriazenes as traceless linkers for DNA-directed synthesis. After reaction of one building block with a building block at another DNA strand the triazene linker is cleaved and reduced with hypophosphorous acid in high yield to leave the aryl group with a hydrogen in place of the triazene i.e. without a functional group trace. It was also demonstrated that alternatively the triazene could be converted to an azide, which was used in a cycloaddition reaction. The linker is generally stable at pH > 7 and could be stored for several months in a freezer without significant degradation. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3ob27504c

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A traceless aryl-triazene linker for DNA-directed
chemistry†
Cite this: DOI: 10.1039/c3ob27504c
Christian Hejesen,a Lars K. Petersen,b Nils Jakob V. Hansenb and Kurt V. Gothelf*a
DNA-directed synthesis of encoded combinatorial libraries of small organic compounds most often
involves transfer of organic building blocks from one DNA strand to another. This requires cleavable
linkers to enable cleavage of the link to the original DNA strand from which the building block is trans-
ferred. Relatively few cleavable linkers are available for DNA-directed synthesis and most often they leave
an amino group at the organic molecule. Here we have extended the application of aryltriazenes as
traceless linkers for DNA-directed synthesis. After reaction of one building block with a building block at
another DNA strand the triazene linker is cleaved and reduced with hypophosphorous acid in high yield
to leave the aryl group with a hydrogen in place of the triazene i.e. without a functional group trace. It
was also demonstrated that alternatively the triazene could be converted to an azide, which was used in
a cycloaddition reaction. The linker is generally stable at pH > 7 and could be stored for several months in
a freezer without significant degradation.
Received 27th December 2012,
Accepted 12th February 2013
DOI: 10.1039/c3ob27504c
www.rsc.org/obc
molecule evolution.10 In these studies we used a succinimidyl
2-(vinylsulfonyl)-ethyl carbonate linker, which is a hetero-
Introduction
The field of DNA-directed chemistry has its origin in chemical bifunctional linker containing a sulfone conjugated alkene in
ligation of DNA strands,1 and during the past decade it has one end and an NHS group in the other end.11 It can selec-
been expanded to a broader range of chemical reactions and tively couple a thiol modified DNA strand with a building
structural designs by Liu et al.2 and others.1,3–5
block containing an amino group. This linker is also cleavable
A variety of covalent linkers are used to attach chemical at elevated pH values releasing the amino moiety of the build-
building blocks to DNA oligonucleotides, and in studies where ing block.
the building block must be transferred from one DNA strand
The purpose of the yoctoreactor technology is to prepare
to another, cleavable linkers are required. The transfer of and screen large combinatorial molecular libraries for com-
building blocks in DNA-directed chemistry has been used to pounds of pharmaceutical relevance in a quick and easy
build DNA encoded combinatorial libraries of small peptides manner in a single tube format.10 For both the above-men-
and other molecules. Liu et al. have reported on programma- tioned linkers, the chemical building block is left with an
ble synthesis of small molecules using DNA-directed amino group after cleavage of the linker. This is very useful for
chemistry.6–9 In these studies a cleavable BSOCOES (bis[2- reactions with another building block; however, for the final
(succinimidooxycarbonyloxy)-ethyl]sulfone) linker was used. building block it is inappropriate to terminate all products
This is a homobifunctional linker that can link two amino resi- with an amino group. To increase the diversity of such
dues and it is cleavable at elevated pH levels releasing the two libraries and to expand the linker chemistry in DNA-directed
formerly tethered amines.
synthesis we have pursued the development of a cleavable
In 2009 we reported on formation of an encoded library of linker that leaves the final building block in a synthesis
short peptides in the yoctoreactor, which utilizes DNA junction without a functional group (a traceless linker). Here we present
structures, e.g. a 3-way junction. In this work, the yoctoreactor such a linker and its fundamental reaction pathway is shown
is used for sequential DNA-directed synthesis for small- in Fig. 1, where the coupled product is released with a hydro-
gen substituent instead of an amino moiety.
The traceless linker studied here is based on triazene chem-
aCenter for DNA Nanotechnology, Department of Chemistry and iNANO,
istry. Aromatic triazenes are fairly common in classical organic
chemistry12,13 and Bräse et al. have pioneered the use of tria-
Aarhus University, Langelandsgade 140, Aarhus C, DK-8000, Denmark.
