Microwave Synthesis of Quaternary Ammonium Salts

The microwave synthesis of several quaternary ammonium salts is described. The synthesis provides comparable or better yields than published methods with reduced reaction times and in the absence of solvent.


Introduction
Fluorescence spectroscopy has become a key technique for the detection and elucidation of biological processes. In particular, cyanine dyes see widespread application as fluorescent probes. They have been used in DNA sequencing, immunoassays, agarose gel and capillary electrophoresis staining [1], DNA analysis in polymerization chain reactions [2,3], in flow cytometry [4], or as fluorescent probes for membrane fluidity [5,6] and membrane potential studies [7]. However, common problems associated with cyanine dyes include their tendency to undergo photobleaching [8], and selfaggregation [9]. This has prompted the development of novel cyanine dyes with increased photostability, Stokes' shift, and quantum yield for use in bio-applications.
Cyanine dyes have traditionally been prepared from a condensation of an N-alkyl heterocyclic quaternary ammonium salt and a bisimine or bisaldehyde (Scheme 1) [9]. N-Alkyl quaternary ammonium salts are used extensively as precursors of near-IR spiropyrans [10] and various cyanine OPEN ACCESS dyes [11]. The salts are synthesized by refluxing reagents with solvents such as chloroform, odichloro-benzene, acetonitrile and ethanol for 6 -48h. One example requires refluxing in acetonitrile for 24 h, then treatment with diethyl ether followed by filtration. The combined filtrates are concentrated and refluxed for an additional 24 h, treated with diethyl ether and filtered [12]. This process was repeated 1-3 times to achieve the published yields (25 -78%). Another method heats the reagents at 80 ο C for 21 h in an ampule tube sealed with a torch [10]. Purification of the salts range from Soxhlet extraction with benzene for 24 h [13] to filtration with cold ether [12]. Scheme 1. General synthesis of heptamethine cyanine dyes.
A simple efficient microwave synthesis of N-alkyl quaternary ammonium salts has now been developed. Reaction times are measured in minutes as opposed to hours and all of the experiments are performed under solvent-free conditions.

Results and Discussion
The reaction of 2,3,3-trimethylindolenine with an array of alkyl halides with varied functionality were studied. The reactions were performed by charging each microwave reaction vial with of 2,3,3trimethylindolenine and an alkyl halide (Scheme 2). Our previously published reaction of ethyl iodide with 2,3,3-trimethylindolenine served as the model system [14]. The microwave reaction conditions were determined using a single-mode microwave system. The temperature was monitored throughout each reaction. The optimized reaction condition was 130  ο C, ramp time: 2:50 min, reaction time: 5:00 min giving a 95% yield.
The scope of the reaction was examined with the coupling of 2,3,3-trimethylindolenine and benzothiazole with iodomethane, iodopropane, bromoethanol, and bromohexanoic acid. The hold time, ramp time, and temperature for each electrophile was studied. The optimized reaction conditions are presented in Table 1. In most cases, the yields were comparable or exceeded the published yields. Most significant is the substantially decreased reaction time and simplicity of the reaction procedure. The yields presented are the yields without resubjection of the filtrates.

Work up and purification
Alkyl salts 1, 2, 3, 6, and 7 were simply filtered and washed with cold ether. The products were pure by NMR analysis and no further purification was necessary. Salts 4, 5, 9 and 10, which all contain hydroxyl groups, did not crystallize right away. The reaction solution containing 4 was concentrated followed by the addition of hexanes. The solution was heated until crystals formed and then filtered. Similarly, salt 5 was recrystallized from acetone. Reaction vials containing 9 and 10 were allowed to sit at room temperature for 2-4 h, after which time crystals formed and could be filtered.

Conclusions
The single mode microwave system has provided substantially decreased reaction times, simplicity of reaction procedure, and comparable or increased reaction yields observed for reactions conducted.

General
All microwave reactions were conducted using the single-mode Biotage Initiator 2.0 (www.Biotage.com). 1 H-and 13 C-NMR spectra were obtained in DMSO-d 6 using a Bruker Avance 400 MHz NMR and were recorded at 400 MHz and 100 MHz, respectively. All reagents and chemicals were obtained from Aldrich Chemical Company (USA) and Alfa Aesar and were used as received.   (5)