6-Aminopenicillanic acid (6-APA) derivatives equipped with anchoring arms

Article abstract of DOI:10.1016/j.tet.2011.10.100

6-APA derivatives were considered as selective labels for the construction of bifunctional linkers dedicated to the oriented immobilization of proteins on materials. Sulbactam-like compounds (i.e., 6-β-sulfonamido-penam sulfones) and penicillin G - like compounds (i.e., para-substituted 6-β- phenylacetamido-penams) were prepared and tested as irreversible inhibitors of representative β-lactamases and D,D-peptidases, respectively. The activity of the modified antibiotics was preserved despite their substitution with various anchoring arms. The (2-nitro-4,5-dimethoxy)-benzyl esters revealed of particular interest due to their capacity to acylate BlaR-CTD without deprotection.

Full text of DOI:10.1016/j.tet.2011.10.100

Tetrahedron 68 (2012) 10818e10826
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6-Aminopenicillanic acid (6-APA) derivatives equipped with
anchoring arms
ˇ
a
a
b
b
a,
*
ꢀ
Annaïck Favre , Jerome Grugier , Alain Brans , Bernard Joris , Jacqueline Marchand-Brynaert
ˇ
a
ꢀ
Institute of Condensed Matter and Nanosciences (IMCN), Universite catholique de Louvain, Batiment Lavoisier, Place Louis Pasteur, L4.02.01, B-1348 Louvain-la-Neuve, Belgium
Centre for Protein Engineering (CIP), Universite de Liege, Batiment B6a, Allee de la Chimie, B-4000 Sart-Tilman, Belgium
ˇ
b
ꢀ
ꢁ
ꢀ
a r t i c l e i n f o
a b s t r a c t
Article history:
6-APA derivatives were considered as selective labels for the construction of bifunctional linkers dedi-
Received 12 September 2011
Received in revised form 20 October 2011
Accepted 27 October 2011
cated to the oriented immobilization of proteins on materials. Sulbactam-like compounds (i.e., 6-
sulfonamido-penam sulfones) and penicillin Gdlike compounds (i.e., para-substituted 6- -phenyl-
acetamido-penams) were prepared and tested as irreversible inhibitors of representative -lactamases
b-
b
b
Available online 4 November 2011
and D,D-peptidases, respectively. The activity of the modified antibiotics was preserved despite their
substitution with various anchoring arms. The (2-nitro-4,5-dimethoxy)-benzyl esters revealed of par-
ticular interest due to their capacity to acylate BlaR-CTD without deprotection.
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Protein array
b
-Lactam antibiotics
Bifunctional linkers
-Lactamases
b
D,D-Carboxypeptidases
1. Introduction
Hence, the active site could be considered as a valuable anchoring
point for the regular arrangement of such proteins on surfaces. This
The ability to pattern surfaces with functional organic (mono)
layers has became the focus of numerous studies because of the
potential applications in various fields, such as microelectronics
and (bio)sensors.1 Several diagnosis methods and drug discovery
techniques are currently based on the immobilization of proteins
on solid supports.2e5 The design of protein-chips requires the de-
velopment of efficient attachment methods, and for the array for-
mat, the concomitant development of patterning methods.6,7
Generally, proteins are fixed in random orientations. However, in-
creasing attention is put on the controlled orientation issue. For
instance, the use of recombinant affinity tags and expressed protein
ligation have been proposed.8
strategy relies upon the availability of dedicated molecular tools, i.e.,
heterotelechelic spacers11 featuring at one terminus a reactive
function for grafting on the support, and at the other terminus a b-
lactam motif susceptible to fix on the serine residue of the active site.
In this article, we fully describe the construction of original 6-APA
(6-aminopenicillanic acid) derivatives designed to interact with BLs
and PBPs in this context of tools for materials science. Accordingly,
the C6 lateral chain typical of antibiotics has been replaced with
different spacer-arms for surface-grafting. The effect of the C6
modifications on the activity of the resulting penicillin-like com-
pounds versus representative target enzymes has been examined.
We are interested in the oriented immobilization of class A and
2. Synthesis
class C b-lactamases (BLs) and Penicillin-Binding Proteins (PBPs) (or
selected mutants thereof) onto metal or silicon supports for the
construction of novel protein-arrays. These proteins are bacterial
serine-enzymes interacting with
teraction leads to acyl-enzyme intermediates, which are very un-
stable in the case of BLs and are naturally quite stable for PBPs. For
2.1. Sulbactam-like compounds
b
-lactam antibiotics.9,10 This in-
6-
b
-Sulfonamido-penam sulfones are structurally related to
-lacta-
sulbactam and behave similarly as suicide inhibitors of
b
mases, the defence enzymes produced by resistant bacteria.12 We
have thus decided to equip the bicylic framework with spacer-arms
positioned on the C6 sulfonamide lateral chain. Our starting ma-
terial was the pivaloyloxymethyl ester (PIV) or the ethoxymethyl
ester (EOM) of 6-APA, which could be deprotected in situ by es-
terases or hydrolysis in wateremethanol medium,13,14 respectively.
Indeed, the chemistry of penam derivatives is easier to perform
with C3 ester compounds than with C3 free acids.15,16 The selected
some
b-lactam compounds the acyl-enzyme intermediate rear-
ranges into permanently stable entities, in the case of so-called
‘suicide-inhibition’ (irreversible inhibition) for both BLs and PBPs.
* Corresponding author. Tel.: þ32 10 47 27 40; fax: þ32 10 47 41 68; e-mail
address: jacqueline.marchand@uclouvain.be (J. Marchand-Brynaert).
0040-4020/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2011.10.100

A. Favre et al. / Tetrahedron 68 (2012) 10818e10826
10819
function for further immobilization on Au and Ag supports was the
disulfide function of thioctic acid (TA).17,18 The two end-motifs,
namely penam-sulfone and TA, were linked via a few ethylene
glycol units (named PEG, for polyethylene glycol) to ensure water-
solubility of the final construct.11
The first strategy we envisaged (method A; Scheme 1) was based
on 6-[(hydroxycarbonyl)methylsulfony]-aminopenicillinate-S,S-di-
oxide (6)19 as key intermediate for the coupling with the TA spacers
(13) independently prepared (Scheme 2A). The pivaloyloxymethyl
(PIV) ester of 6-APA (1a), crystallized as the tosylate salt, reacted
readily with methanesulfonyl chloride and triethylamine to afford
the methanesulfonamide 2 in 60% yield. The oxidation with KMnO4
in aqueous acetic acid, at low temperature, led to the expected
sulfone 4, but in low yield due to loss of product during the work-up.
A similar sequence of reactions was applied using benzyl chlor-
osulfonylacetate freshly prepared from sulfoacetyl dichloride and
benzyl alcohol.12 We isolated 6-[(benzyloxycarbonyl)methyl-
sulfonyl]-aminopenicillinate 3 and the corresponding sulfone 4 in
44% and 96% yields, respectively. Hydrogenolysis was performed in
the presence of N-ethylmorpholine (NEM) to deprotect the benzyl
ester; the salt 6 could be stored in the freezer without degradation.
Hydrogenation of 5 in the absence of NEM, immediately followed by
reaction with N-hydroxysuccinimide (NHS) and dicyclohex-
ylcarbodiimide (DCC) furnished quantitatively the activated ester 7,
which was stable under careful storage conditions.
Scheme 2. Synthesis of spacer-arms (part I).
disappointing results were collected when applying peptide syn-
thesis methods to the coupling of 13 and 6: reactions in the pres-
ence of PyBOP ((benzotriazol-1-yloxy)tripyrrolidinophosphonium
hexafluorophosphate),
EDCI
(1-(3-dimethylamino-propyl)-3-
ethylcarbodiimide hydrochloride) or HBTU (O-(benzotriazol-1-yl)-
N,N,N0,N0-tetramethyluronium hexafluorophosphate), with trie-
thylamine (TEA) or NEM, in dichloromethane (DCM), dime-
thylformamide (DMF) or mixtures of DCE and DMF, at low
temperature (ꢀ20 ꢁC to 0 ꢁC) or room temperature, led to complex
crude mixtures that were very difficult to purify by chromatogra-
phy. Tenuous yields of target-molecules 8 and 9 were recovered by
using HBTU as coupling agent. From the 1H NMR data, a 80:20
mixture of epimers was visible (5,6-cis and 5,6-trans).
Therefore, we considered a second strategy (method B, Scheme
3) based on the coupling of short sulfonated hetero-telechelic PEG
spacers (18, Scheme 2B) to 6-APA, followed by oxidation into sul-
fones and fixation of thioctic acid in the last step. Thus the required
sulfonyl-terminated PEGs were independently prepared in four
steps from the amino-alcohols 14. The N-benzyloxycarbonyl-pro-
tected molecules 15, reacted with ethyl vinylsulfonate (Michael
addition) to furnish 16 in moderate yields (Scheme 2B). Then, the
sulfonate esters were transformed into sodium sulfonates (17) and
sulfonyl chlorides (18) according to a protocol from the literature.20
These reactive species were directly engaged with the 6-APA de-
rivative 1a to afford the sulfonamides 19,20 in good yields (w70%
from 6a,b). KMnO4 oxidation (21,22) followed by hydrogenolysis
led quantitatively to the key-intermediates 23,24, which are stable
only in the form of tosylate salts. The final coupling was performed
with the N-hydroxysuccinimide ester of TA,11 in the presence of
NEM as base. Due to material loss during the chromatographic
purifications, low yields of target-molecules 25,26 were obtained.
From the 1H NMR data, the 5,6-trans epimers were isolated. Indeed
Scheme 1. Synthesis of 6-b-sulfonamido-penam sulfones equipped with a side-chain
for Au (Ag) surface-grafting (method A).
