Using imidazolium-based ionic liquids as dual solvent-catalysts for sustainable synthesis of vitamin esters: Inspiration from bio- and organo-catalysis

Article abstract of DOI:10.1039/c5gc02557e

Vitamin E (VE) has significant biological activities and thus its acylation to increase its stability is of extreme interest. We developed an efficient and sustainable approach using imidazolium-based ionic liquids as dual solvent-catalysts for the esterification between α-tocopherol (the most active form of VE) and succinic anhydride. Although in literature it is reported that lipase can catalyze this reaction, hereby we demonstrate that the reaction observed in DMSO and DMF is catalyzed by the histidyl residues of the protein. Histidine and its analogue containing an imidazole ring were tested as organocatalysts for the production of α-tocopherol succinate. In light of the imidazole organocatalysis, commercially-available 3-alkyl-1-methyl imidazolium ILs [C_nC₁Im][X^-] were investigated as dual solvent-catalysts for the esterification of α-tocopherol with succinic anhydride, and provided satisfactory yields and reaction rates. [C₅C₁Im][NO₃^-] can be recycled by water extraction, instead of organic solvent extraction to separate α-tocopherol succinate from [C₅C₁Im][NO₃^-], with an average yield of 94.1% for 4 subsequent batches, while the catalytic activity of the recycled ILs showed almost no loss after 4 batches. The developed protocol for the synthesis of α-tocopherol esters and IL recycling bears industrial potential due to the ease of use and the efficient recycling.

Full text of DOI:10.1039/c5gc02557e

View Article Online
View Journal
Green
Chemistry
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: Y. Tao, R. Dong, I.
V. Pavlidis, B. Chen and T. Tan, Green Chem., 2015, DOI: 10.1039/C5GC02557E.
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Accepted Manuscripts are published online shortly after
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the Ethical guidelines still
apply. In no event shall the Royal Society of Chemistry be held
responsible for any errors or omissions in this Accepted Manuscript
or any consequences arising from the use of any information it
contains.
www.rsc.org/greenchem

Please do not adjust margins
Green Chemistry
Page 1 of 9
View Article Online
DOI: 10.1039/C5GC02557E
Green Chemistry
Paper
Using Imidazolium-Based Ionic Liquids as Dual Solvent-Catalysts
for Sustainable Synthesis of Vitamin Esters: Inspiration from Bio-
and Organo-Catalysis
Received 00th Oct. 2015,
Accepted 00th Oct. 2015
Yifeng Taoa, †, Ruijuan Dong a, b, †, Ioannis V. Pavlidisc, Biqiang Chen a,*, Tianwei Tan a,*
DOI: 10.1039/x0xx00000x
Vitamin E (VE) has significant biological activities and thus its acylation to increase its stability is of extreme interest. We
developed an efficient and sustainabtle approach using imidazolium-based ionic liquids as dual solvent-catalysts for the
esterification between α-tocopherol (the most active form of VE) and succinic anhydride. Although in literature it is
reported that lipase can catalyze this reactIon, hereby we demonstrate that the reaction observed in DMSO and DMF is
catalyzed by the histidyl residues of the protein. Histidine and analogous containing imidazole ring were tested as
organocatalysts for the production of α-tocopherol succinate. In light of the imidazole organocatalysis, commercially-
available 3-alkyl-1-methyl imidazolium ILs [CnC1Im][X－] were investigated as dual solvent-catalysts for the esterification of
α-tocopherol with succinic anhydride, and provided satisfactory yields and reaction rates. [C5C1Im][NO‒3 ] can be recycled by
water extraction, instead of organic solvents extraction to separate α-tocopherol succinate from [C5C1Im][NO‒3 ], with an
average yield of 94.1% for 4 subsequent batches, while the catalytic activity of the recycled ILs showed almost no loss after
4 batches. The developed protocol for synthesis of α-tocopherol esters and ILs recycle bears an industrial potential due to
the ease of use and the efficient recycling.
www.rsc.org/greenchem
obstacles of biocatalytic approaches especially the
unsatisfactory stability of enzyme for industrial applications
should be overcome2a.
Introduction
The development of sustainable synthesis of chemicals has
strongly required owing to the continuously rising
environmental concerns of conventional chemical approaches.
The use of either biocatalysts or ionic liquids (ILs) as catalysts is
widely deemed to be as the sustainable alternative way to
achieve the concept of green chemistry1. For the cases of
esters synthesis via direct esterification, several green
approaches can be available both from the catalysts and green
Room-temperature ionic liquids become alternative solvents
and catalysts since from an environmental perspective they
offers many advantages including negligible vapor pressure,
designable properties, possibly simplified separation of
products and potential reuse. The majority of ILs reported for
esterification is imidazole or pyridine based derivatives. One
typical and widely used family of this kind ILs is Brønsted acidic
ionic liquids (BAILs); SO3H-fuctionalized imidazolium-based ILs
with acidic counter anion and protonated N-alkylimidazolium
cation has been highlighted as dual solvent-catalysts with
satisfactory conversion rates and selectivity for esterification3,
although the complicate preparation of BAILs may limit their
industrial applications. The produced hydrophobic esters were
immiscible with the hydrophilic ILs so that esters could be
easily separated from ILs by decantation. However,
considering the solubility of substrates in ILs, so far the
synthesized esters were mainly the products of short/medium-
solvents, such as lipase/esterase-mediated catalysis2, using
functionalized ionic liquids as dual solvent-catalysts1b,
1c
.
Lipase/esterase-mediated esterification in non-aqueous
systems possess many advantages compared to the acid/base
catalysis2, as the mild reaction conditions, high selectivity,
creation of less waste, possibility of solvent-free system and
reuse of biocatalyst via immobilization. On other hand, the
chain alcohols with saturated aliphatic acid: ethyl acetate3a,
6
butyl acetate4, glycerol triacetate5 and methyl oleate
.
Compared with ILs, most of these substrates investigated for
esterification were of much lower viscosity7.
