β-Secondary Kinetic Isotope Effects in the Clavaminate Synthase-Catalyzed Oxidative Cyclization of Proclavaminic Acid and in Related Azetidinone Model Reactions

Article abstract of DOI:10.1021/ja992649d

Clavaminate synthase is an Fe(II)/α-ketoglutarate-dependent oxygenase that catalyzes three mechanistically distinct reactions in the course of clavulanic acid biosynthesis. Clavulanic acid is of significant chemical importance as a potent inhibitor/inactivator of β-lactamase enzymes, a prominent means of bacterial resistance to, for example, penicillin. Primary and α-secondary ^T(V/K) kinetic isotope effects have been determined in earlier work for the clavaminate synthase-catalyzed oxidative cyclization of proclavaminic acid, one of the three reactions mediated by this enzyme. In this paper the β-secondary deuterium kinetic isotope effect for this reaction has been determined using remote ³H and ¹⁴C labels in an attempt to distinguish between radical or cationic intermediates in the reaction as suggested by the magnitudes of the primary and secondary α-effects. The presence of the adjacent azetidinone nitrogen and the intervention of an azetinone intermediate, formally antiaromatic in the resonance form of the amide, make interpretation of the low β-secondary effect (1.056 ± 0.002 for dideuteriation at C-3′) problematic. To assist interpretation of this result, a 4-chloroazetidinone model system has been constructed dideuteriated at C-3 identically to proclavaminic acid and bearing remote radiolabels. Reaction of this substrate at 25°C under both radical and solvolysis conditions afforded β-secondary kinetic isotope effect data for direct comparison to the enzymic reaction. The measured effects are similarly small but strongly dependent on the polarity/acidity of the reaction medium. These results are discussed in terms of the commitment to catalysis and the extent to which amide resonance may be favored in the transition state of the oxidative cyclization.

Full text of DOI:10.1021/ja992649d

11356
J. Am. Chem. Soc. 1999, 121, 11356-11368
â-Secondary Kinetic Isotope Effects in the Clavaminate
Synthase-Catalyzed Oxidative Cyclization of Proclavaminic Acid and
in Related Azetidinone Model Reactions
Dirk Iwata-Reuyl,† Amit Basak,‡ and Craig A. Townsend*
Contribution from the Department of Chemistry, The Johns Hopkins UniVersity,
Baltimore, Maryland 21218
ReceiVed July 26, 1999
Abstract: Clavaminate synthase is an Fe(II)/R-ketoglutarate-dependent oxygenase that catalyzes three
mechanistically distinct reactions in the course of clavulanic acid biosynthesis. Clavulanic acid is of significant
chemical importance as a potent inhibitor/inactivator of â-lactamase enzymes, a prominent means of bacterial
resistance to, for example, penicillin. Primary and R-secondary T(V/K) kinetic isotope effects have been
determined in earlier work for the clavaminate synthase-catalyzed oxidative cyclization of proclavaminic acid,
one of the three reactions mediated by this enzyme. In this paper the â-secondary deuterium kinetic isotope
effect for this reaction has been determined using remote 3H and 14C labels in an attempt to distinguish between
radical or cationic intermediates in the reaction as suggested by the magnitudes of the primary and secondary
R-effects. The presence of the adjacent azetidinone nitrogen and the intervention of an azetinone intermediate,
formally antiaromatic in the resonance form of the amide, make interpretation of the low â-secondary effect
(1.056 ( 0.002 for dideuteriation at C-3′) problematic. To assist interpretation of this result, a 4-chloroazetidinone
model system has been constructed dideuteriated at C-3 identically to proclavaminic acid and bearing remote
radiolabels. Reaction of this substrate at 25 °C under both radical and solvolysis conditions afforded â-secondary
kinetic isotope effect data for direct comparison to the enzymic reaction. The measured effects are similarly
small but strongly dependent on the polarity/acidity of the reaction medium. These results are discussed in
terms of the commitment to catalysis and the extent to which amide resonance may be favored in the transition
state of the oxidative cyclization.
Introduction
Scheme 1
The non-heme iron oxygenase clavaminate synthase (CS)
catalyzes three distinct oxidative reactions in the course of
clavulanic acid (6) biosynthesis. These R-ketoglutarate (R-KG)-
dependent steps include (1) a conventional hydroxylation of
deoxyguanidino proclavaminic acid (1, Scheme 1) to guanidino
proclavaminic acid (2), (2) the oxidative cyclization of pro-
clavaminic acid (3) to dihydroclavaminic acid (4) and, (3)
desaturation of the latter to clavaminic acid (5). Remarkably,
CS is a monomeric protein having a single iron binding site at
which all three transformations take place to achieve the
stepwise elevation of chemical potential manifested ultimately
in the potent â-lactamase inhibitor 6 itself.1,2 In each of the CS-
catalyzed reactions an equivalent of R-KG is consumed in the
activation of molecular oxygen and released as succinate and
carbon dioxide. Both the activation of oxygen and the oxidation
of substrate may be regarded, therefore, as irreversible processes.
Mechanistic investigations of clavaminate synthase have taken
place at several levels. The behavior of the iron site during
substrate binding has been monitored by ligand field CD and
MCD measurements to show that the octahedral ferrous resting
state of the enzyme remains 6-coordinate but reorganizes upon
R-KG binding to accommodate bidentate binding of the cofac-
tor.3 Upon the further addition of the substrate, deoxyguanidino
proclavaminic acid (1), however, the site becomes 5-coordinate,
square pyramidal. Presumably the open coordination site on iron
* Address correspondence to this author: Professor C. A. Townsend,
Department of Chemistry, The Johns Hopkins University, 3400 N. Charles
Street, Baltimore, MD 21218. Telephone: 410-516-7444. Fax: 410-516-
8420. E-mail: Townsend@jhunix.hcf.jhu.edu.
† Present address: Department of Chemistry, Portland State University,
P.O. Box 751, Portland, OR 97207.
‡ Present address: Department of Chemistry, Indian Institute of Technol-
ogy, Kharagpur, 721302 India.
(1) Busby, R. W.; Chang, M. D.-T.; Busby, R. C.; Wimp, J.; Townsend,
C. A. J. Biol. Chem. 1995, 270, 4262-4269.
(2) Busby, R. W.; Townsend, C. A. Bioorg. Med. Chem. 1996, 4, 1059-
1064.
(3) Pavel, E. G.; Zhou, J.; Busby, R. W.; Gunsior, M.; Townsend, C.
A.; Solomon, E. I. J. Am. Chem. Soc. 1998, 120, 743-753.
10.1021/ja992649d CCC: $18.00 © 1999 American Chemical Society
Published on Web 11/24/1999

KIEs in OxidatiVe Cyclization of ProclaVaminic Acid
J. Am. Chem. Soc., Vol. 121, No. 49, 1999 11357
Scheme 2
R-secondary effect. A more complex analysis of the changing
3H/14C ratio of residual substrate gave independent determina-
tions of both of these parameters with somewhat larger errors:
primary [V/K] ) 11.9 ( 1.7, R-secondary [V/K] ) 1.12 (
0.07 (Scheme 2).7 In contrast, T[V/K] observed for tritium
substitution at C-3 in 8 was 1.0 ( 0.1, clearly establishing that
the order of reactions is as shown in Scheme 1 and involving
the transient intermediacy of dihydroclavaminic acid (4), whose
structure was determined from small steady-state concentrations
of this species.7,10,11
T
T
The normal primary and R-secondary kinetic isotope effects
determined in the analysis of the oxidative cyclization of
proclavaminic acid (3) to dihydroclavaminic acid (4), although
not maximal, were consistent with a substantial change in
hybridization at the carbon undergoing oxidation from sp3 to
sp2 in the transition state of the reaction. In a related study of
the R-KG-dependent dioxygenase γ-butyrobetaine hydroxylase,
T
T
a primary [V/K] ) 15 and R-secondary [V/K] ) 1.31 were
measured by Blanchard and Englard.12 Like these investigators,
we interpreted our data to discount carbanion or oxenoid C-H
insertion processes in favor of radical or carbenium ion
intermediates in the oxidative cyclization.7
is available to react with dioxygen to initiate the oxidative cycle,4
as has been proposed analogously for isopenicillin N synthase
and supported by NO binding.5 The dynamics of substrate
oxidation were first revealed in kinetic isotope effect experi-
ments to establish the order of the oxidative reactions between
proclavaminic acid (3) and clavaminic acid (5); that is, does
oxidative cyclization precede desaturation or vice versa?
The fact that the CS-catalyzed reactions in Scheme 1 are
sequential and irreversible6 was exploited in V/K kinetic isotope
effect measurements.7 Proclavaminic acid bearing tritium equally
at the two diastereotopic C-4′ positions was prepared (7, Scheme
2), and similarly a specimen with tritium at C-3 (8). Each was
mixed with material radiolabeled with 14C at C-1 (*) to serve
as an internal standard and a kinetically unbiased marker of
the extent of reaction. For a normal kinetic isotope effect
kH/kD > 1, it would be anticipated that the 3H-labeled substrate
relative to the 14C-labeled would be enriched in the unreacted
starting material and depleted in the product as the reaction
progressed. It was shown by Northrup that the competition
between the labeled pairs of substrates is governed by the kinetic
Scheme 3
T
parameter [V/K], which will reflect events only through the
first irreversible step of the reaction.8 Using this method,
discrimination against tritiated molecules should only occur
when hydrogen isotope is present at the position oxidized first.
For the [4′-3H]-proclavaminic acid (7), tritium located on the
R-face (pro-S locus) would be lost during oxidative cyclization9
and would appear as HTO in the medium to provide a measure
of the primary kinetic isotope effect, while tritium located on
the â-face (pro-R) would provide a measure of the R-secondary
kinetic isotope effect in both the product and unreacted substrate.
By monitoring the release of HTO from 7 as function of the
â-Secondary Kinetic Isotope Effect. To examine more
closely the singular CS-mediated cyclization of proclavaminic
acid (3), and to attempt to distinguish between radical and
cationic processes (Scheme 3), we elected to determine the
â-secondary kinetic isotope effect during oxazolidine ring
formation. Alternatively, substrate analogue studies, so revealing
in the investigation of the mechanism of isopenicillin N
synthase,13 were completely unsuccessful in the case of cla-
T
extent of reaction, a [V/K] ) 8.3 ( 0.2 was determined for
the primary kinetic isotope effect. The corresponding appearance
of tritium activity relative to the 14C-internal standard in
clavaminic acid (9) afforded T[V/K] ) 1.06 ( 0.01 for the
(4) Zhou, J.; Gunsior, M.; Bachmann, B. O.; Townsend, C. A.; Solomon,
E. I. J. Am. Chem. Soc. 1998, 120, 13539-13540.
(5) Roach, P. L.; Clifton, I. J.; Hensgens, C. M. H.; Shibata, N.; Schofield,
C. J.; Hajdu, J.; Baldwin, J. E. Nature 1997, 387, 827-830.
(6) Salowe, S. P.; Marsh, E. N.; Townsend, C. A. Biochemistry 1990,
29, 6499-6508.
(10) Baldwin, J. E.; Adlington, R. M.; Bryans, J. S.; Bringhen, A. O.;
Coates, J. B.; Crouch, N. P.; Lloyd, M. D.; Schofield, C. J.; Elson, S. W.;
Baggaley, K. H.; Cassells, R.; Nicholson, N. J. Chem. Soc., Chem. Commun.
1990, 617-619.
(11) Baldwin, J. E.; Adlington, R. M.; Bryans, J. S.; Bringhen, A. O.;
Coates, J. B.; Crouch, N. P.; Lloyd, M. D.; Schofield, C. J.; Elson, S. W.;
Baggaley, K. H.; Cassels, R.; Nicholson, N. H. Tetrahedron 1991, 47, 4089-
4100.
(7) Salowe, S. P.; Krol, W. J.; Iwata-Reuyl, D.; Townsend, C. A.
Biochemistry 1991, 30, 2281-2292.
(8) Northrup, D. B. In Isotope Effects in Enzyme-Catalyzed Reactions;
Cleland, W. W., O’Leary, M. H., Northrup, D. B., Eds.; University Park
Press: Baltimore, MD, 1977; pp 122-152.
(9) Basak, A.; Salowe, S. P.; Townsend, C. A. J. Am. Chem. Soc. 1990,
112, 1654-1656.
(12) Blanchard, J. S.; Englard, S. Biochemistry 1983, 22, 5922-5929.
(13) Baldwin, J. E.; Bradley, M. Chem. ReV. 1990, 90, 1079-1088.