The thioctic spacers were prepared in three steps, from the
commercially available diamines 10: Boc-monoprotection, coupling
to TA with DCC and dimethylaminopyridine (DMAP) as the catalyst,
and Boc-deprotection with trifluoroacetic acid (TFA) gave 13a and
13b in about 90% and 60% yields, respectively. Several attempts of
coupling the fragments 13 and 7 were performed, under various
experimental conditions (different bases, different solvents, varia-
tion of time and temperature), but without success. Similar
the coupling constant of the b-lactamic protons H5 and H6 (see
Table S1 in SD) was in the range of 1.3e1.5 Hz (trans relationship)
instead of 4.3e4.6 Hz (cis relationship). The intrinsic tendency of
penam-sulfones to epimerize15,16 is enhanced in the basic medium
used for the TA coupling.

10820
A. Favre et al. / Tetrahedron 68 (2012) 10818e10826
Scheme 4. Synthesis of spacer-arms (part II) coupled to the side-chain of Penicillin.
6-APA was N-protected using ethyl acetoacetate (31)30 and then
esterified with (2-nitro-4,5-dimethoxy)benzyl bromide and trie-
thylamine in DMF (32). Treatment with p-toluenesulfonic acid
(PTSA) liberated the amine function of the 6-APA ester 33. The
photochemical cleavage31,32 of the (2-nitro-4,5-dimethoxy)benzyl
moiety under UV-irradiation at 420 nm, without destruction of the
penam core has been verified at this stage of the synthesis and also
later on (see Supplementary data). For the next coupling with acids
30a,b, the tosylate salt 33 was neutralized and the corresponding
free amine was added to a pre-formed mixture of acid and DCC in
dichloromethane. Purification by preparative thin layer chroma-
tography afforded the target compounds 34 and 35 in, respectively,
58% and 70% yield (Scheme 5). In order to facilitate the purification
step, we considered the use of water soluble carbodiimides as
coupling agents, namely 1,3-bis(2,2-dimethyl-1,3-dioxolan-4-yl-
methyl)-carbodiimide (BDDC)33 and 1-(3-dimethylamino-propyl)-
3-ethyl carbodiimide hydrochloride (EDCI),34 but without signifi-
cant improvement of isolated yields.
Scheme 3. Synthesis of 6-b-sulfonamido-penam sulfones equipped with a side-chain
for Au (Ag) surface-grafting (method B).
We tested the use of ethoxymethyl (EOM) ester of 6-APA (1b, see
Supplementary data) as starting material for the application of
Schemes 1 and 3. The problems encountered in the PIV series (low
yields, hard purifications) were even more serious in the EOM se-
ries! The synthesis of the target-molecules (i.e., 8,9 and 25,26 in
series b) could not be achieved efficiently. However, some in-
termediates were obtained (see Supplementary data) and consid-
ered for the biological evaluations.
2.2. Benzylpenicillin-like compounds
Benzylpenicillin, also named Pen G: the molecule discovered by
A. Fleming in 1928, is an inhibitor of D,D-carboxypeptidases in-
volved in the bacterial cell-wall biosynthesis. All the semi-synthetic
penicillins differ by the nature of their C6 aminoacyl side-chains.15
Here, we propose to substitute the para-position of the Pen G
benzyl group with the anchoring arms. We considered as starting
material, a photo-cleavable ester derivative of 6-APA, namely the 2-
nitro-4,5-(dimethoxy)-benzyl ester.21 The application of UV light is
a mild technique of deprotection compatible with the penam sta-
bility. This also offers the possibility of surface patterning by irra-
diation through a mask or by lithography.22,23 The selected function
for Si or Ge supports derivatization was an alkene group susceptible
to make a hydrosilylation (hydrogermylation) reaction with re-
duced Si (Ge) surfaces.24,25 This function could also be involved in
thiol-ene click chemistry.26,27 As previously, the link between the
penam motif and the alkene ending was made of a short PEG chain.
Methyl (4-hydroxyphenyl)acetate (28) was used as precursor for
the synthesis of modified Pen G side-chains (Scheme 4). Indeed,
alcohol derivatives (27) can be easily coupled to the phenol moiety
via a Mitsunobu reaction. Tetraethylene glycol was monoalkylated
with 1-bromo-6-alkenes in concentrated sodium hydroxide solu-
tion as previously described in the literature.28 We considered two
different lengths of alkenyl chain, giving the spacers 27a (n¼3) and
27b (n¼9). Their reaction with 28 in the presence of diisopropyl
azodicarboxylate (DIAD) and triphenylphosphine gave the in-
termediates 29a,b, which saponification led to the corresponding
acids 30a,b with 64% and 65% overall yields (three steps). In the
Mistsunobu coupling, the order of addition of reagents could be of
prime importance. Here, we have modified a protocol developed by
Townsend and Salituro29 in the case of nocardicin side-chain syn-
thesis, for obtaining >90% yield of 29a,b. It is worthy of note that
the Williamson coupling between 28 and the mesylates derived
from alcohols 27a,b (K2CO3, CH3CN, reflux, several days) gave low
yields of 29a,b (<30%).
Scheme 5. Synthesis of Penicillin G photo-cleavable ester equipped with a side-chain
for Si (Ge) surface-grafting.
All the synthesized compounds have been characterized by
NMR spectroscopy and HRMS. The 1H and 13C data of the common
penam framework are collected in Tables S1 and S2 (see Supple-
mentary data). The most typical features are summarized here: (i)
the gem-dimethyl substituents at C2 give signals at w32 and
w27 ppm in the penam series, and at w20 and w17 ppm in the
penam-sulfone series (13C NMR data); (ii) the H3 proton is a singlet
at w4.4 ppm in the penam series and at w4.5 ppm in the penam-
sulfone series; (iii) the H5 proton appears as
a doublet
(J5,6¼4.3e4.7 Hz typical of the cis configuration) at 4.5e4.6 ppm for

A. Favre et al. / Tetrahedron 68 (2012) 10818e10826
10821
the 6-APA derivatives, at 5.5e5.6 ppm for the 6-
b
-aminosulfonyl
class A
rivatives (4) are modestly active. The penam precursors (2, 3, 19,
20) showed also anti- -lactamase activity (vs P99). All the com-
b-lactamase BlaP, only the 6-methylsulfonamido de-
derivatives, and 5.4e5.5 ppm for the 6-
rivatives; (iv) the H6 proton features
b
-aminocarbonyl de-
doublet of doublet
a
b
(J5,6¼4.3e4.7 Hz and J6,NH¼9e11 Hz) at w5.5 ppm for the 6-APA
derivatives, at w5.2 ppm for the 6- -aminosulfonyl derivatives,
and 5.6 ppm for the 6- -aminocarbonyl derivatives; (v) after oxi-
dation to sulfone (6- -aminosulfonyl series), H5 and H6 give signals
at 4.8e4.9 ppm and 5.4e5.6 ppm, respectively. The epimerization
leading to 5,6-trans compounds (most probably via the H6 enoli-
sation in basic conditions) is easily detected from the H5eH6
chemical shifts and coupling constant values: J5,6 trans¼0e1.5 Hz
and J6,NH¼8e10 Hz; H5 at 4.9e5.2 ppm and H6 at 5.1 ppm (8-epi-
mer, 25, 26).
pounds in this series were able to form acyl-enzyme complexes
with BlaR-CTD.
b
b
The Table 2 summarizes the inhibition activity of the Penicillin G
derivatives versus BlaR-CTD and R39. Surprisingly, the 2-nitro-4,5-
(dimethoxy)-benzyl esters 34,35 are able to acylate BlaR-CTD, al-
though this ester function cannot be hydrolyzed under the assay
conditions, and the corresponding free acids 36,37, resulting from
the photochemical deprotection, do not acylate BlaR-CTD. The in-
verse situation is observed in the case of R39. The activity of Pen G
is not impaired by the presence of a spacer-arm fixed on the aro-
matic ring.
b
3. Biological evaluation
The capacity of the synthesized molecules to form stable acyl-
enzyme complexes with bacterial enzymes of interest has been
Table 2
Evaluation of Pen G derivatives
verified in vitro. Two b
-lactamases, namely BlaP (class A)35 and P99
(class C),36 were pre-incubated with the different tested com-
pounds (at different concentrations) and the residual enzymic ac-
tivity was determined with nitrocefine, a chromogenic substrate of
b
-lactamases, which hydrolysis rate can be followed in solution by
spectrophotometry at 482 nm. Two penicillin-binding proteins
were also considered: the model D,D-carboxypeptidase R39 (from
Actinomadura)37 and BlaR-CTD.38,39 As above, the tested com-
pounds (at different concentrations) were pre-incubated with the
PBPs, and then fluorescent ampicillin was added in order to label
the residual active enzymes. Reading was made, after denaturation
and SDS-PAGE analysis, with a Phospho-Imager.
The results obtained with the penam-sulfone derivatives are
collected in Table 1. The activities versus BlaP and P99 b-lactamases
are given as percentages of residual enzyme activity for a given final
Entry
R1
R2
Compd
BlaR-CTDb
R39b
1
2
H
H
PIV
þþ
þ
NV-estera
38
(see SD)
þþþ
þþ
3
4
H
H
þþþ
þþþ
þþþ
ꢀ
NV-estera
34
35
5
6
NV-estera
H
þþ
ꢀ
ꢀ
36
(see SD)
þþ
concentration of tested molecule. The activities versus BlaR and
37
(see SD)
R39 are given as scores: ꢀ, inactive; þ, acylation at 100
acylation at 10 M.
M; þþþ, acylation at 1
The 6-sulfonamido-penam sulfones (4, 5, 60, 7) are good in-
hibitors of the class C -lactamase P99 and this activity is not
m
M; þþ,
7
H
ꢀ
þ
m
m
a
b
2-Nitro-4,5-(dimethoxy)-benzyl (i.e., nitroveratryl (NV)) ester.
