"Click-fluors": Modular fluorescent saccharide sensors based on a 1,2,3-triazole ring

Article abstract of DOI:10.1021/jo702584u

(Chemical Equation Presented) A carbohydrate sensing "click- fluor" is reported which displays a nontypical binding preference with sample saccharides. That a fluorescent sensor is generated as a result of triazole formation (click reaction), rather than

Full text of DOI:10.1021/jo702584u

effectively in water is reflected by the number of published
saccharide sensory systems designed around them. Additionally,
boronic acids are known to bind saccharides via covalent
interactions in aqueous media.4-6
The most common interaction is with cis-1,2- or -1,3-diols
of saccharides to form five- or six-membered rings, respectively.
Formation of this cyclic ester leads to an increase in the Lewis
acidity of a central boron atom and it is this property that has
been exploited to create saccharide sensing molecules. Typically,
boronic acids have either been directly or indirectly (via a linker)
connected to fluorophores and the change in boron Lewis acidity
following subsequent complexation to a saccharide triggers a
change in observed fluorescence.5,6
“Click-fluors”: Modular Fluorescent Saccharide
Sensors Based on a 1,2,3-Triazole Ring
David K. Scrafton,† James E. Taylor,† Mary F. Mahon,‡
John S. Fossey,*,† and Tony D. James*,†
Department of Chemistry, The UniVersity of Bath, ClaVerton
Down, Bath, BA2 7AY, UK, and Bath X-Ray Crystallographic
Suite, Department of Chemistry, The UniVersity of Bath,
Bath BA2 7AY, UK
j.fossey@bath.ac.uk; t.d.james@bath.ac.uk
ReceiVed December 3, 2007
The synthesis of boronic acid containing fluorescent sensors
involves either the direct attachment of a boronic acid to a
fluorophore, or the attachment of a boronic acid to an amine to
create boronic acid derivatives such as ortho, meta, and para
sensors 1a-c (Figure 1).7
A carbohydrate sensing “click-fluor” is reported which
displays a nontypical binding preference with sample sac-
charides. That a fluorescent sensor is generated as a result
of triazole formation (click reaction), rather than simply
bringing the sensor and reporter units together via a triazole
linkage, and the potential applicability of the wide range of
easily accessible acetylene units means a simple strategy for
the development of modular fluorescent sensor arrays for
rapid screening of target saccharides will be available.
FIGURE 1. Previously reported amino-sensors 1a ortho, 1b meta,
and 1c para.
We are particularly interested in developing modular ap-
proaches since they offer the possibility of rapidly constructing
sensor molecule libraries. Surprisingly, the Huisgen 1,3-dipolar
cycloaddition,8 to the best of our knowledge, has never been
employed in the construction of boronic acid sensor molecules
even though its importance in fluorescent sensor development
has been proposed.9 Click chemistry has the potential to rapidly
create fluorescent saccharide sensors from boronic acid azides
and any fluorophore bearing a terminal alkyne.
Saccharides are ubiquitous in nature as building blocks for
processes ranging from the production of metabolic energy
through to tissue recognition.1 Despite quantitative analysis and
detection of saccharide-containing biomolecules being of para-
mount importance, reliable and accurate sensors are not widely
available.
The so-called “click reaction”10 forms an aromatic 1,2,3-
triazole ring following the addition of an azide to a terminal
(3) Davis, A. P.; Wareham, R. S. Angew. Chem., Int. Ed. 1999, 38, 2978-
2996.
Many synthetic receptors developed for neutral guests have
relied on noncovalent interactions, such as hydrogen bonding,
for recognition. It is the case, however, that in aqueous systems
neutral guests may become heavily solvated. While biological
systems have the capacity to expel water from their binding
pockets and sequester analytes wholly while exploiting nonco-
valent interactions, synthetic monomeric receptors have not yet
been designed where hydrogen bonding has been able to
compete with bulk water for low concentrations of saccha-
rides.2,3 The capacity of boronic acid receptors to function
(4) Hall, D. G., Ed. Boronic Acids: Preparation and Applications in
Organic Synthesis and Medicine; 2005.
(5) James, T. D.; Phillips, M. D.; Shinkai, S. Boronic Acids in Saccharide
Recognition; RSC Publishing: Cambridge, UK, 2006.
(6) James, T. D.; Shinkai, S. Top. Curr. Chem. 2002, 218, 159-200.
(7) (a) Arimori, S.; Bosch, L. I.; Ward, C. J.; James, T. D. Tetrahedron
Lett. 2001, 42, 4553-4555. (b) Bosch, L. I.; Mahon, M. F.; James, T. D.
