Diol appended quenchers for fluorescein boronic acid

Article abstract of DOI:10.1002/asia.200900386

Fluorescein isothiocyanate is treated with 3-aminophenylboronic acid to provide a fluorescently tagged boronic acid derivative which is used to assess Forster resonance energy transfer (FRET) quenching upon boronate ester formation with a series of bespoke diol appended quenchers. Fluorescence spectroscopy comparison of quenching efficiency between treatment of fluorescein and its boronic acid appended congener with quencher appended diol reveals boronate ester formation (covalently linked) to be the more efficient regime and from the panel of quenchers which also included nucleosides.

Full text of DOI:10.1002/asia.200900386

DOI: 10.1002/asia.200900386
Diol Appended Quenchers for Fluorescein Boronic Acid
Souad A. Elfeky,[a, b] Stephen E. Flower,[b] Naoko Masumoto,[b] FranÅois D’Hooge,[b]
Ludivine Labarthe,[b, c] Wenbo Chen,[a] Christophe Len,[d] Tony D. James,[b] and
John S. Fossey*[a]
Dedicated to the 150th anniversary of Japan–UK diplomatic relations
Abstract: Fluorescein isothiocyanate is
treated with 3-aminophenylboronic
acid to provide a fluorescently tagged
boronic acid derivative which is used to
assess Fçrster resonance energy trans-
fer (FRET) quenching upon boronate
ester formation with a series of be-
spoke diol appended quenchers. Fluo-
rescence spectroscopy comparison of
quenching efficiency between treat-
ment of fluorescein and its boronic
acid appended congener with quencher
appended diol reveals boronate ester
formation (covalently linked) to be the
more efficient regime and from the
panel of quenchers which also included
nucleosides.
Keywords: boronic acid · fluoro-
phore · nucleoside · quencher ·
receptor
Introduction
Boronic acids readily and reversibly form boronate esters
with 1,2- and 1,3-diols under basic conditions, Scheme 1 ex-
emplifies cyclic boronate ester formation from phenyl bor-
onic acid and a generic diol in the presence of hydroxide
ions. Reversible cyclic boronate formation has been exploit-
Scheme 1. Generic phenylboronate ester formation.
[a] Dr. S. A. Elfeky,+ Dr. W. Chen, Dr. J. S. Fossey
School of Chemistry
ed widely in saccharide sensing[1–7] and supramolecular
chemistry[8–10] as well as electrophoresis[11–13] and enantio-
meric excess determinations,[14–17] the higher binding affini-
ties of bidentate diols over monodentate alcohols permits
studies to be performed in alcoholic solutions.[18]
It was reasoned that through judicious choice of boronic
acid and diol derivatives in “molecular beacon” type fluoro-
phore–quencher pairs, similar to the methodology used in
quantitative PCR assays such as Taqman, may be covalently
assembled in solution. That is since FRET efficiency is
strongly dependent on donor–acceptor separation, covalent-
ly linking fluorophore and quencher by means of boronate
ester formation should significantly enhance the observed
quenching. This principle was recently demonstrated by us
for surface appended fluorophores excited by means of the
surface plasmon in an SPR experiment, for a highly engi-
neered system with a specific target function.[19] Solution
studies of this effect, represented generically in Scheme 2,
University of Birmingham
Edgbaston, Birmingham, B15 2TT (UK)
E-mail: j.s.fossey@bham.ac.uk
Homepage: www: http://johnfossey.com
[b] Dr. S. A. Elfeky,+ Dr. S. E. Flower,+ N. Masumoto, Dr. F. D’Hooge,
L. Labarthe, Dr. T. D. James
Department of Chemistry
University of Bath
Claverton Down, Bath, BA2 7AY (UK)
[c] L. Labarthe
Universitꢀ de Poitiers
40, Avenue du Recteur Pineau
F-86022 Poitiers Cedex (France)
[d] Prof. C. Len
Universitꢀ de Technologie de Compiꢁgne (UTC)
ESCOM EA 4297 UTC/ESCOM
1, Allꢀe du rꢀseau Jean-Marie Buckmaster, F-60200 Compiꢁgne,
France.
[+] These authors contributed equally to this work.
