Water soluble and fluorescent copolymers as highly sensitive and selective fluorescent chemosensors for the detection of cyanide anions in biological media

Article abstract of DOI:10.1039/c3ra43497d

We report a simple colorimetric method based on a water soluble polymer (WSP) for the sensitive and selective detection of cyanide in a biological solution. The WSPs were prepared from a radical polymerization of a methacrylate with a cyanide chemosensor moiety based on a coumarin-dicyano-vinyl derivative motif, and the polymer was then rendered water soluble by copolymerizing this hydrophobic monomer with other glyco-conjugated methacrylate derivatives. The lowest concentration for the quantification of cyanide ions was 0.05 μM, and the polymer exhibited nearly no calorimetric response to the presence of other common anions. Therefore, the chemical incorporation of some carbohydrates, as lactose or its usual monosaccharide moieties (glucose, lactose), represents a new method for the implementation of polymers for the detection of cyanide anions in water, exhibiting both good selectivity and efficiency. The Royal Society of Chemistry 2013.

Full text of DOI:10.1039/c3ra43497d

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Water soluble and fluorescent copolymer for highly sensitive and
selective fluorescent chemosensor for cyanide anion detection in
biological medium
Jalal Isaad,*1,2 Fouad Malek,3 and Ahmida El Achari1,2
5
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
We report a simple colorimetric method based on water soluble polymer (WSP) for detection of cyanide
sensitively and selectively in biological solution. The water soluble polymers WSP were prepared from a
radical polymerization of a methacrylate with cyanide chemosensor moiety based on a coumarin ꢀ
10 dicyanoꢀvinyl derivative motif, and the polymer water solubility was achieved by copolymerizing this
hydrophobic monomer with others glycoꢀconjugated methacrylate derivatives. The lowest concentration
for quantification of cyanide ions was 0.05 ꢁM, and other common anions nearly have no colorimetric
response. Therefore, the chemical incorporation of some carbohydrates as lactose or its usual
monosaccharide’s moieties (glucose, lactose) represents a new way for the implementation of the
15 polymers for the cyanide anions detection in water selectively and efficiently.
organic solvents; (ii) show poor selectivity to CN− and, (iii) show
high detection limit. In the recent years, cyanide optical
chemosensors that operate in aqueous solutions are developed, 41ꢀ
Introduction
Considerable efforts have been put into the design and synthesis
of functional molecules that serve as probes or sensors for the
detection of chemically and biologically important ionic
20 species.1,2 Recent studies showed that some toxic anions also act
as severe environmental pollutants and have adverse health
effects. 3ꢀ6 One of the most rapidly acting and powerful poisons is
cyanide CN−. It strongly binds the active site of cytochromeꢀc
and inhibits the mitochondrial electronꢀtransport chain, leading to
25 decreased oxidative metabolism and oxygen utilization.7,8 The
maximum permissive level of cyanide in drinking water is
45
and in our laboratory we developed a novel water soluble
46ꢀ52
50 chemosensors
behaves as a colorimetric cyanide receptor in
water at room temperature. The chemical structure of these water
soluble chemosensors is taken from the structure of our new
glycoconjugated dyeing agents developed recently.53ꢀ59 The water
solubility of these new chemosensor generation was given by the
46ꢀ48
55 incorporation of the saccharidic,
glycerol49, moiety on the
starting organic chemosensor or by the incorporation of the
starting chemodosimeters on an adequate water soluble polymer
as polyvinyl alcohol,50 on a natural cellulose materials,51 through
therefore set at 1.9 ꢂM by the World Health Organization
a
chemical grafting or also by the organic chemosensor
9,10
(WHO).