See Table 1.
b
disturbed by the presence of a spacer-arm (21). In the case of the
Table 1
Evaluation of penam-sulfone derivatives and their penam precursors
Entry
R1
R2
n
Compd
BlaPa
(m
M)
P99a,c
(m
M)
BlaR-CTDb,c
R39b
1
2
3
4
5
6
7
8
Me
Me
Me
Me
PIV
EOM
PIV
EOM
PIV
EOM
PIV
EOM
PIV
PIV
PIV
PIV
0
0
2
2
0
0
2
2
2
2
2
0
0
2
2a
2b
4a
4b
3a
3b
5a
5b
6
10 (103)
80 (102)
35 (103)
30 (102)
90 (103)
80 (102)
90 (103)
80 (102)
90 (103)
15 (10)
50 (102)
10 (10)
20 (10)
25 (102)
25 (10)
25 (102)
0 (102)
þþ
þþþ
ꢀ
þþþ
þþþ
þþ
þþ
ꢀ
BnO2CeCH2e
BnO2CeCH2e
BnO2CeCH2e
BnO2CeCH2e
NEMþeO2CeCH2e
HO2CeCH2e
NHSeO2CeCH2e
CbzNH(CH2CH2O)3CH2CH2e
CbzNH(CH2CH2O)4CH2CH2e
CbzNH(CH2CH2O)3CH2CH2e
þþþ
þþþ
þþþ
þ
þ
9
25 (103)
40 (102)
25 (102)
30 (102)
25 (102)
20 (102)
ꢀ
10
11
12
13
14
60
þþþ
þþþ
þþ
7
19
20
21
PIV
PIV
þþ
þþþ
a
The enzyme was incubated 60 min with the tested compound (10e103
mM final concentration, indicated into parenthesis); then nitrocefine was added for measuring the
residual hydrolytic activity, expressed as percentage (%) of the initial activity. Low value indicates a very active compound.
b
The enzyme was incubated 15 min with the tested compound (1e103
residual active enzyme. The absence of fluorescence indicates a very active compound: ꢀinactive compd;þenzyme acylation at 100
þþþenzyme acylation at 1 M.
In situ ester hydrolysis gave similar results (data not shown).
m
M); then fluorescent ampicillin was added to form a fluorescent acyl-enzyme complex with the
M; þþenzyme acylation at 10 M;
m
m
m
c

10822
A. Favre et al. / Tetrahedron 68 (2012) 10818e10826
4. Conclusion
reported. Rotations were measured at 20ꢁC on PerkineElmer
241HC polarimeter; concentration is given in % (g/100 mL solvent).
HRMS data were obtained from the analytical service of the Uni-
versity College (London, UK). Most of the compounds were isolated
as oils (or gums) and amorphous solids, which decompose under
heating.
We have investigated the synthesis of two series of bifunctional
ligands for the construction of oriented protein-chips using the
penam framework to covalently bind the active site of serine-
enzymes, namely
b-lactamases and Penicillin-Binding Proteins
that we have selected on the basis of an easy access to various active
mutants.40,41 A similar strategy has been recently disclosed by
Funeriu et al.42 for the oriented immobilization of cysteine-
proteases via epoxy-succinyl-based peptides as affinity labels in-
corporated into their bifunctional ligands.
Protocols and spectroscopic characterizations of compounds 11
to 13a,b and 15 to 18a,b described in Scheme 2 (respectively, part A
and part B) are given as Supplementary data (SD). Compounds 1b to
5b (EOM ester series of Scheme 1) are described in SD. Protocols for
27 (Scheme 4) and Pen G esters (Table 2), adapted from the liter-
ature, are given in SD, as well as the free acids 36 and 37 (Table 2)
prepared directly from 6-APA.
Our attempts to incorporate 6-b-sulfonamido-penam sulfones
into bifunctional linkers featuring a thioctic acid terminus were
disappointing. Very poor yields of the final targets were recovered
by using either an amide bond (method A, molecules 8, 9) or an
ether bond (method B, molecules 25, 26) to connect the oligoe-
thyleneglycol spacer. Moreover, due to the enhanced acidity of the
H6 proton in the 6-sulfonamido-penam sulfone moiety (regarding
the 6-APA and penicillin G frameworks), epimerization occurred
during the last step of the total synthesis, leading to 80:20 mixtures
of 5,6-cis and 5,6-trans azetidinones (8, 9) or to exclusively 5,6-trans
azetidinones (25, 26). Thus the idea to use penam-sulfones, like 8,9
and 25,26, as irreversible inhibitors (i.e., suicide-substrates) to label
5.2. Synthesis of 6-b-sulfonamido-penam sulfones of Scheme
1 (first strategy)
5.2.1. Pivaloyloxymethyl 6-
(1a). To a suspension of 6-APA (1 g, 4.64 mmol) in anhydrous DMF
(9 mL) was added triethylamine (906 L, 6.50 mmol) and, after
b-aminopenicillinate, tosylate salt
m
stirring for 30 min, chloromethylpivalate (1.34 mL, 9.28 mmol).
After stirring at 25 ꢁC for 4 h, the mixture was diluted with EtOAc
(70 mL). The precipitate was filtered off and the filtrate was washed
with H2O and brine to remove DMF and unreacted 6-APA. The or-
ganic layer was dried over MgSO4 and concentrated under vacuum.
The yellow oil was solubilised in EtOAc (5 mL) and freshly recrys-
tallised p-toluenesulfonic acid (PTSA) was added (800 mg,
4.64 mmol). The precipitate was filtered and washed with cold
EtOAc. The ammonium salt 1a was obtained as a white solid
b-lactamases and fix these enzymes on supports in a regular
manner, has to be abandoned.
Fortunately, the alternative approach we examined, based on
the benzylpenicillin (i.e., Pen G) framework to label PBPs via stable
acyl-enzyme complexes, was successful. We could prepare the final
targets 34,35 in good yields and without epimerization. The linker,
anchored on the para-position of the aromatic ring of the penicillin
G side-chain, should allow materials derivatization via ‘click’
chemistry or hydrosilylation reaction. The full construct is com-
patible with the UV-deprotection of a masked ester.
From the biological evaluations of several intermediates and
final targets on representative BLs and PBPs, it clearly appeared that
BlaR-CTD (or mutants thereof) is the candidate of choice for the
oriented immobilization on supports. This protein could be effi-
ciently acylated by all tested compounds. Moreover, the depro-
tection of the C3 ester functions was not required to observe BlaR-
CTD inhibition. Indeed, after chemical hydrolysis of the EOM esters
and pre-incubation of the PIV esters with serum (data no shown),
similar results were collected. Yet, the behaviour of the 2-nitro-4,5-
(dimethoxy)-benzyl ester derivatives appeared quite unexpected
because this ester was not susceptible to be in situ deprotected
during the assays. The specific recognition of ortho-nitrobenzyl
esters of various antibiotics by BlaR-CTD is currently under in-
vestigation. The possibility to ‘orthogonally’ label the active site of
PBPs with penam esters (acylation of BlaR-CTD) and penam-acids
(acylation of R39) opens new perspectives for the construction of
oriented protein-arrays.
(1.793 g, 77%) and stored at ꢀ20 ꢁC; IR (film):
n 1778, 1760,
1740 cmꢀ1; 1H NMR (300 MHz, CDCl3):
d
1.22 (s, 9H, Me(tBu)), 1.53
(s, 3H, Me), 1.65 (s, 3H, Me), 4.42 (s, 1H, NCHCO2), 4.60 (d, 1H,
J¼4.3 Hz, NH2CHCH), 5.51 (d, 1H, J¼4.3 Hz, NH2CHCH), 5.77 (d, 1H,
J¼5.5 Hz, OCH2O), 5.88 (d, 1H, J¼5.5 Hz, OCH2O) (pTosOꢀ signals
omitted); 13C NMR (75 MHz, CDCl3):
d 26.7, 26.8, 31.2, 38.6, 62.8,
63.5, 69.5, 70.0, 79.6, 166.7, 176.5, 177.8 (pTosOꢀ signals omitted).
5.2.2. Pivaloyloxymethyl
(2a). To a solution of 1a (191 mg, 0.69 mmol) in anhydrous DCM
(3 mL) at 0 ꢁC, were added triethylamine (97
L, 0.69 mmol) and
methane sulfonylchloride (54
L, 0.69 mmol). After 20 min at 0 ꢁC
6-b-(methylsulfonamido)-penicillinate
m
m
and 5 min at room temperature, water (5 mL) was added. The
aqueous phase was extracted with DCM and the organic phases
were dried over MgSO4 and concentrated. The crude product was
purified by column-chromatography on silica gel (cyclohexane/
EtOAc (1:1), Rf¼0.54) to give 2a as a yellow amorphous solid
(148 mg, 60%); [
a
]
D
þ138.3 (c 3.0, CHCl3); IR (film):
n
3280, 1785,
1765, 1740 cmꢀ1; 1H NMR (300 MHz, CDCl3):
d
1.25 (s, 9H, Me(tBu)),
1.54 (s, 3H, Me), 1.65 (s, 3H, Me), 3.15 (s, 3H, SO2Me), 4.49 (s, 1H,
NCHCO2), 5.17 (dd, 1H, J¼4.5, 10.5 Hz, NHCHCH), 5.61 (d, 1H,
J¼4.5 Hz, NHCHCH), 5.35 (d, 1H, J¼10.5 Hz, NH), 5.79 (d, 1H,
J¼5.5 Hz, OCH2O), 5.88 (d, 1H, J¼5.5 Hz, OCH2O); 13C NMR (75 MHz,
5. Experimental section
5.1. General
CDCl3):
d 26.9, 28.1, 29.4, 38.8, 44.8, 52.5, 64.2, 66.2, 71.2, 80.0,
166.6, 167.0, 176.8.
