Ninth International Electronic Conference on Synthetic Organic Chemistry (ECSOC-9), http://www.mdpi.net/ecsoc-9, November 1-30, 2005


[C002]

Fluorescent Carbostyrils as Labelling Compounds for Biopolymers

Naresh S. BADGUJAR, Georg URAY, Wolfgang STADLBAUER *

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Institut für Chemie, Organic Synthesis Group,
Karl-Franzens-University of Graz
Heinrichstrasse 28, A-8010 Graz (Austria)

E-mail: wolfgang.stadlbauer@uni-graz.at

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Received: 6th October 2005

Contents:

  1. General
  2. N-1 Functionalized Carbostyrils
  3. 6-O Functionalized Carbostyrils
  4. 6-Hydroxycarbostyrils
  5. 6-Acetoxy-Carbostyrils
  6. Experimental
  7. Acknowledgement
  8. References

General
abstract scheme

Fluorescent 6,7-donor - and 4-acceptor substituted carbostyrils have been found to have largely pH independent properties, large Stokes shifts and medium to high quantum yields [1]. Functionalized with O-succinimidyl (OSu) esters labelling of peptides and carbohydrates could be achieved.

N-1 Functionalized Carbostyrils
formula scheme

Regioselective N-alkylation of 6,7-dimethoxy-4-trifluoromethyl-2-quinolone at N-1 with bromoacetate under phase transfer conditions and functionalization of the ester group leads to O-succinimidyl (OSu) esters which react readily in aqueous solution at ambient temperature with aliphatic amino groups of amino acids, peptides, proteins, N-carbohydrates (e.g. aminoglucose) or N-polysaccharides (e.g. chitosan).

diagram01a

Absorption spectra of all N-substituted carbostyrils (including the derivatives already linked to biopolymers) are similar with a maximum at about 370 nm and epsilon-values between 9000 and 10500.
Emission spectra show Stokes shifts of about 60 nm with sufficient quantum yields of about 0.3-0.4 in DMSO and 0.2-0.35 in water.

diagram01b

6-O Functionalized Carbostyrils
formula scheme

The regioselective introduction of the linker group at the 6-O position was achieved by trans-etherification of the 6-methoxy group of 1-alkyl-6,7-dimethoxy-4-trifluoromethyl-2-quinolones, and functionalization of the carboxylate group to the O-succinimidyl (OSu) ester. The OSu ester was used to label amino acids, peptides, proteins (e.g. bovine serum albumine), amino-carbohydrates (e.g. aminoglucose) or amino-polysaccharides (e.g. chitosan) in aqueous solution.
The UV spectra were similar to the N-1 linked carbostyrils (maximum about 370 nm, epsilon-values between 9600 and 13500).

diagram02a

Emission spectra show a similar Stokes shift of about 60 nm, however quantum yields are low (0.03-0.06 in DMSO and 0.02-0.035 in water).

diagram02b

6-Hydroxycarbostyrils
formula scheme

formula scheme

6-Hydroxycarbostyril showed a larger Stokes shift of 85 nm and sufficient emission in DMSO. Quantum yield in water was very low. The smaller curves show the excitation and emission at pH 13 revealing that the excited anion (398 nm) gets reprotonated exhibiting fluorescence of the hydroxy form.

pH-Dependence of the absorption of the hydroxy compound (362 nm) revealed a pKa of about 8.3.

formula scheme

6-Acetoxy-Carbostyrils
formula scheme

formula scheme

6-Acyloxycarbostyrils were synthesized by acetylation of 6-hydroxycarbostryrils and explored as model compounds for labelling purposes. The spectral data of the 6-acetoxycarbostyril show that both excitation and emission spectra suffered a blue-shift of about 20 nm. The epsilon values are rather high (11400 in DMSO and 14600 in water), however the quantum yields are with 0.006-0.007 both in DMSO and water extremely low.

Experimental

Synthesis of N-1-alkylated carbostyrils: N-Alkylation of 6,7-dimethoxy-4-trifluoromethyl-2-quinolone was performed by reaction with bromoacetate and sodium hydroxide in a binary solvent catalyzed by crown ethers at room temperature; the regioisomers were obtained as a 9:1 mixture and separated by dry flash column chromatography. The ester group was hydrolyzed by heating in aqueous sodium hydroxide solution and the acid reacted with N-hydroxysuccinimde in the presence of carbodiimide at room temperature to give O-succinimidyl (OSu) esters. The labeling reactions were performed with aqueous solutions of amino acids, peptides, proteins, amino sugars in slightly basic medium.
Synthesis of O-6-alkylated carbostyrils: Ether cleavage at the 6-O position was performed with hydrobromic acid under reflux, and etherification with bromoacetate in DMF/potassium carbonate at room temperature. The ester group was hydrolyzed by heating in aqueous sodium hydroxide solution and the acid reacted with N-hydroxysuccinimde in the presence of carbodiimide at room temperature to give O-succinimidyl (OSu) esters. The OSu ester was used to label in aqueous solutions amino acids, peptides, proteins, amino sugars in slightly basic medium.
Synthesis of 6-O-acetyl carbostyril: Acylation at O-6 was performed with acetyl chloride in DMF/potassium carbonate at room temperature.

UV/vis spectra: Shimadzu UV/VIS scanning spectrophotometer UV-2101 PC; concentration: 0.01 mg/mL.

Excitation and emission spectra: Shimadzu RF-5001 PC spectrofluorometer (150-W Xe lamp, 6 selectable slits: 1.5, 3, 5, 10, 15, 20 nm, R452-01 photomultiplier; monochromator: ion-blazed holographic concave grating F/2.5); concentration: 0.001 mg/mL.

Determination of quantum yields: emission signals were set in relation to the known signal of quinine under the same conditions.

Acknowledgement

This work was supported by a scholarship of the Austrian Academic Exchange Service for N. S. B.

References

[1] G. A.Strohmeier, W. M. F. Fabian, G. Uray. Helv. Chim. Acta, 2004, 87, 215-226;
G. Uray, K.-H. Niederreiter, F. Belaj, W. M. F. Fabian, Helv. Chim. Acta, 1999, 82, 1408- 1417;
W. M. F. Fabian, K. S. Niederreiter, G. Uray, W. Stadlbauer, J. Mol. Structure, 1999, 477, 209-220.