Selective product amplification of thymine photodimer by recognition-directed supramolecular assistance

Article abstract of DOI:10.1039/b605658j

Two symmetric ditopic supramolecular templates (1 and 2) each presenting two hydrogen bonding recognition subunits were synthesized. Each such subunit comprises the same donor and acceptor pattern, capable of binding a substrate molecule with complementar

Full text of DOI:10.1039/b605658j

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Selective product amplification of thymine photodimer by recognition-directed
supramolecular assistance
W. G. Skene,† Volker Berl,‡ He´le`ne Risler, Richard Khoury§ and Jean-Marie Lehn*
Received 20th April 2006, Accepted 15th August 2006
First published as an Advance Article on the web 30th August 2006
DOI: 10.1039/b605658j
Two symmetric ditopic supramolecular templates (1 and 2) each presenting two hydrogen bonding
recognition subunits were synthesized. Each such subunit comprises the same donor and acceptor
pattern, capable of binding a substrate molecule with complementary hydrogen bonding groups to form
a supramolecular complex. Substrate molecules, such as thymine or uracil derivatives, yield 2 : 1
complexes with the acceptors involving two hydrogen bonds to each subunit with ideal orientation for
subsequent [2 + 2] dimerization upon photoirradiation. Selective syn photoproduct formation and
concomitant suppression of the trans isomer are favored by orientation of the two guest nucleobases
within the template cleft. Complementary donor and acceptor hydrogen bonding induced positioning
of the two substrates and steric hindrance within the template clefts are responsible for the selective
product formation.
investigated in the hope of influencing the photoproduct outcome
Introduction
through molecular recognition.39–43
The [2 + 2] photodimerizations1,2 of thymine and uracil have been
subject to much research effort as their products exhibit high
biotoxicity.3–6 Upon UV irradiation, these cellular nucleobases are
Here we present two such hosts 1 and 2 (Fig. 2) displaying two
recognition subunits that contain each a hydrogen bond donor
=
N–H and a hydrogen bond acceptor –N site. These receptors
known to undergo dimerization, giving products that are directly
are capable of binding two complementary guests of the uracil
responsible for cell death via mutagenic action, suppression of
(4A) or thymine (4B) type (Fig. 3) within their cleft through
hydrogen bonds, thus providing supramolecular assistance44,45 to
the generation of photoproducts upon irradiation as well as to in-
duction of product distribution by preorganization. Owing to our
previous success of controlled photodimerization with 2,46 we
investigated the use of this molecule as a host for biologically-
relevant bases. We describe selective photoproduct amplification
and suppression of normally produced photoproducts for the
biologically relevant thymine nucleobase through formation of
supramolecular hydrogen-bonded complexes of two thymine
derived substrates 4C with the molecular receptors 1 and 2.
Compound 3 was used as scaffold for control experiments.
DNA transformation and/or activation of carcinogenic pathways
by basal cell and squamous cell tumors. The mechanism by
which these toxic photodimers (as well as their derivatives) are
produced, has been extensively investigated in order to suppress
their formation or to repair the site by photochemical or enzymatic
reversible cleavage.7,8 Direct or sensitized photoirradiation of
[2 + 2] precursors, including thymine and uracil, and similar
photodimerization reactions potentially lead to four cycloaddition
products; cis–syn, trans–syn, cis–trans, anti–trans, and anti–cis
(A, B, C, D, respectively, Fig. 1).6,9–11 The distribution of such
products cannot be readily controlled so as to afford a given
photodimer. Typically, specific reaction conditions, such as the use
of various reagents or additives, changes in solvent polarity,4,5,12,13
micelles,14 solid-state irradiation,15–21 additives,22 host–guest in-
clusion complexes,23–26 dipolar interactions,27–30 and precursor
tethering31–38 give only moderate success in controlling the distribu-
tion of photoproducts. Recently, stoichiometric additives, forming
1 : 1 complexes through hydrogen donor/acceptor sites suitable for
complementary hydrogen bonding with the precursors, have been
Results and discussion
Nucleobases of the uracil and thymine type are ideal candidates for
studying selective photoproduct formation through supramolec-
ular control of [2 + 2] photodimerization. They possess two sites
capable of sustaining two hydrogen bonds with complementary
receptors, leading to the formation of hydrogen-bonded complexes
of specific orientation and positioning. Moreover, they readily
undergo [2 + 2] photodimerization with varying distributions
of the possible products A–D (Fig. 1). In view of enforcing
photoproduct selectivity, we investigated whether the molecular
receptors 1 and 2 were capable of forming a specifically oriented
supramolecular adduct with two dimer precursors (Fig. 4). This,
ultimately, would promote selective photoproduct amplification
through geometric control arising from the supramolecular archi-
tecture and steric confinement. The inter-substrate distance in the
2 : 1 supramolecular adducts is suitable for the photodimerization.
