Cyclopropane-1,2-dicarboxylic acids as new tools for the biophysical investigation of O-acetylserine sulfhydrylases by fluorimetric methods and saturation transfer difference (STD) NMR

Article abstract of DOI:10.1080/14756366.2016.1218486

Cysteine is a building block for many biomolecules that are crucial for living organisms. O-Acetylserine sulfhydrylase (OASS), present in bacteria and plants but absent in mammals, catalyzes the last step of cysteine biosynthesis. This enzyme has been dee

Full text of DOI:10.1080/14756366.2016.1218486

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: http://www.tandfonline.com/loi/ienz20
Cyclopropane-1,2-dicarboxylic acids as new tools
for the biophysical investigation of O-acetylserine
sulfhydrylases by fluorimetric methods and
saturation transfer difference (STD) NMR
Giannamaria Annunziato, Marco Pieroni, Roberto Benoni, Barbara
Campanini, Thelma A. Pertinhez, Chiara Pecchini, Agostino Bruno, Joana
Magalhães, Stefano Bettati, Nina Franko, Andrea Mozzarelli & Gabriele
Costantino
To cite this article: Giannamaria Annunziato, Marco Pieroni, Roberto Benoni, Barbara
Campanini, Thelma A. Pertinhez, Chiara Pecchini, Agostino Bruno, Joana Magalhães, Stefano
Bettati, Nina Franko, Andrea Mozzarelli & Gabriele Costantino (2016): Cyclopropane-1,2-
dicarboxylic acids as new tools for the biophysical investigation of O-acetylserine
sulfhydrylases by fluorimetric methods and saturation transfer difference (STD) NMR, Journal
of Enzyme Inhibition and Medicinal Chemistry, DOI: 10.1080/14756366.2016.1218486
To link to this article: http://dx.doi.org/10.1080/14756366.2016.1218486
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ISSN: 1475-6366 (print), 1475-6374 (electronic)
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2016 Informa UK Limited, trading as Taylor & Francis Group. DOI: 10.1080/14756366.2016.1218486
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ORIGINAL ARTICLE
Cyclopropane-1,2-dicarboxylic acids as new tools for the biophysical
investigation of O-acetylserine sulfhydrylases by fluorimetric methods
and saturation transfer difference (STD) NMR
Giannamaria Annunziato1*, Marco Pieroni1*, Roberto Benoni4, Barbara Campanini1, Thelma A. Pertinhez2,3
,
Chiara Pecchini1, Agostino Bruno1, Joana Magalha˜es1, Stefano Bettati4,5, Nina Franko1, Andrea Mozzarelli1,5,6, and
Gabriele Costantino1
2
1Department of Pharmacy, and Department of Biochemical, Biotechnological and Translational Sciences, University of Parma, Parma, Italy,
3Transfusion Medicine Unit, ASMN-IRCCS, Reggio, Emilia, Italy, 4Department of Neurosciences, University of Parma, Parma, Italy, 5National Institute
6
of Biostructures and Biosystems, Rome, Italy, and Institute of Biophysics, CNR, Pisa, Italy
Abstract
Keywords
Cysteine is a building block for many biomolecules that are crucial for living organisms.
O-Acetylserine sulfhydrylase (OASS), present in bacteria and plants but absent in mammals,
catalyzes the last step of cysteine biosynthesis. This enzyme has been deeply investigated
because, beside the biosynthesis of cysteine, it exerts a series of ‘‘moonlighting’’ activities in
bacteria. We have previously reported a series of molecules capable of inhibiting Salmonella
typhimurium (S. typhymurium) OASS isoforms at nanomolar concentrations, using a combin-
ation of computational and spectroscopic approaches. The cyclopropane-1,2-dicarboxylic acids
presented herein provide further insights into the binding mode of small molecules to OASS
enzymes. Saturation transfer difference NMR (STD-NMR) was used to characterize the molecule/
enzyme interactions for both OASS-A and B. Most of the compounds induce a several fold
increase in fluorescence emission of the pyridoxal 50-phosphate (PLP) coenzyme upon binding
to either OASS-A or OASS-B, making these compounds excellent tools for the development of
competition-binding experiments.
O-Acetylserine sulfhydrylase, cyclopropane-
1,2-dicarboxylic acids, cysteine biosynthe-
sis, drug resistance, saturation transfer
difference-NMR
History
Received 28 June 2016
Revised 25 July 2016
Accepted 26 July 2016
Published online 25 August 2016
Introduction
have been identified where OASS-B expression exceeds that of
OASS-A5, including anaerobic conditions that were previously
reported to induce expression of the B isoform6,7. Cysteine
biosynthesis is particularly important for a large number of
microorganisms, especially during infection, when bacteria face
the oxidative stress conditions induced by the host immune
response. Indeed, it has been demonstrated that deletion of genes
belonging to the cysteine biosynthetic pathway leads to a bacterial
phenotype with reduced virulence, compromised fitness and a
susceptibility to antibiotics several fold higher than the wild
strains8,9. The recent disclosure of a number of additional
moonlighting activities of OASS, specifically for OASS-A
isoform, that is, toxin activation in some strains of Escherichia
coli (E. coli)10, gene expression in Bacillus subtilis (B. subtilis)11,
and the involvement of the enzyme in some pathologically
relevant processes as the formation of biofilm and swarming
motility, has further focused the interest of researchers toward
these proteins11–18. These activities span from toxin activation in
contact-dependent growth inhibition of uropathogenic E. coli
strains10, to gene expression in B. subtilis11 and the involvement
of the enzyme in some pathologically-relevant processes as the
formation of biofilm and swarming motility19. Considering that
mammals synthesize cysteine via a metabolic pathway that
involves enzymes different from those present in RSAP, inhibitors
of bacterial cysteine biosynthesis might be able to potentiate
Cysteine biosynthesis is carried out by bacteria and plants through
the so-called reductive sulfate assimilation pathway (RSAP)1,2
.
