Effective Access to Multivalent Inhibitors of Carbonic Anhydrases Promoted by Peptide Bioconjugation

Article abstract of DOI:10.1002/chem.201700241

Multivalency has impressive effects on (bio)molecular recognition, through the simultaneous presentation of multiple copies of a ligand, which can change a weak millimolar binder into a potent nanomolar one. The implementation of multivalency in enzyme inhibition is rather recent, being exemplified by few serendipitous discoveries, and hitherto relying on the random exploration of new multivalent structures as potential enzyme inhibitors. Here, a straightforward and versatile method is reported that enables the construction of multivalent systems for the inhibition of carbonic anhydrases (CA), widespread enzymes that catalyze a fundamental biochemical reaction. Oxime and hydrazone click-type bioconjugation techniques were successfully used for the preparation of tetravalent peptide conjugates tethered with sulfonamide CA inhibitors. The enzyme inhibition assays show that multivalent effects were present with these novel compounds, but also reveal various structural effects provided by the scaffolds. The versatility of this approach may facilitate the exploration of structure–activity relationships for other types of enzyme inhibitors.

Full text of DOI:10.1002/chem.201700241

A Journal of
Accepted Article
Title: Effective Access to Multivalent Inhibitors of Carbonic Anhydrases
Promoted by Peptide Bioconjugation.
Authors: Nasreddine Kanfar, Muhammet Tanc, Pascal Dumy, Claudiu
Supuran, Sébastien Ulrich, and Jean-Yves Winum
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of the final Version of Record (VoR). This work is currently citable by
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published online in Early View as soon as possible and may be different
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To be cited as: Chem. Eur. J. 10.1002/chem.201700241
Link to VoR: http://dx.doi.org/10.1002/chem.201700241
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10.1002/chem.201700241
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Effective Access to Multivalent Inhibitors of Carbonic Anhydrases
Promoted by Peptide Bioconjugation.
Nasreddine Kanfar,[a] Muhammet Tanc,[b] Pascal Dumy,[a] Claudiu T. Supuran,[b] Sébastien Ulrich*[a]
and Jean-Yves Winum*[a]
Abstract: Multivalency has impressive effects on (bio)molecular
recognition, through the simultaneous presentation of multiple copies
of a ligand, which can change a weak millimolar binder into a potent
nanomolar one. The implementation of multivalency in enzyme
inhibition is rather recent, being exemplified by few serendipitous
discoveries, and hitherto relying on the random exploration of new
multivalent structures as potential enzyme inhibitors. We report here
a straightforward and versatile method that enables the construction
of multivalent systems for the inhibition of carbonic anhydrases (CA),
enzyme inhibition is therefore still in its infancy and rest on an
exploratory approach that requires effective and versatile
synthetic methods. The development of multivalent enzyme
inhibitors could be of great interest, not only for improving the
potency of enzyme inhibitors, but also for modulating their
selectivity, especially for enzymes which are dimers or tetramers.
Herein we report the design, synthesis, and evaluation of
multivalent inhibitors of carbonic anhydrases (CAs, EC 4.2.1.1)
that are effectively obtained by using bioconjugation techniques.
It should be mentioned that many CAs are monomeric enzymes,
but some of them are also dimers, or higher oligomers (dimers,
tetramers, etc).[5] Indeed, the CAs are widespread zinc
metalloproteins in higher vertebrates. By catalyzing the
interconversion of CO2 and the bicarbonate ion, the 12
catalytically active isoforms found in humans are involved in
numerous physiological processes such as such as respiration,
pH regulation and fluid formation.[6] Pathological processes can
be induced upon deregulated activity of these isoforms and
many of them are established important therapeutic targets for
diseases such as glaucoma (hCA II, hCA IV, hCA XII),[7] cancer
(hCA IX and hCA XII),[8] and epilepsy (hCA II, hCA VII).[9]
The multivalent approach has been recently implemented in the
field of CA inhibition for improving the inhibitory activity but also
to circumvent the lack of selectivity of conventional inhibitors
against the different hCA isozymes.[3]
Using DNA-encoded libraries, Neri’s group identified bivalent CA
inhibitors of submicromolar potency and proposed, based on
crystallographic data,[10] that the bivalency effect originates from
binding to a secondary polar site located near the active site.[11]
In this context, Whitesides et al. have reviewed the different
types of secondary interactions that can be exploited by bivalent
inhibitors, enabling inhibition potency up to the picomolar
range.[12]
More recently, systems of higher valency have been explored for
the inhibition of carbonic anhydrases. We previously used
fluorescent silica nanoparticles coated with multiple copies (12-
15) of sulfonamide-based CA inhibitors.[13] The results showed
an increase of inhibitory potency (low nanomolar activity, as
previously observed with gold nanoparticles[14]), but also a
specific multivalency effect against the cytosolic isoforms hCA I
and hCA II versus the transmembrane isoforms hCA IX and hCA
XII. The Vincent group described multimeric CA-inhibitors having
different types of zinc binding functions,[15] such as xanthate (3-6
inhibitors)[16] or coumarin (12 inhibitors).[17] Even if weak
multivalent effects were observed for the inhibition of carbonic
anhydrases (2-4 fold valence-corrected increase), improved
potency (in the micromolar range) and selectivity were noticed
compared to the monovalent analogues. These multivalent
systems were constructed from, respectively, polyol and
fullerene scaffolds using the click-type copper-mediated azide-
alkyne cycloaddition (CuAAC) reaction. Supuran’s group
reported the use of different generations of poly(amidoamine)
(PAMAM)-type dendrimer incorporating 4, 8, 16 or 32
widespread enzymes that catalyse
a fundamental biochemical
reaction. Oxime and hydrazone click-type bioconjugation techniques
were successfully used for the preparation of tetravalent peptide
conjugates tethered with sulphonamide CA inhibitors. The enzyme
inhibition assays show that multivalent effects were present with
these novel compounds, but also reveal various structural effects
provided by the scaffolds. The versatility of this approach may
facilitate the exploration of structure-activity relationships for various
types of other enzyme inhibitors.
