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O
MeO2C
CO2Me
NBoc2
i
2
CO Me
H
NBoc2
13
14
ii
iii
MeO2C
CO2Me
NBoc2
15
MeO2C
HO2C
CO2Me
NHBoc
iv, v
CO2H
NH2
6
Scheme 3 Reagents and conditions: i, DIBAL-H, Et
Ph P = CHCO Me, THF, 85%; iii, TFA (1.2 equiv.), CH
LiOH, MeOH, H O, 98%; v, TFA, CH Cl , 87%.
2
O, 278 °C, 86%; ii,
3
2
2
Cl , 82%; iv,
2
2
2
2
as expected, showed no conversion. Subsequent inhibition
studies showed both compounds to be very good but rapidly
reversible inhibitors (not time-dependent) of DAP d-dehy-
drogenase. More detailed kinetic analyses demonstrated that 5
i
is a competitive inhibitor with an inhibition constant (K ) of 5.3
mm. In contrast, a Michaelis–Menten plot for 6, which has the
double bond in the backbone, showed noncompetitive inhibi-
tion with a K value of 44 mm, indicating different binding
i
modes for the two unsaturated analogues.
DAP d-dehydrogenase from Corynebacterium glutamicum
exists as a homodimer with each monomer unit (M ca. 35200)
possessing a similar fold but somewhat different overall
conformation based on crystallographic studies.5 Although
there may be structural differences between the B. sphaericus
and C. glutamicum enzymes,11 their overall mechanism and
inhibition by 4 are similar. Since isoxazoline 4 is a competitive
Fig. 1 (a) Model of 6 (white) docked into homodimeric DAP d-
,10
dehydrogenase (red and blue subunits) from C. glutamicum generated by
best-fit replacement of the isoxazoline 4 in the corresponding enzyme–
inhibitor crystal structure (ref. 5). These inhibitors (4 and 6) have their l-
amino acid center proximal to the NADP moiety (yellow) and therefore
cannot be oxidized by the cofactor. (b) Expansion to show the inhibitor
conformation in the closed active site.
inhibitor with respect to l-tetrahydrodipicolinate 1 (K
i
= 4.2
mm) but a noncompetitive inhibitor with respect to meso-DAP
4
i
(K = 23 mm), it seems likely that the nearly equipotent non-
competitive inhibitor 6 may bind in a similar fashion. This
indicates that interactions of the isoxazoline nitrogen and
oxygen with active site water molecules seen in crystallographic
2
3
R. J. Cox, Nat. Prod. Rep., 1996, 13, 29; G. Scapin and J. S. Blanchard,
Adv. Enzymol. Relat. Areas Mol. Biol., 1998, 72, 279.
H. Misono and K. Soda, J. Biol. Chem., 1980, 255, 10599; S. J.
Maniscalco, S. K. Saha and H. F. Fisher, Biochemistry, 1998, 37,
5
studies may play a minor role in binding. However, 5 is directly
competitive with meso-DAP 3 and is one of the most potent
substrate analogue inhibitors for this enzyme found thus far.
This suggests that 5 may be a true mimic of the imine
intermediate 2, and that normal catalysis by DAP d-dehy-
drogenase can be blocked by conformational changes induced
in the protein by binding of 4 or 6 in the adjacent subunit at a site
normally occupied by THDP 1 (Fig. 1). Additional crystallo-
graphic studies with the unsaturated a-aminopimelic acid
derivatives 5 and 6 may help to clarify the subunit interactions
and the detailed mechanism of this enzyme. Further studies on
1
4585.
4
S. D. Abbott, P. Lane-Bell, K. P. S. Sidhu and J. C. Vederas, J. Am.
Chem. Soc., 1994, 116, 6513.
5 G. Scapin, M. Cirilli, S. G. Reddy, Y. Gao, J. C. Vederas and J. S.
Blanchard, Biochemistry, 1998, 37, 3278.
6
7
8
G. Kokotos, J. M. Padron, T. Martin, W. A. Gibbons and V. S. Martin,
J. Org. Chem., 1998, 63, 3741.
M. A. Walker, K. P. Kaplita, T. Chen and H. D. King, Synlett, 1997,
1
69.
J. E. Baldwin, R. M. Adlington, M. B. Mitchell and J. Robertson,
12
the synthesis of DAP analogues and their interactions with
other DAP enzymes are in progress.
Tetrahedron, 1991, 47, 5901.
9 The preparation of 15 by essentially the same route has been reported
very recently, after our synthesis was complete: J. M. Padron, G.
Kokotos, T. Martin, T. Markidis, W. A. Gibbons and V. S. Martin,
Tetrahedron: Asymmetry, 1998, 9, 3381.
The authors thank Dr Jonathan Parrish for assistance with
molecular modeling. Financial support from the Alberta
Heritage Foundation for Medical Research (Fellowship to A. S.)
and the Natural Sciences and Engineering Research Council of
Canada (Scholarship to J. F. C. and Operating Grant) is
gratefully acknowledged.
1
0 G. Scapin, S. G. Reddy and J. S. Blanchard, Biochemistry, 1996, 35,
1
3 540.
1 H. Misono, M. Ogasawara and S. Nagasaki, Agric. Biol. Chem., 1986,
0, 2729; F. Wang, G. Scapin, J. S. Blanchard and R. H. Angeletti,
1
5
Protein Sci. 1998, 7, 293.
1
2 Y. Gao, P. Lane-Bell and J. C. Vederas, J. Org. Chem., 1998, 63,
Notes and references
2
133.
1
T. D. H. Bugg and C. T. Walsh, Nat. Prod. Rep., 1992, 9, 199; J.-M.
Ghuysen, Int. J. Antimicrob. Agents, 1997, 8, 45.
Communication 9/00297I
556
Chem. Commun., 1999, 555–556