Synthesis of a pentathiophene fluorescent probe, 4&#8217;,3&#8217;&#8217;&#8217;-bis-carboxymethyl-[2,2&#8217;;5&#8217;,2&#8217;&#8217;;5&#8217;&#8217;,2&#8217;&#8217;&#8217;;5&#8217;&#8217;&#8217;,2&#8217;&#8217;&#8217;&#8216;]quinquethiophene-5,5&#8217;

doi:10.1038/nprot.2010.24

Method Article

Synthesis of a pentathiophene fluorescent probe, 4’,3’’’-bis-carboxymethyl-[2,2’;5’,2’’;5’’,2’’’;5’’’,2’’’‘]quinquethiophene-5,5’

https://doi.org/10.1038/nprot.2010.24

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Synthetic chemistry

fluorescent probe

amyloid

prion

synthesis

oligothiophene

Poly and oligo-thiophenes have previously been used for in vitro^1,2, ex vivo^3,4 and in vivo⁵ imaging of protein aggregates. The probe (p-FTAA) has been developed for the purpose of in vivo staining of protein aggregates such as amyloid deposits. It effectively passes the blood brain barrier and imaging can be performed live with two-photon imaging or ex vivo⁵.

The straightforward synthesis of p-FTAA, including two Suzuki couplings, makes it an attractive probe for studies of most diseases involving protein aggregates.

3-Thiopheneacetic acid (Sigma-Aldrich, cat. no. 220639)
Acetyl chloride (Sigma-Aldrich, cat. no. 00990)
Methanol (Merck, cat. no. 1.08325)
N-bromosuccinimide (NBS) (Sigma-Aldrich, cat. no. B81255)
Chloroform (Prolabo, cat. no. 83626.320)
Acetic acid (Sigma-Aldrich, cat. no. A6283)
Silica Gel for flush column chromatograpy (Sigma-Aldrich, cat. no. 22716-6)
2,5-Thiophenediboronic acid (Sigma-Aldrich, cat. no. 470317)
PEPPSI-Ipr® ([1,3-Bis(2,6-Diisopropylphenyl)imidazol-2-ylidene](3-chloropyridyl)palladium(II) dichloride) (Sigma-Aldrich, cat. no. 669032)
Potassiumcarbonate (Sigma-Aldrich, cat. no. 20961-9)
Toluene (Merck, cat. no. 1.08325)
5-(Dihydroxyboryl)-2-thiophenecarboxylic acid (Frontier Scientific, cat. no. C1695)
Sodiumhydroxide (Sigma-Aldrich, cat. no. 45291-2)
Deuterated chloroform (Sigma-Aldrich, cat. no. 151823)
Deuterated DMSO (Sigma-Aldrich, cat. no. 151874)
Deuterated D2O (Sigma-Aldrich, cat. no. 151882)
Thin-layer chromatography plates on glas backing, silica gel 60 F254 (Merck)
Ethylacetate (Prolabo cat. no. 23882.321)
Celite® 545 (Prolabo cat. no. 22552.290)
Trifluoroacetic acid (Merck cat. no. 8.08260)
Diethylether (Prolabo cat. no. 23811.292)
HCl (Scharlau cat. no. AC0741)
Dioxane (Prolabo cat. no. 83620.320)

Magnetic hotplate stirrer
Digital temperature probe
Oil bath
100 and 250 mL round-bottom flasks
Water condenser (to fit neck of flask)
Teflon-coated magnetic stirrer bar
Heat gun
Rotary evaporator (Büchi)
Pyrex chromatographic column (approx. diameter 3 cm)
NMR tubes
VersaFlash® purification system with reversed phase columns (Sigma-Aldrich)

Synthesis of (2-bromo-thiophen-3-yl)-acetic Acid methyl ester (2)

1] To a solution at 0 °C with 3-thiopheneacetic acid (1.42 g, 10 mmol) and methanol (20 mL) add acetic acid (1 mL) slowly.

2] Dilute with toluene and wash with NaHCO3 (sat. aq.) until basic.

3] Dry with MgSO₄, filter and concentrate.

4] Dissolve in 20 mL chloroform/acetic acid (1:1) and cool to 0 °C.

5] Add 1.78 g NBS (10 mmol)

6] After five hours dilute with toluene and wash with NaHCO₃ (sat. aq.) until basic.

7] Dry with MgSO₄, filter and concentrate.

8] Purify on flush column chromatography (toluene).

Synthesis of (3''-methoxycarbonylmethyl-[2,2';5',2'']terthiophen-3-yl)-acetic acid methyl ester (3)

1] Dissolve 2 (2.35 g 10 mmol) and 2,5-thiophenediboronic acid (0.781 g, 4.5 mmol) in 20 mL degassed toluene/methanol (1:1).

2] Add 4.14 g (30 mmol) K₂CO₃ and PEPPSI-Ipr (0.204 g, 0.30 mmol) to the solution and heat at 80 °C for 10 minutes.

3] Filter the reaction mixture on celite, dilute with toluene and wash with NaHCO₃ (sat. aq.).

4] Dry with MgSO₄, filter and concentrate.

5] Purify the crude reaction mixture on a reversed phase column (methanol/H₂O 4:1, 0.05 % trifluoroacetic acid) to give the product.

