Accelerated Hantzsch electrospray synthesis with temporal control of reaction intermediates

Article abstract of DOI:10.1039/c4sc02436b

Complex chemical reactions can occur in electrosprayed droplets on the millisecond time scale. The Hantzsch synthesis of 1,4-dihydropyridines was studied in this way using on-line mass spectral analysis to optimize conditions and characterize the product

Full text of DOI:10.1039/c4sc02436b

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Accelerated Hantzsch electrospray synthesis with
temporal control of reaction intermediates†
Cite this: DOI: 10.1039/c4sc02436b
a
a
ab
*
Ryan M. Bain, Christopher J. Pulliam and R. Graham Cooks
Complex chemical reactions can occur in electrosprayed droplets on the millisecond time scale. The
Hantzsch synthesis of 1,4-dihydropyridines was studied in this way using on-line mass spectral analysis
to optimize conditions and characterize the product mixture. Changing the distance between the
nanospray source and the MS inlet allowed exploration of reaction progress as a function of droplet
time-of-flight. Desolvation of the charged microdroplets is associated with transformation from starting
material to intermediates and eventually to product as the distance is increased. Results of the on-line
experiments require a termination step that discontinuously completes the desolvation process and
allows the generated gaseous ions to be used to characterize the state of the system at a particular time.
The intermediates seen correspond to those known to occur in the bulk solution-phase reaction. Off-
line collection of the sprayed reaction mixture allowed the recovery of 250 mg hꢀ1 of desired reaction
product from a single sprayer, permitting characterization by NMR and other standard methods. A thin
film version of the accelerated reaction is described and it could be controlled through the temperature
of the collection surface.
Received 11th August 2014
Accepted 28th August 2014
DOI: 10.1039/c4sc02436b
www.rsc.org/chemicalscience
spray conditions (on-line) and subsequently collecting product
on a surface (off-line).
Introduction
Accelerated reactions in droplets generated by electrospray
are not to be confused with the use of electrospray and other
droplet-based experiments to monitor the course of a reaction
by mass spectrometry.12–26 An important fact, not sufficiently
noted in the literature, is that under different conditions mass
spectrometry can be used either to follow the course of a bulk
phase chemical reaction or to accelerate a reaction and form
products. The key factor in selecting between conditions which
allow reaction monitoring vs. reaction acceleration is the nature
of the desolvation process: continuous desolvation favors
observation of accelerated reactions in droplets.
It will be evident from this study that accelerated reactions in
electrospray droplets must be followed by a rapid discontinuous
desolvation event (that does not promote reaction) in the ion
source. This freezes out the state of the system. The Hantzsch
reaction has been followed in bulk using electrospray ionization
mass spectrometry (ESI-MS)27,28 but here it is studied as an
accelerated reaction in individual droplets and (more briey) in
thin lms. The choice of the Hantzsch reaction for study was
made to allow comparison between reaction monitoring of bulk
reactions, which has been reported by several authors, and
accelerated reactions in droplets.
This paper is concerned with accelerated chemical reactions
occurring in charged microdroplets. Such reactions have
previously been studied using electrospray and other spray-
based ambient ionization techniques.1–5 Some of these studies
used these rapid reactions to derivatize analytes for improved
chemical analysis. In other cases, the rapid droplet reactions
were used to study the course of chemical reactions and to
identify reaction intermediates.6–8 In still other cases, the
collection and characterization of the product(s) of microscale
synthesis was of interest.9,10 For example, collection of product
in milligram quantities from a Claisen–Schmidt base-catalyzed
condensation was achieved by nanospraying a mixture of an
indanone and an aldehyde under basic conditions.11 Acceler-
ated reactions in conned volumes have been reported to occur
in desorption electrospray ionization (DESI), in dropcasting,
and in nanoelectrospray experiments. The emphasis on nano-
spray in this study, over DESI and other techniques, is because
surface interactions are removed and purely homogeneous
processes in droplets can be explored. Nanospray also allowed
high-yield experiments to be performed by simply optimizing
aDepartment of Chemistry, Purdue University, West Lafayette, IN 47907, USA. E-mail:
cooks@purdue.edu; Fax: +1-765-494-9421; Tel: +1-765-494-5263
bCenter for Analytical Instrumentation Development, Purdue University, West
The Hantzsch synthesis of symmetric 1,4-dihydropyridine
(DHP) derivatives has been of interest for over a century.29
Symmetric DHPs are synthesized from ethyl acetoacetate,
ammonium acetate, and an aromatic aldehyde, as summarized
in Scheme 1.30,31 Many DHP derivatives (9) serve as practical
Lafayette, IN 47909, USA
† Electronic supplementary information (ESI) available: Spectra for all on-line,
off-line, and substituent studies as well as photographs of experimental setups
are supplied. See DOI: 10.1039/c4sc02436b
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yield 5 and for secondary and tertiary amines only the reactants
could be detected. Experiments with various aldehydes showed
predictable results (Tables S4 and S5†).