Elegant works on esterification in ILs have been carried out
because it was found that most ILs do not inactivate enzyme
like polar organic solvents do8. In such system, ILs mostly
served as the solvents while the enzyme acted as the catalysts
so that structural-complex esters could be synthesized. For
This journal is © The Royal Society of Chemistry 2015
Green Chem., 2013, 00, 1-3 | 1
Please do not adjust margins

Please do not adjust margins
Green Chemistry
Page 2 of 9
View Article Online
DOI: 10.1039/C5GC02557E
Paper
Green Chemistry
instance, a clean lipase-catalyzed process for producing flavor Over the past three decades, a remarkable number of works
esters by direct esterification in switchable ILs/solid phases and publications concern lipase/esterase-mediated catalysis of
was described with almost 100% yield and the enzyme activity transesterification or direct esterification since the essential
was practically unchanged during seven consecutive operation paper16 of Zaks and Klibanov on enzymatic processes in organic
cycles9. However, to our knowledge, comparative study among solvents. We initially studied the lipase-mediated esterification
biocatalysis and process using ILs as catalysts has not been of α-tocopherol with fatty acids, aiming to improve α-
carried out so far.
tocopherol stability. For the esterification of α-tocopherol with
Vitamin E (VE) is a major natural antioxidant and an succinic anhydride, solvents have profound effects on the
essential component of biological membranes. The term VE reaction possibility and rates17, due to the different polarity of
covers a group of 8 isoforms: α-, β-, γ-, δ-tocopherol and α-, β-, these two substrates. When screening organic solvents, the
γ-, δ-tocotrienol. Among all these isoforms, α-tocopherol reactions were highly solvent-dependent (Supporting
shows the highest VE activity10. However, VE is unstable and its Information, Table S1), as described in literature14c. Commonly
antioxidant value is reduced by light, air or oxidizing chemicals. applied organic solvents such as hexane, tert-amyl alcohol,
Aiming to improve the stability of VE, acetylation of VE was tert-butanol and acetonitrile are not good media for these
suggested11. The stable derivatives of VE, such as VE succinate, reactions whereas they were efficiently performed in DMSO
have the same biological activity as VE and highlighted as high- and DMF.
12
selective anticancer agents10,
, efficiently inducing the
apoptosis of cancer cells via the mitochondrial route13. The
hydrophobicity of VE due to the long alkyl chain leads to the
immiscibility of VE in most hydrophilic BAILs, resulting in the
low or even no conversion of VE esterification in such ILs. The
esterification of vitamins is a topic that draws significant
attention in the hydrolase community, due to the ability of
lipases to catalyze the acylation in non-conventional media.
Lipase-catalyzed synthesis of VE succinate in molecular
solvents is significantly affected by the solvent due to the
opposite polarity between VE and succinic anhydride: high
yield was obtained in aprotic polar solvents like dimethyl
sulfoxide (DMSO) and N,N-dimethyl formamide (DMF), but
almost no reactions in conventional organic solvents 11, 14. Until
now, the catalytic activity in DMSO and DMF was thought by
the contribution of the lipase, while any possible chemical
acylation reaction was neglected and never reported in these
studies although DMSO and DMF is believed with high
denaturation capacity to the enzyme in most literature15.
Herein, we firstly demonstrated whether the reaction
observed in DMSO and DMF is contributed by the catalytic
activity of lipase or not. We found that the undergoing cause
for such reactions in DMSO is chemical catalysis of histidyl
residue in protein, so that histidine and analogous containing
imidazole ring were tested as organocatalysts for the
production of α-tocopherol succinate. Bearing in mind of green
chemistry, an efficient and sustainable approach using
commercially-available 3-alkyl-1-methyl-imidazolium ionic
liquids as dual solvent-catalysts was investigated. To enhance
the green impact of the IL catalyzed reaction, we developed a
water extraction protocol, instead of organic solvents
extraction method, so that the IL can be reused. The reaction
efficiency, final yield and product separation of these
strategies (Scheme 1) were compared and the distinct
mechanisms of histidine and imidazolium-based ILs catalysis
were proposed.
Scheme 1. Three strategies (1) biocatalysis, (2) organocatalysis and (3) using ionic
liquids as dual solvent-catalysts for the esterification of vitamin
anhydride.
E with succinic
Fig. 1 Comparison of heat-inactivated (120 °C, 20 min) and original lipase catalyzing the
esterification of α-tocopherol succinate in DMSO. TLIM: Lipozyme TLIM; RMIM:
Lipozyme RMIM; CaLB: Candida antarctica Lipase B.
Results and discussion
Plausible lipase-mediated synthesis of vitamin E succinate in
DMSO
2 | Green Chem., 2015, 00, 1-3
This journal is © The Royal Society of Chemistry 2015
Please do not adjust margins

Please do not adjust margins
Green Chemistry
Page 3 of 9
View Article Online
DOI: 10.1039/C5GC02557E
Green Chemistry
Paper
Table 1 Yields of several proteins catalyzing the esterification of α-tocopherol succinate
proper folding of the polypeptide chain, mostly due to the
specific environment that the neighboring residues are
producing. For instance, hydrolases perform their activities via
a catalytic triad usually formed by serine, histidine and
aspartate/glutamate residues20. The results of the previous
paragraph gave an insight that the histidine can be an effective
esterification catalyst for the synthesis of vitamin esters.
Catalysts
Protamine
Pepsin
Yields (%)
0
Percentage of histidine residuea (%)
0.0
0.3
2.7
2.8
2.8
0
Glucose oxidase
Catalase
61.9 ±0.5
65.8 ±0.8
70.0 ±0.6
Table 2. Histidine derivatives/analogues tested as catalysts for esterification of α-
tocopherol with succinic anhydride
Initial rate a
(10-2/min)
Yield after
24hb (%)
Entry
Compound
BSA
1
2
3
10.8 ±0.2
4.4 ±0.1
3.1 ±0.2
98.1 ±1.3 c
97.2 ±1.5 c
91.7 ±1.9 c
a
the values were calculated from the sequence analysis in NCBI
(http://www.ncbi.nlm.nih.gov/protein/).
