Synthesis and photosynthetic inhibition activity of substituted 5-(bis-trifluoromethyl)methyl)-2-aminothiazoles

Article abstract of DOI:10.1016/j.jfluchem.2006.07.007

The reaction of 5,5,5-trifluoro-4-trifluoromethyl-pent-3-en-2-one with aminothiocarbonyls yielded aminothiourea precursors which readily cyclised to the corresponding thiazole derivatives, 5-(bis-trifluoromethyl)methyl)-2-aminothiazoles. The inhibitory po

Full text of DOI:10.1016/j.jfluchem.2006.07.007

Journal of Fluorine Chemistry 127 (2006) 1522–1527
www.elsevier.com/locate/fluor
Synthesis and photosynthetic inhibition activity of
substituted 5-(bis-trifluoromethyl)methyl)-2-aminothiazoles
a,
Cecile Boyer , Giovanni Finazzi , Philippe Laurent , Alois Haas , Hubert Blancou
b
a
c
a
*
´
a
´
Laboratoire OMEMF, UMR 5073, Universite Montpellier 2, Place E. Bataillon, 34095 Montpellier, France
b UMR 7141, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, 75005 Paris, France
c Ruhr-Universita¨t, FNO 034, D-44780 Bochum, Germany
Received 23 December 2005; received in revised form 11 July 2006; accepted 12 July 2006
Available online 16 July 2006
Abstract
The reaction of 5,5,5-trifluoro-4-trifluoromethyl-pent-3-en-2-one with aminothiocarbonyls yielded aminothiourea precursors which readily
cyclised to the corresponding thiazole derivatives, 5-(bis-trifluoromethyl)methyl)-2-aminothiazoles. The inhibitory potency of photosystem II
activity of these thiazoles was evaluated using fluorescence measurement techniques. Two of the compounds showed a good activity in comparison
with the reference compound DCMU, 3-(3,4-dichlorophenol)-1,1-dimethylurea. Calculation of lipophilicity according to Rekker and Frey, and a
corresponding experimental determination are reported.
# 2006 Elsevier B.V. All rights reserved.
Keywords: Gem bis-trifluoromethylated molecules; 2-Aminothiazole; PS II inhibitor; Fluorescence; DCMU; Lipophilicity
1. Introduction
the penetration of cell membranes or by modifying the mode of
binding to a target.
Some aminothiazole herbicides inhibit Photosynthetic
Electron Transport (PET), cf. Dayan et al. [1], Fig. 1, structure
A. The aminothiazoles described are thought to disrupt PET by
binding to the D-1 protein of photosystem II (PS II), displacing
a plastoquinone (QB) from its binding site [2]. Some thiazoles
which can act as herbicides have been patented (Fig. 1,
structure B) [3]. The thiazole ring is a part of many natural
products, such as peptide alkaloids [4], some of which exhibit
antibiotic [5–7] and antifungal activities [8]. Natural products
containing thiazole rings isolated from marine species show
antineoplastic and cytotoxic properties [9]. Cyclopeptides
containing thiazole and dihydrothiazole rings also have
cytotoxic activity [10–12].
This paper describes the syntheses and herbicidal activities
of some new aminothiazoles, containing a bistrifluoromethyl
group. An amido moiety seems to be necessary for the activity
of PS II herbicides [14]. However, from the patent literature [3]
it seemed important to locate a highly fluorinated substituent in
position 5 of the thiazole ring. Therefore a combination of
structural features of compounds A and B, leading to structure
C (Fig. 1) was used to optimize properties.
2. Results and discussion
2.1. Synthesis
The activity of some pesticides can be improved by
substitution of hydrogen with fluorine [13]. As the Van der
Waals radii of fluorine and hydrogen are very close (0.135 and
0.12 nm), the bioisosteric replacement of hydrogen by fluorine
is frequently used to influence biological properties. This has
been particularly useful in pesticides, possibly by improving
5,5,5-Trifluoro-4-trifluoromethyl-pent-3-en-2-one (1) was
synthesized from hexafluoroacetone and triphenylphosphor-
anylidene-2-propanone, using the Abele et al. [15] modification
of the synthetic method, originally described by Plakova et al.
and Pattison [16,17]. A good yield was obtained at room
temperature in the presence of hydroquinone (Scheme 1).
