High isolated yields in thermodynamically controlled peptide synthesis in toluene catalysed by thermolysin adsorbed on Celite R-640

Article abstract of DOI:10.1039/b000797h

An innovative immobilisation method that allows peptide synthesis to be performed even at equimolar concentrations, by controlling water activity, is reported.

Full text of DOI:10.1039/b000797h

High isolated yields in thermodynamically controlled peptide synthesis in
toluene catalysed by thermolysin adsorbed on Celite R-640
Alessandra Basso, Luigi De Martin, Cynthia Ebert, Lucia Gardossi* and Paolo Linda
Dipartimento di Scienze Farmaceutiche, Universit a` degli Studi, Piazzale Europa 1, 34127 Trieste, Italy.
E-mail: gardossi@univ.trieste.it
Received (in Liverpool, UK) 27th January 2000, Accepted 14th February 2000,
Published on the Web, 2nd March 2000
An innovative immobilisation method that allows peptide
synthesis to be performed even at equimolar concentrations,
by controlling water activity, is reported.
Enzymes have proved to be an attractive alternative to chemical
methods in peptide synthesis since several proteases effect
peptide bond formation under mild conditions, with minimum
side-chain protection, and avoiding racemization. In spite of
these benefits, enzymatic methods are not routinely employed,
partly because they suffer from unfavourable thermodynamics
in water.
Scheme 1
There are two distinct mechanisms for protease-catalysed
peptide synthesis: thermodynamically and kinetically con-
trolled synthesis. In principle, replacing water with organic
solvents as a reaction medium should be beneficial for both
types of reaction. However, water is essential for proteases such
as thermolysin and a-chymotrypsin, and various studies
confirmed that these enzymes display no activity when
use of the hydrophobic organic solvent makes the leakage of the
enzyme negligible so that the biocatalyst can be recycled. The
hydrophobic solvent leads to further remarkable advantages in
terms of stability, especially if compared to the detrimental
7
effects of the water miscible solvents largely reported. For
instance, the hydrolases immobilised on Celite R-640 in toluene
were stored in the same solvent at a controlled degree of
hydration without any appreciable decrease of activity for at
least 4 weeks. The same immobilisation technique can also be
employed for the immobilisation of a-chymotrypsin and
penicillin G amidase (PGA), which were shown to be effective
in catalysing amino acid esterification and protection of amino
groups, respectively. Table 1 reports some examples of
syntheses catalysed by the three immobilised enzymes in
1
insufficiently hydrated. Unfortunately, water/solvent mixtures
are not optimal media for peptidases since high percentages of
co-solvents reduce the activity of the enzyme dramatically.
Owing to these two opposite roles of water in enzymatic
peptide synthesis, various approaches have been developed in
order to search for compromises. These include kinetically
controlled syntheses in solvents containing low percentages of
2
water or methods to reverse the equilibrium towards synthesis,
w
toluene at controlled a . The water adsorbed by the porous
such as using an excess of the amino component and extracting/
support provides the enzymes with the hydration necessary to
display their catalytic activity.2 It should be noted that no
reaction was observed when the three native enzymes were
employed in dry toluene or acetonitrile in accordance with our
observation that PGA is also active in organic solvents only
3
precipitating the product.