The RSAP starts with the transport of sulfate inside the cell,
followed by its reduction to bisulfide. Sulfur is then incorporated
into cysteine via the reactions catalyzed by the last two enzymes
of RSAP: serine acetyltransferase (SAT) and O-acetylserine
sulfhydrylase (OASS). The former catalyzes the transfer of an
acetyl group from acetyl-CoA to the hydroxyl of L-serine, leading
to the formation of an activated form of serine, O-acetyl-L-serine
(OAS) and CoA-SH. The latter enzyme catalyzes a two-step
reaction: in the first half-reaction, an a-aminoacrylate is formed
upon b-elimination of substituted L-serine; in the second half-
reaction, the a-aminoacrylate is attacked by sulfide or other sulfur
sources to give L-cysteine3,4. OASS is a pyridoxal 50-phosphate
(PLP)-dependent enzyme that in bacteria is present in two
isoforms, conventionally referred to as OASS-A and OASS-B,
coded by cysK and cysM, respectively. In enteric bacteria, OASS-
A is highly expressed at basal levels, and so far, no conditions
*These authors contributed equally to this work.
Address for correspondence: Marco Pieroni, Department of Pharmacy,
University of Parma, Parma, 43124, Italy. E-mail: marco.pieroni@unipr.it
Barbara Campanini, Deparment of Pharmacy, University of Parma, Parma
43124, Italy. E-mail: barbara.campanini@unipr.it

2
G. Annunziato et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
antibiotic-based therapies20. Over the years, along with other especially useful for the rapid and accurate determination of
groups20,21, we have investigated structural and functional dissociation constants for other classes of ligands that either do
properties of OASS isozymes with the aim of further characteriz- not induce large changes of PLP fluorescence emission intensity,
ing the biological features of these proteins and to explore the or bind with extremely high affinity, that is, behaving as tight
possibility of their inhibition by small molecules. The rational binding inhibitors in the protein concentration range accessible to
design of the first inhibitors was based on the structure of SAT, spectroscopic experiments.
that physiologically inhibits OASS activity upon formation of the
cysteine synthase bioenzyme complex22,23; in particular, it was Methods
considered that the carboxylic moiety of Ile267 of SAT is
Synthetic chemistry
essential for the interaction between the two proteins22–24
.
Therefore, combining a structure-based with a ligand-based The synthesis of title cis-cyclopropane-1,2-carboxylic acid
drug design approach, we planned and synthesized a series of derivatives started from commercially available 3-
cyclopropanecarboxylic acid derivatives that bind to both OASS oxabicyclo[3.1.0]hexane-2,4-dione, which was hydrolyzed with
isoforms at nanomolar concentrations25–27. In order to further either water or ethanol to give, respectively, compounds 2 and 3 in
refine the structure–activity relationships (SAR) around this class good overall yields (Scheme 1). To obtain the trans-cyclopro-
of derivatives, we herein report the design and synthesis of a pane-1,2-carboxylic acid derivatives, diethyl (1R,2R)-cyclopro-
series of cyclopropane-1,2-dicarboxylic acids variously functio- pane-1,2-dicarboxylate was hydrolyzed in the presence of
nalized. Although the binding properties of the previously stoichiometric amount of potassium hydroxide, to give derivatives
reported analogs24–26 could not be improved, the set of com- 5 and 6 in quantitative yields. For the synthesis of substituted
pounds herein presented provides interesting insight into the mode cyclopropane-1,2-dicarboxylic acids (Scheme 2), key intermedi-
of binding of small molecules to OASS enzymes. In particular, ates 13–18 were prepared according to a protocol described by
saturation transfer difference NMR (STD-NMR) was used to McCoy28,29, since the procedures already set by us proved to be
further characterize the molecule/enzyme interactions for both not efficient30,31
.
Therefore, the suitable acrylates and a-halo
OASS-A and B. Interestingly, most of the compounds induce a esters were reacted in the presence of NaH, and the diethyl cis-
several fold increase in fluorescence emission of the pyridoxal 50- cyclopropane-1,2-dicarboxylates were obtained. The subsequent
phosphate (PLP) coenzyme upon binding to either OASS-A or hydrolysis in basic aqueous media afforded the title products 19–
OASS-B, likely due to changes in the polarity of the active-site 25 in good overall yields. Of note, during the cyclopropanation,
microenvironment. This property, together with the modest the solvent plays a key role in the stereochemistry of the resulting
binding affinity, makes these compounds excellent tools for the molecules, with toluene being the best in conferring the desired
development of competition binding experiments. This is cis configuration. Reacting benzyl cyanide and epichlorohydrin in
the presence of sodium amide, at 90 ꢀC in benzene, the
corresponding cyclopropyl alcohol was obtained, that on turn
O
O
O
O
O
underwent basic hydrolysis to give the title compound 28 in 44%
overall yields (Scheme 3). Attempts to synthesize the ethyl ester
of 28 according to other reported procedures27,30 failed to give the
desired compound.
a orb
OR
HO
1
2, R=H
3, R=Et
NMR measurements for STD experiments
NMR spectra were acquired on a Varian (Agilent Technologies,
Santa Clara, CA) Inova 600 NMR Spectrometer at 20 ꢀC, on
samples with a molar excess of 1:300. Ligand concentration was
kept at 3 mM in the presence of 10 mM protein and were dissolved
in phosphate buffer pH 8.0 (5 mM K2PO4, 3% DMSO-d6).
Saturation transfer difference (STD) experiments were col-
lected with 64 K data points in the direct dimension and 512 scans
in a spectral window of 7000 Hz. The water signal was suppressed
by the excitation sculpting method (dpfgse_water). Selective
saturation of the protein resonances (on resonance spectrum) was
performed by irradiating at ꢁ0.9 ppm using Gaussian-shaped
O
O
O
O
c or d
OEt
EtO
OR
HO
5, R=H
6, R=Et
4
Scheme 1. aReagents and conditions: (a) H2O, reflux, 2 h, 98%; (b)
EtOH, pyr, reflux, 12 h, 96%; (c) 1. 6N NaOH, reflux, 12 h, 2. aq. HCl,
pH 2-3, 97%; (d) 14N NaOH (1 equiv.), EtOH, reflux, 15 min, 98%; bFor
complete structures (Table 1).