Distance, temperature and ow-rate effects in the on-line
reaction
Increasing the distance the electrosprayed droplets must travel
to reach the mass analyzer changes the product distribution
dramatically. This shows that the reaction is dependent on
experimental variables that control the extent of desolvation. As
the distance between the ion transfer capillary and the spray
source increases, a shi occurs from starting material 2 and its
simple ammonia adduct 5 towards the Knoevenagel product 4,
its precursor 3, and the Michael addition product 7, while
eventually the DHP 9 and the pyridine 10 are seen. Species 6 is
not seen in the mass spectra, which is consistent with the low
basicity of enamines. Fig. S2 and Table S6† summarize these
distance effects on the reaction progress. Distances were varied
from 4 cm to 100 cm with approximate ight times (estimated
assuming droplets are carried along by the nebulizing gas) of
200 microseconds to 5 milliseconds, respectively. An indepen-
dent set of distance studies was performed using stainless steel
tubes of varying lengths between the spray source and MS inlet.
These tubes cause charge build up and conne the spray radi-
ally, increasing signals several fold. Product ion intensity is still
maximized as distance is increased (Fig. 1 and Table S7†). It is
clear that the extent of reaction is directly dependent on the
distance that the droplets travel.
Increasing the solution ow-rate (without adjusting other
parameters) led to a decrease in the main product 9 and an
increase in intermediates 4, 5, and 7. Working under conditions
in which the extent of reaction was limited (l ¼ 30 cm; room
temperature) and then increasing the temperature of the tube
from ambient to 80 ꢂC resulted in a decrease in intensity of the
early-stage intermediates (5 and 3) and an increase in the late-
stage intermediate (7), Fig. 2. Product was not seen for this
distance/temperature combination.
Scheme 1 Knoevenagel condensation product (4) reacts with the
enamine ester (6) via a Michael addition to form 7, which undergoes
proton transfers and intramolecular enamine formation to give 9. In a
separate step, dehydrogenation yields the pyridine (10). All species are
shown in their charged forms where appropriate.
intermediates in the generation of the corresponding pyridines
(10) through a simple oxidation of the DHP core.32–35 More
information on the importance of the reaction can be found in
the ESI.†
Results
Bulk-phase reaction and analysis
Traditional bulk-phase reactions were performed to determine
the time required for synthesis with and without catalysts, as
well as to measure yields. These reactions were monitored by
absorption spectroscopy and run both at room temperature and
under reux in ethanol, with and without phenylboronic acid
catalyst (10 mol%). Reactions conducted under reux with
phenylboronic acid were complete (ꢁ85% yield) in just under 5
hours. Reactions unaided by catalyst or reux were slower
(Table S1†) and represent the conditions of the ESI sample
before droplet creation and solvent evaporation. Samples under
reux with catalyst represent a traditional synthetic route for
the reaction.
Off-line reaction and analysis of electrosprayed material
Under optimized conditions, the accelerated nanoelectrospray
experiment yielded 250 mg hꢀ1 of the DHP product 9 from a
single sprayer, corresponding to >90% yield. Off-line quanti-
cation of 9 was performed using UV-vis absorption spectroscopy
and NMR proved valuable as an alternative form of structural
characterization (Fig. S1†). The progress of the reaction was also
monitored by TLC aer spraying the reaction mixture onto a
TLC plate.
Effect of reagent choice
The effects of changing the amine or aldehyde reagent were
investigated for both the bulk and accelerated electrospray
Hantzsch reactions. The data are given in Tables S2 and S3.†
Only ammonia or a primary amine can yield a dihydropyridine
and accordingly, both the bulk and the accelerated experiments
Fig. 1 Extent of reaction as a function of distance (l) between the MS
inlet and the spray source. The data are accounted for by desolvation
to give smaller, more rapidly reacting droplets. * denotes known
only yield product 9 when ammonia is the base; primary amines impurities thought to be from the guiding tube.
