10442 J. Am. Chem. Soc., Vol. 123, No. 43, 2001
Kaur et al.
phosphate intermediate in ribonuclease catalysis.19 Analogies
among enzyme inhibitors are the reversible inhibition of
chymotrypsin20 and papain21 by cyclic sulfonates and of
chymotrypsin and thrombin by various lactones.22,23 The â-lac-
tams themselves supply a counterexample, however. They
covalently inhibit bacterial DD-peptidase by acylation of the
active site serine to give the inert complex, 19. This slowly
deacylates by hydrolysis or fragmentation,24 but there is no
evidence of recyclization. This may be due, in part, to the high
thermodynamic barrier to ring closure of bicyclic â-lactams,25
but it may also, in some cases at least, reflect other factors. For
Scheme 8
(Porton Down, Wiltshire, U.K.) and used as received. Chemical reagents
for synthesis were generally purchased from Aldrich Chemical Co.
Lysinoalanine was purchased from Sigma Chemical Co. Salicyloyl
cyclic phosphate 2 and its acyclic analogue 6 were synthesized
previously in this laboratory.10,14
1-Hydroxy-4,5-benzo-2-oxaphosphorinanone(3)-1-oxide Dicyclo-
hexylammonium Salt (5). This compound was prepared by a route
beginning with ethyl o-toluate (Scheme 8). Bromination of this material
with N-bromosuccinimide gave substituted benzylbromide 20. This was
reacted with triethyl phosphite to give phosphonate diester 21 which
was hydrolyzed to diacid 22. The synthesis of 22 was largely as
described by Gasuti et al.29 The acid was cyclized by azeotropic
dehydration to give cyclic acyl phosphonate 5, which was isolated as
the dicyclohexylammonium salt.
example, on acylation of the Streptomyces R61 DD-peptidase
by cephalosporins, rotation around the C6-C7 bond of the
â-lactam occurs (19), presumably to relieve steric stress between
the â-substituents on C6 and C7.26 Recyclization would not
directly be possible from this acyl enzyme conformation. On
the other hand, an acyl enzyme generated from the reaction of
penicillin binding protein 2x of Streptococcus pneumoniae with
cefuroxime appears to be well-placed for recyclization27 (al-
though it should be noted that this structure, like the R61
examples, does not represent the initial acyl enzyme but that of
a subsequent complex where the 3′-leaving group has been
eliminated and a ∆1-dihydrothiazine ring formed). The phosphyl
enzyme intermediates generated from 2 and 5 (18) apparently
retain access to a conformation where recyclization is possible.
A more effective inhibitor than 2 might include functionality
to promote conformational relaxation after phosphylation that
would better hinder or prevent recyclization and regeneration
of the enzyme.
Ethyl o-toluate (20 g, 122 mmol) and N-bromosuccinimide (21.7 g,
122 mmol) were mixed with 81 mL of carbon tetrachloride. Benzoyl
peroxide (20 mg) was added as a catalyst. The flask was irradiated
with visible light from a 150 W tungsten lamp, while the mixture was
heated under reflux for 3 h. At that time, the reaction mixture was
cooled to room temperature. The precipitated solid was removed by
vacuum filtration, and the remaining solvent was removed by rotary
evaporation to give the brominated product 20 as a deep yellow liquid
1
(33.1 g). H (CDCl3) δ 7.90 (d, J ) 7.5 Hz, 1H), 7.49 (t, J ) 7.5 Hz,
1H), 7.48 (t, J ) 7.5 Hz, 1H), 7.40 (d, J ) 7.5 Hz, 1H), 4.99 (s, 2H),
4.42 (q, J ) 7.1 Hz, 2H), 1.44 (t, J ) 7.1 Hz, 3H). This material was
used without further purification.
Acyl phosph(on)ates 2 and 5 thus inhibit the P99 â-lactamase
most likely by phosphylation of the active site. The enzyme is
unable to catalyze the hydrolysis of these species to restore the
enzyme, but the latter can be achieved by a recyclization reaction
which restores the cyclic acyl phosph(on)ates: rescue by return.
These compounds therefore function as reversible inhibitors.
Experiments to expand the kinetic and thermodynamic repertoire
of these molecules in the direction of more effective inhibitors
are in progress. There is evidence that even 2 does enhance
â-lactam activity in in Vitro microbiological assays.28
Ester 20 was heated under reflux with an equimolar amount of
triethyl phosphite at 160 °C for 3 h. The reaction mixture was cooled
overnight and then vacuum distilled to give phosphonate ester 21 (bp
170-174 °C, 0.1 Torr) as a clear yellow liquid in 82% yield (two steps).