Given its acute toxicity, wide availability in massive
60 incorporation into polymeric plastic film based on starch as a
biosourced biopolymer.52 One the other hand, it should be noted
that the area of responsive water soluble polymers (WSP) has
nowadays evolved well beyond the demonstration of novel and
interesting properties. Currently, the exploitation of useful and
65 advanced functions such as drug or gene carriers with triggered
release properties, catalysis, detection and imaging,
environmentally adaptive coatings, and selfꢀhealing materials has
emerged to be a more relevant subject. The capability of facile
manipulation of the solubility, hydrodynamic volume, and chain
70 configuration and conformation of responsive polymers by
external stimuli has indeed enabled the development of
responsive polymeric systems with novel functions. Intuitively,
responsive polymers should play an important role in detection
and sensing applications. However, stimuliꢀresponsive polymerꢀ
75 based detection systems are still in its infancy stage as compared
to the relatively mature field of small molecule probes.17ꢀ40 In this
paper, we designed and synthesized a novel glycoconjugated poly
amounts, and in light of increasing terrorist activity, there is a
30 pressing need for fast, accurate detection of cyanide at the
regulated concentrations in aqueous mediums. Various methods
used previously to analyze cyanide employ titrimetric,11
voltammetric,12 potentiometric13 and electrochemical methods14
as well as ion chromatography.15 However, these methods often
35 require extensive, time consuming procedures that involve the
use of sophisticated instrumentation with high detection limits.
Optical sensors for cyanide, in which a change in color and/or
fluorescence intensity (or emission wavelength) is monitored,
have been studied actively over the past ten years due to their
40 simple, inexpensive, and rapid implementation. Generally, three
approaches for chemosensors 16: (a) the binding siteꢀsignaling
subunit,17ꢀ19 (b) the chemodosimeters,20ꢀ33 or (c) the
displacements34–40 has been extensively reported. However, these
chemosensors suffer from several problems: they (i) act only in
45 pure organic solvents or solutions containing a large amount of
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coumarinꢀcyanocarbon (P2) containing coumarinꢀdicyanoꢀvinyl
for the nucleophilic addition reaction. Meanwhile, because of the
water solubility property of the WSP, the solvated cyanide ions to
reach the guest or the transducer cores, giving rise to the sensing
phenomenon, resulting in higher sensitivity toward cyanide
unit in its main chain as a highly selective and sensitive cyanide
sensor and a carbohydrate, such as galactose as a water soluble
moiety (figure 1). Its design is based on the consideration that the
5
15
20
25
30
dicyanoꢀvinyl group can act as a selective cyanideꢀreactive unit 10 detection
Figure 1: Chemical structure of the monomers and the copolymer P2
2 Results and discussion
cyanide ions to reach the sensory receptor, giving rise to the
sensing phenomenon, resulting in cyanide detection in water. For
this purpose, methacrylic monomer containing a coumarinꢀ
2.1 Monomer synthesis and characterization
As mentioned in the introduction, we hypothesized that the
chemical anchoring of a sensing motif to a hydrophilic polymer 40 dicyanoꢀvinyl derivative (7) as
a
colorimetric cyanide
35 chain could provide a waterꢀrich environment to the sensing
moiety upon polymer swelling, thus permitting the solvated
chemosensor and glycoꢀconjugated methacrylic monomers (S1b)
were prepared as reported in scheme 1.
45
50
55
60
Scheme 1: Synthesis of the monomers. Reagents and conditions: (a) Na2CaP2O7, EtOH/H2O, RT, 1h (b) POCl3, DMF, 50°C, 30 min (c)
Na2CaP2O7, EtOH/H2O, RT, 1h (d) SnCl2, MeOH, reflux, 4h (e,g) THF, methacrylic chloride, RT, 1h (f) 1. DMP, TsOH, 80°C, 8h, 2.
65 NEt3, 15 min, 3. H2O/MeOH, 80°C, 1h.
Compounds
3 and 5 were prepared by Knoevenagel 70 chromeneꢀ3ꢀcarbaldehyde 4 with malononitrile in the case of the
condensation of 2ꢀhydroxyꢀ4ꢀnitrobenzaldehyde1 with diethyl
malonate2 in the case of the compound 3 and 7ꢀnitroꢀ2ꢀoxoꢀ2Hꢀ
compound 5, in the presence of Na2CaP2O7 as a basic catalyst.