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FULL PAPERS
trivial, renders it suitable for investigation in bespoke boro-
nate diol signalling systems, as in Scheme 2.
First, a new boronic acid derivative of fluorescein was
prepared as shown in Scheme 3. The NCS derivative of fluo-
rescein 3 was stirred in dimethylformamide (DMF) with 3-
aminophenylboronic acid 4, at room temperature for 12 h to
furnish the thiourea linked boronic acid fluorescein conju-
gate 5 in 68% yield.
Scheme 2. Schematic representation of boronate ester formation resulting
in an enhanced (static) FRET quenching.
with easy to prepare and operationally simple substrates are
reported herein.
Results and Discussion
Synthesis
Derivatives of fluorescein 1 (depicted in Figure 1) are com-
monly used as biotags and are among the most readily avail-
able and easily accessible commercially available fluoro-
phores. As such they are ideally suited to derivatization and
Scheme 3. Preparation of fluorescein boronic acid 5.
Next, a series of diol appended quenchers, inspired by the
methyl red motif, were prepared. Common to the synthesis
of the quenchers prepared was diol 6, which was prepared
in quantitative yield, Scheme 4 To demonstrate the tunabili-
Figure 1. Structures of fluorescein 1 and methyl red 2.
Scheme 4. Preparation of diol unit 6.
exploitation in various sensing and signalling regimes.
Equally, that methyl red 2 is a routinely used quencher for
fluorescein,[20] and the synthesis of derivatives is relatively
ty of quencher design and ease of diol appended quencher
synthesis, various aniline derivatives were employed in a di-
azotization step utilizing standard conditions to furnish diol
appended quenchers 7, 8, and 9 in good yields, Scheme 5.
Abstract in Japanese:
Scheme 5. Synthesis of diol appended quenchers 7, 8, and 9 and prepara-
1
À
tion of intermediate to 8 (R NH2).
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Diol Appended Quenchers for Fluorescein Boronic Acid
The absorbance spectra of the new diol appended quench-
ers 7, 8, and 9 where recorded and contrasted with that of
methyl red 2 as well as the emission spectra of fluorescein 1
and the boronic acid derivative 5, see Figure 2.
Fluorescence Quenching Experiments
Diol Appended Methyl Red Analogues
With a fluorophore appended boronic acid and a series of
diol appended quenchers in hand, we set about assessing the
influence of boronate ester formation between said quench-
er and fluorophore on quenching ability. That the role of
the diol motif (and thus boronate ester formation) may be
delineated, methyl red itself (2) was used as a control
quencher. This means quenching observed as a result of the
combination of 2 with 5 may be considered as the non-cova-
lent (dynamic) quenching ability of methyl red. Thus,
quenching over and above the non-covalent control should
arise from an increased proximity of the quencher and en-
hanced FRET efficiency, in our case, as a result of boronate
ester formation, that is, a covalent interaction results in an
increased closeness in space (static), as depicted in Figure 3.
The presence of a quencher of suitable absorption charac-
teristics should lead to a reduction in observed fluorescence
intensity (I), thus from a Stern–Volmer plot of I0/I as a func-
tion of quencher concentration one may compare relative
quenching power for a given system (the greater the value
From Figure 2 it can be seen that the emission wavelength
of fluorescein 1 matches the emission wavelength of its bor-
onic acid appended congener 5, thus confirming 1 is a suita-
ble model for comparing interactions of 5 with respect to
boronate ester formation. From comparison of the absorp-
tion maxima wavelengths of quenchers 2, 7, 8, and 9 with
fluorophores 5 and 1 in Figure 2, it can be seen that the best
match between fluoresceinꢃs emission and quencher absorp-
tion is achieved with diol appended quencher 8, decreasing
in the order 7, 9, then 2. Although quencher 8 offers the
best match to fluoresceinꢃs emission, its absorbance spec-
trum displays the lowest maxima of the quenchers studied
suggesting its efficiency as a quencher could be diminished
with respect to 7 and 9.
Figure 2. Absorbance (continuous lines) spectra of 2, 7, 8, and 9 and emission (dotted lines) spectra of 1 and 5 (lex =490 nm).
Chem. Asian J. 2010, 5, 581 – 588
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J. S. Fossey et al.
