Protocol Exchange

Author Information

Subject Terms

Keywords

Abstract

Introduction & Materials

Procedures

Outcomes

Conflicting Financial Interests

Associated Publications

Supplementary Files

Figures

Andreas Åslund

K. Peter Nilsson

Peter Konradsson

Figure 1

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;

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.

- 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.

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

1. Nilsson, K.P.R., Herland, A., Hammarström, P. &amp; 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).

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).

CC BY-NC 3.0

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;

Method Article

Andreas Åslund, K. Peter Nilsson, Peter Konradsson

Andreas Åslund

Linköping University

K. Peter Nilsson

Linköping University

Peter Konradsson

Linköping University

DOI:

10.1038/nprot.2010.24

Download PDF

License:

This work is licensed under a CC BY-NC 3.0 License. Read Full License

Figures

Introduction

Reagents

Equipment

Procedure

Timing

Critical Steps

Troubleshooting

Anticipated Results

References

Acknowledgements

Comments

Version History

CURRENT STATUS: Posted

Version 1

No community comments so far

Metrics

Comments: 0

HTML Views: ...

Subject Areas

Synthetic chemistry

Associated Publications

Efficient imaging of amyloid deposits in Drosophila models of human amyloidoses

by Ina Berg, K Peter R Nilsson, Stefan Thor, +1

Nature Protocols (22 January, 2010)

Method Article

Andreas Åslund, K. Peter Nilsson, Peter Konradsson

Andreas Åslund

Linköping University

K. Peter Nilsson

Linköping University

Peter Konradsson

Linköping University

Badges

Peer Review Timeline

Version History

CURRENT STATUS: Posted

Version 1

No community comments so far

Metrics

Metrics

Comments: 0

HTML Views: ...

Subject Areas

Subject Areas

Synthetic chemistry

DOI & PDF

DOI:

10.1038/nprot.2010.24

Download PDF

License

License:

This work is licensed under a CC BY-NC 3.0 License. Read Full License

Declarations

Figures

Introduction

Reagents

Equipment

Procedure

Timing

Critical Steps

Troubleshooting

Anticipated Results

References

Acknowledgements

Comments

Related Articles