Design and synthesis of an isopenicillin N synthase mimic

Article abstract of DOI:10.1016/j.tet.2004.10.041

Towards the aim of creating a functional mimic of isopenicillin N synthase, a small molecule designed to coordinate around iron(II) and model the enzyme active site has been prepared in nine synthetic steps from 2,6- bis(hydroxymethyl)pyridine, (S)-(+)-ma

Full text of DOI:10.1016/j.tet.2004.10.041

Tetrahedron 61 (2005) 137–143
Design and synthesis of an isopenicillin N synthase mimic
†
Jordan A. Krall, Peter J. Rutledge and Jack E. Baldwin
‡
*
Chemistry Research Laboratory, University of Oxford, Mansfield Road, Oxford OX1 3TA, UK
Received 30 July 2004; revised 20 September 2004; accepted 14 October 2004
Available online 5 November 2004
Abstract—Towards the aim of creating a functional mimic of isopenicillin N synthase, a small molecule designed to coordinate around
iron(II) and model the enzyme active site has been prepared in nine synthetic steps from 2,6-bis(hydroxymethyl)pyridine, (S)-(C)-mandelic
acid and pivaldehyde. One aspartate, two histidines and a water ligand in the natural enzyme are replaced by an a-hydroxy acid, pyridine and
aniline in the model compound. Additionally, a free thiol designed to simulate the enzyme substrate, d-(L-a-aminoadipoyl)-L-cysteinyl-D-
valine, is linked to the ligand by a three carbon chain. We postulate that in the presence of molecular oxygen, the complex formed between
this synthetic ligand and iron(II) will display oxidative chemistry similar to that observed in the active site of isopenicillin N synthase.
q 2004 Elsevier Ltd. All rights reserved.
1. Introduction
crystallographic,4–6 and substrate-analogue turnover
studies7,8 have led to the proposal of a detailed mechanism
for the enzymatic transformation (Fig. 2A).
Isopenicillin N synthase (IPNS) is a non-heme iron(II)-
dependent oxidase that catalyzes the conversion of d-(L-a-
aminoadipoyl)-L-cysteinyl-D-valine (ACV) 1 to isopenicil-
lin N (IPN) 2, the precursor of all penicillin and
cephalosporin antibiotics (Fig. 1).1,2 IPNS uses the full
oxidizing power of molecular oxygen to perform this
remarkable bicyclization, producing 2 equiv of water over
the course of the reaction. Comprehensive spectroscopic,3
Despite the development of a broad understanding of the
IPNS active site, efforts to synthesize an iron-centered
complex that mimics the activity of the enzyme have been
unsuccessful.9 Functional enzyme mimics can be important
tools for advancing mechanistic knowledge as well as for
testing existing hypotheses. Such mimics have recently been
developed for several non-heme iron(II) oxidases including
methane monooxygenase,10 the extradiol-cleaving catechol
dioxygenases11,12 and the a-keto acid-dependent
enzymes.13 A detailed discussion of this topic appears in
a recent review of the mechanisms and mimics of dioxy-
genase enzymes.14
The failure to create an equivalent IPNS mimic can be
attributed primarily to the complexity of the reaction that
this enzyme catalyzes. A major energetic hurdle must be
overcome to hold an ACV-based substrate in the confor-
mation required for the formation of such a strained system.
When ACV is bound in the IPNS active site, multiple
contacts between enzyme and substrate stabilize this
conformation and promote formation of the bicyclic
b-lactam product.5 These stabilizing interactions cannot be
easily replicated in a small model system, and efforts to
mimic the entire reaction cycle of IPNS have consequently
met with little success.9
Figure 1. ACV 1 is converted to IPN 2 by IPNS in the presence of
molecular oxygen.
Keywords: Enzyme mimic; Penicillin; b-Lactam; Isopenicillin N; IPNS.
* Corresponding author. Tel.: C44 1865 275670; fax: C44 1865 285002;
e-mail: jack.baldwin@chem.ox.ac.uk
A more achievable goal is the creation of a model system
that mimics the early steps of the IPNS oxidation with-
out requiring the system to duplicate the high-energy
ring-closures. Recent studies with IPNS and the modified
† Current address: Department of Chemistry and Chemical Biology,
Harvard University, 12 Oxford Street, Cambridge, MA 02138, USA.
‡ Current address: Centre for Synthesis and Chemical Biology, Department
of Chemistry, University College Dublin, Belfield, Dublin 4, Ireland.
0040–4020/$ - see front matter q 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2004.10.041

138
J. A. Krall et al. / Tetrahedron 61 (2005) 137–143
Figure 2. The reaction cycle of IPNS with ACV (A) and ACOV (B). With both substrates, it is proposed that initial hydrogen abstraction by an iron-bound
superoxide results in the formation of a thioaldehyde bound to iron through the sulfur atom. With ACV, the valinyl amide nitrogen atom then attacks the
thioaldehyde to form a b-lactam. In the reaction with ACOV, which lacks this internal nucleophile, the hydroperoxide formed after initial hydrogen abstraction
itself attacks the thioaldehyde, and the resulting hydroxyl group is ultimately oxidized to give a thiocarboxylate.
substrate d-(L-a-aminoadipoyl)-L-cysteine D-a-hydroxyiso-
valeryl ester (ACOV) provide support for a mechanism
involving initial hydrogen abstraction by an iron(III)-bound
superoxide species to give a thioaldehyde and an iron(II)-
hydroperoxide (Fig. 2B).15 In the absence of the substrate-
derived nucleophile that is generated in the reaction of ACV,
the hydroperoxide itself attacks the thioaldehyde, and the
cysteine residue is ultimately oxidized to a thiocarboxylate.
2. Results and discussion
Towards the goal of constructing an IPNS mimic, we
designed a complex small molecule 3 to simulate the iron-
coordinating ligands in the active site of the natural enzyme.
