Mechanistic details of amino acid catalyzed two-component Mannich reaction: experimental study backed by density functional calculations

Article abstract of DOI:10.1007/s00726-019-02798-z

The role of pH-dependent ionic structures of l-amino acids in catalysis has been investigated for the two-component Mannich reactions between dimethyl malonate (DMM)/ethyl acetoacetate (EAA) and imines. As catalysts, l-amino acids performed well, even bet

Full text of DOI:10.1007/s00726-019-02798-z

Amino Acids
https://doi.org/10.1007/s00726-019-02798-z
ORIGINAL ARTICLE
Mechanistic details of amino acid catalyzed two‑component
Mannich reaction: experimental study backed by density functional
calculations
Dixita Rani1 · Lalita Thakur1 · Mayank Khera1 · Neetu Goel1 · Jyoti Agarwal1
Received: 16 May 2019 / Accepted: 2 October 2019
© Springer-Verlag GmbH Austria, part of Springer Nature 2019
Abstract
The role of pH-dependent ionic structures of l-amino acids in catalysis has been investigated for the two-component Mannich
reactions between dimethyl malonate (DMM)/ethyl acetoacetate (EAA) and imines. As catalysts, l-amino acids performed
well, even better than corresponding base catalysts and provided the β-amino carbonyl compounds in very high yields.
Density functional calculations were used to gain the mechanistic insight of the reaction. High catalytic efciency of amino
acids was attributed to the facile formation of carbanion intermediate through barrierless transition state TS1 (−19.43 kcal/
mol) and then its stabilization owing to carbanion interaction with protonated amino acid.
Keywords Zwitterion · l-Glutamic acid · Mannich addition · DFT studies · Amino acids
Abbreviations
plays a pivotal role in the catalytic functioning of enzymes
(Abe et al. 2016; Davie et al. 2007). Consequently, the use of
naturally available amino acids in organocatalysis has gained
attention. Since last two decades, l-Proline and its diverse
derivatives have been established as efcient catalysts for
several organic transformations by enamine/iminium mecha-
nism (Tafda et al. 2018; Panday 2011; Xu and Lu 2008;
Mukherjee et al. 2007). Unfortunately, the literature involv-
ing other amino acids as organocatalysts is relatively lacking
(Valero and Moyano 2016; Jiang et al. 2010; Lombardo et al.
2008; Tsogoeva and Wei 2005; Sakthivel et al. 2001).
Apart from enamine/iminium catalysis, amino acids can
function both as base and acid catalysts owing to their zwit-
terionic form that has –COO− and NH3+ groups. This dual
character of amino acids is likely to have positive impact
on their catalytic efciency, particularly for those organic
processes, which involve the formation of charged interme-
diates. To validate this hypothesis, we planned to investigate
the catalytic activity of amino acids for the two-component
Mannich reaction between dicarbonyl compounds such as
ethyl acetoacetate (EAA)/dimethyl malonate (DMM) and
imines. These dicarbonyl compounds generate activated
nucleophilic carbanions in the presence of base catalysts,
which may attack the imines and result in the C–C bond
formation adjacent to the nitrogen atom.
DMM
EAA
Cb
Dimethyl malonate
Ethyl acetoacetate
Carbanion
AA
Amino acid
Glu
l-Glutamic acid
Methyl ester of l-Glutamic acid
MeGlu
NBocGlu N-Boc-l-Glutamic acid
Glu-H
Protonated l-Glutamic acid
Introduction
Enzymes are well known as highly selective and profcient
catalyst for biochemical reactions (Darwich et al. 2019; Oh
et al. 2018; Cuestal et al. 2014; Milanowski 2013; Patel et al.
1996). The presence of amino acids in enzyme skeleton
Handling Editor: T. Langer.
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s00726-019-02798-z) contains
supplementary material, which is available to authorized users.
* Jyoti Agarwal
jagarwal@pu.ac.in; jyotiagarwa@gmail.com
Since decades, diverse metal and non-metal catalyzed
methodologies have been developed to proceed Mannich
reaction using various active methylene group containing
1
Department of Chemistry and Centre of Advanced Studies,
Panjab University, Chandigarh 160014, India
Vol.:(0123456789)
1 3

