Photoclick postsynthetic modification of DNA

Article abstract of DOI:10.1002/anie.201407874

A new DNA building block bearing a push-pullsubstituted diaryltetrazole linked to the 5-position of 2'-deoxyuridine through an aminopropynyl group was synthesized. The accordingly modified oligonucleotide allows postsynthetic labeling with a maleimide-modified sulfo-Cy3 dye, N-methylmaleimide, and methylmethacrylate as dipolarophiles by irradiation at 365 nm (LED). The determined rate constant of (23± 7) m^-1 s^-1 is remarkably high with respect to other copper-free bioorthogonal reactions and comparable with the copper-catalyzed cycloaddition between azides and acetylenes.

Full text of DOI:10.1002/anie.201407874

Angewandte
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DOI: 10.1002/anie.201407874
Click Chemistry
Hot Paper
“Photoclick” Postsynthetic Modification of DNA**
Stefanie Arndt and Hans-Achim Wagenknecht*
Abstract: A new DNA building block bearing a push–pull-
substituted diaryltetrazole linked to the 5-position of 2’-
deoxyuridine through an aminopropynyl group was synthe-
sized. The accordingly modified oligonucleotide allows post-
synthetic labeling with a maleimide-modified sulfo-Cy3 dye,
N-methylmaleimide, and methylmethacrylate as dipolaro-
philes by irradiation at 365 nm (LED). The determined rate
constant of (23 Æ 7)mÀ1 sÀ1 is remarkably high with respect to
other copper-free bioorthogonal reactions and comparable
with the copper-catalyzed cycloaddition between azides and
acetylenes.
reported not only the photoinduced cycloaddition between
2,5-diphenyltetrazole and methyl crotonate but also the
detection of the 1,3-dipolar intermediate diphenylnitrili-
mine.[15] Recently, Lin and co-workers further developed
this type of reaction for use in water,[16] for peptide cross-
linking,[17] and finally as an important method for the photo-
induced modification of proteins.[18] The “photoclick” cyclo-
addition, however, was never used for the modification of
nucleic acids, probably because of the fact that typical 2,5-
diphenyltetrazoles have to be irradiated at a wavelength
between 250 nm and 310 nm, which strongly overlaps with the
absorption of nucleic acids. Lin and co-workers, however,
described the photoactivatable diaryltetrazole 1, which has an
electron-donating dimethylamino group at one end and an
electron-withdrawing carboxy group at the other end.[19] The
push–pull system of this diaryltetrazole shifts the excitation to
365 nm, which is a wavelength that allows not only selective
excitation outside the absorption range of the nucleic acids
but also the application of LEDs as cheap and efficient light
sources. Herein, we describe the new DNA building block 3,
which bears the diaryltetrazole 1 linked through an amino-
propynyl group to the 5-position of 2’-deoxyuridine, together
with its incorporation into an oligonucleotide by using
automated phosphoramidite chemistry and subsequent “pho-
toclick” modification with a sulfonated Cy3 dye.
The synthesis (Scheme 1) started with 5-(3’’-aminopro-
pynyl)-2’-deoxyuridine (2) as a precursor, which was obtained
from 2’-deoxyuridine according to literature procedures.[15]
The second precursor, the push–pull-substituted diphenyl-
tetrazole 1, was synthesized from the sulfonated hydrazone of
formyl benzoic acid methyl ester and the diazonium salt of p-
N,N-dimethylaminoaniline (see the Supporting Information).
The carboxylic acid moiety of tetrazole 1 was attached to the
aminopropynyl group of nucleoside 2 with the peptide-
coupling reagent HBTU. The resulting nucleoside–tetrazole
conjugate 3 was obtained in 83% yield and then converted
into DNA building block 5 by standard procedures. DNA1
was synthesized from this DNA building block by automated
solid-phase chemistry (with an extended coupling time of 2 ꢀ
300 s for 5) and purified by semipreparative HPLC.
Postsynthetic modification of nucleic acids is a well-estab-
lished method and mainly achieved by copper-catalyzed
cycloadditions between acetylenes and azides.[1,2] Although
successful labeling in live cells by copper-catalyzed “click”-
type reactions has been demonstrated,[3] the reliance on
copper catalysis is problematic. Even trace amounts of copper
ion impurities from the postsynthetic or in vivo modification
represent a major drawback in terms of cytotoxicity.[4] Hence,
current research from our[5] and other[6–11] groups focuses on
the development of bioorthogonal and copper-free “click”-
type alternatives for the modification of nucleic acids. This
includes strain-promoted[5] and other 1,3-dipolar cycloaddi-
tions,[6] Diels–Alder reactions with normal[7] and inverse
electron demand,[8] reductive aminations,[9] thiol-ene addi-
tions,[10] and Suzuki–Miyaura-type coupling reactions.[11]
There are two metal-free bioorthogonal reactions that are
good alternatives, since their reaction rates are comparable to
the rates of copper-catalyzed azide–acetylene cycloadditions
(k ꢀ 10–200mÀ1 sÀ1).[12,13] The first alternative is the inverse
electron demand Diels–Alder reaction of tetrazines and
strained alkenes (k ꢀ 1–104 mÀ1 sÀ1)[8,12,14] and, the second
alternative the light-induced cycloaddition between tetrazoles
and activated alkenes (“photoclick” reaction, k ꢀ
60mÀ1 sÀ1).[12,13]
The latter type is especially attractive since it combines
the speed and specificity of a “click”-type reaction with the
advantages of a photochemical process, in particular spatial
and temporal control. In the 1960s, Huisgen and co-workers
Subsequently, the functional and reactive tetrazole group
of DNA1 was used in a “photoclick” postsynthetic modifica-