The iron center in ACV-bound IPNS is coordinated by one
aspartate (Asp216) and two histidine (His214 and His270
)
residues from the protein in a facial triad arrangement,4 a
motif that is conserved throughout the non-heme mono-
nuclear iron(II) oxidases (Fig. 4).16 A well-ordered water
molecule is bound to iron opposite His214, and ACV binds
via the cysteinyl sulfur trans to His270, giving a square
pyramidal geometry around the metal center. Dioxygen is
believed to initiate catalysis upon binding to iron in the site
opposite Asp216, creating an octahedral geometry that is
maintained throughout the reaction cycle.4,5
We now report the synthesis of a pyridine-based ligand 3
designed to form a pentadentate complex 4 with iron(II)
(Fig. 3). We postulate that in the presence of iron(II), the
complex formed should display oxidative chemistry similar
to that seen in the early stages of the IPNS reaction cycle
(Scheme 1).
Based on the requirements of this system, we designed the
ligand 3 to form a pentadentate complex 4 with iron(II)
while leaving a coordination site vacant for dioxygen
binding (Fig. 3). The framework of this structure is based
on a cobalt complex designed by Chin et al.17 to stereo-
specifically bind amino acids. In our complex, pyridine and
aniline ligands take the place of the histidine residues of
IPNS, while an a-hydroxy acid moiety models both the
Figure 3. Ligand 3 and its complex 4 with iron(II), a proposed mimic for
the IPNS active site.
Figure 4. The active site of IPNS. The iron(II) center is coordinated by
three endogenous protein ligands (His214, Asp216, and His270), a water
molecule, and the thiol sulfur of ACV in a square pyramidal arrangement.
Molecular oxygen binds opposite Asp216, giving the iron center an
octahedral geometry.
Scheme 1. The proposed biomimetic oxidation of the IPNS enzyme mimic
4 upon exposure to molecular oxygen. We propose that the carbon adjacent
to the iron-bound sulfur will be oxidized to a thiocarboxylate in the
resulting complex 18.

J. A. Krall et al. / Tetrahedron 61 (2005) 137–143
139
carboxylate and water ligands. The design includes a thiol
attached to the aniline nitrogen by a three-carbon linker. The
sulfur atom was included to simulate binding of ACV, and it
is at the adjacent carbon that we hope ultimately to achieve
oxidation (Scheme 1).
triphenylphosphine and carbon tetrabromide, giving the
bromide 9 in 81% yield.
At this stage, the a-hydroxy acid moiety was introduced
stereoselectively in the masked form of a [1,3]-dioxolanone.
Seebach and co-workers have shown that cis-2-tert-butyl-5-
substituted-[1,3]-dioxolanones can be stereospecifically
alkylated at the 5-position.19,20 They have also demon-
strated that these cis-2,5-disubstituted dioxolanones can be
synthesized from pivaldehyde and enantiopure a-substi-
tuted-a-hydroxy acids in high diastereomeric purity.
Hydrolysis under either acidic or basic conditions cleaves
the pivaldehyde moiety, leaving an a-disubstituted-a-
hydroxy acid as a single enantiomer.
An important factor to be considered in the design of a
ligand of this nature is the potential for intermolecular side
reactions of the iron complex. Of particular concern is the
propensity of free thiols and ferrous iron to undergo
competing oxidation reactions to give disulfides and
Fe(III)–O–Fe(III) species respectively, in the presence of
atmospheric oxygen. Two phenyl groups are therefore
included in the structure in order to increase steric bulk and
inhibit intermolecular interactions that might render the
complex inactive.
In order to introduce the phenyl group required to bring the
desired steric bulk to the exterior of the mimic, a cis-2,5-
disubstituted dioxolanone was synthesized from pivalde-
hyde 10 and (S)-(C)-mandelic acid 11 (Scheme 4). In the
presence of catalytic triflic acid, these two reagents were
refluxed in pentane with azeotropic removal of water to give
the desired dioxolanone 12,21 which was isolated in
diastereomerically pure form and 81% yield after a single
recrystallization. Deprotonation of the dioxolanone 12 with
LDA followed by addition to the mono-protected bromide 9
gave the 2,5,5-trisubstituted dioxolanone 13 in 84% yield
(Scheme 5). The TBS group was then removed with tetra-n-
butylammonium fluoride in high yield (93%), and the
resulting alcohol 14 was brominated with triphenylphos-
phine and carbon tetrabromide to give the brominated
dioxolanone 15 as a crystalline solid. The stereochemistry
of all dioxolanone products was determined by nOe
spectroscopy, and the solution of an X-ray crystal structure
for the brominated dioxolanone 15 further confirmed that
the (2S,5S)-dioxolanone system had been formed as
anticipated (data not shown).
Initial efforts to synthesize the ligand were based on the
approach taken by Chin et al., who reacted 2,6-bis(bromo-
methyl)pyridine 5 with the enolate of 2-(N-acetylamino)-
diethylmalonate followed by dimethylamine to give a
2,6-hetero-disubstituted pyridine.17 However, this approach
did not prove effective in our hands, and treatment of
2,6-bis(bromomethyl)pyridine 5 with 1 equiv of an appro-
priate nucleophile resulted in a 1:1 mixture of homo-
disubstituted product 6 and starting material (Scheme 2).
This is not surprising as the mono-brominated species is
highly reactive. 2-Bromomethylpyridine was found to be
stable only as an HBr salt, forming polymers at pH 7 (data
not shown). Conversely, 2,6-bis(bromomethyl)pyridine 5 is
stable at neutral pH and can be stored indefinitely in its
deprotonated form.
Scheme 2. Reaction of 2,6-bis(bromomethyl)pyridine 5 with 1 equiv of
nucleophile to give a 1:1 ratio of the 2,6-homo-disubstituted pyridine 6 and
the starting material.
Scheme 4. Conditions: triflic acid (cat), pentane, reflux, 5 h, 81%.
