Fischer Indole Synthesis in the Gas Phase, the Solution Phase, and at the Electrospray Droplet Interface
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Previous reports have shown that reactions occurring in the microdroplets formed during electrospray ionization can, under the right conditions, exhibit significantly greater rates than the corresponding bulk solution-phase reactions. The observed acceleration under electrospray ionization could result from a solution-phase, a gas-phase, or an interfacial reaction. This study shows that a gas-phase ion/molecule (or ion/ion) reaction is not responsible for the observed rate enhancement in the particular case of the Fischer indole synthesis. The results show that the accelerated reaction proceeds in the microdroplets, and evidence is provided that an interfacial process is involved.
KeywordsElectrospray Accelerated reactions Fischer indole synthesis
In the course of spray-based ionization, such as desorption electrospray ionization (DESI)  or paper spray ionization , derivatization reactions can be performed in order to improve the limits of detection for compounds that are difficult to ionize [3, 4, 5]. This is done during DESI by adding a reagent to the primary spray solvent, which desorbs the analyte from the surface and into the secondary droplets, where the reaction occurs during passage to the mass spectrometer (MS) . In paper spray ionization, reactions are often performed by adding reagents to the paper substrate prior to analysis, and the reaction either occurs in the thin film on the paper or in the droplets after they are emitted from the paper tip [7, 8, 9, 10]. Derivatizing reagents for ambient ionization mass spectrometry, organized by specific functional group, have been cataloged in a review article .
Electrospray ionization mass spectrometry (ESI-MS) is gaining recognition as a robust tool for reaction monitoring [11, 12, 13, 14, 15] and, alternatively, as a method for rapid screening of reactions to establish whether products are generated [16, 17]. In the first application, reaction products are simply sampled from the reaction mixture, whereas in the latter, products can be generated during the ESI event. Different conditions (initial droplet size, solvent, concentrations of reagents, droplet flight time, etc.) control these two particular outcomes. Numerous reactions (a score or so) have been found to have significantly faster rates in ESI droplets when compared with the corresponding bulk-phase reactions, and a number of these reactions have been cataloged in recent review articles [12, 18]. Amongst these accelerated reactions are quinolone and isoquinolone synthesis , Hantzsch synthesis of 1,4-dihydropyridines , hydrazone formation [6, 21], and Claisen-Schmidt condensation [22, 23]. The studies of the effects of pH , concentration  and surface activity  (a description of the likelihood of a given species being at the surface of a droplet) in small droplet systems suggested that the underlying cause of reaction rate acceleration lies in surface effects. Studies using acoustically levitated droplets , Leidenfrost levitated droplets , microfluidics , thin films , and “on water” chemistry [30, 31] have helped characterize acceleration phenomena in confined volumes.
It is known that the pH at the surface of electrosprayed droplets decreases [32, 33, 34] during the electrospray ionization (ESI) process. Droplets likely undergo desolvation and fission during their flight time [35, 36, 37, 38]. Correlations between surface activity and reaction acceleration in ESI support the hypothesis that reaction acceleration is connected with surface activity [39, 40]. The main models of ESI for small molecules (the charge residue and ion evaporation models [36, 39, 40, 41]) suggest that at some point in the process of ionization, surface active molecules exist in a partially solvated form. In the charge residue model, solvent molecules continuously evaporate to yield dry ions, so there is presumably a point in the process when ions are partially solvated. In the alternative ion evaporation model, ions exist at the surface of charged droplets, where they are thought to be partially solvated at the air–droplet interface; dry ions are subsequently ejected by Coulombic forces . In some cases, it has been shown that the acceleration of reactions in electrospray droplets requires that the distance between the nESI emitter and the ion transfer capillary be increased well beyond normal operating distances [20, 21, 24]; therefore, observed acceleration factors in chemical reactions under these conditions are thought to be due to reactions of partially solvated ions (increased distance between the nESI emitter and ion transfer capillary corresponds to more time for droplet evaporation as well as time for reaction). However, a gas-phase reaction mechanism is also possible since gas-phase ion/molecule reactions are much faster than solution phase reactions .
All reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) unless otherwise specified. Reactions were prepared by combining 10 μL phenylhydrazine (Eastman, Kingsport, TN, USA), 200 μL acetone, and 50 μL 1.0 M HCl in methanol (prepared by diluting 37% HCl (Mallinckrodt, St. Louis, MO, USA) in methanol to 1.0 M). An excess of acetone was used unlike a standard Fischer indole synthesis. The excess acetone allows the second (and, upon additional reflux of 48 h, the third) acetone addition(s), which compete with and prevent the formation of the standard methyl indole product (4).
Results and Discussion
Collision-Induced Dissociation of 3b
The role of a possible gas-phase reaction in the open air (prior to entering the ion transfer capillary) was then explored. This was done by allowing acetone and phenylhydrazine vapor to mix from two separate, open vials. A corona discharge was struck between a platinum electrode and the ion transfer capillary to facilitate atmospheric pressure chemical ionization (APCI). The full scan mass spectrum showed 3 as the dominant species, indicating that the gas-phase reaction produces only this product. The absence of ions 5 and 6 even in the more selective MS/MS mode is noted. A product ion scan from isolated 3b produces methylindole (4) by elimination of ammonia as expected.
Solution Phase Imine/Enamine Formation
Compound 7 has previously been reported as a product of thermal activation of an intermediate species in the reaction of phenylhydrazine and acetone  when nebulized by electrospray; however, the phase in which it forms (solution/gas phase) had not been delineated. This work shows that a solution-phase mechanism was likely to be responsible for the formation of the precursor ion that fragments to produce 7 in the previous literature.
Reaction Acceleration in Charged Droplets
In an effort to probe reaction at the surface of the droplet, two different surfactants (triton x-100 and pentadecanoic acid) were doped into the reaction mixture. Fresh reaction mixtures were prepared and surfactant was added to the mixture at 1% v/v before analysis. The spectra acquired in the presence of surfactant were of lower quality and contained many interfering peaks; however, the peaks of interest were significant and reproducible in the MS full scan. Surfactants reduced the surface tension of the droplet, creating smaller droplets sooner . This is because the Rayleigh limit (the point at which droplets undergo fission) is reached faster as the surface tension is reduced. Surfactants decrease surface tension and the distance required to achieve equal acceleration effects without surfactants falls from 7 to 3 cm. Interestingly, as the distance was increased further (to the 7 cm distance, which yielded acceleration without surfactant, the spectrum recorded using surfactant was again dominated by 3. This is interpreted as indicating a contribution of a gas-phase reaction to the observed mass spectrum, as the droplets were allowed to completely desolvate, yielding dry reagent ions, which produced the gas phase product, 6. This result was confirmed to be an artifact of the spray process and not due to bulk-phase reaction by returning the sprayer to the 3 mm distance and repeating the distance experiment and acquiring the same result as previously discussed. This observation is significant as it bolsters the hypothesis that the observed acceleration does indeed proceed via a solution-phase mechanism.
The formation of reaction products 5 and 6 can be accelerated by increasing the distance between the nESI emitter and the ion transfer capillary. As this variant of the Fischer indole synthesis forms a different final product in solution as opposed to gas phase, it has been shown that the reaction accelerated in ESI favors the solution-phase product over the ion-molecule gas-phase products. When surfactants are added, acceleration can be achieved at a smaller distance between the ion transfer capillary and the nESI emitter compared with the reactions accelerated without surfactants, pointing to the importance of surface reactions.
The authors acknowledge the financial assistance of the National Science Foundation (CHE-1307264).
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