High-Throughput Experimentation and Continuous Flow Evaluation of Nucleophilic Aromatic Substitution Reactions

Article abstract of DOI:10.1021/acscombsci.9b00212

Nucleophilic aromatic substitution (S_NAr) reactions were optimized using high-throughput experimentation techniques for execution under flow conditions. A total of 3072 unique reactions were evaluated with an analysis time of ～3.5 s per reaction using a system that combines a liquid handling robot for reaction mixture preparation with desorption electrospray ionization (DESI) mass spectrometry (MS) for analysis. The reactions were performed in bulk microtiter arrays with and without incubation. In-house developed software was used to process the data and generate heat maps of the results. This information was then used to select the most promising conditions for continuous synthesis under microfluidic reactor conditions. Our results show that this HTE approach provides robust guidance for narrowing the range of conditions needed for optimization of S_NAr reactions.

Full text of DOI:10.1021/acscombsci.9b00212

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Research Article
High-Throughput Experimentation and Continuous Flow Evaluation
of Nucleophilic Aromatic Substitution Reactions
́
Zinia Jaman, David L. Logsdon, Botond Szilagyi, Tiago J. P. Sobreira, Deborah Aremu, Larisa Avramova,
R. Graham Cooks,* and David H. Thompson*
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ABSTRACT: Nucleophilic aromatic substitution (SNAr) reactions were
optimized using high-throughput experimentation techniques for
execution under flow conditions. A total of 3072 unique reactions were
evaluated with an analysis time of ∼3.5 s per reaction using a system that
combines a liquid handling robot for reaction mixture preparation with
desorption electrospray ionization (DESI) mass spectrometry (MS) for
analysis. The reactions were performed in bulk microtiter arrays with and
without incubation. In-house developed software was used to process the
data and generate heat maps of the results. This information was then
used to select the most promising conditions for continuous synthesis
under microfluidic reactor conditions. Our results show that this HTE
approach provides robust guidance for narrowing the range of conditions
needed for optimization of SNAr reactions.
KEYWORDS: high-throughput experimentation, nucleophilic aromatic substitution, flow chemistry,
desorption electrospray ionization mass spectrometry, DESI-MS
transferred into the mass spectrometer by secondary droplets
that are generated from small splashes of continuously
incoming electrospray droplets. The DESI-MS inlet is then
rastered over the surface using a moving x−y stage to generate
a 2D map of chemical information in the form of full mass
spectra. The thin films and droplets generated in the DESI-MS
process can lead to reaction acceleration.13 This has been
demonstrated previously with amine alkylation,9,14 Suzuki
coupling,15 and aldol reactions.16 In this study, DESI-MS was
also used to analyze the results of the bulk microtiter reactions
run at elevated temperatures that are not easily achieved in the
DESI-MS format. Because the analysis occurs at a rate of ∼3.5
s per reaction mixture, DESI enables a far more rapid
evaluation of reaction outcomes than other techniques, such
as LC-MS, that typically take several minutes for each sample.
It is important to note that we will refer to the droplet/thin
film reactions as desorption electrospray ionization (DESI)
reactions in this Research Article. This is not to be confused
with the DESI-MS analysis of the bulk microtiter reactions.
INTRODUCTION
■
High-throughput experimentation (HTE) allows for the
implementation of large numbers of experiments in parallel,
requiring less labor per experiment,1 and utilizing small
amounts (picogram−nanogram) of material. This technique
can boost lab productivity by rapid generation of compre-
hensive data for the selected transformation.1,2 HTE-based
experiments focused across a range of variables have spread in
biology, drug discovery,3 medicinal chemistry,4,5 and catal-
ysis.6,7 Analysis of the resulting large data sets to extract a
deeper understanding of the chemical transformation, however,
can be a bottleneck in the discovery process. The identification
and optimization of reaction conditions prior to chemical
process development can be accelerated when HTE is coupled
with MS analysis.6,8,9 These impacts are particularly evident in
the pharmaceutical and biopharmaceutical industries where
reduction in the time required to execute each experimental
cycle is a necessity due to the high value of these product
classes.10,11 The HTE method reported herein are based on
two techniques to identify promising reaction conditions for
scale up: (i) desorption electrospray ionization mass
spectrometry (DESI-MS) and (ii) bulk microtiter (small
scale batch) reactions.