FULL PAPERS
15 mm). Quenching should be more extensive as a result of
boronate ester formation owing to the increase proximity of
the fluorophore quencher pair than in the system where no
boronate ester formation is possible. Ratiometric compari-
son of the corresponding I0/I plots is presented in Figure 5,
Figure 3. Proposed structure and FRET quenching mechanism of a boro-
nate ester formed upon reaction of 5 with 7.
of I0/I the better the quencher). The fluorescence spectra of
5 (lex =490 nm, 0.0135 mm, aqueous methanolic PBS, pH 8.2)
was recorded and the obtained arbitrary intensity value of
fluorescence at 520 nm, corresponding to the fluorescein flu-
orophore part of 5, is the initial intensity of fluorescence
(I0). The effect of the presence of diol appended quenchers
7, 8, and 9 as well as methyl red 2 with no diol motif was as-
sessed (0 to 15 mm). Figure 4 shows the relative quenching
ability, I0/I, upon the fluorescence of 5.
Figure 5. The enhancement in quenching as a result of the presence of a
boronic acid in the fluorophore (I0/I for 5)/(I0/I for 1) (represented as
I(1)/I(5)), at 0.0135 mm, for the addition of diol appended quenchers 7, 8,
and 9.
namely (I0/I for 5)/(I0/I for 1). Since both 5 and 1 would give
rise to equivalent levels of dynamic quenching, it is reasona-
ble to assume this comparison reveals the role of the boron-
ic acid unit of 5, which is responsible for initiating the static
component of quenching, whereas 1 has little or no static
component. Although a modified Stern–Volmer equation
may be used to assess static and dynamic quenching,[21] we
suggest our comparison of the corresponding I0/I ratios is in-
dicative of the inherent stability constant of the complexes
formed. For clarity in graphical representations here and
later in this paper we denote this value as I(1)/I(5) since I0
was essentially the same in both cases, although (I0/I for 5)/
(I0/I for 1) was calculated with the individual values for I0 in
each series.
Figure 4. Comparison of relative quenching action observed on
(0.0135 mm) (I0/I) upon addition of non-diol appended 2 and diol bearing
units 7, 8, and 9 at 520 nm (lex =490 nm).
5
The ratiometric comparison graphically represented in
Figure 5 indicates that 8 has a better quenching ability than
7 when the effect of the presence of a boronic acid is
probed (fluorescein 1 versus boronic acid derivative 5), and
9 remains the poorest of the three diol appended quenchers
compared. This apparent disparity with the data presented
in Figures 4 and 5 may be explained by considering that al-
though 7 is the better quencher of boronic acid appended
fluorophore 5, compound 8 has the greater difference be-
tween quenching ability in its action on 1 versus boronic
acid 5, which is consistent with the observation that 8 has a
better match of absorption profile to fluoresceinꢃs emission
but its absorption is not as efficient as that of 7. Equally,
this can explain why 9 is the poorest quencher of the three
(least efficient overlap). As such, it may be inferred that
From Figure 4, a plot of I0/I reveals that, as expected,
methyl red 2 (over the 0 to 15 mm range studied) had the
least quenching effect on 5 (0.0135 mm). Gratifyingly, diol
appended quenchers elicited a stronger quenching response.
Whilst compound 9 gave only a slightly enhanced quenching
effect over 2, compounds 7 and 8 provided stronger quench-
ing, with 7 being the better quencher of those tested under
these conditions.
In Figure 4, diol and non-diol bearing quenchers are com-
pared. Next, the effect of the boronic acid was probed by
comparing the fluorescence quenching action of quenchers
on fluorophores 1 and 5. In two separate experiments, fluo-
rescein 1 or boronic acid derivative 5 (0.0135 mm) were ex-
posed to an increasing concentration of 7, 8, and 9 (0 to
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Diol Appended Quenchers for Fluorescein Boronic Acid
compound 8 has the highest binding affinity for 5 of the diol
appended quenchers compared,[22] and is therefore the
better quencher in terms of the relative difference with and
without boronic acid.