The reactions were performed in anhydrous solvents and under
argon atmosphere. The reagents were purchased from commercial
suppliers and used as received. TLC was performed on Merck silica
gel 60 F254 plates, using an ethanolic solution of phosphomolybdic
acid for the visualization of spots. Column-chromatography was
5.2.3. Pivaloyloxymethyl 1,1-dioxide-6-b-(methylsulfonamido)-pen-
icillinate (4a). The penam 2a (342 mg, 0.83 mmol) dissolved in
a 4:1 mixture of AcOH and H2O (28 mL) was cooled at ꢀ15 ꢁC under
vigorous stirring. KMnO4 (277 mg, 1.74 mmol) dissolved in water
(10 mL) was added very slowly (over about 30 min). The mixture
was further stirred for 1 h at ꢀ10 ꢁC, then 10% H2O2 was added to
discolour the solution. DCM (40 mL) was added. The organic phase
was washed twice with 5% NaHCO3, dried over MgSO4 and con-
centrated. The penam-sulfone 4a was obtained as a colourless solid
made on ‘flash’ silica gel from Merck (40e60 mM). The NMR spectra
were recorded at room temperature on Bruker DPX-500, Bruker
Avance-300, Bruker Avance-250, or Varian Gemini-200 spectrom-
eters. Chemical shifts are reported in parts per million values using
tetramethylsilane as reference. The IR spectra were recorded on
FTIR-8400S Shimadzu apparatus; the most significant bands are
(70 mg, 20%); [
a]
D þ68.0 (c 1.6, CHCl3); IR (film):
n 3315, 1807, 1768,
1743 cmꢀ1; 1H NMR (300 MHz, CDCl3):
d
1.25 (s, 9H, Me(tBu)), 1.45

A. Favre et al. / Tetrahedron 68 (2012) 10818e10826
10823
(s, 3H, Me), 1.61 (s, 3H, Me), 3.15 (s, 3H, SO2Me), 4.54 (s, 1H,
NCHCO2), 4.88 (d, 1H, J¼4.7 Hz, NHCHCH), 5.46 (dd, 1H, J¼4.7,
11.5 Hz, NHCHCH), 5.75 (d, 1H, J¼5.5 Hz, OCH2O), 5.98 (d, 1H,
J¼5.5 Hz, OCH2O), 6.18 (d, 1H, J¼11.5 Hz, NH); 13C NMR (75 MHz,
J¼5.7 Hz, OCH2O), 6.01 (d, 1H, J¼5.7 Hz, OCH2O); 13C NMR (75 MHz,
acetone-d6):
d 8.5, 16.8, 19.3, 26.2, 38.4, 50.9, 51.6, 59.6, 61.0, 63.3,
64.0, 64.1, 66.2, 80.5, 165.6, 167.6, 174.5, 176.3.
Hydrogenation as above in the absence of NEM gave the free
CDCl3): d 17.7, 19.9, 26.7, 29.6, 42.2, 60.3, 63.6, 64.6, 65.5, 80.5, 165.2,
acid 60 (unstable compound), as a colourless oil (44 mg, quantita-
173.2, 176.7.
tive yield); [
d6):
a]
D þ53.40 (c 0.4, CHCl3); 1H NMR (300 MHz, acetone-
d
1.21 (s, 9H, Me(tBu)), 1.45 (s, 3H, Me), 1.57 (s, 3H, Me), 4.40
5.2.4. Pivaloyloxymethyl
6-
b
-[(benzyloxycarbonyl)-methyl-
L,
0.99 mmol) in anhydrous DCM (1 mL) was added at 0 ꢁC the benzyl
alcohol (103 L, 0.99 mmol) in DCM (1 mL). After 1 h, this mixture
was slowly added to a solution of 1a (328 mg, 0.99 mmol) in an-
hydrous DCM (2 mL) previously treated with triethylamine (276 L,
(sharp qAB, 2H, CH2SO2), 4.57 (s, 1H, NCHCO2), 5.32 (d, 1H, J¼4.7 Hz,
NHCHCH), 5.73e5.74 (m, 1H, NHCHCH), 5.82 (d, 1H, J¼5.7 Hz,
OCH2O), 6.01 (d, 1H, J¼5.7 Hz, OCH2O), 7.02 (d, 1H, J¼10.2 Hz, NH);
sulfonamido]-penicillinate (3a). Sulfoacetyl dichloride43 (105
m
m
13C NMR (75 MHz, acetone-d6):
d 17.9, 20.3, 27.3, 39.2, 57.3, 61.3,
63.9, 65.2, 66.7, 81.2, 166.1, 168.2, 174.3, 177.4.
m
1.98 mmol). After 15 min at 0 ꢁC and 1 h at room temperature,
water (4 mL) was added and the aqueous phase was extracted with
DCM. The organic phase was then washed with brine. After con-
centration under vaccum, the crude product was purified by
column-chromatography on silica gel (hexane/EtOAc (3:1), Rf¼0.80
5.2.7. Pivaloyloxymethyl 1,1-dioxide-6- -[succinimidyloxycarbonyl-
b
methylsulfonamido]-penicillinate (7). To a solution of 60 (44 mg,
0.09 mmol) in anhydrous DCM (3 mL) were added successively at
0 ꢁC N-hydroxysuccinimide (10 mg, 0.09 mmol) and DCC (19 mg,
0.092 mmol). After 16 h at room temperature, the solvent was
evaporated and the crude product was triturated with EtOAc. After
a filtration on Celite and evaporation, the product 7 was used
in (1:1)) to give 3a as a colourless oil (240 mg, 44%); [
4.0, CHCl3); IR (film):
1252, 1148, 1107, 1026, 978, 721 cmꢀ1
1.24 (s, 9H, Me(tBu)), 1.53 (s, 3H, Me), 1.61 (s, 3H, Me), 4.22 (d, 1H,
a
]
þ76.4 (c
D
n
1788, 1759, 1713, 1524, 1454, 1339, 1281,
;
1H NMR (300 MHz, CDCl3):
without purification (60 mg, quantitative yield); [
CHCl3); IR (film): 1811, 1782, 1742, 1628, 1574, 1450, 1323, 1157,
1117, 1068, 980, 760 cmꢀ1; 1H NMR (300 MHz, CDCl3):
1.23 (s, 9H,
a]
D þ42.50 (c 0.1,
d
n
J¼15.8 Hz, SO2CHaHbCO), 4.47 (s, 1H, NCHCO2), 4.48 (d, 1H,
J¼15.8 Hz, SO2CHaHbCO), 5.17 (dd, 1H, J¼4.6, 11.0 Hz, NHCHCH),
5.23 (s, 2H, PheCH2), 5.61 (d, 1H, J¼4.6 Hz, NHCHCH), 5.81 (d, 1H,
J¼5.5 Hz, OCH2O), 5.87 (d, 1H, J¼5.5 Hz, OCH2O), 5.93 (d, 1H,
J¼11.0 Hz, NH), 7.36e7.39 (m, 5H, Ph); 13C NMR (75 MHz, CDCl3):
d
Me(tBu)), 1.29 (s, 3H, Me), 1.41 (s, 3H, Me), 2.88 (br s, 4H, NCOC2H4),
4.52 (br s, 3H, NCHCO2, CH2SO2), 4.90 (d, 1H, J¼4.6 Hz, NHCHCH),
5.64 (dd, 1H, J¼4.6, 10.9 Hz, NHCHCH), 5.71 (d, 1H, J¼5.5 Hz,
OCH2O), 5.95 (d, 1H, J¼5.5 Hz, OCH2O), 6.76 (br d, 1H, J¼10.9 Hz,
d
26.7, 26.9, 32.5, 38.8, 57.5, 62.3, 64.8, 67.5, 65.1, 69.9, 79.8, 128.5,
NH); 13C NMR (75 MHz, acetone-d6):
d 17.7, 20.2, 26.4, 27.2, 39.4,
128.7, 134.5, 163.9, 166.2, 172.7, 176.8; HRMS (ESI) m/z: calcd for
55.5, 61.6, 64.4, 65.3, 67.0, 81.5, 160.7, 166.4, 170.1, 174.1, 177.2.
C23H30N2O9S2: 541.1314 [MꢀH]ꢀ. Found: 541.1318.
5.2.8. Coupling of the spacer-arm 13 to 60. To a solution of freshly
prepared 60 (1 equiv) and 13 (1 equiv) in DMF (3 mL, 0.1 mmol)
were added NEM (2.2 equiv) and HBTU (1.2 equiv). The mixture
was stirred for 3 h at room temperature. EtOAc (15 mL) was added
and the organic phase was washed with brine (3ꢂ), dried over
MgSO4 and concentrated. The crude product was purified by
column-chromatography on silica gel (3x), using EtOAc as the el-
uant. About 5% yield of pure 8 (or 9) could be recovered. Compound
5.2.5. Pivaloyloxymethyl
1,1-dioxide-6-b-[(benzyloxycarbonyl)-
methylsulfonamido]-penicillinate (5a). The penam 3a (221 mg,
0.40 mmol) dissolved in a 4:1 mixture of AcOH and H2O (20 mL)
was cooled at ꢀ15 ꢁC, under vigorous stirring. KMnO4 (61 mg,
0.85 mmol) dissolved in water (10 mL) was added very slowly
(30 min). The mixture was further stirred for 1 h at ꢀ10 ꢁC, then
10% H2O2 was added to discolour the solution. DCM (40 mL) was
added. The organic phase was washed twice with 5% NaHCO3, dried
over MgSO4 and concentrated. The penam-sulfone 5a was obtained
as a colourless oil (224 mg, 96%); Rf¼0.46 (hexane/EtOAc (3:1));
8 (gum): IR (film):
n 2958, 2956, 2854, 1801, 1778, 1759, 1647 (br),
1537, 1461, 1327, 1159, 1115 cmꢀ1; 1H NMR (500 MHz, CDCl3):
d 1.24
(s, 9H, Me(tBu)), 1.44, (s, 3H, Me), 1.48 (m, 2H, NHCOC2H4CH2), 1.59
(s, 3H, Me),1.70 (m, 4H, NHCOCH2CH2þNHCOC3H6CH2),1.93 (m,1H,
S2CHCH2), 2.23 (m, 2H, NHCOCH2CH2), 2.45 (m, 1H, S2CHCH2), 3.16
[
a
]
þ105.0 (c 0.2, CHCl3); IR (film):
n
1809, 1774, 1747, 1456, 1358,
D
1325, 1281, 1159, 1115, 978, 732 cmꢀ1
;
1H NMR (300 MHz, CDCl3):
d
1.25 (s, 9H, Me(tBu)), 1.43 (s, 3H, Me), 1.60 (s, 3H, Me), 4.25 (sharp
(m, 2H, S2CH2), 3.49 (m, 4H, CONHCH2), 3.62 (m, 9H, S2CH, OCH2
-
qAB, 2H, SO2CH2CO), 4.50 (s, 1H, NCHCO2), 4.82 (d, 1H, J¼4.6 Hz,
NHCHCH), 5.26 (sharp qAB, 2H, PheCH2), 5.48 (dd,1H, J¼4.6,11.1 Hz,
NHCHCH), 5.74 (d, 1H, J¼5.5 Hz, OCH2O), 5.77 (d, 1H, J¼5.5 Hz,
OCH2O), 6.55 (d, 1H, J¼11.1 Hz, NH), 7.39e7.40 (m, 5H, Ph); 13C NMR
CH2O), 4.15 (d, 1H, J¼14.2 Hz, SO2CH2), 4.20 (d, 1H, J¼14.2 Hz,
SO2CH2), 4.52 (s, 1H, NHCHCO2), 4.99 (d, 1H, J¼4.5 Hz, NHCHCH),
5.55 (dd, 1H, J¼4.5 and 10.8 Hz, NHCHCH), 5.73 (d, 1H, J¼5.5 Hz,
OCH2O), 5.96 (d,1H, J¼5.5 Hz, OCH2O), 6.13 (br t,1H, CONH), 6.75 (d,
(75 MHz, CDCl3):
d
17.6, 20.0, 26.8, 38.8, 57.7, 61.5, 63.6, 64.6, 66.0,
1H, J¼10.8 Hz, SO2NH), 7.21 (br t, 1H, NHCO); Epimer:
d 1.43 (s, 3H,
68.4, 80.6, 128.7, 128.9, 134.4, 163.6, 165.4, 173.1, 176.8; HRMS (ESI)
m/z: calcd for C23H30N2O11S2Na: 597.1189 [MþNa]þ. Found:
597.1197.
