Accelerated tert-butyloxycarbonyl deprotection of amines in microdroplets produced by a pneumatic spray

Article abstract of DOI:10.1016/j.ijms.2018.05.009

Protection and deprotection of organic compounds in multistep reactions using functional groups such as tert-butoxycarbonyl (Boc), is widely performed in synthetic organic chemistry. Reaction rate acceleration studies in spray-based ionization methods (electrosonic spray, paper spray, nanospray) have become increasingly common. Here, we demonstrate reaction rate acceleration of Boc deprotection using easy ambient sonic-spray ionization (EASI), a pneumatic technique which does not involve an applied voltage, in a teaching laboratory setting. The goal of this laboratory exercise was to explore acceleration in a previously unexplored spray-based reaction, while emphasizing in a pedagogic setting the importance of protecting groups for multistep synthesis. Rate acceleration factors of more than an order of magnitude were observed in the uncharged micron-sized droplets generated by EASI. The effect of reaction conditions on reaction acceleration was examined including changes in the type of acid, reagent concentration ratios and syringe pump flow rates. Student knowledge was assessed by pre-laboratory assignments, post-laboratory reports and oral interviews.

Full text of DOI:10.1016/j.ijms.2018.05.009

International Journal of Mass Spectrometry 430 (2018) 98–103
Contents lists available at ScienceDirect
International Journal of Mass Spectrometry
journal homepage: www.elsevier.com/locate/ijms
Full Length Article
Accelerated tert-butyloxycarbonyl deprotection of amines in
microdroplets produced by a pneumatic spray
Patrick W. Fedicka, Ryan M. Baina, Kinsey Baina, Tsdale F. Meharia, R. Graham Cooksa,b,∗
a Department of Chemistry, Purdue University, West Lafayette, IN 47907, United States
b Center for Analytical Instrumentation Development, Purdue University, West Lafayette, IN 47907, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Protection and deprotection of organic compounds in multistep reactions using functional groups such
as tert-butoxycarbonyl (Boc), is widely performed in synthetic organic chemistry. Reaction rate accelera-
tion studies in spray-based ionization methods (electrosonic spray, paper spray, nanospray) have become
increasingly common. Here, we demonstrate reaction rate acceleration of Boc deprotection using easy
ambient sonic-spray ionization (EASI), a pneumatic technique which does not involve an applied volt-
age, in a teaching laboratory setting. The goal of this laboratory exercise was to explore acceleration in a
previously unexplored spray-based reaction, while emphasizing in a pedagogic setting the importance of
protecting groups for multistep synthesis. Rate acceleration factors of more than an order of magnitude
were observed in the uncharged micron-sized droplets generated by EASI. The effect of reaction condi-
tions on reaction acceleration was examined including changes in the type of acid, reagent concentration
ratios and syringe pump flow rates. Student knowledge was assessed by pre-laboratory assignments,
post-laboratory reports and oral interviews.
Received 15 March 2018
Received in revised form 16 April 2018
Accepted 8 May 2018
Available online 9 May 2018
Keywords:
Organic synthesis
Laboratory instruction
Hands-on learning
Mass spectrometry
Reaction acceleration
Amino acids
Boc deprotection
Easy ambient sonic-spray ionization
© 2018 Elsevier B.V. All rights reserved.
1. Introduction
be safely maintained through the subsequent steps to the final
product [7]. The protecting group used in this experiment is the
Multistep organic synthesis involves a sequence of reactions
from starting materials to product, frequently with use of column
chromatography or other means to purify intermediates [1]. There
are many possible reaction routes to any one desired product or
intermediate, and cost, time, and yield are criteria in choosing the
best synthetic route [2]. Throughout a multistep synthesis, func-
tional groups may need to be conserved. A protecting group is often
introduced prior to a particular reaction step and later removed, in
order to preserve a specific group that otherwise would not survive
[3]. To undergraduate chemistry students, knowledge of the use of
protection and deprotection reactions is pivotal to the understand-
ing of multistep synthesis [4–6].
Protecting and deprotecting a functional group adds two addi-
tional steps to a multistep synthetic scheme. There are a battery
of protecting groups, each with requirements that need to be
met for their removal once the molecule has progressed to a
stage in the reaction scheme where the functional group can
tert-butyloxycarbonyl functional group, commonly referred to as
“Boc.”8 It is primarily utilized to protect amines in multistep syn-
theses and is extensively used in peptide synthesis and medicinal
chemistry [9]. The Boc group can be removed by relatively strong
acids, such as hydrochloric acid or trifluoroacetic acid, or by the
combination of heat and milder acids [8].
Time is a major consideration in multistep reactions, so chemists
typically increase the rate of reaction by using elevated temper-
atures. Refluxing is usually used to achieve this increase in rate
without losing sample or solvent [10]. While thermal acceleration
is widely used, another common method of acceleration is through
catalysis [11]. Catalysts accelerate reactions by providing an alter-
native mechanistic pathway with a lower activation energy. More
recently, there has been a growing recognition that reactions can be
accelerated by other means, specifically by performing reactions at
interfaces [12–14]. Modest acceleration has been reported for some
reaction mixtures in microfluidics [15,16], while electrospray ion-
ization [17–20], and other spray methods produce droplets which
sometimes yield very large acceleration factors. In cases of reac-
tions in small confined volumes (i.e. thin films or microdroplets) it
is thought that partial solvation of the reagents at the air-solvent
∗
Corresponding author at: Department of Chemistry, Purdue University, West
Lafayette, Indiana 47907, United States.
E-mail address: cooks@purdue.edu (R.G. Cooks).
https://doi.org/10.1016/j.ijms.2018.05.009
1387-3806/© 2018 Elsevier B.V. All rights reserved.