Received: December 10, 2019
Revised: February 17, 2020
DESI-MS is an ambient ionization technique,12 wherein
electrospray droplets are directed onto a surface generating a
thin film of solvent (∼300 μm in diameter). Analytes present
on the surface desorb from this thin film and are then
https://dx.doi.org/10.1021/acscombsci.9b00212
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Scheme 1. Reaction Formats Employed in This Study
a
Scheme 2. Reagents Used in Round 1 of the HTE Campaign
a
R1 refers to round 1 of the HTE test. A# refers to the amine type, while B# refers to the electrophile type.
Nucleophilic aromatic substitution (SNAr) reactions are
versatile transformations in the organic chemistry arsenal,17
and one of the important reactions used for making
pharmacologically18,19 and biologically active molecules.20−25
B
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a
Table 1. Direct Comparison of SNAr Reactions Using Droplet/Thin Film and Microtiter Approaches
a
(A) The droplet/thin film and (B) bulk microtiter results for the same set of reaction conditions. Experimental conditions: DESI solvent,
methanol; reaction solvent, NMP; base, DIPEA. (C) Same as panel B, but the DESI spray solvent was MeOH+ 1% FA. (D) Same as panel C, but
the reaction solvent was 1,4-dioxane. Each cell is an average of two data points. Green cells represent “yes” reactions (product ion intensity ≥ 150
counts). Red cells represent “no” reactions (product intensity < 150 counts). B12 can form both single and double addition products; the double
addition product can form multiple ions. B12(S) is the singly charged ion of the single addition product, while B12(D) is the sum of the average
intensities of all the double addition ions.
The reaction mechanism of this transformation26,27 involves a
stepwise addition−elimination sequence,28−30 wherein the first
step involves a nucleophilic attack of the substrate to provide a
Meisenheimer complex, followed by the loss of the leaving
group through either catalyzed or noncatalyzed pathways.30−32
The reaction typically involves an amine as the nucleophile,26
although a wide variety of non-nitrogenous nucleophiles may
be used. This study reports HTE evaluation of SNAr reactions
performed in both droplets/thin film and bulk microtiter
formats with analysis by DESI-MS. After HTE evaluation,
validation of the reaction hotspots were performed in flow to
increase confidence in the HTE findings (Scheme 1).
Microfluidic reactions are attractive alternatives for organic
synthesis, since continuous flow methods have shown great
potential to achieve faster and greener transformations,33
including the microfluidic synthesis of active pharmaceutical
ingredients using ESI-MS analysis.34−36 Although the SNAr
reaction is already known in flow,37 we selected a broader
range of substrates for this study in an effort to develop
efficient flow-enabled routes to biologically as well as
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Figure 1. Heat map of 1536 reactions from round 1 of the SNAr HTE using MeOH with 1% FA as DESI spray solvent under droplet/thin film or
bulk microtiter plate conditions at 150 °C for 15 h using four different bases. (A) Reaction solvent, NMP; (B) reaction solvent, 1,4-dioxane. Green
cells represent successful reactions (average product intensity ≥ 150 counts). Red cells represent unsuccessful reactions (average product intensity
< 150 counts).
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pharmaceutically important synthons.15 The preparation of
SNAr reaction mixtures for both the DESI and microtiter HTE
methods was performed in glass-lined 96-well metal plates
using 16 different amines and 13 different aryl halides.
Additional variables, including base, reaction solvent, DESI
spray solvent, temperature, and reaction time, were also
evaluated.
ing (CHRIS) to produce heat maps of the reaction
outcomes.39
We performed two rounds of HTE, differing primarily in the
amine nucleophiles used. A subset of the data from round 1 is
shown in Table 1. Each square in Table 1 represents a unique
reaction condition using DIPEA as base and is an average of
two separate reaction replicates. The peak intensities of the
products in the corresponding full scan mass spectra were used
to evaluate the success or failure of each reaction. A successful
reaction was defined as having a product peak intensity of at
least 150 counts (S/N ∼ 5) in the centroided mass spectrum.
Twelve successful reactions were found for the droplet
reactions, whereas 41 successful reactions were found for the
bulk microtiter transformations (Table 1A and B). There was
no doubt that some of the reactions were favorable under
droplet conditions, however, SNAr reactions typically require
heating,40 so it was not surprising that more “yes” reactions
were observed under the heated bulk reaction conditions
employed. Moreover, MeOH with 1% FA was found to be a
better spray solvent than MeOH due to better product
ionization in the presence of acid41,42 (Table 1C). This change
increased the number of successful reactions detected by 30%
(54 count). It is also worth noting that the reaction worked
better in NMP than 1,4-dioxane (54 “yes” reactions vs 40 “yes”
reactions) (Table 1C and D) since NMP is much more
polar.38 We attribute these finding to the stabilization of the
sigma complex intermediate produced after the addition step.