Tetrahedron Lett. 2004, 45, 2859-2862. (c) Bosch, L. I.; James, T. D. In
Topics in Fluorescence Spectroscopy; Geddes, C. D., Lakowicz, J. R., Eds.;
Springer: New York, 2006; Vol. 11, pp 344-350.
(8) Huisgen, R. In 1,3-Dipolar Cycloaddition Chemistry; Padwa, A., Ed.;
Wiley: New York, 1984.
(9) Zhu, L.; Anslyn, E. V. Angew. Chem., Int. Ed. 2006, 45, 1190-
1196.
† Department of Chemistry.
(10) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed.
2001, 40, 2004-2021.
‡ Bath X-Ray Crystallographic Suite.
(1) Garrett, R. H.; Grisham, C. M. Biochemistry, 2nd ed.; Saunders
College Publishing; Wiley-VCH: London, 1999.
(2) Davis, A. P.; James, T. D. In Functional Synthetic Receptors;
Schrader, T., Hamilton, A. D., Eds.; Wiley-VCH: Weinheim, 2005; pp
45-110.
(11) We have used the phrase “click-fluor” to describe the generation of
a fluorophore from non-fluorescent constituent parts via a so called “click
reaction”. In this case, the triazole ring forms an integral part of the
fluorophore; i.e., a new property is imparted on a molecule conceived by
a “click reaction”.
10.1021/jo702584u CCC: $40.75 © 2008 American Chemical Society
Published on Web 03/13/2008
J. Org. Chem. 2008, 73, 2871-2874
2871

SCHEME 2. Binding of Different Saccharides with Sensor
4 in Aqueous Solution
FIGURE 2. Chemical and X-ray structure of intermediate 5.
SCHEME 1. Five Step Synthesis of “click-fluor” 4
alkyne has the potential to create a fluorescent sensor from
nonfluorescent constituent parts.11
Fluorescent sensor 4 was prepared as outlined in Scheme 1
in 40% yield over five steps. Commercial boronic acid 2 was
pinacol protected and brominated to give boronate ester 3.
Subsequently, a copper(I)-catalyzed azide-alkyne [3 + 2]
cycloaddition developed by Ham was employed,12 enabling the
synthesis of 1,2,3-triazole ring as predominantly the 1,4-
regioisomer. At this point during the synthesis, X-ray quality
crystals of intermediate 5 were obtained (Figure 2).13 The target
sensor 4 was then obtained via a two-step deprotection of the
pinacol ester.14
The fluorescence titrations of 4 (1.67 × 10-6 mol dm-3) with
different saccharides were carried out in a pH 8.21 buffer (52.1
wt % methanol in water with KCl, 0.01000 mol dm-3; KH2-
PO4, 0.002752 mol dm-3; Na2HPO4, 0.002757 mol dm-3).15
The fluorescence spectra of 4 in the presence of D-fructose (0-
2.2 mol dm-3) are shown in Scheme 2 and Figure 3 (upper);
the analogous spectra for the addition of D-glucose, D-galactose,
and D-mannose are available as Supporting Information.
The stability constants (K) of fluorescence sensor 4 with
D-fructose, D-glucose, D-galactose, and D-mannose were calcu-
lated by fitting the emission intensity at 430 nm (λex 276 nm)
vs concentration of saccharides (Figure 3, lower). The stability
constants calculated from these titrations are given in Table 1.16,17
FIGURE 3. Fluorescence spectra of 4 in the presence of increasing
concentrations of D-fructose, with the peak at 300 nm decreasing in
intensity and the peak at 430 nm increasing in intensity, resulting in
an isoemissive point at 360 nm (upper) and binding curves for 9
D-fructose; [ D-glucose; B D-galactose, 2 D-mannose (lower).
(12) Molander, G. A.; Ham, J. Org. Lett. 2006, 8, 2767-2770.
(13) Crystal Data for 5: C21H24BN3O, M ) 361.24, monoclinic, P21/n,
a ) 8.7850(1) Å, b ) 11.7050(2) Å, c ) 18.7420(3) Å, b ) 90.368(1)o, U
) 1927.17(5) Å3, Z ) 4, Dc ) 1.245 g cm-3, m ) 0.08 mm-1, F(000) )
768. Crystal size 0.35 × 0.2 × 0.2 mm, unique reflections 32826 [R(int) )
0.0474], observed [I > 2s(I)] 3497. R1, wR2 (obsd data) ) 0.0565, 0.1013.