Compound 1 is an a,b-unsaturated bistrifluoromethylated
olefin of ‘‘inverted activity’’ and the presence of two electron-
withdrawing CF3 groups direct nucleophilic attack towards the
* Corresponding author. Tel.: +33 467143919; fax: +33 467631046.
E-mail address: cecile.boyer@univ-montp2.fr (C. Boyer).
0022-1139/$ – see front matter # 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.jfluchem.2006.07.007

C. Boyer et al. / Journal of Fluorine Chemistry 127 (2006) 1522–1527
1523
Fig. 1. Known thiazole herbicides A and B and target molecule C.
Table 1
Compounds tested and their relative inhibition activity on photosynthesis for the
unicellular algae Chlamydomonas reinhardtii
Compound Maximal inhibition (r.u.)a Concentration (mM) IC50b(mM)
Scheme 1.
2
1.00
1.00
0.59
0.45
0.70
0.73
0.34
1.00
10
10
0.85
1.82
–
4
5
1.0 ꢀ 103
6
1.0 ꢀ 106
3.0 ꢀ 106
1.0 ꢀ 104
3.0 ꢀ 103
1.0
–
position a to the carbonyl group [18]. A subsequent reaction
with an aminothiocarbonyl compound resulted in the formation
of an isothiourea derivative, which eliminated water and
cyclised forming the desired thiazole derivatives.
7
–
8
–
9
–
DCMU
0.12
Compound 1 reacted with acetyl thiourea in refluxing
ethanol yielding N-(5-(2,2,2-trifluoro-1-trifluoromethyl-ethyl)-
4-methyl-thiazol-2-yl)-acetamide (2). This was purified by
sublimation and chromatography. Analogously the higher
homologue, N-(5-(2,2,2-trifluoro-1-trifluoromethyl-ethyl)-4-
methyl-thiazol-2-yl)-isopropylamide (4), was synthesized from
2-methylpropionyl thiourea (3) (prepared from 2-methyl-
propionyl chloride and thiourea) and 1 by increasing the
reaction time (Scheme 2).
(–) Undetermined, low inhibition at strong concentrations.
a
Inhibition is in relative units. See Section 3 for the calculation method.
Concentration of inhibitor required for 50% inhibition.
b
(3,4-dichlorophenol)-1,1-dimethylurea (DCMU). The proce-
dure is reported in the experimental section. Results for the
Chlamydomonas test are summarized in Table 1.
Even at low concentration (10 mM), compounds 2 and 4
showed 100% inhibition. This means, that in the presence of
these compounds, there is no photosynthetic activity and all the
light energy absorbed is reemitted as fluorescence (and heat).
The IC50 values obtained for compounds 2 and 4 (0.85 and
1.82 mM) are good when compared with DCMU (0.12 mM),
which is one of the best PS II inhibitors known.
To study the influence of bulky groups, such as chlorophenyl
and tertiarybutylphenyl, the corresponding derivatives were
synthesized. As these compounds differ only with respect to the
substituent on the amino group, the aminothiazole 5 was
prepared as a synthon and derivatives were prepared according
to Scheme 3.
The compounds with a chlorophenyl ring as a substituent (6–
8) showed weak to medium activity, which increased from the
ortho to the para position. These compounds were not further
investigated, just as compounds 5 and 9.
2.2. Biological properties
The activity of compounds 2 and 4 have also been evaluated
using Arabidopsis plants, by the method of Barbagallo et al.
[20]. Chlorophyll fluorescence imaging can effectively and
rapidly identify perturbations of leaf metabolism, well before
the onset of any visual effects of leaf morphology or plant
growth. Solutions of inhibitors can be directly sprayed onto the
leaf and the fluorescence recorded photographically. Graphs
2.2.1. Inhibition of photosynthesis
The photosynthesis inhibition activity of compounds 2 and
4–9 were first evaluated using unicellular algae Chlamydomo-
nas reinhardtii, and subsequently using Arabidopsis plants.
