N-Sulfonyloxy-β-lactam inhibitors for β-lactamases

Article abstract of DOI:10.1016/S0040-4020(00)00427-0

Structure-function analysis with a series of N-sulfonyloxy β-lactam molecules as inhibitors of β-lactamases is reported. The best of these compounds acylate the active site of the class A TEM-1 β-lactamase from Escherichia coli rapidly, and resist deacylation. Whereas acylation of the active site of the class C β-lactamase from Enterobacter cloacae was not seen, these compounds function as competitive inhibitors of this enzyme. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(00)00427-0

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 5719±5728
N-Sulfonyloxy-b-lactam Inhibitors for b-Lactamases
Alexey Bulychev,a John R. Bellettini,b Michael O'Brien,b Peter J. Crocker,b Jean-Pierre Samama,c
Marvin J. Millerb and Shahriar Mobasherya,
*
aDepartment of Chemistry, Wayne State University, Detroit, MI 48202, USA
bDepartment of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556, USA
cGroupe de Cristallographie Biologique, Institut de Pharmacologie et de Biologie Structurale du CNRS,
205 route de Narbonne, 31077-Toulouse cedex, France
Received 5 November 1999; accepted 4 February 2000
AbstractÐStructure-function analysis with a series of N-sulfonyloxy b-lactam molecules as inhibitors of b-lactamases is reported. The best
of these compounds acylate the active site of the class A TEM-1 b-lactamase from Escherichia coli rapidly, and resist deacylation. Whereas
acylation of the active site of the class C b-lactamase from Enterobacter cloacae was not seen, these compounds function as competitive
inhibitors of this enzyme. q 2000 Elsevier Science Ltd. All rights reserved.
The emergence of pathogenic microorganisms resistant
to multiple classes of antibiotics is a serious clinical
challenge.1 Among these classes of antibacterials, b-lactam
antibiotics are still the most commonly used more than 50
years after their initial introduction. The success of these
antibiotics is due to their bactericidal activity, good phar-
macokinetics, low host toxicity and their ability to synergize
with other classes of antibiotics such as aminoglycosides. In
light of the fact that there are no replacements for these
versatile antibiotics in the immediate future, it is essential
that their clinical utility be prolonged.
based inhibitors for the class A b-lactamases.6±9 As exem-
pli®ed by compound 1, the inhibitor acylates the active site
serine of b-lactamases, a process that is usually rapid
(Scheme 1). On acylation of the active site, the tosylate is
released from species 2. From that point on, there appears to
be some variations for the ®nal structure of the inhibited
enzyme. We have documented the possibility for structures
3±5 in inhibited b-lactamases.6±8 An a- or b-elimination
would give rise to a species such as 3, which resists deacyl-
ation because it is an a,b-unsaturated ester.6 The iminium
species such as 4 was seen in conjunction with the move-
ment of the ester carbonyl out of the oxyanion hole in the
active site.7 As such, the ester could not undergo the deacyl-
ation step, and the inhibited enzyme enjoyed considerable
longevity. We have also seen the keto species 5 in the
inhibited NMCA b-lactamase from Enterobacter cloacae.8
In this case as well, a movement of the ester carbonyl out of
the oxyanion hole was critical for the inhibition process. The
present report expands on these ®ndings and de®nes further
the properties of this class of enzyme inhibitors.
The most common mechanism for resistance to b-lactam
antibiotics is the ability of bacteria to produce b-lacta-
mases.2,3 These enzymes hydrolyze the b-lactam moiety
in these drugs, a reaction that inactivates the antibiotics.
There are four distinct classes of b-lactamases, of which
class A enzymes are the most common.3 A successful
approach to overcoming the adverse action of these
enzymes has been the use of b-lactamase inhibitors together
with the typical b-lactam antibiotics, such as penicillins.3,4
Unfortunately, this approach has been compromised as well
by the discovery of the new variants of b-lactamases
resistant to inhibition by known inhibitors.5 These new
variants of b-lactamases still remain highly effective in
hydrolyzing b-lactam antibiotics. Therefore, development
of novel b-lactam inhibitors that operate by entirely
different mechanisms is highly desirable.
Results and Discussion
Syntheses
Synthesis of the key intermediate 6 for the preparation of
compounds 7a±f has been described.6 We retained the
phenyl group at position 4 of the azetidinone ring in many
of our compounds since a speci®c hydrophobic interaction
in the active site of class A b-lactamases favored this group.
Furthermore, the stereochemistry at position 4 appeared not
to be important for inhibition,6 so we have a mixture of
isomers at this position with the new compounds. Removal
We have reported previously on the synthesis and mecha-
nism of action of a novel class of monobactam mechanism-
Keywords: b-lactamase; inhibition.
* Corresponding author. Tel.: 1313-577-3924; fax: 1313-577-8822.
0040±4020/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00427-0

5720
A. Bulychev et al. / Tetrahedron 56 (2000) 5719±5728
Scheme 1.
of the Cbz group of 6 by hydrogenolysis over Lindlar's
catalyst (Pd±CaCO3±PbO) afforded the corresponding
N-hydroxy b-lactam, which was treated with a series of
sulfonyl chlorides in the presence of triethylamine to afford
the desired b-lactams 7a±i in high yields (6!7) Scheme 2.
The reaction of the N-hydroxy b-lactam, the product of
hydrogenolysis with pyridine SO3 afforded the correspond-
ing O-sulfonated b-lactam 7j, which was isolated as the
tetra-n-butyl ammonium salt.
these molecules, in conjunction with molecular modeling
with the X-ray structures of b-lactamases, suggested to us
that certain functionalities could be introduced into the basic
design to improve the interactions between the inhibitors
and the target enzyme(s). b-Lactam 8, which contains a
2,3-dimethoxyphenyl group at the C-4 position, was one
such molecule. The methoxy groups of 8 were expected to
have favorable electrostatic interactions with the enzyme,
for example. Compound 9 is another second-generation
compound. The C-3a group of 9 was introduced into the
structure of the inhibitor to retard the deacylation of the
The structure-function analysis of the ®rst generation of
Scheme 2.

A. Bulychev et al. / Tetrahedron 56 (2000) 5719±5728
5721
Scheme 3.
inhibited species. This designed concept ®nds precedent in
earlier work by us.10±14 The synthesis and properties of 9
have already been reported.7
comparisons of these inactivators to one another; the larger
the number, the better the process. The partition ratio (kcat/
kinact) can be evaluated directly, and is an expression of the
ef®ciency of the inactivation process. That is, how readily
the compound experiences turnover versus the rate of
enzyme inactivation: the lower this number, the more
favorable the inactivation process. Once the active site is
modi®ed, one can evaluate the rate constant for recovery of
Compound 8 was synthesized according to Scheme 3.
b-Ketoester 14 was synthesized from 2,3-dimethoxy-
benzoic acid (12) and monomethylmalonate (11)15 using
the Masamune±Brooks reaction.16 The ketone moiety of
14 was reduced with sodium borohydride to afford 15.