R1, wR2 (all data) ) 0.0403, 0.0924. Max peak/hole ) 0.260, -0.205 e
Å-3. Bond lengths (Å), angles (deg) and torsions (deg): N(1)-N(2) 1.3467-
(13), N(2)-N(3) 1.3165(14), B(1)-O(1) 1.3664(16), N(1)-C(7) 1.4681-
(15), B(1)-C(1) 1.5653(17); N(1)-N(2)-N(3) 107.12(9), O(1)-B(1)-C(1)
126.09(11); C(1)-C(6)-C(7)-N(1) 98.90, O(1)-B(1)-C(1)-C(6) 6.83.
(14) Yuen, A. K. L.; Hutton, C. A. Tetrahedron Lett. 2005, 46, 7899-
7903.
The fluorescence enhancements (I/I0) obtained for 4 on the
addition of D-fructose, D-galactose, and D-mannose are 27-, 20-,
and 16-fold, respectively (Figure 3, lower, see the Supporting
Information). We believe that these large fluorescence enhance-
ments can be attributed to fluorescence recovery of the 1,2,3-
triazole fluorophore. Similar fluorescence responses were
(16) The K were analysed in Origin 7 using nonlinear (Levenberg-
Marquardt algorithm) curve fitting. The errors reported are the standard
errors obtained from the best fit.
(17) Cooper, C. R.; James, T. D. J. Chem. Soc., Perkin Trans. 1 2000,
963-969.
(15) Perrin, D. D.; Dempsey, B. Buffers for pH and Metal Ion Control;
Chapman and Hall: London 1974.
2872 J. Org. Chem., Vol. 73, No. 7, 2008

TABLE 1. Stability Constant K (coefficient of Determination; r2) for the Binding of Sensors 1a-c and 4 with Monosaccharides (52.1 wt %
Methanol in Water, pH 8.21, 22 °C)
saccharide
K (mol-1 dm3) (r2) 1a
K (mol-1 dm3) (r2) 1b
K (mol-1 dm3) (r2) 1c
K (mol-1 dm3) (r2) 4
D-fructose
D-glucose
D-galactose
D-mannose
79.2 ( 1.7 (0.99)
6.4 ( 0.4 (0.99)
14.2 ( 0.6 (0.99)
7.9 ( 0.3 (0.99)
212.1 ( 6.9 (0.99)
8.7 ( 1 (0.99)
26.6 ( 1.3 (0.99)
13.9 ( 1.4 (0.99))
128.6 ( 2.6 (0.99)
6.7 ( 0.5 (0.99)
17.7 ( 0.3 (0.99)
16.2 ( 0.8 (0.99)
1.9 ( 0.1 (0.99)
-
1.8 ( 0.3 (0.99)
4.1 ( 0.4 (0.99)
observed for sensors 1a-c.7 In the absence of saccharides the
normal fluorescence of the LE (locally excited) state of the 1,2,3-
triazole donor of sensor 4 is quenched by energy transfer to the
neutral phenylboronic acid acceptor, weakly Lewis acidic boron
center. When saccharides are added, a negatively charged
boronate anion is formed due to the enhancement of the Lewis
acidity of the boron center on saccharide binding. Under these
conditions, energy transfer from the 1,2,3-triazole donor be-
comes unfavorable and fluorescence is recovered. Interestingly
the stability order for sensors 1a-c with fructose > galactose
> mannose > glucose was the “inherent stability order”
observed for all simple boronic acids.4-6,18 However, the
unprecedented stability order with sensor 4 is mannose >
fructose > galactose > glucose. We attribute the deviation in
selectivity to the available hydrogen bond acceptor sites on the
1,2,3-triazole ring of 4 influencing the selectivity among the
saccharides (such additional selectivity is not observed with
sensors 1a-c).
We have reported a simple “click-fluor” we believe that this
strategy will make it possible to develop fluorescent modular
sensor arrays for rapid screening of target saccharides.19 The
two most attractive aspects of “click-fluor” are that a fluorophore
is generated upon triazole formation and the wide availability
of acetylene units. Exploitation of this approach to prepare
multiple receptor sensors is currently underway in our labora-
tories.
and then allowed to cool to room temperature. The mixture was
condensed under reduced pressure until a solid began to form. The
solid was filtered, and the filtrate was condensed further. The crude
product was then purified using flash chromatography on silica gel
(with petroleum ether as eluent) to yield compound 3 as a white
solid (3.74 g, 86%): Rf (6:1, petrol/EtOAc) 0.86; mp 78-80 °C; ν
(CDCl3)/cm-1 2976, 1599, 1444, 1386, 1350, 1324, 1269, 1144,
1102; δH (300 MHz, CDCl3) 1.36 (12H, s), 4.90 (2H, s), 7.22-
7.43 (3H, m), 7.77-7.83 (1H, m); δB (96 MHz, CDCl3) 31.32; δC
PENDENT (75 MHz, CDCl3) 24.9 (+), 33.96 (-), 83.9 (-), 127.6
(+), 130.1 (+), 131.3 (+), 136.4 (+), 144.3 (-); m/z (ES+)
319.0475 ([M + Na]+, C13H18BBrO2Na requires 319.0481).