1
2
1
2
1
2
R
Cl
R
R
R
R
R
RO
OH
EtO
OEt
b or c
a
+
OEt
O
OEt
O
O
O
O
O
7, R1=H
8, R1=CH3
19, R=H R1=CH3 R2=H
20, R=H R1=CH2CH3 R2=H
21, R=H R1=Ph R2=H
22, R=H R1=CH3 R2=CH3
23, R=H R1=Ph R2=CH3
24, R=H R1=Bn R2=CH3
condition c
13, R1=CH3 R2=H
14, R1=CH2CH3 R2=H
15, R1=Ph R2=H
16, R1=CH3 R2=CH3
17, R1=Ph R2=CH3
18, R1=Bn R2=CH3
11, R2=H
12, R2=CH3
9, R1=CH2CH3
10, R1=Ph
11, R1=Bn
25, R=Et R1=Ph R2=H
a
Scheme 2. Reagents and conditions: (a) NaH, dry toluene, 20–40 ꢀC, 36–72 h, 65–78%; (b) 1N KOH, THF/H2O (1:2), 100 ꢀC, 24 h, 62–75%; (c) 14N
NaOH (1 equiv.), EtOH, reflux, 15 min, 64%. For complete structures, see Table 1.
b

DOI: 10.1080/14756366.2016.1218486
New tools for the biophysical investigation of o-acetylserine sulfhydrylases
3
pulses for a saturation time of 3 s. For the reference spectrum (off Results and discussion
resonance), the samples were irradiated at 26.6 ppm. NMR data
Rational design and SAR of OASS inhibitors
were processed and analyzed using MestReNova 8.1 software
(Santiago de Compostela, Spain). Group epitope mapping (GEM) We have recently developed a series of substituted 2-phenylcy-
was calculated setting the highest STD intensity to 100% and all clopropane carboxylic acids that bound with high affinity to both
other STD signals were calculated accordingly.
the isoforms of Salmonella enterica serovar typhimurium OASS
(StOASS)27. The main chemical feature was represented by the
presence of a carboxylic acid moiety linked through a cyclopro-
pane spacer, to a hydrophobic side chain in a trans configuration.
These structures were rationally designed based on the
Haemophilus influenzae (H. influenzae) SAT (HiSAT) native C-
terminal pentapeptide MNLNI, which is the natural inhibitor of
OASS, and the substituent at position C-2 was represented by a
small alkenyl chain, as in the case of compound 29 (Figure 1)25.
In a second round of optimization, the alkenyl chain was
embodied into a phenyl ring30, maintaining the trans stereo-
relationship with the carboxylic moiety. This modification led to
improved synthetic feasibility and stability, and the insertion of a
phenyl ring expanded the scope for further manipulation to
modulate activity and drug-likeness. A vigorous improvement of
compound 30 was obtained through the insertion, at the position
C-1 of the cyclopropane, of hydrophobic appendages such as the
ethyl, the phenylethyl and, in particular, the substituted benzyl.
These modifications culminated in the synthesis of derivative 31
(Figure 1), which showed Kds in the nanomolar range toward both
StOASS-A and B (Figure 1). In this work, we further expand the
SAR around these 2-phenylcyclopropane carboxylic acid deriva-
tives by exploring how substitution at the C-2 or disubstitution at
both C-1 and C-2 of the basic 2-phenylcyclopropanecarboxylic
acid core could affect the binding affinity to StOASS-A and B.
First, we decided to insert a carboxylic moiety at the C-2
position taking into account that: (a) the use of polar moieties
might have been beneficial in order to improve the drug-likeness
of the molecules prepared. It is known that an optimal
hydrophilic–lipophilic balance (HLB) may enable best penetra-
tion across Gram-negative outer membrane33–35; therefore, a polar
moiety lowering the ClogP can be used to facilitate penetration
through S. typhimurium cell wall; (b) serine racemase, a PLP-
dependent enzyme that shows a good structural similarity with
OASS, is inhibited by a series of dicarboxylic derivatives such as
modified malonates36,37; (c) the synthetic protocol allowing to
prepare such compounds is well established and high structural
variability can be obtained starting from easily available mater-
ials. First, we synthesized two close analogs of 30, which are
compounds 25 and 21, in order to assess whether the carboxylic
moiety would better work as an ester or an acid. To maintain the
favorable trans stereo-relationship between the C-2 phenyl ring
and the C-1 carboxylic moiety, the carboxylic moieties were set in
a cis configuration. We were pleased to notice that compound 21
exhibited an affinity comparable to that of the reference
compound 30 toward StOASS-A (21, Kd OASS-A ¼ 7.4 mM; 30,
Kd OASS-A ¼ 9.2 mM), but it was 3-fold higher than parent
compound 30 toward StOASS-B (21, Kd OASS-B ¼ 55 mM; 30,
Kd OASS-B ¼ 148 mM). Also, as expected, both the compounds
had a lower predicted log p than the reference compounds 30
and 31.
Direct determination of ligand-binding affinity to OASS
The binding affinity of ligands to OASS was determined by
monitoring the increase in fluorescence emission of the PLP
coenzyme following excitation at 412 nm24,32. Emission spectra
were recorded as a function of ligand concentration in a
solution containing 0.5–1.0 mM OASS, 100 mM Hepes buffer,
pH 7.0, at 20 ꢀC. Fluorescence measurements were carried out
using a FluoroMax-3 fluorometer (HORIBA), equipped with a
thermostatted cell holder, and spectra were corrected for blank
contribution. The dependence of the fluorescence intensity at
500 nm on ligand concentration was fitted to
isotherm.
a binding
Competitive ligand-binding assays
Competitive ligand-binding assays were carried out in a similar
way to the direct binding assays with the difference that the
titration was carried out on an enzyme solution saturated with a
low-affinity compound and the decrease in the fluorescence
emission of the cofactor was followed as a function of high-
affinity ligand concentration. The dependence of the fluorescence
emission of the cofactor on the ligand concentration was fitted to a
binding isotherm to calculate an apparent dissociation constant
(Kd,app). Kd,app is transformed into the intrinsic Kd by Equation (1)
that takes into account the presence of a competing ligand for
binding to the active site.
