High isolated yields in thermolysin-catalysed synthesis of Z-L-aspartyl-L-phenylalanine methyl ester in toluene at controlled water activity

Article abstract of DOI:10.1016/S0040-4039(01)00452-X

Z-L-Aspartyl-L-phenylalanine methyl and ethyl esters were synthesised enzymatically in toluene by means of the zinc protease thermolysin adsorbed onto Celite R-640, which is a porous support able to control the hydration of the protein. The conversion of the two derivatised amino acids into the desired products was complete, leading to >90% isolated yields. Moreover, working with equimolar concentrations of the reactants no purification steps were required. Thermolysin adsorbed onto Celite R-640 is shown to be a practical tool to synthesise biologically active peptides in organic media.

Full text of DOI:10.1016/S0040-4039(01)00452-X

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 3395–3397
High isolated yields in thermolysin-catalysed synthesis of
Z-
L
-aspartyl-
L
-phenylalanine methyl ester in toluene at
controlled water activity
Luigi De Martin, Cynthia Ebert, Lucia Gardossi* and Paolo Linda
Dipartimento di Scienze Farmaceutiche, Universit a` degli Studi, Piazzale Europa 1, 34127 Trieste, Italy
Received 16 January 2001; revised 8 March 2001; accepted 15 March 2001
Abstract—Z-L-Aspartyl-L-phenylalanine methyl and ethyl esters were synthesised enzymatically in toluene by means of the zinc
®
protease thermolysin adsorbed onto Celite R-640 , which is a porous support able to control the hydration of the protein. The
conversion of the two derivatised amino acids into the desired products was complete, leading to >90% isolated yields. Moreover,
working with equimolar concentrations of the reactants no purification steps were required. Thermolysin adsorbed onto Celite
®
R-640 is shown to be a practical tool to synthesise biologically active peptides in organic media. © 2001 Elsevier Science Ltd.
All rights reserved.
The benefits of using enzymes in organic synthesis and,
in particular, for the preparation of optically pure
drugs are well recognised. In the last few years much
equilibrium constant is shifted towards the hydrolysis
−
1
products (K =1.5 M ), unsatisfactory yields were
eq
1
3
obtained in the dilute aqueous medium. However, over
attention has been paid to protease-catalysed peptide
synthesis by using the reverse of the hydrolytic reaction,
the last few years Halling and co-workers have
obtained very high yields in the thermolysin-catalysed
1
namely thermodynamically controlled synthesis. The
synthesis of Z-L-Gln-L-Leu-NH and Z-L-Asp-L-Phe-
2
enzymatic approach offers some advantages over chem-
ical methods since enzymatic reactions are often per-
formed under mild conditions and without protection
9–11
OMe using aqueous suspensions of the substrates.
An alternative approach could be represented by the
partial substitution of the aqueous medium with an
organic solvent which can shift the thermodynamic
2
of the side chains of the amino acid derivatives. Aspar-
tame (a-L-Asp-L-Phe-OMe) is one of the best known
peptides obtained through enzymatic synthesis. This
dipeptide is an artificial sweetener which is 200 times
sweeter than sucrose and used throughout most of the
12
equilibrium towards synthesis. Therefore, reactions
have been performed in either water/organic solvent
13,14
6,15–17
mixtures
or in two phase systems.
3
world.
In principle, the use of pure organic solvents in enzy-
matic synthesis should be beneficial for the equilibrium
shift, for the recovery of the product and the recycling
The precursor of aspartame (X-L-Asp-L-Phe-OMe,
where X is an N-protecting group) has been synthesised
by several research groups starting from N-protected
18
of the catalyst.
L
-aspartic acid (X-
L
-Asp) and
L-phenylalanine methyl
4–
ester (
7
L-PheOMe) using the zinc protease thermolysin.
Recently we reported that thermolysin is very active in
hydrophobic organic solvents when the hydration of
the protein is controlled by adsorbing the enzyme onto
The first approach reported in the literature included
the thermodynamically controlled synthesis of the pre-
®
19
Celite R-640 rods.
5,8
cursor of aspartame in water. Since in water the
In the present paper we present the thermolysin-
catalysed synthesis of Z- -aspartyl- -phenylalanine
methyl and ethyl esters in a hydrophobic solvent at
L
L
Keywords: aspartame; peptide synthesis; thermolysin; organic solvent;
thermodynamically controlled synthesis; water activity.
controlled water activity (a ), using equimolar concen-
*
Corresponding author. Fax: +39-040-52572; e-mail: linda@
w
univ.trieste.it
trations of the substrates.
0
040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)00452-X