D. Rani et al.
nucleophiles such as cyclic/acyclic ketones, aldehydes,
esters, and so on as Mannich donors (Saranya et al. 2018;
Shi et al. 2018; Cai and Xie 2013; Subramaniapillai 2013;
Verkade et al. 2008). Yet the catalytic processes involving
the EAA/DMM as Mannich donor are sporadically available.
The instability of these enolates is one of the major problems
associated with them. At present, only few catalysts such as
cinchona alkaloids (Lou et al. 2005; Song et al. 2006), pyri-
dine binaphthyldisulfonaic acid (BINSA) salt (Hatano et al.
2008), Li(I) and Mg(II)-binaphthol (BINOL) complexes
(Hatano et al. 2010a, b). Calcium-pyridinebisoxazolidine
(PyBox) complex (Poisson et al. 2010), Zn(OTf)2 (Yang
et al. 2006), and Zn(NO3)2 (Pham et al. 2015) have been
investigated for this reaction. Despite their usefulness, these
catalytic processes sufer from the need of expensive and/
or synthetic catalysts achieved from multi-steps reactions,
moisture sensitive and toxic metal catalysts, and low yields
of products. Contrast to these, amino acids are naturally
available, cheap, and eco-friendly molecules, which does
not require any further modifcation to be used as catalyst.
Thus, as part of our long-term research goal to develop
novel organocatalytic methods (Agarwal and Peddinti 2010,
2011a, b, 2012; Agarwal 2016; Rani et al. 2018), we report
our results for the zwitterionic amino acids catalyzed Man-
nich reaction of imines with dimethyl malonate and ethyl
acetoacetate. Experimental as well as theoretical studies
have been performed to provide the mechanistic insight of
the reaction and to understand the role of pH-dependent
ionic structures of amino acids for their enhanced catalytic
efciency.
soluble substrate molecules through hydrogen bonding with
protic ethanol solvent to provide high yields of the product.
When the same reaction was performed with 10 mol% of
l-Glutamic acid, reaction took 32 h to provide 76% of cor-
responding product 3 (Table 1, entry 7). A blank reaction
was also performed without using any catalyst in EtOH and
only trace amount of the product could be observed (Table 1,
entry 8). Enantioselectivities were also investigated for all
above reactions by HPLC using chiralcel AD-H column
(fow rate=1 ml per minute, 90:10% Hexane: IPA), almost
racemic products were observed in all cases.
It is well known that the reactions involving keto-esters
can be catalyzed by base catalysts; so, to compare the cat-
alytic activity of amino acids with simple bases, we per-
formed this reaction in the presence of triethylamine and
sodium ethoxide as catalyst. In case of NEt3, 29% yield was
observed in 72 h, while in case of NaOEt, no product forma-
tion could be achieved even after 3 days of stirring (Table 1,
entries 9 and 10), It may be due to the fact that its conjugate
base (ethoxide ion) works as good nucleophile also, which
may attack imine and led to the formation of side products.
These results showed the high catalytic efciency of l-Glu-
tamic acid Glu (4) for the studied reaction under mild reac-
tion conditions, i.e. at room temperature.
The pH of reaction mixture was found to be 6.2 and
isoelectric point of l-Glutamic acid is 3.2. Therefore, this
amino acid must be in anionic form in reaction mixture hav-
ing two basic groups, i.e. carboxylate and amine groups. The
pKa value for –NH2 and COO− groups of l-Glutamic acid
are 9.67 and 2.19, respectively; and it can be assumed that
–NH2 must participate preferably to catalyze the reaction. To
investigate this theory, we planned to perform the reaction in
the presence of methyl ester of l-Glutamic acid MeGlu (5)
and NBoc-l-Glutamic acid NBocGlu (6) also as catalysts.
Both of the protected amino acids 5 and 6 cannot exist as
zwitterions/anion/cation and will work as simple base and
acid catalysts, respectively. In case of catalyst 5, reaction
aforded 36% yield in 50 h of corresponding product 3, while
catalyst 6 provided only 12% yield in 48 h (Table 1, entries
11 and 12). These results indicated that –NH2 of MeGlu
(5) participated in catalysis, but not as efective as basic
groups of Glu (4). We believe that better catalytic results
from glutamic acid were obtained due to the fact that both
–COO− and –NH2 groups performed as catalytic sites for the
reaction and facilitated the activation of dimethyl malonate
by generating the carbanion like any other base catalyst. The
ionic form of amino acids provided the extra stabilization of
carbanion intermediate and increased the catalytic efciency
of amino acids than that of any other ordinary base catalyst.
Furthermore to confirm the above hypothesis that
–COO− group had also participated as catalytic site, we
performed the same set of reactions with l-Proline, l-Iso-
leucine, and the corresponding protected amino acids 7–12
Results and discussion
Initial reactions were performed between dimethyl malonate
(1) and imine (2) using l-Glutamic acid as catalyst and vari-
ous solvents were screened. All results are given in Table 1.
In case of non-protic solvents like DMF and DMSO, reac-
tion performed well to aford the corresponding product 3
in 6 and 10 h with 89 and 84% yields, respectively (Table 1,
entries 1 and 2). In dichloromethane and acetonitrile, reac-
tion could not reach to completion even after 30 and 26 h
stirring, which may be due to the insolubility of catalyst and
provided lower yield, i.e. 67 and 51%, respectively (Table 1,
entries 3 and 4). Next reaction was performed in water and
very low yield of 24% was achieved in 72 h (Table 1, entry
5). Here, catalyst was soluble in water, but substrates were
highly insoluble. Then the protic solvent ethanol was used
as reaction medium and reaction took very less time, i.e.
5 h, for completion to provide the excellent yield 95% of
the product 3 (Table 1, entry 6). Though, catalyst was partly
soluble in ethanol, similar to other polar non-protic solvents
like DMF and DMSO, it may come in close proximity to
1 3

Mechanistic details of amino acid catalyzed two-component Mannich reaction: experimental…
Table 1 Optimization of
reaction condition for the two-
component Mannich reaction
Entry
Catalyst
Solvent
Time (h)
Yielda (%)
1
DMF
6
89
84
2
DMSO
DCM
CH3CN
Water
EtOH
EtOH
EtOH
EtOH
EtOH
10
30
26
72
5
3
67
4
51
4
5
24
6
95
7b
8c
9
32
72
72
72
76
–
Trace
29
NEt3
NaOEt
10
Trace
11
12
EtOH
EtOH
50
48
36
12
5
6
Reaction was performed by stirring the solution of dimethyl malonate (1 mM) and imine (1 mM) in 1 mL
of ethanol in presence of 20 mol% of catalyst
aIsolated yields
b10 mol% of catalyst was used
cReaction was performed without catalyst
also. The isoelectric points of l-Proline and l-Isoleucine
results clearly suggested that –COO− group was participat-
ing actively to catalyze the reaction.
are 6.3 and 6.02, respectively and both must exist as zwit-
terions in reaction mixture of pH 6.2. It means they have
only one basic carboxylate group in reaction mixture, as
–NH2 will be converted to –NH3+. As shown in Table 2,
l-Proline (7) provided an excellent yield of 91% in 4 h,
whereas methyl ester of l-Proline (9) aforded 72% yield in
16 h (Table 2, entries 1 and 3). In case of NBoc-l-Proline
(8), only trace amount of product formation was observed
even after 96 h (Table 2, entry 2). Similar trend was real-
ized for isoleucine also. l-Isoleucine (10) and methyl ester
of l-Isoleucine (12) aforded the 88% and 56% yield of the
Mannich product in 4 h and 48 h, respectively (Table 2,
entries 4 and 6). NBoc- l-Isoleucine (11) provided only
trace amount of product in 96 h (Table 2, entry 5). These
Next, the efect of pH was studied on the catalytic ef-
ciency of amino acids. For that purpose l-Aspartic acid
13 was chosen along with l-Glutamic acid, as both have
low pI of 2.77 and 3.22, respectively. At pH 2.0, both the
amino acids existed in reaction mixture as cation bear-
ing acidic property (–COOH and –NH3+) and no product
formation occurred (Table 3, entries 1 and 6). But as the
pH of reaction mixture was increased, they began to con-
vert frst into zwitterionic form (–COO− and –NH3+) and
then into anionic form (–COO− and –NH2); and their basic
character also increased with the increasing pH of reac-
tion mixture. Therefore, the enhancement in basic char-
acter resulted in better catalytic efciency of amino acids
1 3

