Note: many of the syntheses have been carried out multiple times.
(Number in brackets corresponds to the number of attempts. The reactions with the best results have been chosen.)
Attempt to synthetize 2-(2-hydroxyphenyl)-4,5-dihydrothiazole-4-carboxylic acid (1)
The reaction has been carried out under inert atmosphere and with dry solvents.
5.987 g of 2-cyanophenol and 7.997 g of L-cysteine hydrochloride were dissolved in 50 mL of ethanol.
4.200 g of sodium bicarbonate were then added. The mixture was refluxed for 30 minutes. The pH was adjusted to 9 with piperidine after the mixture cooled down.
The mixture was stirred for 12 h at room temperature. 11.0 mL of hot water were added and the mixture was acidified to a pH of 4 using acetic acid.
The precipitate was then washed with cold water and ethanol. Residual solvents were removed under high vacuum.
The washings were acidified again with more acetic acid, which caused more product to precipitate. The workup proceeded as described above.
The product was neither soluble in CDCl3 nor in deuterium oxide, so no NMR spectrum was recorded.
IR-spectroscopy showed a band at 2460 cm -1. This indicates the presence of a free -SH group, which does not belong to the desired product.
Synthesis of 2-(2-hydroxyphenyl)-4,5-dihydrothiazole-4-carboxylic acid (2)
3.000 g of 2-cyanophenol and 8.850 g of L-cysteine were dissolved in a 1:1 mixture of methanol and PBS (0.1 M, pH = 6.4).
The pH of the solution was adjusted to 6.5 with solid potassium carbonate. The solution was stirred at 60 °C for 15 h.
After cooling down, the solvents were removed in vacuo and the residue was dissolved in 150 mL of water.
The pH was adjusted to 2.0 by addition of solid citric acid. 100 mL of DCM were added to the flask. The layers were allowed to separate.
The aqueous layer was extracted three times with 100 mL of DCM. The organic layers were pooled and washed with brine.
DCM was removed in vacuo.
Yield:3.741 g of a pale-yellow, foliaceous solid were obtained. This corresponds to a yield of 68 %.
Synthesis of methyl 2-(2-hydroxyphenyl)-4,5-dihydrothiazole-4-carboxylate (1)
The reaction was carried out under intert atmosphere and with dry solvents.
1.750 g of 2-(2-hydroxyphenyl)-4,5-dihydrothiazole-4-carboxylic acid (see above) were dissolved in 25 mL of methanol.
15 drops of 96 % sulfuric acid were added. The solution was refluxed for 20 h. The mixture was poured into 50 mL of ice cold water.
The aqueous layer was then extracted thrice with diethyl ether. All ether extracts were pooled, washed with a concentrated sodium bicarbonate solution,
dried over sodium sulfate and filtered. Ether was removed in vacuo.
Yield: 1.199 g of a yellow oil were obtained. This corresponds to a yield of 64 %.
Attepted reduction of methyl 2-(2-hydroxyphenyl-4,5-dihydrothiazole-4-carboxylate to 2-(2-hydroxyphenyl)-4,5-dihydrothiazole-4-carbaldehyde 2-(2-hydroxyphenyl)-4,5-dihydrothiazole-4-carbaldehyde(2)
The reaction was carried out under intert atmosphere and with dry solvents
0.320 g of methyl 2-(2-hydroxyphenyl-4,5-dihydrothiazole-4-carboxylate were dissolved in 6.5 mL of diethyl ether. The solution was cooled down to -78 °C.
2.08 mL of a 1.2 molar solution of diisobutylaluminium hydride in toluene was added to the etheric solution at -78 °C. The solution was stirred for 2 h.
The progress of the reaction was controlled with thin layer chromatography (DCM : methanol 9:1)(Rf(product) UV 244/356 nm = 0.81).
2,4-dinitrophenylhydrazine was used to check for the presence of the aldehyde. The reaction was quenched by the addition of 2.0 mL of methanol.