3
396
L. De Martin et al. / Tetrahedron Letters 42 (2001) 3395–3397
-Asp- -Phe-OR (R=Me, Et) catalysed by thermolysin adsorbed
Table 1. Equilibrium conversions in the synthesis of Z-
onto Celite R-640 rods in toluene at controlled aw
L
L
®
a
Acylating agentb Nucleophileb
aw (t0)
aw (equilibrium)
Conversion (%)d
Time (h)
Z-
Z-
Z-
L
L
L
-Asp
-Asp
-Asp
L
L
L
-Phe-OEt
-Phe-OMe
-Phe-OMe
0.78
0.73
0.77
0.81
0.76
0.80
96
99
98
220
240
144
c
a
Experimental conditions: reaction volume: 1 mL of toluene, 30°C, 5 mg/mL of enzyme adsorbed onto 200 mg of Celite R-640® rods.
b
c
8
0 mmol suspended in 1 mL of toluene.
1
60 mmol suspended in 1 mL of toluene.
d
Final conversions were evaluated both by RP-HPLC and by titration of Z-
H NMR and ES–MS.
L
-Asp-CO H with 0.01 M NaOH. Products were characterised by
2
1
It is widely documented that the removal of the
16,17
5,19
product, either by precipitation
or by extraction
from the reaction medium, can be the driving force for
the reaction to reach high conversions in short times. In
the present cases, the product accumulated exclusively
in the liquid phase, and no precipitation was detected
during the reaction course. A typical time course of the
reaction is reported in Fig. 1.
As shown in Fig. 1, the reaction rate is faster at the
beginning since almost 75% of the conversion of the
two substrates into the desired product is achieved in
the first 24 hours. After one day, the concentrations of
the two reactants become extremely low, so that the
reaction rate dramatically slows down. This translated
in complete conversion being obtained only after about
Figure 1. Time course of thermolysin-catalysed Z-
Phe-OEt synthesis in toluene at controlled aw.
L-Asp-L-
2
00 hours.
Enzymatic synthesis of Z-L-Asp-L-Phe-OR
Complete conversions are achievable in shorter times
using a twofold excess of the amino component (98% in
1
44 hours), as reported in Table 1. Nevertheless,
®
Thermolysin was adsorbed onto Celite R-640 directly
in toluene, equilibrated for 24 hours at 30°C in a
thermostatted orbital shaker and gave at equilibrium,
employment of equimolar concentrations of the reac-
tants is recommended, since this enables the recovery
and isolation of the products simply by withdrawing
the organic phase and evaporating the toluene, thus
avoiding any purification step. As a consequence, high
isolated yields are achievable (>90%).
an a value between 0.73 and 0.78. Enzymatic reactions
w
were carried out simply by adding the substrates into
the same vial used for the adsorption and storage of the
†
enzyme. It must be underlined that the substrates were
only partially soluble in toluene, so that the reaction
was performed in an organic solvent suspension of the
two substrates.
In conclusion, the present work describes the thermody-
namically controlled enzymatic synthesis of aspartame
precursors performed in a hydrophobic organic solvent,
leading to complete conversions and to high isolated
yields even when using equimolar concentrations of the
reactants.
The equilibrium conversions for the synthesis of Z-
L-
Asp- -Phe-OR dipeptides are reported in Table 1.
L
The aw values measured at the equilibrium of the
reaction were close to those measured before the start
of the reaction although water is one of the reaction®
products. The absorption properties of Celite R-640
made it possible to shift the equilibrium of the reaction
towards synthesis obtaining quantitative conversions,
even when equimolar amounts of non-activated sub-
strates were employed.
This opens new perspectives for the large-scale produc-
tion of biologically active peptides using peptidases in
low-water media. Moreover, the results presented in
®
this paper confirm that Celite R-640 is an effective
tool for productive biocatalysis in low-water media,
and is potentially applicable to large-scale processes.
Acknowledgements
†
Experimental conditions: 1 mL of toluene, 5 mg of thermolysin
®
adsorbed onto Celite R-640 rods following a procedure previously
reported,19 T=30°, 80 mmol of Z-
Thanks are due to C.N.R. and M.U.R.S.T. (Roma) for
financial support to P.L., and to Universit a` degli Studi
(Trieste) for financial support to L.G.
L
-Asp, 80, 160 mmol of
L
-Phe-
OR·HCl, 100 and 180 mmol Na CO ·10H O were used to obtain the
2
3
2
free amine20 directly in the reaction medium.