D. Rani et al.
Table 2 Results for the
l-Proline, l-Isoleucine, their
methyl esters, and NBoc
derivatives catalyzed Mannich
reaction between DMM and
iminea
Entry
Catalyst
Time (h)
Yieldb (%)
1
4
91
7
pI = 6.3
pH (Reaction Mix.) = 6.2
2
3
4
5
6
96
16
4
Trace
72
8
9
88
10
11
12
96
48
Trace
56
aReaction was performed by stirring the solution of dimethyl malonate (1 mM) and imine (1 mM)
in 1 mL of ethanol in the presence of 20 mol% of corresponding amino acid derivatives as organo-
catalyst
bIsolated yields
and very high yield of the product in very less time was
achieved (Table 3).
methylene proton of dimethyl malonate (1) to generate car-
banion (Cb) and amino acid gets converted into protonated
+
Thus, from all the above experiments, we may confrm
that in reaction mixture (pH 6.2), both the amino (–NH2) and
carboxylate (–COO−) groups of l-Glutamic acid (Glu) have
participated as active catalytic sites followed by stabiliza-
tion of carbanion and contributed to the facile formation of
products.
amino acid A and B, respectively. –NH3 group of proto-
nated amino acid A can stabilize the carbanion directly,
while B can further abstract the proton from solvent to con-
vert the NH2 into –NH3+ and then stabilize the carbanion.
When pH = pI, then like pathway 1a, –COO¯ generate the
carbanion and resulting protonated amino acid C directly
provides the stabilization to Cb (pathway 2). Next, carban-
ion attacks the electrophilic carbon of imine 2 to produce
negatively charged intermediate, which abstracts the proton
from the protonated amino acid. As a result, product for-
mation occurs along with the regeneration of zwitterionic
amino acid to be used for next catalytic cycle. Thus, the
Based on the above experimental results, we proposed
a mechanism as depicted in Fig. 1. It may follow three dif-
ferent pathways to generate the carbanion depending upon
the pH of reaction mixture and the pI of amino acid. When
pH>pI, either the –NH2 (pathway 1a) or the –COO¯ group
(pathway 1b) of anionic amino acid abstracts the active
1 3

Mechanistic details of amino acid catalyzed two-component Mannich reaction: experimental…
Table 3 pH study for amino
acid catalyzed Mannich
reaction of imine with dimethyl
malonate
Sr. No.
1.
Catalyst 20 mol%
pH
2
Time (h)
168
96
Yield (%)
-
2.
4
29
68
<99
<99
-
3.
5
40
4.
8
3.2
4 (pI = 3.22)
5.
10
2
2.5
6.
72
7.
5
45
59
82
91
<99
8.
7
4.5
9.
8
3.8
13 (pI = 2.77)
10.
10
3.0
facile formation of carbanion followed by its stabilization
with protonated amino acid are the key steps for the better
results of amino acids catalyzed Mannich reactions, which is
possible due to the pH-dependent ionic structures of amino
acids.
Present calculations confrm that the reaction proceeds
with the aid of zwitterionic form of amino acid that not
only ofers barrierless TS, but also stabilizes the resulting
carbanion.
The carbanion (Cb) formed in the reaction route may
bind with either the zwitterionic form of amino acid (Glu)
present in the reaction mixture to give Cb-Glu or with
its conjugate acid (Glu-H) formed as a result of reaction
between 1 and Glu to generate Cb-Glu-H. It was noted
that Cb prefers to bind Glu-H, as it imparts more stabiliza-
tion to it (Cb-Glu-H lies 70 kcal/mol lower than Cb-Glu).
This ascertains that the reaction medium requires one mol-
ecule of amino acid for one molecule of DMM. Current
calculations trace that the reaction proceeds through zwit-
terionic amino acid and also cleared the ambiguity con-
cerning the required presence of one or two molecules of
amino acid. The enhanced stability of Cb-Glu-H confrms
the dual role of amino acid and obviates the necessity of
second molecule of amino acid (Fig. 3).
Next, calculations were performed within the formalism
of density functional theory to confrm the signifcance of
pH-dependent ionic structures of amino acids and participa-
tion of –COO− group for their better catalytic efciency. For
that purpose, reaction between DMM (1) with l-Glutamic
acid Glu (4) was investigated by considering three diferent
modes of amino acid: (1) zwitterionic form of the amino
acid Glu (4), (2) methyl ester of l-Glutamic acid MeGlu (5)
and (3) NBoc-protected l-Glutamic acid NBocGlu (6). In
case of zwitterionic amino acid 4, the maxima of the poten-
tial energy scan (PES) was considered for transition state
optimization (Fig. 2a), which led to TS1 (Fig. 2b) charac-
terized by an imaginary frequency of −413.5i cm−1. The
resulting carbanion (Cb) is stabilized owing to its interaction
with protonated amino acid (Glu-H) formed in the reaction.
Interestingly, reaction between 1 and 4 is highly facile as it
involves barrierless TS1 and the interaction between counter
ions Cb and Glu-H imparts enhanced stability to the product
as shown in the energy profle (Fig. 2b).
Encouraged by the above results, we planned to inves-
tigate the catalytic activity of other amino acids also. All
results are shown in Table 4. When, the reaction was per-
formed in the presence of Glycine (14) as catalyst, it pro-
vided slight lower yield, i.e. 75% of the corresponding
product (Table 4, entry 1). In case of l-Leucine (15) and
l-Methionine (16), corresponding product 3 was aforded
in 64% and 32% yields in 4 and 6 h, respectively (Table 4,
entries 2 and 3). Furthermore, when reaction was performed
with l-Alanine (17) and l-Valine (18), both aforded the
product 3 in good yield, i.e. 86% and 67%, in 4 and 5 h
(Table 4, entries 4 and 5).
For the reaction between DMM (1) and MeGlu (5), the
structure corresponding to the maxima of PES (Fig. 2c) was
considered for TS optimization, which led to TS2 (charac-
terized with an imaginary frequency of − 26.9i cm−1) that
imposed a barrier of 47 kcal/mol (Fig. 2d) to the formation
of carbanion (Cb). In the third case, abstraction of hydrogen
from DMM (1) was not feasible with the NBocGlu (6).
1 3