Figure 1 shows the heat map of the round 1 reactions in
both DESI and bulk microtiter format using MeOH with 1%
FA as the spray solvent. In general, electron-donating groups
(EDG) in the amine nucleophile and electron withdrawing
groups (EWG) in the aryl halide substrate favored product
formation.26,27 For these experiments, the most reactive amines
were 1-methylpiperazine (R1-A4) and 3-(2-methylpiperidin-
yl)propan-1-amine (R1-A7), both of which possess electron-
donating groups. Similarly, a strong electron-withdrawing nitro
group in the electrophile (e.g., 1-fluoro-4-nitrobenzene (R1-
B4), 1-chloro-4-nitrobenzene (R1-B5), 1-bromo-4-nitroben-
zene (R1-B6), and 2,4-dichloro-5-nitropyrimidine (R1-B12))
increases the reaction extent for these aryl halides, whereas 4-
bromo-N,N-diethylaniline (R1-B11) did not work well
because of the diethyl amine electron donating group. All
other aryl halides reacted to the same extent. Ortho
substituents in the amine nucleophile or aryl halide substrate
retarded the reaction due to steric hindrance.43 Thus, pyridine-
2,3-diamine, R1-A6, or 2,4-dichloro-5-nitropyrimidine (R1-
B12) did not react well, although (1R,2R)-cyclohexane-1,2-
diamine (R1-A1) reactions were facile since the two adjacent
amino groups are on different faces of the cyclohexane ring. 1-
Methyl-1H-imidazole (R1-A2) did not react (see Tables SI1−
4 for the ion intensities observed in these experiments).
In spite of the structural similarities of piperidine (R1-A3)
and 1-methylpiperazine (R1-A4), R1-A4 always showed better
reactivity than R1-A3 because of the presence of an electron
donating group (EDG) that enhances its nucleophilicity.
Moreover, benzylamine (R1-A5), pyridine-2,3-diamine (R1-
A6), and benzimidazole (R1-A8) showed lower reactivity due
to the presence of an electron-withdrawing aromatic moiety in
these molecules, making them less nucleophilic toward the
addition step in the reaction mechanism.
RESULTS AND DISCUSSION
■
High-Throughput Experimentation (HTE). Two high-
throughput experimentation methods were tested for reaction
evaluation: DESI at room temperature (transformations in
droplets/thin films, used for both synthesis and analysis) and
bulk microtiter plates at elevated temperature (DESI for
analysis only). N-Methyl-2-pyrrolidone (NMP) and 1,4-
dioxane were chosen as the polar aprotic solvents for these
reaction because all the reagents dissolved in these solvents.38
DESI-MS analysis allowed rapid investigation of reactions that
were capable of producing a diverse product profile. The spray
solvents were MeOH or MeOH with 1% formic acid (FA).
Full mass spectra in positive mode were recorded for each
reaction mixture.
Eight amines and 12 aryl halides (Scheme 2) were tested in
the first experiment using different bases in NMP and 1,4-
dioxane. The bulk microtiter HTE reactions were heated for
SNAr reaction product formation at 150 °C for 15 h.
The amine and aryl halide ratios used were 1:1, and the
different bases were used at 2.5 equiv relative to aryl halide. A
Beckman-Coulter Biomek i7 liquid handling robot was used to
prepare the reaction mixtures in either 96-well glass-lined
metal blocks or 384-well plates. A total of 400 μL of the
reaction solution was prepared in each well of the 96-well
plates. Four identical 96-well plates were prepared, each
utilizing one of four different base conditions: N,N-
Diisopropylethylamine (DIPEA), sodium t-butoxide
(NaOtBu), triethylamine (TEA), and no base (control).
Each reaction mixture (40 μL) was then transferred to a
384-well plate (i.e., aliquots from all four 96-well glass-lined
metal plates deposited into one 384-well plate), followed by
transfer of 50 nL of each reaction mixture in the 384-well plate
to a PTFE surface (a porous polytetrafluoroethylene sheet
glued onto a glass support) using a 384-format stainless steel
pin tool. This final transfer is necessary because the reaction
mixtures must be on a planar surface to enable DESI-MS
analysis.
Up to 16 384-well plates can be pinned onto one PTFE
surface (also referred to as the DESI slide) by slightly offsetting
the location of the pins relative to the surface during each
transfer as described by Wleklinski et al.,9 resulting in a total of
up to 6144 spots per DESI slide, including a collection of
rhodamine spots that serve as fiducial markers. It is important
to note that these reaction mixtures were placed onto the
surface prior to any incubation steps for evaluation of the
droplet/thin film reactions. The remaining reaction solutions
in the metal blocks were heated for 15 h at 150 °C to initiate
the bulk reactions. After heating, the well plates were cooled,
and samples of the reaction mixtures were transferred to the
PTFE surface using the same procedure described above.
