Stereoselective synthesis of optically active bicyclic β-lactam carboxylic acids that target pilus biogenesis in pathogenic bacteria

Article abstract of DOI:10.1039/b210551a

Optically active bicyclic β-lactams were synthesized, starting from 2-H-Δ²-thiazolines and Meldrum's acid derivatives. Several methods to accomplish an ester hydrolysis without damaging the β-lactam framework were investigated. A rapid CsOH saponification of the β-lactam methyl esters was developed and protonation of the Cs-carboxylates by Amberlite (IR-120 H⁺) afforded a series of bicycle β-lactam carboxylic acids. Moreover, a convenient method for the synthesis of 2-H-Δ²-thiazolinecarboxylic acid methyl ester 2 was developed. Bicyclic β-lactam carboxylic acids 7a-g and aldehydes 4a-d were screened for their affinity to the bacterial periplasmic chaperone PapD using a surface plasmon resonance technique. β-Lactams substituted with large acyl substituents showed better binding to the chaperone than the native C-terminal peptide PapG8, demonstrating that bicyclic β-lactams constitute a new class of potential bacterial chaperone inhibitors.

Full text of DOI:10.1039/b210551a

View Article Online / Journal Homepage / Table of Contents for this issue
Stereoselective synthesis of optically active bicyclic ꢀ-lactam
carboxylic acids that target pilus biogenesis in pathogenic
bacteria†
Hans Emtenäs,a Marcus Carlsson,a Jerome S. Pinkner,b Scott J. Hultgrenb and
Fredrik Almqvist*a
a Organic Chemistry, Department of Chemistry, Umeå University, SE-901 87 Umeå, Sweden
b Department of Molecular Microbiology, Washington University in St. Louis School of
Medicine, St. Louis, MO 63110, USA. E-mail: fredrik.almqvist@chem.umu.se
Received 29th October 2002, Accepted 20th February 2003
First published as an Advance Article on the web 11th March 2003
Optically active bicyclic β-lactams were synthesized, starting from 2-H-∆2-thiazolines and Meldrum’s acid
derivatives. Several methods to accomplish an ester hydrolysis without damaging the β-lactam framework were
investigated. A rapid CsOH saponification of the β-lactam methyl esters was developed and protonation of the
Cs-carboxylates by Amberlite (IR-120 Hϩ) afforded a series of bicyclic β-lactam carboxylic acids. Moreover, a
convenient method for the synthesis of 2-H-∆2-thiazolinecarboxylic acid methyl ester 2 was developed. Bicyclic
β-lactam carboxylic acids 7a–g and aldehydes 4a–d were screened for their affinity to the bacterial periplasmic
chaperone PapD using a surface plasmon resonance technique. β-Lactams substituted with large acyl substituents
showed better binding to the chaperone than the native C-terminal peptide PapG 8, demonstrating that bicyclic
β-lactams constitute a new class of potential bacterial chaperone inhibitors.
Introduction
β-Lactams have gained substantial interest in the scientific
community not only due to their antibiotic properties1–3 but
also as reactive intermediates and as starting materials in a
wide range of synthetic settings.4,5 The heavy use of antibiotics
during the second half of the last century has resulted in an
increasing number of bacteria being resistant to the drugs
available today.6,7 One way of overcoming this problem involves
development of antibacterial agents against novel targets in the
microorganism.8
A large number of infectious Gram negative bacteria pro-
duce pili, which are a family of extracellular, supramolecular
protein organelles that mediate attachment to host tissue.9 Pilus
assembly is performed by periplasmic chaperones that fold and
transport the subunits to the outer cell membrane where they
are incorporated into a growing pilus.10 Thus, if the chaperone-
subunit complex can be prevented from being formed, coloniz-
ation of host tissue cannot occur.11,12 Moreover, periplasmic
chaperones constitute a highly conserved structure, which can
be found in a large number of pathogenic bacteria such as
B. pertussis, S. typhimurium and H. influenzae responsible for
the diseases whooping cough, gastrointestinal disorder and
meningitis respectively.9,13 In addition, the structures of the
chaperone-subunit complexes are well studied and it has been
shown, both by NMR14,15 and X-ray crystallography,16,17 that
the pili-subunits bind to the chaperone by anchoring of the
carboxy terminus to the side chains of Arg 8 and Lys 112.
Previously, we reported a stereoselective synthesis of optically
active β-lactams, which were designed to be a suitable scaffold
for the development of compounds (termed pilicides) that
inhibit pilus formation in uropathogenic E. coli.18 Bicyclic β-
lactam methyl esters 3 were synthesized in yields as high as
93% by reacting the two key intermediates, the acyl Meldrum’s
acids 1 and 2-H-∆2-thiazoline methyl ester 2 (Fig. 1). These
esters could then be selectively reduced to the corresponding
Fig. 1 Original procedure for the formation of the β-lactam methyl
esters 3 and their corresponding aldehydes 4.
aldehydes 4 by DIBAL-H, which would have potential to form
imines with Lys 112.18
The stereochemistry of the β-lactams synthesized by this
method is different compared to the original penicillin’s, thus
having a chance to withstand enzymatic degradation by
penicillin-resistant bacteria. Other compounds such as bicyclic
2-pyridinones 5 and amino acid derivatives 6 (Fig. 2) are known
† Electronic supplementary information (ESI) available: 13C NMR
spectra of 7(a–g), 10–14, 16(a and d) and 17(a–b). See http://
www.rsc.org/suppdata/ob/b2/b210551a/
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 3 0 8 – 1 3 1 4
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
1308

View Article Online
was constructed. Unfortunately, it turned out to be almost in-
soluble in solvents suitable for the following β-lactam synthesis
(benzene or 1,2-dichloroethane), and no β-lactam was formed.
Therefore the strategy was to synthesize the β-lactam esters,
which then have to be hydrolyzed to generate the carboxylic
acids. Thus, with the methyl esters 3 in hand,18 (Fig. 1) attempts
to hydrolyze 3a by mild saponification (e.g. K2CO3 in MeOH–
H2O 9 : 1) gave in the best case a 1 : 1 mixture of starting
material and product, which was difficult to purify. Prolonged
reaction time resulted in decomposition of the β-lactam ring.