Over the last years growing efforts on the improvement of
the enzymatic processes in anhydrous DMSO and DMF via
protein engineering, immobilization and computational
chemistry were documented. It was reported that the lipases’
activities in neat DMSO can be maintained and even promoted
4
5
1.1 ±0.1
89.1 ±0.6
86.9 ±0.8
0.73 ±0.06
through lipase modification14a,
or immobilization14b,
.
18
19
6 d
0.20 ±0.02
45.7 ±0.6
However, DMSO and DMF, which are known as universal
solvents with high denaturation capacity15a, not only strip
essential water molecules from the enzyme, but also
7
0.25 ±0.03
0.17 ±0.01
45.3 ±0.4
39.8 ±0.4
8 d
dissociate the enzyme tertiary structure leading to enzyme
unfolding and subsequent deactivation15b,
15c
.
This
9 d
10
11
0.11 ±0.01
n.d.e
28.5 ±0.3
n.d. e
conventional wisdom still holds owing to our unexpected
results: several heat-inactivated (120 °C, 20 min) lipases from
different sources can perform the esterification of α-
tocopherol with succinic anhydride in neat DMSO, as shown in
Fig. 1. More interestingly, heat inactivated proteins and
enzymes of other classes, like glucose oxidase and catalase can
perform the reaction (Table 1). Surprisingly, some of the heat-
denatured samples exhibited higher activity than the native
enzymes. It is expected that the unfolding, as result of the
heating process, renders the histidine residues of the protein
core accessible, while in the folded protein only the histidine
residues on the surface of enzyme were available for the
catalysis.
Xanthine; Adenine,
Thymine, Uracil, Guanine
n.d. e
n.d. e
a
initial rate refers to moles of product formatted per moles of catalyst per
b
minute; Conditions: histidine derivatives (30 %, mol of α-tocopherol) in 1 mL
DMF containing 0.2 M α-tocopherol and 0.8 M succinic anhydride was incubated
at 800 rpm, 50 °C in dark and under nitrogen atmosphere for 24 h and yield was
c
d
determined through HPLC analysis, using an external standard; Yield at 9 h;
these catalysts are slightly soluble in DMF; e not detectable.
Reacting L-histidine (30 %, mol towards α-tocopherol) in 1
mL DMF containing 0.2 M α-tocopherol and 0.8 M succinic
anhydride at 50 °C for 24 h led to 40% yield of α-tocopherol
Subsequently, we investigated the amino acid sequences
and found that the activity of the heat inactivated protein was
in line with the histidine content (Table 1); no reaction was
observed for protamine, a protein without histidine residues.
At the same time, enzymes that are not reported to catalyze
esterification reactions, such as glucose oxidase, where quite
efficient on this specific reaction, due to their histidine content.
In summary, these results indicated that the underlying cause
for esterification of α-tocopherol esters in DMSO is not a
lipase-catalyzed reaction, but an organocatalysis.
succinate (Table 2, entry 8). This result was quite remarkable
since histidine is slightly soluble in neat DMF (up to about 0.06
M histidine). The efficiency of histidine-catalyzed esterification
in aprotic polar solvents followed the order: DMSO > DMF >
dimethylacetamide
>
tetrahydro-1,3-dimethyl-
2(1H)pyrimidine > 1,3-dimethyl-2- imidazolidinone. It needs to
be stated that the medium of histidine-mediated esterification
gradually became homogeneous in anhydrous DMSO,
indicating a ring-open reaction of succinic anhydride with
histidine. This was not observed in DMF, thus this solvent is
expected to be more suitable, regarding the efficiency while
minimizing the ring opening of the anhydride.
Organosynthesis of vitamin E succinate
Enzymatic transformations generally have a chemical catalysis
counterpart, but amino acids as the building blocks composing
the protein, rarely fulfill their chemical counterpart roles.
Typically, amino acids exhibit their catalytic potency after
We also compared commercially available histidine
analogues and derivatives under the standard conditions with
30 % mol (towards α-tocopherol) of catalyst (Table 2).
This journal is © The Royal Society of Chemistry 2015
Green Chem., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
Green Chemistry
Page 4 of 9
View Article Online
DOI: 10.1039/C5GC02557E
Paper
Green Chemistry
Histamine (Entry 1) exhibited high initial rate and yield; this shown in Table 3 were selected in our work to avoid the
could be due to the better solubility in DMF, owing to the complicate preparation of functionalized BAILs. This simple ILs
absence of the carboxylic group. Although the final product (α- family was previously reported mostly as the solvents for
tocopherol succinate) was obtained and confirmed by NMR, enzymatic catalysis and already commercially available1b,
23
.
we believed that exposure of histamine to succinic anhydride The high viscosity of ILs limiting the mass transfer rate would
in DMF will form the terminal N-succinoyl derivative as be the first challenge to the use of the ILs during the synthesis
intermediate. It was reported that succinoylhistamine was of α-tocopherol succinate; the resulted ester with a free
formed with a yield of 90% at room temperature within 1.5 h carboxylic group due to the use of anhydride may become
in DMF and a white precipitate formed21. However, in our miscible with ILs, which would increase the difficulty of the
cases, no white precipitate was observed at 50 °C, probably product separation and ILs recycle. Herein, we screened
due to the further usage of the succinic moiety for the several imidazolium based ILs and developed an easy way to
acylation. The possibly formed N-succinoyl derivative arise the reuse the ILs via extraction using water.
question: what is the actual acylation catalyst? We assumed
Table 3 Imidazolium based ionic liquids as dual solvent-catalysts for esterification of
that both the imidazole ring and the amine group are essential
vitamin E with succinate
for the efficient catalysis to occur.