–4
Kitaguchi and Klibanov1a have given an alternative solution
to the problem by demonstrating that water can be replaced
partially by water mimics such as formamide, maintaining the
activity of the enzyme in the organic solvent while minimising
hydrolytic reactions. Finally, promising results have been
obtained by Halling and coworkers by controlling the hydration
of thermolysin in kinetically controlled peptide synthesis in
hexane4 and, more recently, by developing solid-to-solid
peptide synthesis in the presence of minimum amounts of
water.1b
when sufficiently hydrated (a
> 0.4).5
w
w
Table 1 Enzymatic synthesis performed in toluene at a between 0.70 and
0
.75 at 30 °C
Final
conversion
(%)
c
Aiming to overcome the above drawbacks to a larger
application of enzymes in peptide synthesis, the present work
describes a novel approach for the non-covalent immobilisation
of thermolysin in toluene and its application to thermodynam-
ically controlled peptide synthesis at controlled water activity
Enzyme
a
CO H donor
b
Nucleophile
b
t/h
48
144
96
2
Thermolysin/Celite Z-
Thermolysin/Celite Z-
Thermolysin /Celite Z-
PGA /Celite
a-Chymotrypsin /
Celite
L
L
L
-Phe–CO
-Phe–CO
-Phe–CO
2
2
2
H
H
H
L
L
L
L
-Phe–OEt
-Tyr–OEt
98
97
95
98
d
d
-Leu–NH
-Tyr–OEt
2
e
PhCH
2
CO
2
Me
24
(
a
w
). The method exploits the ability of Celite R-640 rods
Fluka) to adsorb large amounts of water ( > 90% of its weight)
constant in a reaction system within wide
e
(
Z- -Phe–CO
L
2
H
MeOH
97
60
and to maintain the a
w
a
Enzyme dissolved in buffer and adsorbed on 200 mg of Celite rods
5,6
defined ranges of water concentrations. Thermolysin was
adsorbed on dry Celite R-640 according to an innovative and
practical technique illustrated in Scheme 1. The enzymatic
aqueous solution was added to the hydrophobic solvent
containing the dry support. Owing to the presence of the
hydrophobic solvent, a uniform coating of the aqueous phase
formed around the Celite rods and the enzymatic solution was
adsorbed on the Celite within 24 h.
according to Scheme 1. b Equimolar concentrations of reactants (80 mmol)
were employed in all reactions. An excess of MeOH (250 mmol) was used
c
only in the esterification of Z-
L
-Phe-CO
2
H. Conversions calculated by
means of RP-HPLC using an internal standard. Isolated yields of Z-
Phe–OEt, Z- -Phe- -Tyr–OEt and Z- -Phe- -Leu–NH2 were 93, 90 and
76%, respectively. Reactions catalysed by a-chymotrypsin and thermolysin
L-Phe-L-
L
L
L
L
2
were monitored also by titrating the Z-L-Phe–CO H with 0.1 M NaOH
solution. d The enzyme was dissolved in 35 mL of buffer, adsorbed on the
Celite rods and equilibrated for 24 h. Afterwards, a buffer solution (35 mL)
The immobilised catalyst suspended in toluene gave, after
2
containing L-Leu–NH was added to the thermolysin/Celite system and
w
equilibration, a values between 0.70 and 0.75. Enzymatic
e
equilibrated for 3 h. The enzyme was dissolved in water before
adsorption.
reactions can be carried out simply by adding the reactants to the
toluene used for the adsorption and storage of the enzymes. The
DOI: 10.1039/b000797h
Chem. Commun., 2000, 467–468
This journal is © The Royal Society of Chemistry 2000
467

View Online
The use of Celite R-640 as a support for thermolysin and a-
chymotrypsin enables peptide bond synthesis and esterification
to be performed using free carboxylic acids since the water
produced during the reaction is adsorbed by the Celite rods.
solvents12–14 are avoided. Moreover, microbial contaminations
are prevented, the support material is stable under the reaction
conditions, and possesses no swelling capacity.
In conclusion, the present work describes the first example, to
the best of our knowledge, of thermodynamically controlled
enzymatic peptide synthesis performed in organic solvent
leading to complete conversions and very high isolated yields
even when equimolar concentrations of the reactants are
employed.
Since it has been already demonstrated that peptidases
catalyse the formation of the bond between oligopeptide
fragments in organic solvents and that PGA is able to remove
phenylacetic groups selectively from amino acids, we are
currently investigating the application of this technique to a
totally enzymatic approach for peptide synthesis competing
with chemical routes.
w
Therefore a was maintained sufficiently low and hydrolytic
reactions were prevented so that nearly quantitative conversions
were achieved even when employing equimolar concentrations
of non-activated, and thus less expensive, substrates. It must be
noted that when native thermolysin was employed in water–
acetonitrile (13% v/v)† in the synthesis of Z-
a maximum of 22% of conversion was obtained in 6 h but
during the reaction course a exceeded 0.90 so that hydrolytic
reactions prevailed, causing a very low final conversion
11%).