Introduction
Multivalency effects have been extensively explored in
biomolecular recognition as a tool to strengthen a non-covalent
association through the simultaneous combination of multiple
interactions.[1] For example, many different types of synthetic
glycoclusters display an affinity for lectins that is enhanced by
several orders of magnitude compared to the single sugar
derivative.[2] The role of multivalency in enzyme inhibition has
received much less attention, most likely because enzymes
usually possess a single active site which should make them
less sensitive to a multivalent presentation of inhibitors. However,
intriguing effects have been recently reported in the area.[3]
Multivalent iminosugar clusters were found to display a greater
potency compared to the monovalent compound toward the
inhibition of certain glycosidases.[4] The mechanistic origin of this
effect is still under debate. It can involve binding to a secondary
site, clustering of the enzymes onto the multivalent compound,
or simply statistical effects that result from the increased local
concentration of inhibitors. The application of multivalency in
[a]
[b]
N. Kanfar, Prof. Dr. P. Dumy, Dr. S. Ulrich, Dr. J.-Y. Winum,
Institut des Biomolécules Max Mousseron (IBMM)
UMR 5247 CNRS, ENSCM, Université de Montpellier,
8 rue de l’Ecole Normale, 34296 Montpellier Cedex (France)
E-mail: sebastien.ulrich@enscm.fr, jean-
yves.winum@umontpellier.fr
Dr. M. Tanc, Prof. Dr. C.T. Supuran
Neurofarba Department ; Section of Pharmaceutical and
Nutriceutical Sciences, Università degli Studi di Firenze ; Via Ugo
Schiff 6, 50019 Sesto Fiorentino, Florence (Italy)
Supporting information for this article is given via a link at the end of
the document.
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10.1002/chem.201700241
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the sulfonamide 1.[25] The nucleophilic substitution of the chlorine
by N-hydroxyphthalimide then furnished compound 2 in 62%
yield. The final deprotection with hydrazine provides the final
sulfonamide-aminooxy compound 3. The sulfonamide-hydrazide
benzenesulfonamides as multivalent CA inhibitors.[18] These
dendrimers, which were synthesized by multiple amide-bond
formation from PAMAM dendrimers and activated esters,
showed excellent improvement of the inhibitory properties
against the different CA isoforms tested compared to the
monovalent inhibitor. Depending on the isoform considered,
weak to important dendritic effects (or multivalent, or generation
effect)[19] were evidenced with these systems.
Despite these significant advances, the multivalent systems
reported up to now still have a limited solubility in aqueous
media, which is crucial when considering potential biomedical
applications. Furthermore, their synthesis involves either the use
of toxic metal catalysts that are difficult to remove, or chemical
reactions that produce by-products. Therefore, it is important to
develop novel synthetic approaches for preparing multivalent CA
inhibitors. In this work, we explore the use of peptide-based
scaffolds and metal-free click-type bioconjugation techniques.
The use of peptides as scaffolds is expected to impart enhanced
water-solubility. We selected hydrazone and oxime ligations[20]
as an effective and facile way of preparing such multivalent
systems. Indeed, these ligations proceed with high yields –
which is a key prerequisite when carrying out multiple ligations in
one pot –, do not require metal catalysts to operate, and only
produce water as a side-product. We show that this synthetic
approach enables the effective preparation of multivalent
systems that act as effective CA inhibitors.
compound
5 was prepared in two steps starting from
hydrocinnamic acid (Scheme 1). The sulfonamide group was
installed by the subsequent reaction with chlorosulfonic acid and
aqueous ammonia. Esterification was carried out in the same
pot and gave compound 4. Finally, the hydrazide 5 was formed
by reaction with hydrazine. These compounds can then be
conjugated with various scaffolds bearing aldehydes.
Scheme 1. Synthesis of CA inhibitors bearing aminooxy and hydrazide groups
for further conjugation by, respectively, oxime and hydrazone ligations. i)
NH4OH, DCM, 95%; ii) N-hydroxyphtalimide, tetrabutylammonium iodide, Et3N,
DCM, 62%; iii) NH2NH2, MeOH, 83%; iv) 1) HSO3Cl, 2) NH4OH, 3) H2SO4,
MeOH, 12% overall; v) NH2NH2, MeOH, 67%.
Results and Discussion
From these synthons, we first prepared monovalent conjugates
9 and 10 (Scheme 2) that will serve as reference for identifying
multivalent effects. Thus, we conjugated sulfonamides 3 and 5
with a glyoxylic aldehyde that mimic the structure present in the
peptide-based multivalent systems. Compound 6, the
succinimide activated ester of protected L-serine,[26] was coupled
to n-butylamine to make compound 7, and the protecting groups
were then removed in acidic conditions to afford unprotected
serine derivative 8. The oxidative cleavage of the amino alcohol,
and the ligation reaction were then subsequently carried out in
one pot to afford monovalent compounds 9 and 10. Note that the
hydrazone ligation and the oxime ligation were carried out in
acidic media, respectively in a pH 5.0 buffer and in a H2O/TFA
99.9/0.1 mixture.
Present in many clinically used drugs, primary sulfonamides R-
SO2NH2 (where
R may be aliphatic or aromatic moiety)
constitute one of the most important class of inhibitors acting as
zinc binding function on the metalloenzymes carbonic
anhydrases.