In addition, attempts to hydrolyze 3a with (Bu3Sn)2O in
benzene,22,23 or 3c with pig liver esterase24,25 were also unsuccess-
ful. Tsuji et al. developed a method for the hydrolysis of benzyl
esters of cephalosporin analogs using AlCl3,26 which seemed
attractive. Thus, the cystein benzyl ester was prepared27 and the
2-H-∆2-thiazoline was synthesized based on the same protocol
as for the methyl ester28 giving the benzyl ester 10 in 37%
overall yield in three steps (Scheme 1). Allowing 10 to react
with the Meldrum’s acid derivative 1c gave the β-lactam 11
26
in 58% yield but all attempts of using AlCl3,26 TiCl4 or
hydrogenation with Pd/C29 to cleave the benzyl-ester were
unsuccessful.
p-Nitrobenzyl (PNB) esters have shown to be hydrolyzable
by using Zn in a phosphate buffer.30 Consequently, the
orthogonally protected cysteine-PNB ester 12 was synthesized
(Scheme 1). After simultaneous acidic removal of the N- and
S-protecting groups, the corresponding cysteine-PNB ester was
converted to the 2-H-∆2-thiazoline-PNB ester 13 in 36% overall
yield (from 12 in three steps) and reaction of this intermediate
with the Meldrum’s acid derivative 1c gave the β-lactam-PNB
ester 14 in 63% yield (Scheme 1). However, this ester also
proved to be difficult to hydrolyze using procedures such as
Zn in phosphate buffer, Zn in HCl (6 M),27 Na2S in THF–
H2O31 or hydrogenation with different catalysts (e.g. Pd/C or
PtO2). At this stage, to be able to find a productive route to
the desired β-lactam carboxylic acids, the need for a practical
procedure to synthesize different 2-H-∆2-thiazoline esters
became critical. In the literature there are a wide range of mild
methods for the hydrolysis of different carboxylic esters32 and
the strategy to prepare cysteine esters via the previous two
routes was time consuming. Thus, we wanted to prepare the
2-H-∆2-thiazoline 2 on a large scale and then perform trans-
esterifications to the desired esters. Scaling up the previous
method28 was not straightforward since flash chromatography
had to be conducted to remove unreacted formylated amino
acid and the 2-Me-∆2-thiazoline methyl ester, which occasion-
ally appeared in the synthesis. By using a modified procedure,
previously used to prepare optically active 2-oxazolines,33 the
Fig. 2 Bicyclic 2-pyridinone 5, amino acid derivative 6 and the hepta-
peptide 8, corresponding to the C-terminus of the pilus adhesion
protein PapG, have shown chaperone binding and chaperone/subunit
inhibitor activity.19 Novel bicyclic β-lactam carboxylic acids 7 targeted
in this article are also designed to have corresponding biological
activity.
to bind to chaperones as free carboxylic acids19 or as their cor-
responding Li-salts.20 This article describes the synthesis of β-
lactam carboxylic acids and the study of their binding affinity
to the periplasmic chaperone PapD by the surface plasmon
resonance technique. Moreover, a new practical and efficient
procedure for the synthesis of the important intermediate, 2-H-
∆2-thiazolinecarboxylic acid methyl ester 2, was developed.
Results and discussion
The first approach was to synthesize the desired β-lactam
carboxylic acids from the 2-H-∆2-thiazoline carboxylic acid and
a Meldrum’s acid derivative. Following a procedure developed
by Walker,21 the ∆2-thiazoline carboxylic acid hydrochloride
Scheme 1 Two different routes to furnish the benzyl- or the p-nitrobenzyl ester 11 and 14 respectively.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 3 0 8 – 1 3 1 4
1309

View Article Online
optically active 2-H-∆2-thiazoline methyl ester 2 could be pre-
pared in quantitative yield without the need for chromato-
graphy (Scheme 2). The enantiomeric ratio (er) was established
1
by H NMR (in the presence of Eu(hfc)3) to be 9 : 1 (corre-
sponding to 80% ee), which also is an improvement compared
to the former procedure.28
Fig. 3 Epimerization of the α-carbon would result in the dia-
stereomers 18.
Although this method had caused racemization in the
1
hydrolysis of 2, the H NMR spectrum of the crude product
showed no undesired diastereomers 18 and the β-lactam moiety
was still intact. Purification by flash chromatography turned
out to be difficult and gave decomposition of the β-lactam
framework. Fortunately, trituration with acetone resulted
in precipitation of the β-lactam carboxylic acids. A series of
optically active bicyclic β-lactam carboxylic acids 7a–g were
synthesized with this method, all in excellent yields >98%
except for the pentyl derivative 7f, which was problematic to
triturate resulting in a moderate yield of 60% (Scheme 4).
Scheme 2 A convenient method for the synthesis of optically active
2-H-∆2-thiazoline methyl ester 2.
The transesterification was performed by first generating the
Cs-carboxylate ( )-15 via a CsOH (0.1 M aq.) saponification.
Thereafter, ( )-15 was sequentially alkylated to furnish the
new esters ( )-16 in moderate to excellent overall yields 57–98%
(Scheme 3).
Scheme 4 Rapid saponification of the methyl esters 3 with CsOH
followed by protonation to give the β-lactam carboxylic acids 7.
Biological evaluation
The β-lactam carboxylic acids 7(a–g) and the β-lactam alde-
hydes 4(a–d) were tested for their capability of binding to the
periplasmic chaperone PapD with surface plasmon resonance
on a Biacore 3000 instrument. This method has earlier proven
to be a good screening device for pilicides, since the chaperone
binding properties have been shown to correlate to the capa-
bility of disrupting a preformed chaperone-subunit complex.19
Normalized responses were calculated using the 2-pyridinone 5
as reference since this was the most efficient pilicide in the
earlier study.19 In this screening β-lactams with a fairly large
acyl substituent showed substantial binding to the chaperone
PapD (Table 1). Interestingly, this was true also for the aldehyde
derivatives 4(a–d). The aldehydes came off the chaperone as
easily as the carboxylic acids in the washing step, which indi-
cates that no imine was formed. Although not as good binders
as the reference compound 5, 7(a–g) still bound better than the
native C-terminal peptide PapG 7 which previously had showed
inhibition of PapD in an ELISA.16 Furthermore, 8(e–f) showed
almost the same affinity as the peptide PapG but the β-lactam
bearing a small methyl substituent 7g showed very weak
binding.
Scheme 3 Synthesis of different 2-H-∆2-thiazoline esters ( )-16 and
their corresponding β-lactam esters ( )-17a and ( )-17b.