Saponi®cation of the methyl ester 15 with 1.0 M sodium
hydroxide and coupling of the resulting acid with O-benzyl-
hydroxylamine using EDC´HCl afforded hydroxamate 16 in
89% yield, which was cyclized to b-lactam 17 in the
presence of carbon tetrachloride, triphenylphosphine, and
triethylamine in 79% yield. Removal of the benzyl group
of 17, followed by tosylation of the N-hydroxy b-lactam
afforded N-tosyloxy b-lactam 8 in 72% yield.
activity (kH O), as a measure of the longevity of the inactive
enzyme species; the smaller this rate constant, the better the
inactivator.
2
The trends that emerged from the kinetic analyses indicated
that there is a strong preference for a hydrophobic moiety at
the sulfonate position for inactivation of the class A TEM-1
b-lactamase. Compound 7j failed to inactivate the enzyme,
whereas compound 7c inactivated the enzyme most
favorably, as discerned from the values for kinact/KI. It is
noteworthy that the rate constants for inactivation of the
enzyme for all compounds that did inactivate the enzyme
are high. The TEM-1 b-lactamase also shows preference for
the phenyl ring at position 4. The change of phenyl to
methyl at this position reduced the ef®ciency of the inacti-
vation process (lower kinact/KI, and higher partition ratio).6
Compounds 18±21, which lack the phenyl group at position
4, did not inactivate the enzyme. They either served as poor
substrates or poor competitive inhibitors for the class A
b-lactamase. Compounds 19, 20 and 21 were found to be
competitive inhibitors for the TEM-1 b-lactamase with
dissociation constants (KI) of 5, 1 and 200 mM, respectively.
Compound 18 was neither a substrate nor an inhibitor for the
TEM-1 b-lactamase, but it inhibited the Q908R enzyme.
The KI values for compounds 18 and 20 with the Q908R
enzyme were 45 and 27 mM, respectively. Compounds 19
and 21 did not inhibit the class C Q908R enzyme. All
inactivators showed desirable partition ratios of less than 10.
To explore the nature of the interactions of the enzyme(s)
with the C-4 substituent, we synthesized four additional
compounds. The syntheses of three of these compounds
(18±20) are given in Scheme 4, and that of compound 16
has been reported earlier.17
Kinetic determinations of enzyme inhibition
The results of our inhibition studies are summarized in
Table 1. The effective inhibitors modify the active site of
the class A TEM-1 b-lactamase essentially instantaneously
on mixing. The rate of this process could not be attenuated
effectively by competition with substrates to permit evalua-
tion of individual parameters for enzyme inhibition, such as
reported by us previously for another type of inhibitor.18
However, we have used three parameters in evaluation of
these compounds with the TEM-1 b-lactamase. The second-
order rate constant for inactivation (kinact/KI) is useful in

5722
A. Bulychev et al. / Tetrahedron 56 (2000) 5719±5728
Scheme 4.
Table 1. Kinetic parameters for inhibition of b-lactamases by monocyclic N-oxy-substituted b-lactams
Compounds
Enzymes
TEM-1
kcat/kinact
Q908R
kinact/KI (M21 s21)£1023
kH O (s21)£103
KI (mM)
2
7aa
7ba
7ca
7d
7e
7f
7.4^0.2
11.2^0.8
72.5^1.4
37.7^1.1
22.6^0.9
13.6^0.7
15.0^0.5
15.8^0.5
5.8^0.3
±
7
5
2
2
4
4
4
2
6
±
±
±
8
7
2.0^0.3
2.1^0.3
1.8^0.2
3.3^0.8
2.7^0.3
3.6^0.5
2.1^0.4
2.7^0.4
1.5^0.8
±
24^5
12^2
65^15
220^55
260^30
680^110
5^1
7g
7h
7i
37^4
110^20
±
45^7
27^9
9^2
520^ 40
7jb
18
20
8
±
±
±
9^1
±
32^3
3.2^0.15
9c
1.6^0.4d
0.013^0.002
a Preliminary results were reported in Ref. 6.
b This compound did neither inactivate nor inhibit the enzymes.
c Results were reported in Ref. 7.
d Recovery of activity was biphasic for compound 9; the two numbers refer to the rates of recovery of activity for each phase.

A. Bulychev et al. / Tetrahedron 56 (2000) 5719±5728
5723
The second-generation inactivator 8 was designed in order
to take advantage of potential electrostatic interactions of
the enzyme with the methoxy substituents in the ring. These
interactions were expected to favor the enzyme modi®cation
process, and indeed they did. Compound 8 modi®ed the
enzyme with a second-order rate constant that was over
three-fold more rapid than that of the parent compound
without the methoxy moieties (i.e., 7b), but regrettably,
the rate of deacylation of this inhibitor from the active site
was faster by the same extent. As reported earlier,
compound 9 inactivated the TEM-1 b-lactamase somewhat
slower than others, re¯ecting an attenuation of the rate of
enzyme acylation due to the presence of the C-3a hydroxy-
ethyl group.7 This compound was designed speci®cally to
undergo deacylation more slowly than the rest of these
inactivators, thereby imparting enhanced longevity of the
inhibited enzyme species. We indeed observed considerably
slower deacylation in this case, which gave a longevity of
the inhibited species of a number of days.7
spectra were obtained on a Jeol JMS-AX505HA mass spec-
trometer. Thin-layer chromatography (TLC) was conducted
on silica gel 60 F254 (0.2 mm thickness, aluminum support)
and the chromatograms were visualized with ultraviolet
light and by dipping in 10% phosphomolybdic acid
(PMA) in ethanol, followed by heating. Flash column
chromatography was performed using silica gel 60 (EM
Science, 230±400 mesh ASTM). Elemental analyses were
performed by M-H-W Laboratories, Phoenix, AZ.
Anhydrous acetonitrile and triethylamine were freshly
distilled from calcium hydride under an atmosphere of nitro-
gen and transferred via syringe or cannula. Bulk grade ethyl
acetate (EtOAc) and Skellysolve B (referred to simply as
`hexanes') were distilled before use. All purchased reagents
were of reagent grade quality and were used without further
puri®cation. Penicillin G and cephaloridine were purchased
from Sigma. The wild-type class A TEM-1 b-lactamase
from Escherichia coli19 and the class C E. cloacae Q908R
b-lactamase20±22 were puri®ed according to literature
methods. All kinetic and spectral measurements were
made on a Hewlett±Packard 8452 or 8453 diode-array spec-
trometer. The enzyme assay and methods for determination
of kinetic parameters were according to published pro-
cedures.6
It is important to note that the sulfonate moiety serves as a
surrogate for the invariant carboxylate of b-lactams in
recognition by the enzyme.6 Compounds that lack it, as in
N-alkoxy or N-carbonate derivatives of our b-lactams, are
recognized by the enzyme with much poorer af®nity or not
at all (data not shown). None of these compounds appeared
to acylate the active-site serine of the class C b-lactamase
(Q908R). However, all of these compounds, except 19 and
21, inhibited the class C enzyme competitively.