Kd, app
½Llow
Kd ¼
ð1Þ
ꢂ
1 þ
Kdlow
[Llow] is the low-affinity ligand concentration and Kdlow is the
dissociation constant for the low-affinity ligand.
Docking studies
Docking studies were performed by means of the Glide9.1 suite
(https://www.schrodinger.com) and using a previously refined
StOASS-A structure26. The protein structure was prepared by
applying the protein preparation protocol available in Maestro9.1
(https://www.schrodinger.com). The grid was cantered on the
corresponding residues defining in StOASS-A, the Ile267-hosting
pocket. The docking runs were carried out using the standard
precision (SP) method; the van der Waals scaling factor of
nonpolar atoms (with an absolute partial charge less than 0.15)
was set to 0.8. Compound 23 was built using the fragment-built
tool available in Maestro9.1 (https://www.schrodinger.com).
Finally, the computationally derived G.E.M were calculated
using an in-house built script.
O
HO
O
OH
a, b
CN
+
Cl
27
26
28
Scheme 3. aReagents and conditions: (a) NaNH2, dry benzene, rt, 16 h; (b) 1N NaOH, reflux, 24 h, 35%.

4
G. Annunziato et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
Figure 1. Evolution of cyclopropanecarboxylic acid derivatives as inhibitors of StOASS-A and StOASS-B.
At this regard, it is worthwhile to point out that inhibition of
both the isoforms of the enzyme is important for granting a
complete inhibition of cysteine biosynthesis in bacteria. Toward
the A isoform, the ethyl ester derivative was almost 10-fold less
active than the acid analog (25, Kd OASS-A ¼ 83 mM), whereas
the reduction in the carboxylic moiety to the corresponding
alcohol (28, Kd OASS-A ¼ 168 mM) led to 20-fold reduction of
the affinity. This finding corroborates the importance of the acid
moiety for an efficient binding to the enzyme. Puzzled by these
results, we synthesized a series of derivatives to establish whether
this cyclopropane-1,2-dicarboxylic acid scaffold could be con-
sidered a chemotype for StOASS inhibition. Based on the
structure of compound 21, a tetrasubstituted cyclopropane-1,2-
dicarboxylic acid was prepared. Compound 23 showed an activity
comparable to that of 21 toward the A isoform (23, Kd OASS-
A ¼ 9.0 mM), and, especially, it was found to be the most active
derivative of this series toward the B isoform (23, Kd OASS-
B ¼ 40 mM). Substitution of the phenyl moiety of 23 with a benzyl
appendage, led to a sharp drop in the affinity toward both the
isoforms (24, Kd OASS-A ¼ 48 mM; Kd OASS-B ¼ 368 mM).
Then, we wanted to reduce the size of the molecules prepared,
as it is known that size, along with the HLB above mentioned, is
an important characteristic in order to improve the penetration
through the outer membrane of Gram-negative bacteria such as S.
Figure 2. Preliminary SAR of cyclopropanecarboxylic acids as StOASS
inhibitors.
cyclopropanecarboxylic acid as the pharmacophore, substituents
at C-2 showing a hydrophilic character must be kept in a cis
stereo-relationship with the C-1 carboxylic group, whereas less
hydrophilic substituents must maintain a trans stereo-relationship.
Fluorescence properties of the OASS/compounds
complexes
typhimurium. However, reducing the steric hindrance of the The enzymatic bioassays for this series of compounds were
substituent at C-2, that is, substituting the phenyl ring with alkyl performed through the fluorimetric determination of the dissoci-
moieties, proved to be detrimental for the affinity. Compound 20, ation constants. This method exploits the increase in the
bearing an ethyl moiety in place of the phenyl, showed an affinity fluorescence emission of the cofactor PLP upon ligand binding
toward StOASS-A in line with the derivatives described thus far at the active site38 and allows the direct determination of
(Kd OASS-A ¼ 8.1 mM), but the affinity toward the B isoform is dissociation constants (i.e. the inhibition constant for competitive
considerably lower than that of 21 and 23 (20, Kd OASS- inhibitors). One interesting property of most compounds reported
B ¼ 173 mM). Finally, mono- and disubstitution with a methyl here is the high fluorescence quantum yield of the complex with
group led to a considerable decrease in affinity toward the A either OASS-A or OASS-B (Table 1). Taking into account that the
isoform (19, Kd OASS-A ¼ 49 mM; 22, Kd OASS-A ¼ 42 mM). formation of the complex between the C-terminal decapeptide of
Further simplification of the structure, as in the case of the SAT and OASS leads to a 4-fold increase in fluorescence
unadorned cyclopropane-1,2-dicarboxylic acids, led to the least emission intensity, likely due to changes in the binding pocket
potent compounds of the series. However, despite the little microenvironment and/or shielding from solvent quenching32,
affinity, synthesis of compounds 2, 3, 5 and 6 allowed to gain some of the compounds identified in this work are far more
additional clues into the SAR of these derivatives. Compounds 3 efficient in exerting such an effect. For example, saturating
and 5 showed low affinity toward the A isoform, with dissociation concentrations of compound 19 and compound 20 lead to a 9- and
constants higher than 1 mM, whereas compounds 2 and 6 had a 10-fold increase in the fluorescence emission of the cofactor of
comparable binding affinity (2, Kd, OASS-A ¼ 215 mM; 6, Kd, the A isoform (Table 1). Due to this special property, most of the
OASS-A ¼ 245 mM). With regard to the stereo-relationship, when compounds identified in this work could be used in competitive
the acid moieties are in a cis configuration, the affinity is higher binding, a titration method where a low-affinity ligand is
with respect to the trans configuration. On a similar vein, when displaced from binding to a specific site by a higher affinity
the ester moiety and the acid are in trans stereo-relationship, the ligand. The physicochemical properties of complexes should be
affinity is higher than that measured on the cis stereoisomer. In a different in order to distinguish the high-affinity complex from
more comprehensive picture (Figure 2), this small set of the low-affinity one by spectroscopic or radiometric methods.
molecules has corroborated the findings partially stemmed This method allows the calculation of dissociation constants for
also from our previous studies25,27
:
assuming the tight ligands circumventing the problems associated with ligand

DOI: 10.1080/14756366.2016.1218486
New tools for the biophysical investigation of o-acetylserine sulfhydrylases
5
Table 1. Dissociation constants and fluorescence emission intensity of the title compounds for StOASS-A and StOASS-B.