2-((4-Phenyl-1H-1,2,3-triazol-1-yl)methyl)phenylboronic Acid
4. Part i: Compound 3 (1.04 g, 3.53mmol) and sodium azide (0.27
g, 4.12mmol) were dissolved in dimethyl sulfoxide (10 mL) and
were stirred at room temperature for 10 min. Ethynylbenzene (0.30
g, 2.94 mmol) and copper(I) iodide (0.17 g, 0.88 mmol) were added,
and the reaction mixture was heated at 80 °C for 4 h. The system
was then heated at 60-70 °C, and the solvents were removed under
high vacuum. Dry dichloromethane (200 mL) was added, and the
resulting suspension was filtered through Celite in order to remove
the inorganic salts. The filtrate was concentrated under reduced
pressure, and the product was obtained by purification using flash
chromatography on silica gel (petroleum ether/ethyl acetate eluent,
6:1). The appropriate fractions were combined, and the solvents
were removed under vacuum to yield 4-phenyl-1-(2-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)-1H-1,2,3-triazole (5) as
a cream solid (0.86 g, 81%): Rf (4:1, petrol/EtOAc) 0.35; mp 99-
101 °C; ν (CDCl3)/cm-1 2978, 1601, 1572, 1484, 1466, 1443, 1382,
1372, 1349, 1322, 1271, 1224, 1144; δH (300 MHz, CDCl3) 1.37
(12H, s), 5.91 (2H, s), 7.24-7.48 (6H, m), 7.77 (1H, s), 7.77-
7.82 (2H, m), 7.92-7.97 (1H, m); δB (96 MHz, CDCl3) 31.79; δC
PENDENT (75 MHz, CDCl3) 24.9 (+), 53.4 (-), 84.2 (-), 119.9
(+), 125.6 (+), 125.7 (+), 127.9 (+), 128.0 (+), 128.8 (+), 129.1
(+), 129.2 (+), 130.8 (-), 131.9 (+), 134.8 (-), 136.7 (+), 140.9
(-), 147.6 (-); m/z (ES+) 384.1845 ([M + Na]+, C21H24BN3O2-
Na requires 384.1859). Part ii: 4-Phenyl-1-(2-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)benzyl)-1H-1,2,3-triazole (0.36 g, 1.0 mmol)
in methanol (7 mL) was added to aqueous potassium hydrogen
difluoride (3 mL, 4.5 M, 13.5mmol) within a plastic beaker. The
resulting white slurry was stirred at room temperature for 15 min,
concentrated in vacuo, and then dissolved in hot acetone. The
mixture was filtered, the filtrate concentrated in vacuo and the
residue recrystallized from a minimal amount of hot acetone and
diethyl ether in order to afford the corresponding potassium
trifluoroborate salt, potassium trifluoro(2-(4-phenyl-1H-1,2,3-triazol-
1-yl)phenyl)borate, as a yellow solid (0.27 g, 78%): mp 242-245
°C; ν (KBr)/cm-1 3435, 3134, 2362, 1610, 1466, 1444, 1431, 1364,
1273, 1234, 1207; δH (300 MHz, CD3OD) 5.79 (2H, s), 7.02-
7.46 (6H, m), 7.61-7.80 (3H, m), 8.15 (1H, s); δB (96 MHz, CD3-
OD) 4.84; δC PENDENT (75 MHz, CD3OD) 55.2 (+), 122.5 (-),
125.7 (+), 126.8 (-), 128.0 (-), 128.1 (-), 129.1 (-), 129.3 (-),
129.5 (-), 130.0 (-), 132.0 (+), 133.89 (-), 133.93 (-), 139.1
(+), 148.8 (+); m/z (ES-) 302.1075 ([M - K], C15H12N3BF3
requires 302.1082). Part iii: Potassium trifluoro(2-(4-phenyl-1H-
1,2,3-triazol-1-yl)phenyl)borate (100 mg, 0.29 mmol) and lithium
hydroxide (24 mg, 1.00 mmol) were added to a solution of water
(5 mL) and acetonitrile (10 mL) and the solution was then stirred
Experimental Section
2-(2-(Bromomethyl)phenyl)-4,4,5,5-tetramethyl-1,3,2-diox-
aborolane 3. Part i: Toluene-2-boronic acid 1 (2.11 g, 15.5mmol)
and pinacol (2.20 g, 18.6mmol) were mixed in toluene (200 mL).