Synthesis of (5,5''-dibromo-3''-methoxycarbonylmethyl-[2,2';5',2'']terthiophen 3-yl)-acetic acid methyl ester (4)

1] Add NBS (0.460 g, 2.04 mmol) to a solution of the trimer (3, 0.382 g, 0.973 mmol) in chloroform/acetic acid (1:1, 5 mL, 0 °C).

2] Stir for 4 hours before dilution with diethyl ether (20 mL)

3] Wash with NaHCO₃ (sat. aq., 20 mL) and finally water (20 mL).

4] Dry with MgSO₄, filter and concentrate.

5] Purify by flash column chromatography (toluene).

Synthesis of 4',3'''-bis-carboxymethyl-[2,2';5',2'';5'',2''';5''',2'''']quinquethiophene-5,5''''-dicarboxylic acid (p-FTAA) (5)

1] Add K₂CO₃ (4.14 g, 30 mmol), PEPPSI-IPr (0.204 g, 0.30 mmol) and 5-(dihydroxyboryl)-2-thiophenecarboxylic acid to a solution of 4 (5.50 g, 10 mmol) in degassed toluene/methanole (1:1, 30 mL).

2] Heat at 80 °C for 10 minutes

3] Add HCl (20 mL, 1 M, aq.) and collect the red precipitate

4] Dissolve the precipitate in boiling dioxane and filter it on celite followed by precipitation by addition of a small amount of water.

5] Dry the precipitate and add two equimolar amounts of NaOH (1 M, aq.)

6] The product is afforded by concentration or freeze drying.

One week

During the cross coupling reactions generating product 5 and 3 both temperature and time should be precisely monitored. If available a microwave reactor can be used.

The purification of product 3 should be performed on reversed phase column for best result.

Low yield:

Repeat reaction with fresh reagents.

Product 2

Typical isolated yield of 2 should be 2.16 g (92 %).

Analytical data:

¹H-NMR (CDCl3) ?: 3.63 (s, 2H), 3.71 (s, 3H), 6.30 (d, 1H, J = 5.7 Hz), 7.33 (d, 1H, J = 5.7 Hz) ¹³C-NMR (CDCl₃) ?: 34.9, 52.2, 111.7, 125.8, 128.7, 133.6, 170.6

Product 3

Typical isolated yield of 3 should be 1.50 g (85 %) as slightly yellow colored oil.

Analytical data:

¹H-NMR (CDCl₃) ?: 3.72 (s, 4H), 3.79 (s, 6H), 7.05 (d, 2H, J = 5.2 Hz), 7.14 (s, 2H), 7.25 (d, 2H, J = 5.2 Hz); ¹³C-NMR (CDCl3) ?: 34.7, 52.3, 124.9, 127.5, 130.5, 130.6, 133.0, 135.9, 171.5; HRMS m/z calcd. for C₁₈H₁₆O₄S₃: [M]+ 392.0211; Found: 392.0228.

Product 4

Typical isolated yield of 4 should be 0.482 g (90 %).

Analytical data:

¹H-NMR (CDCl₃) ?: 3.70 (s, 4H), 3.73 (s, 6H), 7.02 (s, 2H), 7.08 (s, 2H); ¹³C-NMR (CDCl3) ?: 34.5, 52.4, 111.8, 127.2, 127.8, 131.1, 134.1, 134.8, 170.8

Product 5

Typical isolated yield of 5 should be 6.00 g (85 %) as red powder.

Analytical data:

¹H-NMR (DMSO-D6) ?: 3.76 (s, 4H), 7.32 (s, 2H), 7.38 (d, 2H, J = 4.10 Hz), 7.45 (s, 2H), 7.66 (d, 2H, J = 4.10 Hz); ¹³C NMR (D₂O, 45 °C) ?: 38.4, 124.4, 126.6, 129.1, 131.9, 132.0, 134.4, 134.7, 135.5, 139.6, 140.9, 169.9, 179.4; HRMS m/z calcd for C₂₆H₁₅O₈S₅: [M-H]- 614.9371; Found 614.9422

Nilsson, K.P.R., Herland, A., Hammarström, P. & Inganäs, O. Conjugated polyelectrolytes: Conformation-sensitive optical probes for detection of amyloid fibril formation. Biochemistry 44, 3718-3724 (2005).
Åslund, A. et al. Studies of luminescent conjugated polythiophene derivatives: enhanced spectral discrimination of protein conformational States. Bioconjug. Chem. 18, 1860-8 (2007).
Nilsson, K.P.R. et al. Imaging Distinct Conformational States of Amyloid-beta Fibrils in Alzheimer's Disease Using Novel Luminescent Probes. ACS Chem. Biol. 2, 553-560 (2007).
Sigurdson, C.J. et al. Prion strain discrimination using luminescent conjugated polymers. Nature Methods 4, 1023-30 (2007).
Åslund, A. et al. Novel pentameric thiophene derivatives for in vitro and in vivo optical imaging of a plethora of protein aggregates in cerebral amyloidoses. ACS Chem. Biol. 4, 673-84 (2009).

Our work is supported by the Swedish Foundation for Strategic Research (KPRN, PK and AÅ), the Knut and Alice Wallenberg foundation (KPRN), and a grant from the European Union FP-7: "LUPAS" (KPRN).

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Synthesis of a pentathiophene fluorescent probe, 4’,3’’’-bis-carboxymethyl-[2,2’;5’,2’’;5’’,2’’’;5’’’,2’’’‘]quinquethiophene-5,5’

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