11358 J. Am. Chem. Soc., Vol. 121, No. 49, 1999
Iwata-Reuyl et al.
vaminate synthase.14 The â-secondary effect is well understood
to arise principally from the weakening of â-C-H bonds by
hyperconjugation to an adjacent empty (cationic) or partially
filled (radical) p-orbital.15,16 It can be argued from first principles
that this interaction should be greater for a carbenium ion [2
electrons, 2 (or 3) centers] than for a radical owing to Coulombic
repulsion of the unshared electron.17 This being so, one might
expect that a distinction could be made between radical or
carbenium ion formation at C-4′ in the transition state of
oxidative cyclization. However, a number of elements of the
reaction at hand could confound, if not thwart, the envisioned
analysis. (1) No â-secondary kinetic isotope effect determina-
tions have been made in chemical models of radical or cationic
reaction at C-4 of an azetidinone to serve as benchmarks for
the enzymic reaction. We provide these in this paper for
comparison to the enzymic reaction.18 (2) The reaction site lies
adjacent to the â-lactam nitrogen. Delocalization of the nitrogen
lone pair will suppress the magnitude of all isotope effects
measured at the reaction center.15 However, the countervailing
effect of amide resonance, although somewhat reduced in the
four-membered ring, will attenuate the extent of this suppression
(cf. 13 and 14, Scheme 3). A qualitative appreciation for the
net impact of these effects can be inferred from a comparison
of the primary and R-secondary kinetic isotope effects observed
for clavaminate synthase and the somewhat higher values
reported for γ-butyrobetaine hydroxylase.12 Therefore, while
some reduction of the intrinsic isotope effects at C-4′ in
proclavaminic acid is likely to occur through participation by
the adjacent â-lactam nitrogen and the step may not be fully
rate-determining, the primary effect, especially, remains sub-
stantial. (3) That the primary kinetic isotope effect is high is
critical to interpretation of the anticipated â-secondary effect.19
For an enzymic reaction, the commitment to catalysis must be
sufficiently low for the observed effect to approximate the
intrinsic isotope effect before a meaningful comparison can be
made to chemical model reactions. (4) Finally, the envisioned
â-secondary kinetic isotope effect determination relies on a
hyperconjugative interaction of the C-3 hydrogen of the â-lactam
ring. Formally, this participation with the reactive intermediate
(*, Scheme 3) would result in azetinone 16 formation. These
species are known to be extremely unstable owing, presumably,
to the antiaromatic resonance form 15.20
Scheme 4
observation suggested that a â-secondary kinetic isotope effect
would, indeed, be detectable in the CS-catalyzed reaction.
Moreover, it was hoped that chemical model reactions of the
radical and cationic regimes would provide valid comparisons
to the biochemical reaction and, in their observed â-secondary
kinetic effects, take into account all of these considerations
unique to reaction of an azetidinone ring.
Suspecting, therefore, that the â-secondary kinetic isotope
effect was likely to be small, a triple-label experiment was
undertaken to prepare proclavaminic acid 19 dideuteriated (to
maximize the â-effect) or unlabeled at C-3′, with H or 14C,
3
respectively, at remote, nonreacting positions such that the
isotope distribution as a function of the extent of reaction would
appear as a changing H/14C ratio. That is, H would monitor
3
3
the kinetic fate of dideuteriated proclavaminic acid 19, while
14C would track its protiated counterpart.
22
Synthesis of Isotopically Labeled (2S,3R)-Proclavaminic
Acid. A synthesis of enantiomerically pure proclavaminic acid
(3) starting from â-hydroxyornithine reported previously from
this laboratory was not amenable to isotopic substitution at the
desired positions.23 An alternate route was required. The success
of Ojima and co-workers in the asymmetric alkylation of
enantiomerically pure 4′-phenyl glycyl azetidinyl enolates
suggested a similar approach.24 Although phenyl substitution
at C-4′ was not useful for our purpose because of the difficulty
of its removal, the availability of 4-thiophenyl azetidinone in
both the optically pure R- and S-forms,25,26 coupled with its easy
reductive removal, encouraged us to investigate the potential
of asymmetric aldol condensations from the corresponding
glycyl enolates (Scheme 5). Additionally, the introduction of
isotopic labels was conceptually straightforward. Thus deuterium
could be introduced at C-3 of 4-thiophenyl azetidinone by simple
base-promoted exchange, while 3H and 14C could be introduced
into the hydroxyornithyl side chain at several sites. We
recognized the possibility of decreased selectivity in the coupling
reaction owing to both the lower steric profile of thiophenyl
At the outset, it was this last factor that posed the greatest
unknown to analysis of the experimental results. Encouragement
was taken, however, from an earlier observation that the
destabilizing effects imparted by resonance form 15 would not
completely override the observation of a â-secondary kinetic
isotope effect. Tributyltin hydride reduction of azetidinone 18
was qualitatively 2-3 times faster than that of than 17 (Scheme
4). This rate difference was interpreted to arise from the well-
known ability of silyl groups to stabilize radicals and carbenium
ions formed â to them through hyperconjugation effects. 21 This
(14) Iwata-Reuyl, D.; Basak, A.; Silverman, L. S.; Engle, C. A.;
Townsend, C. A. J. Nat. Prod. 1993, 56, 1373-1396.
(15) Sunko, D. E.; Hehre, W. J. Prog. Phys. Org. Chem. 1983, 16, 205-
246.
(16) Melander, L.; Saunders, W. H. Reaction Rates of Isotopic Molecules;
Wiley: New York, 1980; pp 170-201.
(22) All substrates were planned to be equally labeled with deuterium
at both C-3 methylene positions to amplify the â-isotope effect. While the
CS-catalyzed oxidative cyclization is a stereospecific process and the
â-secondary kinetic isotope effect may differ depending on which hydrogen
(deuterium) at C-3′ is syn clinal or gauche to the breaking C-H bond, this
stereochemical distinction will escape analysis. However, if a fully developed
p-orbital exists at the transition state, the effects of both C-3 deuteria will
be the same.
(23) Krol, W. J.; Mao, S.-s.; Steele, D. L.; Townsend, C. A. J. Org.
Chem. 1991, 56, 728-731.
(24) Ojima, I.; Komata, T.; Qiu, X. J. Am. Chem. Soc. 1990, 112, 770-
774.
(17) Symons, M. C. R. Tetrahedron 1962, 18, 333-341.
(18) It is further noted that the hybridization of C-4, while nominally
sp3, will be perturbed to increase p-character to ligands in the strained,
four-membered ring, and increase s-character (increase bond strength) to
the CsH bonds undergoing change in the course of reaction.
(19) Cleland, W. W. In Secondary and SolVent Isotope Effects; Buncel,
E., Lee, C. C., Eds.; Elsevier: Amsterdam, 1987; pp 61-113.
(20) Olafson, R. A.; Morrison, D. S.; Banerji, A. J. Org. Chem. 1984,
49, 2652-2653.
(25) Shibasaki, M.; Nishida, A.; Ikegami, S. J. Chem. Soc., Chem.
Commun. 1982, 1324-1325.
(26) Clauss, K.; Grimm, D.; Possel, G. Liebigs Ann. Chem. 1974, 539-
560.
(21) Lambert, J. B. Tetrahedron 1990, 46, 2677-2689.

KIEs in OxidatiVe Cyclization of ProclaVaminic Acid
J. Am. Chem. Soc., Vol. 121, No. 49, 1999 11359
Scheme 5
Scheme 6a
a Reagents and conditions: (a) sodium benzenesulfinate, H2O, 40 °C; (b) PhSH, (+)-cinchonine, PhH, 40 °C; (c) LiHMDS, -78 f -20 °C,
benzyl [2-14C]bromoacetate or benzyl bromoacetate; (d) LiHMDS, -78 °C, 2h, 22 or 22b; (e) (TMS)3SiH, AIBN, PhH, reflux; (f) DBN, CH2Cl2;
(g) H2, 10% Pd/C, EtOH; (h) TMSCl, Et3N, then LDA, -78 °C, TMSCl, then KF, d4-MeoH; (i) KMnO4; (j) (COCl)2, DMSO, Et3N, -78 °C.
compared to phenyl, and the simultaneous creation of two
stereogenic centers in the aldol reaction. Generation of the
thermodynamic enolates 23 and 24 from the 4S- or 4R-
Table 1. Relative Ratios of Aldol Products Derived from the
Addition of 20 and 21 to Cbz-protected Aldehyde 22
substrate
L-threo
L-erythro
D-threo
D-erythro
thiophenyl azetidinones (20 and 21, respectively) by treatment
with lithium bis(trimethylsilyl)amide at -78 °C followed by
addition of N-Cbz protected aldehyde 22 provided a mixture of
diastereomeric products in the ratios shown in Table 1 in an
overall 70% yield. The reaction showed moderate erythro
selectivity, and the desired L-threo isomer 25a/26a was produced
in only 5.5-11% as one of the minor isomers depending on
which azetidinone was used. Furthermore, the four diastereomers
ran in pairs in a variety of chromatographic conditions with
the L-threo and L-erythro constituting one pair, thus making
20
21
1.0 (25a)
0.5 (26a)
2.0 (25b)
3.0 (26b)
0.5 (25c)
1.0 (26c)
3.0 (25d)
2.0 (26d)
purification of the desired L-isomer difficult. From the remaining
pair, however, the major D-erythro isomer 25d/26d could be
isolated by crystallization from hexane-methylene chloride in
high purity. Reductive removal of the thiophenyl group followed
by epimerization of the protected D-erythro proclavaminate
provided the desired L-threo diastereomer, which could be