This model of pharmacophore function was
therefore selected for this study.[15] On the other hand, based on
our previous experience,[21] we selected cyclic and linear
peptides bearing multiple reactive aldehyde groups as
multivalent scaffolds. While the linear peptide scaffold is most
probably unstructured and quite flexible, its cyclic counterpart is
highly pre-organized – due to b-sheet-type interactions – with
the four chains bearing the reactive aldehyde groups pointing
towards the same direction.[22] These scaffolds have already
been used for preparing, through multiple oxime ligations (up to
84)[23], different types of peptide-based bioconjugates, which
display good aqueous solubility and good stability in vitro and in
vivo.[20, 24]
Thus, we engaged the synthesis of sulfonamide inhibitors
bearing aminooxy and hydrazide functions that will enable the
conjugation through, respectively, oxime and hydrazone
ligations. Both of them are efficient click methods allowing the
bioconjugations to operate chemoselectively in mild conditions
and high yields, which make them an excellent tool for the
preparation of multivalent and multifunctional nanoconstructs for
biological applications. The sulfonamide-aminooxy compound 3
was prepared in three steps (Scheme 1). First, 3-chloro-
propanesulfonyl was reacted with aqueous ammonia to provide
Scheme 2. Synthesis of monovalent sulphonamide-type CA inhibitors. i) n-
butylamine, N,N-diisopropylethylamine, DCM, 84%; ii) TFA/TIS/H2O, 96%; iii)
NaIO4, sodium acetate buffer (100 mM, pH 5.0); iv) hydrazide 5, sodium
acetate buffer (100 mM, pH 5.0), 27% for two steps; v) NaIO4, H2O/TFA
99.9/0.1; vi) aminooxy 3, H2O/TFA 99.9/0.1, 12% for two steps.
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Then, we went on to preparing multivalent systems from the
functionalized peptide scaffolds 11 and 12. While the linear
peptide scaffold 12 is most probably unstructured and quite
flexible, it has been clearly demonstrated that the cyclic peptide
scaffold 11 is highly pre-organized in the solid-state and in
solution – the four arms pointing towards the same direction.[22]
Both peptide scaffolds were prepared by automated solid-phase
peptide synthesis (SPPS) on a 2-chlorotrityl chloride resin, as
previously described.[27] The synthesis of the cyclic peptide
involves in-solution cyclization under high-dilution conditions,
followed by an oxidative cleavage of serine side-chains in order
to unmask the glyoxylic aldehyde groups. The synthesis of the
linear peptide involves acetylation of the N-terminal, cleavage
and deprotection, and oxidative cleavage of serine side-chains.
We then prepared two series of multivalent CA inhibitors: the
first one prepared by oxime ligation from enzyme inhibitor 3
(Scheme 3), and the second one prepared by hydrazone ligation
from enzyme inhibitor 5 (Scheme 4). The oxime ligation was
typically carried out in acidic medium (H2O/TFA 99.9/0.1) and
the acylhydrazone ligation was performed in acidic acetate
buffer at pH 5.0. The tetravalent conjugates were isolated by
semi-preparative reverse-phase HPLC in 30-75% yield, which
correspond to individual reaction yields of 74-93%.
Scheme 4. Synthesis of multivalent CA inhibitors 15 and 16 by hydrazone
ligation from pre-organized and flexible functionalized peptide scaffolds,
respectively 11 and 12. Ligation reactions carried out in acetate buffer (100
mM, pH 5.0).
On the other hand, the corresponding multivalent conjugates 13
and 14 are much less potent inhibitors of hCA IX (as well as
hCA VII and hCA XII). However, an improvement in inhibition
potency when comparing the multivalent system 13 to the
monovalent 10 is observed on hCA II and IV (Table 1). In this
case, the relative potency (rp) increases 13- and 5-fold,
respectively.
Table 1. Results of the CA inhibition assay for the peptide scaffolds 11 and 12,
the monovalent oxime 10, and multivalent oxime conjugates 13 and 14.
Ki (nM)
Cpds
11
hCA I
>10000
>10000
63
hCA II
>10000
>10000
250
hCA IV
>10000
>10000
392
hCA VII
>10000
>10000
>10000
>10000
n/a
hCA IX
>10000
>10000
36
hCA XII
>10000
12
>10000
Scheme 3. Synthesis of multivalent CA inhibitors 13 and 14 by oxime ligation
from pre-organized and flexible functionalized peptide scaffolds, respectively
11 and 12. Ligation reactions carried out in H2O/TFA 99.9/0.1.
10
652
13
89
19
81
421
>10000
rp
0.7
13.2
3.3
4.8
0.1
n/a
In vitro enzyme inhibition assays were used to compare the
potency of the monovalent and multivalent compounds against
the physiologically relevant CA isoforms – the cytosolic hCA I
and II – as well as the transmembrane and tumor-associated
hCA IV, VII, IX and XII. First of all, we observed than no
inhibition is observed with the peptide scaffolds 11 and 12
(Table 1). In the oxime series, we observed that the monovalent
conjugate 10 is a potent CA inhibitor displaying a marked
selectivity for hCAIX (Ki = 36 nM, Table 1).
rp/n
14
0.18
55
1.2
n/a
0.02
481
n/a
51
608
>10000
n/a
-
-
-
rp
1.2
4.9
0.6
0.1
rp/n
0.3
1.2
0.15
n/a
0.02
[a] Errors were in the range of ±10% of the reported values (data not shown).
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For the inhibition of hCA II a positive multivalent effect could be
detected since the relative potency per sulfonamide inhibitor
observed here and operate through an increase in the local
concentration of CA inhibitors. In addition to structural data
about possible sites for secondary interactions,[12] this versatile
methodology should facilitate the development of more
comprehensive structure-activity relationships in this area of CA
inhibition by multivalent systems.
(rp/n) increases by
a factor of 3.3 comparing to the
corresponding monovalent compound that contains the exact
same sulfonamide-type inhibitor.
The multivalent conjugate built from the linear peptide scaffold
(compound 14, Table 1) turns out to be less potent on all CA
isoforms than the multivalent conjugate built from the cyclic
peptide scaffold (compound 13, Table 1).