E-mail: kvg@chem.au.dk; Fax: (+)45 8619 6199
bVipergen, Gammel Kongevej 23A, 1st floor, Copenhagen V, DK-1610, Denmark
zene linkers in solid phase synthesis.14,15 The advantage of
using triazenes is that the products can be cleaved from the
solid support in a traceless manner. Triazenes are furthermore
†Electronic supplementary information (ESI)
available. See DOI:
10.1039/c3ob27504c
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Fig. 2 Synthesis and reductive cleavage of the prototype triazene linker 4 from
the secondary amine 3 and the in situ formed diazonium salt 2a. The linker 4 is
cleaved into the compounds 5 and 3 by the addition of a reductant and an acid.
Fig. 1 Principle of the transfer of building blocks from one strand to another
followed by cleavage of the linker showing the possible cleavage residues from
this work. (X: cleavable linker, Y: residue from the cleavable linker.)
stable towards bases, nucleophiles, reductants and a variety of
metal-catalysts, whereas they are labile towards acids and elec-
trophiles. Triazenes are typically formed by the reaction of an
aryldiazonium salt with a secondary amine. When subjected to
acids or electrophiles, the triazene cleaves and regenerates the
diazonium salt, which can be reduced to the naked arene
(hydrogen) with a mild reductant.16
For the triazene linker described in this work the key issues
addressed are: (a) the triazene linkers must be installed on the
DNA conjugates, (b) the linker must be sufficiently stable
under aqueous conditions at various pH values to function in
DNA-directed chemical reactions efficiently, and (c) the linker
must be cleavable under conditions that are compatible with
DNA and aqueous conditions.
Fig. 3 Showing the resulting states of the triazene 4 prior to acidic cleavage
and 2a and 5 post cleavage of the triazene with their respective 1H NMR
spectra in blue, green and red. The “X” marks a signal from H3PO2 which was
used as a reagent in this case. The disparity between signal intensities in 5 is
caused by partial deuteration at the 3-position.
Several conditions were tested for the cleavage and all sol-
vents and reactants used for the NMR study were deuterated to
1
minimize interference with the signals of interest in H NMR
(Table 1). The cleavage with HSiCl3 is well described as a
highly efficient cleavage and reductive agent for triazenes.16 It
worked very well in neat MeCN, but unfortunately trichlorosi-
lane is incompatible with water, and thus not applicable for
Results and discussion
A model linker 4 was first synthesized by addition of the piper- DNA (entries 1 and 2).
idine derivative 3 to the aryl diazonium salt 2a made in situ
The most suitable reactant for application under aqueous
from 1a as illustrated in Fig. 2 in 57% yield. Subsequently we conditions was hypophosphorous acid, which has previously
have screened for useful reagents to cleave the linker 4 into 5 been used as a reducing agent for diazonium salts (entry 4).17
and 3 under conditions that are not harmful to DNA.
An acid, such as 2,2,2-trifluoroacetic acid (TFA), is only able to
The two steps involved in the cleavage and reduction reac- cleave the linker, but not reduce it (entry 3). To optimize the
1
tion of 4 can be followed by H NMR as shown in Fig. 3. Upon conditions, sodium hypophosphite was used instead, to
treatment with acid, 4 is cleaved back into the diazonium salt enable separate addition of the acid and thereby making it
2a resulting in a downfield shift of the proton signals, because easier to control the pH value and avoid the risk of depurina-
of the decrease in electron density on the aromatic ring tion of DNA (entry 5). It was also attempted to replace the acid
causing decreased shielding. Subsequent addition of a reduc- with an electrophile. We speculated that chloral (2,2,2-
tant led to formation of 5 as verified by the upfield shift.
trichloroacetaldehyde) could react with the terminal nitrogen
Table 1 Examples of various cleavage conditions of the triazene linker in the 1H-NMR study
Entry
Solvent (deuterated)
Cleavage agent
Cleavage conversion
Reductant
Reduction conversion
1
2
3
4
5
6
MeCN
HSiCl3
HSiCl3
4% TFA
D3PO2
4% TFA
Chloral
99%
100%
100%
100%
100%
15%
HSiCl3
HSiCl3
—
D3PO2
NaH2PO2
NaH2PO2
95%
0%
0%
100%
100%
0%
MeCN + 12% D2O
MeCN–D2O 1 : 1
MeCN–D2O 4 : 1
MeCN–D2O 1 : 1
MeCN–D2O 1 : 1
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Table 2 Coupling of various other diazonium compounds to the piperidine
modified oligonucleotide
Diazonium
Entry compound
Calculated
Product Yielda mass
Observed
massb
1
2
3
4
5
O3a
O3b
O3c
O3d
O3e
52%
10%
33%
28%
47%
12 671.92
12 657.90
12 631.90
12 691.82
12 677.88
12 671.4
12 657.6
12 631.3
12 692.4
12 677.9
Fig. 4 General reaction pathway for coupling an aromatic diazonium salt to a
DNA strand via a triazene.
atom of the triazene and cause cleavage. Unfortunately the
cleavage efficiency of chloral was very poor (entry 6), curiously,
however, it improved in the presence of water.