General procedure for the syntheses of compounds 7e±7i
A mixture of compound 6 (0.8 mmol) in methanol (8 mL)
was treated with either 10% Pd/C (15 mg) (for compounds
7e, 7f and 7g) or Lindlar catalyst (15 mg) (Pd±CaCO3±
PbO, Aldrich) under an atmosphere of hydrogen. The
suspension was stirred for 30 min and ®ltered through a
thin layer of celite and concentrated. The resulting crude
N-hydroxy-b-lactam and the appropriate sulfonyl chloride
(1.0 equiv., Aldrich) were dissolved in dry acetonitrile and
cooled to ice±water temperature. Triethylamine (1.0 equiv.)
was added to this mixture dropwise and the reaction mixture
was stirred for 20 min. The reaction mixture was concen-
trated and the residue was subjected to ¯ash column
chromatography (25% ethyl acetate in hexanes) to obtain
the corresponding product.
Conclusions
These monobactam inhibitors modify the active site serine
of class A b-lactamases rapidly. On acylation of the active
site, the acyl-enzyme species undergoes fragmentation,
resulting in enzyme inhibition by three distinct products,
depending on the nature of the funtionalities that have
been incorporated into the inhibitor (species 3, 4, or 5).
The results reported herein for the structure-function
analyses of the sulfonate moiety argue for the requirement
of a hydrophobic functionality, but its size does not appear
to be limiting. The absence of any hydrophobic function-
ality at this position (as in 7j) impairs the ability of the
molecules to inhibit b-lactamases.
(^)-N-(8-Quinolinesulfonyloxy)-4-phenyl-2-azetidinone
(7e). Three fractions were collected. Fraction 1 (21 mg)
contained an unidenti®ed compound. Fraction 2 (191 mg)
contained a mixture of unidenti®ed products and 7e.
Fraction 3 (140 mg) contained 7e as a white solid. Fractions
2 and 3 were recrystallized from ethyl acetate in hexanes to
give a total of 89 mg (35%) of 7e as a white solid:
Experimental
General procedures
mp.1908C; IR (KBr) 1820 cm21 1H NMR (CDCl3,
;
300 MHz) d 9.07 (dd, Jˆ1.8, 4.2 Hz, 1H), 8.51 (dd,
Jˆ1.4, 7.4 Hz, 1H), 8.04 (dd, Jˆ1.4, 8.2 Hz, 1H), 7.60 (t,
Jˆ7.8 Hz, 1H), 7.5 (dd, Jˆ4.3, 8.3 Hz, 1H), 7.26±7.11 (m,
3H), 7.10±6.96 (m, 2H), 4.86 (dd, Jˆ3.4, 6.3 Hz, 1H), 3.18
(dd, Jˆ6.4, 14.4 Hz, 1H), 2.66 (dd, Jˆ3.4, 14.5 Hz, 1H);
HRMS (FAB, MH1) calcd for C18H14N2O4S 355.0753,
found 355.0750; Anal. calcd for C18H14N2O4S: C, 61.01;
H, 3.98; N, 7.90. Found: C, 60.79; H, 4.21; N, 7.72.
Melting points (mp) were determined on a Thomas±Hoover
capillary melting point apparatus in open capillaries and are
uncorrected. Infrared (IR) spectra were obtained on a
Perkin±Elmer 1420 IR spectrophotometer and were cali-
brated with the 1601 cm21 band of polystyrene. Nuclear
magnetic resonance (NMR) spectra were obtained on a
General Electric GN-300, a Varian Unity Plus 300, or a
1
Varian VXR500S spectrometer. H NMR chemical shifts
are reported in parts per million relative to tetramethylsilane
(0.00 ppm). 13C NMR spectra were referenced relative to
the center peak of CDCl3 (77.00 ppm). Fast-atom bombard-
ment (FAB, xenon, 3-nitrobenzyl alcohol matrix) mass
(^)-N-(5-Dimethylamino-1-naphthalenesulfonyloxy)-4-
phenyl-2-azetidinone (7f). The resulting white solid
after the column chromatography was recrystallized with
ethyl acetate in hexanes to yield 96 mg (33%) of 7f as a

5724
A. Bulychev et al. / Tetrahedron 56 (2000) 5719±5728
yellow-brown solid; mp.1908C (dec.); IR (KBr)
1810 cm21 1H NMR (CDCl3, 300 MHz) d 8.56 (d,
evaporated to afford 440 mg (98%) of 7j as a colorless
1
;
oil: IR (neat) 1780 cm21; H NMR (300 MHz, CDCl3) d
Jˆ8.5 Hz, 1H), 8.26 (dd, Jˆ1.2, 6.1 Hz, 1H), 7.48 (tm,
Jˆ8.7 Hz, 2H), 7.20±7.11 (m, 4H), 6.97±6.93 (m, 2H),
4.65 (dd, Jˆ3.3, 6.2 Hz, 1H), 3.18 (dd, Jˆ6.2, 14.5 Hz,
1H), 2.87 (s, 6H) 2.68 (dd, Jˆ3.3, 14.5 Hz, 1H); HRMS
calcd for C19H20N2O4S 396.1222, found 396.1209.
0.98 (t, Jˆ7.2 Hz, 12H), 1.40 (sextet, Jˆ7.2 Hz, 8H),
1.53±1.70 (m, 8H), 2.58 (dd, Jˆ13.2, 2.1 Hz, 1H), 3.14±
3.22 (m, 9H), 5.39 (dd, Jˆ5.4, 2.4 Hz, 1H), 7.23±7.45 (m,
5H); 13C NMR (75 MHz, CDCl3) d 13.62, 19.61, 23.82,
41.96, 58.49, 60.14, 126.67, 128.06, 128.50, 137.71,
162.89; HRMS (FAB) (positive ion) calcd for C16H36N
242.2848, found 242.2841; HRMS (FAB) (negative ion)
calcd for C9H8NO5S 242.0123, found 242.0115.
(^)-N-(Methylethylsulfonyloxy)-4-phenyl-2-azetidinone
(7g). After puri®cation 46 mg (96%) of 7g was obtained as a
light yellow oil: Rf 0.54 (2:3, EtOAc±hexanes); IR (neat)
1795 cm21
;
1H NMR (300 MHz, CDCl3) d 1.51 (d,
Methyl 3-oxo-3-(2,3-dimethoxyphenyl)propanoate (14).