Thus, with these new 2-H-∆2-thiazolines in hand the corre-
sponding heptyl and propargyl β-lactam esters ( )-17a and ( )-
17b were synthesized in 70 and 91% yields respectively. Others
had shown that many heptyl esters can be easily hydrolyzed by
enzymes under relatively mild conditions (acetone–phosphate
buffer pH = 7 at 37 ЊC)34–36 but treating the β-lactam heptyl
ester ( )-17a with a variety of lipases gave no desired carboxylic
acid. Another mild alternative was to use the propargyl ester,
which should be possible to hydrolyze by treatment with
Co2(CO)8 and TFA in CH2Cl2.37 Disappointingly, the pro-
pargylic ester ( )-17b could not be hydrolysed under these
conditions and increasing the temperature or the amount of
Co2(CO)8 gave no improvement and most of the staring
material could be recovered.
Since the CsOH hydrolysis was so successful in the 2-H-∆2-
thiazoline case, we anticipated that it could be fruitful also for
the β-lactam methyl esters provided that the reaction time could
be kept short. Thus, 1 eq. of aqueous CsOH (0.1 M) was added
to a solution of the β-lactam methyl ester 3 in MeOH and the
resulting solution was immediately concentrated under reduced
pressure. After being co-concentrated twice from dry EtOH
followed by dilution in dry EtOH, the β-lactam carboxylic acids
were obtained by protonation with Amberlite (IR-120 Hϩ)
(Fig. 3).
Conclusions
A new practical synthesis of the key building block 2-H-∆2-2-
thiazoline methyl ester 2 was developed which gave excellent
yield and limited racemization. Then, a series of β-lactam esters
was synthesized in good to excellent yields and different
methods for ester hydrolysis were investigated. Among these the
only fruitful protocol was a very fast saponification procedure,
developed in our laboratory, resulting in a series of optically
active β-lactam carboxylic acids. A surface plasmon resonance
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 3 0 8 – 1 3 1 4
1310

View Article Online
Table 1 Affinities of compounds 4(a–d) and 7(a–g) for the PapD
chaperone. Peptide PapG 8 and 2-pyridinone 5 are included for
comparison
(4R)-4,5-Dihydrothiazole-4-carboxylic acid methyl ester (2)
TEA (2.9 mL, 20.6 mmol) was added dropwise to a suspension
of -cystein methyl ester hydrochloride (4000 mg, 23.3 mmol) in
dry 1,2-dichloroethane (23 mL) at 0 ЊC. After stirring for 10 min
the mixture was diluted with 1,2-dichloroethane (30 mL) and
triethyl orthoformate (5.7 mL, 34.6 mmol) was added. After
refluxing the mixture using a Soxhlet with molecular sieves 4 Å
for 2 h a catalytic amount of PTSA was added, and the mixture
was refluxed for another 18 h before reaching rt. Toluene was
added and the mixture was cooled to Ϫ18 ЊC. Precipitated
TEA–HCl was filtrated off and the filtrate was concentrated
yielding 2 as an oil in quantitative yield. [α]2D0 130 (c 1.18,
Norm. response
Compound
R1
R2
for PapDa
7a
7b
7c
7d
7e
7f
7g
4a
4b
4c
4d
5
Ph
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CO2H
CHO
CHO
CHO
CHO
—
35
29
35
46
18
22
2
31
21
34
25
100
25
1-Naphthyl
CH2-1-naphthyl
CH2-2-naphthyl
Cyclohexyl
n-Pentyl
Me
Ph
1-Naphthyl
CH2-1-naphthyl
CH2-2-naphthyl
—
CHCl3); IR (neat) 1741, 1572, 1437, 1340, 1219, 1039, 810 cmϪ1
;
1H NMR (400 MHz, CDCl3) δ 7.99 (d, J = 2.29 Hz, 1H),
5.01–5.11 (m, 1H), 3.78 (s, 3H), 3.39–3.53 (m, 2H); 13C NMR
(100 MHz, CDCl3) δ 170.9, 159.6, 77.7, 52.9, 33.3; HRMS
(EIϩ) Calcd. for C5H7NO2S: 145.0197 Obsd. 145.0186.
ꢀ-Lactam methyl esters 3a–g and ꢀ-lactam aldehydes 4a–d
Prepared according to published procedures.18
8
—
—
a Determined by surface plasmon resonance at a fixed pilicide concen-
tration (30 µM) using a Biacore 3000 instrument. The normalized
responses were calculated using 5 as standard (100 % norm. response)
and all compounds were tested in triplicate in random order.
(4R)-4,5-Dihydrothiazole-4-carboxylic acid benzyl ester (10)
To the suspension of -cystine dibenzyl ester di-tosylate (1640
mg, 2.14 mmol) in dioxane (7.0 mL) and conc. HCl (1.0 mL)
was added zinc dust (600 mg, 9.17 mmol) in many portions over
a period of 15 minutes with vigorous stirring. After stirring at
rt for 1.5 h, undissolved zinc was filtered off and washed with
dioxane and the filtrate was concentrated. The residue was
freeze-dried from water. The residue was dissolved in pyridine
(10 mL) and a previously prepared solution of acetic anhydride
(1.05 eq.) and formic acid (1.2 eq.) was added. After stirring
for 2 h at rt the solution was concentrated. The residue was
dissolved in CHCl3 and the pH was adjusted to neutral with
6 M HCl and the solution was co-concentrated from CHCl3
(3×). The residue was suspended in benzene (100 mL), a cata-
lytic amount of PTSA was added, and the solution was refluxed
overnight using a Dean Stark apparatus. The organic phase was
washed with saturated NaHCO3, water and brine. The water
phase was extracted twice with CH2Cl2 and the combined
organic extracts were dried with Na2SO4, filtered and concen-
trated. Flash chromatography (heptane–ethyl acetate 5 : 1–1 : 1)
gave 10 (350 mg, 37%). [α]2D0 16 (c 4.40, CHCl3); IR (neat) 1747,
1662, 1513, 1392, 1344, 1172, 964, 775, 734 cmϪ1; 1H NMR (400
MHz, CDCl3) δ 8.04 (d, J = 2.38 Hz, 1H), 7.31–7.41 (m, 5H),
5.19–5.30 (m, 2H), 5.10–5.17 (m, 1H), 3.42–3.56 (m, 2H); 13C
NMR (100 MHz, CDCl3) δ 170.1, 159.6, 135.2, 128.5, 128.4,
128.2, 77.7, 67.3, 33.2 HRMS (FABϩ) Calcd. For (M ϩ 1)
C11H12NO2S: 222.0589 Obsd. 222.0591.
screening for the capability of binding to a periplasmic
chaperone, PapD, revealed that β-lactams substituted with
fairly large acyl substituents bound stronger to the chaperone
than the native C-terminal peptide PapG 8. Consequently,
the bicyclic β-lactams can be considered as a new family with
chaperone binding properties and they will be further evaluated
as pilicides in the near future.