The use of Na2CaP2O7 as heterogeneous catalyst in the
2
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Knoevenagel condensation has allowed the isolation of the 10 DMF to give the compound 4. The compound 6 was obtained by
compounds 3 and 5 rapidly (20 min for 3 and 15 for 5) and with
good yield (about 94%). The addition of water (5% in EtOH)
remarkably decreases the reaction time of the Na2CaP2O7 in the
Knoevenagel synthesis of the compounds 3 and 5. In fact, the
the reduction of the nitro group in the presence of SnCl2 inMeOH
and under reflux. In the final step, and in order to prepare the
corresponding monomors 7 and S1b, the compounds 6 and S1a
were reacted with methacryloyl chloride in THF solution and at
5
time reaction goes from 1h to 20 min and from 40 to 15 min in 15 room temperature to afford the corresponding methacrylate
EtOH for 3 and 5 respectively. The Na2CaP2O7 was regenerated,
by calcinations at 500°C during 15 min, and after seven
successive recuperations. Then 3was treated by POCl3 chloride in
monomers. All intermediates and monomers were characterised
1
by 1H and 13C NMR spectroscopy. Figure 2 reports the H NMR
spectra of the monomer 7.
20
25
30
35 Figure 2: 1H NMR spectra of the monomer 7.
2.2 Copolymerization of 7 with S1b
The copolymers P1 were obtained with good yields (60– 95%),
and then, they were deprotected by using TFA solution to afford
the deprotected polymers P2.
The free radical copolymerization of 7 with S1b was performed
in chloroform for 8 h at 65 °C using AIBN as an initiator as
40 reported in scheme 2. Different monomer ratios were considered.
45
50
55
60
65
Scheme 2: Synthesis of the copolymers P1 and P2. Reagents and conditions (a) AIBN, CHCl3, 65°C, 8h (b) TFA, RT, 2h.
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The Table 1 reports the values of mass average molecular
5.28ꢀ3.30 characteristic of the galactoyl moiety. The peaks at δ =
weight (Mw) and number average molecular weight (Mn) and 45 1.78 and 1.67 ppm correspond to the CH2 and CH3 protons of
polydispersity index (I) of copolymers P2aꢀe which are
determined by size exclusion chromatography. The data indicate
that Mw and Mn were found to range from 11600 to 19600 g/mol
and 6400 to 8700 g/mol, respectively. With all these
glycoconjugated polymers with different sugar ratio, we can
compare their solubility in water and their glycidic ratio (Table
1). The water solubility is immediate with the all polymers except
10 the polymers with glycidic ratio under 50% which are insoluble
and poorly soluble, respectively, in water. Therefore we can
conclude that for these polymers P2aꢀe to be soluble, a minimum
glycidic ratio of 50% is required.
ethyl and methyl groups respectively of the coꢀpolymers.
2.3 Fluorescence spectra titration of P2c with various halide
ions
5
The water polymer P2c shows a fluorescence emission band
50 with a maximum at 580 nm with a high fluorescence quantum
yield (0.51) in in buffered aqueous solution (50 mM 50 HEPES,
pH=7.4). Figure 4 reports the fluorescence emission spectra of
the water soluble copolymer P2c in the presence and the absence
−
−
−
of a different anions (CH3COO−, HSO4 , ClO3 , ClO4 , Cl−, Br−,
55 H2PO4 , S2O32−, F−, NO3 , I−, NO2 , SO32−, S2−, C2O42−, SO42ꢀ, N3
−
−
−
ꢀ
, SCNꢀ, CO32ꢀ, HCO3 and CNꢀ). As shown in Figure 3, upon
addition 50 equivalents of CNꢀ, the fluorescence emission band
with a maximum at 581 nm is blue shifted to 500 nm with a slight
decrease of the fluorescence quantum yield (0.35) in buffered
60 aqueous solution. However, upon addition the other metal ions,
no significant changes in the fluorescence emission spectra were
observed both in buffered aqueous solution. This interesting
feature revealed that the copolymer P2c can be used as selective
fluorescent chemosensor for the cyanide anions in water for
65 possible applications of the present system in the anion analysis
in the biological mediums.
ꢀ
Table 1: Molecular weight data and water solubility for the
15 copolymerization of 7 with S1b
20
The chemical structures of coꢀpolymers P2c were confirmed
by spectroscopic methods (IR and 1H NMR).
70
25
75
30
Figure 3: 1H NMR spectrum of the coꢀpolymers P2c
The IR spectra of the copolymers showed strong absorption
bands characteristic of the carbonyls of methacrylate and
35 cinnamate functions at about 1755 cmꢀ1 and 1720 cmꢀ1,
A
weak absorption band at 1613 cmꢀ1 was
80 Figure 4: Fluorescence spectra (λex= 460 nm) of P2c (50 ꢂM)
respectively.