11360 J. Am. Chem. Soc., Vol. 121, No. 49, 1999
Iwata-Reuyl et al.
deprotected to the natural proclavaminate. The yield of the
L-threo isomer using this approach was 15%, but deemed
acceptable in view of the requirements for material of high
chemical and enantiomeric purity.
Scheme 7
Having established a method for the preparation of enantio-
merically pure proclavaminic acid, we proceeded with the
synthesis of the isotopically labeled proclavaminates 34 and 42
(Scheme 6). Reaction of 4-acetoxyazetidinone 27 with sodium
benzenesulfinate in water at 45 °C gave (()-4-phenylsulfonyl
azetidinone 28 in 81% yield.26 Displacement of the phenylsul-
fonyl group with thiophenol in the presence of (+)-cinchonine
as the asymmetric catalyst in benzene at 40 °C provided the
4-thiophenyl azetidinone in 97% yield.25 Fractional crystalliza-
tion from cyclohexane/benzene gave enantiomerically pure (-)-
(4S)-thiophenyl azetidinone (29) in an optical yield of 29%. The
optically impure material could be recycled by oxidizing it back
to 4-phenylsulfonyl azetidinone and repeating the displacement
with thiophenol and (+)-cinchonine. Replacing (+)-cinchonine
with (-)-cinchinidine provided access to (+)-(4R)-thiophenyl
azetidinone.
Scheme 8
For the preparation of 14C-labeled (•) proclavaminate 34, the
(-)-(4S)-isomer 29 was coupled with benzyl [2-14C]bromoac-
etate, prepared from phenyl diazomethane and [2-14C]bromoace-
tic acid, to give the azetidinone 30 in 47% yield.27 Aldol
condensation with N-Cbz-aminopropanal 22 provided the D-
erythro diastereomer 31 in 33% yield after recrystallization.
Because 31 was the last crystalline intermediate in the synthesis,
the specific activity was determined at this point giving an
average value of 1.22 mCi/mmol. Reduction with tris(trimeth-
ylsilyl)silane and AIBN in refluxing benzene28 gave the
protected D-erythro proclavaminate 32 in 68% yield. Epimer-
ization with DBN in CH2Cl2 provided the L-threo isomer 33 in
84% yield, which was deprotected with H2/Pd-C to give
(2S,3R)-[2-14C] proclavaminic acid 34 in essentially quantitative
yield.
labeled proclavaminic acids. In this way, the deuterium contents
of all systems used in the kinetic analysis would be the same.
In view of these requirements, we selected 4′-chloroazetidinone
43. Azetidinones substituted with chlorine at C-4 are known to
undergo a variety of ionic reactions,29 and their reactivities under
radical conditions is well precedented.30,31
Synthesis of the labeled chloroazetidinones is illustrated in
Scheme 7. Preparation of the 14C material (•) was accomplished
in a simple one-step procedure involving chlorinolysis of the
[2-14C]-4′-thiophenyl azetidinone (()-30 with Cl2 in CCl4 to
give the [2-14C]-4′-chloroazetidinone 43 in approximately 60%
yield after crystallization (specific activity 0.131 mCi/mmol).
The synthesis of [3′-2H2,2-3H]-4′-chloroazetidinone 45 involved
introduction of tritium by treating (()-38 with 0.75 equiv of
LiHMDS in THF at -78 °C followed by quenching with [3H]-
H2O in THF to produce 44. Control experiments showed that
no exchange occurred in the â-lactam ring under these condi-
tions. The latter on chlorinolysis gave 45, which had a specific
activity of 0.415 mCi/mmol after recrystallization.
In the preparation of [2H2,3H]-labeled proclavaminic acid 42,
racemic 4-thiophenyl azetidinone (()-29 was first deuteriated
by silylating at C-3 followed by desilylation in anhydrous KF/
d4-MeOH. Four cycles of this procedure provided the dideu-
1
teriated thiophenyl azetidinone 35. High resolution H NMR
To monitor the reactions at different time points, we adopted
the same HPLC assay used previously.7 Initial work revealed
that the chloroazetidinone eluted as a single peak with a retention
time of approximately 8 min in a solvent system of water-
acetonitrile (70:30). We subsequently discovered that the peak
was due not to the chloroazetidinone, but to its hydrolysis
product 47. Hydrolysis of 43 to 46 was very rapid, and the ring
spontaneously opened to 47. However, the reaction went to
completion with no loss of tritium or 14C. Although deuterium
is lost in the hydrolysis, owing to facile exchange during
enolization (Scheme 8), the measured 3H/14C ratio in the
hydrolyzed product would be identical to that in the labeled
chloroazetidinone initially isolated (see Scheme 5). Because of
the simplicity of the reactions and the subsequent ease of
workup, we chose an alkylsilane-mediated radical reduction of
43/45 to the azetidione 48 (Scheme 8) and their acetolysis to
the 4′-acetoxyazetidinone 49 as the model radical and cationic
reactions, respectively. To avoid extrapolations brought about
by the effect of temperature on the magnitude of measured
isotope effects,32 all of the experiments were conducted at 25
spectroscopy revealed an incorporation of 90% deuterium at
each methylene position. An attempt to achieve deuteriation at
C-3 by anion formation with LDA followed by addition of D2O
was largely unsuccessful. The alkaline medium generated after
quenching led to considerable decomposition of the product.
Oxidation of 35 with m-CPBA gave the phenylsulfonyl azeti-
dinone 36, which was further elaborated to provide the (-)-
(4S)-thiophenyl azetidinone 37. Mass spectrometric analysis
gave a deuterium content of 81% H2, 18% H1, and 1% H0
confirming the earlier NMR estimation of ∼90% d/site.
Coupling of the benzyl azetidinyl acetate 38 with [2,3-3H]-N-
Cbz propanal 22a followed by the steps described above
provided the [3′-2H2,2,3-3H]proclavaminic acid 42 with a
specific activity of 2.44 mCi/mmol.
Development of Model Systems. For the best kinetic isotope
effect comparisons to be made, the model and CS-catalyzed
reactions ought to mimic one another as closely as possible.
Important criteria for the model were that it ideally should be
a single compound, which could react by either a radical or
cationic mechanism, and it should be readily synthesized from
a dideuteriated intermediate common to the synthesis of the
2
2
2
(29) Bentley, P. H.; Brooks, G.; Gilpin, M. L.; Hunt, E. Tetrahedron
Lett. 1979, 1889-1890.
(27) Kobayashi, T.; Iwano, Y.; Hirai, K. Chem. Pharm. Bull. 1978, 26,
(30) Neumann, W. P. Synthesis 1987, 665-683.
1761-1767.
(31) Koppel, G. A.; McShane, L.; Jose, F.; Cooper, R. D. G. J. Am.
Chem. Soc. 1978, 100, 3933-3935.
(28) Chatgilialoglu, C.; Griller, D.; Lesage, M. J. Org. Chem. 1988, 53,
3641-3642.
(32) Vogel, P. C.; Stern, M. J. J. Chem. Phys. 1971, 54, 779-796.

KIEs in OxidatiVe Cyclization of ProclaVaminic Acid
J. Am. Chem. Soc., Vol. 121, No. 49, 1999 11361
°C. The radical reaction was run in a quartz test tube under
argon at constant temperature with irradiation provided by a
medium-pressure Hg lamp equipped with a Vycor filter. The
acetolysis reaction was carried out by treating 43/45 with
anhydrous acetic acid at 25 °C under an argon atmosphere.
Termination of the reaction at various time points was achieved
by immediately freezing the sample in dry ice. Experiments with
a second, less polar solvolytic system employing ethanol-THF
(1:1) were carried out in an analogous fashion.
S′ ) (A1 + A2)/A3
) A1/A3 + K
(8)
(9)
or
A1/A3 ) S′ - K
(10)
Therefore
Isotope Effect Determinations in the CS-Catalyzed and
Chemical Model Reactions. Theory. For the general case of
a substrate in an irreversible reaction substituted with a heavy
isotope in macroscopic amounts, the isotope effect, (V/K), is
described for residual substrate by
S ) (S′ - K)/(1 + K)
S′ ) S(1 + K) + K
(11)
(12)
D
K can be easily determined from the experimental design as
K ) (1 - F)M0
(13)
D(V/K) ) ln[(1 - x)(1 + S0)/(1 + Sx)]/
ln[(1 - x)(1 + S0)/(1 + Sx)Sx/S0] (1)
where F is the fraction of deuterium labeling in the substrate
and M0 is the initial apparent D/H mole ratio. Substituting eq
11 for S0 and SX in eq 1 then allows for the calculation of the
isotope effect.
An analogous derivation can be made for calculating the
isotope effect based on product formation, in this case the
relevant equation is
where x is the total fractional extent of reaction and S is the
substrate D/H ratio at the subscripted extent of reaction.16 When
tritium and 14C are used as remote labels for deuterium and
protium, respectively, and deuteriation at the site of interest is
3
complete, that is, every molecule containing H also contains
3
2H, then the H/14C ratio can be used directly as a measure of
D(V/K) ) ln[1 - x(1 + S0)/(1 + Rx)]/
ln[1 - x(1 + S0)/(1 + Rx)(Rx/S0)] (14)
S in eq 1. However, if deuterium labeling is less than 100%,
then there are three substrate species to consider: A1, which
contains deuterium and tritium, A2, which contains protium and
tritium, and A3, which contains protium and 14C. The D/H ratio
is now given by
where Rx is the product D/H ratio at the subscripted extent of
reaction and the other symbols are defined as above. It can be
seen that for calculations based on residual substrate, measure-
ments made after 50% of reaction are the most sensitive to
changes in the 3H/14C ratio, while the corresponding calculations
based on product formation are most sensitive for measurements
made at 0-50% reaction. Furthermore, from a comparison of
the maximum measurable change in specific activity, it is clear
that eq 1 for residual substrate is inherently more sensitive. For
this reason, and in order to make a manageable number of
samplings of the reactions, all of our analyses were based on
calculations of residual substrate specific activities.
S ) A1/(A2 + A3)
(2)
3
while the experimentally measured quantity, the H/14C ratio,
is given by
S′ ) (A1 + A2)/A3
(3)
3
To correlate S′ with S, one first needs to translate the H/14C
ratio, S′, to an apparent D/H mole ratio. The D/H ratio described
by eq 2 is a macroscopic mole ratio of the various deuterated
and protiated substrate molecules, while eq 3, as defined by
3H/14C ratios, is a microscopic quantity and is only a measure
of the apparent macroscopic D/H mole ratio when the specific
Experiment. Incubations of [3′-2H2,4,5-3H]- and [2-14C]-(2S,-
3R)-proclavaminates 42/34 (Scheme 6) with clavaminate syn-
thase were performed in triplicate and the reaction mixtures
analyzed at different time points after separation of the
components by HPLC (see Experimental Section). The extent
of reaction was determined from both the 14C and tritium
distributions in the reaction as noted above. A representative
HPLC radiochromatogram for an incubation of the labeled
clavaminates with CS is shown (Figure 1A). The relevant peaks
are well resolved. Fitting the radiochemical data to eq 1 gave a
3
activities of the H and 14C labeled substrates are equivalent.
3
The conversion of the microscopic H/14C ratios, S′, to the
macroscopic mole ratios, M, is given by
M ) S′(aC/aH)
(4)
where aC equals the specific activity of 14C-labeled substrate
D
â-secondary isotope effect (V/K) ) 1.056 ( 0.002. Superim-
3
and aH equals the specific activity of H-labeled substrate. In
position of the data from three different experiments (Figure
1B) demonstrates that eq 1 is followed well.
the case where the two specific activities are equivalent, then
M is equal to S′. One can then use the fact that the tritium and
14C-labeled substrates bearing hydrogen at the site of interest
(A2 and A3), will show no isotope effect throughout the reaction,
and their ratio (A2/A3), defined as K, will, therefore, remain
constant. It follows then that
A representative radiochromatogram for the radical-mediated
reduction of benzyl [3′-2H2,2-3H]- and [2-14C]-4′-chloroazeti-
dinyl acetate 45/43 (Scheme 7) in acetonitrile-THF is presented
in Figure 2A and a corresponding radiochromatogram for the
acetolysis in Figure 3A. The data are summarized in Figures
2B and 3B, respectively. Fitting the radiochemical data from
triplicate experiments to eq 1 gave a â-secondary isotope effect
of 1.081 ( 0.002 for the radical reaction. Similar data fitting
for the acetolysis reaction gave an unexpectedly low isotope
effect of 1.040 ( 0.002. In contrast, the solvolysis reaction
carried out in the less polar EtOH-THF mixture (Figure 3C)
proceeded with an isotope effect of 1.098 ( 0.005, substantially
S ) A1/(A2 + A3)
(5)
(6)
(7)
) (A1/A3)/(1 + A2/A3)
) (A1/A3)/(1 + K)
and