Experimental
General
All reactions were carried out under an inert atmosphere
with dry solvents under anhydrous conditions, unless otherwise
stated. 1,2-Dichloroethane and CH2Cl2 were distilled from
calcium hydride (and THF from potassium, respectively).
Benzene and toluene were distilled from sodium. TLC was
performed on Silica Gel 60 F254 (Merck) with detection by UV
light and staining with a solution of anisaldehyde (26 mL),
glacial acetic acid (11 mL) and concentrated sulfuric acid
(35 mL) in 95% ethanol (960 mL). Flash column chromato-
graphy (eluents given in brackets) was performed on silica gel
(Matrix, 60 Å, 35–70 µm, Grace Amicon). Centrifugal pre-
parative TLC was performed using rotors coated with silica
gel 60 PF254 containing CaSO4 (Merck). The moving bands
Boc-Cys(Trt)-PNB, (12)
Boc-Cys(Trt)-CO2H (6000 mg, 12.9 mmol) was dissolved in
80% aqueous EtOH (100 mL), and the pH was adjusted to
pH 10 with 30% aqueous Cs2CO3, concentrated at reduced
pressure, then diluted with EtOH (absolute), and concentrated
at high vaccum. The resulting powder was dissolved in DMF
(50 mL) and cooled to 0 ЊC, after which 4-nitrobenzyl bromide
(3300 mg, 15.3 mmol) was added dropwise. After 12 h at rt the
reaction mixture was diluted with water and extracted with
Et2O. The organic phase was dried with Na2SO4, filtered and
concentrated. Flash chromatography (heptane–ethyl acetate
8 : 2–7 : 3) gave 12 ( 7900 mg, 100%). [α]2D0 11 (c 1.7, CHCl3); IR
(neat) 3374, 2966, 1745, 1712, 1606, 1511, 1444, 1346, 1261,
1
were visualized using UV light. The H and 13C NMR spectra
were recorded on a Bruker DRX-400 for solutions in CDCl3
[residual CHCl3 (δH 7.26 ppm) or CDCl3 (δC 77.0 ppm) as
internal standard] or in DMSO-d6 [residual DMSO-d5 (δH 2.50
ppm) or DMSO-d6 (δC 40.0 ppm) as internal standard] at
298 K. First-order chemical shifts and coupling constants were
obtained from one-dimensional spectra. IR spectra were
recorded on an ATI Mattson Genesis Series FTIRTM spectro-
meter. Optical rotations were measured with a Perkin-Elmer
343 polarimeter and are given in 10Ϫ1 deg cm2 g Ϫ1. High reso-
lution mass spectra were recorded on a JEOL SX102 A mass
spectrometer. Ions for FABMS were produced by a beam
of Xenon atoms (6 keV) from a matrix of glycerol and thio-
glycerol. Mass spectra on the β-lactam carboxylic acids 7 were
recorded on a Waters micromassZQ using positive electrospray
(ESϩ).
1
1157, 1012, 844, 738, 698 cmϪ1; H NMR (400 MHz, CDCl3)
δ 8.17 (d, J = 8.69 Hz, 2H), 7.46 (d, J = 8.69 Hz, 2H), 7.33–7.38
(m, 5H), 7.19–7.30 (m, 10H), 5.15–5.30 (m, 2H), 5.04 (d,
J = 8.23 Hz, 1H), 4.26–4.38 (m, 1H), 2.52–2.73 (m, 2H), 1.43
(s, 9H); 13C NMR (100 MHz, CDCl3) δ 170.5, 155.0, 147.7,
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 3 0 8 – 1 3 1 4
1311

View Article Online
144.1, 142.5, 129.4, 128.4, 128.0, 127.0, 123.7, 80.2, 66.9,
65.6, 52.6, 34.0, 28.3 HRMS (FABϩ) Calcd. for (M ϩ Na)
C34H34N2O6SNa: 621.2035 Obsd. 621.2030.
4,5-Dihydrothiazole-4-carboxylic acid 4-nitrobenzyl ester
(( )-16c)
By following the procedure described for the preparation of
( )-16a from ( )-15, ( )-15 (188 mg, 1.30 mmol) and 4-nitro-
benzyl bromide (504 mg, 2.33 mmol) gave ( )-16c (240 mg,
70%) as a white powder. [α]2D0 0 (c 1.23, CHCl3), all spectroscopic
data were in agreement with 13.
(4R)-4,5-Dihydrothiazole-4-carboxylic acid 4-nitrobenzyl ester
(13)
A solution of TFA–H2O–thioanisole–ethanedithiol 175 : 10 : 10
: 5 (200 mL) was added to 12 (7900 mg, 13.2 mmol) and after
stirring for 3 h, AcOH (100 mL) was added and the solution
was concentrated. To the residue was added AcOH (100 mL)
and the white precipitate was filtrated off. The filtrate was con-
centrated and the residue was triturated with Et2O which gave a
solid, crude product which was dissolved in a solution of AcOH
and water and freeze-dried yielding Cys-PNB (3200 mg) as a
powder which was used in the next step without further purifi-
cation. To a solution of Cys-PNB (1450 mg) in pyridine
(7.5 mL) was added a previously prepared solution of acetic
anhydride (1.05 eq.) and formic acid (1.2 eq.) and after stirring
for 2 h at room temperature the solution was concentrated. The
residue was dissolved in CHCl3 and pH was adjusted to neutral
with 6 M HCl and the solution was co-concentrated from
CHCl3 (3×). The residue was suspended in benzene (180 mL),
a catalytic amount of PTSA was added, and the solution was
refluxed overnight using a Dean Stark apparatus. The organic
phase was washed with saturated NaHCO3, water and brine.
