Efficient synthesis of unsymmetrical trisubstituted 1,3,5-triazines catalyzed by hemoglobin

Article abstract of DOI:10.1016/j.mcat.2021.111519

Background: 1,3,5-triazines are important bioactive compounds that have been extensively studied in organic chemistry. In this work, a green and efficient process for the synthesis of unsymmetrical trisubstituted 1,3,5-triazines from isothiocyanate (1) with amidines (2) and 1,1,3,3-tetramethylguanidine (TMG, 3) was developed. Results: Under optimal conditions (isothiocyanate (0.1 mmol), amidines (0.1 mmol), 1,1,3,3-tetramethylguanidine (0.1 mmol), DMSO (1 mL), hemeprotein (heme concentration: 0.05 mol%), TBHP (3 equiv), room temperature, 10 min), high yields of 1,3,5-triazines (81%–96%) could be obtained when HbRb (Hemoglobin from rabbit blood) was used as the catalyst. Conclusion: This enzymatic method demonstrates the great potential for the synthesis of unsymmetrical trisubstituted 1,3,5-triazines and extends the application of enzyme catalytic promiscuity in organic synthesis.

Full text of DOI:10.1016/j.mcat.2021.111519

Molecular Catalysis 505 (2021) 111519
Contents lists available at ScienceDirect
Molecular Catalysis
journal homepage: www.journals.elsevier.com/molecular-catalysis
Efficient synthesis of unsymmetrical trisubstituted 1,3,5-triazines catalyzed
by hemoglobin
Fengxi Lia, Chunyu Wangc, Yaning Xua, Zixian Zhaoa, Jiali Sua, Chenhan Luoa, Yujie Ninga,
,
,
Zhengqiang Lia, Chen Lib *, Lei Wanga *
a Key Laboratory of Molecular Enzymology and Engineering of Ministry of Education, School of Life Sciences, Jilin University, Changchun 130023, China
b Key Laboratory of Zoonosis Research, Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Changchun 130062, China
c State Key Laboratory of Supramolecular Structure and Materials, Jilin University, Changchun 130023, China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Background: 1,3,5-triazines are important bioactive compounds that have been extensively studied in organic
chemistry. In this work, a green and efficient process for the synthesis of unsymmetrical trisubstituted 1,3,5-tri-
azines from isothiocyanate (1) with amidines (2) and 1,1,3,3-tetramethylguanidine (TMG, 3) was developed.
Results: Under optimal conditions (isothiocyanate (0.1 mmol), amidines (0.1 mmol), 1,1,3,3-tetramethylguani-
dine (0.1 mmol), DMSO (1 mL), hemeprotein (heme concentration: 0.05 mol%), TBHP (3 equiv), room tem-
perature, 10 min), high yields of 1,3,5-triazines (81%–96%) could be obtained when HbRb (Hemoglobin from
rabbit blood) was used as the catalyst.
Hemoglobin
Biocatalysis
Synthetic methods
Unsymmetrical 1,3,5-triazines
Oxidative cyclization
Conclusion: This enzymatic method demonstrates the great potential for the synthesis of unsymmetrical trisub-
stituted 1,3,5-triazines and extends the application of enzyme catalytic promiscuity in organic synthesis.
Introduction
trisubstituted unsymmetrical triazines in moderate yields (Scheme 1, B1
and B2) [12,13]. Xu and co-workers designed a base-mediated three--
The core skeleton of 1,3,5-triazines is a very important scaffold in
molecules exhibiting a broad spectrum of biological and pharmacolog-
ical activities, such as anticancer, antiviral, antifungal, antimalarial,
anti-inflammatory, and anti-angiogenesis activities [1–7]. 1,3,5-Tri-
azines and their substitutions are crucial to the generation and escala-
tion of activity. Accordingly, the synthesis of unsymmetrical 1,3,
5-triazines with different bioactive substitutes is significant for
research on organic and pharmaceutical chemistry.
component reaction of imidates, guanidines, and amides/aldehydes to
synthesize unsymmetrical 1,3,5-triazin-2-amines under thermal condi-
tions (Scheme 1, C1) [14]. Vasu et al. presented the one-pot synthesis of
trisubstituted 1,3,5-triazines starting from isothiocyanates, N, N-dieth-
ylamidines, and carbamidines in the presence of mercury (II) chloride to
generate desired 1,3,5-triazines in good to moderate yields (Scheme 1,
D1) [15]. Recently, Liang’s group developed a highly efficient
visible-light-promoted [5 + 1] annulation of biguanides and per-
fluoroalkyl iodides to construct perfluoroalkyl-s-triazines under mild
reaction conditions (Scheme 1, A3) [16]. Another visible-light-promoted
[3 + 1+2] annulation of isothiocyanates, amidines, and guanidines has
been explored by Zhang’s group for the synthesis of unsymmetrical
trisubstituted 1,3,5-triazines in moderate yields (Scheme 1, D2) [17].
However, all these methods suffer from drawbacks such as harsh reac-
tion conditions, toxic metal catalysts, complicated products, difficult
separation, low yields, and long reaction time. Considering these dis-
advantages, developing environmentally friendly and efficient methods
of preparing unsymmetrical trisubstituted 1,3,5-triazines remains
fascinating for researchers of medicinal and synthetic chemistry.
Traditionally, cyanuric chloride is used to synthesize unsymmetrical
trisubstituted triazines, but stepwise replacement of the three chlorines
often produces mixtures of compounds, leading to serious separation
and purification problems [8,9]. Various alternative methods can
reportedly overcome these problems. For example, the acylation/cycli-
zation of biguanides with appropriate esters and the
transition-metal-catalyzed reactions of biguanides and selected alcohols
or dihaloalkenes can enable successful syntheses of trisubstituted un-
symmetrical triazines (Scheme 1, A1 and A2) [10,11]. The
copper-catalyzed cyclization of amidines with a C1 source (N, N-dime-
thylformamide or trialkylamines) has been performed to synthesize
* Corresponding authors.
E-mail addresses: lc2018@jlu.edu.cn (C. Li), w_lei@jlu.edu.cn (L. Wang).
https://doi.org/10.1016/j.mcat.2021.111519
Received 10 September 2020; Received in revised form 3 March 2021; Accepted 4 March 2021
Available online 24 March 2021
2468-8231/© 2021 Elsevier B.V. All rights reserved.