Laboratoire de Chimie Supramole´culaire, ISIS-Universite´ Louis Pasteur, 8,
alle´e Gaspard Monge, BP 70028, 67083, Strasbourg cedex, France. E-mail:
lehn@isis.u-strasbg.fr
† Present address: De´partement de Chimie, Universite´ de Montre´al, C.P.
6128, succ. Centre-Ville, Montre´al, Que´bec, Canada H3C 3J7
‡ Present address: BASF Aktiengesellschaft, Global New Business De-
velopment, Chemical Intermediates for Life Sciences, BASF Aktienge-
sellschaft, CZ/BL-E100, D-67056, Ludwigshafen, Germany
§ Present address: Exelixis Inc., 210 East Grand Avenue, San Francisco,
CA, USA, 94080
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Fig. 1 Potential dimers resulting from [2 + 2] photodimerization of nucleic bases.
Fig. 2 Molecular receptor scaffolds 1 and 2 for selective [2 + 2] photodimerization of thymine derivatives through 2 : 1 supramolecular complex
formation, and control scaffold 3.
The symmetric structure of the 2 : 1 adduct (Fig. 4) is expected to
favor the formation of syn (cis and trans) dimers upon irradiation.
Normally produced anti dimers would be suppressed, since the
corresponding orientation is not possible due to the donor–
acceptor pattern and to steric hindrance within the template cleft.
selected route provided nearly quantitative yield for each step
with the advantage of easy purification of the desired intermediate
dialdehyde 16. Subsequent coupling of 16 according to our
previous method, afforded template 2. In addition, a more rigid
template 21 was synthesized which comprised a dibenzofuran
consisting of a planar backbone. It was unfortunately insoluble
in most organic solvents, in particular those commonly used to
promote intermolecular hydrogen bonding. To circumvent this
short fall, dialkylation in positions 2 and 8 of the dibenzofuran
moiety was pursued, leading to a much more soluble alternative
template 1. The methodology for the synthesis of 1 was similar to
that used for 2, resulting in approximately the same overall yield.
Binding of uracil and thymine derivatives to templates 1 and 2
Previous work has shown the capability of bis-receptors such
as 1 and 2 to efficiently bind substrate molecules via N–H· · ·N
hydrogen bonds between the pyridine nitrogen and the adjacent
NH site and complementary donor and acceptor sites.46–48 The
association constants for the formation of such supramolecular
adducts with one (K1) and two (K2) monomeric substrates or
with a dimer was determined by NMR binding isotherms. These
were obtained by titrating 2 mM host concentrations in CDCl3
with the corresponding nucleobases and monitoring the resulting
Fig. 3 Thymine and uracil derivatives.
Receptor synthesis (Scheme 1)
The synthesis of receptor 2 was achieved in an overall 50%
=
yield from the combined steps from commercial reagents. The
chemical shift changes of the C–H and the –NH proton of
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Fig. 4 Supramolecular complexes arising from multiple hydrogen bonding between receptors 1 and 2 and uracil and photodimer substrates.
=
the templates. Subsequent treatment of the NMR data with the
ChemEqui program49 provides the association constants (Table 1)
corresponding to the binding through hydrogen bond formation
between complementary O, N and N–H sites, as represented by the
structures shown in Fig. 4. The NH signal undergoes a chemical
shift change of d 1.6 ppm and the isotherm measured correlated
well with that determined for the CH proton signal. The binding
isotherms provide the association constants for 1 : 1 and 2 : 1 com-
plexes, which undergo formation of a weak 1 : 1 complex followed
by a 2 : 1 complex upon further substrate addition (Table 1).
The values measured are within the range of complementary
donor/acceptor interactions of the –NH· · ·N( R1)R2 type.41,42
=
For template 1, the photodimer complex 11 exhibits a stronger
binding affinity than the monomer form 10. The donor/acceptor
sites are consequently blocked upon photoproduct formation
and cannot undergo further monomer binding. Conversely, the
binding affinities for the monomer complex 12 are greater than
the photodimer 13. The greater preference for the monomer over
the photoproduct demonstrated for template 2 implies it ultimately
can be used as a photocatalyst for dimerization.