1
2
R
R
O
O
OR
Kd (mM)
OASS B
OH
Fsat/F0x
Comp
Conf*
R
R1
R2
SIy
Log P**
OASS A
OASS A
OASS B
2
3
5
6
19
20
21
22
23
24
25
cis
cis
trans
trans
cis
cis
cis
cis
cis
H
Et
H
Et
H
H
H
H
H
H
Et
H
H
H
H
Me
Et
Ph
Me
Me
Me
Ph
H
H
H
H
H
H
H
Me
Ph
Bn
H
215 ꢃ 9
41000
41000
245 ꢃ 14
49 ꢃ 1
8 ꢃ 1
900 ꢃ 90
41000
4.2
nay
50.6
2.8
6.5
21
7.4
8.3
4.4
7.7
7
na
na
7
9
10
4
7
6
5
2
3
na
7
5
4
6
5
5
7
ꢁ0.09
0.90
640 ꢃ 110
675 ꢃ 9
320 ꢃ 20
173 ꢃ 8
55 ꢃ 3
ꢁ0.09
0.90
ꢁ0.14
0.36
1.08
7 ꢃ 1
42 ꢃ 3
9 ꢃ 1
350 ꢃ 32
40 ꢃ 2
ꢁ0.18
1.04
1.25
cis
cis
48 ꢃ 2
83 ꢃ 2
368 ꢃ 47
41000
3
4
412
2.07
28
168 ꢃ 35
ndô
na
2
4
1.20
O
OH
OH
*Stereochemistry between the carboxylic moieties.
ySI: Selectivity index is intended as the ratio Kd,OASS-B/Kd,OASS-A.
znot attainable.
ônot determined.
xthe ratio between the fluorescence emission intensity at 500 nm upon excitation at 412 nm of the OASS completely saturated with the compound (Fsat
)
and the fluorescence emission intensity of the unbound OASS (F0).
**calculated with molinspiration sotware (http://www.molinspiration.com/services/faq.html).
In Figure 4(A) the spectra of the uncomplexed OASS, OASS
complexed with compound 32 and OASS complexed with
compound 20 are reported. The titration with compound 32 of
an OASS-A solution saturated with compound 20 leads to a
decrease in the fluorescence emission at 500 nm upon excitation
at 412 nm (Figure 4B). The decrease in fluorescence emission is
due to the displacement of compound 20 by compound 32. The
decrease in fluorescence emission can be fitted to a binding
isotherm to determine the apparent dissociation constant (Kd,app
)
that is transformed into the intrinsic dissociation constant (Kd)
using Equation (1). The calculated Kd for compound 32 is
78 ꢃ 6 nM, in very good agreement with the published dissoci-
ation constant of 77 ꢃ 11 nM calculated by fitting to the equation
for tight binding the dependence of the fluorescence emission of
OASS-A on the concentration of compound 32.
Figure 3. Structure of compound 32 and corresponding binding affinities.
STD-NMR analysis
In our previous investigations, we carried out a series of saturation
transfer difference (STD) NMR measurements, in order to define
the interaction between a given ligand and the enzyme39. The
analysis of the STD-NMR spectra allows mapping the ligand
protons directly involved in the interaction with the enzyme40.
Once the interaction is characterized, in order to make the results
readily measurable, the saturation received by the ligand protons
is expressed as group epitope mapping (GEM), setting the highest
saturated proton of each ligand (the closest to the enzyme) as
100%41. The other STD signals were calculated accordingly,
yielding information on the proximity of each proton to the
binding pocket of StOASS-A and StOASS-B. The STD methods
suffer from some restrictions, one of which being the inability to
investigate either very strong or very weak binders. In our
depletion at low ligand concentrations. In fact, the presence of the
low-affinity ligand allows the shifting of the apparent dissociation
constant for the tight-binding ligand to higher values. Provided
the low-affinity inhibitor can be added at saturating concentra-
tions, the apparent dissociation constant for high-affinity ligand
can be calculated and converted to the intrinsic dissociation
constant by Equation (1) (Experimental part). This method is also
convenient when the OASS/ligand complex is nonfluorescent, as
is the case with compounds lacking the carboxylic moiety (BC,
unpublished observation). An example of application of competi-
tive binding assay is given in Figure 4, where the dissociation
constant of the previously identified compound 32 (27) is
determined by competitive binding with compound 20.

6
G. Annunziato et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
Figure 4. Use of compound 20 in a competitive binding assay for the determination of the dissociation constant of compound 32. Panel A: fluorescence
emission spectra of OASS-A upon excitation at 412 nm in the absence of ligands and in the presence of either the low affinity ligand compound 20 or
the high-affinity ligand compound 32. Panel B: displacement titration of OASS-A:compound 20 (960 mM) complex with compound 32. The
concentration of OASS used is 0.5 mM. The solid line represents the fit with a binding isotherm with an apparent dissociation constant of 9.4 ꢃ 0.7 mM.