F. Li et al.
Molecular Catalysis 505 (2021) 111519
Scheme 1. Synthesis of unsymmetrical trisubstituted 1,3,5-triazines catalyzed by hemoglobin.
Biocatalysis is extensively used to synthesize diverse fine chemicals
Experimental
as an efficient and green method with high efficiency, mild reaction
conditions, and environmental friendliness [18]. However, the main
reason limiting the application of biocatalysis is the breadth of reactions
catalyzed by proteins. Thus, discovering new non-natural reactions is
gaining increased research attention [19–21]. In this area, hemoproteins
demonstrate huge potential to catalyze non-natural reactions, and many
related studies have been conducted. Such reactions include oxidative
(hydroxylation, epoxidation, oxidative cyclization, and sulfoxidation)
[22–25] and carbene-mediated reactions [26–29]. New catalytic abili-
ties can further be enhanced by extensive protein engineering [30,31].
Hemoglobin, a hemoprotein responsible for transporting oxygen in the
blood of vertebrates, is low cost, stable, and commercially available,
rendering advantageous is use as an ideal biocatalyst in organic syn-
thesis. In our previous reports, hemoglobin has been utilized to syn-
thesize various nitrogen heterocycles, such as benzoxazoles, indolizines,
and quinoxalines [32–34]. As part of our studies towards the develop-
ment of hemoglobin-catalyzed non-natural reactions, we report herein a
hemoglobin-catalyzed multicomponent reaction to synthesize unsym-
metrical trisubstituted 1,3,5-triazines (Scheme 1). To the best of our
knowledge, this report is the first one on a biocatalysis method of syn-
thesising 1,3,5-triazines. Our hemoglobin-catalyzed protocol can pro-
duce higher yield (81 %–96 %) and shorter reaction time (10 min) than
other methods.
Chemicals
Horseradish peroxidase, Myoglobin from equine heart, Hemoglobin
from porcine blood, Hemoglobin from bovine blood, Cytochrome C from
horse heart muscle, Cytochrome C from bovine heart muscle, Cyto-
chrome C from swine heart muscle was purchased from Shanghai Yuan
Ye Biological Technology Company. TMG, isothiocyanate, amidines
were purchased from Bide Pharmatech Ltd. (Shanghai, China). All the
other chemical reagents were purchased from Shanghai Chemical Re-
agent Company (Shanghai, China). All the commercially available re-
agents and solvents were used without further purification. Proton
nuclear magnetic resonance (1H NMR) spectra were recorded on a 400
MHz spectrometer in CDCl3, and carbon nuclear magnetic resonance
(
13C NMR) spectra were recorded on 100 MHz spectrometer in CDCl3.
Chemical shifts for protons are reported in parts per million downfield
from tetramethylsilane (TMS) and are referenced to residual protium in
the NMR solvent (CHCl3 = δ 7.26 ppm). Chemical shifts for carbon are
reported in parts per million downfield from tetramethylsilane (TMS)
and are referenced to the carbon resonances of the solvent residual peak
(CDCl3 = δ 77.16 ppm). NMR data are presented as follows: chemical
shift (δ ppm), multiplicity (s = singlet, d = doublet, t = triplet, q =
quartet, m = multiplet, br = broad), coupling constant in Hertz (Hz),
integration. The experiments were performed triplicate, and all data
2

