Vanadium(V)-catalyzed epimerization of isoleucine

Article abstract of DOI:10.1016/j.catcom.2016.08.015

All stereoisomers of isoleucine were transformed to the mixtures of the corresponding epimers by epimerization in alkaline aqueous solution. The catalyst was formed in situ by condensation of salicylaldehyde and isoleucine followed by complexation with va

Full text of DOI:10.1016/j.catcom.2016.08.015

Catalysis Communications 86 (2016) 96–99
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Catalysis Communications
journal homepage: www.elsevier.com/locate/catcom
Short communication
Vanadium(V)-catalyzed epimerization of isoleucine
a
b
Lukáš Krivosudský a, , Peter Schwendt , Juraj Filo
⁎
a
Department of Inorganic Chemistry, Faculty of Natural Sciences, Comenius University, Mlynská dolina, Ilkovičova 6, 842 15 Bratislava, Slovakia
Institute of Chemistry, Faculty of Natural Sciences, Comenius University, Mlynská dolina, Ilkovičova 6, 842 15 Bratislava, Slovakia
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 July 2016
Received in revised form 9 August 2016
Accepted 10 August 2016
Available online 13 August 2016
All stereoisomers of isoleucine were transformed to the mixtures of the corresponding epimers by epimerization
in alkaline aqueous solution. The catalyst was formed in situ by condensation of salicylaldehyde and isoleucine
followed by complexation with vanadate. No derivatization of the amino acid was necessary. The
tetrabutylammonium salts of [VO2(N-salicylidene-isoleucinato)]− can be used for diastereomeric separation of
the epimers providing low yields and moderate diastereoselectivities.
© 2016 Elsevier B.V. All rights reserved.
Keywords:
Isoleucine
Epimerization
Racemization
Vanadium
Asymmetric catalysis
1. Introduction
important not only to understand their role in organisms and for poten-
tial medicinal use (e.g. D-allo-Ile is a precursor to isostatine exhibiting
Isoleucine is a branch-chained amino acid carrying two chiral carbon
atoms and thus exists as four stereoisomers (Scheme 1). L-Ile dominates
in biological systems and it is essential in humans. The other stereoiso-
mers were found in animals and in humans in trace amounts [1]. D-Ile
was found in rat urine [2]. L-allo-Ile was found in rats [2] as well as in
human plasma [3] and the level of L-allo-Ile in blood plasma is a sensi-
tive diagnostic marker for Maple Syrup Urine Disease (MSUD) – a reces-
sive deficiency of the branched-chain-ketoacids dehydrogenase
complex [4]. The origin of L-allo-Ile in human plasma is not yet clear:
an in vivo study with L-[13C]-Ile proposed that L-allo-Ile is formed via
retransamination of the corresponding α-keto acid; [5] however, differ-
ent study with L-[15N, 13C]-Ile indicates that the C–N bond is preserved
in an aldimine Schiff base formed with the aldehyde moiety of pyridoxyl
phosphate-aminotransferase enzyme complex which is responsible for
the isomerization [6]. Nevertheless, both mechanisms indicate that the
carbon skeleton of L-allo-Ile is derived from L-Ile and that racemization
of some of its derivative must be involved in the process. D-allo-Ile is
probably more encountered than L-allo-Ile. It was found in human
urine [3], in other mammals [2] and it is incorporated in some biologi-
cally active peptides [7,8,9], e.g. in antibiotic peptides isolated from am-
phibian skin [10,11]. Recently, isoleucine-2-epimerase was isolated
from Lactobacillus otakiensis JCM 15040 bacteria [12]. The enzyme is
the first racemase known to act significantly on L-Ile. In addition,
amino acids often serve as a cheap source of chirality in asymmetric ca-
talysis. The availability of all stereoisomers of isoleucine is therefore
antitumor activity) [13], but also asymmetric catalysis involving differ-
ent isoleucine stereoisomers could lead to chiral products so far inacces-
sible. Thus, several procedures involving multi-step synthesis of D-allo-
Ile from L-Ile and subsequent separation of the epimers exist [14].
Herein is reported a simple and cheap method for epimerization of
isoleucine under alkaline condition catalyzed by the vanadium(V) com-
plex with a Schiff base prepared by condensation of salicylaldehyde and
isoleucine in situ. For the first time, no derivatization of isoleucine is re-
quired for the transformation and the method was successfully used for
all stereoisomers of isoleucine. In addition, a new method for separation
of isoleucine epimers is presented.
2. Experimental
All reagents and solvents were obtained from commercial sources
and used as received. The NMR spectra were recorded with a Varian
VNMRS 600 MHz instrument (600 MHz for 1H, 157.88 MHz for 51V).
Chemical shifts (δ) are given in ppm relative to tetramethylsilane (1H)
and VOCl3 (51V). Samples of isoleucine were dissolved in 0.5 M solution
of KOH in D2O. The diastereomeric ratio was calculated from peak inten-
sities attributed to chemical shifts of hydrogen atoms at α – carbon
atoms of isoleucine.
2.1. Epimerization of isoleucine
A round-bottom flask was loaded with isoleucine (1.31 g, 10 mmol),
NaOH (0.4 g, 10 mmol) and water (5 mL). The mixture was gently heat-
ed until dissolution. Salicylaldehyde (0.011 mL, 0.1 mmol) and NH4VO3
⁎
Corresponding author.
E-mail address: krivosudskyl@fns.uniba.sk (L. Krivosudský).
http://dx.doi.org/10.1016/j.catcom.2016.08.015
1566-7367/© 2016 Elsevier B.V. All rights reserved.

