Oligo switches: Photoresponsive oligonucleotide conjugates by solid-supported click chemistry

Article abstract of DOI:10.1039/c2ra22815g

Photoresponsive oligonucleotides (ONs) incorporating isoxazole-linked azobenzene (AB) moieties were prepared by resin-supported nitrile oxide-alkyne cycloaddition (NOAC) chemistry. The thermal and photochromic properties of the modified ONs were significa

Full text of DOI:10.1039/c2ra22815g

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Oligo switches: photoresponsive oligonucleotide
conjugates by solid-supported click chemistry3{
Cite this: DOI: 10.1039/c2ra22815g
Colin Freeman,*a Joseph S. Vyleb and Frances Heaney*a
Received 8th November 2012,
Accepted 20th November 2012
DOI: 10.1039/c2ra22815g
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Photoresponsive oligonucleotides (ONs) incorporating isoxazole-
linked azobenzene (AB) moieties were prepared by resin-
supported nitrile oxide-alkyne cycloaddition (NOAC) chemistry.
The thermal and photochromic properties of the modified ONs
were significantly influenced by the extent of p-conjugation
between the isoxazole and the AB modules.
available monomers in which the Z-AB isomer has relatively high
thermal stability.
Bioconjugation protocols centered upon copper-catalysed
azide-alkyne cycloaddition (CuAAC) chemistry6 have many positive
attributes and one recent report describes the formation of AB
glycoconjugates.7 However, despite elegant applications to ON
derivitisation,8 this methodology has scarcely been applied to the
introduction of photoswitches to nucleic acid substrates.3b To-
date, both solution-phase and solid-phase catalyst-free nitrile
oxide-alkyne cycloaddition (NOAC) chemistry have been applied to
the conjugation of dyes incorporating AB-chromophores to nucleic
acids.9 However, the low thermal stability of the Z-isomers of these
dyes (except in high concentrations of ethanol) renders them
unsuitable for photomodulated activity switching.
The known relationships between molecular structure, E/
Z-photoisomerisation characteristics and thermal stabilities of
AB moieties have been exploited specifically through the
introduction of substituents capable either of conjugation with
the diazenyl function,10 or of exerting steric demands proximal to
the AB core.3d,11 Here we report the site-specific introduction of
bistable AB photoswitches into ONs by solid-supported ‘‘click’’
NOAC chemistry and the influence of the ensuing, electron
donating, isoxazole-linkers upon their photophysical properties.
The commercial availability of 29-O-propargyl nucleoside
phosphoramidite building blocks, and the precedent for ‘‘click’’
type modifications of substrates incorporating this functionality,12
determined the choice of ‘‘click’’ partners: alkyne-functionalised
ON9s and AB bearing nitrile oxides. The regioisomeric aldehydes 2
served as precursors to the oximes 3, from which the dipoles 4,
were generated as needed (Scheme 1). Lithium aluminium hydride
reduction of 4-(phenylazo)benzoic acid provided the alcohol 1a
and the isomeric 1b was obtained by Mills coupling of meta-
aminobenzyl alcohol with nitrosobenzene. Dess–Martin period-
inane (DMP)-mediated oxidation of the alcohol functional groups
afforded the corresponding aldehydes 2a13 and 2b and subsequent
oximation (yielding 3a and 3b) was readily accomplished under
microwave irradiation. Model ‘‘click’’ reactions were performed in
solution following chloroamine-T (Ch–T)-mediated oxidation of
the oximes to the reactive nitrile oxides 4a and 4b, and in situ
There is considerable interest in the use of biocompatible
wavelengths to elicit reversible control of biomolecular activity.1
In particular, nucleic acid analogues appended with a diverse
range of bistable photoswitches2 including azobenzene (AB)
moieties, have been prepared.3 The photochromic properties of
ABs make them particularly attractive dynamic switches: photo-
isomerisation of the thermodynamically more stable E-form to the
less stable Z-form is accompanied by significant changes in
geometry and dipole moment, and can be reversed by either
heating or irradiation with visible light.4 Successful application of
photoresponsive ABs in addressing biological questions relies
upon both the synthetic accessibility of appropriately functiona-
lised bioprobes, and on key physicochemical characteristics of the
particular switch such as the yield of E/Z-isomerisation and the
lifetime of the Z-isomer under physiological conditions.