Having prepared the bromide 15, the aniline and thiol
functionalities could be introduced. Deprotonation of
N-allylaniline with n-butyllithium in the presence of N,
N0-dimethyl-N, N0-propylene urea (DMPU), followed by
addition to the bromide 15, led to formation of the
unsaturated amine 16 in 94% yield (Scheme 5). The sulfur
functionality was then incorporated by radical addition of
thiolacetic acid to the alkene 16. To this end, a solution of
the unsaturated compound 16 and thiolacetic acid in toluene
was irradiated at 254 nm, and conversion to the thioester 17
proceeded in 56% yield.
In order to overcome this problem, the synthesis was
modified to begin with 2,6-bis(hydroxymethyl)pyridine 7
(Scheme 3). The diol 7 was treated with 1 equiv of sodium
hydride, and the resulting mono-anion reacted with 1 equiv
of tert-butyldimethylsilyl chloride (TBS-Cl) to give the
singly-protected diol 8 in moderate yield.18 Mono-bromina-
tion of the protected diol proceeded smoothly with
The thioester 17 represents a protected form of the target
molecule 3, as unmasking of the thiol and the a-hydroxy
acid by hydrolysis leads to the pentadentate ligand 3. While
it was originally thought that acid-catalyzed hydrolysis
would be more effective in minimizing disulfide formation,
Scheme 3. Conditions: i. NaH, then TBS-Cl, DCM, RT, 5 h, 54%; ii. CBr4,
PPh3, Et2O, RT, 1.5 h, 81%.

140
J. A. Krall et al. / Tetrahedron 61 (2005) 137–143
Scheme 5. Conditions: i. LDA-12 (premixed), THF, K78 8C, 6 h, 84%; ii. TBAF, THF, 0 8C/RT; 1 h, 93%; iii. CBr4, PPh3, DCM, 0 8C/RT, 1.5 h, 81%;
iv. nBuLi-N-allylaniline (premixed), THF, DMPU, K78 8C, 4 h, 90%; v. AcSH, toluene, hv, 4 h, 56%; vi. LiOH, H2O–THF, reflux, 14 h.
the deprotection proceeded more smoothly when run under
basic conditions using dilute lithium hydroxide in tetra-
hydrofuran-water. Mass spectrometric analysis of the crude
product provides strong evidence that the pentadentate
ligand 3 has been formed (HRMS: m/z foundZ423.1737,
required for 3Z423.1742). However, separation of ligand 3
from reaction by-products was problematic, primarily due to
poor solubility properties of the product. As a result, the
NMR spectrum of the crude material is complicated by
residual impurities (identified by HRMS as the starting
material 17 and a partially deprotected product in which
only the thioester has been hydrolyzed), precluding further
characterization of the product by this method. Nonetheless,
it is apparent from the HRMS data that the putative iron
ligand 3 has indeed been formed.
was recrystallized from heptane. Anhydrous dichloromethane
(DCM) was obtained directly before use by refluxing over
calcium hydride under an atmosphere of nitrogen, followed
by distillation under those conditions. Anhydrous tetra-
hydrofuran (THF) and diethyl ether were similarly obtained
by distillation from sodium/benzophenone ketyl. Hexane
and N, N0-dimethyl-N, N0-propylene urea (DMPU) were
dried with calcium hydride, fractionally distilled, and stored
˚
over 4.0 A molecular sieves. Diisopropyl amine was
similarly dried and distilled, and was stored over potassium
hydroxide pellets. ‘Petroleum ether’ refers to the fraction of
light petroleum boiling between 30 and 40 8C and was
distilled prior to use. Concentrations of lithium diisopropyl-
amide and n-butyllithium were determined by titration
against 1,3-diphenylacetone-p-tosyl-hydrazone.
Melting points (mp) were measured using a Cambridge
Instruments Gallene III Melting Point Microscope. Optical
rotations ([a]D) were recorded on a Perkin–Elmer 241
polarimeter at 589 nm. Low-resolution mass spectra were
recorded using a Micromass Platform spectrometer, and
high-resolution mass spectra (HRMS) were recorded on
Micromass Autospec, GCT, and LCT spectrometers.
Infrared spectra were recorded on a Perkin–Elmer Paragon
1000 Fourier Transform spectrometer. Nuclear magnetic
resonance spectra were recorded on Bru¨ker DPX200 and
Bru¨ker DPX400 spectrometers, and nOe spectroscopy was
performed on a Bru¨ker DRX500 spectrometer. Elemental
analysis was performed by Elemental Microanalysis
Limited (Okehampton). Single-crystal X-ray diffraction
data were measured using an Enraf-Nonius KappaCCD
diffractometer (graphite-monochromated Mo Ka radiation,
3. Conclusion
We report the synthesis of a pentadentate ligand 3 to be used
in the construction of a functional mimic of the key
biosynthetic enzyme IPNS. Compound 3 has been prepared
in nine steps from 2,6-bis(hydroxymethyl)pyridine, (S)-(C)-
mandelic acid and pivaldehyde. We propose that the ligand
3 will bind to iron(II) and that in the presence of oxygen, the
resulting complex may function as the first small molecule
mimic of IPNS (Scheme 1). Further work is required to test
this hypothesis.
4. Experimental
4.1. General
˚
lZ0.71073 A) and processed using the DENZO-SMN
package.22
Reactions were carried out under an atmosphere of argon.
