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Peptide LigasesЧTools for Peptide Synthesis.

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Peptide Ligases-Tools
for Peptide Synthesis
Hans-Dieter Jakubke*
Following the first chemical formation of a peptide bond by
Theodor Curtius in 1881 in the laboratory of Hermann Kolbe in
Leipzig, chemical peptide synthesis['] developed over the next
ninety years culminating in the first total synthesis of an enzyme.
Ribonuclease A (RNase A), composed of 124 amino acid
residues, was constructed by condensation of fragments in solution['] as well as by solid-phase synthesis.131Roughly ten years
later Yajima and Fujiic41 synthesized RNase A according to an
improved conventional synthetic strategy and obtained the first
crystalline synthetic enzyme having full enzymatic activity. But
an ideal, universally applicable method for chemical formation
of the peptide bond has still not been found. Since chemosynthetic reactions are typically conducted with residues having
chiral centers, racemization is a constant concern. Even stepwise
chain extension using urethane protecting groups, which are
supposed to prevent racemization, may be affected; chemical
linkage of peptide fragments is yet more unreliable.
A recent paperL5]describes a new synthesis of RNase A, in
which an enzyme was used for the first time to couple fragments
obtained by solid-phase synthesis to form a protein. The idea of
using enzymes to make peptide bonds is about as old as chemical peptide synthesis itself. In 1898, evoking the reversibility of
chemical reactions, van't Hoff predicted that enzymes could be
used to catalyze the formation of peptide bonds. This idea was
first taken up in 1938 by Max Bergmann and confirmed experimentally.
Owing to the stereo- and regiospecificity of enzymes, their
application for the formation of peptide bonds certainly offers
a list of advantages over chemical procedures, for example mild
reaction conditions, no need for permanent protection of functional groups in side chains, racemization-free course of reaction, and simple scale-up after optimization of the process. To
make full use of these advantages, however, a close to universally applicable peptide ligase is needed,[**] which in principle
would display high catalytic efficiency for all theoretically possible combinations of amino acids; in other words, it should be
nonspecific and not affected by the side chain functions of the
amino acids to be coupled. Since an ideal biocatalyst like the
ribosomal peptidyl transferase is not available, and multien[*I
Prof. Dr. H:D. Jakubbe
Lnstitut fur Biochemie
FakultZt fur Biowissenschdften. Pharmdzie und Psychologie der UniversitLt
Talstrasse 3 3 . D-04103 Leipzig (Germany)
Telefdx: Int. code + (341)960-3099
In deviation from EC nomenclature, the term peptide ligase here refers t o a
biocatalyst for the formation of the peptide bond.
C ' h o i i . 1111
Ed. EirRI. 1995, 34. N o . 2
(CI VCH VerluRsgesellschafi
zyme complexes in bacterial peptide synthesis are limited to
specific purposes, only proteases can be used for formation of
the peptide bond. Two different mechanistic strategies for the
application of proteases can be described.L61
In comparison to equilibrium-controlled peptide synthesis,
kinetically controlled synthesisL7]offers the possibility of more
targeted manipulations. Following this approach for reactions
with low enzyme requirements and short reaction times, mechanistic considerations limit the enzymes employed to serine and
cysteine proteases. Unfortunately, proteases are not perfect acyl
transferases; owing to their limited specificity. other undesired
reactions may take place parallel to acyl transfer, for example
hydrolysis of the acyl enzyme 2, secondary hydrolysis of the
peptide product 4, and other undesired cleavages of possible
protease-labile bonds in 1, 3, and 4 (Scheme 1 ) .
Y - N H m - O H
Scheme 1.
To eliminate o r minimize these disadvantages, the enzyme can
be engineered and/or the reaction medium and the mechanistic
features of the process can be adjusted as shown schematically
in Figure 1 .[81 Undesired hydrolytic side reactions may be eliminated by adapting the medium. Protease-catalyzed syntheses in
monophasic organic solvents or in biphasic aqueous-organic
mbH, 0-69451 Weinheim, 1995
0570-0833/95/0202-0175 $ 10.00 + .25:0
Tyr", T Y ~ ' ~ , and AlaZ0,were the closest to matching the
substrate specificity of the subtilisin mutant. A similar strategy
was also used in the synthesis of three variants of RNase A, in
which the two histidine residues His" and His"' at the active
center were exchanged individually and simultaneously for L-4fluorohistidine; the affect of these substitutions in the three
analogues of RNase A was studied in detail.