The water phase was extracted twice with CH2Cl2 and the
combined organic extracts were dried with Na2SO4, filtered and
concentrated. Flash chromatography (heptane–ethyl acetate
3 : 1–1 : 2) gave 13 (550 mg, 35%). [α]2D0 43 (c 1.40 CHCl3);
4,5-Dihydrothiazole-4-carboxylic acid prop-2-ynyl ester (( )16d)
By following the procedure described for the preparation of
( )-16a from ( )-15, ( )-15 (800 mg, 3.04 mmol) and propargyl
bromide (0.60 ml, 5.55 mmol) gave ( )-16d (504 mg, 98%) as
an oil. [α]2D0 0 (c 0.92, CHCl3); IR (neat) 3282, 1745, 1570,
1
1176, 1038, 810 cmϪ1; H NMR (400 MHz, CDCl3) δ 8.05 (d,
2.47 Hz, 1H), 5.11–5.18 (m, 1H), 4.80 (dq, J = 15.55, 2.47 Hz,
2H), 3.46–3.58 (m, 2H), 2.51 (t, J = 2.38 Hz, 1H); 13C NMR
(100 MHz, CDCl3) δ 169.7, 160.1, 77.6, 77.3, 75.7, 53.2,
33.3; HRMS (EIϩ) Calcd. for C7H7NO2S: 169.07975 Obsd.
169.0274.
6-(2-Naphthalen-1-ylacetyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]-
heptane-2-carboxylic acid heptyl ester (( )17a)
By following the published procedure described for the
preparation of 3,18 ( )-16a (810 mg, 3.53 mmol) and 1c
(1440 mg, 4.59 mmol) gave ( )-17a (1100 mg, 70%) as a foam.
[α]2D0 0 (c 1.0, CHCl3); IR (neat) 2972, 2656, 1741, 1676, 1402,
1
1199, 968 cmϪ1; H NMR (400 MHz, CDCl3) δ 7.88 (t, J =
6.95 Hz, 2H), 7.81 (d, J = 8.14 Hz, 1H), 7.34–7.56 (m, 4H),
6.54 (s, 1H), 5.26 (d, J = 6.31 Hz, 1H), 5.10 (s, 1H), 4.14 (s, J =
6.68 Hz, 2H), 4.01 (s, 1H), 3.46–3.53 (m, 1H), 3.21 (d, J = 11.16
Hz, 1H), 1.57–1.67 (m, 2H), 1.20–1.37 (m, 10H), 0.84–0.92
(m, 3H); 13C NMR (100 MHz, CDCl3) δ 169.1, 168.8, 160.7,
133.9, 131.8, 130.5, 128.8, 128.3, 128.1, 126.5, 125.9, 125.4,
123.4, 101.2, 94.1, 66.3, 60.8, 36.7, 32.0, 31.6, 28.7, 28.4, 25.6,
22.5, 14.0; HRMS (EIϩ) Calcd. for C25H29NO4S: 439.18173
Obsd. 440.18897.
IR (neat) 3066, 1749, 1680, 1602, 1512, 1342, 1315, 1180 cmϪ1
;
1H NMR (400 MHz, CDCl3) δ 8.22 (d, J = 8.60 Hz, 2H),
8.06 (d, J = 2.38 Hz, 1H), 7.54 (d, J = 8.78 Hz), 5.33 (s, 1H),
5.15–5.22 (m, 1H), 3.45–3.60 (m, 2H); 13C NMR (100 MHz,
CDCl3) δ 170.1, 160.2, 147.9, 142.5, 128.6, 124.0, 77.7, 65.9,
33.3; HRMS (CIϩ) Calcd. for C11H11N2O4S: 267.0440 Obsd.
267.0443.
4,5-Dihydrothiazole-4-carboxylic acid heptyl ester (( )-16a)
Aqueous CsOH (0.1 M, 68.5 mL) was added dropwise to a
stirred solution of thiazoline methyl ester 2 (1000 mg, 6.85
mmol) in MeOH (200 ml) at rt. The mixture was immediately
concentrated and the residue was co-concentrated four times
from dry EtOH giving the thiazoline Cs-salt as a white foam.
A solution of heptyl bromide (1.50 mL, 9.50 mmol) in DMF
(6 ml) was added dropwise to a solution of the Cs-salt ( )-15
(1390 mg, 5.28 mmol) in DMF (16 ml) at 0 ЊC. After stirring for
2 h the cooling bath was removed and the reaction was allowed
to reach rt overnight. The reaction mixture was diluted with
Et2O and washed with ice cooled water (7×) and the organic
extract was dried with Na2SO4, filtered and concentrated. Flash
chromatography (heptane–ethyl acetate 1 : 1), spinning centri-
fugal preparative TLC (heptane–ethyl acetate 3 : 7), spinning
centrifugal preparative TLC (heptane–ethyl acetate 7 : 3) gave
( )-16a (692 mg, 57%) as an oil. [α]2D0 0 (c 1.05 CHCl3); IR (neat)
2954, 2929, 2858, 2360, 1741, 1571, 1465, 1186 cmϪ1; 1H NMR
(400 MHz, CDCl3) δ 8.04 (d, J = 2.2 Hz, 1H), 5.09 (dt, J =
9.51, 2.38 Hz, 1H), 4.15–4.21 (m, 2H), 3.43–3.55 (m, 2H), 1.61–
1.71 (m, 2H), 1.18–1.38 (m, 10H), 0.81–0.89 (m, 3H); 13C NMR
(100 MHz, CDCl3) δ 170.5, 159.7, 77.6, 66.0, 33.2, 31.6, 28.8,
28.4, 25.6, 22.4, 14.0; HRMS (EIϩ) Calcd. for C11H19NO2S:
229.1136, Obsd. 229.11337.
