Substrate Mimetic-Mediated Reactions
J . Org. Chem., Vol. 66, No. 5, 2001 1631
reagents were of the highest commercial purity. Solvents were
purified and dried by the usual methods.
performed which may additionally reinforce the efforts
for a further optimization of the biocatalyst.
Con st r u ct ion of Mu t a n t Tr yp sin . Recombinant rat
trypsinogen II was prepared from E. coli vector pST.10 This
plasmid codes for the wt-protein from Rattus norvegicus fused
to ADH/GAPDH promoter and R factor leader sequence. Site-
directed mutagenesis was performed by thermocycling in a
mixture of 50 µL contained approximately 50 ng of pST, 125
ng of the appropriate primer, 1.3 units of Pfu DNA polymerase
as well as 20 mM of each desoxynucleotide and the reaction
buffer supplied with the enzyme (coupling parameters: cycle
1, 95 °C, 30 s; cycle 2-17, 95 °C, 30 s (denaturation), 55 °C, 1
min (annealing), 68 °C, 12 min (extension). The oligonucle-
otides GGA GGC AAG GAG TCC TGC CAG, CTG GCA GGA
CTC CTT GCC TCC were used as the primers. After removing
original pST template DNA by incubation with 10 units of
DpnI, the PCR product was transformed into E. coli XL2 blue
ultracompetent cells. The isolated plasmid was then verified
by sequence analysis. The processed trypsinogen construct
was excised using BamHI and SalI, purified from an agarose
gel and ligated into the similarly restricted and purified yeast/
E. coli vector pYT.10 Following a further amplification in E.
coli, the EZ transformation kit (Zymo-Research, Orange, CA)
was employed to transfer the pYT variant to Saccharomyces
cerevisiae (DLMR101). Analogously, wt-trypsin was prepared
using the original pST vector.
E xp r ession a n d P u r ifica t ion of Tr yp sin D189E . S.
cerevisiae transformants were selected and amplified on uracil-
and subsequently leucin-deficient SC media. 20 mL cultures
(Leu-deficient, 2 d incubation) were used to inoculate 1.3 l YPD
medium (10 g L-1 yeast extract, 20 g L-1 peptone, 15 g L-1
glucose). After 4 d incubation, recombinant trypsinogen was
isolated from the supernatant by cation exchange chromatog-
raphy on a Toyopearl SP 650M column (Toso Haas). The
mature trypsin was obtained by treatment with trypsin-free
enteropeptidase (Biozyme) and subjected to affinity chroma-
tography on SBTI agarose. Finally, after dialysis, the protein
was concentrated and stored in 1 mM HCl at 4 °C.
Ch em ica l Syn th eses. Boc-Xaa-4-guanidinophenyl esters
were prepared according to our previously described procedure
by condensation of Boc-Xaa-OH and 4-[N′,N′′-bis(Z)-guanidino]-
phenol.4d The benzoyl-protected esters were synthesized start-
ing with the deprotection of the NR-amino group with triflu-
oroacetic acid and subsequent benzoylation using benzoyl
chloride. Bz-Gly-OGp was prepared by direct condensation of
Bz-Gly-OH with 4-[N′,N′′-bis(Z)-guanidino]phenol. A final
catalytic hydrogenation of the bis(Z)-protected esters resulted
in the 4-N′,N′′-deprotected 4-guanidinophenyl esters. Pen-
tapeptides were synthesized with a semiautomatic batch
peptide synthesizer SP 650 (Labortech AG, Switzerland) using
p-alkoxybenzyl alcohol resin synthesized according to Wang11
and standard Fmoc chemistry. The peptides were precipitated
with dry diethyl- or diisopropyl ether or mixtures of hexane/
ethyl acetate. The identity and purity of all peptides and esters
were verified by analytical HPLC, NMR, thermospray mass
spectroscopy, and elemental analysis. In all cases, satisfactory
analytical data were found ((0.4% for C, H, N).