−
−
−
upon the addition of various (CH3COO−, HSO4 , ClO3 , ClO4 ,
assignable to the CH=C unsaturation of cinnamate groups. A
strong absorption bands characteristic of the glycidic hydroxyl
groups appear at 3140 cmꢀ1. The 1HNMR (figure 3) of the
40 copolymer P2c shows a signals at δ= 7.94 ꢀ 6.83 ppm due to the
aromatic protons. The proton of the CH=C unsaturation is
observed at δ= 8.55 ppm. The presence of the glycidic part on the
copolymers P2c is confirmed by the presence of the signals at
−
−
−
2−
Cl−, Br−, H2PO4 , S2O32−, F−, NO3 , I−, NO2 , SO32−, S2−, C2O4
,
SO42ꢀ, N3 , SCNꢀ, CO32ꢀ, HCO3 and CN) in buffered aqueous
ꢀ
2ꢀ
solution (50 mM HEPES, pH=7.4).
85
In addition to the changed fluorescent behavior caused by the
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tuned DꢀπꢀA structure of fluorophore 7 in the copolymer P2c in
the presence of the cyanide anions, the color change was another
apparent phenomenon. The copolymer P2c contains a coumarin
derivative as the fluorophore and a dicyanoꢀvinyl group as a
with a red color, upon addition of the cyanide anion to the
buffered solution of P2c, The cyanide anions attack the αꢀposition
of the dicyanoꢀvinyl group in the fluorophore 7 of the copolymer
P2c, to generate the stabilized anionic specie of P2c ꢀCN, which
5
putative cyanideꢀdependent reactive subunit. First, the P2c was 10 presented a yellow color (figure 5).
15
−
20 Figure 5: The colour and sensing mechanism of the copolymer P2c in the presence of the cyanide and other anions (CH3COO−, HSO4 ,
−
−
−
−
−
ꢀ
ClO3 , ClO4 , Cl−, Br−, H2PO4 , S2O32−, F−, NO3 , I−, NO2 , SO32−, S2−, C2O42−, SO42ꢀ, N3 , SCNꢀ, CO32ꢀ, HCO32ꢀ).
ꢀ
The fluorescence intensity of the copolymer P2c upon treatment
, SCNꢀ, CO32ꢀ, HCO3 and CNꢀ). The presence of background
with CNꢀ (50 mM) was measured in the presence of 100 mM of
anions did not cause any significant quenching ratio (F/F0)
change of P2 as reported in figure 6.
−
−
−
25 the different anions (CH3COO−, HSO4 , ClO3 , ClO4 , Cl−, Br−,
−
−
−
ꢀ
H2PO4 , S2O32−, F−, NO3 , I−, NO2 , SO32−, S2−, C2O42−, SO42ꢀ, N3
30
35
40
45
50
Figure 6: Quench ratio (F/F0) of the fluorescence intensity of P2c (50 mM) in buffered aqueous solution (50 mM HEPES, pH=7.4) upon
−
−
−
−
the addition of 50 mM of CNꢀ in the presence of 100 mM of background anions (CH3COO−, HSO4 , ClO3 , ClO4 , Cl−, Br−, H2PO4 ,
−
−
ꢀ
55 S2O32−, F−, NO3 , I−, NO2 , SO32−, S2−, C2O42−, SO42ꢀ, N3 , SCN, CO32ꢀ, HCO32ꢀ).
The results reported in the figure 6 showed that the P2c could
The detection limit60 of chemosensor P2c as a cyanide
fluorescent probe was evaluated from the plot of fluorescence
selectively detect CNꢀ in the presence of other anions such as
−
−
−
−
(CH3COO−, HSO4 , ClO3 , ClO4 , Cl−, Br−, H2PO4 , S2O32−, F−, 65 intensity as a function of the CNꢀ concentration as reported in
60 NO3 , I−, NO2 , SO32−, S2−, C2O42−, SO42ꢀ, N3 , SCN, CO3
,
Figure 6. From a plot for the linear region, the detection limit60 of
cyanide with P2c was found to be 1.17 ꢁmol L−1 (Figure 7)
which is comparable with detection limits of the chemosensors 46ꢀ
−
−
ꢀ
2ꢀ
HCO32ꢀ in water.