Then, 9 mL of a saturated ammonium chloride solution were added. The temperature was allowed to rise up to 0 °C and 16 mL of water were added.
Both layers were separated and the aqeuous layer was extracted twice with 50 mL of diethyl ether. The combined organic layers were washed with brine,
dried over sodium sulfate and filtered. The solvents were removed in vacuo the next day. A orange oil was obtained.
The signal of the aldehyde (expected at shifts of around 10 ppm) was missing in the NMR. Further, the NMR confirmed the presence of unreacted methyl 2-(2-hydroxyphenyl)-4,5-dihydrothiazole-4-carboxylate.
Synthesis of 2-(2-hydroxyphenyl)-N-methoxy-N-methyl-4,5-dihydrothiazole-4-carboxamide (3)
505 mg of 2-(2-hydroxyphenyl)-4,5-dihydrothiazole-4-carboxylic acid and 510 mg of 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI-HCl)
were added to a round-bottom flask and dissolved in 15 mL of DCM. The solution was cooled down to 0 °C.
Separately, 336 mg of N,N-diisopropylethylamine were dissolved in 15 mL of DCM. 255 mg of N,O-dimethylhydroxylamine hydrochloride
were suspended in the same solution. The resulting suspension was added to the solution and the mixture was stirred at room temperature for 3 hours.
The reaction progress was monitored with TLC (ligroin 50-70 : ethyl acetate 1 : 2) (Rf(product) UV 244/356 nm = 0.49).
After the reaction was deemed complete, the organic phase was washed twice with a 7 % solution of muriatic acid. The organic phase was dried over sodium sulfate.
The crude product (yield: 297 mg; 50 %) was pooled with the second preparation of this product (113 mg, from 303 mg 2-(2-hydroxyphenyl)-4,5-dihydrothiazole-4-carboxylic acid)
The united products were purified using column chromatography (silica gel mesh 40 - 63, ligroin 50 - 70 : ethyl acetate 1 : 2).
The solvents were removed in vacuo. A yellow oil was obtained, which crystallized in the freezer over night.
Yield: 288 mg (30 %) of an oil which formed light yellow crystals in the freezer.
Synthesis of N-(tert-butoxycarbonyl)-N-methyl-S-trityl-L-cysteine (2)
The reaction was carried out under intert atmosphere and with dry solvents
2.532 g of N-(tert-butoxycarbonyl)-N-methyl-S-trityl-L-cysteine were dissolved in 5 mL of tetrahydrofurane. The solution was
added slowly to a dispersion of 60 % sodium hydride (in mineral oil), dispersed in 10 mL THF. The dispersion was cooled down to 0 °C. After 10 minutes, 2.60 mL
of methyl iodide were added dropwise over the course of 5 minutes. The mixture was stirred for 20 h at room temperature. Reaction progress war monitored with TLC
(ligroin 50 - 70:ethyl acetate 2:1, with 1 % acetic acid) Afterwards, 15 mL of PBS (0.1 M, pH = 7.0) were added to the mixture. Volatile components were removed in vacuo.
The resulting aqueous phase was washed with ligroin 50 - 70 thrice (25 mL), and extracted twice with 10 mL diethyl ether. The pH was adjusted to 3 with HCl,
and the aqueous phase extracted thrice with 20 mL ethyl acetate. Both organic extraacts were washed with brine, dried over sodium sulfate, filtered and the solvents were removed in vacuo.
The crude product was purified using column chromatography (silica gel mesh 40 - 63, ligroin 50 - 70 : ethyl acetate 3 : 2). Solvents were removed in vacuo.
13C-NMR: (100.6 MHz, CDCl3, 25 °C): δ (ppm): 144.7, 129.7, 128.1, 126.9, 31.0, 28.4.
The absence of many signals in the C-NMR is due to the low amount of product measured. Present signals are conform with literature.
Synthesis of N-methyl-L-cysteine (2)
The reaction was carried out under intert atmosphere.
