Improved synthesis of the two-photon caging group 3-nitro-2- ethyldibenzofuran and its application to a caged thymidine phosphoramidite

Article abstract of DOI:10.1021/ol902807q

Chemical Equation Presentation A new and efficient route to the recently reported 3-nitro-2-ethyldibenzofuran caging group was developed. Furthermore, its installation on a thymidine phosphoramidite is described. This caging group is efficiently removed through light-irradiation at 365 nm

Full text of DOI:10.1021/ol902807q

ORGANIC
LETTERS
2010
Vol. 12, No. 5
916-919
Improved Synthesis of the Two-Photon
Caging Group 3-Nitro-2-Ethyldibenzofuran
and Its Application to a Caged Thymidine
Phosphoramidite
Hrvoje Lusic, Rajendra Uprety, and Alexander Deiters*
Department of Chemistry, North Carolina State UniVersity, Raleigh, North Carolina 27695
alex_deiters@ncsu.edu
Received December 4, 2009
ABSTRACT
A new and efficient route to the recently reported 3-nitro-2-ethyldibenzofuran caging group was developed. Furthermore, its installation on a
thymidine phosphoramidite is described. This caging group is efficiently removed through light-irradiation at 365 nm.
Photolabile protecting groups (caging groups) are important
tools in the investigation of biological processes. Cleavage
of caging groups by light (decaging) is a mild and
noninvasive technique that is completely orthogonal to
other chemical processes in a biological system. Moreover,
caged molecules offer a unique way to study biological
systems, because they allow for precise temporal and
spatial control over the chemical and biological (re)activity
of molecules. This approach has been successfully applied
to the photochemical regulation of the activity of small
molecules, peptides, proteins, and oligonucleotides.1-4 Sev-
eral caging strategies for oligomers have been developed:
statistical caging of the DNA or RNA backbone,5-7 incor-
poration of photocleavable linkers into the oligomer
strand,8-11 and incorporation of caged nucleotide building
blocks into oligonucleotides.12-18
All base-caged phosphoramidites developed to date require
UVA irradiation of approximately 360 nm for efficient
decaging. Disadvantages of UVA light include limited tissue
penetration, potential photodamage, and difficulties in achiev-
ing high-precision three-dimensional focusing of the pho-
toexcitation process. A solution to these problems is the
application of two-photon excitation techniques. Under two-
photon irradiation conditions, a molecule that has an absor-
bance maximum at 350 nm can be excited by simultaneously
absorbing two photons of precisely half the energy, i.e. 700
nm.19,20 Light at near-IR or IR wavelengths (between 700
and 1000 nm) exhibits deeper tissue-penetration (e.g., up to
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10.1021/ol902807q  2010 American Chemical Society
Published on Web 01/29/2010

8 mm in bovine muscle tissue),21-23 causes less photodam-
age, and can be focused much more precisely (down to an
effective excitation volume of 1 fL).20,24
sponding phosphoramidites would hydrolyze during the final
step (base deprotection with NH4OH, or MeNH2) of the
automated oligonucleotide synthesis (as observed by us for
similar structures; data not shown), and neither compound
has been converted into a phosphoramidite. Additionally, the
overall yields for the synthesis of Bhc-caged nucleosides
were low due to difficulties associated with the carbamate
formation.
To develop a suitable and easily accessible two-photon
caged phosphoramidite for DNA synthesis, we decided to
combine our 6-nitropiperonyloxymethyl (NPOM) caging
group strategy17,18,34,35 with the recently discovered 3-nitro-
2-ethyldibenzofuran (NDBF) group (1) (Figure 1), which
The main characteristic of a good two-photon caging group
is a high two-photon decaging cross-section δu, which is
defined as a product of a two-photon absorbance cross-
section δa and a two-photon decaging quantum yield Qu.25
Ideally δu should exceed 0.1 Goeppert-Mayer (GM) for
biological experiments in order to avoid potential tissue
damage caused by high laser powers.25-27 The two most
commonly employed two-photon caging groups are 6-bromo-
7-hydroxycoumarin-4-methyl (Bhc), with a δu of 0.72 GM
at 740 nm,25 and 8-bromo-7-hydroxyquinoline (BHQ), with
a δu of 0.59 GM at 740 nm.28 Both have been employed to
cage a variety of substrates, including carboxylic acids,
phosphates, carbonates, carbamates, diols, aldehydes, and
ketones.24,25,27-31 Bhc and BHQ caging groups decage
according to a solvent-assisted heterolysis mechanism.24,26
This makes them unsuitable for direct caging of substrates
with high pKa values,32,33 such as alcohols, phenols, amines,
and amides, hampering their application in the caging of
nucleotide bases. Recently, Furuta and co-workers have
prepared a pair of Bhc-caged nucleosides through the
installation of the Bhc group via a carbamate linkage at the
6-NH2 of deoxyadenosine and the 4-NH2 of deoxycytidine.31
However, the caging group carbamate linkage in the corre-
Figure 1. The NDBF caging group derivatives 1-4.
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decages via a classical Norrish type II mechanism, enabling
efficient caging and decaging of less acidic functional
groups,33 while displaying an excellent δu of 0.6 GM (at
710 nm).
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Despite its excellent photochemical properties and potential
to cage diverse substrates, no application of the NDBF group
has been reported since its discovery. We believe that this
is due to difficulties accessing it synthetically. Reported
syntheses of the alcohol 1 and the bromides 2-3 (Figure 1)
are rather low yielding and often involve laborious and
difficult purification steps.33,36 In addition to developing our
own synthetic path to 1-2, we have also synthesized the
chloromethyl ether 4 (Figure 1), which was modeled after
the NPOM caging group for caging of nitrogen heterocycles
such as nucleotide bases.17,18,34
The previously published synthesis, starting from diben-
zofuran, produced (3-nitrodibenzofuran-2-yl)-ethanol (1) in
4% total yield over 6 steps, and 1-bromo-1-(3-nitrodiben-
zofuran-2-yl)ethane (2) in 10% overall yield over 4 steps.33,36
An alternative synthetic strategy leading to the caging group
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from 1-chloro-2-nitrobenzene and p-cresol, afforded the final
product 3 in only 1% total yield over 6 steps.36
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group 4 (Scheme 1), enabling us to synthesize sufficient
quantities of the caged phosphoramidite 11 to be used in
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Org. Lett., Vol. 12, No. 5, 2010
917