For the introduction of the triazene linker directly onto
DNA we have synthesized a 40-mer oligonucleotide carrying an
internal dT(C6-amino) modifier (O1). Our first approach in
construction of triazene-linked DNA conjugates was to attach a
building block already containing the triazene linker to
amino-functionalized DNA. However, due to problems related
to the presence of two carboxylic acid groups in these deriva-
tives this approach was abandoned. Instead, a strategy in
which the triazene is formed directly on the DNA was
implemented. First, the amino modified DNA O1 was linked to
the N-Fmoc-piperidine-4-carboxylic acid (6) (Fig. 4). The depro-
tected secondary amine conjugate O2 was subsequently sub-
jected to the reaction with the aryldiazonium salts 3a–f at pH 7
in a 1 : 1 H2O–MeCN mixture. The triazenes O3a–f were
formed on DNA without any side reactions and in good to
average yields.
6
O3f
3%
12 672.91
N.D.
a Measured by absorbance following lyophilisation of HPLC purified
samples. b Obtained with ESI-MS.
Six different diazonium salts were linked to the DNA via the
triazene linker as shown in Table 2. In all cases the coupling
reactions were very clean leading to the expected conjugates.
In entry 2 it is demonstrated that the method tolerates the
presence of a free acid group as in 3b. If the ester 3a is applied
it can subsequently be converted into O3b by hydrolysis of the
ester. The coupling to various triazenes shows overall good
yield and purity, although coupling with the free acid 3b
shows lower conversion.
With the DNA–triazene conjugates at hand, we next wanted
to show that building block 3b can undergo a DNA-directed
coupling with a functional group at another DNA strand and
subsequently be fully transferred to the other strand by traceless
cleavage of the triazene linker. The reactions were performed in
the yoctoreactor setup as shown in Fig. 5a, where conjugates
O3b (40 nt), the amino modified O4 (51 nt) and a DNA helper
strand O6 (21 nt) are annealed to form a DNA 3-way junction in
which the reactions occur.10 Both the sequences O3b and O4
Fig. 5 (a) DNA-directed coupling carried out in a yoctoreactor type setup using
a third helper DNA strand (brown) to form the 3-way junction. (b) A detailed
illustration of the reaction between the triazene-linked benzoic acid moiety O3b
and the amino-modified DNA O4, followed by cleavage of the triazene linker
resulting in transfer of the benzoic acid from one strand to the other.
contain a hairpin loop, which principally enables ligation of the mediated
by
4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-
complete 3-way junction structure to one strand, as previously morpholinium chloride (DMTMM) to form the oligonucleotide
shown for the yoctoreactor. The amide coupling reaction is coupling product OP3b-4 (Fig. 5b).18 The reaction is analyzed by
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polyacrylamide gel electrophoresis (PAGE) as shown in Fig. 6.
The potential usefulness of the linker in DNA-directed
The starting oligos are shown in lanes 1–3 while the product of chemistry is highly dependent on the storage stability of the
the reaction before purification is shown in lane 4 and in lane 5 triazene linked conjugates. Therefore, the stability of the linker
after purification. The yield of the amide bond formation was was tested by incubating the cross-linked product at different
estimated to be 38% based on the analysis of the gel band temperatures and different pH values. The samples were
intensities (densitometry), by comparing the band intensity of assayed by PAGE (ESI†) and after analysis of the relative inten-
the cross-linked product with the unreacted DNA.