535 mg of N-(tert-butoxycarbonyl)-N-methyl-S-trityl-L-cysteine (see above) were added to a schlenk round-bottom flask,
followed by the addition of 871 mg of triisopropyl silane, 2600 mg of trifluoroacetic acid and 2.0 mL of DCM. The mixture was stirred for 3 h,
and the disappearance of the starting material controlled with TLC (ligroin 50 - 70 : ethyl acetate 2:1, with 1 % acetic acid).
Volatile components were removed in vacuo, and the remnants washed with 10 mL of diethyl ether thrice.
The resulting oil was azeotroped with toluene twice and placed under high vacuum.
Reduction of 2-(2-hydroxyphenyl)-N-methoxy-N-methyl-4,5-dihydrothiazole-4-carboxamide to 2-(2-hydroxyphenyl)-4,5-dihydrothiazole-4-carbaldehyde (2)
The reaction was carried out under intert atmosphere and with dry solvents
100 mg of 2-(2-hydroxyphenyl)-N-methoxy-N-methyl-4,5-dihydrothiazole-4-carboxamide were dissolved in 5 mL of diethyl ether.
The solution was cooled down to -25 °C. Then, 42 mg of lithium alumanuide were added. The mixture was stirred for 25 minutes.
Reaction progress was monitored with TLC (DCM : methanol 9 : 1). The presence of the aldehyde was confirmed with 2,4-dinitrophenylhydrazine.
After 25 minutes, 2.5 mL of methanol were added to the mixture, followed by the addition of 4 mL of a saturated ammonium chloride solution.
Finally, 5 ml of a 5 % (v/v) sulfuric acid solution were added. The aqueous layer was extracted thrice with ethyl acetate (10 mL each time).
The solvent was removed in vacuo at 20 °C.
Yield: 42 mg of a yellow solid were obtained. This represents a yield of 54 %.
No NMR specra were recorded, as the aldehyde proved too unstable (see third reaction). The product was used immediately.
Synthesis of 2-(2-(2-hydroxyphenyl)-4,5-dihydrothiazol-4-yl)-3-methylthiazolidine-4-carboxylic acid (pyochelin) (2)
42 mg of 2-(2-hydroxyphenyl)-4,5-dihydrothiazole-4-carbaldehyde (see above) were dissolved in 6.5 mL of absolute ethanol.
110 mg N-methyl cysteine were dissolved in 2.0 mL of water and acidified to pH = 1.0 with hydrochloric acid. Both solutions were mixed.
220 mg of potassium acetate were added and the mixture was stirred in the dark over night. The next day, the solution was acidified to pH = 4.5 with hydrochloric acid.
The solution was extracted twice with 20 mL of ethyl acetate and reextracted into a saturated solution of sodium bicarbonate in water.
The aqueous phase was acidified to pH = 4.5, and extracted again twice with ethyl acetate (20 mL each time). Ethyl acetate was removed in vacuo.
The crude oil was dissolved in a minimal amount of DCM, and cyclohexane was added until the solution became cloudy. Solvents were removed in vacuo.
Yield:41 mg of a yellow powder were obtained. This represents a yield of 62 %.
1H-NMR (400 MHz, CDCl3, 25 °C): δ (ppm):7.41 (dd, 3J (H,H) = 7.8 Hz, 4J (H,H) = 1.3 Hz, 1H); 7.37 (m, 1H, H-4); 7.01 (d, 3J (H,H) = 8.3 Hz, 1H); 6.89 (m, 1H, H-5); 4.83-5.10 (m, 1H);
4.37 (m, 1H); 3.84 (m, 1H) 3.23-3.56 (m, 4H, H-9/12), 2.65 (s, 1H, H-16).
No C-NMR data and 2D data were available at this point. The final product is presumed to be present (signals match the reference spectra), but impure.
Synthesis of NH-nor-Pyochelin
Note: the syntheses have been carried out multiple times.
(Number in brackets corresponds to the number of attempts. The reactions with the best results have been chosen.)