Nucleosides as Diol Appended Quenchers
Nucleosides are the basic building blocks of RNA and
DNA, and nucleoside analogues are a crucial part of acute
myeloid leukaemia induction therapy, have been used to
treat lymphoproliferative disorder, and have been used in
tumour therapy. The effectiveness of nucleoside analogues
as antiviral and antitumour agents has also provided impetus
in the development of new approaches to their synthe-
sis.[23–25] For example, the 2’,3’-didehydro-2’,3’-dideoxynu-
cleosides (d4Ns) are a significant class of compounds active
against the human immunodeficiency virus (HIV). In order
to provide more data pertaining to the wider structure-activ-
ity relationship (SAR) a number of analogues have been
synthesized and analyzed.[26–32] As such, nucleosides and
their analogues represent important targets for the develop-
ment of new sensing regimes.[33] Importantly, nucleosides
have been previously shown to interact favorably with bor-
onic acids through boronate ester formation via their 1,2-cis-
diol motif.[34] Therefore, we set about probing the effect of
adding nucleosides A (a), G (b), C (c) and U (d) to 5 to
study the effect of covalently attaching real life quencher an-
alytes to fluorescein derivatives through formation of boro-
nate esters, general formula 10, Scheme 6.
Since compound 8 had the greatest ratiometric quenching
enhancement between its use with boronic acid and non-
boronic acid appended quenchers 1 and 5, the effect of fluo-
rophore concentration was also probed. From this series of
experiments, an estimate of the role of boron and its ester
formation with diol appended quenchers may be obtained.
Using the Stern–Volmer data, the slopes of the lines ob-
tained when I0/I is plot against the volume of quencher 8
added, were determined for a range of fixed fluorophore
concentrations (0.0135, 0.0270, 0.0405, 0.0540, 0.0675,
0.0810, 0.0945, 0.1080, 0.1215, and 0.1350 mm). In this case, I0
was set at the first mixed systems fluorescence. This means
I0 corresponds to the fluorescence of systems containing
5 mm quencher and the difference in relative amounts of
quencher represented as D [quencher], since it was found
that inclusion of the zero quencher concentration value in
this ratiometric comparison skewed the data. Figure 6 repre-
Scheme 6. Formation of 10 a–d upon the addition of nucleosides adeno-
sine A (a), guanosine G (b), cytidine C (c), and uridine U (d) to 5.
Figure 6. I0/I plots for differing concentrations of 5 (upper) or 1 (lower)
(0.0135 to 0.1350 mm) upon increasing concentration of quencher 8 [5 to
25 mm]. I0 set at 5 mm 8.
Figure 7 shows a plot comparing the quenching ability of
nucleosides on boronic acid appended quencher 5 as a ratio
of enhancement over that with fluorescein 1. Data were ma-
nipulated as per Figure 5. Since the nucleosides probed all
present a diol motif in the same geometry, the propensity to
form a boronate ester is the same in each case, and as ex-
pected, the data shown in Figure 5 displays no significant
difference in the ability to form boronate esters. Thus, any
intensity effect arising from more or less efficient energy
transfer between different nucleosides is cancelled out in
such a ratiometric comparison.
sents the data obtained, which shows quencher 8 concentra-
tion on the x axis, against I0/I. It is important to note that
ten concentrations of both 1 and 5 were used and the line
plots represent the average of the ten data series recorded.
Figure 6 reveals a plot of I0/I for increasing concentration
of 8 on various concentrations of 1 and 5 (0.0135 to
0.1350 mm). The former, being essentially a linear relation-
ship, confirms a dynamic quenching, whereas the upward
concave curvature of the later relationship suggests both dy-
namic and static quenching are operating, which is in agree-
ment with boronate ester formation.
Although nucleoside G was a better quencher of both 1
and 5 (data not shown), the ratiometric comparison revealed
that the enhanced quenching upon boronate ester formation
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Experimental Section
Reagents were used as commercially
supplied by Sigma–Aldrich without
further purification unless otherwise
noted. 3-Aminobenzeneboronic acid
was purchased from Frontier Scientif-
ic. Methanol refers to HPLC grade
methanol. Fluorescence spectroscopy
studies were conducted in pH 8.21
aqueous methanolic buffer solution,
which consisted of 52.1 wt% HPLC
grade methanol in deionised water
with
KCl
(0.7456 m),
KH2PO4
(0.3745 m),
and
Na2HPO4
(0.3914 m).[35]
A stock solution of fluorescein boronic
acid (5) was prepared as follows: 5
(0.0527 g, 0.1 mmol) was dissolved in
methanol (10 mL) and made up to
100 mL with aqueous methanolic
buffer (pH 8.2). Then, 4.05 mL of the
solution thus obtained was further di-
luted to 3 L with aqueous methanolic
buffer to give 0.0135 mm solution. A
fluorescein solution was similarly pre-
pared.