Table 1 Association constants determined by NMR titration in CDCl3
from isotherms using ChemEqui49
Complex
K1/M−1a
K2/M−1a
10
11
12
13
30
5
100 10
20 000 700
180 10
240
—
1 100 110
—
5
The binding differences between the two templates can be
ascribed to the different cleft sizes and the intramolecular donor–
acceptor distances. Crystallographic studies (see Fig. 5 and 6)
a K1 and K2 are the ChemEqui b derived values for the A + B → AB and
the A + 2B → AB2 reactions, respectively.
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Scheme 1 Receptor synthesis (A) (i) ethanol, catalytic H2SO4, reflux; (ii) LiAlH4–Et2O (anhydrous); (iii) MnO2–toluene–CH2Cl2, room temperature (1 : 1);
(iv) 2-hydrazinopyridine–CHCl3, room temperature; (v) N,N-dimethyl hydrazine–EtOH, reflux. (B) (i) AlCl3, butyryl chloride, 1,2-dichloroethane,
reflux; (ii) hydrazine hydrate, KOH, triglycol reflux; (iii) TMEDA, n-BuLi in Et2O (anhydrous) −78 ◦C, DMF; (iv) 2-hydrazino pyridine–CHCl3, room
temperature.
show that the sp3 bridgehead of 2 gives it a non-planar shape with
a tighter bite angle. Conversely, the rigid and planar structure of
1 has a large bite angle. These factors, affecting inter-substrate
distances and orientation, are ultimately responsible for the
observed differences in binding affinities. The hydrogen bonding
capabilities of these two receptors is further evidenced by their
crystal state structures. In the solid state, the two receptors
self-associate through their complementary receptor acceptor
sites forming supramolecular networks. The differences in the
receptor orientation and backbone rigidity affect the resulting
supramolecular structures. The non-planar shape of receptor 2,
combined with the small bite angle, allows the complementary
donor–acceptor sites to align so that the thermodynamically stable
structure (Fig. 7 and 8) is a dimer resulting from hydrogen bonding
between the ditopic sites. Conversely, the rigid dibenzofuran
backbone of 1 forces the receptor sites to point outwards. There
is also a slight twist of the dibenzofuran backbone that further
orients the receptor sites out of the plane of the aromatic unit.
The resulting large receptor cleft can accommodate two receptors
side arms through complementary hydrogen bonding, resulting in
a supramolecular ribbon architecture (Fig. 9).
isolated through a sequence of column chromatography, prepara-
tive thin layer chromatography and semi-preparative HPLC. NOE
and COSY 2-dimensional NMR, in addition to analytical HPLC,
allowed for differentiation and subsequent identification of the
three main photoproducts, which were 7A, 7B and 7D. The latter
two were formed in majority. The overall results are consistent with
those described for uracil and thymine derivatives.50–52 Isolation of
sufficient quantities of photodimerization product of the thymine
4C can only be done by sensitized irradiation with acetone. Direct
irradiation at ca. 254 nm is possible, however the dimerization
reaction is photoreversible in the UV region leading to photoprod-
uct decomposition, which reverts back to the starting material.
Furthermore, under direct irradiation, the templates examined act
as light screens and undergo severe photodecomposition, which
does not occur via the triplet-sensitized method.
Photodimerization of 4C with templates 1 and 2
Photosensitized irradiation of a mixture of 2 and two equivalents
of thymine derivative 4C led to the complete suppression of the
anti products illustrated in Fig. 10 (7C and 7D). To confirm
that the formation of the supramolecular complex between the
template and 4C was responsible for the suppression of these
products, trace amounts of acetic acid were added to the reaction
mixture, in order to disrupt the hydrogen bonding and displace the
Photodimerization of thymine 4C
Samples of authentic photodimers were obtained by photosen-
sitized irradiation of thymine derivative 4C in acetone and were
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Fig. 5 Crystal structure of receptor 1.
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Fig. 6 Crystal structure of receptor 2.
promote molecular organization by positioning two substrates
bound thymine substrate. Photoirradiation gave a photoproduct
distribution similar to that in the absence of the template. This
result, coupled with the strong binding constant, demonstrates
the capacity of the bis-receptor 2 to amplify the formation of
photoproducts 7A and 7B. This arises through formation of a
hydrogen bonded 2 : 1 supramolecular complex, with parallel
positioning of the two substrate molecules consistent with syn
[2 + 2] photodimers. The two anti products are suppressed because
the antiparallel precursor alignment required for their formation
is incompatible with the hydrogen bond donor–acceptor site
arrangement and the steric hindrance within the template cleft.
