Design and characterization of mechanism-based inhibitors for the tyrosine aminomutase SgTAM

Article abstract of DOI:10.1016/j.bmcl.2007.11.046

The synthesis and evaluation of two classes of inhibitors for SgTAM, a 4-methylideneimidazole-5-one (MIO) containing tyrosine aminomutase, are described. A mechanism-based strategy was used to design analogs that mimic the substrate or product of the reaction and form covalent interactions with the enzyme through the MIO prosthetic group. The analogs were characterized by measuring inhibition constants and X-ray crystallographic structural analysis of the co-complexes bound to the aminomutase, SgTAM.

Full text of DOI:10.1016/j.bmcl.2007.11.046

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Bioorganic & Medicinal Chemistry Letters 18 (2008) 3099–3102
Design and characterization of mechanism-based inhibitors
for the tyrosine aminomutase SgTAM
Timothy J. Montavon,a Carl V. Christianson,a Grace M. Festin,a
Ben Shenb,c,d and Steven D. Brunera,*
aDepartment of Chemistry, Merkert Chemistry Center, Boston College, Chestnut Hill, MA 02467, USA
bDepartment of Chemistry, University of Wisconsin-Madison, Madison, WI 53705, USA
cDivision of Pharmaceutical Sciences, University of Wisconsin-Madison, Madison, WI 53705, USA
dUniversity of Wisconsin National Cooperative Drug Discovery Group, University of Wisconsin-Madison, Madison, WI 53705, USA
Received 16 October 2007; revised 10 November 2007; accepted 14 November 2007
Available online 19 November 2007
Abstract—The synthesis and evaluation of two classes of inhibitors for SgTAM, a 4-methylideneimidazole-5-one (MIO) containing
tyrosine aminomutase, are described. A mechanism-based strategy was used to design analogs that mimic the substrate or product of
the reaction and form covalent interactions with the enzyme through the MIO prosthetic group. The analogs were characterized by
measuring inhibition constants and X-ray crystallographic structural analysis of the co-complexes bound to the aminomutase,
SgTAM.
Ó 2007 Elsevier Ltd. All rights reserved.
Biosynthetic building blocks based on cinnamates and
OH
OH
b-amino acids are key components of many therapeuti-
cally important natural products.1 These biosynthetic
precursors can be derived from a-amino acids by the ac-
tion of ammonia lyases and aminomutases. The pros-
thetic group 4-methylideneimidazole-5-one (MIO,
Fig. 1) is unique to these classes of enzymes.2 MIO is
a potent electrophile that is formed by the self-conden-
sation of the tripeptide sequence alanine–serine–glycine
in the protein backbone.3 Ammonia lyases catalyze the
elimination of ammonia from aromatic amino acids to
form a,b-unsaturated carboxylic acids, while 2,3-ami-
nomutases promote additional chemical steps resulting
in the net 1,2-amine migration to generate b-amino
acids.4
SgTAM
O2C
O2C
NH2
1
NH2
2
O
N
O
N
N
MIO
N
OH
OH
OH
H+
4
O2C
We have recently solved the first X-ray crystal structure
of an MIO-based aminomutase, SgTAM, demonstrat-
ing that all structurally characterized MIO-containing
enzymes have the same overall protein fold and likely
chemical mechanism.5 SgTAM catalyzes the conversion
O2C
O2C
H
NH2
NH2
NH2
N
O
N
O
N
O
N
N
N
6
3
5
Keywords: Aminomutase; Ammonia lyase; Enediyne; Natural product
biosynthesis; Nonribosomal peptide; b-Amino acid; 4-Methylideneim-
idazole-5-one; MIO.
Figure 1. The mechanism of the MIO-based aminomutase SgTAM.
The overall reaction [L-tyrosine to (S)-b-tyrosine] is boxed. The MIO
prosthetic group is derived from the protein backbone and shown
abbreviated.
*
Corresponding author. Tel.: +1 617 552 2931; e-mail: bruner@
bc.edu
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.11.046