To prepare an NDBF-caged thymidine phosphoramidite
for DNA synthesis, we reacted 5′-O-(4,4′-dimethoxytri-
tyl)thymidine (9, synthesized in one step from thymidine
following a literature procedure37) with the caging group 4
(Scheme 2), using Cs2CO3 in DMF, at 0 °C to rt, obtaining
Scheme 1. Synthesis of NDBF Based Caging Groups 1, 2, and 4
Scheme 2
.
Synthesis of the NDBF-Caged Thymidine
Phosphoramidite 11
mercially available 4-fluoro-2-nitrobenzaldehyde (5) and
2-iodophenol by employing an Ullmann coupling, using
CuBr and K2CO3 in pyridine, at 60 °C, to form the diaryl
ether 6 in 77% yield. The aldehyde 6 was subsequently
alkylated with AlMe3 in DCM, affording the secondary
alcohol 7 in 95% yield. The bottleneck of the synthesis was
the formation of the dibenzofuran ring, which we ac-
complished via a Heck reaction by heating 7 in the presence
of catalytic amounts of Pd(OAc)2, using Cs2CO3 as a base
in DMAc at 80 °C. The caging group 1 was isolated as
the sole regioisomer in 38% yield (28% total yield over three
steps). Unfortunately, attempts to improve the yield of the
ring-closure reaction failed. Substituting Cs2CO3 for alterna-
tive bases (K2CO3, DIPEA, DBU) and changing the solvent
(DMSO, toluene, THF) all provided lower yields. Increasing
the temperature or employing microwave irradiation also
resulted in lower yields, and the formation of multiple side
products hampering purification. Also, cyclization attempts
with the aldehyde 6 led to diminished yields. Subsequently,
the alcohol 1 was converted to a bromide with PBr3 in DCM,
at 0 °C, in 70% yield, thus affording the caging group 2 in
19% over 4 steps. Alternatively, the caging group 1 was
protected as a methylthiomethyl ether 8 in 70% yield, using
Me2S and benzoyl peroxide in CH3CN, at 0 °C. The MTM-
derivative 8 was then converted to the caging group 4,
employing SO2Cl2 in DCM, at 0 °C. The caging group 4
was directly used in the caging of thymidine without
purification.
10 in 73% yield. Gratifyingly, the free 3′-OH group was
unreactive under those conditions, thus obviating additional
protecting group chemistry. The NDBF-caged thymidine 10
was then converted to the phosphoramidite 11 in 80% yield
with 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite,
employing DIPEA as a base in DCM, at rt.
The stability of the NDBF-caged thymidine toward DNA
synthesis was then studied. The installed caging group was
found to be completely stable under several different
conditions commonly used for the removal of protecting
groups from the 5′-OH and the nucleotide bases, as well as
oligonucleotide cleavage from the support resin: (a) con-
centrated NH4OH, 65 °C, 16 h; (b) 33% MeNH2 in EtOH,
55 °C, 16 h; and (c) 5% TFA in DCM, rt, 1 h (see Supporting
The synthetic path to 1, 2, and 4 presented herein is
substantially shorter and offers markedly improved yields.
Our synthesis also avoids the need for laborious purifications
required for the separation of different regiosomers and
multiple reaction side products.
Figure 2. Decaging timecourse of NDBF-thymidine.
918
Org. Lett., Vol. 12, No. 5, 2010

Information).17 Decaging of the NDBF-thymidine was tested
in CH3OH (0.1 mM) by conducting a time course of UV
irradiation over 5 min (hand-held Spectroline ENF-280C UV
lamp, at 365 nm, 23 W) followed by HPLC analysis. It was
found that NDBF-thymidine decages rapidly with a half-
life of about 30 s (see Figure 2 and Supporting Information).
In summary, we have developed an improved synthetic
route to the recently discovered two-photon caging group
3-nitro-2-ethyldibenzofuran (NDBF). Due to its favorable
photochemical fragmentation mechanism, we were able to
apply the NDBF group in the successful caging of a
thymidine phosphoramidite. This caging moiety is stable to
standard DNA synthesis conditions and thus can be readily
incorporated into the synthesis of photocontrollable oligo-
nucleotides for biological applications.
Acknowledgment. Financial support from the National
Institutes of Health (R01GM079114) is acknowledged. A.D.
is the recipient of a Beckman Young Investigator Award, a
Cottrell Scholar Award, an NSF CAREER Award, and a
TEVA USA Scholars Grant.
Supporting Information Available: Synthetic protocols
and analytical data, stability tests and decaging experimentals,
and copies of 1H and 13C NMR spectra of new compounds.
This material is available free of charge via the Internet at
http://pubs.acs.org.
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6513.
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