11362 J. Am. Chem. Soc., Vol. 121, No. 49, 1999
Iwata-Reuyl et al.
Figure 2. (Top) Radiochromatogram for the radical mediated reduction
of benzyl [3′-2H2,2-3H,2-14C]-4′-chloroazetidinyl acetate (45/43) ter-
minated at extent of reaction ) 0.639. The retention times of the
reaction components are as follows: (A) (45/43), 9 min; (B) benzyl
[3′-2H2,2-3H,2-14C]-azetidinyl acetate, 15 min. (Bottom) Relative D/H
ratios of benzyl [3′-2H2,2-3H,2-14C]-4′-chloroazetidyl acetate (45/43)
upon the radical mediated reduction to azetidinone (48) from triplicate
experiments. The curve was drawn using eq 1 with a â-secondary
isotope effect ) 1.081, K ) 0.0975, and R0 ) 0.7997.
Figure 1. (Top) Radiochromatogram for the reaction of CS with [4,5-
3H,3′-2H2,2-14C]-(2S,3R)-proclavaminate (42/34) terminated at extent
of reaction ) 0.306. The retention times of reaction components are
as follows: (A) [3H]water, 2.5 min.; (B) proclavaminate, 12 min; (C)
dihydroclavaminate, 14 min; (D) clavaminate, 17 min. (Bottom)
Relative D/H ratios of [4,5-3H,3′-2H2,2-14C]-(2S,3R)-proclavaminic acid
as a function of extent of reaction upon incubation with CS from
triplicate experiments. The curve was drawn using eq 1 with a
â-secondary isotope effect ) 1.056, K ) 0.1334, and R0 ) 1.047.
Scheme 9
higher than that for the acetolysis reaction. These findings are
summarized in Scheme 9.
Analysis. The experimentally determined â-secondary kinetic
isotope effects for the CS-catalyzed and chemical model
reactions were relatively small and represent the effect of two
deuteria at the remote site. This outcome was anticipated by
the presence of the azetidinone nitrogen adjacent to the site of
reaction, and the antiaromatic character imparted to the â-lactam
ring by the hyperconjugative interaction directly responsible for
expression of the isotope effect itself (cf. 15 and 16, Scheme
3). As all reactions were run at 25 °C and based on a common
synthetic intermediate having the same deuterium content, we
believe the reactions can be justly compared. Particularly
surprising was the observation that solvolysis in acetic acid
should yield a â-secondary isotope effect one-half the magnitude
of a corresponding radical reaction (Scheme 9).
Silanes are known to function as hydride donors under some
conditions.33,34 Although neither the reaction conditions of the
radical model, nor the geometric constraints against chloride
displacement in a four-membered ring seemed to favor such a
process, a control reaction was carried out. Thus, [2-14C]-
chloroazedinone 43 was exposed to tris(trimethylsilyl)silane in
acetonitrile-THF (1:1) and AIBN at 25 °C under an inert
atmosphere, but in the absence of UV irradiation. After 6 h,
only starting material was present, demonstrating that reduction
proceeded, as expected, by a radical mechanism only.
larger for a carbenium ion than the corresponding radical. This
case can be made on the basis of the energetics of the interacting
orbitals17 and rotational barriers at radical and cationic centers.35
Thus, while a special circumstance might apply in the case of
the â-lactam ring, we take the view that the polarity or proton-
donating ability of the medium is unusually important in this
particular instance. That is, hyperconjugation affords the aze-
tinone structure 16 (Scheme 3). In a polar environment the
contribution of structure 14 will be enhanced and hyperconju-
gation will lead to the unstable antiaromatic azacyclobutadiene
configuration 15. That is, the â-secondary kinetic isotope effect
will be maximally suppressed as amide resonance is maximally
stabilized. The overriding importance of this effect is apparent
when the solvolysis is run in a less acidic medium, ethanol-
THF (1:1), and a significantly higher â-secondary isotope effect
is observed. The principal driver of the experimentally deter-
mined kinetic isotope effect, therefore, would appear to be
solvent stabilization of amide resonance, behavior favored by
For a given molecular structure, a compelling argument can
be made that the â-secondary kinetic isotope effect should be
(33) Ojima, I.; Kogure, T. Tetrahedron Lett. 1973, 2475-2478.
(34) Kursanov, D. N.; Loim, N. M.; Baranova, V. A.; Moiseeva, L. V.;
Zalukaev, L. P.; Parnes, Z. N. Synthesis 1973, 420-422.
(35) Radom, L.; Paviot, J.; Pople, J. A. J. Chem. Soc., Chem. Commun.
1974, 58-60.

KIEs in OxidatiVe Cyclization of ProclaVaminic Acid
J. Am. Chem. Soc., Vol. 121, No. 49, 1999 11363
interesting question about the extent of polarization of the lactam
bond during CS-catalyzed oxazolidine ring formation. The low
value of the â-secondary effect suggests that it might be quite
highly polarized. Advantage to catalysis could result from this
phenomenon by favoring the formation of a radical over a
carbenium ion at C-4′ and, thereby, lowering the activation
energy for the difficult catalytic task of removing hydrogen from
an otherwise comparatively unactivated C-H bond of increased
s-character.
Conclusion
While the experimental data make distinction between a
radical and cationic mechanism of oxidative cyclization uncer-
tain, on purely chemical grounds we favor formation of an iron-
oxo intermediate as 52 followed by a homolytic reaction path
in which activated molecular oxygen abstracts the R-hydrogen
atom (4′-proS) from proclavaminic acid (3) in the rate-
determining step to give a radical as 53. The unexpected
observation of an especially small â-secondary deuterium kinetic
isotope effect, however, could point to a high degree of amide
resonance in the transition state of C-4′-H bond cleavage. Such
polarization may favor a radical intermediate as it would be
intrinsically less stabilized than a carbenium ion by hypercon-
jugative interaction leading to the antiaromatic species 15.
Whether this radical species undergoes direct insertion of the
substrate oxygen (path A, Scheme 10),7,37 or by electron transfer
gives the cation/acyliminium 54 followed by ionic cyclization
(path B) cannot be distinguished at present. Such a process may,
or may not, involve coordination of proclavaminate to iron as
shown in Scheme 10.7 Altermatively, transient generation of
an iron-carbon bond in 55 can be visualized. The existence of
an open coordination site on iron in both IPNS5 and CS4 (e.g.,
51) prior to oxygen binding opens the possibility of an agostic
mechanism38 in which the metal inserts into the C-4′-H bond.39
We regard such a reaction path by Fe(II) as highly unlikely.
Finally, a simultaneous process could be advanced in which
C-4′ hydrogen abstraction is synchronous with iron-carbon
bond formation (56, path D). An analogous mechanism has been
proposed for the second oxidative cyclization catalyzed by
IPNS.13,40,41 To the extent that C-H bond breaking and C-Fe
bond forming are synchronous, it would seem unlikely that
reactive intermediates would be stabilized by hyperconjugation,
or that the metal center would be sufficiently close to have an
agostic interaction with a C-3′ hydrogen. Little, if any,
â-secondary kinetic isotope effect would be expected, therefore,
for reaction by this mechanism, and the primary kinetic isotope
effect would be predicted to be small as well. We favor an
explanation for the kinetic behavior of clavaminate synthase
based on the electronic constraints imposed by an azetidinone
Figure 3. (Top) Radiochromatogram for the acetolysis of benzyl [3′-
2H2,2-3H,2-14C]-4′-chloroazetidinyl acetate (45/32) terminated at extent
of reaction ) 0.701. The retention times of the reaction components
are as follows: (A) (45/43), 10 min; (B) benzyl [3′-2H2,2-3H,2-14C]-
4′-acetoxyazetidinyl acetate, 18 min. (Bottom left) Relative D/H ratios
of benzyl [3′-2H2,2-3H,2-14C]-4′-chloroazetidinyl acetate (45/43) upon
the acetolysis to acetoxyazetidinone (49) from triplicate experiments.
The curve was drawn using eq 1 with a â-secondary isotope effect )
1.040, K ) 0.0733, and R0 ) 0.6131. (Bottom right) Relative D/H
ratios of benzyl [3′-2H2,2-3H,2-14C]-4′-chloroazetidinyl acetate 45/43
upon the ethanolysis to ethoxyazetidinone 50 from triplicate experi-
ments. The curve was drawn using eq 1 with a â-secondary isotope
effect ) 1.098, K ) 0.0733, and R0 ) 0.6131.
acetic acid and, to a lesser extent, by ethanol-THF. Superim-
posed on the relatively large variation in â-secondary kinetic
isotope effect brought about by medium effects is the question
of mechanism. Whatever kinetic effect is measured for the
radical, it will likely be larger for carbenium ion formation at
C-4′.
(36) This is likely to be quite variable and not accurately represented by
a macroscopic value (Warshel, A. J. Biol. Chem. 1998, 273, 27035-27038).
Nonetheless, macroscopic dielectrics for active sites have been variously
estimated to range from 4 to 40 with 10 taken as an average value (Warshel,
A. Annu. ReV. Biophys. Biophys. Chem. 1991, 20, 267-298). Jordan has
experimentally determined an ꢀ ) 13-15 in a very recent example (Jordan,
F.; Li, H.; Brown, A. Biochemistry 1999, 38, 6369-6373).
(37) Townsend, C. A. Biochem. Soc. Trans. 1993, 21, 208-213.
(38) Brookhart, M.; Green, M. L. H. J. Organomet. Chem. 1983, 250,
395-408.
(39) The microscopic reverse of this process would be protodemetalation.
For the case of protodemercurations, â-secondary kinetic isotope effects
have been observed to be very low or nonexistent, in contrast to the CS-
catalyzed reaction (Bencivengo, D. J.; Brownawell, M. L.; Li, M.-Y.; San
Filippo, Jr., J. J. Am. Chem. Soc. 1984, 106, 3703-3704).
This being so, it is noteworthy that the â-secondary effect
T
observed for the enzymic reaction (V/K) ) 1.056 ( 0.002 is
smaller than that observed for the radical reaction in acetoni-
trile-THF (1:1; ꢀ ) 36.6 and 7.5, respectively). At least two
factors can account for this difference, although their combined
significance cannot be firmly established. First, the commitment
to catalysis, while probably low, is not accurately known as
the intrinsic primary effect will surely be reduced by the adjacent
azetidinone nitrogen and may be only partially rate-determining.
Even if this were calculable, however, the effective dielectric
of the enzyme active site is not known.36 The apparent sup-
pression of the â-secondary kinetic isotope effect raises the
(40) Baldwin, J. E.; Adlington, R. M.; Marquess, D. G.; Pitt, A. R.; Porter,
M. J.; Russell, A. T. Tetrahedron 1996, 52, 2537-2556.
(41) Baldwin, J. E.; Adlington, R. M.; Marquess, D. G.; Pitt, A. R.;
Russell, A. T. J. Chem. Soc., Chem. Commun. 1991, 856-858.