Template 1 also exhibited photoproduct control similar to
that observed with 2. Due to its poor solubility in acetone, a
mixture of acetone–chloroform was used for the photoirradia-
tions. A control experiment under the same conditions indicated
again photodimerization control with selective product formation
through the ground state supramolecular complex.
˚
within the optimal 6 A distance required for photodimerization.
No effect on photoproduct distribution was therefore expected
and, indeed, none was observed. The selective photoproduct
amplification caused by the receptors 1 and 2 may, therefore, be
ascribed to the formation of a 2 : 1 supramolecular adduct with two
substrate molecules 4C. The hydrogen bonding recognition posi-
tions the two thymine monomers in a favourable pre-photodimer
fashion. The parallel orientation of the two substrates ensures
that only the syn products are formed upon photoirradiation. The
intramolecular distance of the N–H donor sites within the cleft
˚
of 2 was found to be 4.6 A, according to the crystal structure
data.54 This corresponds well with the amplification of the syn
dimers, the intramolecular O acceptor site distances of which are
3.9 A. The pocket could potentially transversally incorporate a
3
˚
single monomer forming a 1 : 1 complex. Under stoichiometric
control, such an arrangement would block the receptor, preventing
selective syn amplification, while the residual monomer would
undergo conventional unselective photodimerization. Given the
templated dimerization data, the 2 : 1 complex predominates and
is responsible for the results observed.
Photoirradiation control experiments
Experiments with the pseudo-template 3 were undertaken to
confirm the role of the hydrogen bonding pattern and the steric
effects for controlled photodimerization.53 This analogue lacks
the NH donor sites, but has a cleft similar to 2 into which two
monomers could potentially be accommodated, on steric grounds,
but without hydrogen bond formation. The photoproduct distri-
bution obtained upon photoirradiation of 4C in the presence of
3 was identical to that observed in the absence of this pseudo
template. The effect of 2-aminopyridine itself was also examined,
because it is capable of forming a supramolecular complex with
a single substrate via its donor and acceptor motifs. It cannot
Experimental
Materials and methods
Unless otherwise stated, all chemicals were purchased from
Aldrich and were used as received. HLPC-spectroscopic grade
solvents were used for HPLC analyses run on a HP 1100 series
HPLC equipped with a diode array detector and an EclipseTM
XDB-C18 reverse phase column. The mobile phase used for
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Fig. 7 Top view of the supramolecular dimer network of receptor 2.
Fig. 8 Side view of the supramolecular dimer network of receptor 2.
separation was 50% methanol–50% water and the retention times Synthesis
2,7-Di-tert-butyl-9,9-dimethyl-9H-xanthene-4,5-dicarboxylic
monitored at 220 nm and absorption maxima were compared to
authentic samples (vide infra). The ChemEqui program was used to
determine the binding constants from the isotherm data obtained
by NMR titration in CDCl3.49 NOE and COSY 2D NMR spectra
were recorded on a Bruker 300 MHz spectrometer.
acid diethyl ester (14). 2,7-Di-tert-butyl-9,9-dimethyl-9H-
xanthene-4,5-dicarboxylic acid (1 g, 2.44 mmol) was dissolved in
ethanol (50 mL). Concentrated sulfuric acid (2 mL) was added
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Fig. 9 Solid-state supramolecular bonding network of receptor 1.
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plug of celite and silica, and then washed with dichloromethane.
The organic layer was evaporated and pure compound (0.79 g,
80%) was obtained as a white powdery solid after recrystallization
from dichloromethane–hexane and from hot toluene–hexane.
Mp 248–249 ◦C. IR (thin film): m = 2958, 2863, 1688, 1606,
1460, 1365, 1296, 1260, 1228, 1167, 930, 902, 868, 851, 748, 704,
1
646 cm−1. H NMR (200 MHz, [D] chloroform): d = 10.68 (s,
2 H), 7.83 (d, J = 2.4 Hz, 2 H), 7.72 (d, J = 2.4 Hz, 2 H), 1.70 (s,
6 H), 1.37 (s, 18 H). 13C NMR (125 MHz, [D] chloroform): d =
188.9, 149.6, 146.7, 130.6, 129.5, 124.1, 123.5, 62.2, 34.8, 32.5,
31.4, 29.7. FAB-MS: m/z 379.3 ([M + H]+, 100%). Anal. calcd for
C25H30O3 (378.50): C 79.33, H 7.99; found: C 79.59, H 8.14.
Fig. 10 Distribution of trans–syn 7B (///), cis–syn 7A ( \ \ \ ) and
trans–anti 7D (≡), isomers, formed by photoirradiation of 4C for 12 h
performed in acetone under in the presence and absence of various addi-
tives. *Denotes reaction undertaken in 2 : 1 (vol%) chloroform–acetone
mixture.