All reagents and solvents were used as received from
manufacturers unless otherwise specified. Trimethylacetal-
dehyde (pivaldehyde) and N-allylaniline were purified by
4.1.1. 2-Hydroxymethyl-6-(tert-butyldimethylsilyloxy-
methyl)pyridine (8).18 Sodium hydride (880 mg of a 60%
dispersion in oil, 21.9 mmol) was washed with petroleum
ether (3!25 mL), and residual solvent was removed under
¨
Kugelrohr distillationat reducedpressure. Triphenylphosphine

J. A. Krall et al. / Tetrahedron 61 (2005) 137–143
141
reduced pressure. The resulting powder was added to a
slurry of 2,6-bis(hydroxymethyl)pyridine 7 (3.01 g,
21.6 mmol) in DCM (25 mL), and this mixture was stirred
at room temperature. After 45 min, a solution of tert-
butyldimethylsilyl chloride (3.30 g, 21.9 mmol) in DCM
(5 mL) was added to the reaction mixture, which was stirred
at room temperature for 5 h. The mixture was diluted with
DCM (250 mL) to dissolve all the solid material, and this
solution was washed with saturated aqueous sodium
bicarbonate (2!125 mL) and saturated brine (125 mL),
and dried with magnesium sulfate. The solvent was
evaporated under reduced pressure to give the crude product
as an orange oil which was purified by column chroma-
tography (petroleum ether/diethyl ether, 1:1) to give the
mono-protected diol 8 as a yellow oil (2.96 g, 54%); Rf 0.25
(petroleum ether/diethyl ether, 1:1); nmax (thin film): 3276
(m, br O–H str), 2955, 2932, 2886, 2857 (s, aliphatic C–H
str), 1598, 1580, 1462 (m, ring str), 1257 (s), 1118, 838 (s,
Si–O str); dH (400 MHz, CDCl3): 0.14 (6H, s, Si(CH3)2),
0.97 (9H, s, SiC(CH3)3), 3.82 (1H, t, JZ5.0 Hz, CH2OH),
4.74 (2H, d, JZ5.0 Hz, CH2OH), 4.84 (2H, s, CH2OSi),
7.10 (1H, d, JZ7.5 Hz, one of CH(Py)3,5), 7.42 (1H, d,
JZ7.5 Hz, one of CH(Py)3,5), 7.71 (1H, t, JZ7.5 Hz,
CH(Py)4); dC (100.6 MHz, CDCl3): K5.37 (Si(CH3)2),
18.35 (SiC(CH3)3), 25.89 (SiC(CH3)3), 63.81 (CH2OH),
65.90 (CH2OSi), 118.40, 118.53 (2!CH(Py)3,5), 137.27
(CH(Py)4), 157.60, 160.30 (2!C(Py)2,6); m/z (APC): 254
(100%, [MH]C); HRMS: found [MH]CZ254.1577,
C13H24NO2Si requires 254.1576.
slurry of (S)-(C) mandelic acid 11 (3.92 g, 25.8 mmol) in
pentane (45 mL). The mixture was stirred at room
temperature while trifluoromethanesulfonic acid (100 mL,
1.1 mmol) was added dropwise, and then heated to reflux for
5 h under Dean-Stark conditions. The reaction mixture was
cooled to room temperature and neutralized with aqueous
sodium bicarbonate (8%, w/v) and then with solid sodium
bicarbonate. During this process, a white precipitate formed
from what had become an orange solution. The pentane was
removed under reduced pressure, and the white solid was
isolated from the aqueous suspension by vacuum filtration.
Two recrystallizations of the solid (ethyl acetate–heptane)
gave the dioxolanone 12 as large white needles (4.54 g,
81%); mp 138–140 8C; Rf 0.20 (petroleum ether/diethyl
ether, 20:1); [a]2D5 C90.0 (chloroform, cZ0.1; lit. [a]2D5
C
88.7,19 chloroform, cZ1.2); nmax (KBr disk): 3070, 3039
(w, aromatic C–H str), 2980, 2960, 2922, 2873 (m, aliphatic
C–H str), 1799 (s, C]O str), 1483, 1457 (m, ring, str), 1206
(s), 1183 (s, C–O–C str); dH (400 MHz, CDCl3): 1.10 (9H, s,
C(CH3)3), 5.25 (1H, d, JZ1.5 Hz, PhCH), 5.34 (1H, d, JZ
1.5 Hz, (CH3)3CCH), 7.38–7.49 (5H, m, 2!CH (Ph)o, 2!
CH(Ph)m, CH(Ph)p); dC (100.6 MHz, CDCl3): 23.63
(C(CH3)3), 34.48 (C(CH3)3), 77.02 (PhCHC]O), 109.32
((CH3)3CCH), 127.06, 128.74 (2!CH(Ph)o, 2!CH(Ph)m),
129.19 (CH(Ph)p) 133.51 (C(Ph)i), 171.86 (C]O); m/z
(TOFC): 238 (100%, [MCNH4]C), 221 (20%, [MH]C),
176 (18%), 152 (25%); HRMS: found [MCNH4]CZ
238.1444, C13H20NO3 requires 238.1443; found C
70.67%, H 7.34%, C13H16O3 requires C 70.89%, H 7.32%.
4.1.2. 2-Bromomethyl-6-(tert-butyldimethylsilyloxy-
methyl)pyridine (9). Carbon tetrabromide (6.70 g,
20.2 mmol) was added as a solid to a solution of the
mono-protected diol 8 (4.70 g, 18.6 mmol) in diethyl ether
(20 mL) and the resulting mixture was stirred at room
temperature. A solution of triphenylphosphine (5.25 g,
20.0 mmol) in diethyl ether (10 mL) was added, and the
reaction mixture was stirred for a further 90 min at room
temperature, during which time a white precipitate formed.