These thermally activated bulk microtiter reaction mixtures
were spotted onto the same PTFE surface as the nonincubated
mixtures to enable direct comparison. The PTFE surface was
then analyzed using DESI-MS and the MS data analyzed using
in-house software called Chemical Reaction Integrated Screen-
The summary of successful SNAr reactions detected upon
analysis of 1536 unique droplet/thin film and bulk reactions is
shown in Table 2. Among all the reactions, 311 “yes” reactions
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Table 2. SNAr Product Formation Outcomes for 96 Reactions As a Function of Reaction Solvent, Base Type, and DESI-MS
Spray Solvent
Scheme 3. Reagents Used in Round 2 of the HTE Campaign
were observed in bulk, while less than half that number were
observed (153) when run in the droplet/thin film format.
Our initial HTE campaign was followed by a second round
of SNAr reactions using a family of biologically active amine
synthons. Transformations in this round employed the same
aryl halides (except that R1-B10 was exchanged for 1-bromo-
4-(trifluoromethyl)benzene, R2-B10) with a different set of
eight amines (Scheme 3). The reaction conditions for round 2
were similar to round 1, except that (i) all reactions were
performed in NMP, (ii) time points of 1, 4, and 15 h at 150 °C
were used for the bulk mictotiter reactions, and (iii) methanol
with 1% FA was the only DESI spray solvent used.
Figure 2 shows the heat maps from round 2. Unfortunately,
this set of reactions did not work well because most of the
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Figure 2. Heat map of 1536 reactions (768 in droplet/thin film and 768 in bulk microtiter at three time points) from round 2 of the SNAr HTE
using MeOH with 1% FA as the DESI spray solvent and NMP as the reaction solvent. (A) Droplet/thin film and bulk microtiter at 150 °C. (B)
Droplet/thin film and bulk microtiter at 200 °C. Green cells represent successful reactions (average product intensity ≥ 150 counts). Red cells
represent unsuccessful reactions (average product intensity < 150 counts).
amines were more electron-deficient than the round 1 amines,
thus making them less nucleophilic toward the addition step.
Only thiophen-2-ylmethanamine (R2-A5) and 2-morpholinoe-
than-1-amine (R2-A6) were comparatively better than the
other amines because of their electron donating moiety. Again,
R1-B4, R1-B5, R1-B6, R1-B7, and R1-B12 worked better due
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a
Table 3. Summary of Product Outcomes (96 reactions) Round 2
DIPEA
bulk
NaOtBu
TEA
no base
bulk
DESI
DESI
bulk
DESI
bulk
DESI
time (h)
150 °C
200 °C
1
8
8
4
9
9
15
6
6
1
8
3
4
7
2
15
7
3
1
11
9
4
11
7
15
9
7
1
11
8
4
9
7
15
6
6
4
7
4
4
7
5
3
6
a
The bulk reaction results are reported for three different time points. Here, droplet experiments were replicates that were done in two different
days.
to the presence of their strong electron-withdrawing nitro and
chloro groups.
positives in droplet/thin film but negatives in bulk microtiter.
This later case is also misguiding, since these positive droplet
reactions outcomes do not translate as positives under bulk
reaction conditions. Possible reasons for these differences are
summarized in Table 4.
Table 3 summarizes round 2 of the SNAr HTE reactions for
both the DESI and bulk microtiter formats at different time
points. Only 18 reactions worked in DESI at 150 °C, whereas
38 reactions worked in bulk after 1 h heating. Efforts to push
the reaction at higher temperatures and longer times (200 °C
for 15 h), did not improve the outcome (Figure 2B). Heating
helped to promote reactions with the most reactive aryl halides
(R1-B1 through R1B7), with the most reactive amine being 2-
morpholinoethan-1-amine, R2-A6; however, very high heating
appeared to promote product degradation. Since most of the
amines were much less nucleophilic, higher reaction temper-
atures did not help the reaction even after prolonged reaction
times at higher temperature (see Tables SI1−4 for detailed
peak intensity values).
Table 4. Possible Reasons for Discrepancy between
Droplet/Thin Film and Bulk Microtiter Reaction
Observations
mechanism
from
to
result
false
thermal degradation in bulk
Q2 Q4
positive
false
positive
false
negative
incomplete bulk reaction (by reaction time or Q2 Q4
temperature)
thermodynamic control in bulk
Q3 Q1/
Q2
Correlation Between DESI and Bulk Reactions. Figure
3 depicts the product intensities in the heated bulk microtiter
Round 2, containing bulk microtiter reaction data at two
temperatures and three reaction times, enabled a more detailed
comparison of droplet/thin film and bulk microtiter reactions.
