Chapter 2. Synthesis and properties of new fluorescent calcium sensors
2. Model compounds
Before we embarked on the synthesis of target compounds Ia-c, we had prepared some model compounds: a) a rhodamine with an alkoxy substituent in the ortho-position to the amino group; b) spiroamides with a ″closed″ rhodol fragment (cf.:[5]).
A conventional synthetic route to rhodols with a carboxy group starts with an acylation of resorcine or m-aminophenol with phthalic anhydride (30) followed by the reaction with a second molecule of m-aminophenol or resorcine (see Scheme 7).
R2N OH
Scheme 7. Various synthetic routes towards xanthene dyes.
3-(N,N-Diethylamino)-4-methoxyphenol (compound 31 in Figure 40) was prepared as a model compound from the commercially available acetanilide 32 which was deacetylated by heating in aqueous HCl under reflux.
Et2N O MeO
31 H
Figure 40. Model compound 31.
The aniline 33 was obtained in good yield and then diazotized. The aryldiazonium hydrogen-sulfate 34 could be converted into the phenol 31 only by prolonged boiling in
aqueous solution.[89] Other methods (adding of the diazonium solution into the boiling 67%
sulfuric acid,[90] or decomposition in the presence of Cu(NO3)2 and Cu2O[91]) failed to provide the required phenol 31.
MeO
Scheme 8. A successful route to the phenol 31.
The phenol 31 was used for the preparation of rhodamine 35 (Scheme 9). Unfortunately, the yield of the dimethoxyrhodamine 35 was only moderate (18%). As a rule, conventional condensation methods require harsh reaction conditions for the second step (Scheme 7): high temperatures (up to ca. 200 °C) and strong acids (e. g. phosphoric acid, 50% aq. sulfuric acid).
For the model reaction (compound 30 + compound 31) the mildest conditions were used:
heating overnight in refluxing propionic acid with p-TosOH•H2O as a catalyst.[92]
MeO
Scheme 9. Synthesis of rhodamine 35.
In order to check the possibility of synthesizing spiroamides from rhodols compounds 39[93] were prepared according to the known method.[93] Towards that end, 3-N,N-diethylaminophenol 36 was acylated with phthalic anhydride 30 in a non-catalytic Friedel-Crafts reaction to produce the benzophenone derivate 37 in good yield. The next step was the reaction of resorcinol 38-H with the benzophenone 37, and this was succeeded by an intramolecular condensation of the intermediate product at high temperature under acid catalysis, yielding 83% of rhodole 39-H.
Et2N OH
Et2N OH O HOOC
HO OR Et2N O O
OOC
R 30, PhCH3,
6 h, reflux
36 37
50% aq. H2SO4 38-H: R = H
38-Me: R = Me
39-H: R = H 39-Me: R = Me
(83%) (48%)
Scheme 10. Synthesis of the model rhodoles 39-H and 39-Me.
For preparation of the other model compound – the rhodole spiroamide 40-H – the carboxy group in compound 39-H would have to be converted it into the corresponding acid chloride,[5] which is impossible due to the presence of the free hydroxy group that may react with an acid chloride or may even be converted into the corresponding aromatic chloride (see Figure 41).
O
Et2N C
O O
l
Figure 41. Possible product upon treatment of compound 39-H with phosphorus oxychloride (POCl3).
To circumvent this problem, compound 39-Me was prepared (Scheme 10) and successfully converted into the acid chloride 41-Me (Scheme 11). The latter reacted with aniline and, in the presence of Et3N, gave the spiroamide 40-Me. Boron trichloride was used to remove the methoxy group[94] to yield the required spiroamide 40-H (Scheme 11).
Et2N O OMe
COO
O OH
Et2N
N Ph O 40-H
Et2N O OMe
COCl
O OMe
Et2N
N Ph O BBr3, CH2Cl2,
–78 °C, 2 h
39-Me 41-Me
POCl3, (CH2Cl)2, reflux, overnight
PhNH2, Et3N, MeCN, 4 h, reflux
40-Me 88%
62%
Scheme 11. Synthesis of the rhodole spiroamide 40-H.
2.1. Spectroscopic properties of the model compounds
For the model compound 35, interesting spectroscopic properties were observed. The fluorescence quantum yield of this compound was found to be only 4.5% in ethanol and 0.9%
in aqueous solution. These values are much lower than those for the similar compound without methoxy groups – Rhodamine B (65% in EtOH and 31% in water).[95]
O
Et2N N 2
MeO OMe
COO O Et
Et2N NEt2
COO
Rhodamine B 35
Figure 42. The structures of Rhodamine B and compound 35.
A sharp decrease in the fluorescence quantum yield may be attributed to the higher rotational dissipation of energy of the molecule in the excited state. The methoxy group in the o-position of the diethylamino group could assist the rotation of the diethylamino group from being coplanar with the aromatic ring into an out-of-plane conformation. Therefore, the fraction of the non-radiative de-excitation processes becomes higher, and then the quantum yield decreases.
Figure 43. Absorption and fluorescence spectra of compound 35 in ethanol (black lines,
=
max
λabs 570 nm, ε = 66500 M-1cm-1, λmaxfl = 596 nm) and in water (red lines, λmaxabs = 568 nm, ε = 47000 M-1cm-1, λmaxfl = 604 nm).
The absorption and emission spectra of compound 35 show bathochromic shifts in comparison with those of Rhodamine B (in ethanol: 542 nm, 568 nm, in water:
553 nm, 581 nm), which can be attributed to the presence of the donor methoxy group.
=
max
λabs λmaxfl =
=
max
λabs λmaxfl =
To check the photoswitching ability of the second series of model compounds 40-H, the samples were illuminated with UV light, and then the emission spectra were recorded.
Figure 44. Emission spectra of compound 40-H in toluene (excitation wavelength: 520 nm).
Black line: sample stabilized in the dark; Red line: sample irradiated for one minute with UV
light, selected with a band-pass filter centered at 366 nm (UG1 – 3 mm, Schott); blue line:
sample allowed to relax in the dark for one minute.
The emission maxima of the open form of the rhodole spiroamide 40-H was observed at 546 nm. The efficiency of the opening reaction of the rhodole spiroamides was very low, and the thermal reclosing was fast. Only in toluene, the closing reaction was slow enough, so that it was possible to accumulate the open form with prolonged irradiation; but the amount of the open form was still very low. Nevertheless, the switching possibility for this kind of compounds was confirmed.
3. Synthesis of rhodole derivatives with a crown-ether moiety fused with