2,7-Di-tert-butyl-9,9-dimethyl-4,5-bis-(pyridin-2-ylhydrazono-
methyl)-9H-xanthene (2). Template 2 was synthesized from 16
as previously described.46
Nꢀ-[2,7-Di-tert-butyl-5-(dimethylhydrazonomethyl)-9,9-dimeth-
and the solution stirred at reflux for 14 h. The solution was
poured onto ice (100 g) and the resulting white precipitate was
collected by filtration. It was washed with cold water until the
wash water was neutral and then dried under vacuum to yield the
product (1.10 g, 96%) as a white powder. Mp 149–150 ◦C. IR (thin
film): m = 2963, 2863, 1724, 1612, 1446, 1393, 1366, 1314, 1243,
yl-8a,10a-dihydro-9H-xanthen-4-yl
methylene]-N,N-dimethyl-
hydrazine (3). The scaffold was prepared from 16 according to
known methods.46
(5-Methyl-2,4-dioxo-3,4-dihydro-2H-pyrimidin-1-yl)acetic acid
benzyl ester (4C). Thymine-1-acetic acid (1.21 g, 6.57 mmol) was
dissolved in 30 DMF with sonication, to which was then added
diimidazol-1-ylmethanone (1.23 g, 7.58 mmol). The slurry was
stirred at room temperature for 10 min until the reagents were
dissolved. Benzyl alcohol was then added (780 ll, 7.56 mmol) and
the solution stirred at room temperature for 12 h. The solvent was
removed under reduced pressure and the paste was suspended in
water. The product was isolated as a white solid (1.67 g, 93%) upon
1
1180, 1146, 1100, 1029, 857, 782 cm−1. H NMR (200 MHz, [D]
chloroform): d = 7.58 (d, J = 2.4 Hz, 2 H); 7.53 (d, J = 2.4 Hz, 2
H), 4.42 (q, J = 7.1 Hz, 4 H), 1.64 (s, 6 H), 1.40 (t, J = 7.2 Hz,
6 H), 1.33 (s, 18 H). 13C NMR (125 MHz, [D] chloroform): d =
166.9, 146.9, 145.4, 130.5, 125.8, 125.6, 120.6, 61.1, 34.8, 34.6,
32.1, 31.5, 14.4. FAB-MS: m/z 467.2 ([M + H]+, 100%). Anal.
calcd for C29H38O5 (466.61): C 74.65, H 8.21; found: C 74.55, H
8.36.
◦
1
filtration. Mp = 201–203 C. H NMR (200 MHz, [D] DMSO):
d = 11.42 5 (s, 1 H), 7.65 (s, 1 H), 7.53 (s, 3 H), 5.57 (s, 2 H),
4.82 (s, 2 H), 1.44 (s, 3 H). 13C NMR (50 MHz, [D] chloroform):
d = 168.09, 164.16, 150.87, 141.41, 135.44, 128.36, 128.12, 127.83,
108.52, 90.18, 66.31, 48.36, 11.78. EI-MS: m/z 574.2 ([M]+, 20%).
Anal. calcd for C14H14N2O4 (274.10): C 61.31, H 5.14, N, 10.21;
found: C 60.90, H 5.12, N 10.39.
(2,7-Di-tert-butyl-5-hydroxymethyl-9,9-dimethyl-9H-xanthen-
4-yl)-1 methanol (15). A solution of 14 (1.1 g, 2.35 mmol) in
diethyl ether (10 mL) was added dropwise to a stirred suspension of
lithium aluminum hydride (LAH, 0.15 g) in diethyl ether (25 mL).