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Conversion of dihydropyridine to pyridine
The nal Hantzsch reaction product is the dihydropyridine
(DHP, 9) but conversion to the pyridine (10) can be achieved by
increasing the temperature of the ion transfer capillary (part of
the atmospheric pressure interface of the mass spectrometer).
The temperature of the ion transfer capillary was used as an
additional experimental variable: as the temperature of the
ꢂ
ꢂ
capillary was raised from 150 C to 350 C, a shi in the spec-
trum occurred (Fig. 4 and Table S9†) with conversion from
reaction product 9 to 10 by dehydrogenation. Both 9 and 10
were observed in three forms – as the [M + H]+, [M + Na]+ and
[M + K]+ ions. The conversion from 9 to 10 was also attempted
off-line by heating both the spray source and the collection
surface, however, no product was observed.
Fig. 2 Chronograms showing relative abundances of compounds 3, 5,
and 7 as the temperature is increased from room temperature to 80 ꢂC
(ꢁ3 min) at l ¼ 30 cm.
Experimental
Spray source
All experiments used the same electrospray ionization source.
The solution mixing and transfer system used three syringes
(Hamilton) and two syringe pumps (Hamilton) with different
ow rates to achieve molar ratios. High-pressure mixers were
used to ensure complete mixing. Minimizing the amount of
time aer the reagents were mixed ensured that reaction
occurred in the course of electrospray and not in the syringe.
Many of the electrospray parameters had to be optimized
including ow rates (gas and reagent), as summarized in Table
S10.† Variables such as reagent and gas ow rate were crucial in
controlling the rate of desolvation, as indicated above. All
reagents were purchased from Sigma-Aldrich (Milwaukee,
Wisconsin).
Distance and temperature effects in the off-line reaction
Experiments were also performed with off-line collection of
droplets and subsequent product analysis. In these experi-
ments, the sprayer can be placed very close to the deposition
surface where it generates a thin lm of solution. At room
temperature only product 9 was observed, while in an analogous
experiment performed with the surface cooled to ꢀ77 ꢂC or ꢀ17
ꢂC no products were seen. Greater sprayer distances did yield
product at these reduced temperatures, because some reaction
occurs in the gas-phase droplets. A plot of the normalized
relative intensity of each species present over a range of
distances is shown in Fig. 3 (see Table S8 for mass spectra†).
Simple dropcasting experiments showed no reaction, illus-
trating the contribution made by small electrospray droplets to
reaction.
Fig.
3
Progress of the off-line cold surface reaction at various Fig. 4 On-line reaction (l ¼ 100 cm) at various ion transfer capillary
distances between the ESI source and the collection surface under temperatures. Spectra on the right are expansions showing 9 and 10.
conditions where reaction at the surface is minimized by operating at Note that 9 and 10 occur as protonated, sodiated, and potassiated
low temperature.
forms. The signal at m/z 282 is due to background.
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Complete desolvation occurs independently of the extent of
prior (droplet-phase) reaction. This explains how the forma-
tion of product can be directly dependent on distance: reac-
tions occur only during the uninterrupted period of slow
(millisecond) droplet desolvation. By contrast with these
accelerated reactions in evaporating droplets, experiments
on reaction monitoring27,28 are performed using conditions
which do not allow this time for desolvation/reaction before
discontinuous desolvation occurs, converting the sampled
reaction mixture into gas-phase ions and allowing bulk
reaction monitoring.
On-line analysis of electrosprayed droplets
The charged droplets emerging from the spray source were
directed pneumatically and electrically towards the inlet capil-
lary of an ion trap MS (Thermo-Finnigan LTQ). Variables
including the distance between the spray source and the ion
transfer capillary, the capillary temperature, the glass/metal
capillary length, the gas ow rate and the reagent ow rate were
controlled as they lead to large changes in reaction yield. Table
S10† gives the optimized values of these parameters.