sities of the bands in the gels; the results of these investi-
The final cleavage and reduction of the purified OP3b-4 were gations are collected in Fig. 7. The three graphs show the
performed in one step to give O5-H (and O2) and this was percentage of non-degraded OP3b-4 over time at three
affected by treatment with 450 mM NaOAc, pH 5 and 50 mM different temperatures (−20 °C; room temperature (19–25 °C);
NaH2PO2. The outcome of the reaction is shown in Fig. 6, lane and 50 °C), and each graph contains data from 5 different pH
6. The yield was approximated to 78% and LCMS analysis values (values 5, 6, 7, 8 and 9).
resulted in a mass of 28 382.3, corresponding well to the calcu-
The linker is generally most stable at high pH values and in
lated mass of 28 383.3. A sample of the non-cleaved residue band particular at −20 °C. At a low pH value the linker degrades
was isolated and identification by LCMS (ESI) was attempted. rapidly at room temperature and with heating, which was
This was, however, not successful indicating that residual expected. From these results it is evident that the linker
material is not simply residual cross-linked material with the should preferably be stored at −20 °C and at pH > 7. In other
expected structure, but more likely several species in small experiments we have stored the cross-linked product for
amounts. However, the amount of residual material was very low. 8 months at −20 °C at pH 7 without seeing any significant
degradation. We therefore believe that the minor degradation
observed at −20 °C in Fig. 7a occurs during the gel loading or
elution.
In addition to the traceless cleavage of the triazene linker
we also explored the opportunity to introduce an azide group.
This was achieved by replacing the sodium hypophosphate
with sodium azide and thus converting the diazonium salt
into an azide (O5-N3). The resulting azide product was
obtained in 72% conversion by densitometry and was identi-
fied by LCMS. It was also attempted to use the classic Sand-
meyer conditions for introduction of halides onto the aromatic
moiety in place of the diazo group, but so far without success.
The mechanism behind the conversion of the diazo group
in the intermediate to the azide is probably due to its unique
mechanism which is not a nucleophilic aromatic substitution.
Rather it is the formation of an azidodiazene or a pentazole by
attack of the azide on the diazonium group. This azidodiazene
or pentazole collapses to form N2 and the azide substitu-
ent.19,20 To further test the presence of the introduced azide,
a Huisgen–Sharpless–Meldal cycloaddition reaction was per-
formed with an alkyne modified fluorophore (7) (Fig. 8).
Fig. 6 A 10.5% denaturing PAGE stained with SYBR® Gold. Lane (1) 21mer
DNA strand (O6). Lane (2) 40mer DNA strand (O3). Lane (3) 51mer DNA strand
The product O5-7 was identified by LCMS and denaturing
(O4). Lane (4) crude reaction mixture from
a DNA cross-linking reaction
PAGE as shown in Fig. 9. The green bands are an overlay from a
fluorescence scan prior to staining the gel, and the black bands
(OP3b-4). Lane (5) purified cross-linked 40 + 51mer DNA. Lane (6) cleavage of
the cross-linked DNA with AcOH and NaH2PO2.
Fig. 7 Graphs showing stability of the triazene linker with varying pH values at (a) −20 °C, at (b) room temperature, and at (c) 50 °C.
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Subsequently, the triazene linker could efficiently be cleaved
and reduced by hypophosphorous acid, leaving the aryl group
with a hydrogen in place of the triazene. The overall process is
a DNA-directed and traceless transfer of an aromatic building
block from one DNA strand to another. We believe this will
find application in DNA-directed synthesis of encoded chemi-
cal libraries. Furthermore, it was shown that the triazene could
also be substituted with an azide, and since the Huisgen–
Sharpless–Meldal 1,3-dipolar cycloaddition reaction of azides
with alkynes is compatible with DNA, the azide approach pro-
vides further opportunities for increasing the diversity in such
libraries.
Fig. 8 Copper catalysed click reaction between the modified oligonucleotide
O5-N3 and an alkyne modified 5-carboxyfluorescein (7), which result in the
product O5-7.
Notes and references
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Fig. 9 A 10% denaturing PAGE stained with SYBR® Gold with a fluorescence
overlay coloured green. Lane (1) 21mer DNA strand (O6). Lane (2) 40mer DNA
strand (O3). Lane (3) 51mer DNA strand (O4). Lane (4) reference 40mer DNA
strand with fluorescein (O). Lane (5) click reaction performed on the azide
cleaved OP3b-4 mixture resulting in O5-7.
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are from the SYBR® Gold stain. It is shown that the upper right-
most band corresponds to the correct product O5-7. To the left
of the product band is a reference band, which is an azide
modified O3 subjected to the same click conditions as O5-N3.
With the results from this gel, it was shown that the azide
functionality was indeed incorporated and fully functional for
chemical modification, and thus vastly increasing the possi-
bility for linker diversity.
17 N. Kornblum, A. E. Kelley and G. D. Cooper, J. Am. Chem.
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Conclusion
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matic building blocks to DNA oligonucleotides containing sec- and S. Tani, Tetrahedron, 1999, 55, 13159–13170.
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