Figure 5. STD effects measurement for compound 23 in complex with StOASS-A (panel A) and StOASS-B (panel B). The relative degree of individual
protons saturation are plotted into the structure.
previous work, this drawback led to evaluate the interaction of a cyclopropane spacer is positioned very close to the surface of
ligand only with StOASS-A27. In this work, we have focused our the binding pocket. Indeed, the GEM values for protons Ha and
attention on compound 23 for the following reasons: (a) the Kds Hb are 47% and 100%, respectively. In addition, the protons of the
for this compound, both toward OASS-A and OASS-B, were in methyl group that is in a cis stereorelationship with Hb, showed a
the suitable interval for STD-NMR experiments (Kd OASS- GEM value of 79%; This corroborates the importance of the
A ¼ 9.0 mM; Kd OASS-B ¼ 40 mM); (b) the structural features of cyclopropane backbone in the interaction of the molecules with
23 allowed for a rapid identification of the peaks and, also, (c) this the target. Therefore, in agreement with the data already reported,
compound was amenable for further chemical manipulation and the cyclopropane is a good scaffold in order to properly arrange
eventually improvement.
the molecule at the binding site. The design of further analogs
The first information derived from this study (Figure 5) is that will rely on maintaining Hb unsubstituted, given its strong
the same molecule exhibits different interaction modes with the proximity to the receptor surface.
two enzymes. Although this result is generally predictable due to
Regarding StOASS-B, it can be seen from the GEM values of
the difference in Kds values; in this case, it was possible to H4 (100%) and H3,5 (89%) that the protons belonging to the
determinate which part of the molecule is mainly involved in the phenyl ring establish the strongest van der Waals interactions with
interaction. It can be speculated that, for StOASS-A, the the receptor surface. This evidence can somehow explain the fact

DOI: 10.1080/14756366.2016.1218486
New tools for the biophysical investigation of o-acetylserine sulfhydrylases
7
that those compounds bearing a phenyl ring at the C-2 of the
cyclopropane core (i.e. 21 and 23) bind to StOASS-B with
dissociation constants in the tens of micromolar range, whereas
compounds in which the phenyl portion is abolished (19, 20 and
22), or in which it is spatially rearranged (i.e. compound 24),
show a several fold lower affinity. Altogether, the possibility to
apply STD-NMR analysis to both the StOASS isoforms has
provided a series of hints about the molecular recognition by each
binding site, and this information can be exploited for the rational
design of analogs able to inhibit the two enzymes in a selective or
unselective fashion.
Computational analysis of 23/StOASS interaction
Since for the most active compounds the main structural motif
(the 2-phenylcyclopropylcarboxylic moiety) is maintained, it can
be reasonably assumed that the binding mode of these derivatives
is similar to that already reported in our previous works25–27. To
ascertain this hypothesis, and to correlate the data obtained from
the STD-NMR experiments and the biochemical evaluation, a
computational analysis of the binding mode of compound 23 with
StOASS-A was performed.
The docking (Figure 6A) clearly shows that the key inter-
actions already described are maintained also with the cyclopro-
pane-1,2-dicarboxylic acids25,27. In particular, (i) the carboxylic
group in position C-1 H-bonds the NH protons of Asn71 and
Thr72 backbone residues; (ii) the carboxylic group in position C-
2 H-bonds Asn69; and (iii) the phenyl ring in position C-2 is
located in a hydrophobic pocket as previously reported, estab-
lishing a ꢀ-ꢀ parallel displaced interaction with Phe143.
Therefore, the docking model that we have herein developed is
in good agreement both with the SAR described in this work and
with that previously reported. Finally, the selected binding mode
also qualitatively recapitulates the experimentally determined
GEM values (Figure 6B). Indeed, comparing the experimental
GEM (green boxes, Figure 6B) with those computationally
derived from the binding mode reported in Figure 6(A) (orange
boxes, Figure 6B), it is clear that Ha and the CH3 hydrogens are
the atoms establishing the most relevant interactions upon
inhibitor binding; the Hb, 2,6H and 3,5H hydrogens show
equivalent contacts with the StOASS-A-active site; on the
contrary, the 4H hydrogen is the least involved in the protein
binding. Therefore, this further corroborates the data derived from
the STD-NMR experiments.
Figure 6. (A) Binding mode of compound 23 into the StOASS-A active
site. Highlighted the Lys41-PLP adduct. (B) Experimentally (left boxes)
vs. computationally (right boxes) derived GEM values. The values were
normalized.
m ¼ multiplet and br ¼ broad signal. HPLC/MS experiments
were performed with an Agilent 1100 series HPLC apparatus,
equipped with a Waters Symmetry C18, 3.5 mm, 4.6 mm ꢄ 75 mm
column and an MS: Applied Biosystem/MDS SCIEX instrument,
with API 150EX ion surce. HRMS experiments were performed
with an LTQ ORBITRAP XL THERMO apparatus. All com-
pounds were tested as 95% purity or higher (by HPLC/MS).
Compounds 242, 343, 544, 645 were prepared according to
published protocols, and the analytical data match with those
already reported.
Experimental part
General information
All the reagents were purchased from Sigma-Aldrich, Alfa-Aesar
and Enamine at reagent purity and, unless otherwise noted, were
used without any further purification. Dry solvents used in the
reactions were obtained by distillation of technical grade mater-
ials over appropriate dehydrating agents. MCRs were performed
using CEM Microwave Synthesizer-Discover model. Reactions
were monitored by thin layer chromatography on silica gel-coated
aluminum foils (silica gel on Al foils, SUPELCO Analytical,
Sigma-Aldrich, St. Louis, MO) at 254 and 365 nm. Where
indicated, intermediates and final products were purified by silica
gel flash chromatography (silica gel, 0.040–0.063 mm), using
appropriate solvent mixtures.
