Benzothiadiazole oligoene fatty acids: fluorescent dyes with large Stokes shifts

Benzothiadiazole oligoene fatty acids: fluorescent dyes with large Stokes shifts Lukas J. Patalag and Daniel B. Werz* Full Research Paper Address: Institut für Organische Chemie, Technische Universität Braunschweig, Hagenring 30, 38106 Braunschweig, Germany Email: Daniel B. Werz* - * Corresponding author Keywords: fatty acid; fluorescence; lipid; membrane; Stokes shift Open Access Beilstein J. Org. Chem. 2016, 12, 2739–2747. doi:10.3762/bjoc.12.270 Received: 27 October 2016 Accepted: 01 December 2016 Published: 14 December 2016 This article is part of the Thematic Series "Lipids: fatty acids and derivatives, polyketides and isoprenoids". Guest Editor: J. S. Dickschat © 2016 Patalag and Werz; licensee Beilstein-Institut. License and terms: see end of document. Abstract Herein, we report on the synthesis and characterization of novel fluorescent fatty acids with large Stokes shifts. Three examples consisting of the same number of carbon atoms and thus of similar chain length are presented differing in their degree of unsaturation. As major fluorogenic contributor at the terminus benzo[c][1,2,5]thiadiazole was used. Respective syntheses based on Wittig reactions followed by iodine-mediated isomerization are presented. The absorption properties are modulated by the number of conjugated C=C double bonds of the oligoene chain ranging from one to three. Large Stokes shifts of about 4900–5700 cm−1 and fluorescence quantum yields of up to 0.44 were observed. Introduction The membrane of living cells consists of a variety of lipids. More than 40 years ago, biological membranes were first described as Fluid Mosaic in which proteins were embedded [1]. During recent decades it became more and more clear that such a simple model is not sufficient to understand membrane dynamics and function. Often membrane domains are formed in which certain lipids, glycolipids or proteins are enriched [2-4]. Such domains – also called lipid rafts – do not only differ in their chemical composition, but also show different physical properties (e.g., differences in membrane thickness and stiffness, different diffusion coefficients etc.) [5,6]. Tools to investi- gate lipid membranes are multifaceted; however, all optical methods are hampered by the missing absorption and fluorescence properties of natural occurring lipid components. Therefore, indirect methods are commonly employed. Either unnatural fluorescent dyes are inserted into the membrane (e.g., pyrene) or the hydrophilic part of lipids is utilized for the covalent attachment of fluorophores. Another possibility is the use of fluorescently labelled antibodies which bind membrane components such as the carbohydrate part of glycolipids [7,8]. A further alternative is to render the lipid and especially the fatty acid part fluorescently active by the introduction of 2739 Beilstein J. Org. Chem. 2016, 12, 2739–2747. fluorescent moieties (Figure 1). Prominent examples in this area are NBD- (nitrobenzoxadiazole) [9,10], BODIPY- (borondipyrromethene) [11,12], BOIMPY- (bis(borondifluoride)-8imidazodipyrromethene) [13] and pyrene-labeled fatty acids [14]. Of course, all these alterations might also affect the membrane structure and its dynamics. While the NBD-fluorophore suffers from unsuitable polarity, a pyrene motif disrupts the unpolar membrane core with high bulkiness. BODIPY and BOIMPY scaffolds on the other hand expose fluoride residues which might be able to interact with polarized H–X bonds. Therefore, we synthesized pentaene and hexaene fatty acids which bear five or six double bonds at the terminus or in the middle of the acyl chain [15]. Their slim shape mimics the natural geometry of a saturated hydrocarbon chain and should therefore only lead to minimal disturbances [16]. Nevertheless, we found that their stability with respect to both, oxygen and strong laser beams, is relatively low. The design of novel fluorescent fatty acids is therefore a challenging tightrope walk between advantageous spectroscopic properties, overall stability and a non-interfering molecular shape. As a promising contribu- tion we designed alternative fatty acids which are constructed as a combination of double bonds and benzo[c][1,2,5]thiadiazole as a relatively unpolar terminal headgroup (Figure 1). Its electron-withdrawing strength adds on the one hand significant stability towards acidic environments and should furthermore trigger a red-shift in absorption. As another strategic goal the fluorescent fatty acids were supposed to be equipped with very similar geometrical parameters differing only in their absorption and emission wavelengths. The grade of unsaturation as the sole geometrical difference thus provides a set of probes to study the effect of rigidified ethene moieties as straight-chain alkane surrogates within biological membranes. Results and Discussion Synthesis The most prominent methods to access oligoene structures are either cross-coupling reactions [17-19] or Wittig-type reactions [20-22]. The advantage of the latter ones is that they are often conducted at low temperatures and therefore are employed for sensitive compounds. However, a drawback of Wittig reactions Figure 1: Examples for previously prepared fluorescent fatty acids and our present work. 2740 Beilstein J. Org. Chem. 2016, 12, 2739–2747. is the fact that the stereochemistry of the emerging double bond strongly depends on the type of substituent used. Aliphatic residues tend to give the (Z)-isomer. If the thermodynamically more stable (E)-isomer is needed, a subsequent isomerization has to take place. To access the benzothiadiazole (BTD) fatty acid 3 with just one conjugated double bond we made use of the Wittig reaction starting with commercially available aldehyde 1. As expected, the (Z)-isomer was the major product; thus, we performed a subsequent cis–trans isomerization with traces of iodine as catalyst (Scheme 1). It proved to be crucial to employ degassed hexane and to ensure a strict exclusion of oxygen. Considering both, the isomerization was finished just by removing the solvent while the yield of compound 3 was not hampered. For a BTD fatty acid analogue of the same length, but of more extended conjugation we made use of the Horner–Wadsworth–Emmons (HWE) reaction. Phosphonate 4 was reacted with the respective aldehyde 1. In a facile three-step one-pot process the emerging α,β-unsaturated ester 5 was immediately converted to the alcohol 6 in 87% yield in the presence of a Lewis acid and DIBAL at low temperatures. MnO2-mediated oxidation afforded the respective aldehyde that was immediately transformed by Wittig reaction. Iodine-catalyzed cis–trans isomerization yielded the desired fatty acid 7 in 35% over three steps (Scheme 2). The analogue with three conjugated double bonds was accessed by a similar route that differs only in the type of the phosphonate being employed as starting material. Since three double bonds are required a tailor-made α,β-unsaturated (...truncated)