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T. J. Montavon et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3099–3102
of L-tyrosine to (S)-b-tyrosine, the first step in the bio-
synthesis of the b-amino acid moiety of the enediyne
antitumor–antibiotic C-1027.4a,b The proposed mecha-
nism for SgTAM, illustrating the role of the MIO pros-
thetic group, is diagrammed in Figure 1. The a-amine of
L-tyrosine adds into the electrophilic moiety via a conju-
gate addition. Formation of this covalent adduct facili-
tates deprotonation of the b-hydrogen and elimination
of ammonia to produce 4-hydroxycinnamate (4). In
the reaction of ammonia lyases, the olefin intermediate
dissociates from the active site and the MIO-adduct re-
leases ammonia.5 Aminomutases retain these intermedi-
ates in the active site, allowing readdition of the MIO-
bound amine nucleophile at the b-position of the sub-
strate. The product (S)-b-tyrosine is ultimately released,
regenerating the prosthetic group.
R
R
R
O
F
F
H
F
+
H
F
SgTAM
NH3
NH2
O
CO2
CO2
CO2
N
N
7a R=OCH3
7b R=F
7c R=H
8a R=OH
8b R=OCH3
8c
R=H
11
β−
SgTAM
trapped tyrosine
mimic
R
R
O
N
H2O
O
N
H
OH
CO2
CO2
O
O
α−
trapped
tyrosine
N
N
mimic
The exploitation of aminomutases with altered specific-
ity is a potential route to produce novel aromatic b-ami-
no acids or natural product derivatives. This can be
accomplished through either the use of aminomutases
as chemoenzymatic tools in vitro or the application of
engineered biosynthesis in vivo.6 Determination of the
substrate scope and enzymatic chemistry is a key step to-
ward this goal. Evaluation of inhibitors through bio-
chemical studies and X-ray crystallography provides a
structural basis for analog binding and recognition. De-
scribed here is the synthesis and characterization of two
classes of mechanism-based inhibitors for the tyrosine
aminomutase SgTAM. The substrate or product ana-
logs were designed to form covalent adducts with the
MIO, mimicking intermediates along the reaction path-
way. We have previously described the synthesis and ini-
tial characterization of a fluorine-substituted analog
(a,a-difluoro-b-tyrosine, 8a) that mimics the product
of this reaction.7 The X-ray crystal structure of 8a
bound to SgTAM provided a structural basis for recog-
nition of the natural substrate L-tyrosine and for the
overall reaction mechanism. Synthesis, biochemical
and structural characterization of additional analogs
based on the structure of 8a are presented along with
9
10
Figure 2. Structures of tyrosine aminomutase inhibitors based on
cinnamate epoxides (7a–c) and a,a-difluoro-b-tyrosine (8a–c). The
proposed binding mode for each inhibitor class is illustrated.
R
R
R
a
b
O
O
-
CO2Et
CO2Et
13a
13b, R=F
, R=H
CO2
12a
, R=OCH3
12b, R=F
12c,
, R=OCH3
7a, R=OCH3 (62%)
7b, R=F
7c
, R=H
(28%, 86%ee)
(37%, 97%ee)
(41%, 95%ee)
(72%)
(70%)
13c
R=H
Scheme 1. Synthesis of epoxide-based inhibitors of SgTAM. Reagents:
(a) 30 mol% Shi’s epoxidation catalyst, oxone, NaHCO3, Na2EDTA,
cat. TBAHS, H2O–CH3CN; (b) KOH, MeOH. Isolated yields and
percent enantiomeric excess are indicated.
where R = OH was not feasible as the final product is
not stable under neutral pH conditions.
Analogs based on the product, (S)-b-tyrosine (8a,
Fig. 2), also form adducts with the MIO.7 The inclusion
of a-fluorines in 8a prevents the reverse reaction [(S)-b-
tyrosine to L-tyrosine] and generates a product-like
intermediate co-complex (Fig. 2, 11). The synthesis of
the a,a-difluoro analogs (8a–c) follows published work
using Ellman’s sulfinamide chemistry to prepare a,a-di-
fluoro-b-phenylalanines.10 The synthesis of 8c is summa-
rized in Scheme 2 as an example. Briefly, the route starts
with benzaldehyde (or 4-substituted benzaldehydes),
a novel class of inhibitors based on cinnamate
epoxides.
Figure 2 illustrates two classes of inhibitors for MIO-
containing enzymes. Analogs based on cinnamate epox-
ides (7a–c) were designed to mimic the para-hydroxycin-
namate intermediate in the reaction while presenting a
reactive functionality in the active site. The design ratio-
nale is based on the similar geometry of the epoxide to
the double bond in para-hydroxycinnamate. In addition,
the stereochemistry of the epoxide mimics that of L-tyro-
sine and (S)-b-tyrosine. The synthesis of epoxide-
containing analogs (7a–c) begins with various para-
substituted ethyl cinnamates (12a–c, Scheme 1).
Enantioselective epoxidation with Shi’s dioxirane cata-
lyst efficiently produced the desired epoxides.8 Saponifi-
cation of the ethyl ester protecting group gave the
desired analogs as potassium salts.9 As opposed to the
free acids, the epoxides were stored as stable potassium
carboxylates. The methoxy- and fluoro-substituents of
7a and 7b were incorporated to be mimics of the phenol
on the natural substrate L-tyrosine. The synthesis of 7
which is condensed with (R)-tert-butylsulfinimine.11
A
Reformatsky-type addition of BrZnCF2CO2Et followed
OHC
c,d
b
a
-
CO2Et
CO2
85%
+H3N
45%,
90%
N
S
H
HN
S
90%de
O
S
F
F
F
F
O
O
8c
NH2
t
Bu
Scheme 2. General synthesis of a,a-difluoro-based inhibitors of
SgTAM. Reagents: (a) CsCO3, DCM; (b) Zn0, ethyl bromodifluoro-
acetate, THF; (c) 6 N HCl; (d) i-PrOH, propylene oxide.