6-(2-Naphthalen-1-ylacetyl)-7-oxo-4-thia-1-azabicyclo[3.2.0]-
heptane-2-carboxylic acid prop-2-ynyl ester (( )-17b)
By following the published procedure described for the
preparation of 3,18 ( )-16d (340 mg, 2.01 mmol) and 1c (816
mg, 2.61 mmol) gave ( )-17b (693 mg, 91%). [α]2D0 0 (c 1.05,
CHCl3); IR (neat) 3288, 1753, 1666, 1398, 1184, 968, 752 cmϪ1
;
1H NMR (400 MHz, CDCl3) δ 7.78–7.91 (m, 3H), 7.34–7.57
(m, 4H), 6.57 (s, 1H), 5.30 (d, J = 6.31 Hz, 1H), 5.11 (s, 1H),
4.74 (d, J = 2.38 Hz, 2H), 4.01 (s, 2H), 3.52 (dd, J = 11.71, 6.40
Hz, 1H), 3.25 (d, J = 11.07 Hz, 1H), 2.49 (s, 1H); 13C NMR (100
MHz, CDCl3) δ 169.2, 168.5, 160.9, 134.0, 131.9, 130.6, 129.0,
128.5, 128.2, 126.7, 126.0, 125.6, 123.6, 101.2, 94.2, 76.8, 76.0,
60.8, 53.6, 36.9, 31.9; HRMS (EIϩ) Calcd. for C21H17NO4S
379.0900 Obsd. 379.0962.
(2R,5S,6R)-6-(Naphthalen-1-ylacetyl)-7-oxo-4-thia-1-aza-
bicyclo[3.2.0]heptane-2-carboxylic acid benzyl ester (11)
By following the published procedure described for the
preparation of 3,18 10 (536 mg, 2.42 mmol) and 1c (904 mg,
2.89 mmol) gave 11 (606 mg, 58%). [α]2D0 7 (c 0.70, CHCl3);
IR (neat) 2973, 2900, 1739, 1662, 1515, 1394, 1180, 1066, 962,
1
773, 732, 694 cmϪ1; H NMR (400 MHz, CDCl3) δ 7.78–7.92
(m, 3H), 7.28–7.57 (m, 9H), 6.55 (s, 1H), 5.32 (d, J = 6.22 Hz,
1H), 5.18 (s, 2H), 5.09 (s, 1H), 4.00 (s, 2H), 3.49 (dd, J = 11.25,
6.22 Hz, 1H), 3.23 (d, J = 11.25 Hz, 1H); 13C NMR (100 MHz,
CDCl3) δ 169.00, 168.98, 160.8, 135.0, 133.9, 131.8, 130.5,
128.9, 128.6, 128.5, 128.4, 128.2, 128.1, 126.5, 125.9, 125.5,
123.5, 101.2, 94.1, 67.8, 60.9, 36.8, 31.9; HRMS (FABϩ) Calcd.
for (M ϩ 1) C25H22NO4S 432.1270 Obsd. 432.1270.
4,5-Dihydrothiazole-4-carboxylic acid benzyl ester (( )-16b)
By following the procedure described for the preparation of
( )-16a from ( )-15, ( )-15 (188 mg, 1.30 mmol) and benzyl
bromide (400 mg, 2.33 mmol) gave ( )-16b (180 mg, 62%) as a
an oil. [α]2D0 0 (c 1.00, CHCl3), all spectroscopic data were in
agreement with 10.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 3 0 8 – 1 3 1 4
1312

View Article Online
(2R,5S,6R)-6-(Naphthalen-1-ylacetyl)-7-oxo-4-thia-1-aza-
bicyclo[3.2.0]heptane-2-carboxylic acid 4-nitrobenzyl ester (14)
ation with acetone 7d was obtained as a powder in quantitative
yield. [α]2D0 1 (c 0.2, DMSO); IR (neat) 2987, 2902, 1708, 1629,
1
1394, 1220, 1058, 968, 757 cmϪ1; H NMR(400 MHz, DMSO-
By following the published procedure described for the
preparation of 3,18 13 (1060 mg, 3.95 mmol) and 1c (1570 mg,
5.03 mmol) gave 14 (1200 mg, 63%). [α]2D0 8 (c 0.60, CHCl3);
IR (neat) 3363, 2971, 2902, 1513, 1392, 1342, 1249, 1056, 734,
696 cmϪ1; 1H NMR (400 MHz, CDCl3) δ 8.21 (d, J = 8.69 Hz,
2H), 7.85–7.91 (m, 2H), 7.82 (d, J = 8.14 Hz, 1H), 7.40–7.56
(m, 5H), 7.37 (d, J = 6.31 Hz, 1H), 6.52 (s, 1H), 5.34 (d, J = 6.22
Hz, 1H), 5.22–5.32 (m, 2H), 5.11 (s, 1H), 4.02 (s, 2H), 3.54
(dd, J = 11.25, 6.22 Hz, 1H), 3.25 (d, J = 11.25 Hz, 1H); 13C
NMR (100 MHz, CDCl3) δ 169.3, 168.9, 160.8, 147.8, 142.2,
133.9, 131.8, 130.4, 128.9, 128.4, 128.2, 126.6, 125.9, 125.5,
123.9, 123.4, 101.1, 94.0, 66.1, 60.8, 36.8, 31.9; HRMS (FABϩ)
Calcd. for (M ϩ 1) C25H21N2O6S 477.1120 Obsd. 477.1120.
d6) δ 7.72–7.94 (m, 4H), 7.34–7.53 (m, 3H), 6.47 (s, 1H), 5.24
(s, 1H), 4.71 (d, J = 5.35 Hz, 1H), 3.73 (s, 2H), 3.09–3.27 (m,
2H); 13C NMR (100 MHz, DMSO-d6) δ 170.9, 167.7, 160.6,
134.2, 133.6, 132.5, 128.6, 128.1, 128.0 (2×), 127.9, 126.8, 126.4,
101.7, 63.1, 95.0, 39.1, 32.9; MS (ESϩ) Calcd. for (M ϩ 1)
C18H16NO4S: 342.08 Obsd. 341.86.
(2R,5S,6R)-6-Cyclohexanecarbonyl-7-oxo-4-thia-1-azabicyclo-
[3.2.0]heptane-2-carboxylic acid (7e)
Compound 7e was prepared from 3e (130 mg, 0.44 mmol)
as described above for the synthesis of 7a from 3a. After
trituration with acetone 7e was obtained as a powder (121 mg,
98%). [α]2D0 27 (c 0.95, DMSO); IR (neat) 3319, 2927, 2852, 1647,
1595, 1414, 1371, 960, 752 cmϪ1; 1H NMR (400 MHz, DMSO-
d6) δ 6.40 (s, 1H), 5.14 (s, 1H), 4.67 (d, J = 6.13 Hz, 1H), 3.21–
3.27 (m, 1H), 3.10–3.16 (m, 1H), 2.07–2.17 (m, 1H), 1.59–1.80
(m, 5H), 1.11–1.28 (m, 5H); 13C NMR (100 MHz, DMSO-d6)
δ 172.4, 170.5, 160.8, 98.5, 94.8, 63.1, 41.2, 33.0, 30.2, 30.0,
25.9, 25.8, 25.7; MS (ESϩ) Calcd. for (M ϩ 1) C13H18NO4S:
