A glucose oxidase-hemoglobin system for efficient oxysulfonylation of alkenes/alkynes in water

Article abstract of DOI:10.1016/j.mcat.2020.111336

Background: β-ketosulfones are important bioactive compounds that have been extensively studied in organic chemistry. In this work, a green and efficient process for the synthesis of β-ketosulfones from alkenes (1) or alkynes (3) with sodium benzenesulfinate (2) was developed. Results: Under optimal conditions (alkenes (0.5 mmol) or alkynes (0.5 mmol), sodium benzenesulfinate (0.5 mmol), water (2 mL), hemoproteins (heme concentration: 0.06 mol%), GOX (42 U/ml), room temperature, 2 h), high yields of β-ketosulfones could be obtained when HgbRb (hemoglobin from rabbit blood) and GOX (glucose oxidase from Aspergillus niger) was used as the catalyst. Conclusion: This enzymatic method demonstrates the great potential for the synthesis of β-ketosulfones and extends the application of dual protein systems in organic synthesis.

Full text of DOI:10.1016/j.mcat.2020.111336

Molecular Catalysis 500 (2021) 111336
Contents lists available at ScienceDirect
Molecular Catalysis
journal homepage: www.journals.elsevier.com/molecular-catalysis
A glucose oxidase-hemoglobin system for efficient oxysulfonylation of
alkenes/alkynes in water
Fengxi Lia, Jiali Sua, Yaning Xua, Jiaxu Liua, Yue Yua, Chunyu Wangc, 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, PR China
b Key Laboratory of Zoonosis Research, Ministry of Education, Institute of Zoonosis, College of Veterinary Medicine, Jilin University, Changchun, 130062, PR China
c State Key Laboratory of Supramolecular Structure and Materials, Jilin University, Changchun, 130023, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Background: β-ketosulfones are important bioactive compounds that have been extensively studied in organic
chemistry. In this work, a green and efficient process for the synthesis of β-ketosulfones from alkenes (1) or
alkynes (3) with sodium benzenesulfinate (2) was developed.
Hemoglobin
Glucose oxidase
Dual-protein system
β-Ketosulfones
Biocatalysis
Results: Under optimal conditions (alkenes (0.5 mmol) or alkynes (0.5 mmol), sodium benzenesulfinate (0.5
mmol), water (2 mL), hemoproteins (heme concentration: 0.06 mol%), GOX (42 U/ml), room temperature, 2 h),
high yields of β-ketosulfones could be obtained when HgbRb (hemoglobin from rabbit blood) and GOX (glucose
oxidase from Aspergillus niger) was used as the catalyst.
Conclusion: This enzymatic method demonstrates the great potential for the synthesis of β-ketosulfones and ex-
tends the application of dual protein systems in organic synthesis.
Introduction
alkylation of sodium sulfinates with phenacyl halides, the acylation of
alkyl sulfones with acid chlorides [23–25], esters or N-acylbenzo-
Enzyme catalysis has attracted considerable interest due to its
exceptional functionality, adaptivity and sustainability [1].
Multiple-enzyme catalysis plays a significant role in the production of
many valuable materials/chemicals at the industrial level because they
facilitate tandem or cascade reactions [2–4]. In the past decade,
exploring novel non-natural reactions has increasingly gained attention
in research [5–8]. Hemoproteins, especially hemoglobin, exhibit huge
potential as catalysts for non-natural reactions due to their low costs,
commercial availability, broad catalytic abilities and good stability
[9–14]. More importantly, the combination of promiscuous catalytic
and specific catalytic abilities of proteins can further expand the appli-
cation range of multiple-enzyme catalysis in organic chemistry [15–17].
As important synthetic intermediates, β-ketosulfones have been
widely used in the construction of natural products, polyfunctionalized
4H-pyrans, quinolines, allenes, ketones and vinylsulfones [18–21].
β-Ketosulfones can be used as antifungal and antibacterial drugs [22].
Given that β-ketosulfones are used in a wide range of applications,
substantial effort has been devoted to their synthesis. Traditionally, the
synthesis of β-ketosulfones is mainly performed through the direct
triazoles [26] and the oxidation of β-ketosulfides or β-hydroxysulfones
with stoichiometric inorganic oxidants [27,28]. The copper-catalyzed
coupling of oxime acetates with sodium sulfinates is used in the syn-
thesis of β-ketosulfones [29]. However, the limited availability of sub-
strates and long reaction time limit the practical application of
β-ketosulfones. In the last decade, as one of the promising synthesis
strategies, the direct oxysulfonylation of alkenes or alkynes has attracted
considerable attention. A variety of homogenous catalytic methods have
been developed based on the sulfonation of alkenes for access of
β-ketosulfones catalyzed by transition metal catalysts (Scheme 1, A)
[30–33]. Lei et al. reported an aerobic oxidative difunctionalization of
alkynes towards β-ketosulfones in the presence of 4 equiv. pyridine
(Scheme 1, B1) [34]. However, when alkenes were employed as pre-
cursors under similar reaction conditions, the only products obtained
were β-hydroxysulfones. Zhou et al. utilized iron salt to catalyze aerobic
oxysulfonylation of terminal alkynes and sodium sulfinates in
MeOH/H2O (v : v = 3 : 1) at 50 ◦C (Scheme 1, B2) [35]. In 2016, Wang
et al. reported the visible light-initiated direct oxysulfonylation of al-
kenes with sulfinic acids in the presence of TBHP (Scheme 1, C1) [36]. In
* 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.2020.111336
Received 1 October 2020; Received in revised form 30 November 2020; Accepted 2 December 2020
Available online 21 December 2020
2468-8231/© 2020 Elsevier B.V. All rights reserved.