Hyd r olysis Stu d ies. All reactions were performed at 25
°C using an assay mixture containing 25 mM Mops buffer, pH
7.6, 0.1 M NaCl, 5 mM CaCl2, and 20% methanol to realize
complete solubility of the substrates. The substrate concentra-
tions were between 0.006 and 4.0 mM and the enzyme
concentrations between 3.1 × 10-6 - 6.2 × 10-7 M. The active
enzyme concentration was determined by active site titration
using 4-nitrophenyl-4′-guanidinobenzoate.12 After thermal
equilibration of the assay mixtures, the reactions were initi-
ated by addition of the enzyme. The rate of reaction was
analyzed by RP-HPLC determining the disappearance of the
substrates for at least 10 different concentrations. For this
Con clu sion
Although, substrate mimetics considerably extend the
synthetic scope of proteases by mediating the acceptance
of nonspecific acyl moieties, the use of this beneficial
strategy is limited to the coupling of peptides lacking
enzyme specific amino acid residues. The presence of site-
specific amino acid moieties, however, inevitably leads
to proteolytic side reactions. Moreover, in the case of wt-
trypsin used for the coupling of an Arg-containing pep-
tide, cleavage behind the specific Arg are even favored
over its coupling with the substrate mimetics. Conse-
quently, the universal use of the substrate mimetics
approach to peptide ligation requires further efforts that
lead to a shift in enzyme activity from the cleavage to
the synthesis reaction. Our results demonstrate the
usefulness of site-directed mutagenesis to design trypsin
species with an enhanced specificity for artificial sub-
strate mimetics. Although the exchange of Asp 189 with
Glu reduces the absolute enzyme specificity, the relative
specificity of trypsin D189E toward acyl-4-guanidinophe-
nyl esters is increased when compared to that of lysyl-
and arginyl-bonds. Thus, compared to the wt-enzyme the
substrate preference of the mutant trypsin is shifted from
Lys and Arg to the substrate mimetics and, therefore,
from the cleavage to the synthesis of peptide bonds.
Similar studies using 4-guanidinophenyl esters of D-
amino acid derivatives reveal, however, that this shift
in specificity should be restricted to the higher specific
L-counterparts. Enzymatic coupling reactions using vari-
ous Bz-L-Xaa-OGp esters and peptides bearing trypsin-
and chymotrypsin-sensitive bonds demonstrated the
usefulness of trypsin D189E for peptide synthesis. While
the proportions of intact peptide product of the whole
peptide product in wt-trypsin-catalyzed coupling of Arg-
and Lys-containing peptides were only about 27% and
62%, in analogous mutant trypsin-mediated syntheses
relative proportions of noncleaved peptide products of
84% and 79% were found. Similar to the wt-enzyme,
trypsin D189E also appears to be practically inactive
toward chymotrypsin-sensitive peptides. In the same way
there was no evidence for an influence of the individual
C-terminal L-amino acid moiety of the substrate mimetics
on the extent of undesired cleavage qualifying trypsin
D189E as a useful and up to now only enzyme capable
of the substrate mimetic-mediated ligation of specific
peptides. However, to make full practical use of this
approach further enzyme optimization will be required
to accomplish a better differentiation between the speci-
ficity constants for the substrate mimetics and for specific
peptide bonds.
Exp er im en ta l Section
Ma ter ia ls. Enzymes for DNA manipulation came from
Boehringer except Pfu DNA polymerase being purchased from
Promega. The oligonucleotide primers were obtained from
MWG-Biotech. Plasmid DNA was isolated with the High Pure
Plasmid Isolation Kit (Boehringer). The QiaQuick Gel Extrac-
tion Kit (Qiagen) was employed for purification of DNA
fragments from agarose gels. Amino acid derivatives, 4-ami-
nophenol, coupling reagents, benzyl chloroformate, S-methyl-
isothiourea, and 4-toluenesulfonic acid were products of Bachem
(Switzerland), Fluka and Merk (Germany), respectively. All
(10) Hedstrom, L.; Szilagyi, L.; Rutter, W. J . Science 1992, 255, 1249.
(11) Wang, S. S. J . Am. Chem. Soc. 1973, 95, 1328.
(12) Bergmeyer, H. U.; Grassl, M.; Walter, H.-E. Samples, Reagents,
Assessments of Results. In Methods of Enzymatic Analysis; Bergmeyer,
H. U., Ed.; Verlag Chemie: Weinheim, 1983; Vol. II, p 319.