Tests were based on fluorescence measurements, as fluores-
cence is inversely related to photosynthetic activity [19]. The
inhibition is calculated relative to the reference inhibitor, 3-
Scheme 2.

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C. Boyer et al. / Journal of Fluorine Chemistry 127 (2006) 1522–1527
Table 2
Calculated log P and RM values for compounds 2 and 4
Compounds
2
4
log P
1.0
2.04
3.59
RM
2.84
then by sublimation, to give 1.76 g (60%) of 2 as a white solid,
mp: 185–189 8C.
1H NMR (CDCl3) : d 2.20 (s, 3H, C( O)CH3); 2.28 (s, 3H,
CH3 thiazole); 4.3 (h, 1H, CH(CF3)2, JHF = 7.89 Hz); 13C NMR
(CDCl3) : d 15.06 (s, 1C, CH3 thiazole); 23.12 (s, 1C, COCH3);
47.72 (h, 1C, C(CF3)2, JCF = 31.3 Hz); 109.03 (s, 1C, C5
thiazole); 122.38 (q, 2C, (CF3)2, JCF = 271.7 Hz) ; 148.9
(C O); 158.9 (s, 1C, C4 thiazole); 167.79 (s, 1C, C2 thiazole);
19F NMR (CDCl3) : d –66,3 (6 F, d, JHF = 5.6 Hz). HR-MS:
307.0356 (C9H9ON2F6S+; calc. 307.0340).
Scheme 3.
obtained by this method are in accordance with those obtained
from Chlamydomonas.
2.2.2. Lipophilicity
3.1.2. Synthesis of 2-methylpropionyl thiourea (3)
To try and explain the variations in activity, the lipophilicity
of the compounds tested was calculated and measured. Based
on general considerations, it has to be assumed that in
comparison to its hydrogen analogue, the presence of fluorine
in a molecule does not necessarily increase its lipophilicity [21–
23]. On the other hand the variation of substituents on the amino
group influences the lipophilicity which is represented by
corresponding log P values.
Thiourea (2 g, 26.3 mmol) was dissolved in toluene
(25 mL). 2-Methylpropionyl chloride (2.74 mL, 2.79 g,
26.3 mmol) was added and the reaction mixture was stirred
and refluxed for 2 h. The reaction flask was connected to a
water trap in order to collect HCl vapors. The crude product
crystallized partially on cooling at room temperature. The
solvent was evaporated in vacuo and the residue was
recrystallized from ethanol to give 2.3 g (60%) of 3 as a white
solid, mp: 114–116 8C (lit. 114.5 8C [28]).
Those were calculated using the Rekker and Mannhold
method [24]. Since Bate-Smith and Westall observed the
correlation between chromatographic properties and log P [25],
measurements were made using reverse phase thin layer
chromatography, according to Frey et al. [26] and Dross et al.
[27] (giving RM values, cf. chapter 3.2.3).
1H NMR (CDCl3) : d 1.24 (d, 2 ꢀ 3H, C( O)CH(CH3)2,
J
HH = 6 Hz); 2.68 (h, 1H, CH(CH3)2, JHH = 6.93 Hz).
3.1.3. Synthesis of N-(5-(2,2,2-trifluoro-1-trifluoromethyl-
ethyl)-4-methyl-thiazol-2-yl)-isopropylamide (4)
We defer only the values obtained for the active compounds
2 and 4 (Table 2). Their lipophilicity is in a median fork in
comparison with the other compounds prepared (5-9,
2.4 < RM < 7).
A mixture of 2-methylpropionyl thiourea 3 (2.2 g, 15 mmol)
and 5,5,5-trifluoro-4-trifluoromethyl-pent-3-en-2-one 1 (3.8 g,
18.4 mmol) in ethanol (30 mL) was stirred at reflux for 2 d.