F. Li et al.
Molecular Catalysis 505 (2021) 111519
Table 1
Table 2
Hemoprotein-catalyzed synthesis of 4a through a multicomponent reaction.a
Effect of solvents on the hemoglobin-catalyzed synthesis of 4a.a
Entry
Catalyst
Isolated yield (%)
Entry
Solvent
Isolated yield (%)
1
HRP
71
46
63
81
74
91
92
81
73
3
1
n-Hexane
26
33
31
85
87
72
65
82
91
47
2
MYO
2
Toluene
3
HbSw
3
Ethyl acetate
Dichloromethane
Acetone
4
HbBv
4
5
HbHm
5
6
HbRb
6
Ethanol
7
Cyt. C Hs
Cyt. C Bv
Cyt. C Sw
Apo-HbRb
Denatured HbRb b
–
7
Acetonitrile
N, N-Dimethylformamide
Dimethylsulfoxide
Water
8
8
9
9
10
11
12
13
14
15
16
10
7
a
Reaction conditions: 1a (0.1 mmol), 2a (0.1 mmol), 3 (1,1,3,3-tetrame-
thylguanidine) (0.1 mmol), HbRb (heme concentration: 0.05 mol%), TBHP (3
equiv.), Solvent (1 mL), room temperature, 10 min.
4
Fe (TPP)Cl c
Hemin c
28 d
32 d
21 d
19 d
c
FeCl3
c
Fe (OTf)3
Abbreviation: HRP (Horseradish peroxidase); MYO (Myoglobin from equine
heart); HbSw (Hemoglobin from swine blood); HbBv (Hemoglobin from bovine
blood); HbHm (Hemoglobin from human); HbRb (Hemoglobin from rabbit
blood); Cyt.C Hs (Cytochrome C from horse heart); Cyt.C Bv (Cytochrome C from
bovine heart); Cyt.C Sw (Cytochrome C from swine heart muscle).
Results and discussion
Initially, we selected the reaction of isothiocyanatobenzene (1a)
with benzimidamide (2a) and 1,1,3,3-tetramethylguanidine (TMG, 3) as
the model to optimise the reaction conditions with tert-butyl hydro-
peroxide (TBHP) as an oxidant. As shown in Table 1, all hemoproteins
demonstrated good catalytic performance at room temperature, and the
catalytic activities primarily depended on the type and origin of he-
moproteins. Among the hemoproteins used, hemoglobin from bovine
blood (HbRb) and cytochrome C from horse heart muscle afforded the
highest yield (91 %–92 %) of triazine with only 0.05 mol% (heme
concentration) catalyst loading (entries 6–7). Considering the protein
cost, we adopted HbRb as the optimum biocatalyst for further study.
When Apo-HbRb or denatured HbRb was used as the catalyst (entries
10–11), the yield was similar to that of the blank experiment (entry 12),
suggesting that the special active conformation and heme center of he-
moglobin were critical in this reaction. Subsequently, we evaluated the
utility of several commercially available ferric porphyrins (Fe (TPP)Cl
and Hemin) and ferric salt catalysts (FeCl3 and Fe (OTf)3). Product 4a
was also detected but in relatively lower yields even with prolonged
reaction time to 1 h (entries 13–16), it was demonstrated different from
Fenton chemistry and there is a more active free radical (Compound I).
Compared with the above chemical catalysts, HbRb exhibited much
higher catalytic efficiency towards this multicomponent reaction. The
loading of HbRb and oxidant ratio were then investigated, and 0.05 mol
% HbRb and 3 equiv. TBHP were found to be ideal (Table S1). We also
investigated the effect of reaction time on products yield, and the results
are shown in Fig. S1. The yield of 4a increased with increasing reaction
time (0–10 min). Nevertheless, longer reaction time did not significantly
increased the yield. Thus, the reactions were conducted with 10 min as
the optimal reaction time for the remainder of this method.
a
1a (0.1 mmol), 2a (0.1 mmol), 3 (1,1,3,3-tetramethylguanidine; 0.1 mmol.),
hemoprotein (heme concentration: 0.05 mol%), TBHP (3 equiv.), dime-
thylsulfoxide (1 mL), room temperature, 10 min.
b
c
Heating HbRb in boiling water for 5 h.
Catalysts loading: 0.5 mol%.
d
Reaction time prolong for 1 h.
were obtained based on the average values. Mass spectra were recorded
on the Bruker MicrOTOF Q II and an Orbitrap Fusion™ Tribrid™ mass
spectrometer (Thermo Scientific, San Jose, CA, U.S.A.) coupled with
HESI ion source.
Preparation of apo Hb
To the ice-cold, salt-free Hb solution containing sufficient 0.1 N HCl
to give pH 2, is added an equal volume of ice-cold methyl ethyl ketone,
and the mixture is shaken for a short time. On standing in the cold,
separation takes place into a ketonic supernatant containing all the
heme, and the aqueous layer containing all the protein, which is dia-
lysed against water to remove the dissolved ketone.
General procedure for the synthesis of 1,3,5-triazines
To a mixture of isothiocyanates (0.1 mmol), 1,1,3,3-tetramethylgua-
nidine (1.0 equiv), in DMSO (1 mL), hemoproteins (heme concentration:
0.05 mol%), benzimidamide hydrochloride (1.0 equiv), was added.
Then 3 equiv. of TBHP was added dropwise into the above mixture and
10 min stirring was allowed at room temperature. The reaction was
monitored by TLC. When the reaction was complete, the crude mixture
was added water and extracted with ethyl acetate. Then the organic
phase was dried over Na2SO4 and concentrated under reduced pressure.
Finally, the desired product was obtained by flash column chromatog-
raphy with petroleum ether/ethyl acetate (4/1) as an eluent. All the
isolated products were well characterized by their 1H, 13C NMR and MS
spectral analysis.
Reaction medium is also an important aspect in a protein-catalyzed
reaction because it influences the distribution of all reactants and in-
termediates and the activity of proteins by varying the protein confor-
mation [35], and the hemoprotein have good stability and long-term
storage in organic solvent [36]. In this study, the effect of reaction
medium on the hemoglobin-catalyzed reaction was investigated, and the
results are presented in Table 2. The yield of 4a was dramatically
affected by reaction medium, and dimethylsulfoxide (DMSO) was the
most suitable solvent for the reaction and DMSO is an environmentally
preferable solvent which off ;ers advantages in terms of health and
3