F. Li et al.
Molecular Catalysis 500 (2021) 111336
Scheme 1. Methods for oxysulfonylation of alkenes/alkynes.
higher tolerance to substrates. To the best of our knowledge, this is the
first report on β-ketosulfone synthesis using proteins as catalysts.
Experimental
Chemicals
Myoglobin from equine heart, Hemoglobin from porcine blood, He-
moglobin from bovine blood, Horseradish peroxidase, Cytochrome c
from bovine heart muscle, Glucose oxidase from A. niger (200 U/mg)
was purchased from Shanghai Yuan Ye Biological Technology Company.
Alkenes, alkynes, sodium benzenesulfinate, were purchased from Bide
Pharmatech Ltd. (Shanghai, China). All the other chemical reagents
were purchased from Shanghai Chemical Reagent Company (Shanghai,
China). All the commercially available reagents and solvents were used
without further purification. Proton nuclear magnetic resonance (1H
NMR) spectra were recorded on a 400 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). 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. 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.
The experiments were performed in triplicate, and all data were ob-
tained based on the average values.
Scheme 2. Oxysulfonylation of alkenes/alkynes catalyzed by dual-
protein system.
2020, they developed a method for the visible light-promoted sulfona-
tion of alkenes catalyzed by a biomimetic photocatalyst of a single-atom
iron site prepared through coupling carbon nitride with hemin (Scheme
1, C2) [37]. However, the long reaction time, excess of oxidant and
hazardous organic solvent limit the practical application of visible
light-promoted methods. Therefore, demand for the development of
mild, efficient and green methods for synthesizing β-ketosulfones has
considerably increased
Herein, we designed a glucose oxidase (GOX)–hemoglobin (Hgb)
system for the synthesis of β-ketosulfones in water through the oxy-
sulfonylation of alkenes/alkynes. Scheme 2 shows that GOX catalyzes
the oxidation of glucose into D-glucono-delta-lactone and hydrogen
peroxide (H2O2) [38]. Hgb can catalyze the oxysulfonylation of alke-
nes/alkynes and sodium sulfinates with H2O2 generated in situ. To a
great extent, this ‘feed-on-demand’ reaction system can prevent the use
of excessive amounts of unstable oxidants. In addition, gluconolactone
spontaneously reacts in water to gluconic acid, which is a high-value
added product, and thus this method has potential practical applica-
tions. Notably, water is used in this reaction because of its low cost and
safety. According to the principles of green chemistry, using water to
replace hazardous solvents is another advantage of organic reactions
[39]. Moreover, sodium sulfinates can be used as substrates to replace
unstable sulfinic acids in this dual-protein system. Compared with pre-
vious methods, this dual-protein system is more efficient, milder and has
Preparation of apo Hgb
To the ice-cold, salt-free Hgb 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.
2