P.W. Fedick et al. / International Journal of Mass Spectrometry 430 (2018) 98–103
99
interface is the cause of reaction rate acceleration [21]. We explore
the use of a spray-based method to accelerate the deprotection of
an amine in this laboratory exercise. Specifically, the deprotection
of Boc-Ala-OH (1) by acid to produce free Ala-OH (2). The deprotec-
tion occurs by initial protonation of the tert-butyl carbamate and
subsequent loss of the cationic butyl group as the carboxylic acid
with production of the free amine.
ation, a modification that is more amenable to a teaching laboratory
environment, while maintaining the mission of bringing cutting-
edge research to the teaching laboratory [32]. This “no-voltage”
spray-based method, also known as easy ambient sonic-spray ion-
ization (EASI), has been the topic of a recent review [33]. The
chemical system of tert-butyloxycarbonyl (Boc) deprotection has
been selected for its common and important use in medicinal
chemistry [9,34] and for the relatively low reagent cost. One learn-
ing objective centered on students considering how experimental
parameters influence the acceleration of the formation of the
deprotected product. Some of these experimental parameters –
variation in the flow rate, concentration of the Boc-protected com-
pound relative to the reactant acid, and the choice of acid itself −
changed the measured rate acceleration factor. Note that the exer-
cise does not measure intrinsic rate constants. The second learning
objective was for students to understand the purpose and impor-
tance of protecting groups in multistep syntheses. By conducting
part of a multistep synthesis in an accelerated fashion, students
learned how and why protecting groups have such an important
role in organic synthesis and how time-saving steps could benefit
synthesis.
Reaction rate acceleration has been demonstrated using on-
line mass spectrometric analysis of reaction mixtures ionized by
electrospray ionization and other spray-based ionization meth-
ods [2,18–20,22–25]. This phenomenon has been highlighted in
recent reviews [21,26,27]. Experiments can be performed using
electrospray ionization to spray and collect appreciable amounts
of material in minutes [17]. Using a continuous thin film variant
of droplet chemistry, Wei et al. collected nearly 100 mg/hr of reac-
tion product with a steady state rate acceleration factor of 100 [28].
We use a variety of electrospray and reaction conditions to explore
both the kinetics of the reaction and the processes of electrospray
reaction rate acceleration. Various factors influence reaction rate
acceleration in electrospray including: solution flow rate, gas flow
rate, collection surface and reagent concentration [21]. Factors such
as solvent evaporation will increase reaction rates but may not
change the rate constant. On the other hand, increasing the sur-
face/volume ratio may increase rate constants if surface reactivity
differs from bulk reactivity. Reaction rate acceleration can be calcu-
lated by comparing the rate for the bulk material to that recorded
using the accelerated method. This is approximated by simply tak-
ing the ratio of product to starting material ratio for the sprayed
material divided by the ratio for the bulk after the same reaction
time. This calculated rate acceleration factor is only approximate as
it assumes equal ionization efficiencies for the reagent and product
as well as assuming the same form of reaction kinetics (Equation
1) [28,29].
2. Experimental
2.1. Chemicals and EASI setup
All chemicals (Boc-Ala-OH, Boc-Ala-OMe, hydrochloric acid
(HCl), and trifluoracetic acid (TFA)) were purchased from Sigma-
Aldrich (St. Louis, MO) except for methanol (MeOH) which was
purchased from Fisher Scientific (Pittsburgh, PA). EASI spray emit-
ters were constructed with fused silica lines with 100-␮m I.D.
and 360-␮m O.D. (PolyMicro, Phoenix, AZ), one tee assembly, one
union assembly, two NanoTight sleeves, and a stainless-steel cap-
illary (IDEX Health and Science, Oak Harbor, WA). To control the
flow of reagent solution, infuse syringe pumps (Standard Infusion
PHD 22/2000, Harvard Apparatus, Holliston, MA) were utilized with
gastight chemseal syringes (Hamilton Robotics, Reno, NV). Nitro-
gen (Indiana Oxygen, Lafayette, IN) was used as the nebulizing gas.
The construction and part numbers can be found in Fig. 1. The reac-
tion mixtures were sprayed into 15 mL Falcon conical centrifuge
tubes (Fisher Scientific, Pittsburgh, PA) from which the bottom had
been removed, so as to avoid pressure build-up, with glass wool in
its place to collect the product.
Equation. 1 Reaction acceleration is determined by the ratio of
ratios of product to reactant of spray and bulk.
The first learning objective of this laboratory exercise was for
students to gain a better understanding of how spray-based reac-
tions can be accelerated when compared to their solution-phase
counterparts. Unlike previously accelerated reaction exercises
performed, developed and implemented by our research group
[21,30,31], this chemical system uses no voltage during the acceler-
Fig. 1. Easy ambient sonic-spray ionization (EASI) droplet generation system consisting of a gastight syringe, fused silica lines, Teflon unions, nanotight sleeves and a
stainless-steel capillary.