The precipitate (triphenylphosphine oxide) was removed by
vacuum filtration, and the filtrate was concentrated under
reduced pressure to give a crude orange oil. The product was
purified by column chromatography (petroleum ether/
diethyl ether, 20:1) to give the bromide 9 as a pale yellow
oil (4.75 g, 81%); Rf 0.20 (petroleum ether/diethyl ether,
19:1); nmax (thin film): 3063 (w, aromatic C–H str), 2955,
2929, 2886, 2856 (m-s, aliphatic C–H str), 1592, 1578,
1460, 1431 (m, ring str),1257 (s), 1123, 838 (s, Si–O str); dH
(400 MHz, CDCl3): 0.13 (6H, s, Si(CH3)2), 0.97 (9H, s,
SiC(CH3)3), 4.52 (2H, s, CH2Br), 4.84 (2H, s, CH2OSi),
7.31 (1H, d, JZ7.5 Hz, one of CH(Py)3,5), 7.46 (1H, d,
JZ7.5 Hz, one of CH(Py)3,5), 7.72 (1H, d, JZ7.5 Hz,
CH(Py)4); dC (100.6 MHz, CDCl3): K5.39 (Si(CH3)2),
18.33 (SiC(CH3)3), 25.89 (SiC(CH3)3), 33.85 (CH2Br),
65.87 (CH2OSi), 119.25, 121.50 (2!CH(Py)3,5), 137.64
(CH(Py)4), 155.56, 161.50 (2!C(Py)2,6); m/z (APC): 318
(80%, [MH]C for 81Br), 316 (100%, [MH]C for 79Br), 268
(82%), 237 (21%, [MH–Br]C), 202 (41%, [MH–
SiC(CH3)3]C for 79Br), 154 (42%); HRMS: found
4.1.4. (2S,5S)-2-tert-Butyl-5-[6-(tert-butyldimethylsilyl-
oxymethyl)pyridin-2-ylmethyl]-5-phenyl-[1,3]-dioxolan-
4-one (13). A solution of the dioxolanone 12 (1.11 g,
5.0 mmol) in THF (25 mL) was cooled to K78 8C and a
solution of lithium diisopropylamide (1.63 M solution in
THF, 3.10 mL, 5.0 mmol) was added. The solution was
stirred at K78 8C for 50 min, then added dropwise via
cannula to a solution of 2-bromomethyl-6-(tert-butyldi-
methylsilyloxymethyl)pyridine 9 (1.59 g, 5.0 mmol) in
THF (25 mL) at K78 8C. The reaction mixture was stirred
at K78 8C for an additional 6 h. It was then poured into half-
saturated aqueous ammonium chloride (40 mL), and the
resulting solution was extracted with diethyl ether (4!
50 mL). The organic phase was dried with magnesium
sulfate, and the solvent was removed under reduced pressure
to give a peach-colored oil. The crude material was purified
by column chromatography (petroleum ether/diethyl ether,
15:2) to yield the trisubstituted dioxolanone 13 as an off-
white crystalline solid (1.93 g, 84%); mp 65–67 8C; Rf 0.15
(petroleum ether/diethyl ether, 8:1); [a]2D5 K37.3 (chloro-
form, cZ0.9); nmax (KBr disc): 3054, 3037 (w, aromatic
C–H str), 2955, 2902, 2855 (m, aliphatic C–H str), 1787 (s,
C]O str), 1578, 1549, 1483, 1460 (m, ring str), 1257 (m),
1192 (s, C–O–C str); dH (400 MHz, CDCl3): 0.13 (6H, s,
Si(CH3)2), 0.86 (9H, s, HCC(CH3)3), 0.97 (9H, s,
SiC(CH3)3), 3.33 (1H, d, JZ14.5 Hz, one of PhCCH2Py),
3.65 (1H, d, JZ14.5 Hz, one of PhCCH2Py), 4.69 (1H, s,
HCC(CH3)3), 4.81 (1H, d, JZ15.5 Hz, one of PyCH2OSi),
4.84 (1H, d, JZ15.5 Hz, one of PyCH2OSi), 6.99 (1H, d,
JZ7.5 Hz, one of CH(Py)3,5), 7.30–7.35 (1H, m, CH(Ph)p),
7.36–7.45 (3H, m, 2!CH(Ph)o, one of CH(Py)3,5), 7.63
(1H, t, JZ7.5 Hz, CH(Py)4), 7.74–7.78 (2H, m, 2!
CH(Ph)m); dC (100.6 MHz, CDCl3): K5.37 (Si(CH3)2),
[MH]CZ316.0729, C13H BrNOSi requires 316.0732.
79
23
4.1.3. (2S,5S)-2-tert-Butyl-5-phenyl-[1,3]-dioxolan-4-one
(12).21 Pivaldehyde 10 (2.65 g, 30.8 mmol) was added to a

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J. A. Krall et al. / Tetrahedron 61 (2005) 137–143
18.36 (SiC(CH3)3), 23.52 (HCC(CH3)3), 25.92
(SiC(CH3)3), 34.86 (HCC(CH3)3), 48.49 (PyCH2CPh),
65.82 (PyCH2OSi), 82.20 (PhCCH2Py), 109.57
(HCC(CH3)3), 118.20, 122.31 (2!CH(Py)3,5), 124.91
(2!CH(Ph)-m), 127.92 (CH(Ph)p), 128.24 (2!CH(Ph)o),
137.06 (CH(Py)4), 139.18 (C(Ph)i), 154.11, 161.17 (2!
C(Py)2,6), 173.28 (C]O); m/z (ESC): 456 (100%, [MH]C);
HRMS: found [MH]CZ456.2572, C26H38NO4Si requires
456.2570; found C 68.50%, H 8.23%, N 3.19%,
C26H37NO4Si requires C 68.35%, H 8.18%, N 3.07%.
3068 (w, aromatic C–H str), 2970, 2905, 2872 (m, aliphatic
C–H str), 1794 (s, C]O str), 1597, 1574, 1482, 1459 (m,
ring str),1210 (m), 1172 (m, C–O–C str); dH (400 MHz,
CDCl3): 0.89 (9H, s, HCC(CH3)3), 3.39 (1H, d, JZ15.0 Hz,
one of PyCH2CPh), 3.66 (1H, d, JZ15.0 Hz, one of
PyCH2CPh), 4.55 (2H, s, PyCH2Br), 5.03 (1H, s,
HCC(CH3)3), 7.04 (1H, d, JZ7.5 Hz, one of CH(Py)3,5),
7.31–7.36 (2H, m, CH(Ph)p) and one of CH(Py)3,5), 7.37–
7.43 (2H, m, 2!CH(Ph)m), 7.62 (1H, t, JZ7.5 Hz,
CH(Py)4), 7.75–7.79 (2H, m, 2!CH(Ph)o); dC
(100.6 MHz, CDCl3): 23.58 (HCC(CH3)3), 33.71 (PyCH2-
Br), 34.96 (HCC(CH3)3), 48.41 (PyCH2CPh), 81.95
(PyCH2CPh), 109.72 (HCC(CH3)3), 121.97, 123.54 (2!