Initial rate a Yield after
Modification of side chains of the imidazole ring (Entries 1-5
and 7-10) resulted in the change of catalyst solubility in the
organic solvents, but also in its reactivity: the presence of a
Entry
ILs
Name
(10-2/min)
1.5 h b (%)
12
13
14
15
[C5C1Im][NO‒3]
[C5C1Im][I‒]
0.69 ±0.12
96.2 ±1.6
carboxylic group led to the decrease of solubility (Entry
6, 8
0.56 ±0.15
0.17 ±0.03
0.33 ±0.06
89.8 ±2.1
44.7 ±1.6
50.8 ±1.5
and 9), while the incorporation of a thiol group diminished any
[C5C1Im][Cl‒]
[C6C1Im][Cl‒]
activity. Interestingly, neither purines nor pyrimidines (Entry
11) exhibited any catalysis, although they possess a similar
structure as imidazole ring. Different kinds of anhydrides and
fatty acids were tested as acyl donor: only anhydrides can be
activated in DMSO or DMF and thus act as the acyl donor, but
none of fatty acids (data not shown). Reactions of vitamin A
and vitamin C with succinic anhydride (Scheme S1) were
further carried out to extend the applications of histidine-
catalyzed esterification. Although that histidine was able to
perform the esterification of both vitamins, no regioselectivity
was observed in the case of vitamin C (Fig. S1).
C6H13
Cl
N
N
16
17
18
19
[C7C1Im][Cl‒]
[C8C1Im][Cl‒]
[C10C1Im][Cl‒]
0.62 ±0.05
0.05 ±0.01
0.20 ±0.01
0.10 ±0.01
57.7 ±2.1
33.8 ±0.9
54.6 ±1.2
28.9 ±0.9
[C5C1Im][PF‒6 ]
[C5C1Im][BF‒4 ]
Although the use of histidine derivatives/analogues as
catalysts for these acylation reactions brings several benefits
related to their simplicity, low price and availability, the
purification of the produced ester for organocatlysis is really
difficult via an organic solvent extraction with from DMSO or
DMF. The recycle of chemical catalysts is not easy but except
for immobilization of the catalysts.
[C5C1Im][BF‒4 ]
[C2C1Im][NTF‒2 ]
[C3NC1Im][NTF‒2 ]
20
n.d.c
n.d. c
a
initial rate refers to moles of product formatted per moles of catalyst per
b
Imidazolium-based ionic liquids as dual solvent-catalysts for
synthesis of vitamin E succinate
minute; Conditions: 4 mM ILs, 0.5 mM α-tocopherol and 1 mM succinic
anhydride was incubated at 800 rpm, 50 °C in dark and under nitrogen
atmosphere for 3 h and yield was determined through HPLC analysis, using an
external standard; c not detectable.
The application of ILs as both solvent and catalysts for the
esterification of α-tocopherol with succinic anhydride become
an alternative sustainable approach since enzymatic process
are not available for these substrates as discussed before.
Considering the importance of ILs properties e.g. dissolving
ability, viscosity, etc. for the possibility and efficiency of α-
tocopherol succinate synthesis, an appropriate choice of the
anion and the cation of ILs is necessary. BAILs containing an
alkane-sulfonic acid group attached to an imidazole or
pyridinium cation and bearing acid counteranions are of
special importance as the most used ILs for the esterification
process because they possess simultaneously the proton
Initial rates and yields of the selected imidazolium based ILs
for the synthesis of α-tocopherol succinate is shown in Table 3.
Investigation of ILs anions (Entry 12-14, 19 and 20) on the
efficiency of α-tocopherol esterification showed that the most
‒
effective nucleophilic anion was NO3 among all the studied
anions although these anions may slightly change the ILs
viscosity. α-Tocopherol was found immiscible with the
‒
‒
hydrophilic ILs containing BF4 and NTF2 , resulting in no
reactivity. It is worthy pointing that the initial rate depends on
the substrates’ concentration: when millimole ratio of α-
tocopherol: Succinic anhydride: ILs was 4: 8: 4, the initial rate
when using [C5C1Im][NO‒3 ] reached 2.86× 10ꢀꢁ min‒1 (See
supporting information Table S1, Entry S5), which is
acidity and the characteristic properties of an ionic liquid3a, 22
.
However, in light of organocatalysis using imidazole as
catalyst, imidazolium based ILs without any modifications as
4 | Green Chem., 2015, 00, 1-3
This journal is © The Royal Society of Chemistry 2015
Please do not adjust margins

Please do not adjust margins
Green Chemistry
Page 5 of 9
View Article Online
DOI: 10.1039/C5GC02557E
Green Chemistry
Paper
1
comparable with the values of organocatalysts. However, difference to the original one from the comparison of H and
respecting to the productivity, the approach using ILs as dual 13C NMR test in DMSO-d6 (SI, Fig. S4). Thus, the developed
solvent-catalysts showed higher efficiency both than protocol for synthesis of α-tocopherol esters and ILs recycle is
biocatalysis and organocatalysis.
practicable and applicable.
The change of substituted alkyl group on imidazole ring of
In order to highlight the applicability of the developed
cations mainly resulted in the change of ILs viscosity: process, we investigated the synthesis of several vitamin
previously study reported that an increase in the van der esters with different acyl donors in [C5C1Im][NO‒3 ]. Vitamin A,
Waals forces primarily contributed to the increase in viscosity C and E could be easily modified by esterification with several
of ILs24. In agreement with this statement, in the 3-alkyl-1- acyl donors; however, it is clear that the formation as an
methylimidazolium [PF‒6 ] series, viscosity increases as the anhydride is
a pre-requirement for the success of the
number of carbon atoms in the linear alkyl group is transformation (Table S3). However, it needs to be mention
increased25.
However,
in
our
work,
3-alkyl-1- that in the case of the vitamin C, where multiple hydroxyl
methylimidazolium [Cl‒] series showed more complicated groups are available, no regioselectivity was observed.
behavior, resulting in the disordered reactivity: there was no
linear relationship between reactivity and the substituted Proposed catalytic mechanism
number of carbon atoms, while [C7C1Im][Cl‒] showed the best
Lipase-mediated esterification occurs via the formation of a
reactivity for α-tocopherol esterification.
tetrahedral acyl-enzyme intermediate, which undergoes a
The reuse of ILs is necessary due to its relative high cost and
nucleophilic attack from the second substrate, leading to the
so far several ILs have been demonstrated with feasible
product formation and release of the product, regenerating
recyclability merely via decantation of esters3c, 5, 26 because
Table 4 Reusability of [C5C1Im][NO‒3 ] ionic liquid for esterification of vitamin E with
hydrophobic esters are mostly immiscible with the used ILs.
