Quantification and Prediction of Imine Formation Kinetics in Aqueous Solution by Microfluidic NMR Spectroscopy

Article abstract of DOI:10.1002/chem.202100874

Quantitatively predicting the reactivity of dynamic covalent reaction is essential to understand and rationally design complex structures and reaction networks. Herein, the reactivity of aldehydes and amines in various rapid imine formation in aqueous sol

Full text of DOI:10.1002/chem.202100874

Accepted Article
Accepted Article
Title: Quantification and Prediction of Imine Formation Kinetics in
Aqueous Solution by Microfluidic NMR
Authors: Youzhen Zhuo, Xiuxiu Wang, Si Chen, Hang Chen, Jie
Ouyang, Liulin Yang, Xinchang Wang, Lei You, Marcel Utz,
Zhongqun Tian, and Xiao-Yu Cao
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To be cited as: Chem. Eur. J. 10.1002/chem.202100874
Link to VoR: https://doi.org/10.1002/chem.202100874
01/2020

10.1002/chem.202100874
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Quantification and Prediction of Imine Formation Kinetics in
Aqueous Solution by Microfluidic NMR
Youzhen Zhuo,[a] Xiuxiu Wang,[a] Si Chen,[a] Hang Chen,[c] Jie Ouyang,[a] Liulin Yang,[a] Xinchang
Wang,*[b] Lei You,*[c] Marcel Utz,*[d] Zhongqun Tian[a] and Xiaoyu Cao*[a]
Dedication ((optional))
[a]
Y.-Z Zhuo, X.-X Wang, S. Chen, J. Ouyang, Dr. L.-L Yang, Prof. Z.-Q Tian, Prof. X.-Y Cao*
State Key Laboratory of Physical Chemistry of Solid Surfaces,
Collaborative Innovation Center of Chemistry for Energy Materials
(iChEM), Key Laboratory of Chemical Biology of Fujian Province
Department of Chemistry, College of Chemistry and Chemical
Engineering, Xiamen University
Xiamen 361005 (P. R. China)
E-mail: xcao@xmu.edu.cn
[b]
Dr. X.-C Wang*
School of Electronic Science and Engineering, Xiamen University
Xiamen 361005 (P. R. China)
E-mail: xcwang@xmu.edu.cn
[c]
[d]
H. Chen, Prof. L. You*
State Key Laboratory of Structural Chemistry, Fujian Institute of Research on the Structure of Matter
Chinese Academy of Sciences, Fuzhou 350002, China
E-mail: lyou@fjirsm.ac.cn
Prof. M. Utz*
School of Chemistry, University of Southampton, Southampton, Hampshire SO17 1BJ, United Kingdom
Email: marcel.utz@soton.ac.uk
Supporting information for this article is given via a link at the end of the document.
Abstract: Quantitatively predicting the reactivity of dynamic covalent
reaction is essential to understand and rationally design complex
structures and reaction networks. In this work, we quantified the
reactivity of aldehydes and amines in various rapid imine formation in
aqueous solution by microfluidic NMR. Investigation of reaction
kinetics allows us to quantify the forward rate constants k+ by an
empirical equation, of which three independent parameters were
introduced as reactivity parameters of aldehydes (SE, E) and amines
(N). Furthermore, these reactivity parameters were successfully used
to predict the unknown forward rate constants of imine formation.
Finally, two competitive reaction networks were rationally designed
based on the proposed reactivity parameters. Our work has
demonstrated the capability of microfluidic NMR in quantifying the
kinetics of label-free chemical reactions, especially rapid reactions
that complete in minutes.
study, which is a prerequisite for predicting product distributions
in DCC reactions.[3b] In a simple system that just involves one
amine and one aldehyde/ketone, conventional spectroscopies,
such as UV-Vis and IR, successfully provide quantitative
information of imine formation[6] by monitoring the absorption of
imine group. However, they are limited in studying muticompent
complex systems that contain multiple amine and
aldehyde/ketone components, owing to the limited structural
resolution of these spectroscopies.
Dynamic covalent chemistry (DCC)[1] has been widely applied in
constructing sophisticated supramolecular structures and
reaction networks.[2] The most frequently used dynamic bond is
imine, which can be formed by condensation between aldehydes
and amines under mild reaction conditions.[3] Precise control over
both thermodynamics and kinetics of imine formation has greatly
promoted the construction of complex structures and chemical
systems.[3b] For instance, Cooper et al. integrated calculations and
experiments to discover 32 new imine-based cages through one-
pot syntheses in chloroform.[4] In aqueous system, imines are also
important building blocks in constructing supramolecular
structures.[5] Nevertheless, fundamental physical-organic
properties of imine formation in aqueous solution need thorough
Scheme 1. Chemical structures of the aldehydes (1-3) and the amines (PrA-
PmA) used in this work.