L. Krivosudský et al. / Catalysis Communications 86 (2016) 96–99
97
Table 1
Vanadium(V)-catalyzed epimerization of isoleucine under alkaline condition.
Entry
A
Yield, %
de, %
B, %
L-Ile
D-Ile
L-allo-Ile
D-allo-Ile
1
2
3
4
99
99
89
87
0
50
x
x
x
50
x
L-Ile
2
51
48
x
49
52
x
D-Ile
4
x
x
L-allo-Ile
D-allo-Ile
12
44
56
de = diastereomeric excess.
conjunction with rising proportion of EtOH and decreasing temperature)
[20]. If equimolar amounts of salicylaldehyde and vanadate are used, the
equilibrium is reached within 1 h. It should be noted here, that it is not
expected that the epimerization shows up at the β – carbon position
under the conditions presented here or in the earlier works. This process
is much slower in Nature than α – epimerization [15] and so far can be
achieved exclusively by enzymes [5,6].
Scheme 1. Stereoisomers of isoleucine.
NH4VO3 can be replaced by another common vanadate (e.g. KVO3,
Na3VO4) or even by a vanadium(IV) compound (VOSO4·nH2O) because
under the reflux in air atmosphere VIV is readily oxidized to VV and the
epimerization proceeds as with NH4VO3. A control reaction without a
vanadium compound resulted in no epimerization. In order to inspect
the effect of transition metal ions on the epimerization under alkaline
condition the reaction was performed with salts of MnII, FeII/III, CoII,
NiII, CuII, ZnII, MoVIO42− and WVIO24−. Indeed, there is a general discus-
sion that metal ions, especially coordinated by Schiff base ligands, can
catalyze racemization of amino acids [21]. However, replacement of
vanadium(V) by other metal ions resulted in no epimerization.
The reaction was performed not only with L-Ile, but also using other
stereoisomers of isoleucine. Thus, D-allo-Ile was transformed to L-Ile, D-
(0.0112 g, 0.1 mmol) were added and the yellow solution (pH = 11)
was heated under reflux 6 h. After this period the heating was carefully
continued without reflux to evaporate approx. 1/2 of the solvent. Then
the solution was cooled to room temperature, 20 mL of EtOH and
0.7 mL of acetic acid were added. The mixture was cooled in a freezer
below 0 °C, white precipitate of isoleucine was filtered off, washed
with EtOH and dried in air.
3. Results and discussion
3.1. Epimerization of isoleucine
Isoleucine itself undergoes epimerization with the reaction half–life
105–107 years (D-allo-Ile/L-Ile ≈0.4) [15,16] what has found application
in dating of biological samples in geochronology [17]. Treatment of L-Ile
in strong alkaline solution (6 M NaOH) 6 h at 100 °C results in formation
of D-allo-Ile in ≈0.7% yield, while in 6 M HCl no conversion is observed
[18]. Thus, the conversion of L-Ile to D-allo-Ile occurs to small extend
only in the alkaline regime. Recently, in the basic crystallization solu-
tions of stereoisomers of NBu4[VO2(N-salicylidene-isoleucinato)] an un-
precedented epimerization of the isoleucine residue was observed by
1H NMR spectroscopy [19]. The formation of the complex anion
(Scheme 2) in situ can be readily used for catalytic acceleration of isoleu-
cine epimerization in alkaline solution.
The epimerization reaction (Table 1) was carried out upon dissolu-
tion of equimolar amounts of isoleucine (A) and NaOH in H2O and addi-
tion of catalytic amounts of salicylaldehyde and NH4VO3 (1 mol%). After
6 h of heating under reflux the equilibrium of epimers (B) was achieved
and isoleucine was recovered in high yields by the addition of EtOH, acid-
ification with concentrated acetic acid and cooling below 0 °C (the solu-
bility of isoleucine in the mixed EtOH/H2O solvent decreases markedly in
Fig. 1. 51V NMR spectra of the reaction mixture at different temperatures; a: HVO42−, b:
HxV2O(74 − x)−, c: [VO2(N-salicylidene-isoleucinato)]−. Slight systematic movement of
the chemical shifts and peak broadening are due to elevated temperature.
Scheme 2. Structural formula of [VO2(N-salicylidene-isoleucinato)]− as revealed by X-ray
crystallography [19].

