Enzymatic synthesis of dipeptides in ionic liquids

Article abstract of DOI:10.2174/157017810790796318

Immobilized protease-catalyzed synthesis of peptides from natural amino acids has been achieved in ionic liquids (ILs). Three ionic liquids BMIM.PF ₆, BMIM.BF₄ and EtPy.BF₄ have been tested; an effective yet simple and practical method has been devised. The product yields are comparable with those seen in molecular solvents. A max yield of 49 % for Boc-Leu-TrpOEt has been obtained. This study shows that the advantages seen in organic solvents can also be achieved in ILs, and use of such medium could provide a viable alternative to organic and aqueous media for bio- catalysis.

Full text of DOI:10.2174/157017810790796318

168
Letters in Organic Chemistry, 2010, 7, 168-171
Enzymatic Synthesis of Dipeptides in Ionic Liquids
Sanjay V. Malhotra*,#, Chengdong Zhang and Hao Wang
Department of Chemistry and Environmental Science, New Jersey Institute of Technology, University Heights, Newark,
NJ, 07010 USA
Received June 09, 2009: Revised October 28, 2009: Accepted November 02, 2009
Abstract: Immobilized protease-catalyzed synthesis of peptides from natural amino acids has been achieved in ionic
liquids (ILs). Three ionic liquids BMIM.PF6, BMIM.BF4 and EtPy.BF4 have been tested; an effective yet simple and
practical method has been devised. The product yields are comparable with those seen in molecular solvents. A max yield
of 49 % for Boc-Leu-TrpOEt has been obtained. This study shows that the advantages seen in organic solvents can also be
achieved in ILs, and use of such medium could provide a viable alternative to organic and aqueous media for bio-
catalysis.
Keywords: Enzymatic synthesis, protease, dipeptides, ionic liquid.
INTRODUCTION
enzymatic catalyst. Also, in place of aqueous and organic
media, we employed hydrophobic IL i.e. BMIM.PF6, and
hydrophilic ILs i.e. BMIM.BF4 and EtPy.BF4. The work
presented in this paper is a new approach to the enzymatic
synthesis of dipeptides.
Peptides represent new opportunities as drugs,
diagnostics reagents, agricultural chemicals and even as
additives in food [1-3]. Therefore, strong interest in the
peptide area is not surprising, and development of
methodologies for obtaining peptides in high yields, good
purity and in large scale production is a field of growing
interest. In water, the equilibrium of amide bond formation
and hydrolysis is generally shifted towards hydrolysis, and
thus enzyme naturally hydrolyzes amide bonds. To
overcome this, there have been advances in the use of non-
aqueous media which have made significant effect on bio-
transformations of interest to chemical and pharmaceutical
industry [4, 5]. It is now well accepted that organic solvents
offer advantages over aqueous medium mainly in
thermodynamically unfavored reactions [4]. However,
despite these advances organic medium suffers from key
drawbacks, such as poor solubility of charged and polar
compounds of high volatility. These limitations have
prompted the design of new reaction media which are non-
aqueous yet help to overcome the drawbacks of organic
solvents. Notable example is the ionic liquid, a new class of
compounds having unique physiochemical properties, and
therefore, studied for various organic transformations, e.g.
hydrogenation, oxidation, substitution, Heck reaction etc. [6-
9]. Since, it is reported earlier that protease has good stability
in alcoholic medium [10], it can catalyze nucleophilic
reactions of both D- & L- amino acids and has been used in
the amidation reactions [11-16]; we decided to study
dipeptide synthesis by using Protease immobilized on Celite.
Use of immobilized enzyme would facilitate the
purification and allows an easy recycling of the
RESULTS AND DISCUSSION
Earlier we reported the use of ionic liquid EtPy.TFA for
the first time as a catalyst for esterification of amino acids,
including unnatural compounds [17]. After N-acetylation,
the N-protected amino acid was dissolved in anhydrous ethyl
or isopropyl alcohol followed by adding the ionic liquid.
Yields were generally good, especially for ethyl esters.
These promising results demonstrated the potential of ionic
liquids for amino acid chemistry, more specifically the
peptide coupling. Motivated by these results, we started to
examine construction of peptide bonds in ionic media by
means of enzymes. Enzymatic reactions in ionic liquids have
been reported earlier [18]. The methods require additional
steps of separation and cleaning of enzymes before next use.
To simplify this process, enzyme in our study was first
immobilized on celite. It was then used repeatedly for
synthesis of peptides by using a host of amino acids shown
in Fig. (1).
In a typical reaction, (see experimental procedure) first
an N-protected amino acid was treated with immobilized
protease resulting in the enzyme coupled intermediate.
Subsequent reaction of the intermediate with C-protected
amino acid gives product dipeptide. A general reaction
scheme is shown in Fig. (2).
Results of various peptide syntheses are shown in Table
1. As can be seen in Fig. (2), once the intermediate is formed
there are two competitive reactions i.e. with water and/or
with C-protected amino acid. Higher yields of products
would be obtained if the reaction with second amino acid is
faster than that with water. Therefore, the nature of reaction
medium could play an important role. For testing, we carried
out a number of syntheses in hydrophobic ionic liquid
*Address correspondence to this author at the Department of Chemistry and
Environmental Science, New Jersey Institute of Technology, University
Heights, Newark, NJ, 07010 USA; Tel: +1 301 846 5141; Fax: +1 301 846
5206; E-mail: malhotrasa@mail.nih.gov
#Current address: Laboratory of Synthetic Chemistry, SAIC-Frederick Inc.,
National Cancer Institute at Frederick, 1050 Boyles Street, Frederick,
Maryland 21702.
1570-1786/10 $55.00+.00
© 2010 Bentham Science Publishers Ltd.