Me), 1.59 (s, 3H, Me), 4.09 (d, 1H, J¼14 Hz, SO2CH2), 4.14 (d, 1H,
J¼14 Hz, SO2CH2), 4.42 (s, 1H, NHCHCO2), 4.93 (br s, 1H, NHCHCH),
5.15 (br d, 1H, J¼8.1 Hz, NHCHCH), 5.74 (d, 1H, J¼5.5 Hz, OCH2O),
5.96 (d, 1H, J¼5.5 Hz, OCH2O), 6.37 (br t, 1H, CONH), 7.12 (br t, 1H,
NHCO), 7.39 (br d, 1H, J¼8.1 Hz, SO2NH); 13C NMR (125 MHz,
5.2.6. Pivaloyloxymethyl 1,1-dioxide-6-b-[hydroxycarbonyl-methyl-
sulfonamido]-penicillinate, N-ethylmorpholine salt (6) and free acid
CDCl3): d 176.8, 173.4, 168.0, 165.4, 161.9, 80.5, 70.5e69.0, 66.3, 64.6,
(60). To a solution of sulfone 5a (50 mg, 0.087 mmol) in EtOH/EtOAc
63.6, 61.5, 59.6, 56.5, 40.3, 39.9, 39.5, 39.2, 38.6, 36.4, 34.6, 28.9,
26.9, 25.4, 20.1, 17.6; Epimer: 69.7, 62.4, 61.3, 19.9, 18.3; MS (ESI): m/
z¼824.95 [MþNa]þ 100%, 802.83 [MþH]þ, 18%. 9: 1H NMR
(4:1) (5 mL) were added successively N-ethylmorpholine (11
mL,
0.087 mmol) and Pd on activated carbon (0.0087 mmol). The
mixture was submitted to hydrogenation for 1 h at room temper-
ature (pH2¼1 atm). Filtration on Celite and concentration under
vacuum gave the salt 6 as a colourless oil (49 mg, 95%); 1H NMR
(300 MHz, CDCl3):
d
1.24 (s, 9H, Me(tBu)), 1.44 (s, 3H, Me), 1.48 (m,
2H, NHCOC2H4CH2), 1.60 (s, 3H, Me), 1.70 (m, 4H,
NHCOCH2CH2þNHCOC3H6CH2), 1.92 (m, 1H, S2CHCH2), 2.22 (m, 2H,
NHCOCH2CH2), 2.48 (m, 2H, S2CHCH2), 3.17 (m, 2H, S2CH2),
(300 MHz, acetone-d6):
d
1.21 (s, 9H, Me(tBu)), 1.33 (t, 3H, J¼6.7 Hz,
Me-morph), 1.45 (s, 3H, Me), 1.56 (s, 3H, Me), 3.04e3.11 (m, 6H,
NHþ(morph)-CH2, CH2eCH3), 3.92e3.93 (m, 4H, O(morph)-CH2),
4.06 (sharp qAB, 2H, SO2CH2CO), 4.53 (s, 1H, NCHCO2), 4.35 (d, 1H,
J¼4.7 Hz, NHCHCH), 5.66 (d, 1H, J¼4.7 Hz, NHCHCH), 5.82 (d, 1H,
3.45e3.57 (m, 4H, CONHCH2), 3.60e3.70 (m, 13H, S2CH, OCH2
-
CH2O), 4.12 (d, 1H, J¼14.5 Hz, SO2CH2), 4.23 (d, 1H, J¼14.5 Hz,
SO2CH2), 4.52 (s, 1H, NHCHCO2), 5.03 (d, 1H, J¼4.5 Hz, NHCHCH),
5.54 (dd, 1H, J¼4.5 Hz and 10.7 Hz, NHCHCH), 5.74 (d, 1H, J¼5.5 Hz,

10824
A. Favre et al. / Tetrahedron 68 (2012) 10818e10826
OCH2O), 5.97 (d, 1H, J¼5.5 Hz, OCH2O), 6.24 (br t, 1H, CONH), 6.75
(d, 1H, J¼10.7 Hz, SO2NH), 7.36 (br t, 1H, NHCO); 13C NMR (75 MHz,
SO2NH), 7.30e7.37 (m, 5H, Ph); 13C NMR (75 MHz, CDCl3):
d 17.5,
20.0, 26.8, 38.8, 40.9, 54.5, 60.9, 63.5, 64.4, 65.1, 66.1, 66.6, 69.6,
69.9, 70.1, 70.3, 70.7, 80.5, 128.1, 128.2, 128.5, 136.7, 156.5, 165.5,
173.9, 176.8; HRMS (ESI) m/z: calcd for C30H45 N3O14S2Na: 758.2241
[MþNa]þ. Found: 758.2250.
CDCl3): d 176.8, 173.3, 168.0, 165.4, 162.1, 80.6, 70.1e69.2, 66.3, 64.5,
63.5, 61.6, 59.4, 56.5, 40.2, 39.9, 39.1, 38.8, 38.4, 36.2, 34.6, 29.7,
26.8, 25.4, 20.0, 17.6; MS (ESI): m/z¼869.04 [MþNa]þ 100%, 846.9
[MþH]þ 25%.
Compound 22 was similarly prepared from 20 (116 mg,
0.15 mmol) and KMnO4 (51 mg, 0.325 mmol). After work-up, the
penam-sulfone 22 was recovered as a colourless oil (120 mg, 95%);
5.3. Synthesis of 6-
3 (second strategy)
b-sulfonamido-penam sulfones of Scheme
[
a
]
65.4 (c 0.81, CHCl3); IR (film):
n
1807, 1778, 1755, 1711, 1520,
1456, 1325, 1259, 1149, 1107, 1026, 976, 737 cmꢀ1 1H NMR
(300 MHz, CDCl3):
1.22 (s, 9H, Me(tBu)), 1.39 (s, 3H, Me), 1.55 (s,
D
;
5.3.1. Coupling of the spacer-arm 18 to 1a. To a solution of 1a
d
(127 mg, 0. 25 mmol) in DCM (3 mL) at 0 ꢁC, was added triethyl-
3H, Me), 3.38 (m, 1H, SO2CH2CH2), 3.39e3.41 (m, 2H, CONHCH2),
3.47 (m, 1H, SO2CH2CH2), 3.55e3.63 (m, 14H, OCH2CH2O), 3.82 (m,
1H, SO2CH2CH2), 3.93 (m, 1H, SO2CH2CH2), 4.47 (s, 1H, NCHCO2),
4.96 (d, 1H, J¼4.6 Hz, NHCHCH), 5.10 (s, 2H, CH2Ph), 5.46 (br s, 1H,
O2CNH), 5.58 (dd, 1H, J¼4.6, 11.1 Hz, NHCHCH), 5.70 (d, 1H,
J¼5.4 Hz, OCH2O), 5.94 (d, 1H, J¼5.4 Hz, OCH2O), 6.33 (d, 1H,
J¼11.1 Hz, SO2NH), 7.32e7.36 (m, 5H, Ph); 13C NMR (75 MHz,
amine (71
mL, 0.5 mmol). After 10 min, a solution of sulfonyl-
chloride 18a (104 mg, 0.25 mmol) in DCM (2 mL) was added. After
10 min at 0 ꢁC and 40 min at room temperature, the mixture was
washed by water and brine, dried over MgSO4 and concentrated.