L. De Martin et al. / Tetrahedron Letters 42 (2001) 3395–3397
3397
References
10. Erbeldinger, M.; Ni, X.; Halling, P. J. Biotechnol. Bioeng.
999, 63, 316–321.
1
1
2
3
. Faber, K. Biotransformations in Organic Chemistry;
Springer-Verlag: Berlin, 1997.
11. Erbeldinger, M.; Ni, X.; Halling, P. J. Biotechnol. Bioeng.
2001, 72, 69–76.
. Nagayasu, T.; Miyanaga, M.; Tanaka, T.; Sakiyama, T.;
Nakanishi, K. Biotechnol. Bioeng. 1994, 43, 1108–1117.
. Oyama, K.; Irino, S.; Hagi, N. In Methods in Enzymol-
ogy; Colowick, S. P.; Kaplan, N. O., Eds. Production of
Aspartame by Immobilized Thermoase. Academic Press:
San Diego, 1987; Vol. 136, pp. 503–516.
1
1
2. Halling, P. J. Biotechnol. Bioeng. 1990, 35, 691–701.
3. Miyanaga, M.; Tanaka, T.; Sakiyama, T.; Nakanishi, K.
Biotechnol. Bioeng. 1995, 46, 631–635.
14. Lee, G. I.; Nakai, S.; Clark-Lewis, I. Food Res. Int. 1994,
27, 483–488.
15. Persichetti, R. A.; St. Clair, N. L.; Griffith, J. P.; Navia,
M. A.; Margolin, A. L. J. Am. Chem. Soc. 1995, 117,
4
5
6
7
. Oyama, K.; Kihara, K.; Nonaka, Y. J. Chem. Soc.,
Perkin Trans. 2 1981, 356–360.
. Isowa, Y.; Ohmori, M.; Ichikawa, T.; Mori, K. Tetra-
hedron Lett. 1979, 28, 2611–2612.
2
732–2737.
6. Murakami, Y.; Yoshida, T.; Hirata, A. Biotechnol. Lett.
998, 20, 767–769.
7. Murakami, Y.; Hayashi, S.; Takehara, A.; Hirata, A.
Biotechnol. Tech. 1999, 13, 165–168.
8. Ebert, C.; Gardossi, L.; Linda, P. J. Mol. Catal. B:
Enzym. 1998, 5, 241–244.
9. Basso, A.; De Martin, L.; Ebert, C.; Gardossi, L.; Linda,
P. Chem. Commun. 2000, 467–468.
1
1
1
1
1
. Oyama, K.; Nishimura, S.; Nonaka, Y.; Kihara, K.;
Hashimoto, T. J. Org. Chem. 1981, 46, 5241–5242.
. Oyama, K. In Biocatalysis in Organic Media; Lane, C.;
Tramper, J.; Lilly, M. D., Eds. Enzymatic Synthesis of
Aspartame in Organic Solvents. Elsevier Science: Amster-
dam, The Netherlands, 1987; pp. 209–224.
8
. Stoineva, I. B.; Galunsky, B. P.; Lozanov, V. S.; Ivanov,
I. P.; Petkov, D. D. Tetrahedron 1992, 48, 1115–1122.
. Erbeldinger, M.; Ni, X.; Halling, P. J. Biotechnol. Bioeng.
20. Basso, A.; Braiuca, P.; De Martin, L.; Ebert, C.; Gar-
dossi, L.; Linda, P. Tetrahedron: Asymmetry 2000, 11,
1789–1796.
9
1
998, 59, 68–72.
.
.