D. Rani et al.
Fig. 1 Proposed mechanism for
the amino acid catalyzed Man-
nich reaction
Then, we chose aromatic amino acids as the next cat-
egory to study the catalytic activity. First, we performed
the reaction with 20 mol% of catalyst loading of l-Tyrosine
(19) and l-Phenylalanine (20) and both provided moderate
results, as they aforded the products of 43% and 37% yield
in 4 h, respectively (Table 4, entries 6 and 7). It means that
the presence of aromatic ring in amino acid reduced their
catalytic activity, which may be due to the steric hindrance
caused by aromatic rings. Furthermore, when reaction was
catalyzed with l-Tryptophan (21), improved yield of the
product was obtained (Table 4, entry 8).
Next, reactions were performed in the presence of l-Histi-
dine (22) and l-Arginine (23). Both the amino acids contain
basic side chain and they provided the lower yield of the
product, i.e. 42% and 41%, respectively, in 5 h (Table 4,
entries 9 and 10). Furthermore, reaction was performed with
polar uncharged amino acids. Thus, when l-Serine (24) and
L-Cysteine (25) were investigated as catalyst, both of them
provided the lower yield of the corresponding product 3,
i.e. 43% and 63% in 6 and 4 h (Table 4, entries 11 and 12).
Further, we studied the catalytic activity of amino acids
for the reaction between ethyl acetoacetate (26) and imine
1 3

Mechanistic details of amino acid catalyzed two-component Mannich reaction: experimental…
Fig. 2 a Potential energy scans (PES) and b energy profle for the reaction between dimethyl malonate (1) and zwitterionic l-Glutamic acid Glu
(4); c PES and d energy profle diagram for the reaction between dimethyl malonate (1) and methyl ester of l-Glutamic acid MeGlu (5)
(2). For this reaction too, l-Proline (7) and l-Glutamic acid
(4) provided the best results. l-Proline aforded the corre-
sponding product 27 of 73% yield in 6 h and L-Glutamic
provided of 75% yield in 6 h (Table 5, entries 1 and 2). In
case of Glycine, 59% yield of the corresponding product 27
was obtained in 7 h (Table 5, entry 3). l-Tyrosine (19) gave
very low chemical yield due to the presence of aromatic side
chain (Table 5, entry 4), i.e. 40% in 7 h. l-Histidine (22) and
l-Serine (24) provided moderate chemical yield (Table 5,
entries 5 and 6).
example, when dimethyl malonate (1) was reacted with
N-(4-bromobenzylidene)benzenamine (28) in the presence
of l-Glutamic acid, reaction took 6 h to complete and pro-
vided the product 30 in 72% yield. In similar manner, ethyl
acetoacetate (26) also aforded the corresponding product
32 in good yield, i.e. 64%. Next, the reaction was car-
ried out with dimethyl malonate (1) and N-(4-chloroben-
zylidene) benzenamine (29), reaction showed only 25%
conversion of the reactants into corresponding product 31
in 5 h and aforded 21% isolated yield of it. Reaction was
stirred for prolonged time (24 h), but it had not shown any
further progress.
Next, the substrate scope was studied for this method-
ology and imines 28 and 29 were allowed to react with
donors 1 and 26. All results are reported in Scheme 1. For
1 3

D. Rani et al.
Fig. 3 Carbanion (Cb) stabilized by Glu-H instead of second molecule of Glu
Table 4 Amino acid catalyzed
Mannich reaction with dimethyl
malonate (1)
Entry Amino acids, R
Time (h) Yielda (%)
75
1.
Glycine
H
5
14
2.
L-leucine
4
64
15
SCH3
3.
4.
L-Methionine
L-Alanine
6
4
32
86
16
17
CH3
5.
L-Valine
5
67
18
6.
7.
L-Tyrosine
4
4
43
37
19
20
L-Phenylalanine
8.
L-Tryptophan
5
59
21
N
H
9.
L-Histidine
L-Arginine
5
5
42
41
22
23
NH2
10.
N
NH2
H
11.
12.
L-Serine
6
4
43
63
24
25
L-Cysteine
Reaction was performed by stirring the solution of dimethyl malonate (1 mM) and
imine (1 mM) in 1 mL of ethanol in the presence of 20 mol% of corresponding
amino acid as organocatalyst
aIsolated yields
1 3