1H NMR (CDCl3) δ 7.91 (d, J ) 7.7 Hz, 1H), 7.44 (d, J ) 7.7 Hz,
1H), 7.41 (t, J ) 7.7 Hz, 1H), 7.30 (t, J ) 7.7 Hz, 1H), 4.39 (q, J )
7.6 Hz, 2H), 4.00 (quint, J ) 7.6 Hz, 4H), 3.82 (d, J ) 23 Hz, 2H),
1.41 (t, J ) 7.6 Hz, 3H), 1.21 (t, J ) 7.6 Hz, 6H).
The phosphonate 21 was added to 500 mL of 6 M HCl and the
mixture heated under reflux for 3 days. The HCl/H2O was removed by
rotary evaporation, and the white solid thus obtained was recrystallized
from water in 62% yield. The product diacid 22 exhibited a broad
Experimental Section
1
melting point of 194-202 °C (lit.29 mp 177 °C). H (D2O) δ 7.83 (d,
Materials. The â-lactamase of Enterobacter cloacae P99 was
purchased from the Centre for Applied Microbiology and Research
J ) 8.1 Hz, 1H), 7.56 (t, J ) 8.1 Hz, 1H), 7.41 (m, 2H), 3.57 (d, J )
21.9 Hz, 2H). νmax (KBr) 1659s (CdO), 1271s (PdO).
Solid acid 22 (200 mg) was heated under reflux in 50 mL xylene in
a Dean and Stark apparatus. The solid dissolved in boiling xylene, and
drops of water were noted in the collection arm of the apparatus after
5 h, signifying cyclization. At this stage, an equimolar amount of
dicyclohexylamine was added to the reaction mixture. A colorless salt
precipitated as the solution was cooled. The solid was recrystallized
from cyclohexane/benzene, yielding the amine salt of pure cyclized
(19) Eftink, M. R.; Biltonen, R. L. In Hydrolytic Enzymes; Neuberger,
A., Brocklehurst, K., Eds.; Elsevier: Amsterdam, 1987; Chapter 7.
(20) Heidema, J. H.; Kaiser, E. T. J. Am. Chem. Soc. 1970, 92, 6050.
(21) Campbell, P.; Kaiser, E. T. Biochem. Biophys. Res. Commun. 1972,
48, 866.
(22) Hedstrom, L.; Moorman, A. R.; Dobbs, J.; Abeles, R. H. Biochem-
istry 1984, 23, 1753.
(23) Weir, M. P.; Bethell, S. S.; Cleasby, A.; Campbell, C. J.; Dennis,
R. J.; Dix, C. J.; Finch, H.; Harren, J.; Mooney, C. J.; Patel, S.; Chi-Man,
T.; Ward, M.; Wonacott, A. J.; Wharton, C. W. Biochemistry 1998, 37,
6645.
(24) Fre`re, J.-M.; Joris, B. Crit. ReV. Microbiol. 1985, 11, 299.
(25) Page, M. I. AdV. Phys. Org. Chem. 1987, 23, 165.
(26) Kuzin, A. P.; Liu, H.; Kelly, J. A.; Knox, J. R. Biochemistry 1995,
34, 9532.
1
product 5. Melting point 163-165 °C. H NMR (CDCl3) δ 9.06 (br,
2H), 8.21 (d, J ) 8 Hz, 1H), 7.51 (t, J ) 8 Hz, 1H), 7.38 (t, J ) 8 Hz,
1H), 7.28 (d, J ) 8 Hz, 1H), 3.29 (d, J ) 18 Hz, 2H), 2.82 (br, 2H),
1.1-2.0 (m, 20H). 31P NMR (2H2O) δ 10.52 (t). νmax (KBr) 1713s
(CdO), 1251s (PdO). Anal. Calcd for C20H29NO4P: C, 63.31; H, 7.97;
N, 3.69; P, 8.16. Found: C, 63.40; H, 8.08; N, 3.64; P, 8.80. ESMS
(H2O) m/z 560.9 (M + 2Cl2H23NH+) 560.8.
(27) Gordon, E.; Mouz, N.; Due´e, E.; Dideberg, O. J. Mol. Biol. 2000,
299, 477.
(28) Besterman, J. M.; Rahil, J.; Pratt, R. F. PCT Int. Appl.; Chem. Abstr.
1999, 131, 82947.
(29) Garuti, L.; Farranti, A.; Roberti, M.; Katz, E.; Budriesi, R.; Chiarini,
A. Pharmazie 1992, 47, 295.