A stirred solution of 720 mg (6.10 mmol) of monomethyl-
malonate (10)23 in 7 mL of anhydrous THF was charged
with 3.0 mL (3.0 mmol) of a 1.0 M solution of dibutylmag-
nesium in heptane (Aldrich) at 2788C under an atmosphere
of argon. A white precipitate formed after 10 min of
reaction, at which time the mixture was allowed to warm
to room temperature, followed by an additional 1.5 h of
stirring. At this point, the solvent was evaporated to afford
a white solid.
Jˆ6.9 Hz, 6H), 2.84 (dd, Jˆ14.3, 2.9 Hz, 1H), 3.35 (dd,
Jˆ14.3, 5.9 Hz, 1H), 3.69 (heptet, Jˆ6.9 Hz, 1H), 5.12
(dd, Jˆ5.7, 2.7 Hz, 1H), 7.32±7.46 (m, 5H); 13C NMR
(75 MHz, CDCl3) d 16.33, 16.81, 42.36, 53.76, 62.65,
126.79, 129.01, 129.26, 135.17, 164.88; HRMS (FAB)
calcd for C12H16NO4S (MH1) 270.0800, found 270.0825.
(^)-N-(trans-2-Phenylethenesulfonyloxy)-4-phenyl-2-
azetidinone (7h). Column chromatography afforded 94 mg
(93%) of 7h as a light yellow oil, which crystallized upon
standing: Rf 0.31 (1:4, EtOAc±hexanes); mp 90±92 8C; IR
(neat) 1800 cm21; 1H NMR (300 MHz, CDCl3) d 2.83 (dd,
Jˆ14.4, 3.2 Hz, 1H), 3.332 (dd, Jˆ14.4, 6.1 Hz, 1H), 5.08
(dd, Jˆ6.1, 3.2 Hz, 1H), 6.73 (d, Jˆ15.5 Hz, 1H), 7.28±
7.55 (m, 10H), 7.65 (d, Jˆ15.5 Hz, 1H); 13C NMR
(75 MHz, CDCl3) d 42.37, 62.13, 119.54, 126.87, 128.78,
128.92, 129.13, 131.49, 132.05, 135.34, 147.92, 165.46;
HRMS (FAB) calcd for C17H16NO4S (MH1) 330.0800,
found 330.0766.
A 980 mg portion of carbonyldiimidazole (6.04 mmol) was
added to a stirred solution of 1.0 g (5.5 mmol) of 2,3-
dimethoxybenzoic acid 12 (Aldrich) in 7 mL of anhydrous
THF at ice-water temperature under an atmosphere of
argon. The reaction was allowed to warm to room tempera-
ture, and was aged for 2 h. The acyl imidazolide (13)
solution was transferred via a cannula over to the mag-
nesium salt and the mixture was stirred for 40 h at ambient
temperature under an atmosphere of argon. Stirring became
dif®cult due to the precipitation of salts. The reaction was
diluted by addition of portions of ether and 1.0 M HCl, and
the layers were separated. The aqueous layer was extracted
with ether (3£), and the combined ether portions were
washed with saturated NaHCO3, brine, dried (MgSO4), the
suspension was ®ltered and the solvent was evaporated to
afford a light yellow oil. The oil was puri®ed on a
Chromatotron device (2 mm silica gel plate), eluting with
20% EtOAc in hexanes to afford 655 mg (50%) of 14 as a
colorless oil: Rf 0.60 (2:3, EtOAc±hexanes); IR (neat) 1745,
(^)-N-((4-Phenylazo)benzenesulfonyloxy)-4-phenyl-2-
azetidinone (7i). The orange solid obtained after chromato-
graphic puri®cation was recrystallized from ethyl acetate in
hexanes to provide 16.7 mg (13%) of 7i as an orange solid,
mp 120±1218C (dec.). IR (KBr) 1800 cm21 1H NMR
;
(CDCl3, 300 MHz) d 8.07±8.03 (m, 1H), 8.0±7.95 (m,
4H), 7.59±7.56 (m, 3H), 7.35±7.31 (m, 3H), 7.28±7.24
(m, 3H), 4.96 (dd, Jˆ3.3, 6.2 Hz, 1H), 3.28 (dd, Jˆ6.2,
14.6 Hz, 1H), 2.81 (dd, Jˆ3.3, 14.6 Hz, 1H); HRMS
(FAB, MH1) calcd for C21H17N3O4S 408.1018, found
408.1043. UV (CH3CN) lmax (trans) 321 nm (e
2.4£104 L/mol cm); lmax (cis) 434 nm (e 1.2£103 L/
mol´cm).
1
1675 cm21; H NMR (300 MHz, CDCl3) d 3.75 (s, 3H),
3.90 (s, 3H), 3.92 (s, 3H), 4.01 (s, 2H), 6.99±7.15 (m,
2H), 7.30±7.40 (m, 1H) (a small amount of the enol
tautomer was also observed); 13C NMR (75 MHz, CDCl3)
d 49.59, 52.12, 56.01, 61.22, 116.80, 121.49, 123.94,
131.48, 149.09, 152.83, 168.24, 193.79 (a small amount of
the enol tautomer was also observed); HRMS (FAB) calcd
for C12H15O6 (MH1) 239.0919, found 239.0954.
(^)-2-Oxo-4-phenyl-1-azetidinyl sulfate, tetra-n-butyl
ammonium salt (7j). A stirred solution of 276 mg
(0.928 mmol) of b-lactam 66 in 10 mL of methanol was
charged with 266 mg of the Lindlar catalyst (Pd±CaCO3±
PbO, Aldrich), and stirred for 45 min under an atmosphere
of hydrogen. The catalyst was removed by ®ltration and the
solvent was evaporated to afford a colorless oil. The oil was
dissolved in 3.6 mL of pyridine and 443 mg (2.78 mmol) of
pyridine SO3 was added. The solvent was evaporated after
5.5 h of stirring at room temperature, the residue was
dissolved in 75 mL of a 0.5 M KH2PO4 solution, and the
resultant solution was washed with EtOAc to remove any
organic-soluble impurities. Tetra-n-butylammonium hydro-
gen sulfate (280 mg, 0.825 mmol) was added to the aqueous
solution, which was washed with CH2Cl2 and CHCl3. The
combined organic layers were dried over anhydrous
MgSO4, the suspension was ®ltered, and the ®ltrate was
Methyl (^)-3-hydroxy-3-(2,3-dimethoxyphenyl)propa-
noate (15). Sodium borohydride (384 mg, 10.2 mmol) was
added to a stirred solution of 4.84 g (20.3 mmol) of b-keto
ester 14 in 140 mL of methanol at ice-water temperature
under an atmosphere of nitrogen. Brine (100 mL) was
added after 1 h and the reaction was stirred at room tempera-
ture for 10 min. The reaction was diluted by addition of
portions of EtOAc and water, and the layers were separated.