11364 J. Am. Chem. Soc., Vol. 121, No. 49, 1999
Iwata-Reuyl et al.
Scheme 10
using a 1 dm cell at 25 °C, with concentrations expressed in g/100
mL. Melting points were determined in open capillary tubes with a
Thomas-Hoover Uni-Melt melting point apparatus and are uncorrected.
Microanalysis were performed by Atlantic Microlab, Inc. of Norcross,
GA. A Cahn 25 Automatic Electrobalance was used for the accurate
weighing of micro-samples (50 µg - 4 mg). Radioactive samples were
quantitated by liquid scintillation counting using a Beckman LS 5801
Liquid Scintillation Counter with Opti-Fluor (Packard) scintillation fluid.
All MPLC (flash) chromatography was carried out using Merck
Kieselgel 60 (230-400 mesh) silica gel. Radial chromatography was
performed on a Chromatotron (Harrison Research), using rotors
prepared with silica gel PF-254 with CaSO4‚0.5H2O as binder. TLC
analysis was performed using Analtech Uniplate TLC plates (cat #
21521) with basic KMnO4 (1% in 6.5% Na2CO3/0.2% KOH for
visualization. HPLC chromatography was performed with a Waters 600
multisolvent delivery system equipped with a Rheodyne injector and a
Waters 490 programable multiwavelength detector. Fractions were
collected with an Isco Cygnet automatic fraction collector. Reaction
temperatures for kinetic isotope effect experiments were maintained
with a Lauda RM-6 circulating bath. Photochemically initiated reduc-
tions were performed in a quartz test tube with illumination from a
Hanovia medium-pressure mercury lamp ( no. 679A36) equipped with
a Vycor sleeve. Micro-samples were concentrated under high vacuum
in a Savant SVC 100D circulating speed vacuum.
Synthesis of (2S,3R)-[2-14C]-Proclavaminic Acid (34). 4-Phenyl-
sulfonyl-azetidinone (28). 4-Acetoxyazetidinone (27, 13.15 g, 101.8
mmol), H2O (120 mL), and sodium benzenesulfinate (20.05 g, 122.2
mmol were stirred in an oil bath (40 °C) for 17 h. The product was
partioned between ethyl acetate (200 mL) and H2O (80 mL additional)
and the aqueous layer back-extracted with ethyl acetate (3 × 200 mL).
The combined organic layers were washed with saturated brine (2 ×
600 mL), dried (anhydrous Na2SO4), and concentrated in vacuo to give
a white solid. Recrystallization from ethyl acetate/petroleum ether gave
the desired product as white prisms (15.13 g, 71.61 mmol, 70%): mp
155.5-157 °C [lit.26 mp 156-157 °C].
ring and, to a lesser extent, to commitment effects. The
alternative rationale implied by synchronous C-H bond break-
ing and C-Fe bond forming would bypass the formation of
discrete radical or ionic intermediates. As a consequence, the
attendant rehybridization at C-4′ would be avoided, and, hence,
expression of kinetic isotope effects would be markedly
decreased. While this view cannot be strictly excluded, we
regard it as less likely based on the magnitude of the primary
kinetic isotope effect observed, the three model reactions
presented and the effect that amide resonance may play in the
transition state of the CS-catalyzed reaction.
Experimental Section
All air- or moisture-sensitive reactions were run under an inert
atmosphere (Ar or N2) in flame- or oven-dried glassware with magnetic
stirring unless otherwise noted. Moisture-sensitive reagents were added
to reaction vessels by dry syringes equipped with oven-dried needles
through rubber septa. Reaction temperatures refer to bath temperatures.
Tetrahydrofuran (THF) and diethyl ether (Et2O) were freshly distilled
from Na/benzophenone ketyl; dichloromethane (CH2Cl2) was freshly
distilled from CaH2. All other solvents and reagents for air- or moisture-
sensitive reactions were used as received or dried by standard procedures
(Perrin, D. D.; Armarego, W. L. F.; Perrin, D. R. Purification of
Laboratory Chemicals; Pergamon: Oxford, 1980.)
1H and 13C NMR spectra were obtained on a Varian XL/VXR-400
or a Bruker AMX 300 NMR spectrometer. Chemical shifts of hydrogen
resonances are reported on the δ-scale and referenced to tetramethyl-
silane (0.0 ppm), deuteriochloroform (7.26 ppm), or acetone-d6 (2.23
ppm). Coupling constants are reported in Hertz. Carbon-13 chemical
shifts are also reported on the δ-scale and referenced to deuteriochlo-
roform (77.0 ppm), or p-dioxane-d8 (66.5 ppm). Low- and high-
resolution mass spectrometric data were obtained on a VG Instruments
70-S GC/MS at 70 eV and are tabulated as m/z (intensity relative to
the base peak). IR spectra were obtained on a Perkin-Elmer 1600 Series
FT-spectrophotometer (CHCl3 solution or a KBr disk). Ultraviolet-
visible spectra were obtained on Beckman DU 70 spectrophotometer.
Optical rotations were obtained with a Perkin-Elmer 141 polarimeter
(4S)-4-Thiophenyl-azetidinone (29). To a solution of the azetidinone
28 (15.13 g, 71. 61 mmol) in benzene (1.5 L) were added (+)-
cinchonine (25.30 g, 85.94 mmol and thiophenol (25 mL, 24 mmol),