It also reveals why there are conflicting findings in reaction
outcomes (i.e., Q1 and Q4). For a bulk microtiter reaction, the
reaction time and temperature are among the most important
variables; however, the droplet/thin film reaction format
cannot reliably approximate these conditions. For example,
reaction A (Figure 4) showed only positive hits at 150 °C, but
only negatives at 200 °C, whereas the opposite behavior is
Figure 3. Correlation plot for the comparison of droplet/thin film and
bulk data from 831 unique SNAr products in round 1. Q1: 186 points.
Q2: 124 points. Q3: 491 points. Q4: 30 points.
reactions of round 1 as a function of product intensities
obtained in DESI. This correlation plot gives a visual
representation of the agreement between the droplet/thin
film and bulk reactions. Because a comprehensive statistical
correlation analysis is beyond the scope of this study, this
figure shows that a simple threshold intensity analysis provides
a binary “yes”−“no” information space and splits the
correlation plot into four important quadrants. Q2 and Q3
are the regions of good agreement between droplet/thin film
and bulk microtiter outcomes. The reactions in Q1 are
positives in bulk microtiter and negative in droplet/thin film.
In the context of droplet reaction-guided bulk reaction design,
these reactions would have been missed. Q4 points are
Figure 4. Two representative examples of heated bulk reaction
outcomes showing the negative impact of thermal degradation
(reaction A) and the positive effects of heating (reaction B). Reaction
A: R2-A6, R1-B12(D); base, NaOtBu in NMP. Reaction B: R2-A6,
R1-B3; base, DIPEA in NMP. The dashed lines are only to guide the
eye.
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observed in the case of reaction B. Hence, in 50% of these bulk
reactions, there is a discrepancy between the observed
outcomes for droplet/thin film DESI and bulk microtiter
reactions. Ideally, the optimum droplet/thin film and optimum
bulk conditions should be comparable.
Reaction A is an example of thermal degradation. The
reactions at 200 °C were all negatives, and although there are
only positives at 150 °C, the product intensity decreases with
time. Since this reaction was a “yes” in the droplet/thin film
format, the thermal decomposition explains some of the false
positives in that the product is detectable in DESI but is
potentially degraded before the bulk microtiter experiment is
terminated and assayed.
In sharp contrast, reaction B in bulk showed positives at high
temperature (200 °C) and negatives at lower temperature (150
°C), and this reaction was a “no” under droplet/thin film
conditions. The second SNAr data set contains 13 such
reactions (bulk reactions with more “yes” outcomes at 200 °C
than at 150 °C, which were all negatives under droplet/thin
film conditions). One of the proposed mechanisms of reaction
acceleration is the lowering of the activation energy barrier,
which is a kinetic effect.13 Hence, an explanation for some false
negatives can be that the high temperature in bulk might have
shifted the chemical equilibrium toward product.
of amines and aryl halides in NMP (Figure 3). DIPEA (2.5
equiv) was used as base since it showed the most promising
results for both rounds 1 and 2. Reactions in 1,4-dioxane were
not possible in flow because of the low solubility of the base in
this solvent, resulting in reactor clogging.
Formation of the expected products in flow was confirmed
by TLC and electrospray ionization-mass spectrometry (ESI-
MS). We also found that the results of the microfluidic
reactions were comparable to bulk and droplet screening
experiments. Scheme 4 shows the “yes” reactions from HTE
that were conducted in flow, including amines with EWGs that
were found to produce successful reactions. The reaction of 4-
chloro-6-ethyl-5-fluoropyrimidine (R1-B7) with 3-(2-methyl-
piperidin-1-yl)propan-1-amine (R1-A7) and 1-methylpipera-
zine (R1-A4) always produced the SNAr product. Since 1-
methyl-1H-imidazole (R1-A2) does not have a reactive amine
site, it is not surprising that it did not participate in an SNAr
reaction. It should be noted that there is a possibility of
obtaining a false positive result in MS for the reaction between
1-methyl-1H-imidazole (R1-A2) and 4-bromo-N,N-diethylani-
line (R1-B11) because the m/z of the aryl halide starting
material M+2 peak and the product m/z are same. Despite this,
we were able to confirm that no product was formed from this
reaction because the ratio of the bromine isotope peaks
intensities always remained in a 1:1 ratio. 2-Morpholinoethan-
1-amine, R2-A6, was the only reactive amine in the round 2
reaction set; continuous flow conditions also showed that this
amine produced a positive result. We also examined two
negative outcomes identified by HTE and found almost no
product peak when those reaction conditions were evaluated
under continuous flow (Scheme 5, see SI for spectra).