Quencher stock solution preparation
exemplified by 8: A stock solution was
prepared by dissolving
8 (0.0495 g,
Figure 7. Plot ratiometrically comparing quenching ability of nucleosides on boronic acid appended quencher
5 as a percentage enhancement over that with fluorescein 1.
0.1 mmol) in methanol (1.0 mL) made
up to 10 mL with aqueous methanolic
buffer (pH 8.2). Then 1.0 mL of this
solution was made up to 10 mL with
is the same for A, G, C, and U, as judged by the essentially
coincident plots obtained, Figure 7. This implies the binding
strength of the nucleosides to the boronic acid of 5 is the
same in each case, which one may expect since the 1,2-cis-
diol binding motif is the same in each case. This demon-
strates that such ratiometric comparison may be a viable es-
timator for relative binding constant information.
the aqueous methanolic buffer to give a 1 mm. Other quencher solutions
were similarly prepared.
Nucleosides experiments: Fluorophores 1 and 5 were used at a concen-
tration of 0.0675 mm, prepared by diluting 10 mL of 20 mm solutions in
aqueous methanolic buffer to 3 mL. Nucleoside stock solutions of
125 mm were prepared also in aqueous methanolic buffer.
Instrumentation
Fluorescence spectroscopy measurements were performed using a Gilden
Photonics FluoroSENS SENS-9000 instrument, with Starna Silica
(quartz) cuvettes with 10 mm path lengths and four polished faces. Data
was collected processed with the FluoroSENS 1.6 software package. Nu-
clear magnetic resonance spectroscopy measurements were performed
using Bruker AVANCE 300 and 250 spectrometers in CDCl3, CD3OD, or
CD3OD+D2O (2 drops). 1H NMR spectra were recorded at 300 or
250 MHz, 13C{1H} at 75 or 63 MHz, and 11B{1H} at 96 MHz. Chemical
shifts (d) are expressed in parts per million and are reported relative to
the residual solvent peak or tetramethylsilane as an internal standard and
coupling constants (J) are given in Hertz.
Conclusions
Three diol appended quenchers were synthesized and their
interactions with a fluorescein derivative of a boronic acid,
which was also synthesized, were probed. Boronate ester
formation between diol and boronic acid was shown to en-
hance quenching by comparison with control systems.
Methyl red was employed as a control quencher and fluores-
cein itself as a control fluorophore. That the quenchers syn-
thesized had different absorption profiles and they elicited
different responses with fluorescein appended boronic acid
demonstrates the ready tuneability of this motif. The fluo-
rescein boronic acid prepared was also shown to interact
with nucleosides through boronate ester formation, and a
common binding affinity for nucleosides was demonstrated.
Synthesis
3-(3-(3’,6’-Dihydroxy-3-oxo-3H-spiro[isobenzofuran-1,9’-xanthene]-5-
yl)thioureido)phenylboronic acid (5): 3-Aminobenzeneboronic acid (4)
(0.35 g, 2.57 mmol) was added to a solution of fluorescein isothiocyanate
isomer I (3) (1.00 g, 2.57 mmol) in DMF (5 mL). The reaction mixture
was stirred at room temperature for 12 h, then poured into methanol
(10 mL). The solvents were removed in vacuo the residue was then dis-
solved in the minimum amount of fresh methanol. Chloroform was
added and the product was obtained as a bright orange precipitate
(920 mg, 68% yield). 1H NMR (300 MHz, CDCl3): dH =7.97 (1H, d, J=
2.7), 7.74 (1H, dd, J=8.5 and 2.7 Hz), 7.71 (1H, s), 7.6 (1H, d, J=
8.5 Hz), 7.36 (1H, m), 7.19–7.15 (2H, m), 6.63–6.59 (5H, m), 3.35 ppm
(2H, NH). 13C{1H} NMR (75 MHz, D2O): dC =182.2, 179.9, 160.0, 159.9,
141.9, 141.8, 139.7, 133.1, 132.9, 132.1, 131.7, 131.5, 129.7, 128.2, 127.1,
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Diol Appended Quenchers for Fluorescein Boronic Acid
126.6, 123.6, 123.4, 115.5, 114.9, 104.7, 104.5 ppm. 11B{1H} NMR (D2O,
96 MHz): dB =19.17 ppm. HRMS (ESI)À: m/z calcd for C27H18BN2O7S:
525.0933 [MÀH]À; found: 525.0946.