CH(Py)3,5), 124.91 (2!CH(Ph)o), 127.98 (CH(Ph)p),
128.29 (2!CH(Ph)m), 137.55 (CH(Py)4), 139.29 (C(Ph)i),
155.48, 156.39 (2!C(Py)2,6), 173.28 (C]O); m/z (ESC):
4.1.5. (2S,5S)-2-tert-Butyl-5-(6-hydroxymethylpyridin-2-
ylmethyl)-5-phenyl-[1,3]-dioxolan-4-one (14). A solution
of the protected dioxolanone 13 (397 mg, 0.87 mmol) in
THF (8 mL) was cooled to 0 8C, and TBAF (1.0 M solution
in THF, 1.75 mL, 1.75 mmol) was added. The solution was
stirred at 0 8C for 5 min and at room temperature for 70 min.
It was then poured into water (10 mL) and extracted with
DCM (3!20 mL). The organic layer was washed with
saturated brine (10 mL), dried with magnesium sulfate, and
the solvent was removed under reduced pressure to give a
runny yellow oil. The crude product was purified by column
chromatography (petroleum ether/diethyl ether, 1:1) to give
the alcohol 14 (275 mg, 93%) as a viscous pale yellow oil;
Rf 0.20 (petroleum ether/diethyl ether, 1:1); [a]2D5 K57.8
(chloroform, cZ0.8); nmax (thin film): 3422 (s, br, O–H str),
3064, 3029 (m, aromatic C–H str), 2974, 2935, 2908, 2874
(s, aliphatic C–H str), 1790 (s, C]O str), 1594, 1578, 1483,
1459 (s, ring str),1178 (s, C–O–C str); dH (400 MHz,
CDCl3): 0.88 (9H, s, HCC(CH3)3), 3.39 (1H, d, JZ14.5 Hz,
one of PhCCH2Py), 3.66–3.72 (2H, m, one of PhCCH2Py
and CH2OH), 4.73 (2H, d, JZ4.5 Hz, PyCH2OH), 4.85 (1H,
s, CHC(CH3)3), 7.01 (1H, d, JZ7.5 Hz, one of CH(Py)3,5),
7.11 (1H, d, JZ7.5 Hz, one of CH(Py)3,5), 7.31–7.36
(1H, m, CH(Ph)p), 7.37–7.42 (2H, m, CH(Ph)m), 7.59 (1H, t,
JZ7.5 Hz, CH(Py)4), 7.71–7.75 (2H, m, CH(Ph)o); dC
(100.6 MHz, CDCl3): 23.49 (HCC(CH3)3), 34.88
(HCC(CH3)3), 47.81 (PyCH2CPh), 63.98 (PyCH2OH),
81.91 (PhCCH2Py), 109.44 (HCC(CH3)3), 118.81, 122.91
(2!CH(Py)3,5), 124.94 (2!CH(Ph)o), 128.03 (CH(Ph)p),
128.26 (2!CH(Ph)m), 137.10 (CH(Py)4), 128.74 (C(Ph)i),
154.28, 158.49 (2!C(Py)2,6), 173.24 (C]O); m/z (ESC):
364 (25%, [MCNa]C), 342 (100%, [MH]C); HRMS: found
[MH]CZ342.1696, C20H24NO4 requires 342.1705.
406 (100%, [MH]C for 81Br), 404 (95%, [MH]C for 79Br);
79
23
HRMS: found [M]CZ403.0787, C20H BrNO3 requires
403.0783.
4.1.7. (2S,5S)-5-{6-[(N-Allyl-N-phenylamino)methyl]-
pyridin-2-ylmethyl}-2-tert-butyl-5-phenyl-[1,3]-dioxo-
lan-4-one (16). N-Allylaniline (442 mg, 3.32 mmol) in THF
(25 mL) was cooled to 0 8C, and nBuLi (2.26 M in hexanes,
1.52 mL, 3.44 mmol) was added. The solution was stirred
for 30 min, after which DMPU (445 mg, 3.47 mmol) was
added. Stirring was continued at 0 8C for another 15 min,
before the lithium amide solution thus formed was added
dropwise via cannula to a solution of the bromide 15
(1.39 g, 3.4 mmol) in THF (25 mL) at K78 8C. The
resulting solution was stirred at K78 8C for 4 h and
then quenched by pouring into half-saturated ammonium
chloride (40 mL). This mixture was extracted with diethyl
ether (3!100 mL), and the organic phase was washed
with brine (100 mL) and dried with magnesium sulfate.