Experimental Section
In the hydrazone series, we found that the monovalent
conjugate 9 is a very good inhibitor of hCA IX (Ki = 9 nM, Table
2), with a good selectivity among the isoforms tested. Multivalent
effects were observed with conjugate 15 on hCA isoforms II, IV
and XII (rp/n > 1, Table 1). Again, the multivalent conjugate
made of the cyclic scaffold (compound 15) shows an improved
potency when compared to the multivalent conjugate made of
the linear scaffold (compound 16, Table 2).
General
All reagents and solvents were of commercial quality and used without
further purification. TLC analyses were performed on silica gel 60 F254
plates (Merck), and visualized either under 254nm UV illumination, or by
ninhydrin staining. Melting points were determined on a Büchi Melting
Point 510. 1H and 13C NMR spectra were recorded on Brucker Avance
400 instruments and were referenced with respect to the residual solvent
peak. Data are reported as follows: chemical shift (δ in ppm), multiplicity
(s for singlet, d for doublet, t for triplet, m for multiplet, dd for doublet of
doublet and br of broad), coupling constant (J in Hertz) and integration.
Flash chromatography was performed on silica gel (40–63 μm)
purchased from Merck. HPLC analyses were performed on a Waters
HPLC 2695 (EC Nucleosil 300-5 C18, (125 × 3 mm) column, Macherey-
Nagel) equipped with a Waters 996 DAD detector. The following linear
Table 2. Results of the CA inhibition assay for the monovalent compound 9,
and the corresponding multivalent conjugates 15 and 16, in the hydrazone
series.
Ki (nM)[a]
Cpds
9
hCA I
48
hCA II
619
53
hCA IV
811
80
hCA VII
97
hCA IX
9
hCA XII
817
103
7.9
gradients of solvent
B
(acetonitrile) into solvent
A
(100 mM
triethylammonium acetate/acetonitrile 95/5) were used: 0 to 95% of
solvent B in 45 min. Retention times (tR) are given in minutes. Semi-
preparative RP-HPLC was performed on a Waters 515 HPLC (VP
Nucleodur HTec C18, 7 μm, (250 × 21) column, Macherey-Nagel)
equipped with a Waters 2487 detector. Method A: linear gradient of
acetonitrile into (100 mM triethylammonium acetate/acetonitrile 95/5): 0%
to 40% in 45 min. Method B: the following linear gradient of
acetonitrile/TFA 99.9/0.1 into H2O: 0% to 40% in 45 min. MALDI-TOF
and high resolution mass spectrometry analyses were carried out on an
Ultraflex III and a Waters MicromassQ ToF mass spectrometer (positive
mode).
15
329
0.1
34
100
0.1
rp
11.7
2.9
10.1
2.5
2.8
rp/n
16
0.02
460
0.1
0.7
0.02
10
1.9
89
428
1.9
75
938
0.9
rp
6.9
1.3
0.9
rp/n
0.02
1.7
0.5
0.3
0.2
0.2
Synthesis
[a] Errors were in the range of ±10% of the reported values (data not shown).
3-chloropropane-1-sulfonamide (1). 1.2 mL of ammonium hydroxide
solution (25% in water) was added to a solution of 3-chloro-propane
sulfonyl chloride (0.14 mL; 1.1 mmol; 1 equiv.) in DCM (1.2 mL). After
stirring 3 hours, the solvent was removed under reduced pressure. The
residue was triturated in DCM, and the precipitate was filtered and
washed with DCM. The filtrate was then concentrated in vacuo to give a
colourless oil. Yield: 95 %; 1H NMR (400 MHz, CDCl3) d 5.25 (s, 2H,
SO2NH2), 3.70 (t, J = 6.2 Hz, 2H, CH2-SO2NH2), 3.35 (t, J = 6.2 Hz,
2H,Cl-CH2), 2.37 – 2.26 (m, 2H, CH2-CH2-CH2); 13C NMR (101 MHz,
CDCl3) δ 52.55 (CH2-SO2NH2), 42.65 (Cl-CH2), 27.07 (CH2-CH2-CH2).
Conclusions
We reported here a straightforward approach that enables the
effective preparation of multivalent CA inhibitors from
functionalized peptide scaffolds. The enzyme inhibition assays
revealed that multivalent effects occur specifically with a couple
of CA isoforms, and more specifically hCA II. This phenomenon
could be due to the existence of a secondary binding site as
previously suggested, or by the ability of certain isoforms to
cluster favorably onto the multivalent system. We also observed
structural differences, the nature of the scaffold – cyclic versus
linear – having a surprising effect on the potency of these CA
inhibitors. The fact that pre-organized conjugates 13 and 15
show an enhanced potency compared to conjugates 14 and 16
seem to indicate that the structural fit between CAs and the
multivalent inhibitor play an important role. Thus, it is possible
that statistical effects are at the origin of the multivalent effects
3-((1,3-dioxoisoindolin-2-yl)oxy)propane-1-sulfonamide (2). To
a
solution of compound 1 (0.3 g; 1.9 mmol; 1 equiv.) in DMF (2 mL) were
added successively N-hydroxyphtalimide (0.22 g; 2.2 mmol; 1.2 equiv.),
tetrabutylammonium iodide (0.85 g; 2.2 mmol; 1.2 equiv.) and
triethylamine (0.18 mL; 0.22 mmol; 1.2 equiv.). After refluxing overnight,
the mixture was filtered and the precipitate washed with AcOEt. The
mixture was then washed twice with a saturated solution of NaHCO3, and
brine. The organic layer was dried over anhydrous sodium sulfate,
filtered and concentrated under vacuum. The resulting powder was re-
cristallized from ethanol to give a white solid. Yield: 62 %; mp: 181°C;
HR-ESI-MS [M+H]+ calculated for [C11H13N2O5S]+: 285.0545 found
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285.0550; 1H NMR (400 MHz, DMSO-d6) d 7.86 (s, 4H, CHar), 6.89 (s, 2H,
SO2NH2), 4.25 (t, J = 6.4 Hz, 2H, O-CH2), 3.22 – 3.19 (m, 2H, CH2-
SO2NH2), 2.14 – 2.03 (m, 2H, CH2-CH2-CH2). 13C NMR (101 MHz,
DMSO-d6) d 163.67 (C=O), 134.97 (CHar), 129.04 (CHar), 123.41 (CHar),
76.47 (O-CH2), 51.68 (CH2-SO2NH2), 23.40 (CH2-CH2-CH2).