The aqueous layer was extracted with EtOAc (3£) and the
combined organic layers were washed with brine, dried over
MgSO4, the suspension was ®ltered, and the solvent was
evaporated to afford a colorless oil. The oil was chromato-
graphed on silica gel eluting with 35% EtOAc in hexanes to

A. Bulychev et al. / Tetrahedron 56 (2000) 5719±5728
5725
afford 3.94 g (81%) of 14 as a colorless oil: Rf 0.47 (2:3,
HRMS (FAB) calcd for C18H20NO4 (MH1) 314.1392,
found 314.1409.
1
EtOAc±hexanes); IR (neat) 3500, 1735 cm21; H NMR
(300 MHz, CDCl3) d 2.60±2.84 (m, 2H), 3.43 (br s, 1H),
3.70 (s, 3H), 3.85 (s, 3H), 3.86 (s, 3H), 5.38 (dd, Jˆ9.0,
4.0 Hz, 1H), 6.80±6.90 (m, 1H), 7.01±7.10 (m, 2H); 13C
NMR (75 MHz, CDCl3) d 42.06, 51.58, 55.54, 60.57,
65.74, 111.63, 118.11, 124.01, 135.89, 145.50, 152.18,
172.70; HRMS (FAB) calcd for C12H16O5 (M1z) 240.0998,
found 240.1007.
(^)-N-p-Toluenesulfonyloxy-4-(2,3-dimethoxyphenyl)-2-
azetidinone (8). Lindlar catalyst (Pd±PbO±CaCO3, 60 mg)
and 10% Pd/C (5 mg) were added to a stirred solution of
60 mg (0.19 mmol) of b-lactam 17 in 5 mL of absolute
methanol at room temperature under an atmosphere of
hydrogen, and the mixture was stirred for 1 h. The catalyst
was removed by ®ltration and the solvent was evaporated in
vacuo to afford a colorless oil. The oil was dissolved in
2 mL of anhydrous dichloromethane and 40 mg
(0.21 mmol) of p-toluenesulfonyl chloride and 20 mL
(0.25 mmol) of anhydrous pyridine were added at room
temperature under an atmosphere of nitrogen. The solvent
was evaporated after 45 min of stirring and the residue was
dissolved in EtOAc/1.0 M HCl. The layers were separated
and the organic portion was washed with saturated
NaHCO3, brine, dried (Na2SO4), ®ltered and the solvent
was evaporated to afford a light brown oil. The oil was
radially chromatographed (1 mm silica gel plate) eluting
with 30% EtOAc in hexanes to afford 52 mg (72%) of 8
as a colorless oil, which solidi®ed upon standing: mp
104±1058C (dec.) (EtOAc±hexanes); Rf 0.43 (2:3,
(^)-O-Benzyl-3-hydroxy-3-(2,3-dimethoxyphenyl)propa-
nohydroxamate (16).
A stirred solution of 1.56 g
(6.49 mmol) of b-hydroxy ester 15 in 6 mL of THF was
mixed with 7.8 mL (7.8 mmol) of a 1.0 M NaOH solution.
The reaction mixture was diluted with ether after 30 min of
stirring at room temperature and the layers were separated.
The aqueous layer was acidi®ed to pH 5 using 3.0 M HCl.
O-benzylhydroxylamine hydrochloride (1.35 g, 8.46 mmol)
was added and the pH was adjusted to 4.5 by the addition of
saturated NaHCO3 solution. EDC´HCl (1.87 g, 9.75 mmol)
was added to the mixture in three portions over a period of
1 h while maintaining the pH of the reaction between 4 and
5. The reaction was stirred for an additional 30 min and was
extracted with EtOAc, washed with 1.0 M HCl, saturated
NaHCO3, brine, dried (Na2SO4), ®ltered and the solvent
evaporated to afford a light yellow oil. The oil was chroma-
tographed on silica gel eluting with 60% EtOAc in hexanes
to afford 1.28 g (60%) of 16 as a colorless oil, which
solidi®ed upon standing. An analytical sample was obtained
by recrystallization from EtOAc±hexanes: mp 88±908C; Rf
0.24 (1:1, EtOAc±hexanes); IR (KBr) 3475, 3365,
EtOAc±hexanes); IR (KBr) 1790 cm21
;
1H NMR
(300 MHz, CDCl3) d 2.46 (s, 3H), 2.81 (dd, Jˆ14.1,
3.0 Hz, 1H), 3.18 (dd, Jˆ14.1, 6.2 Hz, 1H), 3.85 (s, 3H),
3.87 (s, 3H), 5.22 (dd, Jˆ6.0, 3.0 Hz, 1H), 6.82 (dd, Jˆ7.8,
1.5 Hz, 1H), 6.92 (dd, Jˆ8.4, 1.5 Hz, 1H), 7.05 (t, Jˆ8.1 Hz,
1H), 7.34 (d, Jˆ7.8 Hz, 2H), 7.84 (d, Jˆ8.4 Hz, 2H); 13C
NMR (75 MHz, CDCl3) d 21.80, 41.54, 55.77, 58.18,
60.90, 113.05, 119.76, 124.19, 128.29, 129.06, 129.93,
130.93, 146.24, 147.63, 152.60, 165.39; HRMS (FAB)
calcd for C18H19NO6S (M1z) 377.0933, found 377.0935.
1
1680 cm21; H NMR (300 MHz, CDCl3) d 2.34±2.64 (m,
2H), 3.81 (s, 3H), 3.83 (s, 3H), 4.10 (d, Jˆ4.5 Hz, 1H), 4.81
(d, JABˆ11.7 Hz, 1H), 4.86 (d, JABˆ11.7 Hz, 1H), 5.21±
5.34 (m, 1H), 6.80±6.90 (m, 1H), 6.97±7.12(m, 2H), 7.33
(br s, 5H), 8.85 (br s, 1H) (a small amount of the hydro-
ximate was also observed); 13C NMR (75 MHz, CDCl3) d
40.47, 55.64, 60.70, 66.18, 78.14, 111.70, 118.21, 124.26,
128.46, 128.64, 129.17, 135.13, 136.02, 145.37, 152.23,
169.58 (a small amount of the hydroximate was also
observed); HRMS (FAB) calcd for C18H22NO5 (MH1)
332.1498, found 332.1483. Anal. calcd for C18H21NO5:
C, 65.24; H, 6.39; N, 4.23. Found: C, 65.33; H, 6.44; N,
4.21.
t-Butyl 4(S)-hydroxy-6-oxo-6-(propene-3-oxyamino)hexa-
noate (23). To a suspension of 850 mg (7.8 mmol) of
O-allylhydroxylamine hydrochloride in 17 mL of anhy-
drous CH2Cl2 at 08C under an atmosphere of nitrogen was
added 3.9 mL (7.8 mmol, 2 M solution in hexanes, Aldrich)
of trimethylaluminum over 10 min. The solution was
warmed to room temperature for 1 h and then cooled to
08C. Methyl ester 22 (1.0 g, 4.3 mmol) in 4 mL of anhy-
drous CH2Cl2 was added dropwise to the reaction mixture.