F. Li et al.
Molecular Catalysis 505 (2021) 111519
Table 3
Scope of isothiocyanate compounds.a
a
Reaction conditions: 1 (0.1 mmol), 2a (0.1 mmol), 3 (0.1 mmol), HbRb (heme concentration: 0.05 mol%), TBHP (3 equiv.), DMSO (1 mL), room temperature, 10
min.
safety [37]. The stability of HbRb in DMSO were then investigated. The
isothiocyanatobenzenes with an electron-withdrawing group on the
benzene ring afforded higher yields than those with an electron-
donating group on benzene. Steric effects were also found to affect the
yield of the triazines as demonstrated by the ortho-substituted iso-
thiocyanates, producing a diminished yield of the products compared
with their meta- and para-substituted counterparts. Furthermore,
aliphatic isothiocyanates (isothiocyanatomethane and 2-isothiocyanato-
propane) were successfully utilized for this reaction to produce the
corresponding products in excellent yields (4o–4p).
results indicated the good stability of HbRb in DMSO and confirmed an
active role of the protein in the reaction instead of free hemin released
from the protein (Figs. S2-S3). Furthermore, we scaled up the
hemoglobin-catalyzed reaction by 500-fold under the optimum condi-
tions [DMSO (500 mL), isothiocyanatobenzene (1a, 50 mmol), benzi-
midamide (2a, 50 mmol), 1,1,3,3-tetramethylguanidine (3, 50 mmol),
HbBv (heme concentration of 0.1 mol %), and TBHP (3 equiv.) at room
temperature]. The yield of the corresponding triazine 4a reached 95 %
in a short reaction time (10 min). This result indicated the high potential
of the efficient and green method for practical application.
To further demonstrate the applicability of the method, the scope of
amidines 2 in this hemoglobin-catalyzed reaction was also explored. All
substituted benzamidines were reacted smoothly with iso-
thiocyanatobenzene 1a and TMG 3 smoothly to afford the correspond-
ing triazines in high yields. As shown in Table 4, the substituents had
weak electronic effects and strong steric effects on the reaction. The
para-substituted(5b-5 h) substances afforded the products in higher
yields than the ortho- (5i-5k) or meta-substituted(5 L-5 m) benzami-
dines. Meanwhile, aliphatic amidines (acetimidamide and cyclo-
propanecarboximidamide) were subjected to this reaction system and
Under the optimum reaction conditions, the scope and generality of
the reaction with respect to the isothiocyanates 1, amidines 2, and TMG
3
were investigated. Firstly,
a
range of substituted iso-
thiocyanatobenzenes 1 were examined in the presence of 2a and 3. As
shown in Table 3, isothiocyanatobenzenes with various substituents on
the aromatic ring were well tolerated, affording the desired triazines
(4b–4n) in high yields (81 %–94 %). The electronic effects of the sub-
stituents had
a
certain influence on the reaction, and
4