The mixture was subsequently refluxed for 1.5 h. Additional LAH
was added (0.5 g) and the suspension heated to reflux for 1 h to
drive◦ the reaction to completion. The mixture was then cooled
to 0 C and ice water was added dropwise until the evolution of
hydrogen ceased. 10% Sulfuric acid was added until the precipitate
of AI(OH)3 was dissolved. The ether layer was removed and the
aqueous layer was washed twice with ether (50 mL). The combined
organic extracts were dried over MgSO4, evaporated to dryness
and chromatographed (SiO2) with 30% ethyl acetate–hexane to
provide a white powder (0.89 g, 99%). Mp 199–200 ◦C. IR
(thin film): m = 3261, 2951, 2863, 1464, 1413, 1362, 1326, 1297,
1-(4-tert-Butylbenzyl)-1H-pyrimidine-2,4-dione (4D). To
a
suspension of uracil (1.00 g, 9.00 mmol) in 20 mL anhydrous
acetonitrile was added N,O-bis-trimethylsilyacetamide (5.6 g,
7.0 mL, 28 mmol). The mixture was stirred at room temperature
until complete dissolution of the initial solid. To this was added
tert-butylbenzyl bromide (2.04 g, 9 mmol) and the mixture was
refluxed for 12 h, at which time the solvent was evaporated and
the residue purified by column chromatography (SiO2) with 5%
methanol–dichloromethane. The product was obtained as a white
1
1279, 1223, 1143, 1033, 903, 883, 852, 763, 664 cm−1. H NMR
◦
1
solid. Mp = 165–166 C. H NMR (200 MHz, [D] chloroform):
d = 9.65 (d, J = 7.9 Hz, 1 H), 7.39 (d, J = 8.3 Hz, 2 H), 7.22
(d, J = 8,3 Hz, 2 H), 7.16 (d, J = 7.9 Hz, 1 H), 5.69 (d, J =
7.9 Hz, 1 H), 4.88 (s, 2 H), 1.31 (s, 9 H). 13C NMR (50 MHz, [D]
chloroform): d = 164.0, 151.8, 151.4, 144.1, 132,1. 128.0, 126.2,
102.7, 51.0, 34.7, 31.3. EI-MS: m/z 258.2 ([M]+, 60%). Anal. calcd
for C15H18N2O2 (258.32): C 69.74, H 7.02, N 10.84; found: C 69.99,
H 7.24, H 11.06.
(200 MHz, [D] chloroform): d = 7.42 (d, J = 2.2 Hz, 2 H), 7.20 (d,
J = 2.2 Hz, 2 H), 4.66 (s, 4 H), 4.49 (br. s, 2 H), 1.67 (s, 6 H), 1.36
(s, 18 H). 13C NMR (125 MHz, [D] chloroform): d = 146.9, 145.3,
129.7, 127.2, 125.0, 122.5, 62.2, 34.6, 32.3, 31.7. FAB-MS: m/z
382.2 ([M]+, 57%). Anal. calcd for C25H34O3 (382.54): C 78.49, H
8.96; found: C 78.46, H 9.18.
(2,7-Di-tert-butyl-9,9-dimethyl)-9H-xanthene-4,5-dicarbaldehyde
(16). To a solution of 15 (1.00 g, 2.61 mmol) in toluene (150 mL)
and dichloromethane (50 mL) was added MnO2 (5 g). The
reaction was followed by TLC and was complete after 24 h of
stirring at room temperature followed by refluxing for 2 h. The
finely dispersed MnO2 suspension was filtered through a short
1-(8-Butyryldibenzofuran-2-yl)-butan-1-one (17). Dry alu-
minum chloride (21.42 g, 161 mmol) was added in small por-
tions over 1 h to a well stirred solution of butyryl chloride
(17.12 g, 161 mmol) in 1,2-dichloroethane (65 mL). A solution of
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dibenzofuran (10.4 g, 61.8 mmol) in 1,2-dichloroethane (20 mL)
was added dropwise to the mixture over 30 min while at 50 ◦C. The
mixture was subsequently heated to reflux for 12 h, cooled to
30 ◦C, and poured slowly into ice water (150 mL). Concentrated
hydrochloric acid (25 mL) was added and the mixture was further
stirred for another hour. The organic layer was separated and
the aqueous layer extracted with dichloromethane. The combined
organic extracts were dried, evaporated and chromatographed
(SiO2; 10% dichloromethane–hexane) to give the title compound
(9.9 g, 52%) as a slightly brown solid. Mp 125–127 ◦C. IR
(thin film): m = 2960, 2872, 1680, 1631, 1596, 1564, 1467, 1412,
2,8-Dibutyl-4,6-bis-(pyridin-2-ylhydrazonomethyl)dibenzofuran
(1). To a flask containing 19 (0.24g, 0.713 mmol) in chloroform
(4 mL) was added a solution of 2-hydrazinopyridine (0.18 g,
1.57 mmol) in chloroform (1 mL). The solution was stirred
overnight at room temperature after which the product precipi-
tated as a yellow–grey solid (0.35 g, 96%) which was collected by
filtration, washed with small amounts of chloroform and hexane,
and then dried under the high vacuum. Mp 248–249 ◦C. IR (thin
film): m = 3186, 2923, 2839, 1602, 1572, 1515, 1442, 1305, 1182,
1155, 1106, 1078, 990, 767 cm−1.