Substrate binding at the active site['] plays a crucial role in
protease-catalyzed peptide bond formation. Unfortunately, a
simple C-N ligase is not capable of developing different substrate binding regions, for example by induced fit, for the structurally diverse amino acid side chain functionalities. According
to Linus Pauling the action of an enzyme depends on the complementarity of the active site to the transition state structure of
a reaction, as shown in Scheme 2 for peptide bond formation.
Catalytic antibody approach
Peptide ligases
Design of new
Site-directed mutagenesis
Modulation of
substrate specificity
Fig. 1. Possibilities for engineering enzymatic peptide synthesis at different levels.
systems proceed with minimal proteolytic side reactions. Since
the reactants are better soluble in organic media, the chemical
and enzymatic steps can be accomodated. Side reactions can
also be eliminated and yields of peptides increased by direct
reduction of the water content of the reaction medium, for example in syntheses in frozen aqueous systems and reactions in
dense media. Another option in modifying a kinetically controlled reaction is manipulating the leaving group so that the
enzyme reacts exclusively with the acyl donor 1 and not with the
peptide product 4 or the amino component 3. Then even
protease-labile products can be obtained in good yields. Two
recent reviews give an overview of the current possibilities for
The term enzyme engineering[''] (see Fig. 1) describes a range
of techniques from deliberate chemical modification to remodeling a wild-type enzyme by gene technology. "Subtiligase", a
mutant of subtilisin BNP, was prepared by Jackson et al.15]by
protein design and used in a further total synthesis of RNase A.
This work, which combines solid-phase synthesis of oligopeptides and enzymatic coupling of these fragments, demonstrates
the state of the art in peptide synthesis and proves impressively
the potential of enzymes for the formation of peptide bonds.
Average yields of roughly 75 % were obtained in the fragment
condensations. The excellent leaving group of the Phe-NH,modified carboxamido methyl ester, which even in unmodified
form was found to be a very good acyl donor in other syntheses,r''l and the use of a considerable excess of acyl donor ensured that most of the side reactions were suppressed. The total
yield after the five fragment condensations was 15 YO,and after
the folding of the final product, 8%. Thus starting with 100 mg
of the initial peptide (residues 98-124), RNase A can be obtained on a 10-mg scale. The fragments (77-97, 64-76, 52-63,
21 - 51, 1-20) were chosen such that the C-terminal residues,
Q VCH Verlagsgewtlschafi mbH, 0-69481 Wein/w.trn,1995
Scheme 2.
Given the fact that the enzyme should not bind the substrate
very strongly but must stabilize the transition state considerably, it is unlikely that a relatively simple enzyme can act as a
universal peptide ligase. This premise was recently confirmed by
reports by Hirschmann et al.["] and by Jacobsen and S ~ h u l t z " ~ ]
on peptide bond formation with catalytic antibodies (abzymes) .
Analogues of the transition state were used as haptens to induce
antibodies with the correct arrangement of catalytic groups. The
idea behind both strategies is illustrated in Scheme 2 by the
transition state analogue 6 synthesized by Hirschmann et aI.["]
When these haptens are injected in animals, their immune systems generate antibodies against them. Since the structure of the
tetrahedral intermediate 5 is also greatly affected by the amino
acid side chains R' and R2,it is doubtful whether a universally
applicable catalytic antibody can be generated for peptide bond
formation. The structures of the transition state analogues 7,
8a, and 8 b were changed deliberately, in particular by incorporation of a cyclohexyl group, such that the abzymes that catalyzed the reaction of 9 and 10 would have broad substrate
specificity (Scheme 3).
The abzyme-catalyzed dipeptide syntheses gave all possible
stereoisomers in yields of 44 to 94%. The abzymes did not
catalyze the hydrolysis of either the dipeptide products or the
activated esters employed. Jacobsen and S c h ~ l t z Ielicited
' ~ ~ antibodies against a neutral phosphonate diester transition state
analogue, which significantly accelerated the coupling of an Nuacylalanine azide with a phenylalanine derivative relative to the
0570-0833/95/0202-0176S fU.U#+.25/0
Angen,. Cliem. Inl. Ed. Engl. 1995, 34, No. 2
currently is, and this perspective emphasizes the importance of
the chemoenzymatic synthesis of RNase A.
Keywords: catalytic antibodies . ligases . peptide synthesis .