Ethanol was evaporated and the crude reaction product was
purified by column chromatography (eluent: pentane/diethy-
lether, 30:70) to give 2.9 g (57%) of 4 as a white solid, mp: 136–
140 8C.
3. Experimental
3.1. Chemistry
1H NMR (CDCl3) : d 1.22 (d, 2 ꢀ 3H, CH(CH3)2,
J
HH = 6.93 Hz); 2.28 (s, 3H, CH3 thiazole); 2.57 (h, 1H,
NMR spectra were recorded on Brucker AC 300 (1H 300.13;
13C 75.47; 19F 282.40 MHz) instrument. Chemical shift values
are given in ppm relative to tetramethylsilane. High-resolution
mass spectra (HRMS) were recorded on JEOL JMS SX 120A
instrument. Melting points were determined on a Stuart
Scientific apparatus and are uncorrected.
CH(CH3)2, JHH = 6.93 Hz); 4.31 (h, 1H, CH(CF3)2,
HF = 7.89 Hz); 9.5 (br., 1H, NH); 13C NMR (CDCl3) : d
13.91 (s, 1C, CH3 thiazole); 16.5 (s, 1C, CH3) ; 16.74 (s, 1C,
CH3); 33.07 (s, 1C, C(CH3)2); 45.38 (h, 1C, C(CF3)2,
J
J
CF = 30.94 Hz); 106.72 (s, 1C, C5 thiazole) ; 120.15 (q, 2C,
(CF3)2, JCF = 282.3 Hz); 146.45 (C O) ; 157.0 (s, 1C, C4
thiazole) ; 172.91 (s, 1C, C2 thiazole); 19F NMR (CDCl3) : d –
3.1.1. Synthesis of N-(5-(2,2,2-trifluoro-1-trifluoromethyl-
ethyl)-4-methyl-thiazol-2-yl)-acetamide (2)
66,3 (6 F, d,
JHF = 8.4 Hz). HR-MS: 335.0634
(C11H13ON2F6S+; calc. 335.0653).
A mixture of acethyl thiourea (1.18 g, 10 mmol) and 5,5,5-
trifluoro-4-trifluoromethyl-pent-3-en-2-one1(1.97 g,9.5 mmol)
in ethanol (30 mL) was refluxed with stirring for 4 d. Ethanol was
evaporated and the crude reaction product was purified firstly by
column chomatography (eluent: pentane/diethylether, 10:90),
3.1.4. Synthesis of 4-methyl-5-(bis trifluoromethyl)methyl-
2-aminothiazole (5)
A mixture of thiourea (2.95 g, 38.8 mmol) and 5,5,5-
trifluoro-4-trifluoromethyl-pent-3-en-2-one 1 (4 g, 19.4 mmol)

C. Boyer et al. / Journal of Fluorine Chemistry 127 (2006) 1522–1527
1525
in ethanol (25 mL) was stirred at reflux for 2 h. A white
precipitate appeared when cooling at room temperature
(remaining thiourea). The solution was filtered and the solvent
was removed in vacuo. The residue was washed with water, and
then extracted three times with diethylether. The organic phase
was dried over Na2SO4, filtered and evaporated to dryness. The
yellowish solid obtained was purified by sublimation to give
4.5 g (87%) of 5 as a white solid, mp : 104–108 8C (lit. : 106–
107 8C [18]).