45 mL) at 08C, was added, dropwise, a chilled solution of sodium nitrate
(0.2m, 1.2 equiv, 9.4 mmol). Sulphamic acid was added to quench any re-
maining nitrite. After 30 min a solution of 6 (1.35 g, 0.95 equiv, 7.5 mmol)
in methanol and aqueous hydrochloric acid (1m) (2:1, 45 mL) was added
dropwise to the reaction mixture at 08C. Solid sodium acetate was added
to the solution until pH 4 and allowed to stir overnight. The solution was
extracted with dichloromethane (3ꢄ100 mL). The combined organic
layers were dried over sodium sulphate and concentrated in vacuo to
3-(Methyl
(phenyl)amino)propane-1,2-diol (6): (Æ)Glycidol (9.3 mL,
140 mmol, 1.5 equiv) was added to a solution of N-methylaniline (10 g,
93 mmol) in methanol (100 mL). The mixture was heated to reflux for
48 h. On cooling the reaction mixture was concentrated in vacuo to give
the desired product as colourless oil, which was deemed pure by inspec-
tion of the 13C{1H} NMR spectrum and no further purification was re-
quired. 1H NMR (250 MHz, CDCl3): dH =7.19 (2H, t, J=6.8 Hz), 6.79–
6.70 (3H, m), 3.97 (1H, ddd, J=12.0, 4.3, and 4.3 Hz), 3.95–3.25 (4H, m),
2.95 ppm (3H, s). 13C{1H} NMR (63 MHz, CDCl3): dC =150.0, 129.3,
117.5, 113.2, 103.8, 69.7, 64.3, 55.9, 39.4 ppm.
1
give the product as a yellow/orange powder (94% yield, 2.25 g). H NMR
(300 MHz, CDCl3): dH =7.78 (2H, d, J=9.1 Hz), 7.70 (2H, d, J=8.7 Hz),
7.40 (2H, d, J=8.7 Hz), 7.20–7.14 (2H, m), 3.02–3.92 (1H, m), 3.53–3.39
(4H, m), 3.05 ppm (3H, s). 3C{1H} NMR (75 MHz, CDCl3): dC =152.3,
151.9, 150.5, 135.6, 129.8, 125.6, 119.5, 112.2, 70.3, 64.6, 55.4, 40.0 ppm.
(E)-3-(Methyl(4-((4-nitrophenyl)diazenyl)phenyl)amino)propane-1,2-diol
(7): To a stirred solution of 4-nitroaniline (1.00 g, 7.20 mmol) in a mixture
of methanol, water, and aqueous hydrochloric acid (5m) (1:1:1, 30 mL) at
08C, was added, dropwise, a chilled solution of sodium nitrate (0.2m,
1.2 equiv, 8.7 mmol). Sulphamic acid was added to quench any remaining
nitrite. After 30 min, a solution of 6 (1.25 g, 0.95 equiv, 6.9 mmol) in
methanol and aqueous hydrochloric acid (1m) (2:1, 30 mL) was added
dropwise to the reaction mixture at 08C. Solid sodium acetate was added
to the solution until pH 4 and allowed to stir overnight. The solution was
extracted with dichloromethane (3ꢄ100 mL). The combined organic
layers were dried over sodium sulphate and concentrated in vacuo to
give the product as a dark red powder (89% yield, 2.02 g). 1H NMR
(300 MHz, CDCl3): dH =8.22 (2H, d, J=9 Hz), 7.82 (2H, d, J=9 Hz),
7.79 (2H, d, J=9 Hz), 6.73 (2H, d, J=9 Hz), 3.92–3.85 (1H, m), 3.60–
3.37 (4H, m), 3.02 ppm (3H, s). 13C{1H} NMR (75 MHz, CDCl3): dC =
160.1, 157.2, 151.1, 148.0, 130.9, 129.0, 126.9, 115.9, 74.1, 68.3, 59.1,
44.0 ppm. HRMS (ESI)+: m/z calcd for C16H18N4O4: 331.1406 [M+H]+;
found: 331.1399.