Despite the fact that the poor solubility of peptides has often
L
-Phe-
L
-Tyr–OEt,
w
(
been viewed as a restriction for enzymatic reactions in organic
solvents, results in Table 1 indicate that thermodynamically
controlled peptide enzymatic synthesis in hydrophobic solvents
can be carried out employing substrates having very low
solubility and which are present in the reaction medium mainly
as a suspension. This observation is in accord with previous
We thank CNR and MURST (Roma) for financial support to
P. Linda.
Notes and references
4
studies concerning kinetically controlled peptide synthesis and
≈ 0.7312d in MeCN.
†
13% v/v of H O enables operation at a
2 w
the solid-to-solid synthesis.1
b,c
‡ The three synthesised dipeptides precipitated upon formation owing to
their low solubility in toluene. The toluene was removed and the solid
residue was washed with MeCN. The organic solutions were combined,
filtered and taken to dryness obtaining products having > 98% purity by
HPLC. Products of reactions catalysed by PGA and a-chymotrypsin are
soluble in toluene and were isolated by removing the organic phase and
evaporating the solvent.
The dipeptide Z-
L
-Phe-
L
-Leu–NH
2
was synthesised follow-
-Leu–NH
ing an alternative strategy since the poorly soluble
L
2
was previously dissolved in an aqueous buffer and then
adsorbed on the Celite rods where thermolysin had been already
immobilised. However, this second method led to lower isolated
yields (76%) probably owing to some adsorption of the product
on the Celite rods.
1
(a) H. Kitaguchi and A. M. Klibanov, J. Am. Chem. Soc., 1989, 111,
The time required to achieve complete conversion is mainly
affected by the fact that equimolar concentrations of the
reactants were used in all peptide synthesis, so that the reaction
rate slows down dramatically at the end of the reaction when the
concentrations of the two reactants become extremely low.
Complete conversions are achievable in shorter times using a
9
1
272; (b) M. Erbeldinger, X. Ni and P. J. Halling, Biotechnol. Bioeng.,
998, 59, 68; (c) P. Kuhl, U. Eichhorn and H.-D. Jakubke, Biotechnol.
Bioeng., 1995, 45, 276; (d) P. Clap e´ s and P. Adlercreutz, Biochim.
Biophys. Acta, 1991, 1118, 70.
2 K. Ohiama, S. Nishimura, Y. Nonaka, K.-I. Kihara and T. Hashimoto,
J. Org. Chem., 1981, 46, 5242; P. Clap e´ s, E. Pera and J. L. Torres,
Biotechnol. Lett., 1997, 19, 1023; Y. Murakani, S. Hayashi, A. Takehara
and A. Hirata, Biotechnol. Tech., 1999, 13, 165; R. A. Persichetti, N. L.
St. Clair, J. P. Griffith, M. A. Nava and A. Margolin, J. Am. Chem. Soc.,
two fold excess of the amino component ( > 98% yield of Z-
Phe- -Tyr–OEt in 48 h). Nevertheless, employing equimolar
L
-
L
concentrations of reactants is advisable since this enables the
recovery and isolation of the products very simply and, most
importantly, avoiding any purification step.‡ As a consequence,
remarkably high isolated yields (93 and 90%) are achievable.
This is a factor of major importance in peptide synthesis,
especially when various subsequent synthetic steps are re-
quired.
1
995, 117, 2732.