KIEs in OxidatiVe Cyclization of ProclaVaminic Acid
J. Am. Chem. Soc., Vol. 121, No. 49, 1999 11365
and the mixture was stirred at 40 °C under argon for 41 h. The mixture
was filtered through a pad of silica gel washing with benzene to remove
the cinchonine, the filtrate concentrated in vacuo, and the residue applied
to a column of silica gel (60 g, benzene). The column was eluted with
benzene until thiophenol was no longer detected in the eluent, and
elution was continued with ethyl acetate to recover the desired product.
Concentration in vacuo gave a white solid, which when recrystallized
from benzene/cyclohexane and methylene chloride/petroleum ether,
gave optically pure 29 (3.724 g, 20.77 mmol, 29%) as white needles
and optically impure product (8.342 g, 46.54 mmol, 65%) as white
plates. The optically pure material gave the following physical and
spectral data: mp 68-69 °C [lit.25 mp 58-60 °C]; [R]D ) -137° (c
425.1713; Anal. Calcd for C29H30N2O5S: C 65.15, H 5.66, N 5.24, S
6.00; found: C 65.00, H 5.61, N 5.18, S 5.90.
Benzyl (2R,3R)-[2-14C]-5-(N-Benzyloxycarbonyl)-amino-3-hydroxy-
2-(2′-oxoazetidin-1′-yl)pentanoate (32). Thiophenyl â-lactam 31 (48.8
mg, 0.091 mmol), tris(trimethylsilyl)silyl hydride (75 µL, 0.24 mmol),
and AIBN (∼5) were disolved in dry, distilled benzene (4 mL) and
the solutionwas degassed by bubbling Ar for 30 min. The reaction
mixture was brought to reflux with stirring. Additions of tris-
(trimethylsilyl)silyl hydride (3 × 75 µL) and AIBN (3 × 5 mg) were
made over the next 15 h, and the reaction was terminated after 19 h.
The benzene was removed in vacuo and the residue purified via radial
chromatography (1 mm silica gel; ethyl acetate:hexane, 1:4 to 100%
ethyl acetate), giving 26.3 mg (0.062 mmol, 68%) of the desired product
as a colorless oil. Radioinactive and nondeuteriated material prepared
similarly gave the following physical and spectral data: [R]D ) -24°
1
) 1.0, CHCl3) [lit.25 [R]D ) -105°]; H NMR (CDCl3) δ 7.49-7.37
(m, 5H, ArH), 6.27 (b, 1H, NH), 5.03 (dd, J ) 2.4, 4.9 Hz, 1H, H-4),
3.40 (ddd, J ) 1.9, 5.0, 15.3 Hz, 1H, H-3), 2.96 (dd, J ) 2.4, 15.3 Hz,
1H, H-3); 13C{1H} NMR (CDCl3) δ 166.1, 133.4, 131.3, 129.3, 128.6,
54.2, 45.4; MS m/z 179 (M+, 7%), 119, 110, 77, 70 (100%); accurate
mass 179.0406, calcd for C9H9NOS 179.0405.
1
(c 1.7, CHCl3); H NMR (CDCl3) δ 7.36 (m, 10H, ArH), 5.22 (ABq,
J ) 12.0 Hz, 2H, CH2Ph), 5.16 (bs, 1H, NH), 5.09 (s, 2H, CH2Ph),
4.65 (bs, 1H, OH), 4.18 (m, 1H, H-3), 4.08 (br, 1H, H-2), 3.43 (m,
1H, H-5), 3.32 (m, 2H, H-4′), 3.25 (m, 1H, H-5), 2.95 (t, J ) 4.0 Hz,
2H, H-3′), 1.87 (m, 1H, H-4), 1.78 (m, 1H, H-4); 13C{1H} NMR
(CDCl3) δ 168.5, 168.0, 156.8, 136.4, 135.0, 128.5, 128.3, 128.1, 127.9,
127.8, 69.1, 67.1, 66.5, 61.7, 39.6, 37.8, 36.2, 33.4; MS m/s 219, 148,
128, 108, 91 (100%), 79, 65; accurate mass 426.1799, calcd for
C23H26N2O6 426.1791.
Benzyl [2-14C]-(4′S)-(4′-thiophenyl-2′-oxoazetidin-1′-yl) acetate
(30). A solution of azetidinone (-)-29 (161.8 mg, 0.9027 mmol) in
THF (13 mL) was cooled to -78 °C, and a solution of LiHMDS (950
µL, 1.0 M in hexane) was added dropwise via syringe in 5 min. After
50 min, [2-14C]-methyl bromoacetate (196.1 mg, 0.8561 mmol) in THF
(5 mL) was added dropwise via syringe over 10 min. The reaction
was warmed to -20 °C over 45 min and stirred for 1 h at -20 °C.
After the reaction was quenched with a solution of acetic acid in THF,
ethyl acetate (100 mL) was added, and the mixture was washed with
1 N HCl (100 mL), 5% NaHCO3 (100 mL) and saturated brine (2 ×
100 mL). The organic layer was dried (anhydrous Na2SO4) and
concentrated in vacuo, and the residue was purified by radial chroma-
tography (1 mm silica gel; ethyl acetate:hexane, 1:19 to 1:1) to give
the desired product (131.3 mg, 0.4011 mmol, 47%) as a colorless oil.
Radioinactive material prepared similarly gave the following spectral
data: [R]D ) +98.5° (c 0.72, CHCl3) IR (CHCl3) 3016, 1767, 1405,
Benzyl (2S,3R)-[2-14C]-5-(N-Benzyloxycarbonyl)-amino-3-hydroxy-
2-(2′-oxoazetidin-1′-yl)pentanoate (33). To D-erythro diastereomer 32
(26.3 mg, 0.062 mmol) in CH2Cl2 (4 mL) was added DBN (10 µL,
0.08 mmol), and the reaction was stirred for 1 h. Silica gel (∼500 mg)
was added to terminate the reaction, and the mixture filtered through
a pad of silica gel, washing with ethyl acetate. The filtrate was
concentrated in vacuo to give 22.0 mg (0.052 mmol, 84%) of the
protected proclavaminic acids in ratio of > 4:1 favoring the desired
(2S,3R) (threo) diastereomer. Radioinactive and nondeuteriated mate-
rial prepared similarly gave the following physical and spectral data:
1
1
1387, 1345, 1260, 1230, 1188, 941 cm-1; H NMR (CDCl3) δ 7.41-
[R]D ) +22.3° (c 0.85, CHCl3); H NMR (CDCl3) δ 7.33 (m, 10H,
7.28 (m, 10H, ArH), 5.22 (dd, J ) 2.3, 5.1 Hz, 1H, H-4′), 5.11 (ABq,
J ) 12.1 Hz, 2H, CH2Ph), 4.32 (ABq, J ) 18.2 Hz, 1H, H-2), 3.78
(ABq, J ) 18.2 Hz, 1H, H-2), 3.18 (dd, J ) 5.1, 15.2 Hz, 1H, H-3′â),
2.88 (dd, J ) 2.3, 15.2 Hz, 1H, H-3′R); 13C{1H} NMR (CDCl3) δ 167.7,
165.3, 134.8, 133.8, 130.1, 129.3, 128.7, 128.6, 128.5, 67.3, 58.9, 36.6,
41.1; MS m/z 327(M+, 0.3%), 218 (M+ - SPh, 9%), 181, 176, 135,
109, 91 (100%); accurate mass 327.0931, calcd for C18H17NO3S,
327.0929.
ArH), 5.22 (ABq, J ) 12.0 Hz, 2H, CH2Ph), 5.13 (br, 1H, NH), 5.07
(s, 2H, CH2Ph), 4.47 (d, J ) 8.2 Hz, 1H, OH), 4.27 (m, 1H, H-3), 4.14
(d, J ) 3.1 Hz, 1H, H-2), 3.44 (m, 3H, H-5 and H-4′), 3.28 (m, 1H,
H-4′), 3.00 (t, J ) 4.0 Hz, 2H, H-3′), 1.68 (m, 2H, H-4); 13C{1H} NMR
(CDCl3) δ 169.3, 168.5, 157.1, 136.3, 135.1, 128.6, 128.5, 128.3, 128.2,
128.1, 128.0, 68.9, 67.4, 66.8, 61.8, 40.3, 37.7, 36.3, 34.4; MS m/z
219, 148, 128, 108, 91 (100%), 79, 65; accurate mass 426.1799, calcd
for C23H26N2O6 426.1791.
(2S,3R)-[2-14C]-Proclavaminic Acid (34). Hydrogenation of dia-
stereomer 33 (22.0 mg, 0.052 mmol) in ethanol (4 mL) containing 10%
Pd/C (∼5 mg) by bubbling hydrogen through the Ar degassed solution
for 30 min with stirring. The catalyst was removed by filtration and
the filtrate concentrated to approximately 0.5 mL in vacuo. The material
was purified twice via reverse phase HPLC, first using a Partisil ODS-3
C-18 column, then using a Spheresorb S5 ODS-2 C-18 column, both
with H2O as eluent. Radioinactive and nondeuteriated material prepared
in a similar manner gave the following physical and spectral data: mp
153-154 °C; [R]D ) +6.85° (c 0.35, H2O) [lit.23 +7.3° (c ) 1.0, H2O)];
Benzyl (2R,3R,4′S)-[2-14C]-5-(N-Benzyloxycarbonyl)-amino-3-hy-
droxy-2-(4′-thiophenyl-2′-oxoazetidin-1′-yl)pentanoate (31). Azeti-
dinone 30 (131.3 mg, 0.4011 mmol) in THF (10 mL) was cooled to
-78 °C, a solution of LHMDS (480 µL, 1.0 M in hexane) was added
dropwise via syringe and the reaction stirred for 2 h. Cbz-aldehyde
22b (202.9 mg, 0.9791 mmol) in THF (3 mL) was then added dropwise
and the mixture stirred a further 2 h. After the reaction was quenched
by the addition of acetic acid in THF, the mixture was diluted with
ethyl acetate (70 mL) and washed with 1 N HCl (50 mL), 5% NaHCO3
(50 mL), and saturated brine (50 mL). The organic layer was dried
(anhydrous Na2SO4) and concentrated in vacuo, and the residue was
purified via radial chromatography (2 mm silica gel; ethyl acetate:
hexane, 1:19 to 1:1), to give the starting azetidinone 30 (25.0 mg, 0.076
mmol, 19%), and the desired D-erythro 31 (59.1 mg, 0.111 mmol, 28%)
as a white solid. Recrystallization from ethyl acetate/cyclohexane
provided the product as fine, white needles (48.8 mg, 0.091 mmol,
23%), with a specific activity of 1.22 mCi/mmol. Radioinactive and
nondeuteriated material prepared similarly gave the following physical
and spectral data: mp 102-103 °C; [R]D ) +73 ° (c 1.4, CHCl3); IR
(CHCl3) 3446, 3006, 2950, 1748, 1735, 1509, 1261, 1233, 1216, 1188,
1
IR (KBr) 3373, 2940, 1703, 1634, 1378, 773 cm -1; H NMR (D2O/
acetone) δ 4.15 (ddd, J ) 3.9, 5.5, 9.4 Hz, 1H, H-4′), 4.00 (d, J ) 5.5
Hz, 1H, H-2), 3.50 (m, 1H, H-4′), 3.43 (m, 1H, H-4′), 3.07 (m, 2H,
CH2NH2), 2.93 (t, J ) 4.0 Hz, 2H, H-3′), 1.89 (m, 2H, H-4); 13C{1H}
NMR (D2O/dioxane) δ 175.3, 173.2, 70.0, 63.5, 41.2, 38.1, 36.2, 32.0;
CI-MS m/z 203 (MH+, 100%), 185, 167, 143, 115; accurate mass
(MH+) 203.1037, calcd for C8H15N2O4 203.1037.
Synthesis of (2S,3R 3′-2H2,4,5-3H]-Proclavaminic Acid (42). [2,3-
3H]-N-benzyloxycarbonyl-3-aminopropanol (22a). To NaHCO3 (377.1
mg, 4.489 mmol) and â-alanine (181.0 mg, 2.032 mmol) in a 50 mL
flask was added a solution of [2,3-3H]-â-alanine (5 mCi) in 5 mL of
0.01 N HCl followed by H2O washings (5 mL) and dioxane (7.5 mL).
The solution was cooled in an ice bath, and benzyl chloroformate (450
µL, 3.15 mmol) dissolved in dioxane (2.5 mL) was added in
approximately 15 min. The mixture was stirred for 45 min, warmed to
room temperature, and stirred a further 3 h. The dioxane was removed
in vacuo, and the resulting solution was transferred to a separatory
funnel, diluted with 5% NaHCO3 (30 mL), and washed with ethyl
1
1040 cm-1; H NMR (CDCl3) δ 7.36-7.28 (m, 15H, ArH), 5.21 and
5.12 (ABq, J ) 11.2 Hz, 2H, CH2Ph), 5.11 (s, 2H, CH2Ph), 5.22 (bs,
1H, NH), 4.96 (bs, 1H, OH), 4.88 (dd, J ) 2.4, 5.1 Hz, 1H, H-4′),
4.20 (m, 1H, H-3), 3.99 (bs, 1H, H-2), 3.45-3.31(m, 2H, H-5), 3.34
(dd, J ) 5.1, 15.4 Hz, 1H, H-3′â), 2.89 (dd, J ) 2.1, 15.3 Hz, 1H,
H-3′R), 2.05-1.88 (m, 2H, H-4); MS m/z 425 (M+ - SPh, 2.2), 407
(M+ - SPh - H2O), 383 (M+ - SPh - CH2CO), 327, 219, 218, 176,
91 (100); accurate mass 425.1717, calcd for C23H25N2O5 (M+ - SPh)