After stirring for 18 h at room temperature, the solution was
cooled to 08C, and the reaction was quenched by the
addition of 25 mL of 10% citric acid and stirred for 1 h.
The aqueous layer was extracted with CH2Cl2, washed
with 10% citric acid, saturated NaHCO3, brine, dried over
MgSO4, and the suspension was ®ltered. The ®ltrate was
evaporated to dryness and the residue was recrystallized
from EtOAc±hexanes to give 780 mg (66%) of 23 as a
white solid: mp 74±768C; IR (KBr) 1730, 1660, 1170,
(^)-N-Benzyloxy-4-(2,3-dimethoxyphenyl)-2-azetidinone
(17). A stirred solution of 566 mg (1.71 mmol) of hydroxa-
mate 16 in 15 mL of anhydrous acetonitrile was charged
with 500 mL (5.18 mmol) of carbon tetrachloride, 360 mL
(2.58 mmol) of triethylamine, and 672 mg (2.56 mmol) of
triphenylphosphine at room temperature under an atmos-
phere of nitrogen. The solvent was evaporated after 12 h
to afford a dark brown solid. The solid was chromato-
graphed on silica gel eluting with 30% EtOAc in hexanes
to afford 425 mg (79%) of 17 as a light yellow oil: Rf 0.33
1
940 cm21; H NMR (300 MHz, CDCl3) d 1.45 (s, 9H),
1.60±1.86 (m, 2H), 2.16±2.48 (m, 2H), 2.41 (t, Jˆ7.0 Hz,
2H), 3.86 (d, Jˆ3.3 Hz, 1H), 3.92±4.13 (m, 1H), 4.40 (d,
Jˆ6.2 Hz, 2H), 5.26±5.47 (m, 2H), 5.84±6.18 (m, 1H), 8.84
(br s, 1H); 13C NMR (75 MHz, CDCl3) (27.97, 31.72, 40.21,
67.75, 77.19, 80.65, 120.37, 132.07, 169.71, 173.44;
[a]2D0ˆ120.88 (cˆ1, CDCl3); HRMS (EI) calcd for
C9H15NO5 (M12C4H8) 217.0950, found 217.0940. Anal.
calcd for C13H23NO5: C, 57.13; H, 8.48; N, 5.12. Found:
C, 57.13; H, 8.52; N, 5.02.
1
(30% EtOAc in hexanes); IR (neat) 1775 cm21; H NMR
(300 MHz, CDCl3) d 2.59 (dd, Jˆ13.5, 2.4 Hz, 1H), 3.06
(dd, Jˆ13.7, 5.6 Hz, 1H), 3.77 (s, 3H), 3.86 (s, 3H), 4.87
(dd, Jˆ5.7, 2.4 Hz, 1H), 4.90 (d, JABˆ11.4, 1H), 4.98 (d,
JABˆ11.4 Hz, 1H), 6.85±6.93 (m, 2H), 7.02±7.10 (m, 1H),
7.30±7.36 (m, 5H); 13C NMR (75 MHz, CDCl3) d 41.27,
55.27, 55.69, 60.82, 77.77, 112.33, 118.50, 124.31, 128.44,
128.70, 128.92, 130.61, 135.13, 147.20, 152.53, 164.64;

5726
A. Bulychev et al. / Tetrahedron 56 (2000) 5719±5728
(4R)-1-(2-Propenoxy)-4-(2-(1,1-dimethylethoxycarbonyl)-
ethyl)-2-azetidinone (24). To a stirred solution of 2.80 g
(10.2 mmol) of hydroxamate 23 in 56 mL of anhydrous
acetonitrile at room temperature under an atmosphere of
nitrogen was added 5.0 mL (52 mmol) of carbon tetra-
chloride, 1.9 mL (14 mmol) of triethylamine, and 3.22 g
(12.3 mmol) of triphenylphosphine. After stirring at room
temperature for 36 h the solvent was evaporated to afford a
brown solid. The solid was chromatographed on silica gel
eluting with 30% EtOAc in hexanes to afford 2.2 g (85%) of
24 as a yellow oil: Rf 0.43 (2:3, EtOAc±hexanes); IR (neat)
HRMS (EI) calcd for C16H20N2O4 (M1) 304.1423, found
304.1430.
(4R)-1-p-Toluenesulfonyloxy-4-(2-benzyloxycarbonylamino-
ethyl)-2-azetidinone (18). To a stirred solution of 618 mg
(2.03 mmol) of b-lactam 26 in 10 mL of anhydrous aceto-
nitrile at room temperature under an atmosphere of nitrogen
was added 30 mg (0.11 mmol) of triphenylphosphine,
190 mL (2.28 mmol) of pyrrolidine, and 89 mg (0.077
mmol) of tetrakis(triphenylphosphine)palladium (0). The
reaction was shielded from light. After stirring for 3 h at
room temperature the solvent was evaporated to afford a
yellow oil. The oil was dissolved in EtOAc and was
extracted with 5% Na2CO3. The combined basic extracts
were acidi®ed to pH 5.0 using 3 M HCl, and then was
extracted with CH2Cl2 (3£). The combined organic layer
was washed with brine, dried (Na2SO4), ®ltered and the
solvent was evaporated to afford 417 mg of a yellow oil.