Enzymatic Synthesis of Dipeptides in Ionic Liquids
Letters in Organic Chemistry, 2010, Vol. 7, No. 2
169
O
O
O
OH
OH
H2N
N
OH
NH
NH2
NH2
Glycine (Gly)
O
Leucine (Leu)
NH2
Histidine (His)
O
O
HO
OH
HN
N
OH
OH
OH
H
NH2
NH2
NH2
Serine (Ser)
Arginine (Arg)
Norleucine (Nle)
O
O
Cl
O
HO
OH
NH2
H2N
OH
NH2
Tryptophan (Trp)
Phenylalanine (Phe)
Threonine (Thr)
Fig. (1). Structures of amino acids studied for peptide synthesis.
Cp-A2-NH2
Np-A1-A2-Cp
Np-A1-E
Np-A1-OH
+
E
H2O
+
H2O
Np-A1-OH
Fig. (2). Enzymatic synthesis of dipeptide (Np: N-terminal protected amino acid, Cp-C-terminal protected amino acid, and E-protease).
Table 1. Enzymatic Synthesis of Dipeptides in Ionic Liquids (37oC, 5 % Water Content, 48 h)
Serial #
Amino Acid-1
Amino Acid-2
Peptide
Ionic Liquid
Yield (%)
1.
2.
Boc-Leu
Boc-Leu
Boc-Leu
Boc-Leu
Boc-Leu
Boc-Gly
Boc-Gly
Boc-Leu
Boc-Arg
Boc-Arg
Boc-Arg
Boc-His
Boc-His
Boc-His
Boc-His
TrpOEt
GlyOEt
Boc-Leu-TrpOEt
Boc-Leu-GlyOEt
Boc-Leu-GlyOEt
Boc-Leu-SerOMe
Boc-Leu-SerOMe
Boc-Gly-TrpOEt
Boc-Gly-TrpOEt
Boc-Leu-Phe(Cl)OEt
Boc-Arg-SerOEt
Boc-Arg-NleOMe
Boc-Arg-Phe(Cl)OEt
Boc-His-SerOMe
Boc-His-TrpOEt
Boc-His-ThrOMe
Boc-His-Phe(Cl)OEt
BMIM.PF6
BMIM.PF6
EtPy.BF4
49
22
40
38
40
19
26
45
12
36
7
3.
GlyOEt
4.
SerOMe
SerOMe
TrpOEt
BMIM.PF6
BMIM.BF4
BMIM.PF6
EtPy.BF4
5.
6.
7.
TrpOEt
8.
Phe(Cl)OEt
SerOEt
BMIM.PF6
BMIM.PF6
BMIM.PF6
BMIM.PF6
BMIM.BF4
EtPy.BF4
9.
10.
11.
12.
13.
14.
15.
NleOMe
Phe(Cl)OEt
SerOMe
TrpOEt
25
31
18
44
ThrOMe
Phe(Cl)OEt
EtPy.BF4
EtPy.BF4
BMIM.PF3 and also in hydrophilic ILs i.e. BMIM.BF4 and
EtPy.BF4.
as solvent lower yield of Boc-Leu-GlyOEt is seen (entry 2).
This is because GlyOEt has low solubility in BMIM.PF6.
When same reaction is carried out in a hydrophilic IL
EtPy.BF4 the yield is significantly higher. Similarly, a
comparison can also be seen in the synthesis of Boc-Leu-
As data show the solubility of amino acid-2 in solvent
affects the peptide yield. For example, high yield of Boc-
Leu-TrpOEt is obtained in BMIM.PF6 while in the same IL