When using [C5C1Im][NO‒3 ] as dual solvent-catalyst for α-
succinate
tocopherol esterification in its first batch, the reaction mixture
tends to spontaneously separate into two layers, and the
recycled yield of ILs was around 72.5±1.8%; unfortunately and
surprisingly, in the second batch, homogenous mixture was
observed even after centrifugation under 10,000 g for 30 min.
1-alkoxymethylimidazolium lactates was synthesized and
lactate could serve as the anion27. Herein, we assumed that
the resulted free carboxylic group of α-tocopherol succinate
owing to the use of succinic anhydride may act as anion to
coordinating with imidazolium cations. Consequently, this ionic
liquid became miscible with the reactants.
Initial rate
(10-2/min)
Yield after
1.5 h (%)
Recycled yield
Runs
of ionic liquid (%)
1
2
3
4
0.69 ±0.12
0.67 ±0.08
0.66 ±0.09
0.66 ±0.05
96.2 ±1.6
95.8 ±1.2
93.8 ±2.3
91.5 ±1.0
95.2 ±2.1
96.3 ±1.8
92.8 ±1.5
93.5 ±0.8
the free enzyme28. In the catalytic triad of lipases, the basic
nitrogen of histidine abstracts a proton from serine to activate
it as nucleophile. As discussed before, the approach using
lipase as catalyst for the esterification of vitamin E with
succinic anhydride was not contributed by the lipase but the
imidazole ring of histidyl residue in proteins. Thus, here we
discuss the mechanism based on imidazole catalysis.
Hence, extraction of products by solvents became an
alternative choice. Firstly, we used organic solvents, like 10
volumes ethyl acetate or diethyl ether to extract VE and α-
tocopherol succinate from the IL while succinic anhydride was
firstly removed by centrifugation because it became insoluble
in ILs with the decrease of temperature. Although high isolated
yield with 92.1±1.2% of α-tocopherol succinate could be
obtained, the extraction by organic solvents is not compatible
to the concept of green chemistry. Water as solvent is strong
environmentally favorable with respect to safety, cost and
sustainability. To our delight, [C5C1Im][NO‒3 ] is totally miscible
with water while the other reactants are not. After the
removal of succinic anhydride by centrifugation, addition of 2-
3 times volume water into the reaction mixture recycled
almost 95.2±2.1% ionic liquid after removing water via
Imidazole is known as an ester hydrolysis catalyst29.
Imidazole carbamates and ureas acting as catalysts and
substrates mediated the chemoselective esterification and
amidation of carboxylic acids in acetonitrile30. Peptides or
polymers containing histidine residues were reported to have
the hydrolytic activity on p-nitrophenyl esters31 and the
activity on Michael additions32, aldol reactions33,
oligomerisation34, etc. However, the proposed mechanisms for
these catalysts were remarkable different from the one of the
lipase-catalyzed reaction. Imidazole carbamates catalyzed
esterification was supported to a mechanism involving an
evaporation under 80
Pa).
°C and reduced pressure (around 100
activated ester intermediate30b
.
After the removal of water, the reusability of [C5C1Im][NO‒3 ]
ionic liquid was investigated. As summarized in Table 4, about
94 ± 2 % is the recovery of the IL after all batches tested. Initial
rate of α-tocopherol converted into α-tocopherol succinate for
4 batches was almost similar but yield was slightly decreased
due to the loss of ionic liquid in each batch. Meanwhile, the
recovered [C5C1Im][NO‒3 ] ionic liquid showed no structural
Being cognizant of the early reported works30b, 31a, 35, we
currently propose this histidine-mediated esterification via
acyl-imidazole intermediate (Scheme 2). The imidazole moiety
is expected to be partly deprotonated in DMSO or DMF since
the pKa of imidazole in DMSO is 18.636. As a result of the
participation of the electron pair of the amide nitrogen in the
This journal is © The Royal Society of Chemistry 2015
Green Chem., 2013, 00, 1-3 | 5
Please do not adjust margins

Please do not adjust margins
Green Chemistry
Page 6 of 9
View Article Online
DOI: 10.1039/C5GC02557E
Paper
Green Chemistry
π-electron system of the ring, tautomeric forms in five- Candida sp. was produced by Beijing CTA New Century
membered N-heterocycles happen37, leading to the possibility Biotechnology Co., Ltd. Lipases including Lipozyme TLIM,
that nucleophilic attack can be alternatively accomplished by Lipozyme RMIM, Candida antarctica lipase B were purchased
one of the electronegative nitrogen atoms in the imidazole from Novozymes®. The other proteins were commercially
ring. The first step of the substitution pathway, involving available. All 3-alkyl-1-methyl-imidazolium ionic liquids with a
nucleophilic attack by the imidazole ring on the anhydride purity of 99% were purchased from the center for Centre for
carbonyl and the leaving of the other carbonyl group forms the Green Chemistry and Catalysis, Lanzhou Institute of Chemical
acyl-imidazole intermediate. The subsequent transition begins Physics, Chinese Academy of Sciences (Lanzhou, China).
with a SN2 nucleophilic attack on the carbonyl by the hydroxyl Methanol of chromatographic grade was purchased from
group of α-tocopherol and ends with the formation of final Sigma-Aldrich. The other solvents and salts of analytical grade
product through expelling the catalyst. The amine group of were obtained from Beijing Chemical Factory.
histamine may be properly coordinating the acyl-imidazole
intermediate, resulting in the significant increase of reaction
rate.
Understanding the mechanism of catalysis by ILs at the
molecular level is crucial for the rational design of ILs due to
the impossibly experimental study even a small fraction of the
potential cations-anions combinations38, but the mechanism of
nucleophilic substitution by ILs seems more complicate. It was
recognized that C2 of 1,3-alkyl-imidazolium anions is positively
charged due to the electron deficit in the C=N bond whereas
the other carbons are practically neutral38-39. This resulting
acidity of C2 hydrogen atom is the key to understanding the
mechanism of ILs catalysis.