Dibenzofuran-4,6-dicarbaldehyde (20). Dibenzofuran (5.77 g,
34.3 mmol) and TMEDA (11.96 g, 102.9 mmol) were dissolved in
dry diethyl ether (200 mL). A 1.6 M solution of n-butyllithium
(64.1 mL, 102.9 mmol) was added dropwise and the mixture
refluxed for 16 h. DMF (7.9 mL, 102.9 mmol) was added over
a period of 10 min at 0 ◦C, and then the mixture was stirred
further for 24 h at room temperature. The mixture was poured
into cold 1 M hydrochloric acid (150 mL) and the aqueous
layer was extracted 3 times with dichloromethane. The combined
organic extracts were washed with water and brine, dried over
MgSO4, and chromatographed (SiO2; dichloromethane) to yield
the product (4.63 g, 60%) as a white crystalline solid. As a side
product, dibenzofuran-4-carbaldehyde could be obtained. Mp
224–225 ◦C. IR (thin film): m = 2839, 1682, 125, 1601, 1479, 1435,
1422, 13393, 1336, 1259, 1227, 1186, 1131, 1056, 996, 844, 814,
1
1368, 1306, 1248, 1116, 1011, 997, 900, 845, 753 cm−1. H NMR
(200 MHz, [D6] DMSO): d = 9.00 (d, 41 = 1.4 Hz, 2 H); 8.15 (dd,
31 = 8.7 Hz, 41 = 0.9 Hz, 2 H); 7.81 (d, 31 = 8.7 Hz, 2 H); 3.13
(t 31 = 7.1 Hz, 4 H); 1.68 (sm, 4 H); 0.98 (t 31 = 7.4 Hz, 6 H).
13C NMR (50 MHz, [D6] DMSO): d = 198.9; 158.5; 132.7; 128.1;
123.5; 122.5; 111.9; 17.3; 13.6. FAB-MS: m/z 309.1 ([M + H]+,
100%). Anal. calcd for C20H2003 (308.37): C 77.90, H 6.54; found:
C 77.80, H 6.52.
2,8-Dibutyldibenzofuran (18). To 17 (9.75 g, 31.6 mmol),
hydrazine hydrate (9.5 g, 10 mL, 190 mmol) and finely ground
potassium hydroxide (14.28 g, 254 mmol) were suspended in
triglycol (150 mL) and heated to reflux for 2 h. The excess hydrazine
and water were subsequently distilled off. After cooling to below
90 ◦C, water (300 mL) was added and the mixture was extracted
with diethyl ether and dichloromethane (twice, 200 mL, portions).
The combined organic extracts were evaporated and the residue
applied to a column (SiO2; 7% dichloromethane–hexane) to afford
the product (2.6 g, 29%) as a highly viscous translucid oil. IR (thin
film): m = 3030, 2959, 2928, 2867, 1869, 1485, 1468, 1417, 1337,
1362, 1318, 1274, 1246, 1207, 1187, 1103, 1091, 1118, 1024, 931,
869, 811, 747 cm−1. 1H NMR (200 MHz, [D6] DMSO): d = 7.91
(s, 2 H); 7.54 (d, 3J = 8.4 Hz, 2 H); 7.30 (d, 3J = 8.4 Hz, 2 H); 2.72
(t, 3J = 7.5 Hz, 4 H); 1.63 (sm, 4 H); 1.35 (sm, 4 H); 0.91 (t, 3J =
7.2 Hz, 6 H). 13C NMR (50 MHz, [D6] DMSO): d = 154.1; 136.9;
127.6; 123.5; 120.1; 111.0; 34.6; 33.5; 21.6; 13.7. FAB-MS: m/z
280.1 ([M]+, 100%). Anal. calcd for C20H2403 (280.40): C 85.67, H
8.63; found: C 85.73, H 8.90.
1
782, 720 cm−1. H NMR (200 MHz, [D] chloroform): d = 10.70
(s, 2 H); 8.25 (dd, 3J = 7.7 Hz, 4J = 1.0 Hz, 2 H); 8.05 (dd, 3J =
4
3
7.7 Hz, J = 1.0 Hz, 2 H); 7.56 (t, J = 7.7 Hz, 2 H). FAB-MS:
m/z 225.0 ([M + H]+, 100%).
4,6-Bis-(pyridin-2-ylhydrazonomethyl)dibenzofuran (21). To
20 (0.24 g, 0.713 mmol) in chloroform (4 mL) was added a solution
of 2-hydrazinopyridine (0.18 g, 1.57 mmol) in chloroform (1 mL).