3
R. Didziapetris, B. Drabnig, V. Schellenberger, H.-D. Jakubke and V.
Svedas, FEBS Lett., 1991, 287, 31; V. Kasche and B. Galusky,
Biotechnol. Bioeng., 1995, 45, 261; M. Haensler, S. Thust, P. Klossek
and G. Ullman, J. Mol. Catal. B: Enzymatic, 1999, 6, 95; G. Lye and
J. M. Woodley, TIBTECH, 1999, 17, 395; M. Erbeldinger, X. Ni and P.
J. Halling, Biotechnol. Bioeng., 1999, 63, 316.
4
5
6
P. Kuhl, P. J. Halling and H.-D. Jakubke, Tetrahedron Lett., 1990, 31,
Reactions reported in Table 1 catalysed by a-chymotrypsin
and PGA are also of practical use in peptide synthesis since they
are potentially useful for the preparation and protection of
activated amino acids suitable for kinetically controlled peptide
synthesis. It is noteworthy that a-chymotrypsin catalysed the
5
231.
L. De Martin, C. Ebert, G. Garau, L. Gardossi and P. Linda, J. Mol.
Catal. B: Enzymatic, 1999, 6, 437.
A. Basso, L. De Martin, C. Ebert, L. Gardossi and P. Linda, J. Mol.
Catal. B: Enzymatic, 2000, 8, 245.
7 J. Partridge, B. D. Moore and P. J. Halling, J. Mol. Catal. B: Enzymatic,
1999, 6, 11.
8 P. J. Halling, Enzyme Microb. Technol., 1994, 16, 178.
complete esterification of Z-
L
-Phe, despite it being previously
reported that the enzyme accepts preferentially the N-acetyl
amino acids,1d which, however, are deprotected with diffi-
9
E. Wehtje, H. de Wit and P. Adlercreutz, Biotechnol. Tech., 1996, 10,
47.
culty.
9
The novelty of our method lies not only in the synthetic
results obtained but also in the immobilisation technique
developed. The use of Celite R-640 allows control of the water
activity, rather than water concentration, during the whole
process, thus controlling effectively both the enzyme activity
and the reaction equilibrium. No devices, such as hydrated
salts,8 equilibration with the atmosphere at known relative
1
0 (a) E. Wehtje, I. Svensson, P. Adlercreutz and B. Mattiasson,
Biotechnol. Tech., 1993, 7, 873; (b) S. J. Kwon, K. M. Song, W. H.
Hong and J. S. Rhee, Biotechnol. Bioeng., 1994, 46, 393.
1 S. Bloomer, P. Adlecreutz and B. Mattiasson, Enzyme Microb. Technol.,
1
1
992, 14, 546; M. Otamiri, P. Adlercreutz and B. Mattiasson,
Biotechnol. Bioeng., 1994, 44, 73; P. Mensah, J. L. Gainer and G. Carta,
Biotechnol. Bioeng., 1996, 20, 434.
9
10
12 (a) J. Partridge, G. A. Hutcheon, B. D. Moore and P. J. Halling, J. Am.
Chem. Soc., 1996, 118, 12873; (b) M. C. Parker, B. D. Moore and A. J.
Blaker, Biocatalysis, 1994, 10, 269; (c) T. Ke and A. M. Klibanov,
Biotechnol. Bioeng., 1998, 57, 746; (d) J. Partridge, P. J. Halling and
B. D. Moore, Chem.Commun., 1998, 841.
3 K. Dabulis and A. M. Klibanov, Biotechnol. Bioeng., 1993, 41, 566.
4 P. Adlercreutz, Eur. J. Biochem., 1991, 199, 609; J. Kim and B. G. Kim,
Biotechnol. Bioeng., 1996, 50, 687.
w
humidity, or more complex methods for adjusting the a of
the system are required; also there is no need to remove the
water produced during the process.11 Furthermore, the im-
mobilisation method fulfils the fundamental requirements for a
larger application to biotransformations since the procedure is
simple and inexpensive, it provides very high reproducibility in
terms of enzyme and water content so leading to reproducible
activity and quantitative adsorption yields. Detrimental effects
caused by the removal of water under vacuum or with polar
1
1
Communication b000797h
468
Chem. Commun., 2000, 467–468