100
P.W. Fedick et al. / International Journal of Mass Spectrometry 430 (2018) 98–103
Table 1
Reactions that students performed using six sets of conditions, altering flow rate,
acid type, and acid to Boc-Ala-OH ratio.
Student’s Reaction Conditions
Flow Rate
HCl
TFA
5 ␮l/min
5 ␮l/min
20 ␮l/min
10:1 HCl to Boc-Ala-OH
1:1 HCl to Boc-Ala-OH
10:1 HCl to Boc-Ala-OH
10:1 TFA to Boc-Ala-OH
1:1 TFA to Boc-Ala-OH
10:1 TFA to Boc-Ala-OH
2.2. Experimental parameters
This three-hour laboratory exercise was performed by 15 under-
graduate students enrolled in an organic chemistry II honors
section. The students were divided into three groups, and each
group performed the same set of six reactions using a range of
conditions, with varying flow rates, acids and ratios of acid to Boc-
Ala-OH (Table 1). No voltage was applied to the EASI spray emitter.
2.3. Mass spectrometry
Fig. 2. Full scan positive ion mode mass spectra. The blue box indicates the Ala-OH
which has had the Boc group removed, whereas the green indicates the reactant Boc-
Ala-OH. The reactant peak has been scaled by a factor of ten to aid in visualization.
The orange box indicates the intermediate, which the students did not use in their
calculations. (a) Spray reaction utilizing HCl in a 10:1 ratio of acid to Boc-Ala-OH at a
flow rate of 5 ␮L/minute. (b) Corresponding bulk reaction mixture. Ion signals at m/z
104 and 148 are the product and intermediate of a side reaction (esterification of the
carboxylic acid of the Boc-Ala-OH) and for the purpose of the teaching laboratory
exercise were not brought to the attention of the student. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version
of this article.)
All spectra were recorded in positive ion mode using a LTQ XL
ion trap mass spectrometer (Thermo Instruments, San Jose, CA). All
mass spectra recorded for product analysis utilized nanospray ion-
ization as the ionization source at 2.0 kV and set 3 mm from the inlet
to the vacuum system. The nanospray emitters were constructed
from borosilicate glass capillaries (1.5 mm o.d., 0.86 mm i.d., Sutter
Instrument Co.) that were pulled to a tip using a Flaming/Brown
micropipette puller (Sutter Instrument Co. model P-97, Novato, CA,
U.S.A.).
Table 2
Increase in ratio of the acid to the Boc-Ala-OH causes the product to form at an
accelerated rate. Similarly, both acids had the highest acceleration rate with an EASI
flow rate of 5 ␮L/min. TFA in a 10:1 ratio to Boc-Ala-OH at a flow rate of 5 ␮L/min
had the largest acceleration factor overall.
3. Results and discussion
3.1. Accelerated reactions
Student Reaction Acceleration Factors for Boc-Ala-OH for Each Spray Condition
To determine the acceleration factor for spray conditions rela-
tive to bulk conditions, students performed reactions under each
of the six conditions listed in Table 1 in both bulk and spray. Upon
completion of the spray reactions, the product was extracted from
the glass wool by running 2 mL of MeOH through the tube. Reac-
tions that were conducted under bulk conditions were all allowed
to react for a time equal to the entire spray and sample workup. Both
the spray based and bulk reactions were analyzed by nanospray
ionization mass spectrometry. Fig. 2 shows student-collected spec-
tra of both the spray and the bulk conditions using HCl in a 10:1
ratio acid to Boc-Ala-OH, with a flow rate of 5 ␮L/min condition.
The reactant was observed at m/z 190, corresponding to protonated
Boc-Ala-OH, and the product was observed at m/z 90, correspond-
ing to protonated Ala-OH with removal of the Boc protecting group.
The carbamic acid intermediate in the Boc deprotection process
Acid
Mole Ratio of Acid to
Boc-Ala-OH
Flow Rate
Acceleration Factor
1:1
10:1
1.2
18
HCl
5 ␮L/min
20 ␮L/min
5 ␮L/min
12
2.0
35
TFA
1:1
10:1
20 ␮L/min
4.2
was also observed at m/z 134, which corresponds to the loss of
the tert-butyl group and formation of protonated carbamic acid
(Scheme 1).
Students collected spectra for each condition and calculated the
acceleration factors (uncorrected for ionization efficiencies) of each
Scheme 1. Deprotection of Boc-Ala-OH (1) using strong acid to form the amino acid (2) via the intermediate carbamic acid.