NMR (400 MHz, CDCl3) d 6.55 (br, 1H, CH2-NH), 5.43 (br, 1H, CH-NH),
4.10 (s, 1H, CH), 3.83 – 3.71 (m, 1H, CH-CH2), 3.34 (s, 1H, CH-CH2),
3.26 (dd, J = 12.8, 6.6 Hz, 2H, CH2-NH), 1.44 (3, 11H, CH2-CH2-CH2,
NH-Boc), 1.39 – 1.26 (m, 2H, CH2-CH3), 1.17 (s, 9H, O-tBu), 0.91 (t, J =
7.3 Hz, 3H, CH2-CH3). 13C NMR (101 MHz, CDCl3) d 170.62 (CH-C=O),
155.58 ((C=O)O-tBu), 73.92 (C(CH3)3) 62.00 (CH-CH2), 39.20 (CH2-NH),
38.70 (CH), 31.65 (CH2-CH2-CH2), 28.40 (C(CH3)3), 27.51 (C(CH3)3),
20.03 (CH2-CH3), 13.74 (CH2-CH3). HR-ESI-MS [M+H]+ calculated for
[C16H33N2O4]+: 317.2440, found 317.2440.
3-(aminoxy)propane-1-sulfonamide (3). Methylhydrazine (36 µL; 0.7
mmol; 2 equiv.) was added to a solution of compound 2 (0.1 g; 0.35
mmol; 1 equiv.) in EtOH (1 mL). After stirring at room temperature for 4
hours, the mixture was concentrated under vacuum and the residue was
purified on silica gel using AcOEt/EtOH (9/1) as eluent. The expected
compound was obtained as a colourless oil. Yield: 83 %; 1H NMR (400
MHz, DMSO-d6) d 6.78 (s, 2H, SO2NH2), 6.00 (br, 2H, O-NH2), 3.58 (t, J
= 6.3 Hz, 2H, O-CH2), 3.01 – 2.93 (m, 2H, CH2-SO2NH2), 1.97 – 1.85 (m,
2H, CH2-CH2-CH2); 13C NMR (101 MHz, DMSO-d6) d 72.64 (O-CH2),
51.67 (CH2-SO2NH2), 22.98 (CH2-CH2-CH2); HR-ESI-MS [M+H]+
calculated for [C3H11N2O3S]+: 155.0490, found 155.0492.
(2S)-N-butyl-2-amino-3-hydroxypropanamide (8). Compound 7 (0.69
g; 2.2 mmol; 1 equiv.) was added to 28 mL of a solution of TFA/TIS/H2O
(95/2.5/2.5) and stirred for 4 hours at room temperature. The mixture was
then concentrated under vacuum and the residue was triturated with
Et2O. The resulting precipitate was filtered and washed with Et2O to give
compound 8 as a white solid. Yield: 96 %; mp: 99°C; 1H NMR (400 MHz,
DMSO-d6) d 8.36 (t, J = 5.4 Hz, 1H, CH2-NH), 8.11 (s, 2H, CH-NH2), 5.52
(br, 1H, CH2-OH), 3.80 – 3.60 (m, 3H, CH-CH2), 3.11 (dd, J = 12.5, 6.8
Hz, 2H, CH2-NH), 1.46 – 1.35 (m, 2H, CH2-CH2-CH2), 1.31 – 1.22 (m, 2H,
CH2-CH3), 0.87 (t, J = 7.3 Hz, 3H, CH2-CH3); 13C NMR (101 MHz,
DMSO-d6) d 166.60 (CH-C=O), 60.61 (CH-CH2), 54.66 (CH), 38.44 (CH2-
NH), 30.93 (CH2-CH2-CH2), 19.42 (CH2-CH3), 13.62 (CH2-CH3); HR-ESI-
MS [M+H]+ calculated for [C7H17N2O2]+: 161.1290, found 161.1288.
Methyl 3-(4-sulfamoylphenyl)propanoate (4). Hydrocinnamic acid (3.0
g; 19.9 mmol; 1 equiv.) was slowly added to chlorosulfonic acid (7.9 mL;
119.4 mmol; 6 equiv.) at 0°C. The reaction mixture was stirred at 0°C (ice
bath) for 1 hour and then warmed to room temperature and stirred for 3
hours. The suspension was filtered and the solid was washed with cold
water, then dried in vacuo. The resulting solid was added to a solution of
ammonium hydroxide (3 mL, 25% in water) and stirred at 60°C overnight.
After addition of 2 mL of water, the mixture was acidified to pH 2-3 with a
3M aqueous solution of HCl. After concentration under vacuum the
obtained yellow solid was refluxed 3 hours in a solution of 1.8 mL sulfuric
acid in 40 mL of methanol. The solvent was then removed under reduced
pressure and the residue obtained was triturated with Et2O to afford a
white precipitate, which was filtered, washed with Et2O and dried in
vacuo. Yield: 12%; mp: 109°C; 1H NMR (400 MHz, DMSO-d6) d 7.72 (d, J
= 8.4 Hz, 2H, CHar), 7.41 (d, J = 8.4 Hz, 2H, CHar), 7.29 (s, 2H, SO2NH2),
3.56 (s, 3H, O-CH3), 2.91 (t, J = 7.5 Hz, 2H, CH2-CH2-C=O), 2.67 (t, J =
7.5 Hz, 2H, CH2-CH2-C=O); 13C NMR (101 MHz, DMSO-d6) d 172.29
(C=O), 145.23 (Car-SO2NH2), 142.19 (Car-CH2), 128.79 (CHar), 125.74
(CHar), 51.40 (O-CH3), 34.37 (CH2-CH2-C=O), 29.95 (CH2-CH2-C=O);
HR-ESI-MS [M+H]+ calculated for [C10H14NO4S]+: 244.0644 found
244.0645.