To a stirred solution of the oil in 10 mL of anhydrous
CH2Cl2 at room temperature under an atmosphere of nitro-
gen was added 320 mL (2.30 mmol) of triethylamine, and
470 mg (2.47 mmol) of p-toluenesulfonyl chloride. After
stirring at room temperature for 1.5 h, the solvent was
evaporated to yield a yellow solid. The solid was chromato-
graphed on silica gel eluting with 40% EtOAc in hexanes to
afford 428 mg (50%) of 18 as a colorless oil: Rf 0.57 (3:2,
EtOAc±hexanes); IR (neat) 1795, 1710 cm21; 1H NMR
(300 MHz, CDCl3) d 1.85±2.00 (m, 1H), 2.06±2.20 (m, 1H),
2.46 (s, 3H), 2.50 (dd, Jˆ14.9, 2.9 Hz, 1H), 2.89 (dd, Jˆ14.4,
6.0 Hz, 1H), 3.21±3.45 (m, 2H), 4.02±4.18 (m, 1H), 4.98 (br t,
1H), 5.09 (d, JABˆ12.4 Hz, 1H), 5.13 (d, JABˆ12.4 Hz, 1H),
7.36 (d, Jˆ7.8 Hz, 2H), 7.36 (s, 5H), 7.87 (d, Jˆ8.4 Hz); 13C
NMR (75 MHz, CDCl3) d 21.78, 32.64, 37.49, 38.12, 57.80,
66.75, 128.05, 128.15, 128.50, 129.10, 129.98, 130.26,
136.30, 146.52, 156.39, 165.08; HRMS (FAB) calcd for
C20H22N2O6S (MH1) 419.1277, found 419.1281.
1
1775, 1725 cm21; H NMR (300 MHz, CDCl3) d 1.46 (s,
9H), 1.82±1.97 (m, 1H), 2.05±2.19 (m, 1H), 2.36 (dd,
Jˆ13.7, 2.5 Hz, 1H), 2.35 (t, Jˆ7.7 Hz, 2H), 2.79 (dd,
Jˆ13.7, 5.2 Hz, 1H) 3.85±3.96 (m, 1H), 4.36±4.50 (m,
2H), 5.30±5.45 (m, 2H), 5.93±6.10 (m, 1H); 13C NMR
(75 MHz, CDCl3) d 27.44, 27.55, 30.87, 37.70, 56.12,
76.43, 80.05, 120.08, 131.93, 163.40, 171.15; [a]2D0ˆ
215.08 (cˆ1, CDCl3); HRMS (FAB) calcd for C9H14NO4
(MH12C4H8) 200.0923, found 200.0914.
(4R)-1-(2-Propenoxy)-4-(3-propanoic acid)-2-azetidinone
(25). To a stirred solution of 4.30 g (16.8 mmol) of 24 in
4 mL of anhydrous CH2Cl2 at 08C under an atmosphere of
nitrogen was added 1.8 mL of anisole and 12 mL of TFA.
After stirring for 2 h at 08C, toluene (10 mL) was added and
the solvents were evaporated. The residue was dissolved in
EtOAc and extracted with a saturated NaHCO3 solution.
The combined aqueous layers were acidi®ed to pH 3.0
using 3 M HCl, and the mixture was extracted with
CH2Cl2 (3£). The combined organic extracts were dried
(Na2SO4), ®ltered and the solvent was evaporated to afford
2.4 g (72%) of 25 as a yellow oil: Rf 0.24 (1:1, EtOAc±
hexanes); IR (neat) 3090, 1750, 1735 cm21 1H NMR
;
(300 MHz, CDCl3) d 1.88±2.04 (m, 1H), 2.10±2.23 (m,
1H), 2.39 (dd, Jˆ13.8, 2.4 Hz, 1H), 2.51 (t, Jˆ7.4 Hz,
2H), 2.83 (dd, Jˆ13.8, 5.2 Hz, 1H), 3.88±3.98 (m,
1H), 4.35±4.50 (m, 2H), 5.27±5.44 (m, 2H), 5.92±
6.17 (m, 1H), 8.57 (br s, 1H); 13C NMR (75 MHz,
CDCl3) d 27.60, 29.87, 37.51, 56.54, 77.22, 121.11,
132.13, 164.03, 177.45; [a]2D0ˆ220.08 (cˆ1, CDCl3); MS
(EI) m/z 200 (MH1).
1-Benzyloxy-4-[2-[2-(trimethylsilyl)ethoxycarbonylamino]-
ethyl]-2-azetidinone (29). To a stirred solution of 401 mg
(1.61 mmol) of carboxylic acid 2724 in 5 mL of anhydrous
MeCN was added 240 mL (1.72 mmol) of triethylamine
followed by 380 mL (1.76 mmol) of diphenylphosphoryl
azide (DPPA). The mixture was stirred under an atmosphere
of nitrogen at 90±958C for 1 h. 2-(Trimethylsilyl)ethanol
(460 mL, 3.22 mmol) was added and the solution was stirred
at 90±958C for 19 h. The mixture was allowed to cool, and
the solvent was evaporated to afford a brown oil. The oil was
chromatographed eluting with 40% EtOAc in hexanes to
afford 282 mg (48%) of carbamate 29 and 16 mg (4%) of
isocyanate 28, both as colorless oils. Compound 29: IR
(4R)-1-(2-Propenoxy)-4-(2-benzyloxycarbonylaminoethyl)-
2-azetidinone (26). To a stirred solution of 228 mg
(1.14 mmol) of 25 in 4 mL of toluene under an atmosphere
of nitrogen was added 270 mL (1.25 mmol) of diphenyl-
phosphoryl azide and 180 mL (1.29 mmol) of triethylamine.
After stirring at 808C for 20 min, 240 mL (2.32 mmol) of
benzyl alcohol was added. After stirring for 19 h at 808C,
the reaction was cooled and the solvent was evaporated to
afford a brown oil. The oil was chromatographed on silica
gel eluting with 55% EtOAc in hexanes to afford 162 mg
(47%) of 26 as a yellow oil: Rf 0.30 (3:2, EtOAc±hexanes);
1
(neat) 1770, 1710 cm21; H NMR (300 MHz, CDCl3) d
0.03 (s, 9H), 0.94 (t, Jˆ4.5 Hz, 2H), 1.55±1.69 (m, 1H),
1.70±1.84 (m, 1H), 2.32 (dd, Jˆ13.7, 2.4 Hz, 1H), 2.76 (dd,
Jˆ13.7, 5.2 Hz, 1H), 3.05±3.21 (m, 2H), 3.45±3.56 (m,
1H), 4.12 (t, Jˆ8.2 Hz), 4.94 (d, JABˆ11.2 Hz, 1H), 4.98
(d, JABˆ11.2 Hz, 1H), 7.35±7.46 (m, 5H); 13C NMR
(75 MHz, CDCl3) d 21.75, 17.45, 32.92, 37.33, 37.60,
55.69, 62.61, 77.88, 128.33, 128.73, 129.06, 134.84,
156.49, 163.66; MS (FAB) m/z 377 (MH12C2H4).