Off-line collection of electrosprayed droplets
The ndings of the off-line distance studies show that the
distances required to yield product under cold collection
conditions are signicantly greater than those when product
is collected under ambient conditions. These data suggest
that a second factor contributes to reaction rate enhance-
ment in conned volume solutions. In addition to des-
olvation in microdroplets, desolvation from thin lms on
collection surfaces must also contribute. The rate enhance-
ment effects occurring in droplets are shown by the distance
effects of both the on-line and the cold collection off-line
experiments. The lack of dependence on distance in the
ambient off-line collection at room temperature suggests that
the reaction is accelerated all the way to product 9 in the thin
lm conned volume on the surface, while the distances are
such that there is little opportunity for reaction in the evap-
orating droplet.
The spray source was housed in an airtight poly(methyl meth-
acrylate) box to minimize release of solvent vapor into the air
and to provide the ability to explore moisture- and air-sensitive
reactions (photographs in ESI†). Spray variables were those that
optimized the on-line reaction, except that the effects of
distance were systematically explored. Product collection was
performed at room temperature on a grounded colander sitting
atop a rotating beaker to increase surface area. Experiments
were done under ambient conditions or using temperature-
controlled surfaces (grounded copper foil cup oating atop a
ꢂ
dry-ice/2-propanol bath, ꢁꢀ77 C and a dry-ice/ethylene glycol
ꢂ
bath, ꢁꢀ17 C) to explore the role of conned volumes on the
surface. Off-line mass spectral analysis showed that product
ꢂ
ꢂ
formation decreased at ꢀ77 C/ꢀ17 C vs. room temperature.
Quantitative and structural analysis of the collected spray
product (as well as the products of the bulk reactions) was
performed using a Cary 100 UV-vis spectrophotometer (l ¼ 378
nm, molar absorptivity 5.2 ꢃ 103 L molꢀ1 cmꢀ1
)
and NMR
36
Conclusion
(Bruker ARX 400 with 5 mm QNP probe), respectively [1H NMR
(CDCl3): d 1.26(6H), 2.32(6H), 2.33(3H), 4.08(4H), 4.99(1H),
5.67(1H), 7.20(5H)]. TLC analysis of the spray product was done
by directly spraying onto a TLC plate with a ꢁ1 mm diameter
hole cut in a piece of paper in between the sprayer and the plate
(ꢁ2 cm) to dene the spot size.
An ESI-based droplet method was used to compare the accel-
erated Hantzsch synthesis in sprayed droplets with the parallel
bulk-phase reactions. On-line chemical analysis in real-time
allowed optimization of the sprayer conditions so that slow
desolvation and its accompanying accelerated reaction could
occur before the sudden and discontinuous desolvation event
in the ion source. The on-line experiments allow the explora-
tion of reaction progress as a function of distance between the
sprayer and the ion transfer capillary. This distance controls
the progress of the reaction and the degree of desolvation of
the droplets. The discontinuity in desolvation before mass
analysis in the on-line distance experiments requires a sudden
completion of desolvation with release of free gas-phase ions
and truncation of reaction. This event occurs in the atmo-
Bulk-phase reactions
Bulk-phase reactions were performed for comparison with
reactions occurring in the electrosprayed droplets. These reac-
tions were run at both 10 mM in ethanol and as specied by
Debache et al.37 Both experiments yielded similar data.
Discussion
Occurrence of the same intermediate species in the bulk- spheric pressure interface or ion source. Accelerated reactions
phase Hantzsch reaction and the accelerated reaction also occur in thin lms and they can be suppressed by control
occurring in droplets establishes the underlying similarity in of the surface temperature. The experiment as a whole illus-
reaction mechanism. Exploration of both distance and trates the role both types of conned volumes (desolvating
temperature effects for the electrospray reaction emphasizes microdroplets and thin lms) have in accelerating reaction
the role that droplet desolvation plays in reaction progress on progress. Off-line experiments using charged droplets yield
the millisecond timescale. A particularly important nding is product for this reaction at rates as high as 250 mg hꢀ1. The
that the reported observations require a discontinuity in the study explains the apparently contradictory fact that the same
droplet desolvation process. This discontinuity in the evap- process – droplet formation, evaporation and ion mass anal-
oration of solvent must occur in the MS inlet with sudden ysis – can be used to monitor the course of a bulk-phase
conversion of the solvated ions in the droplets into free gas- reaction or to accelerate reactions of the same reagents in
phase ions without contributing to reaction acceleration. microdroplets.
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