The crude product was purified by column-chromatography on
silica gel (EtOAc) to give 19 (127 mg, 71%) as a yellow oil; Rf¼0.25
(EtOAc); [
a
]
D
þ62.4 (c 0.17, CHCl3); IR (film):
n
1786, 1757, 1705,
1H
1.21 (s, 9H, Me(tBu)), 1.50 (s, 3H, Me), 1.63
CDCl3): d 17.5, 20.0, 26.8, 38.8, 40.9, 54.6, 60.9, 63.5, 64.4, 65.1, 66.1,
1529, 1456, 1331, 1279, 1254, 1145, 1111, 1026, 982, 733 cmꢀ1
NMR (300 MHz, CDCl3):
;
66.6, 69.7, 70.0, 70.2, 70.3, 70.4, 70.5, 70.7, 80.5, 128.0, 128.1, 128.5,
136.7, 156.5, 165.5, 174.0, 176.8; HRMS (ESI) m/z: calcd for
C32H50N3O15S2: 780.2683 [MþH]þ. Found: 780.2676.
d
(s, 3H, Me), 3.27 (m, 1H, SO2CH2CH2), 3.38e3.41 (m, 2H, CONHCH2),
3.57e3.70 (m, 11H, SO2CH2CH2, OCH2CH2O), 3.86e3.88 (m, 2H,
SO2CH2CH2), 4.43 (s, 1H, NCHCO2), 5.10 (s, 2H, CH2Ph), 5.20 (dd, 1H,
J¼3.6 Hz, 10.0 Hz, NHCHCH), 5.44 (br s, 1H, O2CNH), 5.52 (d, 1H,
J¼3.6 Hz, NHCHCH), 5.75 (d, 1H, J¼5.3 Hz, OCH2O), 5.86 (d, 1H,
J¼5.3 Hz, OCH2O), 5.95 (d, 1H, J¼10.0 Hz, SO2NH), 7.30e7.33 (m, 5H,
5.3.3. Hydrogenolysis of penam-sulfones 21,22. To a solution of 21
(65 mg, 0.088 mmol) in EtOH/EtOAc (4:1) (6.5 mL) were added Pd
on activated carbon (0.0088 mmol) and p-toluenesulfonic acid
(15 mg, 0.088 mmol). The mixture was submitted to hydrogenation
for 1 h at room temperature (pH2¼1 atm). Filtration on Celite,
washing with acetone and concentration under vacuum gave the
deprotected product 23 (n¼2) as a colourless oil, which was used
without further purification.
Ph); 13C NMR (75 MHz, CDCl3):
d 26.8, 26.9, 31.5, 38.8, 40.9, 53.8,
62.0, 64.6, 65.1, 66.6, 67.9, 69.7, 69.9, 70.2, 70.3, 79.8, 128.1, 128.2,
128.5, 136.6, 156.5, 166.4, 173.4, 176.8; HRMS (ESI) m/z: calcd for
C30H45N3O12S2Na: 726.2342 [MþNa]þ. Found: 726.2369.
Compounds 20 was similarly prepared from 1a (145 mg,
The deprotected compound 24 (n¼3) was similarly prepared. In
both cases, the complete disappearance of the Cbz group was ver-
ified by 1H NMR.
0.028 mmol)
chromatography afforded 20 (171 mg, 79%) as a yellow oil; [
þ110.0 (c 0.17, CHCl3); IR (film): 1788, 1759, 1713, 1524, 1454, 1338,
1281, 1252, 1148, 1107, 1026, 978, 721 cmꢀ1 1H NMR (300 MHz,
CDCl3):
1.23 (s, 9H, Me(tBu)), 1.52 (s, 3H, Me), 1.64 (s, 3H, Me), 3.30
and
18b
(146 mg,
0.31 mmol).
Column-
a
]
D
n
;
5.3.4. Coupling of 23,24 to thioctic acid. To a solution of 23 (PTSA
salt) (68 mg, 0.088 mmol) and N-hydroxysuccinimide ester of TA
(26 mg, 0.088 mmol) in anhydrous DMF (3 mL) was added N-eth-
ylmorpholine (0.176 mmol). The mixture was stirred for 24 h at
room temperature. EtOAc was added (5 mL). The organic phase was
washed with brine (6ꢂ), dried over MgSO4 and concentrated. The
crude product was purified by column-chromatography on silica
d
(m, 1H, SO2CH2CH2), 3.40e3.43 (m, 2H, CONHCH2), 3.56e3.66 (m,
15H, SO2CH2CH2, OCH2CH2O), 3.91e3.92 (m, 2H, SO2CH2CH2), 4.44
(s, 1H, NCHCO2), 5.11 (s, 2H, CH2Ph), 5.22 (dd, 1H, J¼4.4, 10.0 Hz,
NHCHCH), 5.40 (br s, 1H, O2CNH), 5.55 (d, 1H, J¼4.4 Hz, NHCHCH),
5.77 (d,1H, J¼5.5 Hz, OCH2O), 5.87 (d,1H, J¼5.5 Hz, OCH2O), 5.92 (d,
1H, J¼10.0 Hz, SO2NH), 7.30e7.36 (m, 5H, Ph); 13C NMR (75 MHz,
gel to give 25 as a yellow oil (12 mg, 18%); Rf¼0.27 (EtOAc); [
þ51.2 (c 0.2, CHCl3); IR (film): 1801, 1772, 1757, 1659, 1634, 1541,
1468, 1423, 1331, 1277, 1149, 1115, 978, 735 cmꢀ1 1H NMR
(300 MHz, CDCl3):
1.24 (s, 9H, Me(tBu)), 1.43 (s, 3H, Me), 1.45e1.47
a]
D
CDCl3):
d
26.8, 26.9, 31.6, 38.8, 40.9, 53.8, 62.1, 64.6, 65.2, 66.6, 68.0,
n
69.7, 70.0, 70.2, 70.4, 70.5, 79.8, 128.1, 128.2, 128.5, 136.6, 156.5,
166.4, 173.4, 176.9; HRMS (ESI) m/z: calcd for C32H49N3O13S2Na:
770.2605 [MþNa]þ. Found: 770.2595.
;
d
(m, 2H, NHCOC2H4CH2), 1.59 (s, 3H, Me), 1.67e1.69 (m, 4H,
NHCOCH2CH2, NHCOC3H6CH2), 1.94 (m, 1H, S2CHCH2), 2.25 (t, 2H,
J¼7.4 Hz, NHCOCH2CH2), 2.47 (m, 1H, S2CHCH2), 3.05e3.22 (m, 2H,
S2CH2), 3.37 (m, 1H, SO2CH2CH2), 3.42e3.49 (m, 3H, SO2CH2CH2,
CONHCH2), 3.59e3.97 (m, 11H, OCH2CH2O, S2CH), 3.96e3.98 (m,
2H, SO2CH2CH2), 4.43 (s, 1H, NCHCO2), 5.12 (dd, 1H, J¼1.3, 10.1 Hz,
NHCHCH), 5.14 (d, 1H, J¼1.3 Hz, NHCHCH), 5.73 (d, 1H, J¼5.4 Hz,
OCH2O), 5.98 (d, 1H, J¼5.4 Hz, OCH2O), 6.48 (br s, 1H, CONH), 7.02
5.3.2. Oxidation of the penams 19,20. The penam 19 (43 mg,
0.06 mmol) dissolved in a 4:1 mixture of AcOH and H2O (4 mL) was
cooled at ꢀ15 ꢁC, under vigorous stirring. KMnO4 (20 mg,
0.13 mmol) dissolved in water (2 mL) was added very slowly
(30 min). The mixture was further stirred for 1 h at ꢀ10 ꢁC, then
10% H2O2 was added to discolour the solution. DCM (15 mL) was
added. The organic phase was washed twice with 5% NaHCO3, dried
over MgSO4 and concentrated. The penam-sulfone 21 was obtained
(d, 1H, J¼10.1 Hz, SO2NH); 13C NMR (75 MHz, CDCl3):
d 18.2, 20.0,
25.4, 26.8, 28.9, 34.6, 36.1, 38.4, 38.8, 38.9, 40.2, 52.1, 56.5, 61.6, 62.5,
63.2, 65.2, 68.1, 69.5, 69.7, 69.8, 70.1, 70.5, 80.4, 165.3, 168.6, 173.5,
176.8; HRMS (ESI) m/z: calcd for C30H50N3O13S4: 788.2227 [MꢀH]-.
Found: 788.2234.
as a colourless oil (42 mg, 90%); Rf¼0.46 (hexane/EtOAc (3:1)); [
þ72.1 (c 0.19, CHCl3); IR (film): 1809, 1776, 1757, 1705, 1521, 1456,
1325, 1281, 1149, 1113, 1028, 980, 737 cmꢀ1 1H NMR (300 MHz,
CDCl3):
1.23 (s, 9H, Me(tBu)), 1.39 (s, 3H, Me), 1.55 (s, 3H, Me), 3.37
a]
D
n
;
d
Compound 24 was similarly treated to furnish 26 as a yellow oil
(m, 1H, SO2CH2CH2), 3.39e3.40 (m, 2H, CONHCH2), 3.43 (m, 1H,
SO2CH2CH2), 3.55e3.62 (m, 10H, OCH2CH2O), 3.82 (m, 1H,
SO2CH2CH2), 3.92 (m,1H, SO2CH2CH2), 4.47 (s,1H, NCHCO2), 4.88 (d,
1H, J¼4.6 Hz, NHCHCH), 5.11 (s, 2H, CH2Ph), 5.50 (br s, 1H, O2CNH),
5.56 (dd, 1H, J¼4.6, 11.2 Hz, NHCHCH), 5.69 (d, 1H, J¼5.4 Hz,
OCH2O), 5.94 (d, 1H, J¼5.4 Hz, OCH2O), 6.36 (d, 1H, J¼11.2 Hz,
(10% yield after column-chromatography); Rf¼0.27 (EtOAc); [
þ64.8 (c 0.15, CHCl3); IR (film): 1803, 1774, 1755, 1660, 1635, 1540,
1469 cmꢀ1; 1H NMR (500 MHz, CDCl3): 1.22 (s, 9H, Me(tBu)), 1.40
(s, 3H, Me), 1.45e1.50 (m, 2H, NHCOC2H4CH2), 1.58 (s, 3H, Me),
1.67e1.73 (m, 2H, NHCOCH2CH2), 1.74 (br s, 2H, NHCOC3H6CH2),
1.90e1.96 (m, 1H, S2CHCH2), 2.22 (t, 2H, J¼7.5 Hz, NHCOCH2CH2),
a]
D
n
d

A. Favre et al. / Tetrahedron 68 (2012) 10818e10826
10825
2.44e2.51 (m, 1H, S2CHCH2), 3.13 (m, 1H, S2CH2), 3.21 (m, 1H,
S2CH2), 3.30e3.36 (m, 1H, SO2CH2CH2), 3.40e3.52 (m, 3H,
SO2CH2CH2, CONHCH2), 3.58e3.83 (m, 15H, OCH2CH2O, S2CH),
3.92e4.01 (m, 2H, SO2CH2CH2), 4.39 (s, 1H, NCHCO2), 5.07 (dd, 1H,
J¼1.5, 10.1 Hz, NHCHCH), 5.29 (d, 1H, J¼1.5 Hz, NHCHCH), 5.70 (d,
1H, J¼5.4 Hz, OCH2O), 5.96 (d, 1H, J¼5.4 Hz, OCH2O), 6.39 (br s, 1H,
CONH), 7.04 (d, 1H, J¼10.1 Hz, SO2NH); 13C NMR (75 MHz, CDCl3):
5.4.2. Coupling of 30 to the photo-cleavable ester of 6-APA. 2-Nitro-
4,5-(dimethoxy)-benzyl 6-
-aminopenicillinate (330): 6-APA (1 g,
4.6 mmol) and triethylamine (1.30 mL, 9.2 mmol) in DCM (10 mL)
were stirred at room temperature until total dissolution. Methyl
b
acetoacetate (502 mL) was added and the mixture was stirred for
3 h. The solvent was evaporated and the crude 31 was dissolved in
dry DMF (10 mL) with 4,5-dimethoxy-2-nitrobenzyl bromide
(1.28 g, 4.6 mmol). The mixture was stirred overnight, then diluted
with EtOAc (100 mL). The solution was washed with brine (3ꢂ),
dried over MgSO4 and concentrated under vacuum. The solid res-
idue (32) was dissolved in acetone (10 mL) with p-toluenesulfonic
acid monohydrate (967 mg). Within 15 min a solid appeared (33). It
was filtered and washed with EtO2. Finally, crude 33 was solubi-
lized with triethylamine (1.30 mL) in DCM (50 mL) and stirred at
room temperature for 2 h. A solution of NaOH 1 M was added and
the organic layer was separated. The aqueous layer was extracted
with DCM. The combined organic layer was washed with brine,
dried over MgSO4 and concentrated under vacuum to give the free
d
18.3, 20.0, 25.5, 27.0, 29.0, 34.8, 36.4, 38.6, 39.3, 40.3, 40.2, 52.6,
56.6, 61.7, 62.5, 63.2, 65.4, 68.3, 69.7, 69.9, 70.0, 70.2, 70.4, 70.5, 70.7,
80.5, 165.5, 169.3, 173.2, 176.9.