Microfluidic Evaluation. After identifying reaction
hotspots from HTE, we tested some of the positive DESI
findings via flow reaction conditions (Figure 5, Table 5) to
CONCLUSION
■
This investigation used a robotic technique to execute SNAr
reactions in 96-well arrays that were coupled with fast DESI-
MS analysis to boost the speed of reaction optimization for the
preparation of biologically important synthons. Extremely high
throughputs can potentially be achieved using DESI because
both synthesis and analysis can occur simultaneously; however,
in the case of the SNAr transformations studied herein, we
found that many reactions required incubation at elevated
temperatures to observe product. Even with the added time of
incubation, the analysis times reachable with DESI (∼3.5 s/
sample) still results in sample throughputs that far exceed
traditional techniques. A total of 16 amines and 13 aryl halides
were used for HTE evaluation, yielding 1536 unique reactions
each in droplet mode and bulk microtiter modes using four
different bases in two different solvents. The outcome of 3072
individual reactions in this HTE campaign produced a total of
170 successful droplet reactions and 351 successful bulk
microtiter reactions in a total experimental time of
approximately 3 h. The expected impact of electron donating
and withdrawing substituents on SNAr reaction outcomes were
observed in our HTE. A few of the positive and negative
reactions identified by HTE were evaluated under continuous
flow conditions. Those findings revealed that the positive
conditions identified by HTE were true positives and the same
was true for negative reaction conditions. Although many
unsuccessful reaction conditions were identified by HTE, these
negative results are highly valuable in that they can support
machine learning efforts.44,45 Since negative data is rarely
published, the resulting gaps in the data available impedes the
progress of groups trying to develop machine learning
Figure 5. Continuous SNAr reactions in flow using a Chemtrix glass
chip reactor, SOR 3225: A = amine; B = aryl halide; C = DIPEA.
a
Table 5. Summary of SNAr Reactions in Flow
amines
(μL/min)
aryl halides
(μL/min)
base
(μL/min)
residence time temperature
Tr (min)
(°C)
6.67
3.33
1.11
0.67
6.67
3.33
1.11
0.67
6.67
3.33
1.11
0.67
0.5
1
3
100/150
100/150
100/150
100/150
5
a
Chemtrix Reactor Chip: 3225. Reactor volume: 10 μL. Pressure:
Ambient pressure.
check the validity of the high-throughput results. In these
microfluidic experiments, the reactions were run at 30 s, 1, 3,
and 5 min residence times at 100 and 150 °C using a 1:1 ratio
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Scheme 4. Microfluidic Evaluation of Select “Yes” Reactions from the HTE Study
Scheme 5. Microfluidic Evaluation of Two “No” Reactions
from the HTE
large arrays of rationally designed experiments and rapidly
identify the most important reaction parameters for faster
optimization of microfluidic reactions while also eliminating
wasted effort spent exploring failed reaction conditions.
Moreover, it makes it possible to derive multidimensional
hypotheses that can be explained from easily collected huge
data sets. Increasing the number of successful reactions can
facilitate the population of libraries with more compounds for
physicochemical and biological evaluation. Further, by
applying this process to other common important class of
reactions, it may accelerate library synthesis and the
identification of optimal conditions for challenging substrates,
suggesting that this approach is an important new tool in the
repertoire of the synthetic chemist.
EXPERIMENTAL SECTION
■
All chemicals and reagents were purchased from Sigma-Aldrich
(St Louis, Missouri) and used without any purification.
High-Throughput Reaction Conditions. High-through-
put SNAr experimentation in bulk was performed in 96-well
metal block assemblies (Analytical Sales and Services, Inc., NJ,
algorithms that can predict the success or failure of organic
reactions.
The power of HTE in both the droplet/thin film and bulk
microtiter reaction modes enables chemists to rapidly perform
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USA). The reaction mixtures were prepared in 1 mL glass
inserts of the 96-well metal block. All the reagent transfers and
mixing were performed using a Beckman Coulter i7 liquid
handling robot. The stock solutions were 111 mM for amines
and aryl halides, and the base stock solution concentration was
1.25 M in NMP or 1,4-dioxane. The final reaction
concentrations were 50 mM (1 equiv) for both the amines
and aryl halides and 125 mM (2.5 equiv) for the bases. All
solutions were prepared in appropriate solvent and added to
the 96-well plate in a ratio of 9: 9: 2 (amine: aryl halide: base).