N-butyl-2-(2-(3-(4-sulfamoylphenyl)propanoyl)hydrazono)acetamide
(9). Compound 8 (0.1 g; 0.6 mmol, 1 equiv.) was dissolved in 60 mL of
aqueous buffer (100 mM sodium acetate, pH 5.0) and then NaIO4 (0.14
g; 0.66 mmol, 1.1 equiv.) was added. The resulting mixture was stirred 3
hours at room temperature, and then quenched with Na2S2O3 (0.1 g; 0.66
mmol, 1.1 equiv.). Hydrazide 5 (0.3 g; 1.2 mmol, 2 equiv.) was then
added and the solution was stirred overnight at room temperature. The
mixture was extracted twice with AcOEt and the organic layer was
washed with brine, then dried over anhydrous sodium sulfate and filtered.
The filtrate was concentrated under vacuum and the residue was purified
on silica gel using AcOEt/Cyclohexane (8/2) as eluent. The expected
compound 9 was obtained as a white solid. Yield: 27 %; mp: 119°C; 1H
NMR (400 MHz, DMSO-d6) d 10.5 (br, 1H, CH=N), 8.20 (t, J = 5.9 Hz,
1H, CH2-NH-C=O), 7.72 (d, J = 8.3 Hz, 2H, CHar), 7.43 (dd, J = 13.7, 8.2
Hz, 2H, CHar), 7.25 (s, 2H, SO2NH2), 3.13 (dd, J = 13.3, 6.8 Hz, 2H, CH2-
NH), 3.04 – 2.87 (m, 4H, CH2-CH2-C=O), 1.49 – 1.35 (m, 2H, CH2-CH2-
CH2), 1.27 (dt, J = 14.5, 7.3 Hz, 2H, CH2-CH3), 0.87 (t, J = 7.3 Hz, 3H,
CH2-CH3); δ 13C NMR (101 MHz, DMSO-d6) d 173.99 (N-NH-C=O),
162.42 (CH2-NH-C=O), 145.59 (Car-SO2NH2), 141.78 (Car-CH2), 136.96
(CH=N), 128.92 (CHar), 125.66 (CHar), 38.02 (CH2-NH), 32.80 (CH2-CH2-
C=O), 31.26 (CH2-CH2-CH2), 29.46 (CH2-CH2-C=O), 19.68 (CH2-CH3),
13.68 (CH2-CH3); HR-ESI-MS [M+H]+ calculated for [C15H23N4O4S]+:
355.1440, found 355.1445.
4-(3-hydrazinyl-3-oxopropyl)benzenesulfonamide (5). A solution of
compound 4 (0.6 g; 2.6 mmol; 1 equiv.) in methanol (27 mL) was
refluxed for 7 hours with hydrazine monohydrate (1.2 mL; 26.2 mmol, 10
equiv.). Then the reaction mixture was cooled at 0°C for 30 min until
formation of a precipitate. The latter was filtered, washed with methanol
and dried in vacuo. The solid was re-cristallized from ethanol to give the
expected compound as a white solid. Yield: 67%; mp: 128°C; 1H NMR
(400 MHz, DMSO-d6) d 9.01 (s, 1H, NH-NH2), 7.75 (d, J = 8.4 Hz, 2H,
CHar), 7.37 (d, J = 8.4 Hz, 2H, CHar), 7.29 (s, 2H, SO2NH2), 4.18 (br, 2H,
NH-NH2), 2.88 (t, J = 7.6 Hz, 2H, CH2-CH2-C=O), 2.35 (t, J = 7.6 Hz, 2H,
CH2-CH2-C=O); 13C NMR (101 MHz, DMSO-d6) d 170.65 (C=O), 145.77
(Car-SO2NH2), 142.16 (Car-CH2), 128.83 (CHar), 125.79 (CHar), 34.60
(CH2-CH2-C=O), 30.74 (CH2-CH2-C=O). HR-ESI-MS [M+H]+ calculated
for [C9H14N3O3S]+: 244.0756, found 244.0760.
N-butyl-2-((3-sulfamoylpropoxy)imino)acetamide (10). Compound 10
was prepared following the same procedure as for compound 9 but using
oxyamine 3 (0.1 g; 0.65 mmol, 1 equiv.) in 65 mL of a mixture H2O/TFA
(99.9/0.1). The expected compound was obtained as a yellow oil. Yield:
12 %; 1H NMR (400 MHz, DMSO-d6) d 8.27 (t, J = 5.7 Hz, 1H, NH), 7.56
(s, 1H, CH), 6.83 (s, 2H, SO2NH2), 4.22 (t, J = 6.4 Hz, 2H, O-CH2), 3.18 –
3.08 (m, 2H, CH2-SO2NH2), 3.07 – 2.99 (m, 2H, CH2-NH), 2.08 – 2.03 (m,
J = 12.2, 6.5 Hz, 2H, O-CH2-CH2), 1.48 – 1.36 (m, 2H, CH2-CH2-CH2),
1.33 – 1.19 (m, 2H, CH2-CH3), 0.87 (t, J = 7.3 Hz, 3H, CH2-CH3); 13C
NMR (101 MHz, DMSO-d6) d 160.83 (NH-C=O), 144.37 (CH=N), 72.35
(O-CH2), 51.09 (CH2-SO2NH2), 38.19 (CH2-NH), 30.97 (CH2-CH2-CH2),
23.64 (O-CH2-CH2), 19.50 (CH2-CH3), 13.60 (CH2-CH3); HR-ESI-MS
[M+H]+ calculated for [C9H20N3O4S]+: 266.1175, found 266.1176.