1H NMR (DMSO) : d 2.40 (s, 3H, CH3 thiazole); 5.9 (h, 1H,
CH(CF3)2, JHF = 7.5 Hz); 7.58 (d, 1H, arom., JHH = 7.9 Hz);
7.71 (dd, 1H, arom., JHH = 1.06 Hz, JHH = 7.24 Hz); 8.02 (d,
1H, arom., JHH = 7.78 Hz); 8.14 (s, 1H, arom.); 13C NMR
(CDCl3) : d 14.78 (s, 1C, CH3 thiazole); 45.43 (h, 1C, C(CF3)2,
J
CH = 30.2 Hz); 107.45 (s, 1C, C5 thiazole); 123.01 (2 ꢀ q, 2C,
(CF3)2, JCF = 280.75 Hz); 126.88 (s, 1C, arom.); 127.92 (s, 1C,
arom.); 130.56 (s, 1C, arom.); 132.5 (s, 1C, arom.); 133.4 (s,
1C); 133.7 (s, 1C); 159.8 (s, 1C); 164.33 (s, 1C); 19F NMR
3
1H NMR (CDCl3): d 2.2 (s, 3H, CH3 thiazole); 4.28 (h, 1H,
7-CH, JHF = 7.9 Hz); 5.0 (br., 2H, NH2); 13C NMR (CDCl3) : d
14.95 (s, 1C, CH3 thiazole); 47.68 (hept, 1C, C(CF3)2,
(CDCl3) : d –65.6 (6 F, d, JHF = 8,5 Hz). HR-MS: 403.0100
(C14H10ON2ClF6S+; calc. 403.0107).
J
CH = 30.9 Hz); 102.79 (s, 1C, C5 thiazole); 122.52 (2 ꢀ q,
3.1.7. Synthesis of 4-chloro-N-(5-(2,2,2-trifluoro-1-
2C, (CF3)2, JCF = 284.5 Hz); 150.86 (s, 1C, C4 thiazole);
168.13 (s, 1C, C2 thiazole); 19F NMR (CDCl3) : d –66,7 (6 F, d,
trifluoromethyl-ethyl)-4-methyl-thiazol-2-yl)-benzamide (8)
4-Methyl-5-(bis trifluoromethyl)methyl-2-aminothiazole 5
(1.3 g, 4.9 mmol) was dissolved in THF (25 mL). Triethyla-
mine (0.75 mL, 0.54 g, 5.4 mmol) was added and the reaction
mixture was stirred. 4-Chlorobenzoyl chloride (0.69 mL,
0.95 g, 5.4 mmol) was added dropwise, resulting in a turbid
suspension. After stirring 0.5 h at room temperature, the
precipitate (HCl.Et3N) was filtered off and the solvent was
removed in vacuo. The residue was washed with water, and then
extracted with diethylether. The organic phase was dried over
Na2SO4, filtered and evaporated to dryness. The brown-orange
oil was purified by column chromatography (eluent: light
petroleum/diethylether, 70:30) to give 0.91 g (46%) of 8 as a
white solid, mp: 145–150 8C.
J
HF = 8,5 Hz). HR-MS: 265.0222 (C7H7N2F6S+; calc.
265.0234).
3.1.5. Synthesis of 2-chloro-N-(5-(2,2,2-trifluoro-1-
trifluoromethyl-ethyl)-4-methyl-thiazol-2-yl)-benzamide (6)
4-Methyl-5-(bis trifluoromthyl)methyl-2 aminothiazole 5
(0.9 g, 3.4 mmol) was dissolved in THF (25 mL). Triethyla-
mine (0.52 mL, 0.38 g, 3.7 mmol) was added and the reaction
mixture was stirred. The 2-chlorobenzoyl chloride (0.65 g,
0.5 mL, 3.7 mmol) was added drop by drop, resulting in a
turbid suspension. After stirring for 0.5 h at room temperature,
the precipitate (HCl.Et3N) was filtered and the solvent was
removed in vacuo. The residue was washed with water, then
extracted by diethylether. The organic phase was dried over
Na2SO4, filtered and evaporated to dryness. The orange residue
was purified by sublimation to give 0.7 g (51%) of 6 as a white
solid, mp: 120–130 8C.