Synthesis of 2‘-(2-Hydroxyphenyl)-2‘-thiazoline-4‘-carboxylic acid (carboxylic acid)
A round bottom flask was charged with 3.000 g 2-hydroxybenzonitrile and 8.855 g (R)-cysteine hydrochloride. The reactants were dissolved in 113 mL methanol and 113 mL 0.1 M PBS (pH 6.4).
Solid potassium carbonate was added until the pH was at 6.5. The mixture was stirred at 60 °C over night. The solution was concentrated under reduced pressure and the yellow crude was diluted with 150 mL of water.
The pH was adjusted to 2.0 by the addition of solid citric acid. The mixture was extracted with DCM. The resulting organic layers were pooled and dried over sodium sulfate and filtered. The solvent was
removed under reduced pressure.
Yield: 3.825 g (17.30 mmol, 68 %) of a yellow solid were obtained.
Synthesis of methyl 2-(2-hydroxyphenyl-4,5-dihydrothiazole-4-carboxylate (ester) (2)
The reaction was carried out under inert athmosphere and with dry solvents.
A round bottom flask was charged with 1.501 g of 2‘-(2-Hydroxyphenyl)-2‘-thiazoline-4‘-carboxylic acid. 24 mL of methanol were added. Ten drops of sulfuric acid were added and the solution was stirred at 60 °C for 17.5 h.
The solvent was concentrated under reduced pressure. The residue was then dissolved in 35 mL of ethyl acetate. The pooled organic layers were washed twice with 10 mL of water and with 10 mL of brine.
The solvent was removed under reduced pressure.
Yield: 0.8440 g (3.560 mmol, 53 %) of a yellow oil were obtained.
Reduction of methyl 2-(2-hydroxyphenyl-4,5-dihydrothiazole-4-carboxylate to 2-(2-hydroxyphenyl)-4,5-dihydrothiazole-4-carbaldehyde (aldehyde) (2)
The reaction was carried out under intert atmosphere and with dry solvents.
0.312 g of methyl 2-(2-hydroxyphenyl-4,5-dihydrothiazole-4-carboxylate were dissolved in 6.0 mL of absolute diethyl ether.
To the solution were added 6.2 mL diisobutylaluminium hydride in toluene (1.2 M) at -78 °C. The solution was stirred for 2 h.
The progress of the reaction was controlled with thin layer chromatography (DCM : methanol 9:1). The presence of the
aldehyde was confirmed using 2,4-dinitrophenylhydrazine. The reaction was then quenched by addition of 2.0 mL of methanol at -78 °C.
Then, 10 mL of a saturated ammonium chloride solution were added. The temperature was allowed to rise up to 0 °C and 16 mL of water were added.
Both layers were separated and the aqeuous layer was extracted thrice with 15 mL of diethyl ether. The combined organic layers were washed with brine,
dried over sodium sulfate and filtered. The solvents were removed in vacuo. A yellow solid was obtained.
Yield:
44 mg of a yellow solid were obtained. This corresponds to a yield of 17 %.
No analysis was conducted because the aldehyde was too unstable and thus used directly in the next step.
Synthesis of 2-(2-(2-hydroxyphenyl)-4,5-dihydrothiazol-4-yl)thiazolidine-4-carboxylic acid (N-demethylated pyochelin) from the ester (2)
44 mg of the freshly synthetized aldehyde (see above) were dissolved in 36 mL of ethanol and 12 ml of water. 328 mg of
L-cysteine hydrochloride and 620 mg of potassium acetate were added. The solution was stirred overnight at room temperature.
Afterwards, the ethanol was removed in vacuo and water was added to the residue. The pH was adjusted to 5 with solid citric acid.
The aqueous layer was then extracted twice with ethyl acetate. The combined organic layers were dried over sodium sulfate and then filtered.
The solvent was removed in vacuo. A yellow oil was obtained. The oil was dissolved in a minimal amount of DCM.
Cyclohexane was added dropwise, until the solution became cloudy. The solvents were removed in vacuo.
Yield:
53 mg of a yellow powder were obtained. This corresponds to a yield of 81 %.