(2S)-N-butyl-2-([tert-butoxycarbonyl]amino)-3-(tert-butoxy)
propanamide (7). In a solution of Boc-Ser(Otbu)-succinimide (1 g; 2.8
mmol; 1 equiv.) in DCM (16 mL) was added n-butylamine (0.36 mL; 3.6
mmol; 1.3 equiv.) and N,N-diisopropylethylamine (1.9 mL; 11 mmol; 4
equiv.). After stirring at room temperature overnight, the mixture was
washed successively with 1N aqueous solution of HCl, saturated solution
of NaHCO3, and brine. The organic layer was dried over anhydrous
sodium sulphate and filtered. The filtrate was concentrated under
vacuum to get compound 7 as a white solid. Yield: 84 %; mp: 71°C; 1H
General procedure for the synthesis of oxime conjugates 13 and 14.
8 equiv. (per peptide scaffold) of aminooxy compound 3 were added,
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10.1002/chem.201700241
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from a 200 mM stock solution in H2O/DMF (9/1), onto a 1 mM solution of
scaffold 11 or 12 in H2O/TFA (99.9/0.1). The mixture was stirred
overnight at room temperature then purified on semi-preparative RP-
HPLC using the method B (see general conditions) to afford a white solid
after freeze-drying.
Keywords: multivalency • enzyme inhibition • carbonic
anhydrase • bioconjugation
[1]
a) E. Mahon, M. Barboiu, Org. Biomol. Chem., 2015, 13, 10590-10599;
b) C. Fasting, C. A. Schalley, M. Weber, O. Seitz, S. Hecht, B. Koksch,
J. Dernedde, C. Graf, E. W. Knapp, R. Haag, Angew. Chem. Int. Ed.,
2012, 51, 10472-10498; c) M. Mammen, S. K. Choi, G. M. Whitesides,
Angew. Chem. Int. Ed., 1998, 37, 2755-2794.
Oxime conjugate 13. Yield: 31 %; HPLC: tR = 9.91 min; MALDI-ToF (α-
cyano-4-hydroxycinnamic acid): calculated for [C64H110N22O26S4 + Na]+
1753.66, found 1753.69.
[2]
[3]
O. Renaudet, R. Roy, Chem. Soc. Rev., 2013, 42, 4507-4844.
N. Kanfar, E. Bartolami, R. Zelli, A. Marra, J.-Y. Winum, S. Ulrich, P.
Dumy, Org. Biomol. Chem., 2015, 13, 9894-9906.
[4]
a) C. Matassini, C. Parmeggiani, F. Cardona, A. Goti, Tet. Lett., 2016,
57, 5407-5415; b) R. Zelli, J.-F. Longevial, P. Dumy, A. Marra, New J.
Chem., 2015, 39, 5050-5074; c) P. Compain, A. Bodlenner,
ChemBioChem, 2014, 15, 1239-1251; d) S. G. Gouin, Chem. Eur. J.,
2014, 20, 11616-11628.
Oxime conjugate 14. Yield: 75 %; HPLC: tR = 8.23 min; MALDI-ToF (α-
cyano-4-hydroxycinnamic acid): calculated for [C66H114N22O28S4 + Na]+
1813.69, found 1813.65.
[5]
[6]
C. T. Supuran, Biochem. J., 2016, 473, 2020-2032.
V. Alterio, A. Di Fiore, K. D'Ambrosio, C. T. Supuran, G. De Simone,
Chem. Rev., 2012, 112, 4421-4468.
A. Scozzafava, C. T. Supuran, Subcell Biochem., 2014, 75, 349-359.
C. T. Supuran, J. Y. Winum, Future Medicinal Chemistry, 2015, 7,
1407-1414.
A. Thiry, J. M. Dogne, C. T. Supuran, B. Masereel, Curr. Pharm. Des.,
2008, 14, 661-671.
General procedure for the synthesis of hydrazone conjugates 15
and 16. 8 equiv. (per peptide scaffold) of hydrazide 5 were added, from a
200 mM stock solution in H2O/DMF (9/1), onto a 1 mM solution of
scaffold 11 or 12 in aqueous buffer (100 mM sodium acetate, pH 5.0).
The mixture was stirred overnight at room temperature and then purified
on semi-preparative RP-HPLC using method A (see general conditions)
to afford a white solid after freeze-drying.
[7]
[8]
[9]
[10] A. Casini, F. Abbate, A. Scozzafava, C. T. Supuran, Bioorg. Med. Chem.
Lett., 2003, 13, 2759-2763.
[11] a) N. Krall, F. Pretto, D. Neri, Chem. Sci., 2014, 5, 3640-3644; b) F.
Buller, M. Steiner, K. Frey, D. Mircsof, J. Scheuermann, M. Kalisch, P.
Buhlmann, C. T. Supuran, D. Neri, ACS Chem. Biol., 2011, 6, 336-344.
[12] V. M. Krishnamurthy, G. K. Kaufman, A. R. Urbach, I. Gitlin, K. L.
Gudiksen, D. B. Weibel, G. M. Whitesides, Chem. Rev., 2008, 108,
946-1051.
Hydrazone conjugate 15. Yield: 35 %; HPLC: tR = 13.24 min; MALDI-
ToF (α-cyano-4-hydroxycinnamic acid): calculated for [C88H122N26O26S4 +
Na]+ 2109.77, found 2109.81.
[13] N. Touisni, N. Kanfar, S. Ulrich, P. Dumy, C. T. Supuran, A. Mehdi, J.-Y.
Hydrazone conjugate 16. Yield: 61 %; HPLC: tR = 12.25 min; MALDI-
ToF (α-cyano-4-hydroxycinnamic acid): calculated for [C90H126N26O28S4 +
Na]+ 2169.79, found 2169.84.
Winum, Chem. Eur. J., 2015, 21, 10306-10309.
