Synthetic Chemistry

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Authors: Andreas Åslund, K. Peter Nilsson & Peter Konradsson

Introduction

Poly and oligo-thiophenes have previously been used for in vitro (1,2), ex vivo (3,4) and in vivo (5) 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 vivo (5).

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

Reagents

  1. 3-Thiopheneacetic acid (Sigma-Aldrich, cat. no. 220639)
  2. Acetyl chloride (Sigma-Aldrich, cat. no. 00990)
  3. Methanol (Merck, cat. no. 1.08325)
  4. N-bromosuccinimide (NBS) (Sigma-Aldrich, cat. no. B81255)
  5. Chloroform (Prolabo, cat. no. 83626.320)
  6. Acetic acid (Sigma-Aldrich, cat. no. A6283)
  7. Silica Gel for flush column chromatograpy (Sigma-Aldrich, cat. no. 22716-6)
  8. 2,5-Thiophenediboronic acid (Sigma-Aldrich, cat. no. 470317)
  9. PEPPSI-Ipr® ([1,3-Bis (2,6-Diisopropylphenyl) imidazol-2-ylidene)(3-chloropyridyl)palladium(II) dichloride) (Sigma-Aldrich, cat. no. 669032)
  10. Potassiumcarbonate (Sigma-Aldrich, cat. no. 20961-9)
  11. Toluene (Merck, cat. no. 1.08325)
  12. 5-(Dihydroxyboryl)-2-thiophenecarboxylic acid (Frontier Scientific, cat. no. C1695)
  13. Sodiumhydroxide (Sigma-Aldrich, cat. no. 45291-2)
  14. Deuterated chloroform (Sigma-Aldrich, cat. no. 151823)
  15. Deuterated DMSO (Sigma-Aldrich, cat. no. 151874)
  16. Deuterated D2O (Sigma-Aldrich, cat. no. 151882)
  17. Thin-layer chromatography plates on glas backing, silica gel 60 F254 (Merck)
  18. Ethylacetate (Prolabo cat. no. 23882.321)
  19. Celite® 545 (Prolabo cat. no. 22552.290)
  20. Trifluoroacetic acid (Merck cat. no. 8.08260)
  21. Diethylether (Prolabo cat. no. 23811.292)
  22. HCl (Scharlau cat. no. AC0741)
  23. Dioxane (Prolabo cat. no. 83620.320)

Equipment

  1. Magnetic hotplate stirrer
  2. Digital temperature probe
  3. Oil bath
  4. 100 and 250 mL round-bottom flasks
  5. Water condenser (to fit neck of flask)
  6. Teflon-coated magnetic stirrer bar
  7. Heat gun
  8. Rotary evaporator (Büchi)
  9. Pyrex chromatographic column (approx. diameter 3 cm)
  10. NMR tubes
  11. VersaFlash® purification system with reversed phase columns (Sigma-Aldrich)

Procedure

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.

Timing

One week

Critical Steps

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.

Troubleshooting

  • Low yield:
    • Repeat reaction with fresh reagents

Anticipated Results

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

References

  1. 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).
  2. Åslund, A. et al. Studies of luminescent conjugated polythiophene derivatives: enhanced spectral discrimination of protein conformational States. Bioconjug. Chem. 18, 1860-8 (2007).
  3. 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).
  4. Sigurdson, C.J. et al. Prion strain discrimination using luminescent conjugated polymers. Nature Methods 4, 1023-30 (2007).
  5. 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).

Acknowledgements

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).

Figures

Scheme 1. Proposed synthetic route to p-FTAA (5)

Fig 1

Associated Publications

Efficient imaging of amyloid deposits in Drosophila models of human amyloidoses, Ina Berg, K Peter R Nilsson, Stefan Thor, and Per Hammarström, Nature Protocols 5 (5) 935 - 944 29/04/2010 doi:10.1038/nprot.2010.41

Author information

Andreas Åslund, K. Peter Nilsson & Peter Konradsson, Linköping University

Source: Protocol Exchange (2010) doi:10.1038/nprot.2010.24. Originally published online 26 January 2010.

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