170 Letters in Organic Chemistry, 2010, Vol. 7, No. 2
Malhotra et al.
Table 2. Comparison Enzymatic Reaction in Ionic Liquid and Organic Solvent (37 oC, 5 % Water Content, 48 h)
Peptides
Yield (%)
Yield (%)
(Ionic Liquid)
(Organic Solvent)
Boc-Leu-TrpOEt
Boc-Leu-SerOMe
Boc-Leu-Phe(Cl)OEt
Boc-His-TrpOEt
49
(BMIM.PF6)
38
21
(Acetone)
30
(BMIM.PF6)
45
(Acetone)
55
(EtPy.BF4)
31
(Ethyl Acetate)
64
(EtPy.BF4]
26
(Ethyl Acetate)
7
Boc-Gly-TrpOEt
(EtPy.BF4)
(Acetone)
SerOMe (entry 4 and 5) and Boc-Gly-TrpOEt (entry 6 and
7).
7.2), triethylamine, anhydrous ethanol, toluene, ethyl acetate,
acetone, and Celite were from Aldrich. Protease (alcalase
from Bacillus Licheniformis) was from Sigma Company. All
Boc- protected amino acids and amino acid ester were
purchased from Sigma Company. LC-MS analysis was
carried out by HPLC (waters) system at detection of 254 nm
with flow rate of 1.0ml/min. The mobile phase consisted of
acetonitrile/water (50/50 % v/v).The column was Alltech
C18 5ꢀm, 4.6x250mm. All the products were confirmed by
mass spectroscopy (LCQ Advantage). 1H NMR analysis was
performed on Varian 300 MHz instrument.
Since, protease has also been by used in the peptide
synthesis using organic solvent [1], we decided to compare
the synthesis of few representative dipeptides in both ionic
liquids and organic solvents. Results are shown in Table 2.
Earlier, one report suggested the possibility of peptide
synthesis in anhydrous ethanol [15], however, in our hand,
reaction did not occur when ethanol was used as solvent. We
believe this is due to the denaturalization of enzyme and lose
of its catalytic activity. Also, the polarity of ionic liquids can
be tuned as opposed to the fixed polarity of known molecular
solvents, which suggests the possibility of enzymatic
reactions using ILs, where the most polar solvents are
ineffective. This could extend many enzyme catalyzed
reactions to a wide polarity range. Further, it should be noted
that mass spectrometric analysis of the solvent extraction
showed the presence of dipeptide product which could
account for lower yields. This observation is similar to
earlier report on peptide synthesis [19]. Overall yields in our
study in ionic liquid medium are comparable to those
reported in organic medium [15]. Lastly, reusability of ionic
liquid was tested with recycled BMIM.PF6 for the synthesis
of Boc-Leu-TrpOEt and yield of 48 %, 42% and 41 % were
obtained for three consecutive runs. This further establishes
the potential of ILs for peptide synthesis.
Ionic Liquid Preparation
Synthesis of (EtPy.BF4)
EtPy.BF4 was synthesized according to Zhao’s method
[9] and characterized by NMR and FTIR.
A general synthetic procedure could be described as
follows: Tetrafluoroboric acid (0.2mol) was slowly added to
stirred slurry of silver (ꢁ) oxide (0.1 mol) and distilled water
(50 ml). To avoid photodegradation of silver (ꢁ) oxide, the
reaction mixture was fully covered with aluminum foil. The
reaction mixture was stirred continuously until the reaction
was complete, which was indicated by the formation of a
solution. A solution of N-ethyl-pyridinium bromide (0.2
mol) was added to the reaction mixture. As reaction took
place and ILs formed, a yellow precipitate of silver (ꢁ)
bromide started to be observed. The mixture was stirred at
room temperature until no precipitate formed. The
precipitate of silver (ꢁ) bromide was filtered off, and then the
solvent was removed by rotary evaporation under vacuum at
about 65 °C. The resulting ionic liquids were put in an oven
overnight at 65 °C to remove the moisture.
CONCLUSIONS
Our study has demonstrated the utility of ionic liquids in
the synthesis of peptide catalyzed by immobilized protease.
Both hydrophobic and hydrophilic ILs are found to be
effective for this reaction. Results also suggest that both
organic solvents and ionic liquids media could be useful for
peptide synthesis. However, systematic investigation should
be done to find the most suitable medium for large scale
synthesis.
Synthesis of BMIM.BF4
Tetrafluoroboric acid (13.3 ml, 0.173mol) was slowly
added to stir slurry of silver (I) oxide (20.0 g, 0.0863 mol) in
50 ml distilled water over 10 minutes. To avoid photo
degradation of Ag2O, the reaction mixture was covered with
aluminum foil. Until the Ag2O was fully reacted, a solution
of BMIM.Cl (30.15 g, 0.1726 mol) in 150 ml distilled water
was added to the reaction mixture and stirred at room
temperature for 3 h. The yellow precipitate was filtered off,
EXPERIMENTAL
General
Materials and reagents were of highest commercially
available grades and used as obtained. Phosphate buffer (pH