tion with commercially available sulfo-Cy3 dye 6, which bears
a maleimide functionality at the end a short alkyl linker.
Unexpectedly, the cycloaddition did neither work with
nucleoside 4 in water/MeCN nor with single-stranded (ss)
DNA1 in DMSO or water/DMSO mixtures. The reaction
occurred only in standard aqueous buffer solution (50 mm
sodium phosphate buffer, 250 mm NaCl, pH 7), which is
clearly an advantage for future in vivo applications. The
reaction can be followed by UV/Vis absorption spectroscopy
(Figure 1), since the characteristic absorption of the tetrazole
[*] Dipl.-Chem. S. Arndt, Prof. Dr. H.-A. Wagenknecht
Institute of Organic Chemistry
Karlsruhe Institute of Technology (KIT)
Fritz-Haber-Weg 6, 76131 Karlsruhe (Germany)
E-mail: Wagenknecht@kit.edu
[**] Financial support from the Deutsche Forschungsgemeinschaft
(ERACHEM Wa 1386/15-1) and the KIT is greatly acknowledged.
Supporting information for this article (synthetic details, images of
NMR and MS analyses, experimental procedures of “photoclick”
modification, and determination of rates) is available on the WWW
under http://dx.doi.org/10.1002/anie.201407874.
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Figure 1. Top: UV/Vis absorption of starting material DNA1 with 6
compared with that of the “photoclick” product DNA3; DNA1
(2,5 mm), 6 (1.05 equiv, 2.63 mm). Bottom: Time-dependent absorption
changes at 355 nm (DNA1) and 400 nm (DNA3), and the time-
dependent fluorescence of DNA3, lexc =357 nm. All measurements
were performed in 50 mm sodium phosphate buffer, 250 mm NaCl,
pH 7, 208C.
formation of the pyrazoline chromophore in the product
DNA3. The product was additionally identified by MALDI-
TOF mass spectrometry. The isosbestic point at labs = 378 nm
(see the Supporting Information) shows that this “photoclick”
modification proceeds without observable intermediates, and
supports the idea that the nitrilimine intermediate is very
short-lived and reacts very fast with the maleimide function-
ality of 6. The modified DNA3 was again purified by
semipreparative HPLC, identified by MALDI-TOF MS, and
quantified by UV/Vis absorption at labs = 260 nm (using
e(6)260 nm = 3400mÀ1 cmÀ1) to obtain yields for the “photo-
click” modification. Product formation was determined after
irradiation for 30 min at 365 nm by an LED. Interestingly, the
yield of purified DNA3 increased from 8% to 15% (with
1.05 equiv 6) if double-stranded DNA1 was used as the
starting material, and further to 34% (with 5 equiv 6). Longer
irradiation times were not productive, since photochemical
degradation of the diaryltetrazole group competes with the
desired photoinduced cycloaddition. The observation that
double-stranded DNA1–2 works better than single-stranded
DNA1 can be rationalized by the assumption that the regular
double-helical structure makes the tetrazole functional group
more accessible for the reaction with 6 than does the
corresponding single strand, where the diaryltetrazole func-
tionality is “hidden” in the random coil. The “photoclick”
cycloaddition of tetrazoles to form pyrazolines is known to be
a fluorogenic reaction.[20,21] However, the pyrazoline fluores-
cence of DNA3 is rather weak (see the Supporting Informa-
Scheme 1. Synthesis of tetrazole-modified DNA building block 5 and
postsynthetic modification of DNA1 with sulfo-Cy3 dye 6: a) HBTU,
HOBt·H2O, (iPr)2NEt, DMF, RT, 20 h; 83%; b) DMT-Cl, pyridine, RT,
3 days; 60%; c) b-cyanoethyl-N,N-diisopropylchlorophosphoramidite,
DIPEA, RT, 24 h; 99%; d) solid-phase synthesis under standard
conditions; e) 6, LED l=365 nm, 10 mm sodium phosphate buffer,
250 mm NaCl, pH 7, 208C, 30 min. DIPEA=diisopropylethylamine,
DMT=dimethoxytrityl, HBTU=N,N,N’,N’-tetramethyl-O-(1H-benzo-
triazol-1-yl)uronium hexafluorophosphate, HOBt=1-hydroxybenzo-
triazole.
chromophore of DNA1 occurs as a side band between 300 nm
and 400 nm, with a broad maximum at 355 nm. Irradiation of
the solution by LEDs at a wavelength of 365 nm resulted in
the absorption at 355 nm decreasing and the appearance of
a new absorption at 400 nm, which can be assigned to the
2
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tion). The fluorescence (upon excitation at 357 nm) is
dominated by the strong fluorescence intensity of the sulfo-
Cy3 dye. The time-dependent increase in the fluorescence
intensity of DNA3 formed during the “photoclick” modifi-
cation (Figure 1, bottom) tracks well with the observed
Keywords: bioorthogonality · cycloaddition · oligonucleotides ·
photochemistry · tetrazoles
.
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and comparable with the copper-catalyzed cycloaddition
between azides and acetylenes (k ꢀ 10–200mÀ1 sÀ1).[12,13]
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postsynthetic modification of the oligonucleotide DNA1 with
the sulfo-Cy3 dye 6. The corresponding synthetic DNA
building block 5 carries the diaryltetrazole unit as the
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inside living cells. Moreover, it provides the advantages of
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Received: August 1, 2014
Revised: September 12, 2014
Published online: && &&, &&&&
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Communications
Click Chemistry
S. Arndt,
H.-A. Wagenknecht*
&&&& — &&&&
“Photoclick” Postsynthetic Modification
of DNA
A light click away: The diaryltetrazole
group at the 5-position of 2’-deoxyuridine
allows the photoinduced postsynthetic
modification with maleimide-functional-
ized sulfo-Cy3 dye.
4
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