T. J. Montavon et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3099–3102
3101
by global deprotection yields the desired b-tyrosine
analogs.
Binding of the designed inhibitors (7a–c and 8a–c) was
evaluated by determination of their inhibition constants
(IC50) with SgTAM. The natural substrate L-tyrosine
was incubated with the enzyme and various concentra-
tions of analogs. The relative binding efficiency was
measured using an HPLC-based assay and OPA deriva-
tization of the starting L-tyrosine and product (S)-b-
tyrosine.4b The results (Fig. 3) show all analogs inhibit
the reaction in a concentration-dependent manner and
the designed substitutions are tolerated by the enzyme.
To observe adequate turnover in the HPLC assay, a
high concentration (0.5 mg/mL) of enzyme and sub-
strate L-tyrosine (75 lM) was necessary, resulting in
IC50 values in the mM range.
To determine the structural basis of inhibition, the co-
complex structures of each of the inhibitors were deter-
mined through X-ray crystallography. The structures
from each of the two inhibitor classes gave very similar
binding modes, therefore the structures of 7b and 8b are
illustrated to represent their respective classes (Fig. 4).
The structure of the epoxide analog 7b confirms that
the analog forms a covalent interaction with the electro-
philic MIO through the epoxide oxygen. Electron den-
sity for
corresponding to the addition of water as illustrated in
a
b-hydroxyl is clear in the maps,
Figure 2. The binding mode of 7b is analogous to that
Figure 4. X-ray crystal structures of representative inhibitors bound in
the active site of the aminomutase SgTAM. The inhibitors (dark gray),
active site amino acid residues (light gray), and MIO prosthetic group
(dark red) are illustrated as sticks.
R
R
O
F
H
F
+
H
NH3
predicted for amino acids bound to MIO-based en-
zymes. The presence of the 4-fluoro group in 7b pushes
the aryl ring slightly away from the recognition elements
His93 and Tyr415. These residues are predicted to form
hydrogen bonds with the substrate 4-hydroxyl.12 The
2,3-diol observed in the crystal structure is a single dia-
stereomer (2R,3S) and results from attack and inversion
at the 3-position of 7. To provide evidence that the ring
opening is enzyme-catalyzed, 7c was incubated under
standard assay conditions with and without SgTAM.
Substrate analog 7c was converted to the corresponding
diol when incubated in the presence of SgTAM, but was
recovered intact after a 24-incubation in buffer without
enzyme.13 Therefore, the hydrolysis of the epoxide ring
was considered to be enzyme-catalyzed as shown in Fig-
ure 2. The observed diol can be generated either through
an SN1 (as shown) or SN2-type mechanism. The result is
a structure that is a close mimic of the starting adduct
between L-tyrosine and MIO.
CO2
R
CO2
Analog
Analog
IC50 (mM)
R
IC50 (mM)
7a
7b
7c
8a
8b
8c
OMe
F
H
3.0
11.6
5.8
OH
OMe
H
4.8
5.3
7.3
100
90
80
70
60
50
40
In addition to the previously reported structure of 8a
bound to SgTAM, the biochemical evaluation of the
analogs bearing substitutions at the 4-position suggests
these will also form a covalent co-complex. The struc-
tures of 8b and 8c bound to SgTAM were solved (see
Fig. 4) and both displayed a similar binding mode.
The 4-methoxy analog interacts with the two residues
30
0
2
4
6
8
10
12
14
[inhibitor, 7a] (mM)
Figure 3. IC50 measurements for synthetic inhibitors of SgTAM.
Experiments were done in triplicate and a representative graph is
illustrated.