RNase A . subtilisin
German version: Angew. Chem. 1995. 107, 189
X= NH, R= -(CH,),COOH
8a X= 0, R= -(CH,),COOH
8b X=O, R=Me
Ac-Xrn-ONp + H-Tv-NH, 7 - A c - X a a - T r p - N H 2
(Xaa = Val,Leu, Phej
Scheme 3
uncatalyzed reaction. The application of catalytic antibodies
exploits the fundamental property of the immune system, generating binding sites in the folds of the antibodies which are
analogous to the active sites in enzymes. Proof of this is provided by the determination of the three-dimensional structure of a
catalytic antibody developed for peptide cleavage.['41The active
site of this antibody contains a Ser-His dyad and thus has great
similarities with the active sites of serine proteases.
These sensational findings will certainly initiate a new era in
the construction of peptide ligases. Now with the emergence of
catalytic antibodies for peptide synthesis, the arsenal of methods is augmented, and the prospects for future progress are
bright. However, extensive development is needed before this
approach is applied as routinely as enzymatic peptide synthesis
[ l ] K.-H. Altmann, M. Mutter, Chem. Unsrrer Zeit 1993. 27, 274-286.
[2] R. Hirschmann. R. F. Nutt, D . F. Veber, R. A. Vitali, S. L. Varga, T. A. Jacob.
F, W. Holly, R. G . Denkewalter, J. Am. Chem. Soc. 1969. Y1. 507-508.
[3] B. Gutte, R. B. Merritield, J. Am. Chem. SOC.1969. 91, 501 -502.
[4] H. Yajima, N. Fujii. J. Am. Chern. Soc. 1981, 103. 5867 -5871.
[ 5 ] D. Y Jackson. J. Vurnier, C. Qudn, M. Stanley, J. Tom. J. A. Wells, Science
1994,266, 243-247.
161 Selected reviews: a) J. S. Fruton, Adi,. Enzjmol. Relur. A r m s Mol B i d . 1982,
239--306, b) H:D. Jakubke, P. Kuhl. A. Konnecke, Angew. Chem. 1985, 97.
79-87; Angew. Chem. Inr. Ed. Engl. 1985. 24, 85-93: cj W. Kullmann, Enzymalic Peplide Sj,nfhesis.CRC. Boca RdtOn, FL. USA. 1987: d) H:D. Jakubke
in The Pepfides;Ano/mis, Synthesis. Bio/og.c.(Eds.: S. Udenfriend, J. Meienhofer), Band 9, Academic Press, New York. 1987, pp. 103- 165; e) K . Moribara,
Trends Biorechnol. 1987, 5. 164-170; f) C.-H. Wong, K:T. Wang, E.rperientiu
1991, 47. 1123-1129: g) A. J. Andersen, J. Fromsgdard. P. Thorbeck. S. Aasmul-Olsen. Chim. Oggi 1991, Y(3). 17-24: ibid. 1991. 9(4), 17-23.
[7] V. Schellenberger, H.-D. Jakubke, A n g m . Chem. 1991. 103. 1440- 1452;
Angew. Chem. Inf. Ed. Engl. 1991. 30, 1437--1449.
Chin. Chem. Soc. 1994, 41. 355-370.
[S] H.-D. Jakubke. .l
[9] J. Bongers, E. P. Heimer, Peptides 1994. 15. 183-193.
[lo] C.-H. Wong, Chimiu 1993, 47. 127-132.
[ l l ] P. Kuhl. U. Zacharias. H . Burckhardt. H.-D. Jakubke. Monu/sh. Chem. 1986,
117, 1195-1204.
[12] R. Hirschmann. A. B. Smith 111. C. M. Taylor. P. A. Benkovic, S. D. Ttdylor,
K . M. Yager. P. A. Sprengeler, S . J. Benkovic, Science 1994, 265. 234-237.
[I31 J. R. Jacobsen. P. G . Schultz, Proc. Null. Acad. Sci. U S A 1994,91,5888-5892.
[14] G . W. Zhou, J. Guo, W. Hudng. R. J. Fletterick, T. S. Scdnlan. Science 1994.
265, 1059-1064.
Ingham, J. et al.
Chemical Engineering Dynamics
Modelling with PC Simulation
1994. XX, 701 pages with experimentation with the pro430 figures.
vision of 85 accompanying
Hardcover. DM 276.4
computer-based simulation
examples (Part 2) supplied
OS 2153.4 sFr. 256.-.
on diskette.
ISBN 3-527-28577-6
In this book, the reader is
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A n g w . (%em lnr. Ed Engl. 1995. 34. No. 2
The treatment employed in
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