Current methodologies for the synthesis of AB–ON conjugates
typically rely upon unique monomers requiring optmisation of
both coupling and deblocking conditions. Although post-synthetic
coupling reactions (both in solution and on support) have been
described, the accessibility of these approaches remains limited by
the use of orthogonal deprotection conditions or labile linkages.5
Thus, the goal of this work was the development of a general
preparative route to AB-derivatised ONs from commercially-
aDept. of Chemistry, National University of Ireland, Maynooth, Co. Kildare, Ireland.
E-mail: mary.f.heaney@nuim.ie; Fax: +353 1708 3815; Tel: +353 1708 3802
bSchool of Chemistry and Chemical Engineering, Queen’s University Belfast, David
Keir Building, Stranmillis Road, Belfast BT95AG UK. E-mail: j.vyle@qub.ac.uk;
Fax: (+44) 28-9097-6524
Electronic Supplementary Information (ESI) available: Experimental procedure,
3
NMR, MALDI-TOF, kinetic and photochemical data. See DOI: 10.1039/c2ra22815g
{ The manuscript is dedicated to the memory of H. Gobind Khorana.
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Fig. 1 HPLC traces of (a) 29-O-propargyl uridine and the crude AB-NOAC products
(b) 8a and (c) 10a.
Scheme 1 (a) Synthesis of azo-oximes 3a and 3b. (i) DMP, DCM, 1 h, 97% (2a)
71% (2b). (ii) NH2OH?HCl, pyridine, EtOH, MW (Pmax = 300 W, T = 120 uC), 1 h, (3a)
94%, (3b) 88%. (b) Model NOAC reactions. (i) [p-CH3C6H4SO2NCl]Na (Ch–T), EtOH/
H2O (3 : 1), PhOCH2CCH, 40 uC, 1 h, 89% (5a) 46% (5b).
To demonstrate compatibility of the isoxazole linkage with
standard automated DNA synthesis, three synthetic cycles were
performed on the supported isoxazole 7a to yield the putative,
fully-protected tetramer 9a. MALDI-TOF MS and HPLC (Fig. 1c)
characterisation of the product following cleavage and deprotec-
tion (10a) indicated that the structural integrity of the isoxazole
linked-AB was maintained.
reaction with the dipolarophile phenyl propargyl ether.
Regioselective formation of the 3,5-disubstituted isoxazoles 5a
and 5b was optimal after 1 h stirring in aqueous ethanol (75% v/v)
1
at 40 uC. No degradation products were observed (by H NMR),
thus supporting compatibility of the Ch–T protocol with the azo-
functionality.
ONs modified at the 59-terminus, or at a predetermined
internal position, were constructed employing the 29-O-propargyl
uridine phosphoramidite 12 as a building block. The terminally-
modified pentamer 59-X-T4-CPG (13) was an excellent substrate for
NOAC ‘‘click’’ chemistry and the crude product 15a (Scheme 2b)
was characterised by HPLC and MALDI-TOF MS.
The introduction of a para-AB moiety mid-sequence was
investigated using a ‘‘click-and-grow’’ as well as the more
frequently demonstrated ‘‘grow-and-click’’14 strategy (routes A or
B respectively, Scheme 2b). Thus, the ‘‘clicked’’ pentamer 14a was
The optimised ‘‘click’’ reaction conditions were subsequently
applied to detritylated, CPG-supported 29-O-propargyl uridine (6,
Scheme 2a); conversion to the putative isoxazole-linked AB-
nucleoside 7 followed agitation of a suspension of 6 in aq.
ethanol (75% v/v) at rt in the presence of a premixed solution of
oxime 3a and Ch–T (30 eq. each). Cleavage from the resin under
standard conditions yielded AB-conjugated uridine 8a as a y1 : 3
mixture of Z : E-isomers as judged by RP-HPLC (Fig. 1b).