1H-NMR and 13C-NMR spectra were recorded on a BRUKER
AVANCE spectrometer at 300 and 100 MHz, respectively, with
1
TMS as internal standard. H-NMR spectra are reported in this
order: multiplicity and number of protons. Standard abbreviations
indicating the multiplicity were used as follows: s ¼ singlet,
d ¼ doublet, dd ¼ doublet of doublets, t ¼ triplet, q ¼ quadruplet,
General procedure for the synthesis of esters 13–18
To a stirred suspension of sodium hydride (60% suspension in
mineral oil, 1.1 equiv) in anhydrous toluene (2 mL/mmol), the
suitably substituted ethyl 2-halocarboxylate (1 equiv) and ethyl 2-
acrylate (1 equiv) were added under nitrogen atmosphere at room
temperature. The reaction mixture is allowed to stir at a
temperature maintained between 20 and 40 ꢀC until consumption
of the starting reagents according to TLC (usually 36–72 h). The
reaction is quenched by cautious addition of a small amount of
methanol (1 mL) and then poured into ice-water and extracted
with Et2O (3 ꢄ 10 mL). The combined organic layers are then
washed with brine, dried over MgSO4, and concentrated under
reduced pressure. The crude residue was purified by flash

8
G. Annunziato et al.
J Enzyme Inhib Med Chem, Early Online: 1–10
chromatography eluting with a mixture of petroleum ether/ethyl under reduced pressure, to afford the title compound as a white
acetate in variable proportion, to give the desired product as solid in yields ranging from 62% to 75%. The products underwent
a white solid in yields ranging from 65% to 78%. Analytical biological assays without any further purification. Following a
data for compounds 13 and 16 matched the data already similar procedure, but using equimolar amount of KOH and EtOH
published46.
as the solvent, title compound 25 was obtained from 15 in 64%
yield. Analytical data for compounds 19 and 22 matched those
already published46.
Cis (ꢃ)-diethyl 1-methylcyclopropane-1,2-dicarboxylate13
Purified by flash column chromatography, eluent petroleum ether/
ethyl acetate 90:10. Yield: 68% (pale oil); 1H-NMR (CDCl3
300 MHz): ꢁ 1.15 (s, 3H); 1.21–1.29 (m, 6H); 1.32–1.36 (m, 1H);
1.63–1.66 (m, 1H); 2.00–2.12 (m, 1H); 4.12–4.19 (m, 4H); LRMS
(ESI) calculated for C10H16O4 ([M-H]-) 200.10; found 200.23.
Cis (ꢃ)-1-methylcyclopropane-1,2-dicarboxylic acid19
Purified by filtration and washed with Et2O. Yield: 70%
(white powder); 1H-NMR (CDCl3 300 MHz): ꢁ 1.13 (s, 3H);
1.23–1.26 (m, 1H); 1.51–1.56 (m, 1H); 2.13–2.17 (m, 2H).
13C-NMR (100.6 MHz, CDCl3): 33.21; 31.41; 23.54; 16.54.
HRMS (ESI) calculated for C6H8O4 ([M-H]-) 144.0425;
found 144.1388.
Cis (ꢃ)-diethyl 1-ethylcyclopropane-1,2-dicarboxylate14
Purified by flash column chromatography, eluent petroleum ether/
ethyl acetate 90:10. Yield: 65% (pale oil); 1H-NMR (CDCl3
300 MHz): ꢁ 1.11 (t, J ¼ 8.34, 3H); 1.21–1.29 (m, 6H); 1.34–1.38
(m, 1H); 1.97–2.06 (m, 2H); 2.15–2.21 (m, 2H); 4.12–4.19 (m,
4H); LRMS (ESI) calculated for C11H18O4 ([M-H]-) 214.12;
found 214.16.
20
Cis (ꢃ)-1-ethylcyclopropane-1,2-dicarboxylic acid
Purified by filtration and washed with Et2O. Yield: 73% (white
1
powder); H-NMR (CDCl3 300 MHz): ꢁ 1.11 (t, J ¼ 8.34, 3H);
1.26–1.31 (m, 1H), 1.97–2.06 (m, 2H), 2.15–2.21 (m, 2H). 13C-
NMR (100.6 MHz, CDCl3): 33.32; 31.17; 21.48; 18.49; 11.59.
HRMS (ESI) calculated for C7H10O4 ([M-H]-) 158.0679; found
158.1500.
Cis (ꢃ)-diethyl 1-phenylcyclopropane-1,2-dicarboxylate15
Purified by flash column chromatography, eluent petroleum ether/
1
ethyl acetate 90:10. Yield: 77% (yellow oil); H-NMR (CDCl3
Cis (ꢃ)-1-phenylcyclopropane-1,2-dicarboxylic acid21
300 MHz): ꢁ 1.23–1.30 (m, 6H); 1.41–1.45 (m, 1H); 2.24–2.27 (m,
2H); 4.23–4.27 (m, 4H); 7.36ꢁ7.43 (m, 5H); LRMS (ESI)
calculated for C15H18O4 ([M-H]-) 262.12; found 262.30.
Purified by filtration and washed with Et2O. Yield: 75% (white
powder); 1H-NMR (CDCl3 300 MHz): ꢁ 1.40–1.44 (m, 1H); 2.24–
2.27 (m, 2H), 7.33ꢁ7.39 (m, 5H). 13C-NMR (100.6 MHz, CDCl3):
129.29; 128.21; 126.87; 33.32; 31.17; 21.48. HRMS (ESI)
calculated for C15H18O4 ([M-H]-) 206.0625; found 206.1912.
Cis (ꢃ)-diethyl 1,2-dimethylcyclopropane-1,2-dicarboxylate16
Purified by flash column chromatography, eluent petroleum ether/
ethyl acetate 80:20. Yield: 78% (pale oil); 1H-NMR (CDCl3
300 MHz): ꢁ 1.15 (s, 6H); 1.31–1.36 (m, 6H); 2.21–2.25 (m, 2H);
4.21–4.26 (m, 4H); LRMS (ESI) calculated for C11H18O4 ([M-
H]-) 214.12; found 214.16.
Cis (ꢃ)-1,2-dimethylcyclopropane-1,2-dicarboxylic acid22
Purified by filtration and washed with Et2O. Yield: 75% (white
1
powder); H-NMR (CDCl3 300 MHz): ꢁ 1.12 (s, 6H); 2.21–2.25
(m, 2H). 13C NMR (100.6 MHz, CDCl3): 33.32; 31.17; 21.48;
17.58. HRMS (ESI) calculated for C7H10O4 ([M-H]-) 158.0606;
found 158.1245.