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T. J. Montavon et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3099–3102
(Tyr415 and His93) important in tyrosine recognition.
Hydrogen bonding interactions are maintained, and
the methyl group of the methoxy substrate displaces a
bound water molecule that is present in the enzyme
structure when the 4-hydroxy or the 4-fluoro inhibitor
is covalently bound.
References and notes
1. For examples see: (a) Van Lanen, S. G.; Oh, T. J.; Liu, W.;
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Shen, B.; Bruner, S. D. Biochemistry 2007, 46, 7205.
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Overall, the results provide a structural basis for cova-
lent catalysis and substrate recognition by MIO-based
enzymes. Of particular note is the novel use of epoxide
analogs as mechanism-based trapping reagents to pro-
vide structural insights into L-tyrosine binding and rec-
ognition. The structures of the bound inhibitors
support a mechanism of catalysis via an amino/MIO ad-
duct and the utilization of Tyr63 as a catalytic base to
promote the observed 1,2 amino shift catalyzed by
SgTAM.7 The L-tyrosine analog (7b) interacts with
one face of the MIO while the product-like analog (8b)
forms a bond with the opposite face. This finding sup-
ports a catalytic mechanism in which the amine bound
to the MIO (5, Fig. 1) rotates while the substrate re-
mains relatively fixed, resulting in the observed 1,2-
amine shift and inversion of configuration at C3.
Acknowledgments
8. Wu, X. Y.; She, X.; Shi, Y. J. Am. Chem. Soc. 2002, 124,
8792.
We thank Heather A. Cooke for helpful discussions and
critical reading of the manuscript, Dr. Y. Li for provid-
ing the C-1027-producing S. globisporus strain, and A.
Orville and the staff at the Brookhaven NSLS PXRR
for assistance with X-ray data collection. This work is
supported in part by funds from Boston College and
the Damon Runyon Cancer Research Foundation
DRS-41-01 (S.D.B.) and NIH Grants CA78747 (B.S.).
9. Experimental procedures and complete characterization of
all novel compounds are included in the supplemental
section accompanying this letter.
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Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.
2007.11.046.
13. Refer to the supplemental section for assay conditions.