F. Li et al.
Molecular Catalysis 500 (2021) 111336
continuous addition of H2O2 combined with HgbRb in this reaction
resulted in a low yield of β-ketosulfone 4a (Entry 11–12). When the
reaction was performed in air, yield decreased to 62 % (Entry 13) and a
low yield was obtained when the reaction was conducted under a ni-
trogen atmosphere in the presence of H2O2 (Entry 14). This result in-
dicates that oxygen is necessary not only for the generation of H2O2
catalyzed by GOX but also for the oxdative steps in the synthesis of
β-ketosulfones catalyzed by Hgb. Hence, oxygen was used to improve
the catalytic efficiency of this reaction system for further investigation.
To determine the reaction involving a radical pathway, we added
TEMPO, a radical scavenger, to this reaction (Entry 15). Accordingly,
only trace product was determined. We optimized the reaction condi-
tions, such as the substrate ratio, protein concentration and glucose
dosage (data shown in Table S1).
Table 1
Synthesis of β-ketosulfones by a dual-protein system (GOX and hemoprotein) a.
Entry
GOX
Hemoprotein
Isolated yield (%)
1
GOX
HgbRb
HgbBv
HgbSw
MYO
91
2
GOX
61
3
GOX
52
4
GOX
69
5
GOX
Cyt C. Bv
HRP
75
6
GOX
71
7
GOX
Heminb
Apo-HgbRb
–
HgbRb
HgbRb
HgbRb
HgbRb
HgbRb
HgbRb
9
8
GOX
NDc
ND
ND
41
9
GOX
10
11
12
13
14
15
–
H2O2 (stepwise)d
H2O2 (continuous)d
GOXe
66
62
GOXf
10
Under optimal conditions, the scope and limitations of the dual-
protein system were evaluated. The result is summarized in Table 2. A
variety of aromatic alkenes containing electron-donating (Me, OMe) or
electron-withdrawing groups (F, Cl, Br) on aryl rings reacted with so-
dium benzenesulphinate efficiently and afforded the corresponding
products with satisfactory yields. The reaction was not obviously
affected by the electronic effects of the substituents in substituted sty-
renes, and steric hindrances influenced the yields. Compared with the
yields obtained after meta- and para-substituted counterparts were used
(4b-4 g), a lower yield was obtained when ortho-substituted styrene (4h-
4i) was selected as substrate. Internal alkyne (β-methylstyrene) reacted
with sodium benzenesulphinate (4 j) in a higher yield than those ob-
tained by other methods [33,34]. Unfortunately, aliphatic alkenes, such
as dodec-1-ene and vinylcyclohexane, did not work in the reaction
(4k-4l). Subsequently, some substituted sodium benzenesulphinates
bearing either electron-donating groups (Me) or electron-withdrawing
groups (F, Cl, Br) were used as substrates for the production of the
desired β-ketosulfones in high yields (4m-4p). Moreover, when sodium
aliphatic sulfinates (Me, Et) were used as substrates, the reaction pro-
ceeded smoothly and generated high yields (4q-4 r). However, no ex-
pected product was obtained when sodium trifluomethanesulfonate was
selected as the substrate (4 s).
GOXg
Trace
a
1a (0.5 mmol), 2a (0.5 mmol), H2O (2.0 mL), glucose (1.1 mmol), GOX (42
U/mL), hemoprotein (heme concentration: 0.06 mol %), room temperature,
oxygen, 2 h.
b
Using 0.6 % mmol.
c
d
Not detected.
H2O2 (10 % aqueous solution, 1.0 mmol).
e
Air.
f
g
N2 instead O2.
Added 3 equiv. TEMPO; Abbreviation: MYO (Myoglobin from equine heart);
HgbSw (Hemoglobin from swine blood); HgbBv (Hemoglobin from bovine
blood); HgbRb (Hemoglobin from rabbit blood); Cyt.C Bv (Cytochrome C from
bovine heart); GOX (Glucose oxidase from A. Niger); HRP (Horseradish
peroxidase).
General procedure for the synthesis of β-ketosulfones
To a mixture of alkenes/alkynes (0.5 mmol), sodium benzenesulfi-
nate (0.5 mmol), glucose (1.1 mmol) in water (2 ml), hemoproteins
(heme concentration: 0.06 mol%), GOX (42 U/ml), was added. The re-
action mixture was then stirred at room temperature in a round bottom
flask for 2 h, with oxygen added at a rate of 1 mL/min. The reaction was
monitored by TLC. When the reaction was complete, the crude mixture
was extracted with ethyl acetate. Then the organic phase was dried over
sodium sulfate and concentrated under reduced pressure. Finally, the
desired product was obtained by flash column chromatography with
petroleum ether/ethyl acetate (4/1) as an eluent. All the isolated
products were well characterized by their 1H spectral analysis.
Subsequently, we determined whether alkynes can be reacted with
sodium aryl sulphinates to produce corresponding β-ketosulfones
(Table 3). As expected, substituted phenylethyne can be reacted with
sodium benzenesulphinate in moderate yields under the dual-protein
system (4b-4i). However, all the used alkynes exhibited lower reac-
tivity in this dual-protein-catalyzed reaction. Aliphatic alkynes did not
yield corresponding products (4k-4l). Compared with reactions of sty-
rene in Table 2, similar phenomena were observed when sodium aryl
sulphinates (4m-4p) and sodium aliphatic sulfinates (4q-4 s) were
reacted with phenylethyne. Considering the good substrate generality
and tolerance to various functional groups, this dual-protein-catalyzed
method is more attractive in the practical synthesis of β-ketosulfones
To gain insight into the reaction mechanism, 18O-labeling experi-
ment was performed to elucidate the origin of the carbonyl oxygen atom
of β-ketosulfones (Scheme 3). As demonstrated in eqn.1, the reaction of
1a and 2a under 18O2 for 2 h and 18O-4a were detected. In contrast, only
16O-4a was obtained when the reaction was conducted in H128O (eqn.2).
The experimental result shows that the carbonyl oxygen atom of
β-ketosulfone is derived from dioxygen (MS spectrum, see Fig S1). To
determine the kinetic parameters for HgbRb activity on this protein-
catalyzed reaction, we performed the steady-state reactions under the
following conditions (HgbRb heme concetration: 0.02 mol%, 100 mM
1a, phosphate buffered solution (pH 7.4), 100 mM H2O2, air, 25 ◦C) by
Results and discussion
Initially, styrene (1a) and sodium benzenesulphinate (2a) were
adopted as model substrates for the synthesis of β-ketosulfones in the
GOX-Hgb system. Generally, GOX from A. niger is well known for its
broad applicability as H2O2 producer [40]. Therefore, we attempted to
investigate the effect of hemoprotein under an oxygen atmosphere. As
shown in Table 1, all the hemoproteins possessed catalytic ability to a
certain extent, and hemoglobin from rabbit blood (HgbRb) exhibited the
highest catalytic performance in this reaction (Entry 1). Notably, when
hemin and GOX were combined as catalysts, only a 9 % yield was ob-
tained (Entry 7). No corresponding product was obtained when hemo-
globin, GOX or apo-HgbRb was used as the catalyst (Entry 8–10). These
results indicate that the active conformation and heme center of he-
moglobin play key roles in the synthesis of β-ketosulfones. Compared
with the in situ feeding mode of H2O2 generated by GOX, the stepwise or
3