Additional solvent was used instead of base for the “no base”
condition. For DESI-MS HTE, 384-well plates were prepared
from the 96-well plates using the robot; a 384 pin tool was
used to transfer the final reagent mixtures (50 nL) onto PTFE
slides.
For bulk microtiter HTE, the plates were heated in a
customized heating block at 150 or 200 °C for varying times.
The cover on top of the glass inserts (top of the metal block)
was made using a chemically resistant perfluoroalkoxy (PFA)
film. Double silicone rubber mats were used on top of the PFA
film, providing a tight seal that enables solution heating above
the boiling point with less than 5% solvent loss and no cross
talk between wells. After heating, the plates were cooled to
room temperature, and loaded back onto the deck of the liquid
handling robot to prepare 384-well plates. The reaction
mixtures were pinned onto the same DESI slide as described
above before and after heating using the same transfer method.
Liquid Handling Robot. Samples in 96-well aluminum
blocks fitted with glass vial liners (Analytical Sales and Services,
Inc., NJ, USA) or 384-well polypropylene plates (Analytical
Sales and Services, Inc., NJ, USA) were prepared both for
DESI and bulk HTE using a Biomek i7 (Beckman Coulter,
Inc., Indianapolis, IN) liquid handling robot. A 384-tip head
was used to transfer a single volume of 384 samples under the
same speed of aspiration and dispensing conditions. Also, the
heights of pipetting at the source and destination positions,
pattern of pipetting, etc. remained constant for each transfer.
An 8-channel head provided more flexibility in the amount of
liquid transferred. Moreover, the 8-channel tip head provided
better flexibility in terms of the layout of source and
destination platforms, speed, pipetting height, and reaction
stoichiometry. The i7 deck is also capable of accommodating
all necessary labware including robotic tips, plates, reservoirs,
etc., for assembling one reaction step. Chemically resistant
polypropylene and disposables tips (Beckman Coulter, Inc.,
Indianapolis, IN) were used to make the reaction mixtures.
The reservoirs of reagents solutions were the polypropylene
multiwell plates and reservoirs, as well as custom-made Teflon
reservoirs. Development of new transfer methods and
validation were done using the Biomek point-and-click
programming tool.
was used to prepare the DESI slide using reagents that were
pipetted into standard polypropylene 384-well plates. Porous
polytetrafluoroethylene (PTFE) sheets (EMD, Millipore
Fluoropore, Saint-Gobain) were glued (Scotch Spray mount)
onto glass slides (Foxx Life Sciences) to make the DESI slides.
No signs of interference from the glue were observed. The
reagents were mixed, and rhodamine B dye in a separate
reservoir was added to the robotic deck as a fiducial marker.
The liquids (50 nL) were deposited onto a porous PTFE
surface using the magnetic pin tool at 3072 spot densities. A
linear ion trap mass spectrometer (LTQ XL; Thermo
Scientific, San Jose, CA) equipped with a commercial DESI-
imaging source (DESI 2D source, Prosolia Inc., Indianapolis,
IN) was used to collect the DESI-MS data. Xcalibur v.3.0
software was used to control the instrument and run the
worklists for DESI-MS data acquisition. The DESI spray angle
was 56° using MeOH or MeOH with 1% formic acid (FA) as
spray solvent and with an applied voltage of 5 kV. Mass spectra
were collected in positive ion mode over the m/z range of 50−
500. The DESI-MS imaging lateral resolution was 350 μm.
This was achieved using a stage speed of 4376 μm/s and an
instrument scan time of 80 ms. The time to acquire data from
one DESI slide was approximately 3 h, resulting in an analysis
time of ∼3.5 s per reaction mixture. For data processing, data
were visualized using in-house software designed9 to automati-
cally search for the m/z values of reactants, intermediates, and
byproducts. The analysis using the in-house software generates
a heat map indicating “yes/no” output for each spot on the
PTFE surface of the DESI slide.
DESI-MS Analysis Software (CHRIS). The Chemical
Reaction Integrated Screening (CHRIS) tool is an in-house
software suite developed to automatically control the DESI
system (mass spectrometer, solvents system, and Prosolia
DESI 2D stage) and search the captured data for m/z values
that correspond to the starting materials, intermediates,
byproducts, and products. CHRIS generates a yes/no report,
through a web interface and displays the mass spectrum of any
spot, as well as spreadsheets with the intensity for the selected
molecules, the possible contaminants, or unknown byproducts
as guided by the user.