1H NMR (CDCl3) : d 1.99 (s, 3H, CH3 thiazole); 4.3 (h, 1H,
CH(CF3)2, JHF = 7.84 Hz); 7.37 (d, 1H, arom., JHH = 8.57 Hz);
7.82 (d, 1H, arom., JHH = 8.28 Hz); 11 (1H, br., NH); 13C NMR
(CDCl3) : d 13.53 (s, 1C, CH3 thiazole); 47.13 (h, 1C, CH(CF3)2,
J
CH = 30.187 Hz) ; 108.26 (s, 1C, C5 thiazole); 121.17 (2 ꢀ q,
1H NMR (CDCl3): d 2.16 (s, 3H, CH3 thiazole); 4.33 (h, 1H,
CH(CF3)2, JHF = 7.9 Hz); 7.22-7.48 (m, 2H, arom.); 7.78 (dd,
1H, JHH = 0.7 Hz, JHH = 7.3 Hz); 7.89 (d, 1H, JHH = 7.3 Hz);
13C NMR (CDCl3) : d 14.37 (s, 1C, CH3 thiazole); 47.7 (h, 1C,
C(CF3)2, JCF = 31.7 Hz); 109.23 (s, 1C, C5 thiazole); 127.39 (s,
1C, arom.); 130.81 (s, 1C, arom.); 130.84 (s, 1C, arom.); 131.7
(s, 1C); 132.9 (s, 1C, arom.); 149.29 (s, 1C); 159.34 (s, 1C);
164.12 (s, 1C); 170.22 (s, 1C); 19F (CDCl3) : d –66,2 (6 F, d,
2C, (CF3)2, JCF = 280.0 Hz); 127.12 (s, 1C, arom.); 128.14 (s,
1C, arom.); 130.22 (s, 1C, arom.); 131.69 (s, 1C, arom.); 138.74
(s, 1C); 147.41 (s, 1C); 159.16 (s, 1C); 163.39 (s, 1C); 169.86 (s,
1C); 19F NMR (CDCl3): d –66,3 (6 F, d, JHF = 8,5 Hz). HR-MS:
403.0090 (C14H10ON2ClF6S+; calc. 403.0107).
3.1.8. Synthesis of 4-tert-butyl-N-(5-(2,2,2-trifluoro-1-
trifluoromethyl-ethyl)-4-methyl-thiazol-2-yl)-benzamide (9)
4-Methyl-5-(bis trifluoromethyl)methyl-2-aminothiazole 5
(1.3 g, 4.9 mmol) was dissolved in THF (10 mL). Triethyla-
mine (0.9 g, 9 mmol) and 4-tertiobutylbenzoyl chloride (1.06 g,
5.4 mmol) in THF (3 mL) were added and the mixture was
warmed to reflux for 24 h. After evaporating in vacuo, the
brown oil residue was purified by colomn chromatography
(eluent: light petroleum/diethylether, 60:40). The yellowish oil
obtained crystallized to give 1.3 g (62%) of 9 as a yellowish
solid, mp: 135–140 8C.
J
403.0107).
HF = 8,5 Hz). HR-MS: 403.0101 (C14H10ON2ClF6S+; calc.
3.1.6. Synthesis of 3-chloro-N-(5-(2,2,2-trifluoro-1-
trifluoromethyl-ethyl)-4-methyl-thiazol-2-yl)-benzamide (7)
4-Methyl-5-(bis trifluoromethyl)methyl-2-aminothiazole 5
(0.62 g, 2.3 mmol) was dissolved in THF (15 mL). Triethyla-
mine (0.36 mL, 0.26 g, 2.6 mmol) was added and the reaction
mixture was stirred. The 3-chlorobenzoyl chloride (0,33 mL,
0,45 g, 2,6 mmol) was added drorwise, resulting in a turbid
suspension. After stirring 0.5 h at room temperature, the
precipitate (HCl.Et3N) was filtered off and the solvent was
removed in vacuo. The residue was washed with water, and then
extracted with diethylether. The combined organic layers were
dried over Na2SO4, filtered and evaporated to dryness. The
orange residue was purified by sublimation to give 0.69 g (73%)
of 7 as a white solid, mp: 201–205 8C.