Cis (ꢃ)-diethyl 1-methyl-2-phenylcyclopropane-1,2-
dicarboxylate17
Purified by flash column chromatography, eluent petroleum ether/
ethyl acetate 95:5. Yield: 77% (pale oil); 1H-NMR (CDCl3
300 MHz): ꢁ 1.17 (s, 3H); 1.30–1.35 (m, 6H), 2.17–2.21 (m, 2H);
4.31–4.36 (m, 4H); 7.32–7.38 (m, 5H). LRMS (ESI) calculated for
C16H20O4 ([M-H]-) 276.14; found 276.33.
Cis (ꢃ)-1-methyl-2-phenylcyclopropane-1,2-dicarboxylic acid23
Purified by filtration and washed with Et2O. Yield: 69% (white
1
powder); H-NMR (CDCl3 300 MHz): ꢁ 1.21 (s, 3H); 2.12–2.15
(m, 2H); 7.31–7.37 (m, 5H). 13C NMR (100.6 MHz, CDCl3):
129.18; 128.33; 126.67; 31.17; 17.58. HRMS (ESI) calculated for
C12H12O4 ([M-H]-) 220.0712; found 220.2035.
Cis (ꢃ)-diethyl 1-benzyl-2-methylcyclopropane-1,2-
dicarboxylate18
Purified by flash column chromatography, eluent petroleum ether/
ethyl acetate 90:10. Yield: 71% (yellowish solid); 1H-NMR
(CDCl3 300 MHz): ꢁ 1.15 (s, 3H), 1.34–1.38 (m, 6H), 1.93–2.00 Purified by filtration and washed with Et2O. Yield: 62% (white
Cis (ꢃ)-1-benzyl-2-methylcyclopropane-1,2-dicarboxylic acid24
1
(m, 2H); 2.19–2.23 (m, 2H); 4.35–4.39 (m, 4H); 7.48–7.56 (m, powder); H-NMR (CDCl3 300 MHz): ꢁ 1.21 (s, 3H); 1.94–2.03
5H); LRMS (ESI) calculated for C17H22O4 ([M-H]-) 290.15; (m, 2H); 2.21–2.25 (m, 2H); 7.41–7.49 (m, 5H). 13C-NMR
found 290.30.
(100.6 MHz, CDCl3): 131.123; 129.56; 126.32; 33.54; 17.34;
11.23. HRMS (ESI) calculated for C13H14O4 ([M-H]-) 234.0985;
found 234.1158.
General procedure for the synthesis of derivatives 19–24
To a solution of the substituted ethyl cyclopropanedicarboxylic
ester (1 equiv), dissolved in a mixture of THF/water (1:2) at 0 ꢀC,
a solution of aqueous potassium hydroxide 1N (1 mL/mmol) was
Cis (ꢃ)-2-(ethoxycarbonyl)-2-phenylcyclopropanecarboxylic
acid25
added dropwise. The reaction mixture was stirred at 50 ꢀC until Purified by flash column chromatography eluting petroleum
1
consumption of the starting material according to the TLC, and ether/ethyl acetate 90:10. Yield: 64% (white powder); H-NMR
then acidified to pH 3 with 1N HCl. The aqueous phase was (CDCl3 300 MHz): ꢁ 1.21–1.25 (t, J ¼ 7.54, 3H); 1.65–1.67 (m,
extracted with Et2O (3 ꢄ 10 mL), and the combined organic layers 1H); 2.33–2.35 (m, 2H); 4.11–4.16 (m, 2H); 7.33–7.37 (m, 5H).
were washed with brine, dried over MgSO4, and concentrated 13C NMR (100.6 MHz, CDCl3): 130.12; 129.67; 127.44; 33.54;

DOI: 10.1080/14756366.2016.1218486
New tools for the biophysical investigation of o-acetylserine sulfhydrylases
9
32.23; 17.54; 11.13. HRMS (ESI) calculated for C13H14O4 ([M- Acknowledgements
H]-) 234.0920; found 234.2102.
The Centro Interdipartimentale Misure ‘‘G. Casnati’’ is kindly acknowl-
edged for the contribution in the analytical determination of the
molecules synthesized.
Synthesis of 2-(hydroxymethyl)-1-phenylcyclopropane-1-car-
boxylic acid28
Declaration of interest
A solution of benzyl cyanide (2.0 g, 17.1 mmol) was added
dropwise over 30 min to a suspension of NaNH2 (1.54 g,
39.3 mmol) in benzene at 0 ꢀC. After stirring for 3 h at room
temperature, a solution of epichloroydrin (1.53 g, 16.6 mmol) was
added to the reaction mixture over 45 min, using an ice-bath to
keep the temperature in a range between 20 and 40 ꢀC. After
consumption of the limiting reagent, monitored by TLC, 1N
NaOH (16.6 mL) is cautiously added dropwise to the reaction
mixture that is allowed to react at 90 ꢀC overnight. After cooling,
the benzene was decanted, and the acqueous phases was extracted
with dichloromethane (3 ꢄ 20 mL), acidified with 2N HCl to pH 2
and extracted again with ethyl acetate (3 ꢄ 10 mL). The combined
organic layers were then washed with brine, dried over MgSO4
and concentrated under reduced pressure. The crude residue was
purified by flash chromatography eluting with petroleum ether/
ethyl acetate (90:10), to give the desired product as a white solid
in 35% overall yield. 1H-NMR (CDCl3 300 MHz): ꢁ 1.74–1.78 (m,
1H); 1.88–1.94 (m, 2H); 3.98–4.03 (m, 2H); 7.44–7.58 (m, 5H).
13C-NMR (100.6 MHz, CDCl3): 130.24; 129.86; 128.21; 33.61;
32.84; 15.16. HRMS (ESI) calculated for C11H12O3 ([M-H]-)
192.0898; found 192.2011.
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of this article.
The work described in this paper was partly carried out under the
MSCA-ITN-2014-ETN project INTEGRATE (grant number 642620).
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