284.10 Obsd. 283.95.
(2R,5S,6R)-6-Benzoyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-
2-carboxylic acid (7a)
Aqueous CsOH (0.1 M, 10 mL) was added to a stirred solution
of β-lactam methyl ester 3a (300 mg, 1.03 mmol) in MeOH
(40 mL) at room temperature and the solution was immediately
concentrated. The residue was co-concentrated twice from dry
EtOH giving a yellow oil. The resulting yellow oil was dissolved
in EtOH and pH was adjusted to pH = 4 with Amberlite
(IR-120 Hϩ). The amberlyst was filtrated off and the filtrate
was concentrated and the residue was triturated with acetone
to give 7a as a powder in quantitative yield. [α]2D0 57 (c 1.48,
DMSO); IR (neat) 3365, 1639, 1593, 1408, 1373, 1273 cmϪ1; 1H
NMR (400 MHz, DMSO-d6) δ 7.72–7.77 (m, 2H), 7.42–7.52
(m, 3H), 6.63 (s, 1H), 6.05 (s, 1H), 4.73 (d, J = 6.10 Hz,
1H), 3.28–3.34 (m, 1H), 3.19 (d, J = 9.80 Hz, 1H); 13C NMR
(100 MHz, DMSO-d6) δ 170.3, 162.3, 160.9, 131.7, 129.3, 126.7,
99.0, 95.4, 63.6, 33.3; MS (ESϩ) Calcd. for (M ϩ 1) C13H12-
NO4S: 278.05 Obsd. 277.93.
(2R,5S,6R)-6-Hexanoyl-7-oxo-4-thia-1-azabicyclo[3.2.0]-
heptane-2-carboxylic acid (7f)
Compound 7f was prepared from 3f (160 mg, 0.56 mmol) as
described above for the synthesis of 7a from 3a. After tritur-
ation with acetone 7f was obtained as a powder (91 mg, 60%).
[α]2D0 13 (c 0.36, DMSO); IR (neat) 3278, 2924, 2854, 1645, 1593,
1
1414, 1373, 1273, 968 cmϪ1; H NMR (400 MHz, DMSO-d6)
δ 6.50 (s, 1H), 5.28 (s, 1H), 5.00 (d, J = 6.40 Hz, 1H), 3.41 (dd,
J = 10.98, 6.40 Hz, 1H), 3.17 (d, J = 10.98 Hz, 1H), 2.21 (t, J =
7.50 Hz, 2H), 1.42–1.50 (m, 2H), 1.20–1.31 (m, 4H), 0.83–0.89
(t, J = 6.68 Hz, 3H); 13C NMR (100 MHz, DMSO-d6) δ171.2,
170.1, 160.6, 99.9, 94.3, 61.4, 32.7, 32.2, 31.0, 25.8, 22.2, 14.3:
MS (ESϩ) Calcd. for (M ϩ 1) C12H18NO4S: 272.10 Obsd.
272.07.
(2R,5S,6R)-6-Naphthalenyl-7-oxo-4-thia-1-azabicyclo[3.2.0]-
heptane-2-carboxylic acid (7b)
Compound 7b was prepared from 3b (300 mg, 0.88 mmol)
as described above for the synthesis of 7a from 3a. After tritur-
ation with acetone 7b was obtained as a powder in quantitative
(2R,5S,6R)-6-Acetyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-
2-carboxylic acid (7g)
yield. [α]2D0 16 (c 0.9, DMSO); IR (neat) 1563, 1508, 1338 cmϪ1
;
1H NMR (400 MHz, DMSO-d6) δ 7.96–8.18 (m, 3H), 7.51–7.73
(m, 4H), 6.94 (s, 1H), 5.7 (s, 1H), 4.74 (d, J = 5.85 Hz, 1H),
3.17–3.37 (m, 2H); 13C NMR (100 MHz, DMSO-d6) δ 170.1,
164.0, 160.6, 133.7, 131.6, 130.6, 130.6, 129.3, 128.6, 127.8,
126.9, 125.7, 125.5, 104.3, 95.6, 63.1, 33.4; MS (ESϩ) Calcd. for
(Mϩ1) C18H16NO4S: 328.06 Obsd. 327.88.
Compound 7g was prepared from 3g (190 mg, 0.83 mmol)
as described above for the synthesis of 7a from 3a. After
trituration with acetone 7g was obtained as a powder in quanti-
tative yield. [α]2D0 24 (c 1.0, DMSO); IR (neat) 3294, 2949, 1645,
1
1593, 1412, 1367, 964, 748 cmϪ1; H NMR(400 MHz, DMSO-
d6) δ 6.46 (s, 1H), 5.22 (s, 1H), 4.69 (d, J = 6.04 Hz, 1H), 3.18–
3.27 (m, 1H), 3.12–3.17 (m, 1H), 1.92 (s, 3H); 13C NMR
(100 MHz, DMSO-d6) δ 170.8, 165.7, 160.6, 101.0, 94.5, 63.2,
33.0, 19.3; MS (ESϩ) Calcd. for (M ϩ 1) C8H10NO4S: 216.03
Obsd. 215.95.
(2R,5S,6R)-6-(Naphthalen-1-ylacetyl)-7-oxo-4-thia-1-azabi-
cyclo[3.2.0]heptane-2-carboxylic acid (7c)
Compound 7c was prepared from 3c (300 mg, 0.84 mmol) as
described above for the synthesis of 7a from 3a. After tritur-
ation with acetone 7c was obtained as a powder in quantitative
yield. [α]2D0 Ϫ1 (c 0.1, DMSO); IR (neat) 3313br, 3056, 2923,
1714, 1596, 1373, 1095, 779 cmϪ1; 1H NMR(400 MHz, DMSO-
d6) δ 8.06 (d, J = 8.13 Hz, 1H), 7.83–7.98 (m, 2H), 7.41–7.61
(m, 4H), 6.51 (s, 1H), 5.01 (s, 1H), 4.71 (d, J = 6.73 Hz, 1H),
3.99–4.14 (m, 2H), 3.22–3.29 (m, 1H), 3.16 (d, J = 10.27 Hz,
1H); 13C NMR (100 MHz, DMSO-d6) δ 170.8, 167.9, 160.5,
133.9, 132.2, 132.0, 129.0, 128.3, 128.2, 126.9, 126.4, 126.2,
124.5, 101.5, 95.0, 62.9, 36.2, 32.9; MS (ESϩ) Calcd. for
(M ϩ 1) C18H16NO4S: 342.08 Obsd. 341.86.