1
IR (neat) 3320, 1765, 1715, 1530, 1250 cm21; H NMR
(300 MHz, CDCl3) d 1.74±1.90 (m, 1H), 1.96±2.20 (m,
1H), 2.37 (dd, Jˆ13.7, 2.0 Hz, 1H), 2.81 (dd, Jˆ13.7,
5.1 Hz, 1H), 3.33 (q, Jˆ6.6 Hz, 2H), 3.81±3.93 (m, 1H),
4.35±4.50 (m, 2H), 4.99 (br s, 1H), 5.10 (s, 2H), 5.25±5.44
(m, 2H), 5.90±6.07 (m, 1H), 7.30±7.40 (m, 5H); 13C
NMR (75 MHz, CDCl3) d 33.28, 37.66, 37.80, 55.54,
66.59, 77.00, 120.91, 127.95, 128.05, 128.39, 131.95,
136.26, 156.29, 163.92; [a]2D0ˆ29.28 (cˆ1, CDCl3);
(^)-1-p-Toluenesulfonyloxy-4-[2-[2-(trimethylsilyl)eth-
oxycarbonylamino]ethyl]-2-azetidinone (19). To a stirred
solution of 258 mg (0.708 mmol) of b-lactam 29 in 10 mL

A. Bulychev et al. / Tetrahedron 56 (2000) 5719±5728
5727
of methanol under an atmosphere of nitrogen was added
References
71 mg of 10% Pd/C. A hydrogen atmosphere was applied
to the ¯ask. After stirring under hydrogen for 1.5 h, the
catalyst was ®ltered and the solvent was evaporated to
afford the crude N-hydroxy b-lactam as a colorless oil,
which was used without further puri®cation. To a stirred
solution of the N-hydroxy b-lactam in 8 mL of anhydrous
CH2Cl2 was added 99 mL (707 mmol) of triethylamine and
135 mg (707 mmol) of TsCl. After stirring at room tempera-
ture under an atmosphere of nitrogen for 45 min, the solvent
was evaporated. The residue was chromatographed eluting
with 25% EtOAc in hexanes to afford 269 mg (89%) of 19,
as a colorless oil which crystallized upon cooling: mp 76±
1. Cohn, M. L. Science 1992, 257, 1050; Neu, H. C. Science 1992,
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NY, 1998; pp 71±98.
1
788C (EtOAc±hexanes); IR (KBr) 1775, 1710 cm21; H
NMR (300 MHz, CDCl3) d 0.04 (s, 9H), 0.98 (t,
Jˆ8.5 Hz, 2H), 1.82±1.98 (m, 1H), 2.05±2.20 (m, 1H),
2.47 (s, 3H), 2.48 (dd, Jˆ11.2, 3.3 Hz, 1H), 2.92 (dd,
Jˆ14.4, 6.0 Hz, 1H), 3.19±3.45 (m, 2H), 4.10±4.25 (m,
1H), 4.14 (t, Jˆ8.6 Hz, 2H), 4.85 (br s, 1H), 7.38 (d,
Jˆ8.3 Hz, 2H), 7.88 (d, Jˆ8.4 Hz, 2H); 13C NMR
(75 MHz, CDCl3) d 21.60, 17.63, 21.72, 32.72, 37.25,
38.14, 57.80, 63.00, 129.05, 129.92, 130.23, 146.44,
156.73, 165.12; HRMS (FAB) calcd for C16H25N2O6SSi
(MH12C2H4) 401.1203, found 401.2040.
4. Bush, K. Clin. Microbiol. Rev. 1988, 1, 109; Bush, K. Antibiotic
and Chemotherapy; Churchill Livingstone: Edinburgh, UK, 1997;
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Â
Â
 Â
(^)-1-p-Toluenesulfonoxy-4-(4-methyl-3-pentenyl)-2-
azetidinone (20). To a stirred solution of 85 mg (0.40
mmol) of N-acetoxy-b-lactam 3025 in 3 mL of 2:1
MeOH±H2O was added 107 mg (1.0 mmol) of Na2CO3.
After stirring for 4 h at 258C, the apparent pH of the solution
was adjusted to ,5 with 1 M HCl and then the solution was
extracted three times with CH2Cl2. The extracts were
combined, dried, and concentrated to afford the crude
N-hydroxy b-lactam as a dark yellow oil. This crude product
was used directly in the next reaction.
T.; Peduzzi, J.; CanicËa, M. M.; Paul, G.; Nevot, P.; Barthelemy,
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Â
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To a stirred solution of the crude N-hydroxy b-lactam in
2.0 mL of anhydrous CH2Cl2 was added 85 mg (0.45 mmol)
of TsCl and 66 mL (0.82 mmol) of pyridine. After stirring
for 1 h at 258C, the mixture was concentrated on a rotary
evaporator and then chromatographed eluting with 1:4
EtOAc±hexanes to afford 83 mg (64%, 2 steps) of N-tosyl-
oxy b-lactam 20 as a colorless oil: Rf 0.45 (3:7, EtOAc±
hexanes); IR (TL) 1800 cm21; 1H NMR (300 MHz, CDCl3)
d 1.50±1.63 (m, 1H), 1.60 (br s, 3H), 1.69 (br s, 3H), 1.94±
2.11 (m, 3H, CH2CHv and one of CH2CH2CHv), 2.45 (dd,
Jˆ14.3 and 3.2 Hz, 1H), 2.47 (s, 3H), 2.84 (dd, Jˆ14.3,
5.9 Hz, 1H), 3.94±4.03 (m, 1H), 5.06 (tm, Jtˆ7.2 Hz, 1H),
7.38 (app. d, Jˆ8.0 Hz, 2, ArH), 7.88 (app. d, Jˆ8.4 Hz, 2H);
13C NMR (75 MHz, CDCl3) d 17.61, 21.75, 23.77, 25.60,
31.90, 38.06, 59.56, 122.39, 129.08, 129.89, 130.65,
132.94, 146.27, 165.36; HRMS (FAB) calcd for
C16H22NO4S (MH1) 324.1270, found 324.1274.
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Acknowledgements
19. Zafaralla, G.; Manavathu, E. K.; Lerner, S. A.; Mobashery, S.
Biochemistry 1992, 31, 3847.
The Notre Dame group gratefully acknowledges support
from the NIH (GM25845) and Eli Lilly & Company, as
well as the use of the NMR facilities of the Lizzadro
Magnetic Resonance Research Center at Notre Dame. The
work at Wayne State University was supported by the NIH.
20. The E. cloacae P99 and Q908R b-lactamases differ only in
four amino acids, positions of which are far from the active site.
Therefore, it is believed that these variations would not affect
the activity nor the structure of the Q908R enzyme, compared

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A. Bulychev et al. / Tetrahedron 56 (2000) 5719±5728
to P99. Moreover, the kinetic pro®les of both enzymes are
very similar. The Q908R enzyme has not been crystallized to
date, hence we have used the structure of the P99 enzyme in our
modeling.
Organic Synthesis Collective; Wiley: New York, 1990; Vol. VII,
pp 323±325.
24. (a) Guzzo, P. R.; Teng, M.; Miller, M. J. Tetrahedron 1994,
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