98
L. Krivosudský et al. / Catalysis Communications 86 (2016) 96–99
vanadium is distributed into monovanadate, HVO24− (−537 ppm)
[22], and a second species with the chemical shift at −558 ppm. A li-
gand concentration study (Fig. 2) revealed that this species is present
in the solution regardless of the presence of the isoleucine sodium salt
(3% of V at 0 M Ile). Increasing concentration of the isoleucine sodium
salt results in profound formation of this species (26% of V at 2 M Ile).
The actual conditions (pH = 11, −logc(V) = 1.7) represent a medium
in which concurrent presence of HVO24− and HxV2O7(4 − x)− is possible
[23]. We assume that the relatively high concentration of
HxV2O(74 − x)− in the reaction mixture is a consequence of the ionic
strength effect on the composition of vanadate solutions [22].
The third signal in the spectrum of the catalytic system at
≈−540 ppm (Fig. 1) appearing at elevated temperature is assigned to
the catalyst, [VO2(N-salicylidene-isoleucinato)]− [19]. Subsequent
cooling of the solution results in disappearance of this peak. The
equilibrated coexistence of vanadates and [VO2(N-salicylidene-
isoleucinato)]− allows formation and disintegration of the vanadium
Schiff base complex so the epimerization can proceed in a catalytic
manner. A minor peak at −568 ppm attributed to linear tetravanadate
5−
HV4O
arises after 1 h of refluxing [22]. No reaction intermediates
13
were detected.
3.3. Separation of epimers
The diastereomers of the complex involved in the epimerization ex-
hibit different solubility of their tetrabutylammonium salts: stereoiso-
mers NBu4[VO2(N-salicylidene-L/D-isoleucinato)] are less soluble then
NBu4[VO2(N-salicylidene-L/D-allo-isoleucinato)] and can be separated
from various mixtures by fractional crystallization [24]. The first product
of crystallization (yield ≈20–50%, Table 2) contains predominantly L- or
D-Ile and can be directly decomposed back to the amino acid (Entries 3
and 4). As for the separation of L- and D-allo-Ile, the first crystals are re-
moved by filtration and L- or D-allo-Ile is recovered immediately from
the diastereomerically enriched mother liquor (Entries 1 and 2). This
separation method offers only low yields and moderate diastereomeric
purity due to the fast epimerization at high vanadium concentration
(see Appendix A for further details).
Fig. 2. 51V NMR spectra representing a ligand concentration study at the following
conditions: c(V) = 0.02 M; pH = 11; c(Ile) = 0 M, 0.2 M, 0.6 M, 1 M, 1.4 M and 2 M
(bottom up). The pH was adjusted to 11 with 2 M NaOH. a: HVO24−, b: HxV2O7(4 − x)−
.
Ile to L-allo-Ile and vice versa (Table 1). From the practical point of view,
in addition to the conversion of L-Ile to D-allo-Ile, the transformation of
L-allo-Ile into D-Ile is obviously reasonable, because L-allo-Ile is cheaper
and more prevalent in Nature than D-Ile. The vanadium-catalyzed
epimerization yields approx. 1:1 mixture of the corresponding epimers
within 6 h in all cases. The second naturally occurring amino acid with
two chiral carbon atoms threonine does not epimerize under the same
conditions.
4. Conclusion
In conclusion, a facile method for preparation of epimer mixtures of
isoleucine was reported. The method is based on catalytic epimerization
of isoleucine in alkaline solution and requires no derivatization of the
amino acid. The catalyst, vanadium Schiff base complex anion [VO2(N-
salicylidene-isoleucinato)]−, can be easily prepared in situ and its pres-
ence in the catalytic system was corroborated by 51V NMR spectroscopy.
For the first time, a catalytic epimerization of all isoleucine stereoiso-
mers into the corresponding mixtures of epimers was described. The
tetrabutylammonium salts of [VO2(N-salicylidene-isoleucinato)]− may
3.2. Speciation in the catalytic system
The composition of the reaction mixture was investigated by 51V
NMR spectroscopy (Fig. 1). In a fresh solution at room temperature
Table 2
Diastereomeric resolution of epimer mixtures.
Entrya
Mixture of epimers
NBu4[VO2(N-salicylidene-isoleucinato)]
Isoleucine
Yield of the first fraction, %
de, %
Stereoisomer
Yield, %b
de, %
Dominant isoleucine stereoisomer
1
2
3
4
36
64
50
66
60
50
32
22
24
52
58
74
78
L-Ile + D-allo-Ile
D-Ile + L-allo-Ile
L-allo-Ile + D-Ile
D-allo-Ile + L-Ile
D-allo-Ile
L-allo-Ile
D-Ile
L-Ile
41
D-Ile
27
D-Ile
29
L-Ile
L-Ile
a
Entries correspond with Table 1.
Yield of the individual epimer.
b

L. Krivosudský et al. / Catalysis Communications 86 (2016) 96–99
99
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Acknowledgment
This work was supported by the Grant Agency of the Ministry of Ed-
ucation, Science, Research and Sport of the Slovak Republic and Slovak
Academy of Sciences VEGA project no. 1/0336/13.
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Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.catcom.2016.08.015.
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