Mechanistic details of amino acid catalyzed two-component Mannich reaction: experimental…
Table 5 Mannich reaction of
imine and asymmetric ester
Entry
1.
Amino acids, R
L-Glutamic acid
Time (h)
6
Yielda(%)
75
4
2.
3.
4.
5.
6.
L-Proline
Glycine
6
7
7
6
6
73
59
40
64
68
7
H
14
19
22
24
L-Tyrosine
L-Histidine
L-Serine
Reaction was performed by stirring the solution of ethyl acetoacetate (1 mM) and imine (1 mM) in
1 mL of ethanol in presence of 20 mol% of catalyst
aIsolated yield
Scheme 1 Mannich reaction of
imine with dimethyl malonate
ester using glutamic acid
acid can be reused to catalyze the reaction with slight loss
Reusability of catalysts
of its catalytic efciency.
To check the stability of catalyst in recyclability experi-
ments, the IR spectrum of the recovered l-Glutamic acid
after each run and 1H NMR spectra (D2O, 500 MHz) after
each alternate run were recorded and compared with that
of pure l-Glutamic acid (Fig. 5). In IR spectra, no signif-
cant change in the functional groups of l-Glutamic acid
was observed. In 1H NMR also, similar spectra for l-Glu-
tamic acid after frst, third, and ffth cycles were observed
having no change in chemical shifts for diferent protons
Next, the reusability of l-Glutamic acid as organocatalyst for
Mannich reaction was also investigated by performing the
reaction between dimethyl malonate (1) and imine (2) under
optimized reaction conditions. The catalysts were recovered
after the completion of the reaction by fltration and washed
with dichloromethane to remove residual organic compound.
The catalyst was dried well and used for subsequent reac-
tions. The experimental results are demonstrated by bar dia-
gram as depicted in Fig. 4. It was observed that l-Glutamic
1 3

D. Rani et al.
Conclusions
We have demonstrated the mechanism of amino acid cata-
lyzed two-component Mannich reaction through experimen-
tal as well as computational studies. This work highlights
many interesting facts. It has been observed that Glu initi-
ates the reaction through barrierless transition state and sta-
bilizes also the resulting carbanion. While the corresponding
base, i.e. MeGlu, proceeds the reaction through a transition
state with barrier of 47 kcal/mol. Thus, amino acids catalyze
the reaction very efciently and provide the products in high
yields under mild conditions. This study further clears the
ambiguity concerning the required presence of one or two
molecules of amino acid. The enhanced stability of Cb-Glu-
H confrms the dual role of amino acid and obviates the
necessity of second molecule of amino acid.
We believe that this work provides the better understand-
ing of the catalytic functioning of amino acids and as amino
acids are the integral part of enzymes; this study may pro-
vide an insight for developing novel small catalysts that
mimic enzymes for organic processes.
Fig. 4 Bar diagram to show the reusability of l-Glutamic acid (Glu,
4) for the reaction of dimethyl malonate (1) with imine (2) under the
optimized reaction conditions
1
in comparison to the H NMR of pure l-Glutamic acid.
No other organic impurities due to the reaction substrates/
products were also present in any of the NMR spectrum.
Thus, it can be concluded that there was no disparity in the
structures of l-Glutamic acid after multiple recyclability
experiments, which proves to retain its catalytic activity.
Experimental section
The optimization of all the molecular species involved
in the reaction mechanism has been realized by perform-
ing density functional calculations without imposing any
Fig. 5 a IR spectra of the recovered l-Glutamic acid after each run. b 1H NMR (D2O, 500 MHz) spectra of the recovered l-Glutamic acid after
each alternate run
1 3