A Dean-Stark head was fitted, and the reaction mixture was heated
under reflux for 2 h. The mixture was allowed to cool to room
temperature and then was washed with water (3 × 100 mL),
condensed under reduced pressure, and dichloromethane (100 mL)
was added. This was washed with water (2 × 100 mL), dried over
sodium sulfate, and filtered, and the solvents removed under reduced
pressure to yield 4,4,5,5-tetramethyl-2-o-tolyl-1,3,2-dioxaborolane
as a colorless oil (3.23 g, 96%); ν (film)/cm-1 2979, 1602, 1490,
1438, 1381, 1347, 1312, 1273, 1214, 1146; δH (300 MHz, CDCl3)
1.21 (12H, s), 2.42 (3H, s), 6.99-7.22 (3H, m), 7.14-7.22 (1H,
m); δB (96 MHz, CDCl3) 32.57; δC PENDENT20 (75 MHz, CDCl3)
22.7 (+), 25.4 (+), 83.8 (-), 125.2 (+), 130.2 (+), 131.3 (+),
136.4 (+), 145.3 (-); m/z (ES+) 241.1370 ([M+Na]+, C13H19-
BO2Na requires 241.1376). Part ii: 4,4,5,5-Tetramethyl-2-o-tolyl-
1,3,2-dioxaborolane (3.20 g, 14.7mmol), N-bromosuccinimide (3.92
g, 22.0mmol), azobisisobutylonitrile (0.03 g, 1 mol %), and
acetonitrile (100 mL) were refluxed at 90 °C for 2 h under nitrogen
(18) Lorand, J. P.; Edwards, J. O. J. Org. Chem. 1959, 24, 769-774.
(19) Edwards, N. Y.; Sager, T. W.; McDevitt, J. T.; Anslyn, E. V. J.
Am. Chem. Soc. 2007, 129, 13575-13583.
(20) The PENDANT technique results in primary and tertiary carbon
atoms havid a different phase to secondary and quaternary carbon atoms.
The phase is represented in the following manner: (+) positive phase; (-)
negative phase. Homer, J.; Perry, M. C. J. Chem. Soc., Chem. Commun.
1994, 373-374.
J. Org. Chem, Vol. 73, No. 7, 2008 2873

at room temperature for 24 h. The solution was then acidified with
saturated ammonium chloride (8 mL) and hydrochloric acid solution
(2 mL, 1 M) and then extracted with ethyl acetate (3 × 20 mL).
The combined organic extracts were washed with hydrochloric acid
solution (2 × 20 mL, 0.5 M), dried over sodium sulfate, and filtered,
and the solvents were removed under reduced pressure to afford
the title compound 3 as a white solid (0.062 g, 77%): ν (CDCl3)/
cm-1 3580, 3141, 1600, 1448, 1397, 1280, 1253; δH (300 MHz,
CDCl3) 5.88 (2H, s), 7.00-7.85 (9H, m), 7.92-8.20 (1H, m); δB
(96 MHz, CD3OD) 31.00; δC PENDENT (75 MHz, CD3OD) 55.8
(-), 122.7 (+), 127.1 (+), 129.3 (+), 129.8 (+), 130.4 (+), 130.6
(+), 131.0 (+), 131.6 (+), 132.0 (-), 134.0 (+), 136.1 (+), 140.0
(-), 149.5 (-); m/z (ES-) 302.1069 ([M - H + Na], C15H14BN3O2-
Na requires 302.1077).
Acknowledgment. We are grateful to the University of Bath,
the EPSRC (D.K.S. studentship), and The Leverhulme Trust
(J.S.F. F00351P Project Grant) for support. Dr. Stephen Flower
and Dr. Franc¸ois D’Hooge are thanked for helpful discussions.
Supporting Information Available: General procedures, fluo-
rescence data, and tables of XRD data and X-ray data (CIF). This
materialisavailablefreeofchargeviatheInternetathttp://pubs.acs.org.
JO702584U
2874 J. Org. Chem., Vol. 73, No. 7, 2008