Hansen et al.
bridge may distort the binding region away from the
bound conformation. Several examples of naturally oc-
curring, macrocyclic inhibitors of serine peptidases are
known, including cyclotheonamide A9,10 and the cyano-
peptolides.11-13 Interesting as these molecules are, in
many respects they are more complex than traditional
inhibitors and they provide limited insight into how to
design simpler macrocycles.
Structure-based design approaches are high-risk and
imprecise, and they often lead to challenging targets for
synthesis.3,14-19 The modest success we have had in
enhancing the affinity of peptidase inhibitors by design-
ing macrocyclic derivatives based on the bound structures
of acyclic analogues has not only underscored the poten-
tial payoff but also illustrated the design challenge. For
example, the phosphonates 1and 2 bind to the aspartic
peptidase penicillopepsin almost identically,5 yet the
macrocycle has 420-fold greater affinity.19 In contrast, the
cyclic and acyclic naphthalene analogues 3 and 4 differ
by only a factor of 10 in affinity18 and they do not bind to
the enzyme in quite the same way.20
of which would be the difficulty in preparing these
structures in an automated fashion. For this reason, we
have developed an inverse approach: screening acyclic
product-like molecules to identify analogues that are
readily cyclized by the peptidase.21 The acyclic substrates
are readily synthesized with terminal amine and car-
boxylic acid or carboxylate ester groups, as required for
different peptidases. If the bridging moiety allows the
peptidic part of the structure to interact favorably with
the enzyme, under appropriate conditions, the enzyme
will catalyze peptide bond formation via a macrocyclic
transition state. Since both the forward (hydrolysis) and
reverse (amide synthesis) reactions catalyzed by a pep-
tidase involve the same enzyme intermediate,22 linear
derivatives that are readily cyclized by the enzyme should
point to bridging units that facilitate formation of this
transition state and, thus, would be good candidates for
incorporation into a transition state analogue inhibitor.
A variety of strategies is available for inducing peptide
bond formation by a peptidase, including using activated
precursors, lowering the pH, and reducing the water
content of the medium.22 In addition to our preliminary
work,21 there are two earlier reports of the use of enzymes
to synthesize cyclic peptides (thermolysin23 and subtili-
gase24), although not in the context of library screening.
More recently, TycC thioesterase has been used to create
a library of cyclic peptide antibiotic products.25
A colorimetric, on-bead assay is used to identify
analogues that are cyclized by the enzyme (Figure 1). The
substrates are designed with an ester moiety as a
cleavable linker between the amine and carboxyl ends
of the acyclic substrate. A dye molecule and the point of
attachment to the resin particle are also located on
opposite sides of the ester linkage. Thus, after treatment
with the enzyme and subsequent saponification of the
ester linkage, the dye molecule remains associated with
the resin only if the lactam amide bond has been formed,
i.e., if the substrate has been cyclized. By simple visual
inspection of the beads, competent substrates can be
distinguished from poor substrates. This method facili-
tates the rapid identification of optimal scaffolds for the
construction of transition state analogue inhibitors. This
screening strategy was first tested using trypsin on a
derivative of the cyanopeptolin A90720A, a potent inhibi-
tor of trypsin.21 In this report, we describe the design and
synthesis of smaller, novel macrocycles that incorporate
the features of the screening strategy and are synthesized
with serine, aspartic, and metallopeptidases.
The idea of screening a library of diverse macrocyclic
structures offers complementary challenges, not the least
(9) Lee, A. Y.; Hagihara, M.; Karmacharya, R.; Albers, M. W.;
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