Mechanistic details of amino acid catalyzed two-component Mannich reaction: experimental…
symmetry restrictions. The hybrid B3LYP exchange corre-
lation functional (Becke 1993; Lee et al. 1988) in combina-
tion with 6-31G+(d, p) as instigated in Gaussian 09 (Frisch
et al. 2009) has been employed for geometry optimizations.
Dispersion efects have been taken into consideration using
Grimme’s D3 approach (Grimme et al. 2010). The station-
ary points and frst-order saddle points have been character-
ized by frequency calculations that also provided zero-point
vibrational energy (ZPE) and thermal corrections. The mini-
mum energy mechanistic route was fgured out by carrying
potential energy scan (PES) calculations. The geometry cor-
responding to the maxima of PES was considered for tran-
sition state (TS) optimization according to TS Berny algo-
rithm (Chéron et al. 2012). The change in Gibbs free energy
(∆G) has been calculated according to below equation:
3, 4, 5. Progress of the reaction was checked using TLC.
After completion of the reaction, ethanol was evaporated
and 5 ml of water was added. Then the product was extracted
with dichloromethane. The combined organic layers were
dried over anhydrous Na2SO4 and evaporated to aford crude
product, which was purifed by simple washing with small
amount of cold ethanol to obtain pure product. The collected
product was found to be pure enough and subjected to stand-
ard analytical techniques such as NMR, IR and elemental
analysis.
Dimethyl 2‑(phenyl(phenylamino)methyl)malonate (3)
White solid; m.p. 107–109 °C; IR (KBr) υmax: 3391 (–NH),
1745 (C=O), 1727 (COOMe), 1515 (Ar, C=C), 1494 (Ar,
C–H), 748 (monosubstituted aromatic ring), 691 (monosub-
stituted aromatic ring) cm−1; 1H NMR (300 MHz, CDCl3):
δ 3.56 (s, 3 H, OCH3), 3.59 (s, 3H, OCH3), 3.79 (d, J=5.4,
1 H, CH(COOMe)2), 5.11 (d, J = 5.7, 1 H, CHNH), 5.17
(br, 1 H, NH), 6.48 (d, J=7.8, 2 H, ArH), 6.55 (t, J=7.2,
1 H, ArH), 6.92–7.02 (m, 2 H, ArH), 7.07–7.30 (m, 5 H,
ArH) ppm; 13C NMR (100 MHz, CDCl3): δ 52.4, 52.7, 57.0,
57.9, 113.8, 117.9, 126.6, 127.6, 128.6, 129.0, 139.5, 146.4,
167.5, 168.4 ppm. Elemental analysis: C18H19NO4 (313.13);
calcd. C 68.99, H 6.11, N 4.47; found C 68.98, H 6.13, N
4.45.
ΔG = ꢀGProduct − ꢀGReactant.
(1)
The zero-point vibrational energy ZPVE results from the
vibrational motion of molecular systems at 0 K, it is calcu-
lated as a sum of contributions from all i vibrational modes
(harmonic oscillator model) of the molecular system.
Experimental details
Organic solvents were dried by standard methods. Commer-
cially available reagents were used without further purifca-
tion. All amino acids were purchased from AVRA Synthesis
Private Limited and none of the used amino acids was in the
form of hydrochlorides. All reactions were monitored by
TLC on aluminum plates coated with silica gel. TLC plates
were visualized with the short-range UV lamp (254 nm) or
in an iodine chamber. FTIR spectra were recorded in the
spectral region of 4000–500 cm−1 using PerkinElmer (RXI)
spectrophotometer (PerkinElmer, Beaconsfeld, Buck, UK.)
equipped with AgCl windows. The 1H NMR spectra were
recorded at 300 MHz on JEOL FT-NMR and on 400 MHz
BRUKER AVANCE FT-NMR in DMSO or CDCl3 using
trimethylsilane (TMS) as internal standard. Chemical shift
(δ) of NMR spectra were given in parts per million (ppm)
with respect to TMS. 13C NMR spectra were recorded at
100 MHz on BRUKER AVANCE FT-NMR spectrophotom-
eter in CDCl3 using TMS as internal standard. The following
abbreviations were used in NMR, s = singlet, d = doublet,
t=triplet, q=quartet, m=multiplet, and br=broad spectra.
Ethyl 3‑oxo‑2‑(phenyl(phenylamino)methyl)butanoate (27)
Yellow viscous liquid; IR (KBr) υmax: 3372 (NH), 1732
(C=O–OMe), 1604 (COOEt), 1513 (Ar C=C), 1358 (Ar
C–H), 748 (monosubstituted Ar), 691 (monosubstituted
1
Ar) cm−1; H NMR (300 MHz, CDCl3): δ 1.00–1.08 (m,
3H, CH3), 2.10 (s, 3 H, COCH3), 3.81 (d, J=6, 1 H, CH),
3.96–4.02 (m, 2 H, COOCH2CH3), 4.69 (br, 1H, NH), 5.07
(d, J=9, 1 H, CHNH), 6.49 (d, J=9, 2 H, ArH), 6.54–6.59
(m, 1 H, ArH), 6.96–7.01 (m, 2 H, ArH), 7.08–7.27 (m, 5 H,
ArH) ppm. 13C NMR (100 MHz, CDCl3): 13.8, 29.9, 57.4,
61.6, 65.5, 113.9, 115.0, 118.1, 126.8, 128.9, 129.1, 139.8,
146.3, 167.4, 203.1 ppm. Elemental analysis: C19H21NO3
(311.15); calcd. C 73.29, H 6.80, N 4.50; found C 73.23, H
6.85, N 4.45.
Dimethyl 2‑((4‑bromophenyl)(phenylamino)methyl)
malonate (30)
Procedure for amino acid catalyzed two‑component
Mannich reaction
Pale yellow solid; m.p. 119–123 °C; IR (KBr) υmax: 3369
(NH), 1726 (C = O), 1602 (COOMe), 1518 (Ar, C = C),
1431 (Ar, C–H), 825 (p-substituted Ar), 696 (monosub-
In a 25 ml single-neck round bottomed fask, the solution
of imine (0.5 mM), ethyl acetoacetate or dimethyl malonate
(0.5 mM) and l-amino acid (0.1 mM, 20 mol%) in 1 ml of
ethanol was taken. The resulting reaction mixture was stirred
at room temperature for the time mentioned in Tables 1, 2,
1
stituted Ar) cm−1. H NMR (300 MHz, CDCl3): δ 3.59
(s, 3 H, OCH3), 3.61 (s, 3 H, OCH3), 3.72 (d, J = 5.4,
CH(COOMe)2), 5.04 (d, J = 5.4, 1 H, CHNH), 5.22 (br,
1 H, NH), 6.42–6.47 (m, 2 H, ArH), 6.56 (t, J = 7.2, 1
1 3