[14] M. Stiti, A. Cecchi, M. Rami, M. Abdaoui, V. Barragan-Montero, A.
Scozzafava, Y. Guari, J. Y. Winum, C. T. Supuran, J. Am. Chem. Soc.,
2008, 130, 16130-16131.
[15] J. Y. Winum, C. T. Supuran, J. Enzyme Inhib. Med. Chem., 2015, 30,
CA inhibition assay. An Applied Photophysics stopped-flow instrument
has been used for assaying the CA catalyzed CO2 hydration activity.
Phenol red (at a concentration of 0.2 mM) has been used as indicator,
working at the absorbance maximum of 557 nm, with 20 mM Hepes (pH
7.5) as buffer, and 20 mM Na2SO4 (for maintaining constant the ionic
strength), following the initial rates of the CA-catalyzed CO2 hydration
reaction for a period of 10-100 s. The CO2 concentrations ranged from
1.7 to 17 mM for the determination of the kinetic parameters and
inhibition constants. At maximum the concentrations used in the assay
system were: 9.2 nM for hCA I; 7.8 nM for hCA II; 10.2 nM for hCA IX
and 14.1 nM for hCA XII. For each inhibitor at least six traces of the initial
5-10% of the reaction have been used for determining the initial
velocity.[28] The uncatalyzed rates were determined in the same manner
and subtracted from the total observed rates. Stock solutions of inhibitor
(0.1 mM) were prepared in distilled-deionized water and dilutions up to
0.01 nM were done thereafter with distilled-deionized water. Inhibitor and
enzyme solutions were preincubated together for 15 min - 2 h (or longer,
i.e., 4-6 h) at room temperature (at 4°C for the longer incubation periods
than 15 min) prior to assay, in order to allow for the formation of the E-I
complex. The inhibition constants were obtained by non-linear least-
squares methods using PRISM 3, and represent the mean from at least
three different determinations.
321-324.
[16] M. Abellan-Flos, M. Tanc, C. T. Supuran, S. P. Vincent, J. Enzyme
Inhib. Med. Chem., 2016, 31, 946-952.
[17] M. Abellán-Flos, M. Tanç, C. T. Supuran, S. P. Vincent, Org. Biomol.
Chem., 2015, 13, 7445-7451.
[18] a) F. Carta, S. M. Osman, D. Vullo, A. Gullotto, J. Y. Winum, Z.
AlOthman, E. Masini, C. T. Supuran, J. Med. Chem., 2015, 58, 4039-
4045; b) F. Carta, S. M. Osman, D. Vullo, Z. AlOthman, C. T. Supuran,
Org. Biomol. Chem., 2015, 13, 6453-6457.
[19] A.-M. Caminade, A. Ouali, R. Laurent, C.-O. Turrin, J.-P. Majoral, Chem.
Soc. Rev., 2015, 44, 3890-3899.
[20] S. Ulrich, D. Boturyn, A. Marra, O. Renaudet, P. Dumy, Chem. Eur. J.,
2014, 20, 34-41.
[21] a) R. Zelli, E. Bartolami, J. F. Longevial, Y. Bessin, P. Dumy, A. Marra,
S. Ulrich, RSC Adv., 2016, 6, 2210-2216; b) E. Bartolami, Y. Bessin, N.
Bettache, M. Gary-Bobo, M. Garcia, P. Dumy, S. Ulrich, Org. Biomol.
Chem., 2015, 13, 9427-9438.
[22] a) S. Peluso, T. Ruckle, C. Lehmann, M. Mutter, C. Peggion, M. Crisma,
Chembiochem, 2001, 2, 432-437; b) P. Dumy, I. M. Eggleston, G.
Esposito, S. Nicula, M. Mutter, Biopolymers, 1996, 39, 297-308.
[23] B. Thomas, N. Berthet, J. Garcia, P. Dumy, O. Renaudet, Chem.
Commun., 2013, 49, 10796-10798.
[24] a) D. Boturyn, E. Defrancq, G. T. Dolphin, J. Garcia, P. Labbe, O.
Renaudet, P. Dumy, J. Peptide Sci., 2008, 14, 224-240; b) Y. Singh, G.
T. Dolphin, J. Razkin, P. Dumy, ChemBioChem, 2006, 7, 1298-1314.
[25] T. Tsuri, N. Haga, T. Matsui, S. Kamata, H. Kakushi, K. Uchida, Chem.
Pharm. Bull., 1992, 40, 75-84.
[26] S. Foillard, M. O. Rasmussen, J. Razkin, D. Boturyn, P. Dumy, J. Org.
Chem., 2008, 73, 983-991.
[27] E. Bartolami, Y. Bessin, V. Gervais, P. Dumy, S. Ulrich, Angew. Chem.
Int. Ed., 2015, 54, 10183-10187.
[28] R. G. Khalifah, The Journal of Biological Chemistry, 1971, 246, 2561-
Acknowledgements
2573.
We thank the Ligue contre le Cancer (comité des Pyrénées-
Orientales) and the LabEx CheMISyst (ANR-10-LABX-05-01) for
funding. N.K. thanks the French Ministère de la Recherche for a
doctoral fellowship.
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10.1002/chem.201700241
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Entry for the Table of Contents (Please choose one layout)
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The application of multivalency in
enzyme inhibition has been recently
exemplified by a few serendipitous
Nasreddine Kanfar, Muhammat Tanc,
Pascal Dumy, Claudiu T. Supuran,
Sébastien Ulrich* and Jean-Yves
Winum*
discoveries. We report here
a
straightforward and versatile method
that enables the effective construction
of multivalent peptide-based systems
Page No. – Page No.
Effective Access to Multivalent
Inhibitors of Carbonic Anhydrases
Promoted by Peptide Bioconjugation
for
the
inhibition
of
carbonic
anhydrases. The enzyme inhibition
assays show that multivalent effects
were present with these novel
compounds.
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