As shown in scheme 3, the imidazolium ILs initiates the
esterification by donating the C2 proton to the oxygen atom of
anhydride, inducing the electrophilic activation of anhydride
Scheme 2. Proposed acyl-imidazole mechanism of the histidine-catalyzed
carbonyl. The subsequent necleophilic attack on carbonyl
esterification of vitamin E with succinic anhydride.
following a SN1 mechanism by the alcohol was proposed for ILs
catalyzed synthesis of biodiesel40 or lipophilic esters41 in
previous literatures, in which the nucleophilic role of anions
was neglected. Chakraborti et al.42 proposed an “electrophile
nucleophile dual activation” role of the [C4C1Im][CH3COO－] in
catalyzing O-tert-butoxycarbonylation of 2-naphthol. They
concluded that counteranions also involved in the cooperative
hydrogen bonds and charge-charge interactions with both
substrates. Welton et al.43 proposed that the hydrogen bond
basicity of the ILs, controlled by the anions, is the dominant
factor in determining the esterification rate. In view of these
crucial issues and considering the weak necleophilic attack
ability of phenol hydroxyl group of α-tocopherol due to the
shielding effect of two neighbor methyl groups, we promote
that the anions in ILs performs the necleophilic attack on
anhydride carbonyl in the first step, forming an anion-
anhydride intermediate after the leaving of the other carbonyl Scheme 3. Proposed mechanism for esterification of α-tocopherol with succinic
anhydride using 3-alkyl-1-methylimidazolium ionic liquids as dual solvent-catalysts.
group, and then the second step following a SN2 mechanism
starts with a nucleophilic attack on the carbonyl by the
Esterification reactions
hydroxyl group of α-tocopherol and ends with the formation of
final product through expelling the anion.
For biocatalysis, 40 mg protein was added into 1 mL DMSO
containing 0.2 M α-tocopherol and 0.8 M succinic anhydride at
50 °C for 24 h; for organocatlysis, reacting L-histidine or its
analogues (30 %, mol towards α-tocopherol) in 1 mL DMF
containing 0.2 M α-tocopherol and 0.8 M succinic anhydride
was carried out at 50 °C for 24 h; for experiments using ILs as
dual solvent-catalysts, a standard mixture containing 0.5 mmol
α-tocopherol, 1 mmol succinic anhydride and 4 mM ionic
liquids was used and the reactions were carried out at 50 °C
Experimental
General aspects
Vitamin
E containing 96% α-tocopherol and succinic
anhydride with a purity of 99% were obtained from Aladdin
Industrial Inc. (Shanghai, China). Lipase formulation from
6 | Green Chem., 2015, 00, 1-3
This journal is © The Royal Society of Chemistry 2015
Please do not adjust margins

Please do not adjust margins
Green Chemistry
Page 7 of 9
View Article Online
DOI: 10.1039/C5GC02557E
Green Chemistry
Paper
for 3 h. All reactions were investigated in dark and under catalysts for these acylation reactions brings several benefits
nitrogen atmosphere. Samples were obtained at scheduler related to their simplicity, low price and availability, the
time and yield was determined through HPLC analysis (Thermo purification of the produced ester for organocatlysis is really
Scientific Dionex Ultimate 3000 equipped with an AcclaimTM difficult via an extraction with organic solvent from DMSO or
C18 column), using an external standard. Initial rate refers to DMF. The recycle of chemical catalysts is not easy but it can
moles of product formatted per moles of catalyst per minute become even more appealing by immobilizing the catalyst to
when yield was under 20%. α-Tocopherol succinate yield was be able to reuse it, or even use cheap sources of histidine, such
calculated from the ratio between observed and theoretical as commercial soymeal.
production of α-tocopherol succinate.
Bearing in mind of green chemistry and in light of the
imidazole organocatalysis, we developed an efficient and
sustainable strategy using 3-alkyl-1-methylimidazolium ILs as
dual solvent-catalysts for the synthesis of α-tocopherol
succinate, with satisfactory yields and reaction rates.
[C5C1Im][NO‒3 ] ionic liquid can be recycled by water extraction
with an average regeneration yield of 94.1%. Initial rate of α-
Recyclability of ionic liquids
The usability of biocatalysts and organocatalysts were not
investigated due to the difficulty of the catalysts recycle.
Considering the relative high cost of ILs, the recyclability study
of ILs is important and necessary; however, decantation of
products for recycling ILs was not available for our selected ILs.
To our delight, [C5C1Im][NO3‒ ] is totally miscible with water
while the reactants are not. After the removal of succinic
anhydride by centrifugation at room temperature, the reaction
mixture was strongly mixed after the addition of 2-3 times
volume deionized water. After removing water via evaporation
tocopherol converted into α-tocopherol succinate for
4
batches was almost similar but yield was slightly decreased
due to the small amount loss of ionic liquid in each batch,
indicating that the catalytic activity of recycled ILs was
unaffected. The developed protocol for synthesis of VE esters
and ILs recycle is practicable and applicable.
under 80 °C and reduced pressure (around 100 Pa), the
reusability of [C5C1Im][NO‒3 ] ionic liquid was investigated under
Although imidazole ring is one of the main structural part
composing histidine and cations of imidazolium ILs, the
catalytic mechanism significantly differs from histidine-
catalyzed to imidazolium ILs catalyzed esterification. We
proposed an acyl-imidazole intermediate for histidine
catalyzed esterification, whereas for ILs catalyzed
esterification, the role of anions of imidazolium ILs was
promoted as performing the nucleophilic attack while the
esterification was initiated by donating the proton of C2 on
imidazole ring to the oxygen atom of anhydride.
the standard procedure.