2.4
Detection limit calculation
53
developed in our laboratory or in the literature.20ꢀ33 According
70 to the World Health Organization (WHO), cyanide concentrations
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lower than 1.9 ꢁmol L−1 are acceptable in drinking water,10 which
meant that the water soluble polymer P2c based fluorescent 55 Resolution Mass Spectra (HRMS) were recorded on
electrospray ionisation (ESI, +c or −c technique). High
method is sensitive enough to monitor cyanide concentration in
drinking water.
aLTPOrbitrap mass spectrometer from Thermo Electron
Corporation under ESI (+c) technique. Mass spectrometric
analysis is quoted in the m/z form. Elemental analyses were
recorded on a Perkin Elmer 240 C Elemental Analyzer. The
60 derivatives S1a was prepared following our previous procedure
[49].
5
4.2 The chemosensor 7 concentration determination
The initial concentration of the fluorophore 7 C0 and its
concentration C1 after the copolymerisation (after separation
65 from the copolymerisation solution) were determined by using
HPLC (C18 column, reversedꢀphase). The quantitative
determination of the fluorophore was carried out by UV
detector settled at 494 nm. The concentration of the
fluorophore 7 in the obtained copolymers is C0 ꢀ C1.
10
Figure 7: Calibration curve of P2c –CNꢀ in HEPES (pH=7.4).
The excitation wavelength was 460 nm. The concentration of
70 4.3 Intermediates and monomers
15 the chemosensor P2 was 50 ꢀM
4.3.1 Synthesis of 7ꢀnitroꢀ2Hꢀchromenꢀ2ꢀone (3)
3. Conclusions
2ꢀhydroxyꢀ4ꢀnitrobenzaldehyde
1 (1.00 g, 5.95 mmol),
diethylmalonate (2) (0.96 g, 5.95 mmol) and Na2CaP2O7
catalyst (0.2 g) were combined in 10 mL of ecoꢀcompatible
75 EtOH/water: 95/5 and stirred at room temperature for 20 min.
Then, the catalyst was removed by filtration and washed with
ethyl acetate. After concentration of the filtrate, the residue
was purified by reꢀcrystallization using EtOH leading to 7ꢀ
A novel water soluble polymer P2c for the cyanide anions
chemosensing was prepared and characterised. The
chemosensor P2c shows a remarkably high ability to detect
20 selectively the cyanide anions with a visible colorimetric and
fluorometric changes in biological solution. The detection
limit for the CNꢀ was calculated to be 1.17 ꢁmol L−1 which
meant that the water soluble polymer P2c based fluorescent
method is sensitive enough to monitor cyanide concentration
25 in drinking water.
1
nitroꢀ2Hꢀchromenꢀ2ꢀone 3 (1.07 g, 94%) as yield. H NMR
80 (200 MHz, CDCl3) δ= 8.16ꢀ8.10 (m, 3H), 7.93 (d, 1H), 6.29
(d, 1H) ppm. 13C NMR (50 MHz, CDCl3) δ= 161.3, 154.7,
147.4, 143.5, 129.4, 126.3, 120.1, 113.0, 112.3 ppm. MS
(ESI): m/z = 192.21 [M + 1] +. C9H5NO4 (191.02): C, 56.55;
H, 2.64; N, 7.33 found, C, 56.63; H, 2.77; N, 7.42.
4.