1
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thermodynamic control. Our work has successfully quantified the
reactivities of imine formation and demonstrated the capability of
μF-NMR in characterizing dynamic covalent chemistry.
In our previous work, we tried to study the kinetics of imine
formation in aqueous solution by conventional NMR,[10] for
instance aldehyde 1 and aliphatic amines. But the reactions
already finished when the first NMR spectrum was recorded at 5
min. Thus, we first mixed 1 with n-butylamine (BuA) to analyze
the feasibility of monitoring imine formation in aqueous solution
using μF-NMR. After injecting an aqueous solution of 1 (20 mM)
and BuA (20 mM) into a microfluidic chip by syringe pumps
(Figure 1a), the time-resolved 1H-NMR spectra (Figure 1b) clearly
showed that the reaction finished at 180 s. As the reaction went
on, the peak area of reactant (HCHO at 9.45 ppm) gradually
decreased, whereas the peak area of product (HCHN at 7.8 ppm)
gradually increased. Integration of those peaks was plotted to give
the profile of the yield against time (Figure 1c), as well as
equilibrium constant K. Similarly, condensation of 1 with several
types of amines, such as aliphatic amine PrA-PeA, hydroxy or
methoxy containing amine MomA-AeA, and aromatic amine PhA
were also investigated (See Supporting Information, Fig. S2-11†).
The kinetic curves were first fitted by a second-order equation,
which is commonly used for imine formation in organic solution.
Taking the condensation of 1 and AeA for example, no obvious
linear relationship between 1/[CHO] and time was observed
(Figure 2b), except for the early stage of the reaction.
Figure 1. (a) Schematic diagram of fluid flow process in the microfluidic chip.
(b) time-resolved 1H NMR spectra and (c) yield plot of reaction between 1 (10
mM) and BuA (10 mM) at different reaction time in D2O at 25 ℃. Error in
experiments: ±5%.
Nuclear magnetic resonance (NMR) is a powerful tool to study
organic reactions, because it provides valuable high-resolution
structural information.[7] Over the past decade, thermodynamics
and kinetics of imine formation with time scales ranging from
hours to days have been investigated by conventional NMR,[8]
such as the time-dependent distribution of imine reaction
network[9] and the influence of weak n-π* interaction[10] in
protecting imine bonds. But it fails to characterize the kinetics of
aqueous imine formation finished within minutes for two reasons.
First, important kinetic profiles at the early stage of reactions are
missing because it takes at least 3 min to acquire the first NMR
spectrum. Second, poor sensitivity of NMR requires the
accumulation of scans to achieve an acceptable signal-to-noise
ratio (S/N), leading to poor time resolution. The combination of
flow systems with NMR[11] provides enchanced NMR-based
techniques, such as flow NMR[12] and microfluidic NMR (μF-
NMR),[13] which significantly improve the time resolution.[14] For
instance, Wensink et al. reported the first work on monitoring the
kinetics of fast imine formation using microfluidic NMR.[15] But due
to the low sensitivity of the planar coil, a high concentration of
reactants (4.95 M) was required. Oosthoek-de Vries et al.[16] have
used a highly efficient stripline NMR flow probe to study the
kinetics of fast acetylation of benzyl alcohol on a time scale of
seconds at 500 mM.
We hypothesized that at the early stage, the concentration of
products is quite low, and the reverse reaction rate is
approximately zero. The total reaction rate equals the forward
reaction rate (r ≈ r+), which obeys the second-order equation. As
the reaction proceeds, the concentration of [CHN] together with
the rate of imine hydrolysis increase, leading to the deviation from
the second-order reaction process. Therefore, imine formation
should be considered as a reversible reaction in aqueous solution.
Kln(2Kx + 4KC0 + 1 - 2KC0 - 1)
√
A = -
4KC + 1
√
0
Kln(2Kx - 4KC0 + 1 - 2KC0 - 1)
√
+
= k+t + C
(1)
4KC + 1
√
0
The 2-1 reversible reaction equation (1) was attempted to fit the
kinetics of imine formation (page S8 in Supporting Information)
Recently, our group has successfully employed microfluidic
NMR (μF-NMR) to investigate the kinetics of a multi-component
host-guest supramolecular system.[17] Taking the advantage of
high sensitivity of double striplines in our design,[18] we have
reduced the concentration of reactants to as low as 2 mM. Herein,
we applied μF-NMR to characterize the kinetics of a variety of fast
imine formation in aqueous solution. Time-resolved NMR spectra
allow us to systematically investigate the kinetics and establish
the kinetic models. We then employed an empirical equation to
quantify the reactivity of aldehydes and amines. The collected
parameters can be directly used to predict the forward reaction
rate constant. We also designed two competitive imine-based
networks based on their different reactivities and demonstrated
the effect of amines and aldehydes on the kinetic and
Figure 2. (a) The reaction of 1 with AeA. The plot of the fitting curve by (b) the
second-order kinetic equation and (c) the 2-1 reversible kinetic equation. Error
in experiments: ±5%.