Scheme 2 Synthesis of (a) 39-modified AB–ON conjugate 10a, (b) 59-modified AB–ON conjugate 15a and internally-modified AB–ON conjugates 18a,b by Routes A and B.
(i) 3, Ch–T, aq. EtOH (75% v/v), rt, 18 h; (ii) 40% (w/w) aqueous CH3NH2, 65 uC, 30 min.
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enabled Arrhenius and Eyring parameters to be determined
(Table 2). Consistent with previous observations on the relative
stability of substituted AB modules3e,10,11 the meta-linked Z-18b is
significantly more stable than its para-analogue Z-18a. The lack of
mesomeric interaction between a meta-substituent and the azo
functionality contributes to the long half-life of Z-18b (114 h at 37
uC). In contrast, the para-relationship between these units in Z-18a
is assumed to have extended p-conjugation, resulting in attenu-
ated activation energy for Z A E-thermal isomerisation and a
reduced half-life of 30.3 h at physiological temperature.
In conclusion, we have demonstrated the concept of solid
phase ‘‘click’’ cycloaddition for site-specific incorporation of
photoswitches to nucleic acids using ON-alkynes and AB-
substrates bearing reactive nitrile oxides para- or meta-to the azo
functionality. In all cases, the ‘‘click’’ reaction was clean and
regioselective. The thermal stability of the photoswitch was
significantly influenced by the extent of p-conjugation between
the isoxazole linker and the photochromic AB moiety. The
enhanced thermal stability of Z-18b, where the AB is ligated to
the ON through an isoxazole linker meta-to the azo group, suggests
that such moieties may be successfully employed in photoswitch-
ing applications.
Table 1 Photophysical characterisation of 18a and 18b
lmax/nm
xE
E-isomer
Z-isomer
Compound
p–p*
n-p*
n-p*
DA-PSS
Irr-PSS
18a
18b
336
323
433
438
433
428
85%
81%
27%
17%
extended following further rounds of phosphoramidite coupling
under standard SPS conditions to give the corresponding
nonomer 17a. Alternatively, following growth of the ON-alkyne
16, resin-supported click ligation to 4a (generated in situ) also
yielded 17a. Significantly, the efficiency of the ‘‘click’’ reactions
appeared to be independent of the alkyne position; conjugation to
the 39- or 59-termini, or at an internal position (yielding 10a, 15a or
18a respectively) all gave essentially quantitative coupling.
Employing the putative nitrile oxide 4b, the meta-AB moiety was
also successfully introduced by the ‘‘grow-and-click’’ strategy. RP-
HPLC analysis of crude, deprotected oligomers bearing internal
modifications (18a, 18b) showed a well-resolved mixture of E-
(major) and Z-(minor) isomers which were desalted and purified to
yield individual samples of E-18a and E-18b.
Acknowledgements
Both para- and meta-isomers of the isoxazole-linked AB
chromophore conform to Rau’s classification15 of ‘‘AB-like’’
moieties in that they show large energy gaps between the p–p*
and n–p* transitions (Table 1) and thus have relatively high
switching efficiencies upon irradiation at 366 nm (to the irradiated
photostationary state; Irr-PSS) or above 400 nm (to the dark-
adapted photostationary state; DA-PSS). Monitoring the loss of UV-
absorption arising from the E-AB p–p* transition at 320–340 nm,
extremely rapid E/Z-photoisomerisation of both 18a and 18b was
observed such that switching to the Irr-PSS was essentially
complete after 10 s using a medium pressure Hg lamp with a
band-pass filter (although irradiation for 10 min was used to
maintain consistency with related 29-amido-linked photoswitch-
able analogues5b). Using a cut-off filter to remove incident
radiation below 400nm, the DA-PSS was achieved within 2 min.
The compositions of these photostationary states were analysed
using RP-HPLC from which the mole fractions of E-isomers (xE)
were calculated (Table 1).
The work was supported by the Science Foundation of Ireland
(Programme code 05/PICA/B838/NSs) and the Irish Research
Council of Science and Engineering (Embark Postgraduate
Research Scholarship to CF, Programme code RS/2007/48)
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