11366 J. Am. Chem. Soc., Vol. 121, No. 49, 1999
Iwata-Reuyl et al.
acetate (50 mL) to remove excess benzyl chloroformate. The aqueous
layer was then acidified with 6 N HCl, diluted with an equal volume
of saturated brine, and extracted with ethyl acetate (2 × 80 mL). The
combined organic layers were dried (anhydrous Na2SO4) and concen-
trated in vacuo to give a white solid. After drying under high vacuum,
the residue was dissolved in THF (11 mL) and the solution cooled in
an ice bath. Triethylamine (295 µL, 2.11 mmol) was then added
dropwise, and after the mixture stirred for 30 min, a solution of ethyl
chloroformate (215 µL, 2.25 mmol) in THF (2 mL) was added
dropwise. The reaction mixture was stirred for 2 h and filtered through
a fritted funnel containing Celite to remove precipatated triethylam-
monium hydrochloride. The filtrate and THF washings (20 mL) were
taken up in a syringe and added dropwise to a 0 °C solution of sodium
borohydride (215.0 mg, 5.69 mmol) in H2O (20 mL). When the addition
was complete, additional sodium borohydride (114 mg, 3.02 mmol)
was added, and the reaction stirred for 5 h, warming to room
temperature. The reaction mixture was transferred to a separatory funnel,
diluted with ethyl acetate (120 mL) and then washed with 1 N HCl
(100 mL), 5% NaHCO3 (100 mL), and brine (100 mL). The organic
layer was then dried (anhydrous Na2SO4) and concentrated in vacuo
to give a white solid, which was recrystallized from ethyl acetate/
petroleum ether to give the product as white needles (236.2 mg, 1.129
mmol, 56% from â-alanine). Radioinactive material prepared in a
similar manner gave the following physical and spectral data: mp 44-
45 °C; IR (CHCl3) 3630, 3450, 3015, 2950, 1706, 1520, 1250, 1225,
1135, 1072, 1028 cm-1; 1H NMR (CDCl3) δ 7.35 (m, 5H, ArH), 5.11
(s, 2H, CO2CH2), 5.02 (bs, 1H, NH), 3.68 (t, J ) 5.6 Hz, 2H, CH2-
OH), 3.37 (m, 2H, CH2NH), 2.51 (bs, 1H, OH), 1.70 (m, 2H, C-2);
13C{1H} NMR (CDCl3) δ 136.4, 128.5, 128.1, 128.0, 66.7, 59.4, 37.7,
32.4; MS m/z 209 (M+, 2%), 108, 91(100%), 79, 65; accurate mass
209.1054, calcd for C11H15NO3 209.1052.
[3,4-3H]-N-benzyloxycarbonyl-3-aminopropanal (22b). Cbz-
alcohol 22a (236.2 mg, 1.129 mmol), in CH2Cl2 (5 mL) was added
dropwise to a -78 °C solution oxalyl chloride (110 µL, 1.26 mmol) in
CH2Cl2 (3 mL) and DMSO in CH2Cl2 (2.5 mL). The reaction was stirred
a further 15 min, then triethylamine (790 µL, 5.67 mmol) was added
dropwise in 1.5 min. The mixture was stirred an additional 5 min at
-78 °C, warmed to room temperature over 45 min, diluted with ethyl
acetate (60 mL) and washed with 1 N HCl (40 mL), 5% NaHCO3 (40
mL), and brine (2 × 40 mL). The organic layer was dried (anhydrous
Na2SO4) and concentrated in vacuo to give a pale yellow oil.
Crystallization from ethyl acetate/cyclohexane afforded the product
(176.3 mg, 0.8508 mmol, 76%) as fine white needles, with a specific
activity of 2.45 mCi/mmol. Radioinactive material prepared in a similar
manner gave the following physical and spectral data: mp 59-60 °C;
1H NMR (CDCl3) δ 9.80 (s, 1H, C-1), 7.35 (m, 5H, ArH), 5.12 (bs,
1H, NH), 5.10 (s, 2H, CO2CH2), 3.50 (q, 2H, J ) 6.0 Hz, CH2NH),
2.73 (t, 2H, J ) 5.74 Hz, CH2CO); 13C{1H} NMR (CDCl3) δ 201.1,
157.1, 136.3, 128.4, 128.0, 127.9, 66.6, 43.9, 34.4; MS m/z 207 (M+,
3%), 108, 91 (100%), 79, 65; accurate mass 207.0900, calcd for C11H13-
NO3 207.0895.
ArH), 6.01 (b, 1H, NH), 4.77 (d, J ) 2.4 Hz, 1H, H-4), 2.72 (dd,
J ) 0.9, 2.4 Hz, 1H, H-3), 0.14 (s, 9H, TMS); 13C {1H} NMR (CDCl3)
δ 168.6, 133.4, 132.1, 129.4, 128.7, 56.5, 50.09, -3.0; MS m/z 251
(M+, 0.5%), 236, 208, 193, 182, 151, 142; accurate mass 251.0802,
calcd for C12H17NOSSi 251.0800. Anal. Calcd. for C12H17NOSSi: C,
57.33; H, 6.82; N, 5.57; S 12.75. Found C, 57.35; H, 6.84; N 5.53; S,
12.66. Cis isomer: 7.30 (m, 5H, ArH), 6.02 (b, 1H, NH), 5.22 (d, J )
5.2 Hz, 1H, H-3), 3.23 (1H, dd, J ) 1.5, 5.2 Hz, 1H, H-4), 0.31 (s,
9H, TMS); 13C {1H} NMR (CDCl3) δ 168.8, 134..6, 131.4, 129.4, 127.7,
58.7, 49.5, -1.2; MS m/z 236 (M - CH3, 0.14%), 179, 167, 151, 142,
109, 70 (100%); accurate mass 236.0565, calcd for C11H14NOSSi
(M - CH3) 236.0565; Anal. Calcd for C12H17NOSSi: C, 57.33; H,
6.82; N, 5.57; S, 12.75. Found: C, 57.29; H 6.85; N, 5.55; S, 12.67.
A mixture containing both cis- and trans-azetidinones (9.5 g, 37.85
mmol) was dissolved in d4-MeOH (6 mL) under argon. The solution
was then transferred to another flask containing dry KF (2.2 g, 1 equiv)
under argon at 0 °C. After stirring at 0 °C for 5 min, the solution was
warmed to room temperature over a period of 10 min and filtered
through silica gel with EtOAc. The combined filtrate and washings
were evaporated to leave an oil from which 4-thiophenyl azetidinone
was crytallized as a white solid from hexanes and EtOAc having 46%
deuteriation at C-3 per site (6.2 g, 92%). The procedure of silylation
at C-3 and desilylation in deuterated methanol was repeated 4 times
and the level of deuteriationat C-3 was raised to ∼90% per site.
[3-2H2]-4-Phenylsulfonyl-azetidinone (36). To [3-2H2]-4-thiophenyl
azetidinone 35 (1.02 g, 5.64 mmol) in acetone at 0 °C was added 2 N
H2SO4 (4 mL) followed by the slow addition of KMnO4 (1.3 g, 8.2
mmol) in water (25 mL). After 1 h at room temperature, the mixture
was filtered through Celite, which was washed with EtOAc. The
combined filtrate and washings were concentrated to ∼50 mL and then
partitioned between EtOAc and water (50 mL each). The aqueous layer
was reextracted with EtOAc (50 mL). The combined EtOAc layers
were dried (Na2SO4) and evaporated to leave the title compound as a
white crystalline solid (1.1 g, 93%).
(4S)-[3-2H2]-4-Thiophenyl-azetidinone (37). The title compound
was prepared following the same procedure as that described for the
preparation of 29.
Benzyl (4′S)-[3-2H2]-(4′-Thiophenyl-2′-oxoazetidin-1′-yl) Acetate
(38). The title compound was prepared following the same procedure
as that described for the preparation of 30.
Benzyl (2R,3R,4′S)-[3′-2H2,4,5-3H]-5-(N-Benzyloxycarbonyl)-
amino-3-hydroxy-2-(4′-thiophenyl-2′-oxoazetidin-1′-yl)pentanoate
(39). The dideuteriated azetidinone 38 (206.0 mg, 0.6254 mmol) in
dry THF (6 mL) was cooled to -78 °C, and LHMDS (750 µL, 1 M in
hexanes) was added dropwise. The reaction was stirred 2 h, and a
solution of the tritiated aldehyde 22b in THF (6 mL) was added
dropwise in 10-15 min. After 2 h, the reaction was quenched with
excess acetic acid in THF (400 µL, 2.0 mmol). After stirring for a few
min at -78 °C, the mixture was warmed to room temperature,
transferred to a separatory funnel with ethyl acetate (60 mL), and
washed with 1 N HCl (2 × 50 mL), 5% NaHCO3 (50 mL), and brine
(50 mL). The organic layer was dried (anhydrous Na2SO4) and
concentrated in vacuo, and the crude material was purified by radial
chromatography (1 mm silica gel; 10% ethyl acetate in hexanes to
100%). Recrystallization from ethyl acetate/cyclohexane gave 88.5 mg
(0.165 mmol, 19%) of the desired D-erythro diastereomer 39 as fine
white needles, with a specific activity of 2.44 mCi/mmol. For physical
and spectral data of radioinactive material prepared in an analogous
manner, see 31 above.
Benzyl (2R,3R)-[3′-2H2,4,5-3H]-5-(N-benzyloxycarbonyl)-3-hydroxy-
2-(2′-oxoazetidin-1′-yl)pentanoate (40). The thiophenyl â-lactam 39
(88.5 mg, 0.165 mmol) and AIBN (∼10 mg) were dissolved in dry
benzene (5 mL) and tris(trimethylsilyl)silyl hydride (220 µL, 0.713
mmol) was added. The solution was sparged with Ar for approximately
40 min, and the reaction heated to reflux. Additions of tris(trimethyl-
silyl) hydride (3 × 220 µL) and AIBN (3 × 10 mg) were made over
approximately 10 h, and the reaction terminated after 16.5 h. The
benzene was removed in vacuo, and the residue purified by radial
chromatography (1 mm silica gel; ethyl acetate:hexane, 1:4 to 100%
ethyl acetate) to give 58.1 mg (0.136 mmol, 82%) of the desired product
[3′-2H2]-4-Thiophenyl-2-azetidinone (35). To a solution of racemic
4-thiophenyl azetidinone 29 (5.053 g, 28.19 mmol) in THF (55 mL)
at 0 °C under Ar, diisopropylamine (4.35 mL, 31.0 mmol) was added
dropwise. The reaction was stirred for 1 h and filtered through a fritted
funnel with THF (45 mL) to remove the precipitated diisopropylam-
monium hydrochloride. The clear filtrate was cannulated dropwise to
a -78 °C solution of LDA [prepared from disopropylamine (5.9 mL,
49.1 mmol) and n-BuLi (28.2 mL, 1.5 M in hexane)]. When the
addition was complete, the reaction was stirred for 20 min, and trimethyl
silyl chloride (6.5 mL, 51.2 mmol) was added dropwise. After the
mixture stirred 2 h, the reaction was stopped by the addition of 5 M
acetic acid (12 mL) in THF and warmed to room temperature. The
reaction mixture was diluted with EtOAc (400 mL) and washed with
1 N HCl (2 × 300 mL), 5% NaHCO3 (350 mL), and brine (2 × 300
mL). The organic layer was dried (Na2SO4) and concentrated in vacuo,
and the crude mixture was purified by flash chromatography to give,
after recrystallization from CH2Cl2/pentane, the trans (2.125 g, 30%)-
and cis (3.442 g, 49%)-isomers as white needles. trans-Isomer: mp
90-90.5 °C; IR (CHCl3) 3401, 3008, 1751, 1479, 1334, 1253, 1091,
1
859, 847 cm-1; H NMR (CDCl3) 7.45 (m, 2H, ArH), 7.34 (m, 3H,