2
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The fitting resulted in an excellent correlation coefficient R2
(0.9992) (Figure 2c). Moreover, besides k+ and K, reverse
constant k− can also be acquired by calculation. For comparison,
we also introduced aldehydes 2m, 2p, and 3 to investigate the
substituent effect on the reaction kinetic. All kinetic data were
summarized in Table 1.
Table 2. The values of reactivity parameter N of amines.
Amine
MomA
AeA
PrA
PeA
1.20
BuA
1.24
N [a]
−1.23
−0.63
0.83
[a] Error in experiments: ±5%.
Table 1. Summary of the equilibrium constant K, rate constant k+, and k- of imine
formation.
[a]
[a]
[b]
Aldehydes
Amines
K (M−1
)
k+ (M−1·s −1
)
k- (s−1
)
568
2.30
3.47
4.05×10−3
3.24×10−3
3.76×10−3
1.47×10−3
1.07×103
884
3.32
Figure 3. Plots of reactivity parameter N versus lnk+ of the reaction of (a) 2m
and (b) 2p with amines. Error in experiments: ±5%.
361
0.531
0.291
4
325
8.96×10−
We investigated the kinetics of imine formation between various
aldehydes and amines, and tried to correlate to their molecular
structures. The k+ of four aldehydes with each amine decreased
in an order of 3＞1＞2m＞2p, which is in accordance with the
order of the electronic effect of substituents on the aldehydes.
Likewise, the k+ of reactions of amines with each aldehyde
follow an order of n-butylamine > n-pentylamine > n-propylamine
> 2-hydroxyethylamine > 2-methoxy-ethylamine. But the pKa,
HOMO energy levels and electron density of these amines (Figure
S43-45†) showed no linear positive correlation to their k+.
Therefore, the basic molecular properties (pKa, HOMO energy
levels and electron density) of the reactants are insufficient to
predict the r+.
[c]
[c]
14.8
-
-
3
516
643
269
287
302
1.60
2.22
3.10×10−
3
3.45×10−
3
1.98
7.36×10−
3
0.552
0.352
1.92×10−
3
1.17×10−
[c]
[c]
5.36
-
-
3
127
158
181
53.9
52.4
0.593
0.849
0.788
0.223
0.154
4.67×10−
Table 3. Predicted and experimental values of rate constant k+ for the reactions
of aldehydes and PmA.
3
5.37×10−
Predicted
value
Experiment
value
Amine
Aldehydes
Error
3
4.35×10−
3
4.14×10−
0.705
-
-
M−1·s −1
3
2.94×10−
E=0 SE=1
[c]
[c]
1.83
-
-
3
2.68×103
4.34×103
3.24×103
2.83×103
1.32×103
3.74
4.56
4.37
1.39
0.842
1.40×10−
0.686
0.676
1.46%
M−1·s−1
M−1·s −1
3
1.05×10−
E= −0.18 SE= 0.74
3
1.35×10−
N=−0.35
4.91×104
6.38×104
0.272
0.261
4.21%
6.12%
M−1·s −1
M−1·s −1
E= −1.51 SE= 0.70
E= 1.02 SE= 0.67
[c]
[c]
28.3
-
-
1.56
1.47
[a] Error in experiments: ±5%. [b] Error in experiments: ±10%. [c] the
concentration is below the detection limit.
M−1·s −1
M−1·s −1
3
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Figure 4. (a) Kinetic selectivity for the reaction of 1 with PeA and MomA. (b) Kinetic trace for imine formation from the reactions of aldehyde 1 with the amine PeA
and MomA separately (dotted) and a mixture of the amine PeA and MomA (Solid). (c) Kinetic selectivity for the reaction of 3 and 2m with PrA and PmA. (d) Kinetic
plots of the evolution of the compounds generated from a mixture of equal amounts of components 2m + 3 + PrA + PmA as a function of time. The size of circle
means the relative content of different products. The concentration of reactants was 10 mM in all cases. Error in experiments: ±5%.
To unambiguously quantify the reaction rate constants of imine
formation, we employed an empirical equation proposed by
Mayr.[19] This equation relies on three basic assumptions: (1) the
reactivity of amines and aldehyde are independent; (2) the
reactivity of amines is defined as N (Nucleophilicity); (3) the
aldehyde has two parameters: the reactivity of aldehyde
(electrophilicity defined as E), and the sensitivity to amine (defined
as SE). Equation (2) was fitted with the forward reaction rate
constant k+ to quantify the three reactivity parameters:
In addition, the equation (2) can also be used to predict the
unknown experimental k+ using the aforementioned reactivity
parameters. Taking phenylmethanamine (PmA) as an example,
its k+ with different aldehydes were predicted using equation (2).