The solvent was removed under reduced pressure to give a
yellow oil, which was purified by column chromatography
(petroleum ether/diethyl ether, 6:1), giving the alkene 16
(1.43 g, 94%) as a viscous, colorless oil; Rf 0.10 (petroleum
ether/diethyl ether, 8:1); [a]2D5 K44.5 (chloroform, cZ1.0);
nmax (thin film): 3062, 3040 (w, aromatic C–H str), 2962,
2934, 2907, 2836 (m, aliphatic C–H str), 2361 (w, overtone
of C–O–C str), 1792 (s, C]O str), 1643 (w, C]C str),
1599, 1576, 1506, 1482 (m-s, ring str), 1178 (s, br, C–O–C
str); dH (400 MHz, CDCl3): 0.90 (9H, s, C(CH3)3), 3.40 (1H,
d, JZ14.5 Hz, 1 of PyCH2C), 3.72 (1H, d, JZ14.5 Hz, 1 of
4.1.6. (2S,5S)-5-(6-Bromomethylpyridin-2-ylmethyl)-2-
tert-butyl-5-phenyl-[1,3]-dioxolan-4-one (15). A solution
of the alcohol 14 (1.45 g, 4.2 mmol) in DCM (30 mL) was
cooled to 0 8C, and a solution of carbon tetrabromide
(1.54 g, 4.6 mmol) in DCM (5 mL) was added to it via
cannula. A solution of triphenylphosphine (1.23 g,
4.7 mmol) in DCM was then added via cannula, and the
resulting yellow solution was stirred at 0 8C for 5 min, and
then at room temperature for 90 min. The reaction mixture
was diluted with diethyl ether (300 mL), causing the
precipitation of triphenylphosphine oxide. The white
precipitate was removed by vacuum filtration, and the
solvent was evaporated under reduced pressure to give a
thick yellow oil. This crude material was purified by column
chromatography (petroleum ether/diethyl ether, 9:2) to give
the bromide 15 (1.39 g, 81%) as a white crystalline solid;
mp 101–102 8C; Rf 0.20 (petroleum ether/diethyl ether,
4:1); [a]2D5 K58.4 (chloroform, cZ0.9); nmax (KBr disc):
PyCH2C), 4.11 (2H, ddd, X of ABMX, JXMZ5.0 Hz, JXA
Z
1.5 Hz, JXBZ1.5 Hz, NCH2CHCH2), 4.63 (2H, s, PyCH2N),
4.80 (1H, s, (CH3)3CCH), 5.22 (1H, ddt, B of ABMX,
JBMZ10.0 Hz, JABZ1.5 Hz, JBXZ1.5 Hz, Htrans of NCH2-
CHCH2), 5.25 (1H, ddt, A of ABMX, JAMZ17.0 Hz, JAB
Z
1.5 Hz, JAXZ1.5 Hz, Hcis of NCH2CHCH2), 5.94 (1H, ddt,
M of ABMX, JAMZ17.0 Hz, JBMZ10.0 Hz, JMXZ5.0 Hz,
NCH2CHCH2), 6.65–6.75 (3H, m, 2!CH(Ph–N)o, CH(Ph–
N)p), 7.01 (1H, d, JZ7.5 Hz, one of CH(Py)3,5), 7.14 (1H, d,
JZ7.5 Hz, one of CH(Py)3,5), 7.16–7.23 (2H, m, 2!
CH(Ph–N)m), 7.32–7.38 (1H, m, CH(Ph–C)p), 7.39–7.45
(2H, m, 2!CH(Ph–C)m), 7.53 (1H, t, JZ7.5 Hz, CH(Py)4),
7.77–7.81 (2H, m, 2!CH(Ph–C)o); dC (100.6 MHz,
CDCl3): 23.57 (C(CH3)3), 34.88 (C(CH3)3), 48.41
(PyCH2C), 53.83 (NCH2CHCH2), 56.19 (PyCH2N), 82.17
(PhCCH2Py), 109.60 ((CH3)3CCH), 112.28 (2!CH(Ph–
N)o), 116.65 (NCH2CHCH2), 116.70 (CH(Ph–N)p), 119.11,

J. A. Krall et al. / Tetrahedron 61 (2005) 137–143
143
122.50 (2!CH(Py)3,5), 124.96 (2!CH(Ph–C)o), 127.98
Acknowledgements
(CH(Ph–C)p), 128.27 (2!CH(Ph–C)m), 129.18 (2!
CH(Ph–N)m), 133.39 (NCH2CHCH2), 137.15 (CH(Py)4),
139.13 (C(Ph–C)i), 148.38 (C(Ph–N)i), 155.22, 159.20 (2!
C(Py)2,6), 173.24 (C]O); m/z (APC): 479 (15%, [MC
Na]C); 457 (100%, [MH]C); HRMS: found [MH]CZ
457.2497, C29H33N2O3 requires 457.2491.
We would like to thank the Rhodes Trust for funding J.A.K.
We would also like to thank Dr. B. Odell for nOe
measurements, Dr. A. Cowley and Dr. D. Watkins for
X-ray crystallography, Dr. N. Oldham, Mr R. Procter and
Dr. J. Kirkpatrick for mass spectrometry, and Dr. R.
Adlington for helpful discussions.
4.1.8. Thiolacetic acid S-(3-{[6-((2S,4S)-2-tert-butyl-5-
oxo-4-phenyl-[1,3]-dioxolan-4-ylmethyl)pyridin-2-yl-
methyl]phenylamino}propyl) ester (17). To a solution of
the alkene 16 (248 mg, 0.54 mmol) in toluene (1.25 mL) in
a quartz flask was added thiolacetic acid (130 mg,
1.7 mmol). Argon was bubbled through the solution for
20 min to remove dissolved oxygen. The flask was then
sealed and the solution was irradiated with 4!8 W bulbs at
254 nm for 4 h. The solvent was removed under reduced
pressure to give a crude yellow oil which was purified by
column chromatography (petroleum ether/diethyl ether,
2:1), giving the thioester 17 as a yellow oil (162 mg,
56%); Rf 0.30 (petroleum ether/diethyl ether, 2:1); [a]2D5 K
48.4 (chloroform, cZ0.5); nmax (thin film): 3092, 3062,
3028 (w, aromatic C–H str), 2961, 2933, 2873 (aliphatic
C–H str), 1790 (s, lactone C]O str), 1693 (s, thioester
C]O str), 1599, 1576, 1505, 1456 (s, ring str), 1195 (s),
1177 (s, C–O–C str); dH (400 MHz, CDCl3): 0.89 (9H, s,
HCC(CH3)3), 1.98 (2H, quint, JZ7.5 Hz, NCH2CH2CH2S),
2.36 (3H, s, SC(]O)CH3), 2.95 (2H, t, JZ7.0 Hz, NCH2-
CH2CH2S), 3.38 (1H, d, JZ14.5 Hz, one of PyCH2CPh),
3.54 (2H, t, JZ7.5 Hz, NCH2CH2CH2S), 3.69 (1H, d, JZ
14.5 Hz, one of PyCH2CPh), 4.59 (2H, s, PyCH2N), 4.79
(1H, s, (CH3)3CCH), 6.61–6.65 (2H, m, 2!CH(Ph–N)o),
6.67–6.72 (1H, m, CH(Ph–N)p), 6.98 (1H, d, JZ7.5 Hz, one
of CH(Py)3,5), 7.04 (1H, d, JZ7.5 Hz, one of CH(Py)3,5),
7.16–7.21 (2H, m, 2!CH(Ph–N)m), 7.31–7.37 (1H, m,
CH(Ph–C)p), 7.38–7.44 (2H, m, 2!CH(Ph–C)m), 7.49 (1H,
t, JZ7.5 Hz, CH(Py)4), 7.74–7.79, (2H, m, 2!CH(Ph–
C)o); dC (100.6 MHz, CDCl3): 23.57 (HCC(CH3)3), 26.56
(NCH2CH2CH2S), 27.27 (NCH2CH2CH2S), 30.63
(SC(]O)CH3), 34.88 (HCC(CH3)3), 48.34 (PyCH2CPh),
50.72 (NCH2CH2CH2S), 56.73 (PyCH2N), 82.15 (PyCH2-
CPh), 109.53 (HCC(CH3)3), 112.24 (2!CH(Ph–N)o),
116.67 (CH(Ph–N)p), 119.12, 122.53 (2!CH(Py)3,5),
124.96 (2!CH(Ph–C)o), 127.97 (CH(Ph–C)p), 128.25
(2!CH(Ph–C)m), 129.25 (2!CH(Ph–N)m), 137.11
(CH(Py)4), 139.05 (C(Ph–C)i), 147.76 (C(Ph–N)i), 155.17,
158.92 (C(Py)2,6), 173.15 (O–C]O), 195.47 (S–C]O);
m/z (ESC): 533 (100%, [MH]C); HRMS: found [MH]CZ
533.2477, C31H37N2O4S requires 533.2474.