Acknowledgements
JSF thanks the Royal Society for a Research Grant (2007/R2), the Lever-
hulme Trust (JSF F/00351/P) and the University of Birmingham for sup-
port. SAE acknowledges Cairo University (NILES-LAMPA). Ryosuke
Matsubara (M.I.T.) is thanked for help with finalizing the manuscript.
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3-((4-((E)-(2,5-Dimethoxy-4-((E)-(4-
nitrophenyl)diazenyl)phenyl)diazenyl)phenyl)ACTHNUGRTNEUNG(methyl)amino)propane-
1,2-diol (8): To a stirred solution of 4-nitroaniline (5.00 g, 36 mmol) in a
mixture of methanol, water, and aqueous hydrochloric acid (5m) (1:1:1,
150 mL) at 08C, was added, dropwise, a chilled solution of sodium nitrate
(0.2m, 1.2 equiv, 43.5 mmol). Sulphamic acid was added to quench any re-
maining nitrite. After 30 min a solution of 2,5-dimethoxyaniline (5.28 g,
0.95 equiv, 34.5 mmol) in methanol and aqueous hydrochloric acid (1m)
(2:1, 150 mL) was added dropwise to the reaction mixture at 08C. Solid
sodium acetate was added to the solution until pH 4 and allowed to stir
overnight. The solution was extracted with dichloromethane (3ꢄ
200 mL). The combined organic layers were dried over sodium sulphate
and concentrated in vacuo to give (E)-2,5-dimethoxy-4-((4-nitrophenyl)-
diazenyl)aniline as a deep red powder (80% yield, 8.3 g). To a stirred so-
lution of (E)-2,5-dimethoxy-4-((4-nitrophenyl)diazenyl)aniline (2.00 g,
6.6 mmol) in a mixture of methanol, water, and aqueous hydrochloric
acid (5m) (1:1:1, 30 mL) at 08C, was added, dropwise, a chilled solution
of sodium nitrate (0.2m, 1.2 equiv, 7.9 mmol). Sulphamic acid was added
to quench any remaining nitrite. After 30 min a solution of 6 (1.14 g,
0.95 equiv, 6.3 mmol) in methanol and aqueous hydrochloric acid (1m)
(2:1, 30 mL) was added dropwise to the reaction mixture at 08C. Solid
sodium acetate was added to the solution until pH 4 and allowed to stir
overnight. The solution was extracted with dichloromethane (3ꢄ
100 mL). The combined organic layers were dried over sodium sulphate
and concentrated in vacuo to give the product as a metallic appearance
black/red powder (70% yield, 2.18 g). 1H NMR (300 MHz, CDCl3): dH =
8.30 (2H, d, J=9.1 Hz) , 7.90 (2H, d, J=9.1 Hz), 7.40 (1H, s), 7.26 (1H,
d, J=7.2 Hz) , 7.23 (1H, d, J=7.2 Hz), 6.82–6.74 (2H, m), 6.35 (1H, s),
4.05–3.99 (1H, m), 3.98 (3H, s), 3.90 (3H, s), 3.78 (1H, dd, J=11.4 and
3.3 Hz), 3.56 (1H, dd, J=11.4 and 5.5 Hz) , 3.43 (1H, dd, J=14.5 and
8 Hz), 3.30 (1H, dd, J=14.5 and 5.5 Hz), 2.96 ppm (3H, s). 3C{1H} NMR
(75 MHz, CDCl3): dC =157.7, 156.7, 150.5, 147.5, 144.9, 142.4, 134.3,
129.7, 125.1, 123.0, 118.1, 113.7, 98.2, 97.8, 70.0, 64.7, 57.2, 56.4, 56.3,
39.8 ppm.
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Chem. Asian J. 2010, 5, 581 – 588