First, the PmA reacted with 1 to derive its reactivity parameter N
to be −0.35. Then the k+ of PmA with 2m, 2p, and 3 were
calculated using equation (2), as listed in Table 3. The errors
between the predicted and experimental k+ were 1.46%, 4.21%,
and 6.12%, respectively. Hence, equation (2) can predict the
experimental k+ of imine formation once the reactivity parameters
of corresponding aldehydes or amines are derived.
lnk+ = SE (E + N)
(2)
Furthermore, the derived reactivity parameters of aldehydes
and amines allow us to rationally design and investigate the
kinetic selectivity in dynamic covalent chemistry. The designed
competitive reaction system contains two amines with large
difference in reactivity and an aldehyde: n-pentylamine (PeA,
N=1.20, 10 mM) and 2-methoxy-ethylamine (MomA, N=−1.23, 10
mM), and aldehyde 1 (10 mM). As expected, at the early stage of
the reaction (within 200 s), 1 reacted more quickly with PeA than
MomA (Figure 4b), as the concentration of 1·PeA reached a
maximum of 66% at 200 s. This means amine with larger N
dominates in the kinetic selection of its corresponding product. At
the end of the reaction (after 200 s), the system gradually reached
thermodynamic equilibrium, and due to competition from MomA,
the ratio of 1·PeA decreased to 58% at 480 s. Moreover, the time
First, aldehyde 1 was used as a reference, and its SE and E
were assumed to be 1 and 0, respectively. Then, the reactivity
parameters N of amines were calculated by substituting their
corresponding reaction rate constants k+ between 1 into the
equation (2) (Table 2). With all the N of amines derived, the
reactivity parameters (SE and E) of other aldehydes can also be
quantified. The k+ of aldehyde 2m and 2p with other amines were
measured. Plotting N of amines against lnk+ of 2m and 2p with
these amines gave the reactivity parameters of 2m and 2p (Figure
3). This demonstrated the applicability of empirical equation (2) in
quantifing experimental k+ with calculated reactivity parameters,
although the physical meaning of those parameters is not
straightforward.
4
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for competitive system to reach its thermodynamic equilibrium is
determined by the slowest process. In this case, the slower
formation of 1·MomA determines the time to reach the
thermodynamic equilibrium of the system (Figure 4b). Hence, our
proposed reactivity parameters can be used to design the kinetic
selection. For instance, if one wants to accelerate an imine
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We further designed a more complex [2×2] competitive system
(Figure 4c), which contains two amines (PrA and PmA) and two
aldehydes (2m and 3). At the early stage of reaction (30 s), 3·PrA
formed rapidly as a major product (Figure 4d, orange), along with
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In summary, we have quantified the reactivity parameters of a
series of aldehydes and amines for imine formation in aqueous
solution using μF-NMR. The high time-resolution NMR spectra
unambiguously gave the kinetic profiles of imine formation in
aqueous solution as a 2-1 reversible reaction. More importantly,
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systems ([1×2] and [2×2]) based on their differences in reactivity
parameters. Kinetic investigation of competitive systems revealed
that the kinetic selection was determined by the reactivity of
amines, whereas thermodynamics distribution was determined by
the stability of aldehydes. Our work demonstrated the capability
of μF-NMR in studying the kinetics of fast chemical reactions, and
quantification of the reactivity parameters of imine formation.
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Acknowledgements
This work is supported by the NSFC (No. 91427304, 21573181,
91227111, 21971216, 21722304, 21672214, and 21971217), the
Top-Notch Young Talents Program of China, and the
Fundamental Research Funds for the Central Universities of
China (No. 20720160050). Xinchang Wang and Liulin Yang also
thank the support from Nanqiang Young Top-notch Talent
Fellowship in Xiamen University, and Lei You thanks the Key
Research Program of Frontier Sciences (QYZDB-SSW-SLH030)
of the CAS for funding. Development and construction of the NMR
probe were supported in part by a Marie Curie Career Integration
Grant to MU by the European Commission (Project μF-NMR) and
by the EU H2020 Project "TISuMR".
Keywords: Dynamic covalent chemistry • Imines • Microfluidic
NMR • Dynamic combinatorial library • Kinetics
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10.1002/chem.202100874
Chemistry - A European Journal
COMMUNICATION
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10.1002/chem.202100874
Chemistry - A European Journal
COMMUNICATION
Table of Contents
We investigated the imine formation in aqueous solution finished within several minutes through microfluidic NMR. The forward rate
constants k+ were quantifiedby an empirical equation containing three independent parameters: reactivity parameters of aldehydes (SE,
E) and amines (N). Those results allow the prediction of the unknown forward rate constant k+ and rational design of two reaction
networks exhibiting kinetic selectivity.
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