P.W. Fedick et al. / International Journal of Mass Spectrometry 430 (2018) 98–103
101
Scheme 2. Deprotection of Boc-Ala-OMe (3) using strong acid to form the amino acid ester (4) via the intermediate carbamic ester.
Table 3
Increase in ratio of the acid to the Boc-Ala-OMe causes the product to form at an
accelerated rate. Similarly, the highest acceleration rate with an EASI flow rate of
5 ␮L/min. The larger acceleration factors may provide the justification of spending
more on the reagents to more clearly demonstrate the fundamentals of reaction
acceleration to students.
Reaction Acceleration Factors for Boc-Ala-OMe for Each Spray Condition
Acid
Mole Ratio of Acid to
Boc-Ala-OMe
Flow Rate
Acceleration Factor
1:1
59
HCl
5 ␮L/min
10:1
216
183
20 ␮L/min
eration rate for the students to observe and may increase their
understanding of the concept. Similarly, depending on the time
allotted for the laboratory exercise comparing both the Boc-Ala-
OH and the Boc-Ala-OMe could be a worthwhile learning exercise
to discuss side reactions and by-products.
Fig. 3. Full scan positive mode spectra. The blue box indicates the Ala-OMe from
which the Boc group is removed, the green indicates the reactant Boc-Ala-OMe. The
orange box indicates the intermediate. (a) The spray reaction utilizing HCl in a 10:1
ratio of acid to Boc-Ala-OMe at a flow rate of 5 ␮L/minute. (b) Mass spectrum for
corresponding bulk reaction mixture. (For interpretation of the references to colour
in this figure legend, the reader is referred to the web version of this article.)
3.2. Laboratory evaluation and assessment
The students were required to answer pre-laboratory questions
based on the background information provided on spray-based
reaction acceleration and the importance of Boc protection and
deprotection. A week after the completion of the laboratory
exercise, students turned in post-laboratory questions and data
manipulations to assess their comprehension of the experiment.
Both the pre-laboratory and post-laboratory questions are avail-
able in the Supporting Information. In an ungraded exercise a week
after the completion of the laboratory exercise, the students were
interviewed orally to assess their understanding of mass spectrom-
etry, reaction rate acceleration, and the experiment in which they
participated. Students were interviewed in three groups of 5 to
maximize participation and promote discussion that may not have
been elicited through individual written or verbal prompting [35].
Table 4 summarizes the responses to the five questions that the
students were asked to answer.
As seen in Table 4, all the students thought this was a worthwhile
experiment, sharing excitement about the novelty of this experi-
ment since this reaction had not previously been reported in spray.
For example, one student stated that this was an “. . .alternative
method that may be more commonly used in the future. . . We
are ahead of the game.” In their post-laboratory reports, the stu-
dents most commonly suggested multiplexing the spray emitters
to increase product formation. The students showed a fundamen-
tal understanding on how reactions are accelerated by spray-based
methods and how to calculate acceleration factors based on their
pre-laboratory, post-laboratory, and verbal interviews.
condition (Table 2). They then compared the acceleration factors for
each condition to determine which provided the largest accelera-
tion factor compared to its bulk counterpart. Every condition that
the students utilized demonstrated spray-based acceleration. The
most significant acceleration was observed from the TFA acid condi-
tion in a 10:1 acid to Boc-Ala-OH ratio, with a flow rate of 5 ␮L/min
during spray reaction. While not explored by the students in this
laboratory exercise, the deprotection of Boc-Ala-OH led to a side
reaction where the carboxylic acid on the alanine was converted
into the methyl ester (3). This reaction was accelerated to form the
deprotected Ala-OMe (4) as seen in Scheme 2.
To determine the acceleration of deprotection of the Boc-Ala-
OMe without the convoluting effect of the methylation of the
carboxylic acid in-situ, the same experiment was performed with
using Boc-Ala-OMe as the starting material. Fig. 3 shows the spray
and bulk spectra for bulk conditions for the HCl acid condition
in a 10:1 ratio acid to Boc-Ala-OMe, with a flow rate of 5 ␮L/min
condition.
There is a larger acceleration factor as seen in Table 3 likely due
to the removal of the intermediate step of the methyl-ester forma-
tion. The trend in acceleration factor for Boc-Ala-OMe is the same as
Boc-Ala-OH. Boc-Ala-OMe was not used initially in the teaching lab-
oratory exercise because it costs three times as much as Boc-Ala-OH
and the fundamentals of spray-based reaction acceleration were
still demonstrated. However, if budget allows, the authors suggest
utilizing the Boc-Ala-OMe as the reactant as it yields a larger accel-