1H-NMR (400 MHz, acetone-d6, 20 °C): δ [ppm] = 7.52 (m, 2H), 7.04 (m, 2H), 5.62(dd, 1H), 3.88 (s, 3H), 3.84 (d, 2H), 3.25 (s, 3H). Trace amount of product signals were present in the spectrum.
Synthesis of 2-(2-hydroxyphenyl)-N-methoxy-N-methyl-4,5-dihydrothiazole-4-carboxamide (Weinreb-amide) (3)
A round-bottom flask was charged with 300 mg of 2-(2-hydroxyphenyl)-4,5-dihydrothiazole-4-carboxylic acid and 285 mg of 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI-HCl). Both solids were dissolved in 15 mL of DCM and the solution was cooled down to 0 °C.
In a seperate flask 300 mg of N,N-diisopropylethylamine was dissolved in 15 mL of DCM. 157 mg of N,O-dimethylhydroxylamine hydrochloride
were suspended in the solution. The suspension was added to the solution and the mixture was stirred at room temperature for 4 hours.
The reaction progress was monitored with TLC (ligroin 50-70 : ethyl acetate 1 : 2) (Rf(product) UV 244/356 nm = 0.49).
After the reaction was deemed complete, the organic phase was washed with 5 % aqueous HCl. The organic phase was dried over sodium sulfate.
The crude product was purified using column chromatography (silica gel mesh 40 - 63, ligroin 50 - 70 : ethyl acetate 1 : 2).
The solvents were removed in vacuo. A yellow oil was obtained, which crystallized in the freezer over night. Yield: 96 mg of a yellow solid were obtained. This corresponds to a yield of 28 %.
Reduction of 2-(2-hydroxyphenyl)-N-methoxy-N-methyl-4,5-dihydrothiazole-4-carboxamide to 2-(2-hydroxyphenyl)-4,5-dihydrothiazole-4-carbaldehyde (aldehyde) (2)
The reaction was carried out under intert atmosphere and with dry solvents
100 mg of 2-(2-hydroxyphenyl)-N-methoxy-N-methyl-4,5-dihydrothiazole-4-carboxamide have been dissolved in 5 mL of diethyl ether. The solution was cooled down to - 20 °C.
40 mg of lithium tetrahydridoaluminate(III) were added to the solution at once. The mixture was stirred for 20 minutes at -20 °C. The reaction was monitored
by TLC (DCM : methanol 9 : 1) and checked for aldehyde presence with 2,4-dinitrophenylhydrazine. After 20 minutes, 2.5 mL of methanol were added at -20 °C,
followed by the addition of 5 mL of saturated ammonium chloride solution and 5 % (v/v) sulfuric acid solution. The aqueous phase was extracted with 10 mL ethyl acetate
thrice. The combined organic phases were dried over sodium sulfate and filtered. Ethyl acetate was removed in vacuo.
Yield:
64 mg of a yellow powder was obtained. This corresponds to a yield of 82 %.
As already mentioned, the aldehyde was too unstable to conduct analysis and ths used immediately.
Synthesis of 2-(2-(2-hydroxyphenyl)-4,5-dihydrothiazol-4-yl)thiazolidine-4-carboxylic acid (N-demethylated pyochelin) from the amide (2)
64 mg of 2-(2-hydroxyphenyl)-4,5-dihydrothiazole-4-carbaldehyde (see above) were dissolved in 6.0 mL ethanol and 1.5 mL of water. 83 mg L-cysteine hydrochloride
and 208 mg potassium acetate were added. The solution was stirred overnight (16 h) in the dark. The ethanol was then removed in vacuo and the residue diluted with water.
The pH of the aqueous phase was set to 5 by addition of solid citric acid. The aqueous phase was then extracted with ethyl acetate thrice. All the organic extracts were pooled,
dried over sodium sulfate and filtered. Ethyl acetate was removed in vacuo. The resulting yellow oil was then dissolved in a minimal amount of DCM, and cyclohexane was added
until the solution became cloudy. The solvents were removed in vacuo.