Experimental
4.1 Materials
85 4.3.2
carbaldehyde (4)
Synthesis
of
7ꢀnitroꢀ2ꢀoxoꢀ2Hꢀchromeneꢀ3ꢀ
All chemicals were reagent grade (Aldrich Chemical Co.) and
were used as purchased without further purification. Thin
30 layer chromatography (TLC) analysis was performed using
Fluka aluminium foils coated with 25 mm particle size silica
gel matrix F254. TLC development involved either UV (254
and 366 nm) or visible light inspection, followed by either
treatment with an acid solution of pꢀanisaldehyde or a basic
35 solution of KMnO4 and heating. Flash column
chromatography was performed on Merck silica gel 60
(particle size 0.040 ꢀ 0.063 nm, 230 – 400 mesh ASTM)
according to the procedure of Still.63 Uvꢀvisible spectra were
recorded on a Caryꢀ4000 Varian spectrophotometer, using
40 either 0.1 or 1 cm quartz cuvettes. Infraꢀred spectra were
recorded in a KBr disk on a Perkin ElmerꢀSpectrum BX FTIR
Fresh distilled DMF (2 mL) was added dropwise to POCl3 (2
mL) at 50 °C with N2 atmosphere and stirred for 30 minutes to
yield a red solution. This solution was combined with a
90 portion of 3 (1.00 g, 5.24 mmol, dissolved in 10 mL DMF) to
yield a scarlet suspension. The mixture was stirred at 60 °C
overnight and then poured into 70 mL of ice water. NaOH
solution 2M was added to adjust the pH of the mixture to
yield large amount of precipitate. The crude product was
95 filtered, thoroughly washed with water, dried and
recrystallized in absolute ethanol to give 3 (0.80 g, 69%) as
1
yield. H NMR (200 MHz, CDCl3) δ= 9.98 (s, 1H), 8.38 (s,
1H), 8.17ꢀ8.11 (m, 3H) ppm. 13C NMR (50 MHz, CDCl3) δ=
187.5, 159.5, 154.6, 147.5, 147.1, 134.7, 129.3, 124.4, 120.4,
100 112.8 ppm. MS (ESI): m/z = 220.17 [M + 1] +. C10H5NO5
(219.02): C, 54.81; H, 2.30; N, 6.39found, C, 54.88; H, 2.39;
N, 6.46.
1
system. Absorptions are quoted in wavenumbers (cmꢀ1). H
and 13C NMR spectra were recorded at 200 MHz 1H (50.0
MHz 13C) on a Varian Gemini spectrometer. Spin resonances
45 are reported as chemical shifts (d) in parts per million (ppm)
and referenced to the residual peak as an internal standard of
the solvent employed, as follow: CDCl3 7.27 ppm (1H NMR),
77 ppm (13C NMR, central band), DMSOd6 2.50 ppm (1H
NMR, central band), 39.5 ppm (13C NMR, central band).
50 Spin multiplicity is showed by s=singlet, d=doublet, t=triplet,
q=quartet, m=multiplet, br=broad. Coupling constants J are
4.3.3
Synthesis of 2ꢀ((7ꢀnitroꢀ2ꢀoxoꢀ2Hꢀchromenꢀ3ꢀyl)
methylene) malononitrile (5)
105 7ꢀnitroꢀ2ꢀoxoꢀ2Hꢀchromeneꢀ3ꢀcarbaldehyde 4 (1.00 g, 4.54
mmol), Malononitrile (0.73 g, 4.54 mmol) and Na2CaP2O7as
catalyst (0.2 g) were combined in 15 mL of ecoꢀcompatible
EtOH/water: 95/5 and stirred at room temperature for 15 min.
Then, the catalyst was removed by filtration and washed with
reported in Hertz. Mass spectra were recorded on
a
ThermoScientific LCQꢀFleet mass spectrometer under
6
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ethyl acetate. After concentration of the filtrate, the residue
Na2SO4 and filtered, the filtrate was concentrated under the
was purified by reꢀcrystallization using EtOH leading to 5 60 reduce pressure and the residue was purified by Flash
1
(1.12 g, 95%) as yield. H NMR (200 MHz, CDCl3) δ= 8.16ꢀ
8.11 (m, 3H), 7.77 (s, 1H), 7.53 (s, 1H) ppm. 13C NMR (50
MHz, CDCl3) δ= 165.5, 161.7, 154.2, 147.6, 138.6, 129.3,
124.3, 120.2, 119.6, 116.4, 112.7, 105.5 ppm. MS (ESI): m/z
chromatography (AcOEt/PE: 3:2) to afford S1b in 86%) as
yield. 1H NMR (200 MHz, CDCl3): see table 2 for the glycidic
part and δ = 6.14 and 5.90 (2d, 2H, CH2=), 2.08 (s, 3H), 1.51,
1.45, 1.34, 1.33 [4s, each 3 H, 2 x C(CH3)2] ppm. 13C NMR
5
= 268.35 [M + 1] +. C13H5N3O4 (267.03): C, 58.44; H, 1.89; 65 (50 MHz, CDCl3): δ = 167.3, 136.8, 125.2, 109.1, 108.3 [2 x
N, 15.73 found, C, 58.52; H, 1.94; N, 15.80.