References and notes
1. Samson, S. M.; Belagaje, R.; Blankenship, D. T.; Chapman,
J. L.; Perry, D.; Skatrud, P. L.; VanFrank, R. M.; Abraham,
E. P.; Baldwin, J. E.; Queener, S. W.; Ingolia, T. D. Nature
1985, 318, 191–194.
2. Cooper, R. D. G. Bioorg. Med. Chem. 1993, 1, 1–17.
3. Que, L. Jr.; Ho, R. Y. N. Chem. Rev. 1996, 96, 2607–2624.
4. Roach, P. L.; Clifton, I. J.; Fulop, V.; Harlos, K.; Barton, G. J.;
Hajdu, J.; Andersson, I.; Schofield, C. J.; Baldwin, J. E. Nature
1995, 375, 700–704.
5. Roach, P. L.; Clifton, I. J.; Hensgens, C. M. H.; Shibata, N.;
Schofield, C. J.; Hajdu, J.; Baldwin, J. E. Nature 1997, 387.
6. Burzlaff, N. I.; Rutledge, P. J.; Clifton, I. J.; Hensgens,
C. M. H.; Pickford, M.; Adlington, R. M.; Roach, P. L.;
Baldwin, J. E. Nature 1999, 401, 721–724.
7. Baldwin, J. E.; Bradley, M. Chem. Rev. 1990, 90, 1079–1088.
8. Baldwin, J. E.; Schofield, C. J. In The Biosynthesis of b-
Lactams; Page, M. I., Ed.; Blackie Academic and Professional:
London, 1992; pp 1–78.
9. (a) Fung, K. W. D. Phil. Thesis, University of Oxford, 1993.
(b) Chu, C. D. Phil. Thesis, University of Oxford, 1999.
10. Tshuva, E. Y.; Lee, D.; Bu, W.; Lippard J. Am. Chem. Soc.
2002, 124, 2416–2417.
11. Lin, G.; Reid, G.; Bugg, T. D. H. J. Chem. Soc., Chem.
Commun. 2000, 1119–1120.
12. Lin, G.; Reid, G.; Bugg, T. D. H. J. Am. Chem. Soc. 2001, 123,
5030–5039.
13. Hegg, E. L.; Ho, R. Y. N.; Que, L. Jr. J. Am. Chem. Soc. 1999,
121, 1972–1973.
14. Bugg, T. D. H. Tetrahedron 2003, 59, 7075–7101.
15. Ogle, J. M.; Clifton, I. J.; Rutledge, P. J.; Elkins, J. M.;
Burzlaff, N. I.; Adlington, R. M.; Roach, P. L.; Baldwin, J. E.
Chem. Biol. 2001, 8, 1231–1237.
16. Hegg, E. L.; Que, L. Jr. Eur. J. Biochem. 1997, 250, 625–629.
17. Chin, J.; Lee, S. S.; Lee, K. J.; Park, S.; Kim, D. H. Nature
1999, 401, 254–257.
4.1.9. (2S)-2-Hydroxy-3-(6-{[(3-mercaptopropyl)phenyl-
amino]methyl}pyridin-2-yl)-2-phenylpropionic acid (3).
Lithium hydroxide (15 mg, 0.63 mmol) was dissolved in
water (2.5 mL) and a solution of the thioester 17 (106 mg,
0.20 mmol) in THF (2.5 mL) was added. The solution was
then heated at reflux for 16 h and the solvent was removed
under reduced pressure to give the deprotected molecule 3
as an off-white solid; m/z (ESC): 423 (100%, [MH]C);
HRMS: found [MH]CZ423.1737, C24H27N2O3S requires
423.1742.
18. McDougal, P. G.; Rico, J. G.; Oh, Y. I.; Condon, B. D. J. Org.
Chem. 1986, 51, 3388–3390.
19. Seebach, D.; Naef, R. Helv. Chim. Acta 1981, 64, 2704–2708.
20. Seebach, D.; Naef, R.; Calderari, G. Tetrahedron 1984, 40,
1313–1324.
21. Grover, P. T.; Bhongle, B. N.; Wald, S. A.; Senanayake, C. H.
J. Org. Chem. 2000, 65, 6283–6287.
22. Otwinowski, Z.; Minor, W. Methods Enzymol. 1997, 276,
307–326.