5.4. Synthesis of benzylpenicillin derivatives of Scheme 5
5.4.1. Synthesis of the O-substituted 4-hydroxyphenylacetic side-
chains 29,30. Mitsunobu coupling: To the mixture of DIAD (429 mL,
2.16 mmol) and triphenylphosphine (568 mg, 2.16 mmol) in anhy-
drous THF (5 mL) was added at room temperature methyl 4-
hydroxyphenylacetate 28 (264 mg, 1.58 mmol). The mixture was
stirred for 30 min, then 27a or 27b (1.44 mmol) was added. After
stirring for 20 h, the mixture was concentrated and purified by flash
chromatography on silica gel (cyclohexane/EtOAc (1:1)) to give 29a
amine (330) as a yellow solid (86% yield); Mp¼81 ꢁC; IR (film):
n
3338, 1778, 1743, 1522 cmꢀ1 1H NMR (200 MHz, CDCl3):
; d 1.47 (s,
3H, Me), 1.61 (s, 3H, Me), 3.92 (s, 3H, OMe), 3.95 (s, 3H, OMe), 4.41
(s, 1H, NCHCO2), 4.54 (d, 1H, J¼5.7 Hz, NHCHCH), 5.47 (d, 1H,
J¼5.7 Hz, NHCHCH), 5.53 (s, 2H, CH2Ph) 6.98 (s, 1H, MeOCCH(Ph)),
or 29b (>90% yield) as a translucent oil. Compound 29a: IR (film):
n
1745 cmꢀ1 1H NMR (200 MHz, CDCl3):
;
d
1.65 (quint, 2H, J¼8.5 Hz,
]CHCH2CH2), 2.08 (q, 2H, J¼8.5 Hz, ]CHCH2CH2), 3.44 (t, 2H,
J¼8.3 Hz, ]CHC2H4CH2), 3.53e3.68 (m, 17H, OCH2CH2Oþ OMe),
3.81 (s, 2H, PhCH2), 4.08 (t, 2H, J¼5.6 Hz, PhOCH2), 4.95 (dd, 1H,
J¼1.8, 10.3 Hz, CH2]), 5.01 (dd, 1H, J¼1.8, 16.9 Hz, CH2]), 5.79 (ddt,
1H, J¼16.9,10.3, 6.7 Hz, CH]), 6.85 (d, 2H, J¼8.8 Hz, Ph), 7.16 (d, 2H,
7.68 (s, 1H, NO2CCH(Ph)); 13C NMR (50 MHz, CDCl3):
d 27.3, 31.6,
56.8, 63.1, 64.2, 64.4, 70.2, 70.5, 77.9, 108.8, 110.6, 125.7, 140.6, 149.0,
153.9, 167.8, 178.1; Anal. Calcd for C17H21N3O7S: C 49.63, H 5.14, N
10.21; found: C 49.61, H 5.13, N 9.98%.
Coupling of the side-chains 30 to 330: The side-chain 30a or 30b
(0.396 mmol) in dry DCM (5 mL) was treated with dicyclohex-
ylcarbodiimide (90 mg, 0.436 mmol) at room temperature for
30 min. After filtration on Celite, 330 (163 mg, 0.396 mmol) was
added to the solution. The mixture was stirred for 6 h, then con-
centrated and purified by preparative TLC (DCM/EtOAc (1:1)) to
furnish 34 or 35 as a yellow oil (58% and 70% yield, respectively);
J¼8.8 Hz, Ph); 13C NMR (50 MHz, CDCl3):
d 28.8, 30.1, 40.2, 51.8, 67.5,
69.7, 70.1, 70.6, 70.8, 114.5, 114.8, 126.2, 130.1, 138.2, 157.9, 172.1;
HRMS (ESI) m/z: calcd for C22H34O7Na: 433.2202 [MþNa]þ. Found:
433.2213. Compound 29b: IR (film):
n ;
1745 cmꢀ1 1H NMR
(250 MHz, CDCl3):
d 1.24e1.42 (m, 12H, ]CHC2H4C6H12), 1.51 (m,
2H, ]CHCH2CH2), 1.95 (q, 2H, J¼7.1 Hz, ]CHCH2CH2), 3.39 (t, 2H,
J¼6.7 Hz, alkCH2O), 3.36e3.67 (m, 17H, OCH2CH2Oþ OMe), 3.78 (s,
2H, PhCH2), 4.05 (t, 2H, J¼5.4 Hz, PhOCH2), 4.90 (dd, 1H, J¼1.8,
10.3 Hz, CH2]), 4.94 (dd, 1H, J¼1.8, 16.9 Hz, CH2]), 5.75 (ddt, 1H,
J¼16.9, 10.3, 6.7 Hz, CH]), 6.81 (d, 2H, J¼8.7 Hz, Ph), 7.12 (d, 2H,
34: IR (film):
CDCl3): 1.40 (s, 3H, Me), 1.55 (s, 3H, Me), 1.65 (m, 2H, ]
n ;
3328, 1775, 1740, 1512 cmꢀ1 1H NMR (500 MHz,
d
CHCH2CH2), 2.08 (q, 2H, J¼6.6 Hz, ]CHCH2CH2), 3.44 (t, 2H,
J¼6.8 Hz, ]CHC2H4CH2), 3.54e3.71 (m, 14H, OCH2CH2O), 3.83 (s,
2H, PhCH2), 3.94 (s, 3H, OMe), 3.95 (s, 3H, OMe), 4.09 (t, 2H,
J¼4.7 Hz, PhOCH2), 4.38 (s, 1H, NCHCO2), 4.92 (dd, 1H, J¼1.8, 9.0 Hz,
CH2]CH), 4.98 (dd, 1H, J¼1.8, 16.9 Hz, CH2]CH), 5.47 (d, 1H,
J¼4.2 Hz, NHCHCH), 5.53 (s, 2H, OCH2Ph), 5.62 (dd, 1H, J¼9.0,
4.2 Hz, NHCHCH), 5.78 (ddt, 1H, J¼16.9, 9.0, 6.6 Hz, CH]), 6.11 (d,
1H, J¼9.0 Hz, NH) 6.88 (d, 2H, J¼8.6 Hz, Ph), 6.96 (s, 1H,
MeOCCH(Ph)), 7.14 (d, 2H, J¼8.6 Hz, Ph), 7.69 (s, 1H, NO2CCH(Ph));
J¼8.7 Hz, Ph); 13C NMR (62.5 MHz, CDCl3):
d 21.7, 25.8, 28.7, 28.9,
29.2, 33.6, 40.0, 51.7, 67.2, 69.5, 69.8, 70.3, 71.3, 113.9, 114.5, 126.0,
130.0, 139.0, 157.7, 172.1; HRMS (ESI) m/z: calcd for C28H46O7Na:
517.3141 [MþNa]þ. Found: 517.3146.