C(CH3)2], 95.7, 70.5, 70.1, 69.8, 65.4, 63.2, 25.4, 25.3, 24.4,
23.9 [2 x C(CH3)2], 18.7 ppm. MS (ESI): m/z = 329.36 [M +
1] +. C16H24O7 (328.15): C, 58.52; H, 7.37 found, C, 58.63; H,
7.44.
4.3.4 Synthesis of 2ꢀ ((7 – amino – 2 – oxo ꢀ 2H –
10 chromen – 3 ꢀyl) methylene) malononitrile (6)
To a solution of 5 (1. 00g, 3.74 mol) in methanol (10 ml),
SnCl2 (2 mol)] was added and the resulting mixture was
stirred under reflux. After the completion of the reaction
(monitored by TLC), the reaction mixture was filtered through
15 celite. The filtrate was evaporated under vacuum and the
residue was taken into chloroform, washed twice with 80%
saturated brine solution and finally with water. The organic
layer was dried over anhydrous sodium sulphate and
evaporation of the organic layer was followed by purification
20 either by column chromatography (AcOEt/PE: 10/3, Rf=0.47)
to yield 6 in 84/ as yield. 1H NMR (200 MHz, CDCl3) δ= 7.75
(s, 1H), 7.54 (s, 1H), 7.32 (d, 1H), 6.59 (d, 1H), 6.31 (s, 1H),
6.22 (s, 2H) ppm. 13C NMR (50 MHz, CDCl3) δ= 165.4,
161.5, 156.3, 152.3, 138.7, 129.3, 119.5, 116.6, 110.5, 108.4,
25 105.4, 100.9 ppm. MS (ESI): m/z = 238.45 [M + 1] +.
C13H7N3O2 (237.03): C, 65.82; H, 2.97; N, 17.71 found, C,
65.92; H, 3.11; N, 17.85.
70 Table 2: 1H NMR spectroscopic data (δ, ppm; J, Hz) in
CDCl3 of the glycide portion for protected 6ꢀOꢀDꢀgalactoyl
(S1b) derivatives.
75
4.3.7
Synthesis of the polymers P1aꢀb
Five copolymerization experiments were performed using 7
and S1b as coꢀmonomers. Various monomer molar ratios S1b/7
(aꢀ 30/70, bꢀ 40/60, cꢀ 50/50, dꢀ 70/30 and eꢀ 80/20) were
80 considered. Appropriate amounts of monomers, initiator
(AIBN, 1% compared to 7), and chloroform (5 ml) were
introduced in a roundꢀbottom flask fitted with a condenser.
The mixture was placed under nitrogen atmosphere, and then
stirred at 65 °C for 8 h. The polymers formed were
85 concentrated by evaporation and purified by precipitation with
methanol (5 mL) and diethyl ether (100 mL). After drying in
vacuum, the polymers P1aꢀe were obtained as a red solid.
4.3.5
Synthesis of Nꢀ(3ꢀ(2,2ꢀdicyanovinyl)ꢀ2ꢀoxoꢀ2Hꢀ
chromenꢀ7ꢀyl) methacrylamide (7)
30 To a solution of the dye 6 (1.00g, 4.21 mol) in 15 mL of THF,
methacrylic chloride (0.44g, 4.21 mol) was added and the
mixture was stirred at room temperature for 1 h. TLC showed
the formation of one major spot at (Rf. 0.53, dichloromethane
/ methanol: 10:0.20). The reaction mixture was evaporated to
35 dryness under reduced pressure. The residue was dissolved in
chloroform (20mL) and washed with solution of HCl 5%
(20mL) and water (3 x 20mL). The organic solution was dried
over Na2SO4 and filtered, the filtrate was concentrated under
the reduce pressure and the residue was purified by Flash
40 chromatography (dichloromethane/methanol: 10:0.20) to
1
Yield: 65–90%. H NMR (200 MHz, DMSOd6) δ= 8.57 (s, ꢀ
CH=Cꢀ), 8.11 (s, NH), 7.96 (s, ArꢀH), 7.58ꢀ7.54 (m, ArꢀH),
90 7.01ꢀ6.89 (m, ArꢀH), 6.71 (s, ArꢀH), 5.57 (m, glyꢀH), 4.61 (m,
glyꢀH), 4.37ꢀ4.08 (m, glyꢀH ), 1.79 (s, CH2), 1.69 (s, CH3),
1.52, 1.45, 1.32, 1.31 [4s, each 3 H, 2 x C(CH3)2] ppm. 13C
NMR (50 MHz, DMSOd6) δ= 176.5, 175.8 (CO), 165.6
(=CH), 161.6 (Ar C), 156.4 (Ar C), 139.2 (Ar =CH). 138.9
95 (Ar C), 128.2 (Ar C) 119.5 (=C−CH=), 109.2, 108.6 [2 x
C(CH3)2], 118.4 (Ar C), 113.4 (Ar C), 111.7 (Ar C), 105.7
(N≡C−C), 95.4, 70.6, 70.3, 69.5, 65.6, 63.0, 43.6 (C−CH3),
38.7, 37.5 (CH2), 25.5, 25.2, 24.6, 23.6 [2 x C(CH3)2] 22.5,
22.1 (CH3), ppm.