Acknowledgements
We thank Einar Nilsson at Lund University for HRMS
analysis. This work was funded by grants from the Knut
and Alice Wallenberg Foundation, the foundation for tech-
nology transfer in Umeå, the Swedish Research Council and
J. C. Kempe Foundation.
References
1 A. G. Brown, Pure Appl. Chem., 1987, 59, 475–484.
2 Chemistry and Biology of β-lactam Antibiotics, eds. R. B. Morin and
M. Gorman, Academic Press: New York, 1982, Vol. 1–3.
3 D. Nathwani and M. Wood, J. Drugs, 1993, 45, 866–894.
4 The Organic Chemistry of β-Lactams, ed. G. I. Georg, VCH, New
York, 1993.
(2R,5S,6R)-6-(Naphthalen-2-ylacetyl)-7-oxo-4-thia-1-azabi-
cyclo[3.2.0]heptane-2-carboxylic acid (7d)
Compound 7d was prepared from 3d (200 mg, 0.56 mmol)
as described above for the synthesis of 7a from 3a. After tritur-
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 3 0 8 – 1 3 1 4
1313

View Article Online
5 The Chemistry of β-Lactams, M. I. Page, Blackie Academic and
Professional, New York, 1996.
6 C. M. Henry, Chem. Eng. News, 2000, 78, 41.
7 C. Walsh, Nature, 2000, 406, 775–781.
8 C. Stephens and L. Shapiro, Chem. Biol., 1997, 4, 637–641.
9 S. J. Hultgren, Escherichia Coli Salmonella typhimurium, 1996,
2730.
20 H. Emtenas, K. Ahlin, J. S. Pinkner, S. J. Hultgren and F. Almqvist,
J. Comb. Chem., 2002, 4, 630–639.
21 J. Walker, J. Chem. Soc., C, 1968, 1522–1526.
22 C. J. Salomon, E. G. Mata and O. A. Mascaretti, J. Org. Chem.,
1994, 59, 7259–7266.
23 E. G. Mata and O. A. Mascaretti, Tetrahedron Lett., 1988, 29, 6893–
6896.
10 S. J. Hultgren, S. Abraham, M. Caparon, P. Falk, J. W. Stgeme and
S. Normark, Cell, 1993, 73, 887–901.
24 E. Alvarez, T. Cuvigny, C. H. Dupenhoat and M. Julia, Tetrahedron,
1988, 44, 119–126.
25 M. Ohno, Y. Ito, M. Arita, T. Shibata, K. Adachi and H. Sawai,
Tetrahedron, 1984, 40, 145–152.
26 T. Tsuji, T. Kataoka, M. Yoshioka, Y. Sendo, Y. Nishitani, S. Hirai,
T. Maeda and W. Nagata, Tetrahedron Lett., 1979, 2793–2796.
27 L. Zervas and I. Photaki, J. Am. Chem. Soc., 1962, 84, 3887–3897.
28 F. Almqvist, D. Guillaume, S. J. Hultgren and G. R. Marshall,
Tetrahedron Lett., 1998, 39, 2293–2294.
29 W. H. Hartung and R. Simonoff, Org. React., 1953, VII, 263–326.
30 T. Kumagai, S. Tamai, T. Abe, H. Matsunaga, K. Hayashi, I. Kishi,
M. Shiro and Y. Nagao, J. Org. Chem., 1998, 63, 8145–8149.
31 S. R. Lammert, A. I. Ellis, R. R. Chauvette and S. Kukolja, J. Org.
Chem., 1978, 43, 1243–1244.
32 T. W. Greene and P. G. M. Wuts Protective Groups in Organic
Synthesis. 3rd Edn, John Wiley & Sons, New York, 1999.
33 K. Kamata, I. Agata and A. I. Meyers, J. Org. Chem., 1998, 63,
3113–3116.
34 P. Braun, H. Waldmann and H. Kunz, Synlett, 1992, 39–40.
35 P. Braun, H. Waldmann, W. Vogt and H. Kunz, Synlett, 1990,
105–107.
11 S. Langermann, S. Palaszynski, M. Barnhart, G. Auguste,
J. S. Pinkner, J. Burlein, P. Barren, S. Koenig, S. Leath, C. H. Jones
and S. J. Hultgren, Science (Washington, D. C.), 1997, 276, 607–611.
12 J. A. Roberts, B.-I. Marklund, D. Ilver, D. Haslam, M. B. Kaack,
G. Baskin, M. Louis, R. Moellby, J. Winberg and S. Normark,
Proc. Natl. Acad. Sci. U. S. A., 1994, 91, 11889–11893.
13 D. L. Hung, S. D. Knight, R. M. Woods, J. S. Pinkner and
S. J. Hultgren, Embo J., 1996, 15, 3792–3805.
14 K. F. Karlsson, B. Walse, T. Drakenberg, S. Roy, K. E. Bergquist,
J. S. Pinkner, S. J. Hultgren and J. Kihlberg, Bioorg. Med. Chem.,
1998, 6, 2085–2101.
15 M. Pellecchia, P. Sebbel, U. Hermanns, K. Wuthrich and
R. Glockshuber, Nat. Struct. Biol., 1999, 6, 336–339.
16 M. J. Kuehn, D. J. Ogg, J. Kihlberg, L. N. Slonim, K. Flemmer,
T. Bergfors and S. J. Hultgren, Science, 1993, 262, 1234–1241.
17 G. E. Soto, K. W. Dodson, D. Ogg, C. Liu, J. Heuser, S. Knight,
J. Kihlberg, C. H. Jones and S. J. Hultgren, Embo J., 1998, 17,
6155–6167.
18 H. Emtenas, G. Soto, S. J. Hultgren, G. R. Marshall and
F. Almqvist, Org. Lett., 2000, 2, 2065–2067.
19 A. Svensson, A. Larsson, H. Emtenas, M. Hedenstrom, T. Fex,
S. J. Hultgren, J. S. Pinkner, F. Almqvist and J. Kihlberg,
ChemBioChem, 2001, 2, 915–918.
36 D. Sebastian and H. Waldmann, Tetrahedron Lett., 1997, 38,
2927–2930.
37 Y. Fukase, K. Kukase and S. Kusumoto, Tetrahedron Lett., 1999, 40,
1169–1170.
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 1 3 0 8 – 1 3 1 4
1314