Enzymatic Synthesis of Dipeptides in Ionic Liquids
Letters in Organic Chemistry, 2010, Vol. 7, No. 2
171
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Purification of Ionic Liquids
The ionic liquids were purified according to the literature
procedure [20] with following modification. Ionic liquid was
re-dissolved in acetonitrile or methanol and any excess of Br-
or Cl- was removed by adding silver nitrate drop by drop till
no more halide was left. Neutral alumina was added to the
solution, and stirred over night to absorb un-reacted trace
imidazole or pyridinium reactant. After evaporation under
vacuum, clear ionic liquid product was obtained.
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and
enzymatic
resolution
of
L-(+)-homophenylalanine
hydrochloride. Enantiomer, 2002, 7, 1.
Immobilized enzyme was filtered off after the reaction,
BMIM.PF6 was washed by 5% citric acid, NaHCO3 followed
by water and the dried under vacuum at 60oC. For
hydrophilic ionic liquids EtPy.BF4 and BMIM.BF4, after
separating enzyme, 10ml methanol was added with small
amount of neutral alumina, shaking over night. After
evaporating, purified ionic liquid was dried under vacuum
for reuse.
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esters using ionic liquid N-ethyl pyridinium trifluoroacetate.
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Enzyme-Celite was prepared according to reported
procedure [1]. A 2 ml aqueous solution of protease (15
units/mg) was added to a mixture of 2 g Celite and5 ml
buffer solution (pH 7). It was mixed thoroughly and then
dried overnight under vacuum at room temperature.
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[15]
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Representative Procedure for Enzymatic Synthesis
The N-Boc protected acyl alcohol (1 equiv.) and
nucleophile (1.5 equiv.) were taken in 5 ml ionic liquid.
When the amine component was in hydrochloride form,
equal amount of triethylamine was added. 5% (v/v) buffer
was added as water content. The reaction was started by
adding 1g immobilized enzyme. After being shaken at 37ꢀC
for 48 hours, 20 ml toluene was used to extract product from
ionic liquid continuously over two days. The extract solution
was washed with 5 % citric acid, followed by water and 5 %
NaHCO3. After the toluene was evaporated under vacuum,
the residue was dissolved in 2ml methanol. The reaction
progress was monitored by thin-layer chromatographic
analysis performed on a silica gel pre-coated plate in ethyl
acetate liquid phase. Amino acids were tested by ninhydrin.
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