1
afford 7 in 71%) as yield. H NMR (200 MHz, CDCl3) δ=
8.51 (s, 1H), 7.84 (s, 1H), 7.78 (s, 1H), 7.58ꢀ7.55 (m, 3H),
5.85ꢀ5.60 (2d, 2H, CH2=), 1.77 (s, 3H), ppm. 13C NMR (50
MHz, CDCl3) δ= 165.6, 163.4, 161.4, 156.1, 141.3, 138.8,
45 135.2, 128.5, 119.7, 118.5, 118.1, 116.8, 113.6, 111.7, 105.6,
40.2, 21.9 ppm. MS (ESI): m/z = 306.42 [M + 1] +.
C17H11N3O3 (305.08): C, 66.88; H, 3.63; N, 13.76 found, C,
66.93; H, 3.69; N, 13.82.
100 4.3.8
Synthesis of the polymers P2aꢀe
A solution of P1aꢀb (1.00 g) in 90% aqueous TFA (15 mL)
was stirred at room temperature for 3 h. The violet solution
was repeatedly coꢀevaporated with toluene (5 x 25 mL) at
reduced pressure to give the final product P2aꢀe (0.78 g) as a
105 red powder consisting of a mixture of αꢀ and βꢀpyranosic
anomers in a ratio of 50:50, calculated on the basis of the
4.3.6
6ꢀOꢀ(methyl
methacrylate)
ꢀ1,2:3,4ꢀdiꢀOꢀ
50 isopropylideneꢀαꢀDꢀgalactopyranose (S1b)
1
relative Cꢁ1 signal intensities. H NMR (200 MHz, D2O) δ=
To a solution of S1a (1.00g, 3.84 mmol) in 15 mL of THF,
methacrylic chloride (0.41g, 3.84 mmol) was added and the
mixture was stirred at room temperature for 1h. TLC showed
the formation of one major spot at (Rf. 0.49, AcOEt/PE: 3:2).
55 The reaction mixture was evaporated to dryness under
reduced pressure. The residue was dissolved in chloroform
(20 mL) and washed with solution of HCl 5% (20mL) and
water (3 x 20mL). The organic solution was dried over
8.55 (s, ꢀCH=Cꢀ), 8.13 (s, NH), 7.94 (s, ArꢀH), 7.57ꢀ7.52 (m,
ArꢀH), 7.00ꢀ6.85 (m, ArꢀH), 6.72 (s, ArꢀH), 5.28ꢀ5.20 (d, glyꢀ
110 H), 4.78 (d, glyꢀH), 3.83ꢀ3.61 (m, glyꢀH), 3.47ꢀ3.31 (m, glyꢀ
H), 1.78 (s, CH2), 1.67 (s, CH3) ppm. 13C NMR (50 MHz,
D2O) see Table 3 for the glycidic part and δ= 176.4, 175.6
(CO), 165.5 (=CH), 161.3 (Ar C), 156.7 (Ar C), 139.4 (Ar
=CH). 138.6 (Ar C), 128.5 (Ar C) 119.4 (=C−CH=), 118.2
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