F. Li et al.
Molecular Catalysis 505 (2021) 111519
Table 4
Scope of amidines.a
a
Reaction conditions: 1a (0.1 mmol), 2 (0.1 mmol), 3 (0.1 mmol), HbRb (heme concentration: 0.05 mol%), TBHP (3 equiv.), DMSO (1 mL), room temperature, 10
min.
provided 5n and 5o in high yields.
and quickly affords intermediate 6, which is oxdized by compound II to
the produce corresponding radical intermediates 6′. Radical interme-
diate 2a′ further reacts with radial intermediate 6′ to generate a key
intermediate 7 through the radical coupling process. This intermediate
is then further converted to the product 4a by the elimination of H2S and
intramolecular cyclization.
To probe the mechanism of this reaction, some control experiments
were performed, and the results are shown in Scheme 2. Firstly, addition
of the radical scavenger TEMPO (3 equiv.) dramatically decreased the
yield of 4a (Eq. 1). This result implied the involvement of a radical
pathway in this hemoglobin-catalyzed reaction. No corresponding
triazine 4a was obtained when TBHP was absent in this system (Eqs. 2
and 3), and only 4% yield of 4a was observed (Eq. 4) in the presence of
TBHP without hemoglobin. These results indicated that hemoglobin
obviously accelerated the reaction and TBHP was necessary to maintain
the catalytic ability of hemoglobin. Moreover, the reaction of iso-
thiocyanatobenzene 1a with TMG 3 afforded compound 6 in 80 % yield
without hemoglobin and oxidant (Eq. 5), and it was also detected during
this hemoglobin-catalyzed reaction. Therefore, compound 6 was a key
intermediate in this reaction pathway.
In conclusion, this work demonstrated an efficient and environ-
mentally friendly strategy for the construction of unsymmetrical
trisubstituted 1,3,5-triazines at room temperature. This work presented
a new hemoglobin capable of catalyzing the synthesis of 1,3,5-triazines
with higher yield (81 %–96 %) and shorter reaction time (10 min) than
ever reported, thereby demonstrating the huge potential of hemoglobin
as an optimum source of novel biocatalysts for new non-natural
reaction.
Based on literature and our experimental results [17,38–41], a
possible mechanism of the hemoglobin-catalyzed synthesis of trisubsti-
tuted 1,3,5-triazines was proposed (Scheme 3). Initially, the heme of
hemoglobin is converted to compound I in the presence of TBHP. This
potent oxidant intermediate facilitates hydrogen atom transfer (HAT)
from the substrate 2a to the cofactor, generating the Fe (IV)-hydroxide
species (compound II) and the radical intermediates 2a′. Then, the re-
action of isothiocyanatobenzene 1a and TMG 3 occurrs spontaneously
CRediT authorship contribution statement
Fengxi Li: Conceptualization, Methodology, Formal analysis, Soft-
ware. Chunyu Wang: Software. Yaning Xu: Investigation, Resources.
Zixian Zhao: Visualization. Jiali Su: Writing - original draft. Chenhan
Luo: Writing - review & editing. Yujie Ning: Data curation. Zheng-
qiang Li: Software. Chen Li: Supervision. Lei Wang: Supervision.
5

F. Li et al.
Molecular Catalysis 505 (2021) 111519
Scheme 2. Control experiments.
6

F. Li et al.
Molecular Catalysis 505 (2021) 111519
Scheme 3. Plausible mechanism of hemoglobin - catalyzed reaction.
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Declaration of Competing Interest
The authors report no declarations of interest.
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