Purification
Purification of vitamin
E
succinate for biocatalysis and
(a) 1
organocatalysi approaches was proceeded as following
:
mL of the reaction medium was added to 2 mL diethyl ether
and incubated overnight under ‒18 °C; (b) after centrifugation
at 8000
added to 1.5 mL deionized water, and then mixed by vortex for
1 min; (c) after centrifugation at 8000 g for 2 min, the
×g for 2 min, the organic phase was removed and
×
Moreover, we have highlighted that the lipase-catalyzed
esterification reactions taking place in aprotic polar solvents
should be treated with caution, as the histidines of the protein
(not necessarily the one of the active site) could perform the
reaction as chemical catalysts. Thus, we would suggest that in
such works, the imidazole concentration after purification of
lipases via His-Tag should be titrated, in order to be able to
exclude the potential of chemical catalysis via the imidazole
used for the elution of the protein.
organic phase was carefully transferred into a new tube and
then dried with Na2SO4; (d) after the removal of solvent in a
reduced-pressure rotary evaporator, 400 μL n-hexane were
added to dissolve the unreacted α-tocopherol further
purification of the final isolated ester because α-tocopherol
succinate is not soluble in n-hexane; (e) Centrifuging at 8000
×
g for 2 min again, the liquid was discarded and after dried by
nitrogen, white powder was obtained for further
characterization.
For the approach using ILs as dual solvent-catalysts, white
solid was obtained after the removal of anhydride and the
recycle of ILs. 500 μL n-hexane were added to dissolve the
unreacted α-tocopherol further improving the purity of final
ester.
Acknowledgements
The authors acknowledge the financial support of the National
Basic Research Program of China (973 program:
2013CB733600), and of the National Natural Science
Foundation of China (21436002, 21576019). The authors
especially thank Prof. Dr. Uwe T. Bornscheuer (Dept. of
Conclusions
Biotechnology
&
Catalysis, Institute of Biochemistry,
Comparative study among bio-, organocatalysis and using ILs
as dual solvent-catalysts for the direct esterification of vitamin
E with succinic anhydride was carried out. Each approach
possesses its own advantages and disadvantages. It is
demonstrated that lipase-catalyzed esterification in DMSO or
DMF was not attributed to the catalytic activity of lipase, but
the chemical catalysis of the histidyl residue in protein.
Although the use of histidine derivatives/analogues as
Greifswald, Germany) for valuable comments and fruitful
discussions on this work.
References
1 (a) S. Wenda, S. Illner, A. Mell and U. Kragl, Green Chem, 2011,
13, 3007-3047; (b) H. Olivier-Bourbigou, L. Magna and D.
This journal is © The Royal Society of Chemistry 2015
Green Chem., 2013, 00, 1-3 | 7
Please do not adjust margins

Please do not adjust margins
Green Chemistry
Page 8 of 9
View Article Online
DOI: 10.1039/C5GC02557E
Paper
Green Chemistry
Morvan, Appl Catal A gen, 2010, 373, 1–56; (c) Q. Zhang, S. 20 (a) L. Brady, A. M. Brzozowski, Z. S. Derewenda, E. Dodson, G.
Zhang and Y. Deng, Green Chem, 2011, 13, 2619-2637.
2 (a) M. T. Reetz, J Am Chem Soc, 2013, 135, 12480-12496; (b) A.
I. Brígida, P. F. Amaral, M. A. Coelho and L. R. Gonçalves, J Mol
Catal B Enzym, 2014, 101, 148-158.
Dodson, S. Tolley, J. P. Turkenburg, L. Christiansen, B. Huge-
Jensen and L. Norskov, Nature, 1990, 343, 667-770; (b) J. D.
Schrag, Y. Li, S. Wu and M. Cygler, Nature, 1991, 351, 761-764.
21 H. Marcel, Org Biomol Chem, 2013, 11, 5162-5172.
3 (a) A. C. Cole, J. L. Jensen, I. Ntai, K. L. T. Tran, K. J. Weaver, D. 22 A. R. Hajipour and F. Rafiee, Org Prep Proced Int, 2010, 42
C. Forbes and J. H. Davis, J Am Chem Soc, 2002, 124, 5962- 285-362.
5963; (b) T. Joseph, S. Sahoo and S. Halligudi, J Mol Catal A 23 J. P. Hallett and T. Welton, Chem Rev, 2011, 111, 3508-3576.
chem, 2005, 234, 107-110; (c) X. Han, H. Du, C. Hung, L. Liu, P. 24 X. Wu, L. M. Wang, R. A. Nieman and C. A. Angell, J Phys
,
Wu, D. Ren, S. Huang and S. Liu, Green Chem, 2014, 17, 499-
Chem B, 2003, 107, 11749-11756.
508.
25 D. S. V. and B. R. A., Chemphyschem, 2002, 3, 161-166.
4 H. Zhu, F. Yang, J. Tang and M. He, Green Chem, 2003, 1, 38-39. 26 H. Zhang, F. Xu, X. Zhou, G. Zhang and C. Wang, Green Chem,
5 X. Liu, H. Ma, Y. Wu, C. Wang, M. Yang, P. Yan and U. Welz-
2007,
9, 1208-1211.
Biermann, Green Chem, 2011, 13, 697-701.
27 J. Pernak, I. Goc and I. Mirska, Green Chem, 2004, 6, 323-329.
6 A. H. M. Fauzi and N. A. S. Amin, Energ Convers Manage, 2013, 28 H. Beer, G. Wohlfahrt, J. Mccarthy, D. Schomburg and R.
76, 818-827. Schmid, Protein Eng, 1996, , 507-517.
7 (a) S. Zhang, N. Sun, X. He, X. Lu and X. Zhang, J Phys Chem Ref 29 W. P. Jencks and J. Carriuolo, J Biol Chem, 1959, 234, 1272-
9
Data, 2006, 35, 1475-1517; (b) G. Yu, D. Zhao, L. Wen, S. Yang
and X. Chen, AIChE Journal, 2012, 58, 2885-2899.
1279.
30 (a) S. T. Heller and R. Sarpong, Org Lett, 2010, 12, 4572-4575;
(b) S. T. Heller and R. Sarpong, Tetrahedron, 2011, 67, 8851-
8859.