Microfluidic System. All microfluidic validation reactions
were performed using a Labtrix S1 system (Chemtrix, Ltd.,
Netherlands). The system was described previously in Jaman
et al.15 The micro reactor 3225 is made of glass and used for all
conducted reactions. The staggered orientated micro reactor
(SOR) chip 3225 (four inlets and one outlet, volume 10 μL)
have channel width 300 μm and channel depth 120 μm. The
Labtrix unit is enabled to pump five syringes into the
microreactor positioned on a heating and cooling unit. All
the gastight glass syringes were bought separately from
Hamilton Company (Hamilton, Reno, Nevada). All operations
are controlled via a ChemTrix GUI software, connected to the
Labtrix S1 casing using a USB cable.
Customized Heating Block. Home built heating devices
made of aluminum heater blocks containing four, 100 W
cartridge heaters were fabricated to accommodate standard size
96-well plates. A CNi series temperature controller (Omega
Engineering) enabled precise temperature control and a solid-
state relay was used to modulate the 120 Vac power to the
heaters.15 The heating blocks tolerate temperatures ranging
from −20 to 200 °C.
DESI-MS Analysis. DESI-MS analysis was performed
following the previously published method of Wleklinski et
al.9 However, in this work, the density of reaction spots was
3072 spots/plate instead of 6144/plate. The Biomek i7 robot
Microfluidic Reaction Conditions. Solutions of amines
(100 mM, 1 equiv) and aryl halides (100 mM, 1 equiv) in
NMP were loaded individually into two separate 1 mL
Hamilton gastight glass syringes (Hamilton Company, Reno,
NV). DIPEA (150 mM, 1.5 equiv) solution in NMP was
loaded into another 1 mL Hamilton gastight glass syringe.
Each solution was continuously dispensed into the SOR 3225
reactor to engage the reactants. All the SNAr reactions were run
at 100 and 150 °C using residence times of 30 s, 1 min, 3 min,
and 5 min. The products were collected without quenching
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and stored at −80 °C. TLC analyses were performed at the
end of the reactions and the findings confirmed by subsequent
ESI-MS analysis after extraction in ether and dilution into
methanol.
wrote the manuscript. R.G.C. and D.H.T. secured the project
funding.
Notes
The authors declare no competing financial interest.
Analysis of Microfluidic Reactions. A Thermo Fisher
TSQ Quantum Access MAX mass spectrometer connected to a
Dionex Ultimate 3000 Series Pump and WPS-3000 Autosam-
pler (Thermo Fisher Scientific, Waltham, MA) was used to
acquire electrospray ionization mass spectra (ESI-MS) of the
samples. The analysis was performed in full scan mode,
monitoring each analysis in both positive and negative ion
modes. The optimized parameters for the ESI source and MS
are as follows: spraying solvent, MeOH; spray voltage +5 kV
(positive mode) and −5.0 kV (negative mode); capillary
temperature, 250 °C; sheath gas pressure, 20; scan time, 0.5 s;
Q1 peak width (fwhm), 0.70 Th; micro scans, 1. The
autosampler settings were as follows: MS acquire time, 2
min; sample injection volume, 1 μL. The data from MS
spectrometer was processed using Thermo Fisher Xcalibur
software.
ACKNOWLEDGMENTS
■
We thankfully acknowledge the financial support from the
Defense Advanced Research Projects Agency (Award no.
W911NF-16-2-0020) and the Purdue University Center for
Cancer Research NCI CCSG (P30 CA023168). Ryan Hilger
of the Amy Facility at Purdue University, Department of
Chemistry, is also acknowledged for fabricating the custom
heating block. We also thank Christina R. Ferreira, Sangeeta
Pandey, and Andy Koswara for helpful discussions.
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acscombsci.9b00212.
■
sı
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AUTHOR INFORMATION
Corresponding Authors
■
R. Graham Cooks − Department of Chemistry, Purdue
University, West Lafayette, Indiana 47907, United States;
orcid.org/0000-0002-9581-9603; Email: cooks@
purdue.edu
David H. Thompson − Department of Chemistry, Purdue
University, West Lafayette, Indiana 47907, United States;
orcid.org/0000-0002-0746-1526; Email: davethom@
purdue.edu
Authors
Zinia Jaman − Department of Chemistry, Purdue University,
West Lafayette, Indiana 47907, United States
David L. Logsdon − Department of Chemistry, Purdue
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Tiago J. P. Sobreira − Department of Chemistry, Purdue
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Deborah Aremu − Department of Chemistry, Purdue University,
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Larisa Avramova − Department of Chemistry, Purdue
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