KIEs in OxidatiVe Cyclization of ProclaVaminic Acid
J. Am. Chem. Soc., Vol. 121, No. 49, 1999 11367
as a colorless oil. For physical and spectral data of radioinactive material
prepared in an analogous manner, see 32 above.
layer was dried (anhydrous Na2SO4) and concentrated in vacuo, and
the residue was purified by radial chromatography (1 mm silica gel;
ethyl acetate:hexane, 1:3) to give 45.0 mg (0.162 mmol, 81%) of the
desired product as a colorless oil. IR (CHCl3) 3028, 1779, 1749, 1405,
Benzyl (2S,3R)-[3′-2H2,4,5-3H]-5-(N-Benzyloxycarbonyl)-amino-
3-hydroxy-2-(2′-oxoazetidin-1′-yl)pentanoate (41). The D-erythro
diastereomer 40 (58.1 mg, 0.136 mmol) in CH2Cl2 (5 mL) was treated
with freshly distilled DBN (17 µL, 0.14 mmol. After stirring at room
temperature for 2 h, the mixture was filtered through silica gel washing
with ethyl acetate. Concentration of the filtrate in vacuo and purification
of the residue by radial chromatography (1 mm silica gel; ethyl acetate:
hexane, 1:4 to 1:1) gave 39.8 mg (0.093 mmol, 69%) of the desired
L-threo diastereomer 41 as a colorless oil and 9.2 mg (0.022 mmol,
16%) of the D-erythro diastereomer. For physical and spectral data of
radioinactive material prepared in an analogous manner, see 33 above.
1
1381, 1351, 1236, 1224, 1194, 1043, 929 cm-1; H NMR (CDCl3) δ
7.36 (m, 5H, ArH), 6.06 (dd, J ) 1.5, 4.2 Hz, 1H, H-4′), 5.17 (ABq,
J ) 14.0 Hz, 2H, CH2Ar), 4.22 (ABq, J ) 18.0 Hz, 1H, CH2CO2),
3.94 (ABq, J ) 18.0 Hz, 1H, CH2CO2), 3.33 (dd, J ) 4.1, 15.1 Hz,
1H, H-3′cis), 3.06 (d, J ) 14.7 Hz, 1H, H-3′trans), 2.07 (s, 3H, CH3);
13C{1H} NMR (CDCl3) δ 171.6, 168.0, 165.7, 135.0, 128.7, 128.6,
128.4, 76.5, 44.5, 42.9, 20.7; MS m/z 277 (M+, 0.13%), 235, 217, 189,
173, 143, 142, 100, 91 (100%); accurate mass 277.0957, calcd for
C14H15NO5 277.0950.
Determination of the â-2° Kinetic Isotope Effect in the Reaction
of (2S,3R)-[3′-2H2,4,5-3H]- and (2S,3R)-[2-14C]-Proclavaminic Acid
34 and 42, Respectively with Clavaminate Synthase. Incubations
were run in Wheaton glass mini-vials equipped with a spin vane at 25
°C and contained, in a volume of 280 µL or 468 µL, 50 mM MOPS
buffer (pH 7.0), 0.5 mM DTT, 0.1 mM sodium ascorbate, 0.01 mM
ferrous ammonium sulfate, 3 mM R-KG, 0.3-0.35 mM [3′-2H2; 4,5-
3H]-(2S, 3R)-proclavaminic acid, and 0.15-0.2 mM [2-14C]-(2S,3R)-
proclavaminic acid. The 3H/14C ratios were approximately 2.0-4.0. A
40 µL aliquot was removed for the time zero point and added to 10 µL
of 4 mM EDTA. The reaction was initiated with the addition of 0.52
mg of protein. Six aliquots of 40 µL were removed and terminated
with 10 µL of 4 mM EDTA at 0.5, 2, 6, 9, 20, and 30 min, with
additional enzyme (0.13 mg) added at 7 min. The samples were frozen
in dry ice until immediately prior to HPLC analysis.
(2S, 3R)-[4,5-3H,3′-2H2]-Proclavaminic acid (42). Protected pro-
clavaminate 41 (20.0 mg, 0.047 mmol) in ethanol (4 mL) was
hydrogenated over 10% Pd/C (∼5 mg). The solution was degassed with
bubbling Ar for approximately 30 min followed by The catalyst was
removed by filtration through a 0.22 µm filter with H2O, and the ethanol
removed in vacuo. Purification was as described for 34 above. For other
physical and spectral data of radioinactive, nondeuteriated material,
see 34 above.
Synthesis of Benzyl [2-3H,3′-2H2]-2-(4′-Chloro-2′-oxoazetidin-1′-
yl)acetate and Benzyl [2-14C]-2-(4′-Chloro-2′-oxoazetidin-1′-yl)-
acetate (43) and (45). Benzyl [2-14C]-2-(4′-Chloro-2′-oxoazetidin-
1′-yl)acetate (43). To (()-[2-14C]azetidinone 30 (200 mg, 0.611 mmol)
in CCl4 (3 mL) was added a freshly prepared solution of Cl2 in CCl4
(3 mL, 1% w/v) at 0 °C. After stirring for 20 min, the mixture was
concentrated in vacuo, the residue was triturated with hexane (2 × 10
mL), and the hexane was removed by pipet. The residual oil was
purified by radial chromatography (1 mm silica gel; ethyl acetate:
hexane, 1:4) followed by recrystallization from CH2Cl2/petroleum ether
to give approximately 15 mg of the product as fine white needles having
a specific activity of 0.131 mCi/mmol. Unlabeled material prepared in
an analogous manner gave the following physical and spectral data:
mp 70-71 °C; IR (CHCl3) 3018, 1776, 1747, 1384, 1262, 1228, 1193,
Analysis of the incubation mixture at various time points was carried
out on a Whatman Partisil ODS-3 10 mm C-18 column (4.6 × 250
mm), with isocratic elution of an ion-pairing solution containing 0.1%
TFA/0.01% sodium octane sulfonate (OSA). Samples were eluted at
1.5 mL/min while collecting 13 drop (∼30 s) fractions directly into 7
mL glass mini-scintillation vials. Optifluor liquid scintillation cocktail
(6.25 mL) (Packard) was added for counting. After subtracting
appropriate blank valuesfor each channel, the dpm values for the
relevant fractions were added. The extent of reaction was determined
1
933 cm-1; H NMR (CDCl3) δ 7.37 (m, 5H, ArH), 5.82 (dd, J ) 1.3,
4.0 Hz, 1H, H-4′), 5.19 (ABq, J ) 12.2 Hz, 2H, CH2Ar), 4.35 (ABq,
J ) 18.2 Hz, 1H, CH2CO2), 3.75 (ABq, J ) 18.2 Hz, 1H, CH2CO2),
3.63 (dd, J ) 4.0, 15.3 Hz, 1H, H-3′cis), 3.24 (d, J ) 15.3 Hz, 1H,
H-3′trans); 13C{1H} NMR (CDCl3) δ 167.5, 163.9, 134.8, 128.8, 128.5,
67.6, 66.6, 50.3, 40.9; MS m/z 253 (M+, 2.0%), 118, 91 (100%), 76,
65, 55, 43, 39; accurate mass 253.0508, calcd for C12H12ClNO3
253.0506.
3
from both the 14C and H distribution as the total dpm in the product
3
divided by the total dpm recovered in the injection. The data on H/
14C ratios as a function of extent of reaction were computer fit to eq 1
using a modified nonlinear least-squares regression program and by
assuming constant variance. Values are reported with their standard
errors.
Determination of the â-2° Kinetic Isotope Effect in the Solvolysis
Reaction of Benzyl [2-14C]-2-(4′-chloro-2′-oxoazetidin-1′-yl)acetate
(43) and Benzyl [2-3H,3′-2H2]-2-(4′-chloro-2′-oxoazetidin-1′-yl)ac-
etate (45) with Acetic Acid. A dry Wheaton glass mini-vial equipped
with a dry spin vane and rubber septum was charged with 300-430
µg of benzyl [2-14C]-2-(4′-chloro-2′-oxoazetidin-1′-yl)acetate (43) and
275-310 µg of benzyl [2-3H,3′-2H2]-2-(4′-chloro-2′-oxoazetidin-1′-yl)-
acetate (45). The 3H/14C ratios were approximately 2-3. Dry THF (500
µL) was added, and the solution was transferred by syringe to a second
dry mini-vial equipped with a spin vane. A 30 µL aliquot was removed
for the time zero point, and the THF in the reaction vial removed under
a dry stream of Ar or N2. Dry, freshly distilled acetic acid (500 µL,
distilled from acetic anhydride), equilibrated to 25 °C, was then added
by syringe. The reaction was run under a dry atmosphere of N2 or Ar
with stirring. Seven to eight aliquots of 30 µL were removed at various
times up to 6 h, and the samples were frozen in dry ice until immediately
prior to HPLC analysis. The solvent was then removed in a Speed-
Vac, the residue taken up in 30 µL of CH3CN, and 25 µL analyzed by
HPLC.
Benzyl [2-3H,3′-2H2]-2-(4′-Thiophenyl-2′-oxoazetidin-1′-yl)acetate
(44). THF (2 mL) and a solution of LiHMDS (150 µL, 1.0 M in hexane)
was cooled to -78 °C, dideuterated azetidinone (()-38 (47.9 mg, 0.146
mmol) in THF (3 mL) added dropwise. The reaction was stirred for
45 min, quenched by the addition of a solution of [3H]H2O (20 µL, 1.0
Ci/mL) in THF (100 µL), and warmed to room temperature. The
mixture was filtered through a pad of silica gel and anhydrous Na2SO4
with ethyl acetate, and the filtrate was concentrated in vacuo to give a
yellow oil. The residue was purified by radial chromatography (1 mm
silica gel; ethyl acetate:hexane, 1:3) to give 37.4 mg (0.114 mmol, 78%)
of the tritiated product as a colorless oil.
Benzyl [2-3H,3′-2H2]-2-(4′-Chloro-2′-oxoazetidin-1′-yl)acetate (45).
The labeled azetidinone 44 (134.4 mg, 0.4080 mmol) in CCl4 (3 mL)
was cooled to 0 °C, and a freshly prepared solution of Cl2 in CCl4 (3
mL, 1% w/v) added. After stirring 20 min, the reaction mixture was
concentrated in vacuo, the residue triturated with hexane (2 × 10 mL),
and the hexane removed by pipet. The residual oil was purified by
radial chromatography (1 mm silica gel; ethyl acetate:hexane, 1:4)
followed by recrystallization from CH2Cl2/petroleum ether to give
approximately 10 mg of the product as fine white needles with a specific
activity of 0.415 mCi/mmol. For physical and spectral data of
radioinactive material prepared in an analogous manner, see 43 above.
Analysis of the reaction mixture a various time points was carried
out on a Whatman Partisil ODS-3 10 µm C-18 column with elution at
1 mL/min, using the following gradient: 80% H2O and 20% CH3CN
to 50% H2O in 15 min, held at 50% for 10 min, then back to 80% H2O
in 15 min. The column eluent was collected in approximately 30 s
fractions directly into 7 mL glass mini-scintillation vials, and 6.25 mL
of Optifluor liquid scintillation cocKtail (Packard) added for counting.
Benzyl 2-(4′-Acetoxy-2′-oxoazetidin-1′-yl)acetate (49). Chloro-
azetidinone 43 (51.0 mg, 0.201 mmol) and glacial acetic acid (3 mL)
were treated with triethylamine (30 µL), and after stirring for 2 h, the
reaction mixture was diluted with ethyl acetate (60 mL) and washed
with 5% NaHCO3 (2 × 50 mL) and brine (2 × 50 mL). The organic
3
Extent of reaction and data on H/14C ratios as a function of reaction
were calculated as described above.

11368 J. Am. Chem. Soc., Vol. 121, No. 49, 1999
Iwata-Reuyl et al.
Determination of the â-2° Kinetic Isotope Effect in the Solvolysis
Reaction of Benzyl [2-14C]-2-(4′-Chloro-2′-oxoazetidin-1′-yl)acetate
(43) and Benzyl [2-3H, 3′-2H2]-2-(4′-Chloro-2′-oxoazetidin-1′-yl)-
acetate (45) with Ethanol. A dry Wheaton glass mini-vial equipped
with a dry spin vane and rubber septum was charged with 320-480
µg of benzyl [2-14C]-2-(4′-chloro-2′-oxoazetidin-1′-yl)acetate 43 and
340-420 µg of benzyl [2-3H, 3′-2H2]-2-(4′-chloro-2′-oxoazetidin-1′-
µg of benzyl [2-14C]-2-(4′-chloro-2′-oxoazetidin-1′-yl)acetate 43 and
270-360 µg of benzyl [2-3H, 3′-2H2]-2-(4′-chloro-2′-oxoazetidin-1′-
3
yl)acetate 45. The H/14C ratios were approximately 3. A solution of
dry, degassed CH3CN:THF (350 µL, 1:1; CH3CN dried at reflux over
CaH2, then distilled; degassing achieved with five cycles of freeze/
pump/thaw) was added followed by tris(trimethylsilyl)silyl hydride (150
µL) and a solution of AIBN (20 µL, ∼40 mM) in dry, degassed CH3-
CN:THF (1:1). After stirring, the solution was transferred to a dry quartz
test tube equipped with a dry stir bar and rubber septum. A 30 µL
aliquot was removed for the time zero point and the reaction photolyzed
at 25 °C with a medium-pressure mercury lamp equipped with a Vycor
sleeve. Two to four additions of AIBN (20 µL) were made during the
course of the reaction. Six aliquots of 30 µL were removed at various
times up to 6 h and the samples frozen in dry ice until immediatly
prior to HPLC analysis. The solvent and silyl hydride were then
removed in a Speed-Vac, the residue taken up in 30 µL CH3CN, and
25 µL analyzed by HPLC.
3
yl)acetate 45. The H/14C ratios were approximately 2-3. Dry THF
(300 µL) was added, and, after stirring thoroughly, the solution (250
µL) was transferred by syringe to a second dry mini-vial equipped with
a spin vane. A 15 µL aliquot was removed and added to 15 µL of H2O
for the time zero point. Dry, denatured ethanol (235 µL, Aldrich),
equilibrated to 25 °C, was then added. Seven to eight aliquots of 30
µL were removed and added to 30 µL at various times up to 3 h, and
the samples were frozen in dry ice until prior to HPLC analysis, when
they were thawed and immediately injected.
Analysis of the reaction mixture was carried out as described
immediately above for solvolysis in acetic acid. Data analysis was
identical to that described above for the clavaminate synthase reaction.
Analysis of the reaction mixture was carried out as described above
for solvolysis in acetic acid. Data analysis was identical to that described
above for the clavaminate synthase reaction.
Determination of the â-2° Kinetic Isotope Effect in the Photo-
chemically Initiated Reduction of Benzyl [2-3H,3′-2H2]-2-(4′-Chloro-
2′-oxoazetidin-1′-yl)acetate 45 and Benzyl [2-14C]-2-(4′-Chloro-2′-
oxoazetidin-1′-yl)acetate 43. A dry Wheaton glass mini-vial equipped
with a dry spin vane and rubber septum was charged with 260-310
Acknowledgment. We are grateful to the National Institutes
of Health for financial support of this work (AI14937).
JA992649D