102
P.W. Fedick et al. / International Journal of Mass Spectrometry 430 (2018) 98–103
Table 4
Student responses to post-laboratory verbal interviews.
Comparison of Student Post-Laboratory Verbal Interview Questions and Responses
Questions/Statements for Responsea
Groups, Nb Response Characterization – Students in the Group:
1
3
3
Thought the experiment was a valuable laboratory exercise.
Stated that data processing was difficult. (Although stated to be
difficult all the students correctly determined their acceleration
factors.)
Suggested that only one variable should be assigned to a group and
then the data pooled.
This is probably the first time that EASI has been used to spray and
collect accelerated reaction products in an undergraduate
teaching laboratory. Could you please explain why you believe
this was or was not a valuable laboratory experiment? How could
this experiment be improved?
1
2
3
3
3
Described the overall experimental setup.
Please describe the collection set-up, EASI
parameters, as well as syringe pump
parameters.
Explained why the bottom of the collection tube was removed to
allow gas flow and the use of glass wool as a collection mechanism.
Described the effect of flow rate on the syringe pump had on the
rate of acceleration and droplet size.
Described surface effects of the partially desolvated droplets.
Described the change in activation energy in partially desolvated
droplets.
3
3
2
Why are reactions able to be accelerated by
spray-based methods? What factors contribute
to this?
4
5
Please explain how you calculate acceleration factors?
3
3
Stated that it was a ratio of the bulk and spray based methods’
ratio of intensities of the product and reactants.
Described how both the bulk and sprayed products are analyzed
using nanospray so the effect is negated.
Why does the nanospray analysis not effect the acceleration?
a
Graduate TAs verbally interviewed students.
The 15 total students were assigned to 3 groups of 5 each.
b
4. Conclusions
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Acknowledgments
Financial support was provided by the U.S. Department of
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Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.ijms.2018.05.009.

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