D. Rani et al.
H, ArH), 6.93–7.03 (m, 2 H, ArH), 7.09–7.20 (m, 3 H,
ArH), 7.31–7.35 (m, 2 H, ArH) ppm; 13C NMR (100 MHz,
CDCl3): δ 52.6, 52.9, 56.5, 57.6, 113.8, 118.2, 121.5,
128.5, 129.1, 131.7, 138.6, 145.0, 167.3, 168.2 ppm. Ele-
mental analysis: C18H18BrNO4 (391.04); calcd. C 55.12, H
4.63, Br 20.37, N 3.57; found C 55.08, H 4.66, Br 20.35,
N 3.54.
dichloromethane to remove residual organic compound. The
catalyst was dried well and used for subsequent reactions.
Associated content
1
Copy of H and 13C NMR spectra of all the products and
reaction coordinates of all optimized structures have been
provided in supporting information.
Dimethyl 2‑((4‑chlorophenyl)(phenylamino)methyl)
malonate (31)
Author contributions The manuscript was written through contribu-
tions of all the authors. All the authors have given approval to the fnal
version of the manuscript.
Pale yellow solid; m.p. 120–122 °C; IR (KBr) υmax
:
3387 (NH), 1746 (COCH3), 1601 (Ar, C = C), 1495 (Ar,
C–H stretch), 748 (monosubstituted Ar), 696 (monosub-
Funding We are grateful to DST, Delhi (Grant no(s). IFA-
13-CH-101 and EMR/2017/000453) and UGC, Delhi (Grant no. F.30-
94/2015(BSR) for providing fnancial assistance. D.S. thanks UGC,
New Delhi for the award of the research fellowship.
1
stituted Ar) cm−1; H NMR (400 MHz, CDCl3): δ 3.67
(s, 3 H, OCH3), 3.68 (s, 3 H, OCH3), 3.84–3.88 (m, 1H,
CH(COOMe)2), 5.17 (d, J = 5.6, 1 H, CHNH), 5.27 (br,
1 H, NH), 6.54–6.58 (m, 2 H, ArH), 6.65–6.70 (m, 1 H,
ArH), 7.06–7.13 (m, 2 H, ArH), 7.21–7.26 (m, 2 H, ArH),
7.39–7.44 (m, 2 H, ArH) ppm; 13C NMR (100 MHz, CDCl3):
δ 52.4, 52.7, 57.0, 57.9, 113.9, 117.9, 126.6, 127.6, 128.6,
129.0, 139.5, 146.4, 167.5, 168.4 ppm. Elemental analysis:
C18H18ClNO4 (347.09); calcd. C 62.16, H 5.22, Cl 10.99, N
4.03; found C 62.13, H 5.26, Cl 10.94, N 4.00.
Compliance with ethical standards
Conflict of interest Authors declare that there is no confict of interest.
Research involving human participants and/or animals This article
does not contain any studies with human participants or animals per-
formed by any of the authors.
Ethyl 2‑((4‑bromophenyl)(phenylamino)
methyl)‑3‑oxobutanoate (32)
References
Abe T, Hashimoto Y, Zhuang Y, Ge Y, Kumano T, Kobayashi M
(2016) Peptide bond synthesis by a mechanism involving an
enzymatic reaction and a subsequent chemical reaction. J Biol
Chem 291:1735–1750
Yellow solid; m.p. 114–117 °C; IR (KBr) υmax: 3355 (NH),
1732 (COCH3), 1707 (COOEt), 1600 (Ar, C=C), 1488 (Ar,
C–H stretch), 864 (p-substituted aromatic ring), 693 (mono-
substituted Ar) cm−1; 1H NMR (300 MHz, CDCl3): δ 1.03
(t, J=7.2, 3 H, CH3), 2.09 (s, 3 H, COCH3), 3.77 (d, J=6,
1 H, CHCOOEt), 3.92–4.05 (m, 2 H, OCH2CH3), 4.67 (br,
1 H, NH), 5.04 (d, J=7.5, 1 H, CHNH), 6.43–6.49 (m, 2 H,
ArH), 6.50–6.59 (m, 1H, ArH), 6.94–7.00 (m, 2 H, ArH),
7.09–7.28 (m, 4 H, ArH) ppm. 13C NMR (100 MHz, CDCl3):
δ 13.9, 29.9, 57.4, 61.7, 65.5, 113.9, 118.2, 126.8, 127.7,
128.6, 129.1, 139.8, 146.3, 167.4, 203.1 ppm. Elemental
analysis: C19H20BrNO3 (391.04); calcd. C 58.47, H 5.17,
Br 20.47, N 3.59; found C 58.44, H 5.19, Br 20.46, N 3.55.
Agarwal J (2016) Progress in aminosugar derived asymmetric cataly-
sis. Org Bio Chem 14:10747–10762
Agarwal J, Peddinti RK (2010) Highly efcient and solvent-free
direct aldol reaction catalyzed by glucosamine-derived proli-
namide. Tetrahedron 21(15):1906–1909
Agarwal J, Peddinti RK (2011a) Asymmetric Michael addition cata-
lysed by sugar-based prolinamides in solvent-free conditions.
Tetrahedron Lett 52:117–121
Agarwal J, Peddinti RK (2011b) Glucosamine-based primary amines
as organocatalysts for the asymmetric aldol reaction. J Org
Chem 76:3502–3505
Agarwal J, Peddinti RK (2012) Synthesis and characterization of
monosaccharide derivatives and application of sugar-based
prolinamides in asymmetric synthesis. Eur J Org Chem
32:6390–6406
Becke AD (1993) Density-functional thermochemistry. III. The role
of exact exchange. J Chem Phys 98:5648–5652
Reusability of catalysts
Cai X, Xie B (2013) Recent advances on organocatalysed asymmet-
ric Mannich reactions. ARKIVOC 2013:264–293
Chéron N, Jacquemin D, Fleurat-Lessard P (2012) A qualitative fail-
ure of B3LYP for textbook organic reactions. Phys Chem Chem
Phys 14:7170–7175
To investigate the reusability of l-Glutamic acid, reactions
were performed between dimethyl malonate (1) and imine
(2) under optimized reaction conditions in presence of
20 mol% of l-Glutamic acid. After completion of the reac-
tion, catalyst was recovered by fltration and washed with
Cuestal SM, Furnham N, Rahman SA, Furnham N, Rahman SA, Sil-
litoe I, Thorntonet JM (2014) The evolution of enzyme function
in the isomerases. Curr Opin Struct Biol 26:121–130
1 3