1H NMR (CDCl3) : d 1.26 (s, 3H, C(CH3)3); 1.27 (s, 3H,
C(CH3)3); 1.28 (s, 3H, C(CH3)3); 2.01 (s, 3H, CH3 thiazole);
4.3 (h, 1H, CH(CF3)2, JHF = 7.89 Hz); 7.42 (d, 2H, arom.,
J
HH = 8.52 Hz); 7.8 (d, 2H, arom., JHH = 8.56 Hz); 13C NMR
(CDCl3) : d 14.45 (s, 1C, CH3 thiazole); 31.01 (s, 3C, 3 ꢀ CH3);
35.17 (s, 1C, C(CH3)3); 47.69 (h, 1C, C(CF3)2, JCF = 30.9 Hz);
109.2 (s, 1C, C5 thiazole); 126.01 (s, 1C, arom.) ; 127.74 (s, 1C,
arom.) ; 128.61 (s, 1C, arom.) ; 148.67 (C O) ; 157.24 (s, 1C,

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C. Boyer et al. / Journal of Fluorine Chemistry 127 (2006) 1522–1527
C4 thiazole) ; 160.16 (s, 1C, arom.) ; 165.31 (C2 thiazole); 19
NMR (CDCl3): d –66,3 (6 F, d, JHF = 8,5 Hz). HR-MS:
F
fluorescence were measured with a home made fluorimeter
camera.
425.1151 (C18H19ON2F6S+; calc. 425.1122).
3.2.3. Lipophilicity measures
3.2. Biological test methods
3.2.1. Chlamydomonas tests
Chromatographical evaluation of lipophilicity was realized
using commercially available TLC aluminium sheets (size
20 cm ꢀ 20 cm, silicagel RP-18 F254s; MERCK). 3 mL of a
methanolic solution of the test compounds were applied to the
plate. The developing solvent was 100/0, 90/10, 80/20, 75/25,
70/30 and 60/40 mixtures of MeOH/H2O. Measuring the
distance of the substance maximum from front and start affored
Rf values. RM values have been calculated according to Bate-
Smith and Westall [25]:
3.2.1.1. Strains and culture conditions. Chlamydomonas
reinhardtii wild-type (from strain 137 C) were grown at
24 8C in tris-acetate-phosphate (TAP)-supplemented medium
[29] under 6 mE (E = mol photon/m2/s) white light. The cells
were harvested during exponential growth (ꢁ2 ꢀ 106 cells/
mL), centrifuged (at 20 8C, for 5 min, 4000 RP speed) and
resuspended in minimal medium [29].
RM ¼ log½ð1=RFÞ ꢂ 1ꢄ
3.2.1.2. Fluorescence measurements. Fluorescence measure-
ments were performed at room temperature on a home-built
fluorimeter: samples were excited using a light source at
590 nm with ꢁ60 mE intensity. The fluorescence response was
detected in the far red region of the spectrum.
A linear correlation plot of % of MeOH versus RM gave RM
values for 100% H2O.
Acknowledgements
´
We thank Mrs. Tuccio-Lauricella, Universite d’Aix-Mar-
´
seille I and M. Baston, Universite de Lausanne for helpful
3.2.1.3. Samples. Compounds were tested at different con-
centrations (100 to 0.1 mM). Stock solutions with a concentra-
tion of 0.1 M were obtained by dissolving the tested compounds
in ethanol. Solutions of different concentrations were obtained
by diluting the stock solution with water. Final aliquots tested
were obtained by mixing the solution containing the inhibitor
(at proper concentration) with 1 mL algae suspension.
comments and Prof. Dr. Mr. E. Peach, Acadia University,
Canada, for reading the manuscript and valuable discussions.
This work was partially supported by the European Community
(TMR contract no. ERBFMRXCT 970 120).
References
3.2.1.4. Test progress and inhibition calculations. The fluor-
escence measurement provides F0, Fm and F** values. The first
fluorescence measure is one of a sample of pure algae (‘‘white’’)
and algae with DCMU (‘‘white DCMU’’). Then, primary to the
mixture inhibitor/algae test (providing Fm inhibitor) and mixture
DCMU/algae test(providing Fm DCMU), foreach concentration
of inhibitor, the pure algae solution was first tested in order to
acquire a ‘‘control result’’ (providing F0 control). The inhibition
value results from the calculation of:
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