The solution was stirred overnight at room temperature and the
product precipitated as a yellow–grey solid (0.35 g, 96%) that was
collected by filtration, washed with small amounts of chloroform
and hexane, and then dried under high vacuum. Mp >250 ◦C. IR
(thin film): m = 3198, 2982, 1594, 1570, 1510, 1423, 1331, 1277,
1175, 1115, 1083, 1143, 983, 770, 736 cm−1. 1H NMR (200 MHz,
[D6] DMSO): d = 11.19 (s, 2 H); 8.54 (s, 2 H); 8.15 (d, 3J = 4.3 Hz,
2 H); 7.93 (d, 4J = 1.6 Hz, 2 H); 7.80 (d, 4J = 1.6 Hz, 2 H); 7.70
2,8-Dibutyldibenzofuran-4,6-dicarbaldehyde (19). To a solu-
tion of 18 (1 g, 3.56 mmol) and TMEDA (1.66 g, 14.3 mmol)
in dry diethyl ether (25 mL) was added a hexane solution of
n-butyllithium (1.6 M, 8.9 mL, 14.3 mmol). After refluxing for
16 h, DMF (1.25 mL, 16.1 mmol) was added over a period of
5 min while cooling at 0 ◦C, The solution was then warmed to
room temperature and allowed to stir for 24 h. The mixture was
then poured into cold 1 M hydrochloric acid (30 mL) and the
acidic aqueous layer was extracted twice with dichloromethane.
The combined organic extracts were washed with water and brine,
dried over MgSO4, and then purified by chromatography (SiO2;
10% ethylacetate–hexane) to afford the product (0.54 g, 45%) as
3
4
3
(td, J = 7.9 Hz, J = 1.7 Hz, 2 H); 7.37 (d, J = 8.3 Hz, 2 H);
6.81 (t, J = 7.9 Hz, 2 H). 13C NMR (50 MHz, [D6] DMSO):
3
d = 159.0; 152.6; 147.8; 138.1; 132.2; 124.1; 123.6; 123.3; 120.9;
120.2; 106.6. FAB-MS: m/z 407.1 ([M + H]+, 100%). Anal. calcd
for C24Hl8N60 (406.44): C 70.92, H 4.46, N 20.68; found: C 73.34,
H 4.40, N 20.62.
Photodimerization
Photodimers. The photodimers were isolated according to the
following procedure. In a Schlenk line was added 200 mg of 4C
which was dissolved in 60 ml acetone with sonication. The flask
was subjected to four cycles of freeze–pump–thaw, sealed, and
then irradiated for 12 h with a 400 W tungsten lamp. The solvent
was subsequently removed and the yellow oil purified on silica
with neat dichloromethane. The apolar fractions were collected
and then purified by semi-preparative HPLC to yield 7A. The
polar fractions were collected, concentrated, and then isolated by
1
a white solid. H NMR (200 MHz, [D] chloroform): d = 10.53
4
4
(s, 2 H); 7.91 (d, J = 1.6 Hz, 2 H); 7.73 (d, J = 1.6 Hz, 2 H);
3
2.77 (t, J = 7.7 Hz, 4 H); 1.66 (sm, 4 H); 1.36 (sm, 4 H); 0.94
(t, J = 7.2 Hz, 6 H). 13C NMR (50 MHz, [D] chloroform): d =
3
187.6; 155.2; 138.6; 127.3; 126.2; 124.6; 120.8; 35.2; 33.8; 22.2;
13.8. FAB-MS: m/z 336.2 ([M]+, 100%).
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preparative TLC with 98% chloroform–methanol to afford 7B and
7D. Absolute assignment of the photoproducts was done by COSY
and NOE 1H-NMR and compared to uracil dimer analogues.55,56
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(s, 2 H), 4.28 (d, J = 17.8 Hz, 2 H), 4.1 (m, 2 H), 3.92 (d, J =
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acid benzyl ester (7B). 1H NMR (300 MHz, [D6] DMSO): d =
10.77 (s, 2 H), 7.37 (s, 10 H), 5.16 (s, 4 H), 4.44 (d, J = 17.8 Hz, 2
H), 3.96 (s, 2 H), 3.47 (d, J = 17.4 Hz, 2 H), 1.25 (s, 6 H).
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Acknowledgements
The authors acknowledge Dr D. Hickman and Prof. F. Schaper or
helpful suggestions and Prof. T. Carell for a gift of authentic uracil
dimers 9A and 9B. WGS acknowledges the Natural Sciences and
Engineering Research Council Canada and the Universite´ Louis
Pasteur for financial support.
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˚
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3
˚
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Org. Biomol. Chem., 2006, 4, 3652–3663 | 3663
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