Saponification: The ester 29a or 29b (2.29 mmol) was treated
with NaOH 1 M (2 mL) in MeOH (5 mL) at room temperature for
2 h, then the mixture was acidified by addition of HCl 1 M. The
aqueous phase was extracted with EtOAc (3ꢂ). The organic layer
was washed with brine, dried over MgSO4 and concentrated to
furnish 30a or 30b as a translucent oil (>90% yield). Compound
13C NMR (125 MHz, CDCl3):
d 26.5, 28.6, 30.0, 31.2, 42.3, 56.3, 56.4,
58.5, 64.2, 67.3, 67.8, 69.5, 69.9, 70.3, 70.4, 70.5, 70.7, 108.2, 111.1,
114.5, 115.1, 125.1, 125.7, 130.4, 138.1, 140.2, 148.6, 153.4, 158.2, 166.9,
170.6, 173.6; HRMS (ESI) m/z: calcd for C38H51N3O13SNa: 812.3040
[MþNa]þ. Found: 812.3010; 35: IR (film): 3328, 1774, 1740,
30a: IR (film): n d 1.65
3200, 1710 cmꢀ1; 1H NMR (200 MHz, CDCl3):
(quint, 2H, J¼8.5 Hz, CH2), 2.08 (q, 2H, J¼8.5 Hz, CH2), 3.44 (t, 2H,
J¼8.3 Hz, CH2), 3.53e3.68 (m, 14H, CH2), 3.81 (s, 2H, CH2Ph), 4.08 (t,
2H, J¼5.6 Hz, CH2CH2O), 4.90e5.03 (m, 2H, CH2]), 5.79 (ddt, 1H,
J¼16.9, 10.3, 6.7 Hz, CH]), 6.85 (d, 2H, J¼8.8 Hz, Ph), 7.16 (d, 2H,
1512 cmꢀ1 1H NMR (500 MHz, CDCl3):
; d 1.24e1.38 (m, 12H, ]
CHC2H4C6H12), 1.32 (s, 3H, Me), 1.53 (s, 3H, Me), 1.54 (m, 2H, ]
CHCH2CH2), 2.00 (q, 2H, J¼6.6 Hz, ]CHCH2CH2), 3.41 (t, 2H,
J¼6.8 Hz, alkCH2O), 3.54e3.71 (m, 14H, OCH2CH2O), 3.83 (s, 2H,
PhCH2), 3.94 (s, 3H, OMe), 3.95 (s, 3H, OMe), 4.09 (t, 2H, J¼4.9 Hz,
PhOCH2), 4.38 (s, 1H, NCHCO2), 4.89 (dd, 1H, J¼1.8, 9.0 Hz, CH2]
CH), 4.96 (d, 1H, J¼1.8, 16.9 Hz, CH2]CH), 5.47 (d, 1H, J¼4.2 Hz,
NHCHCH), 5.53 (s, 2H, OCH2Ph), 5.62 (dd, 1H, J¼9.0, 4.2 Hz,
NHCHCH), 5.78 (ddt, 1H, J¼16.9, 9.0, 6.6 Hz, CH]), 6.14 (d, 1H,
J¼9.0 Hz, NH) 6.88 (d, 2H, J¼8.6 Hz, Ph), 6.96 (s, 1H, MeOCCH(Ph)),
7.14 (d, 2H, J¼8.6 Hz, Ph), 7.69 (s, 1H, NO2CCH(Ph)); 13C NMR
J¼8.8 Hz, Ph); 13C NMR (50 MHz, CDCl3):
d 28.9, 30.3, 40.2, 67.9,
69.8, 70.2, 70.7, 70.9, 114.8, 114.9, 125.8, 130.4, 138.3, 158.1, 176.7;
HRMS (ESI) m/z: calcd for C21H32O7Na: 419.2046 [MþNa]þ. Found:
419.2013. 30b: IR (film):
n ;
3200, 1710 cmꢀ1 1H NMR (250 MHz,
CDCl3):
d 1.24e1.42 (m, 12H, CH2), 1.51 (m, 2H, CH2), 1.95 (q, 2H,
J¼7.1 Hz, CH2C]C), 3.39 (t, 2H, J¼6.7 Hz, CH2), 3.36e3.67 (m, 14H,
CH2), 3.78 (s, 2H, CH2Ph), 4.05 (t, 2H, J¼5.4 Hz, CH2CH2O),
4.85e4.96 (m, 2H, CH2]), 5.75 (ddt, 1H, J¼16.9, 10.3, 6.7 Hz, CH]),
6.81 (d, 2H, J¼8.7 Hz, Ph), 7.12 (d, 2H, J¼8.7 Hz, Ph); 13C NMR
(125 MHz, CDCl3): d 26.5, 28.6, 30.0, 31.2, 42.2, 56.2, 56.4, 58.5, 64.2,
(62.5 MHz, CDCl3):
d
21.9, 26.0, 28.9, 29.1, 29.3, 29.4, 33.7, 40.1, 67.5,
67.3, 67.8, 69.5, 69.8, 70.3, 70.4, 70.6, 71.3, 108.2, 111.1, 113.9, 115.1,
125.0, 125.7, 130.4, 139.0, 140.1, 148.5, 153.3, 158.1, 167.0, 170.6,
173.5; HRMS (ESI) m/z: calcd for C44H63N3O13SNa: 896.3979
[MþNa]þ. Found: 896.3946.
69.7, 70.0, 70.5, 70.8, 71.5, 114.0, 114.8, 125.9, 130.3, 139.1, 158.0,
176.4; HRMS (ESI) m/z: calcd for C27H44O7Na: 503.2985 [MþNa]þ.
Found: 503.2953.

10826
A. Favre et al. / Tetrahedron 68 (2012) 10818e10826
5.5. Biological evaluations
References and notes
1. Cremer, P. S. J. Am. Chem. Soc. 2011, 133, 167e169 and the volume: special issue
on Surface Patterning.
2. Zhu, H.; Snyder, M. Curr. Opin. Chem. Biol. 2001, 5, 40e45 and 2003, 7, 55e63.
3. Phizicky, E.; Bastiaens, P. I.; Zhu, H.; Snyder, M.; Fields, S. Nature 2003, 422,
208e215.
5.5.1. Assay with BlaP (class A b-lactamase). BlaP (5 mL, solution of
42 ng/mL) in buffer (50
1 mg/mL, pH 7.2) and tested molecule (5
concentrations) were incubated at 30 ꢁC (multi-well plate) for
60 min. Nitrocefine (50 L, at 100 M) was added just before
m
L, NaPi buffer 50 mM, with ovalbumin
mL solution at different
4. Cheung, C. L.; Camarero, J. A.; Woods, B. W.; Lin, T. W.; Johnson, J. E.; De Yoreo,
J. J. J. Am. Chem. Soc. 2003, 125, 6848e6849.
m
m
reading at 482 nm (Power-Wave microplate-reader, BioTek). The
nitrocefine hydrolysis by the residual active enzyme (i.e., BlaP non
acylated by the tested compound) gives immediately a coloured
product. Pre-incubation of EOM esters with buffer (4 h, 20 ꢁC for
ester hydrolysis) afforded similar results (incubation with the en-
zyme for 15 min).
5. Bronze, M. S.; Greenfield, R. A. Drug Discovery Today 2003, 8, 740e745.
6. Lee, K.-B.; Park, S.-J.; Mirkin, C. A.; Smith, J. C.; Mrksich, M. Science 2002, 295,
1702e1705.
7. Kim, K.; Yang, H.; Jon, S.; Kim, E.; Kwak, J. J. Am. Chem. Soc. 2004, 126,
15368e15369 and references cited therein.
8. Camarero, J. A.; Kwon, Y.; Coleman, M. A. J. Am. Chem. Soc. 2004, 126,
14730e14731 and references cited therein.
9. Hubschwerlen, C., Chapter 7.17 In Comprehensive Medicinal Chemistry, 2nd ed.;
Taylor, J. B., Triggle, D. J., Eds.; Elsevier: Oxford, UK, 2007; pp 479e517.
ꢀ
10. Marchand-Brynaert, J.; Brule, C. In Comprehensive Heterocyclic Chemistry, 3rd
5.5.2. Assay with P99 (class C
400 ng/mL) in buffer (50 L, HEPES buffer 50 mM with NaCl 200 mM
L solution at
b-lactamase). P99 (5 mL, solution of
ed.; Katritzky, A. R., Ramsden, C. A., Scriven, E. F. V., Taylor, R. J. K., Eds.; Peni-
cillins; Elsevier: Oxford, UK, 2008; Vol. 2, pp 173e238; (CHC III); Chapter 2.03.
11. Moreau, J.; Marchand-Brynaert, J. Eur. J. Org. Chem. 2011, 1641e1644.
12. Vanwetswinkel, S.; Fastrez, J.; Marchand-Brynaert, J. J. Antibiot. 1994, 47,
1041e1051.
m
and ovalbumin 1 mg/mL, pH 8) and tested molecule (5
m
different concentrations) were incubated at 30 ꢁC (multi-well plate)
for 60 min. Reading at 482 nm is performed as above. Previous hy-
drolysis of EOM esters (see above) gave similar results.
13. Vanwetswinkel, S.; Carlier, V.; Marchand-Brynaert, J.; Fastrez, J. Tetrahedron
Lett. 1996, 37, 2761e2762.
14. Yadav, J. S.; Ganganna, B.; Buhnia, D. C.; Srihari, P. Tetrahedron Lett. 2009, 50,
4318e4320.
5.5.3. Assay with R39 D,D-carboxypeptidase. R39 (5
100 g/mL) in buffer (5 L, NaPi buffer 50 mM with NaCl 200 mM
and ovalbumine 1 mg/mL, pH 8), water (4 L) and tested molecule
(5
L solution at different concentrations) were incubated at 25 ꢁC
during 15 min (Table 1) or 90 min (Table 2). Fluorescent ampicillin
(1 L,100 M) was added and after 5 min or 15 min, the protein was
denaturated and deposited on the SDS-PAGE 15% gel. After migra-
tion, the fluorescence was measured with a Phospho-Imager (Mo-
lecular Image FX equipment and Quantity One Software, Biorad,
Hercules, CA, USA). The fluorescence intensity is proportional to the
residual active protein (i.e., protein non acylated by the tested
compound).
mL, solution of
15. The Chemistry of
b-lactams; Page, M. I., Ed.; Blackie Academic and Professional:
m
m
London, 1992.
m
16. The Organic Chemistry of
b
-lactams; Gerog, G. I., Ed.; VCH: New York, NY, 1992.
17. Dreesen, L.; Silien, C.; Volcke, C.; Sartenaer, Y.; Thiry, P. A.; Peremans, A.; Gru-
gier, J.; Marchand-Brynaert, J.; Brans, A.; Grubisic, S.; Joris, B. ChemPhysChem
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This work was supported by the Inter-university Attraction Pole
(IAP program P6/19 PROFUSA), the Walloon Government (Waleo 2
program OPARRAYand Waleo 3 program RAPARRAY) and the Fonds
de la Recherche Scientifique (F.R.S.-FNRS, Belgium). Ir S. Grubisic is
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NMR data, are provided as Supplementary data. Supplementary
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