Yield
49 mg of a yellow powder was obtained. This corresponds to a yield of 51 %.
The product was a mixture of three diastereomers.
Synthesis of 4,5-Dihydro-2-(2,4-dihydroxyphenyl)thiazole-4(R)-carboxylic acid (Desferrithiocin)
The reaction was carried out under inert atmosphere.
Two solutions were prepared:
Solution 1: 1.50 g 2,4-dihydroxybenzonitrile were dissolved in 48 mL of methanol and degassed twice with the freeze-pump-thaw method.
Solution 2: 2.64 g (R)-cysteine hydrochloride and 1.46 g sodium bicarbonate were dissolved in 30 mL 0.1 M PBS (pH 6.4).
Both solutions were combined and degassed twice using the freeze-pump-thaw method.
Subsequently, the reaction mixture was heated at 80 ° C for 6 hours.
The mixture was stirred for 16.5 hours and then the pH was adjusted to 2 with 12 ml of a 1N hydrochloric acid solution in water.
Thereafter, the precipitate was washed three times with 40 mL of distilled water and then twice with 40 mL of ethanol. The residual solvents were removed in vacuo.
To find how efficient the product is, several photometric kinetic studies were conducted. First, the iron complexation of all synthesized siderophores was explored to have a baseline to compare other measurements with. The final goal was to find out if the complexation is necessary beforehand or if an already tried approach, applying gallium and siderophores directly, is the efficient route to go. This measurement was a bit of a failure since the premisse was followed that gallium/iron are bound to our siderophores in a 1:2 ratio. Which is true but only if the siderophores are provided in excess. In this experiment we added twice as many siderophores as ions to our respective solution which defiled the results.
Nor-Pyochelin Iron Complexation
Desferriothicin Iron Complexation
Even though the experimental setup was flawed we can deduce some results from the presented results. Two premisses need to be set. As Mislin et. al. showed the 2:1 (siderophore, ion) complex forms only when we have a vast overrepresentation of siderophores, secondly the kinetics for the second siderophore binding is very slow and the binding is weak.
The first thing that can be deduced is that obviously desferriothicin does not experience a shift in its absorption during complex formation, or that the complex formation is so fast that we were unable to monitor it at all.
Secondly we can clearly see that the NH-nor-pyochelin signal intensifies when the iron complex forms, even though the complexation seems to proceed very quickly as the signal does not experience a later shift.
Gallium Complexation
After the experiments to observe the iron chelation with our siderophores, gallium chelation experiments with all three siderophores were conducted. This was dont in order to measure the specific affinity for gallium in comparison to iron, the native chelate ion. It is very important to check if the gallium is not expelled from the complex due to a vastly higher affinity of the siderophores to iron. If utilized as an antibiotic, the surrounding media will be very scarce of iron, still many of the experiments were conducted in iron containing media to find out if potential bounce back mechanisms after gallium extraction from the siderophores took place.
Pyochelin Gallium Complexation
Nor-Pyochelin Gallium Complexation
K. L. Rinehart, A. L. Staley et al., J. Org. Chem. 1995, 60, 2786-2791.
M. R. Vippila, P. K. Ly, G. D. Cuny, J. Nat. Prod. 2015, 78, 2398-2404.
P. Weng, G. J. Hong, M. R. Wilson, E. P. Balskus, J. Am. Chem. Soc., 2017, 139 (8), pp 2864–2867.
Y. Segade, M. A. Montaos, J. Rodríguez, C. Jiménez, Org Lett. 2014 Nov 7;16(21):5820-5823.
S. Noël, L. Guillon, I. J. Schalk, G. L. A. Mislin, Org. Lett., 2011, 13 (5), pp 844–847.
A. Zamri, I. J. Schalk, F. Pattus, M. A. Abdallah, Bioorganic & Medicinal Chemistry Letters, 2003, 13, 1147-1150.
A. Zamri, M. A. Abdallah, Tetrahedron, 2000, 56, 249-256.