Mechanistic details of amino acid catalyzed two-component Mannich reaction: experimental…
Darwich AS, Burt HJ, Rostami-Hodjegan A (2019) The nested
enzyme-within-enterocyte (NEWE) turnover model for pre-
dicting dynamic drug and disease efects on the gut wall. Eur J
Pharm Sci 131:195–207
Panday SK (2011) Advances in the chemistry of proline and its deriva-
tives: an excellent amino acid with versatile applications in asym-
metric synthesis. Tetrahedron 22:1817–1847
Patel MT, Nagarajan R, Kilara A (1996) Lipase-catalyzed biochemi-
cal reactions in novel media: a review. Chem Eng Commun
152:365–404
Davie EAC, Mennen SM, Xu Y, Miller SJ (2007) Asymmetric cataly-
sis mediated by synthetic peptides. Chem Rev 107:5759–5812
Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheese-
man JR, Scalmani G, Barone V, Mennucci B, Petersson GA,
Nakatsuji H, Caricato M, Li X, Hratchian HP, Izmaylov AF,
Bloino J, Zheng G, Sonnenberg JL, Hada M, Ehara M, Toyota K,
Fukuda R, Hasegawa J, Ishida M, Nakajima T, Honda Y, Kitao
O, Nakai H, Vreven T, Montgomery JA, Peralta JE, Ogliaro F,
Bearpark M, Heyd JJ, Brothers E, Kudin KN, Staroverov VN,
Kobayashi R, Normand J, Raghavachari K, Rendell A, Burant JC,
Iyengar SS, Tomasi J, Cossi M, Rega N, Millam JM, Klene M,
Knox JE, Cross JB, Bakken V, Adamo C, Jaramillo J, Gomperts
R, Stratmann RE, Yazyev O, Austin AJ, Cammi R, Pomelli C,
Ochterski JW, Martin RL, Morokuma K, Zakrzewski VG, Voth
GA, Salvador P, Dannenberg JJ, Dapprich S, Daniels AD, Farkas
O, Foresman JB, Ortiz JV, Cioslowski J, Fox DJ (2009) Gaussian
09. Gaussian Inc, Wallingford
Pham K, Huang X, Zhang W (2015) One-pot fuorination and Man-
nich reactions of 1,3-dicarbonyl compounds. Tetrahedron Lett
56:1998–2000
Poisson T, Tsubogo T, Yamashita Y, Kobayashi S (2010) Asymmetric
Mannich reaction of malonates with imines catalyzed by a chiral
calcium complex. J Org Chem 75:963–965
Rani D, Singla P, Agarwal J (2018) ‘Chitosan in water’ as an eco-
friendly and efcient catalytic system for knoevenagel condensa-
tion reaction. Carbohydr Polym 202:355–364
Sakthivel K, Notz W, Bui T, Barbas CF (2001) Amino acid catalyzed
direct asymmetric aldol reactions: a bioorganic approach to cata-
lytic asymmetric carbon-carbon bond-forming reactions. J Am
Chem Soc 123:5260–5267
Saranya S, Harry NA, Krishnan KK, Kumar A (2018) Recent devel-
opments and perspectives in the asymmetric Mannich reaction.
Asian J Org Chem 7:613–633
Grimme S, Antony J, Ehrlich S, Krieg H (2010) A consistent and
accurate ab initio parametrization of density functional disper-
sion correction (DFT-D) for the 94 elements H-Pu. J Chem Phys
132:154104–154119
Shi Y, Wang Q, Gao S (2018) Recent advances in the intramolecular
Mannich reaction in natural products total synthesis. Org Chem
Front 5:1049–1066
Hatano M, Maki T, Moriyama K, Arinobe M, Ishihara K (2008) Pyri-
dinium 1,1′-binaphthyl-2,2′-disulfonates as highly efective chiral
brønsted acid-base combined salt catalysts for enantioselective
Mannich-type reaction. J Am Chem Soc 130:16858–16860
Hatano M, Horibe T, Ishihara K (2010a) Chiral lithium(I) binaphtho-
late salts for the enantioselective direct Mannich-type reaction
with a change of syn/anti and absolute stereochemistry. J Am
Chem Soc 132:56–57
Song J, Wang Y, Deng L (2006) The Mannich reaction of malonates
with simple imines catalyzed by bifunctional cinchona alkaloids:
enantioselective synthesis of β-amino acids. J Am Chem Soc
128:6048–6049
Subramaniapillai SG (2013) Mannich reaction: a versatile and con-
venient approach to bioactive skeletons. J Chem Sci 125:467–482
Tafda UI, Uzairu A, Abechi SE (2018) Mechanism and rate constant
of proline-catalysed asymmetric aldol reaction of acetone and
p-nitrobenzaldehyde in solution medium: density-functional
theory computation. J Adv Res 12:11–19
Hatano M, Horibe T, Ishihara K (2010b) Magnesium(II)-binaphtho-
late as a practical chiral catalyst for the enantioselective direct
Mannich-type reaction with malonates. Org Lett 12:3502–3505
Jiang Z, Yang H, Han X, Luo J, Wong WM, Lu Y (2010) Direct asym-
metric aldol reactions between aldehydes and ketones catalyzed
Tsogoeva SB, Wei S (2005) (S)-Histidine-based dipeptides as organic
catalysts for direct asymmetric aldol reactions. Tetrahedron
16:1947–1951
by
Valero G, Moyano A (2016) Cooperative efects between arginine and
glutamic acid in the amino acid-catalyzed aldol reaction. Chiral-
ity 28:599–605
l-tryptophan in the presence of water. Org Biomol Chem
8:1368–1377
Lee C, Yang W, Parr RG (1988) Development of the colle-salvetti
correlation-energy formula into a functional of the electron den-
sity. Phys Rev B 37:785–789
Verkade JMM, Van Hemert LJC, Quaedfieg PJLM, Rutjes FPJT (2008)
Organocatalysed asymmetric Mannich reactions. Chem Soc Rev
37:29–41
Lombardo M, Easwar S, Pasi F, Trombini C, Dhavale DD (2008) Pro-
tonated arginine and lysine as catalysts for the direct asymmetric
aldol reaction in ionic liquids. Tetrahedron 64:9203–9207
Lou S, Taoka BM, Ting A, Schaus SE (2005) Asymmetric Mannich
reactions of β-keto esters with acyl imines catalyzed by cinchona
alkaloids. J Am Chem Soc 127:11256–11257
Xu LW, Lu Y (2008) Primary amino acids: privileged catalysts in
enantioselective organocatalysis. Org Biomol Chem 6:2047–2053
Yang YY, Shou WG, Wang YG (2006) Synthesis of β-amino carbonyl
compounds via a Zn(OTf)
2-catalyzed cascade reaction of anilines
with aromatic aldehydes and carbonyl compounds. Tetrahedron
62:10079–10086
Milanowski P (2013) Enzyme catalysis and the outcome of biochemical
reactions. J Proteom Bioinform 06:132–141
Mukherjee S, Yang JW, Hofmann S, List B (2007) Asymmetric enam-
ine catalysis. Chem Rev 107:5471–5569
Publisher’s Note Springer Nature remains neutral with regard to
jurisdictional claims in published maps and institutional afliations.
Oh SW, Pereira A, Zhang T, Li T, Lane A, Fu J (2018) DNA-mediated
proximity-based assembly circuit for actuation of biochemical
reactions. Angew Chem Int Ed 57:13086–13090
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