F. Li et al.
Molecular Catalysis 500 (2021) 111336
Table 2
Oxysulfonylation of alkenes catalyzed by dual-protein systema.
a
1 (0.5 mmol), 2 (0.5 mmol), H2O (2.0 mL), glucose (1.1 mmol), GOX (42 U/mL), HgbRb (heme concentration: 0.06 mol %), room temperature, oxygen, 2 h.
varying the concentration of 2a. The results are shown in Fig S2-S3, and
the obtained kinetic parameters, Vmax and Km are listed in Fig S3.
On the basis of our preliminary results and previous reports [41–43],
we propose a reasonable mechanism for this dual-protein-catalyzed re-
action (Scheme 4). Initially, H2O2 is formed from the reaction of
dioxygen with the FAD (flavin adenine dinucleotide) cofactor of GOX.
Then, the heme of hemoglobin is converted to compound I in the pres-
ence of H2O2, which can produce a sulfonyl radical (II). Thereafter,
sulfonyl radical (II) is added to alkenes or alkynes, generating a reactive
radical (III or III’). Subsequently, peroxy radical (IV or IV’) is generated
from radical (III or III’) captured by dioxygen. The peroxy radical (IV)
can react with radical (III) to produce oxyl radical (V). Finally, the
hydrogen radical abstraction of radical V furnishes corresponding β-keto
sulfone 4. Peroxy radical (IV’) is converted to an intermediate VI, then
undergoes reduction and isomerisation to produce β-ketosulfone 4.
Overall, we successfully constructed an efficient dual-protein system
for the synthesis of β-ketosulfones in water. This method has several
distinct advantages over non-enzymatic methods, such as strong
oxidant-free, high efficiency, broad substrate scope, mild and
environmentally-benign reaction conditions. Therefore, this dual-
protein system offers great prospects for organic chemistry applica-
tions. To enhance the reusability of protein and decrease the cost of this
dual-protein system, the co-immobilization of these two proteins is
ongoing in our laboratory and will be reported in the future.
CRediT authorship contribution statement
Fengxi Li: Conceptualization, Methodology, Formal analysis, Soft-
ware. Jiali Su: Investigation, Resources. Yaning Xu: Visualization, Data
curation. Jiaxu Liu: Writing - original draft. Yue Yu: Writing - review &
editing. Chunyu Wang: Software. Zhengqiang Li: Software. Chen Li:
Supervision. Lei Wang: Supervision.
Declaration of Competing Interest
The authors report no declarations of interest.
4

F. Li et al.
Molecular Catalysis 500 (2021) 111336
Table 3
Oxysulfonylation of alkynes catalyzed by dual-protein systema.
a
2 (0.5 mmol), 3 (0.5 mmol), H2O (2.0 mL), glucose (1.1 mmol), GOX (42 U/mL), HgbRb (heme concentration: 0.06 mol %), room temperature, oxygen, 2 h.
Scheme 3. Labelling experiments.
Scheme 4. The plausible mechanism of this dual-protein system.
5

F. Li et al.
Molecular Catalysis 500 (2021) 111336
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Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.mcat.2020.111336.
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