8 (a) S. Park and R. J. Kazlauskas, Curr Opin Biotech, 2003, 14
,
432-437; (b) C. Aouf, E. Durand, J. Lecomte, M. C. Figueroa-
Espinoza, E. Dubreucq, H. Fulcrand and P. Villeneuve, Green 31 (a) K. G. Byler, Y. Li, R. A. Houghten and K. Martinez-Mayorga,
Chem, 2014, 16, 1740-1754.
Org Biomol Chem, 2013, 11, 2979-2987; (b) G. Chadha and Y.
Zhao, Org Biomol Chem, 2013, 11, 6849-6855; (c) Y. Liu, X.
Meng, J. Li and X. Li, Colloid Surface A, 2013, 436, 839-845; (d)
9 (a) P. Lozano, J. M. Bernal and A. Navarro, Green Chem, 2012,
11, 3026-3033; (b) P. Lozano, J. M. Bernal, E. Garcia-Verdugo,
G. Sanchez-Gomez, M. Vaultier, M. I. Burguete and S. V. Luis,
Green Chem, 2015, 17, 3706-3717.
H. Yang and J. C. Sherman, Bioorg Med Chem Lett, 2013, 23
,
1752-1753.
10 K. N. Prasad, B. Kumar, X.-D. Yan, A. J. Hanson and W. C. Cole, 32 K. Akagawa, N. Sakai and K. Kudo, Angew Chem Int Edit, 2015,
J Am Coll Nutr, 2003, 22, 108-117.
54, 1822-1826.
11 P. Torres and D. Reyes, Process Biochem, 2008, 43, 145–153.
12 (a) Y. Zhang, J. Ni, E. M. Messing, E. Chang, C.-R. Yang and S.
33 S. Bayat, B. A. Tejo, A. B. Salleh, E. Abdmalek, Y. M. Normi and
M. B. A. Rahman, Chirality, 2013, 25, 726-734.
Yeh, P Natl Acad Sci USA, 2002, 99, 7408-7413; (b) N. Duhem, 34 R. Wieczorek, M. Dörr, A. Chotera, P. L. Luisi and P.-A.
F. Danhier and V. Préat, J Control Release, 2014, 182, 33-44. Monnard, Chembiochem, 2013, 14, 217-223.
13 (a) J. Truksa, L. Dong, J. Rohlena, J. Stursa, M. Vondrusova, J. 35 L. Mandell, J. Moncrief and J. Goldstein, Tetrahedron, 1963,
Goodwin, M. Nguyen, K. Kluckova, Z. Rychtarcikova and S. 19, 2025-2030.
Lettlova, Antioxid Redox Sign, 2015, 22, 883-900; (b) B. Yan, M. 36 F. G. Bordwell, Accounts Chem Res, 1988, 21, 456-463.
Stantic, R. Zobalova, A. Bezawork-Galeta, M. Stapelberg, J. 37 (a) W. F. Reynolds, I. R. Peat, M. H. Freedman and J. R. Lyerla,
Stursa, K. Prokopova, L. Dong and J. Neuzil, BMC Cancer, 2015,
15, 401-412.
J Am Chem Soc, 1973, 95, 328-331; (b) H. Saito, Y. Tanaka and
S. Nagata, J Am Chem Soc, 1973, 95, 324-328.
14 (a) C. Yin, Z. Cong and M. Gao, Chinese J Chem Eng, 2011, 19
,
38 W. Hermann, Angew Chem Int Edit, 2008, 47, 654-670.
135-139; (b) Y. Hu, X. Jiang, S. Wu, L. Jiang and H. Huang, 39 R. S. Mohan, S. Chowdhury and J. L. Scott, Tetrahedron, 2007,
Chinese J Catal, 2013, 34, 1608-1616; (c) X. Jiang, H. Yi, L. Jiang,
J. Gong and H. Huang, Chem Res Chinese U, 2013, 29, 223-226.
15 (a) Y. L. Khmelnitsky, V. V. Mozhaev, A. B. Belova, M. V.
63, 2363–2389.
40 K. Li, Z. Yang, J. Zhao, J. Lei, X. Jia, S. H. Mushrif and Y. Yang,
Green Chem, 2015, DOI: 10.1039/c5gc00976f.
Sergeeva and K. Martinek, Eur J Biochem, 1991, 198, 31-41; (b) 41 A. Arfan and J. P. Bazureau, Org Process Res Dev, 2005,
U. R. Desai and A. M. Klibanov, J Am Chem Soc, 1995, 117 743-748.
3940-3945; (c) P. P. Wangikar, P. C. Michels, D. S. Clark and J. S. 42 A. K. Chakraborti and R. Sudipta Raha, J Am Chem Soc, 2009,
Dordick, J Am Chem Soc, 1997, 119, 70-76. 131, 6902-6903.
16 (a) A. Zaks and A. M. Klibanov, Science, 1984, 224, 1249-1251; 43 (a) T. P. Wells, J. P. Hallett, C. K. Williams and W. Tom, J Org
9,
,
(b) A. Zaks and A. M. Klibanov, P Natl Acad Sci USA, 1985, 82
,
Chem, 2008, 73, 5585-5588; (b) C. Lorna, F. Ruben, L. N
Llewellyn, L. M. Veronica and W. Tom, J Org Chem, 2006, 71
3192-3196.
,
17 A. M. Klibanov, Nature, 2001, 409, 241-246.
8847-8853; (c) G. Ranieri, J. P. Hallett and T. Welton, Ind Eng
Chem Res, 2007, 47, 638-644.
18 (a) P. Yedavalli and N. M. Rao, Protein Eng Des Sel, 2013, 26
,
317-324; (b) M. T. Reetz, P. Soni, L. Fernandez, Y. Gumulya and
J. D. Carballeira, Chem Commun, 2010, 46, 8657-8658.
19 J. Ge, D. Lu, J. Wang and Z. Liu, Biomacromolecules, 2009, 10
,
1612-1618.
8 | Green Chem., 2015, 00, 1-3
This journal is © The Royal Society of Chemistry 2015
Please do not adjust margins

Page 9 of 9
Green Chemistry
View Article Online
DOI: 10.1039/C5GC02557E
96x63mm (600 x 600 DPI)